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List of Contributors
Mahmoud Abu-Shakra Autoimmune Rheumatic Diseases Unit Soroka Medical Center Ben-Gurion University Beer-Sheva Israel
Anat Achiron Multiple Sclerosis Center Sheba Medical Center Tel-Hashomer Israel Miranda K. Adelman Arizona Arthritis Center The University of Arizona Tucson, AZ USA Marina Afanasyeva Department of Physiology and Biophysics Faculty of Medicine University of Calgary Calgary, Alberta Canada Jun Akaogi Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA Jorge Alcocer-Varela Department of Immunology and Rheumatology Instituto Nacional de Ciencias M6dicas y Nutrici6n Salvador Zubir~in Mexico City Mexico
Amedeo Amedei Department of Internal Medicine University of Florence Florence Italy
Ben J. Appelmelk Department of Medical Microbiology Vrije Universiteit, Medical School Amsterdam The Netherlands
Anabel Aron-Maor Center for Autoimmune Diseases Department of Medicine 'B' Sheba Medical Center Tel Hashomer Israel
Ronald A. Asherson Rheumatic Diseases Unit University of Cape Town School of Medicine Cape Town South Africa
Leonard H. van den Berg Department of Neurology University Medical Center Utrecht Utrecht The Netherlands Mathijs P. Bergman Department of Medical Microbiology Vrije Universiteit, Medical School Amsterdam The Netherlands
~
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M. Blank Internal Medicine 'B' The Center of Autoimmune Diseases Sheba Medical Center Tel Hashomer Israel
Stefano Bombardieri Rheumatology Unit Department of Intemal Medicine University of Pisa, Medical School Pisa Italy
Milan Buc Department of Immunology School of Medicine Bratislava Slovak Republic
James Bussei Pediatric Hematology/Oncology Weill Medical College of Cornell University New York, NY USA
Irun R. Cohen Department of Immunology The Weizmann Institute of Science Rehovot Israel
Pascal Cohen Department of Internal Medicine H6pital Cochin University of Paris Paris France
J.C. Crispin Acufia Department of Immunology and Rheumatology Instituto Nacional de Ciencias M6dicas y Nutrici6n Salvador Zubirdn Mexico City Mexico
Edecio Cunha-Neto Laboratory of Immunology Heart Institute (Incor) University of Sao Paulo, School of Medicine S~o Paulo Brazil
Franceseo Cainelli Section of Infectious Diseases Department of Pathology University of Verona Verona Italy
Maurizio Cutolo Department of Internal Medicine University of Genova Genova Italy
Ricard Cervera Department of Autoimmune Diseases Institut Clinic d'Infeccions i Immunologia Hospital Clinic Barcelona, Catalonia Spain
Michael David Rabin Medical Center Department of Dermatology Petah-Tiqva Israel
Alexander J. Chou Pediatric Hematology/Oncology Weill Medical College of Comell University New York, NY USA
Mario M. D'Elios Department of Internal Medicine University of Florence Florence Italy
viii
Gianfranco Del Prete Department of Internal Medicine University of Florence Florence Italy
A.M. Denman Northwick Park Hospital Harrow England Barbara Detrick School of Medicine The Johns Hopkins University Baltimore, MD USA Andrea Doria Division of Rheumatology Department of Medical and Surgical Science University of Padova Padova Italy C. Dugu6 Laboratory of Immunology Brest University Medical School Brest France
Clodoveo Ferri Rheumatology Unit Department of Internal Medicine University of Modena, Medical School Modena Italy
Johan FrostegArd Department of Medicine Unit of Rheumatology Karolinska Hospital Stockholm Sweden
Pier Franca Gambari Division of Pdaeumatology Department of Medical and Surgical Science University of Padova Padova Italy
Marjorie A. Garvey National Institute of Mental Health Department of Health and Human Services Bethesda, MD USA
Malarvizhi Durai Department of Microbiology and Immunology University of Maryland, School of Medicine Baltimore, MD USA
M. Eric Gershwin Division of Rheumatology, Allergy and Clinical Immunology University of California at Davis Davis, CA USA
Alan Ebringer Division of Life Sciences King's College University of London London UK
Anna Ghirardello Division of Rheumatology Department of Medical and Surgical Science University of Padova Padova Italy
Miriam Eisenstein Department of Chemical Services The Weizmann Institute of Science Rehovot Israel
Roberto Giacomelli Department of Internal Medicine University of L'Aquila L'Aquila Italy
ix
Serena Guiducci Department of Rheumatology University of Florence Florence Italy
Simone Gonqaives Fonseca Laboratory of Immunology Heart Institute (Incor) University of S~o Paulo, School of Medicine Sao Paulo Brazil Luiza Guilherme Heart Institute (InCor) University of S~o Paulo, School of Medicine Sao Paulo Brazil Lo'ic Guillevin Department of Internal Medicine H6pital Cochin University of Paris Paris France David A. Hailer Laboratory of Molecular Immunology Center for Neurologic Diseases Brigham and Women's Hospital and Harvard Medical School Boston, MA USA Giilen Hatemi Department of Internal Medicine Division of Rheumatology Cerrahpasa Medical School Istanbul University Istanbul Turkey Xiao-Song He Division of Rheumatology, Allergy and Clinical Immunology University of California at Davis Davis, CA USA
John J. Hooks Laboratory of Immunology National Eye Institute National Institutes of Health Bethesda MD USA
Luca Iaccarino Division of Rheumatology Department of Medical and Surgical Science University of Padova Padova Italy
Steven Jacobson Viral Immunology Section NIH/NINDS Bethesda, MD USA
M.D. Jansen Department of Neurology University Medical Center Utrecht Utrecht The Netherlands
Luis J. Jara Clinical Research Unit Hospital de Especialidades Centro M6dico La Raza, IMSS Mexico City Mexico Hee-Sook Jun Center for Immunologic Research and Department of Microbiology and Immunology The Chicago Medical School North Chicago, IL USA Jorge Kalil Heart Institute- InCor University of S~o Paulo, School of Medicine S~o Paulo Brazil
Efstathia K. Kapsogeorgou Department of Pathophysiology Medical School National University of Athens Athens Greece
Dimitrios A. Liakos Department of Pathophysiology Medical School National University of Athens Athens Greece
Leo Kei Iwai Laboratory of Immunology Heart Institute (Incor) University of Sao Paulo, School of Medicine S~o Paulo Brazil
Dong-Gyun Lim Laboratory of Molecular Immunology Center for Neurologic Diseases Brigham and Women's Hospital and Harvard Medical School Boston, MA USA
Kindra M. Kelly Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA
Burkhard Ludewig Kantonal Hospital St. Gallen Research Department St. Gallen Switzerland
Ilan Krause Department of Medicine E Rabin Medical Center Petah-Tikva Israel
John J. Marchalonis Microbiology and Immunology The University of Arizona Tucson, AZ USA
Philippe Krebs Kantonal Hospital St. Gallen Research Department St. Gallen Switzerland Yoshiki Kuroda Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA Aaron Lerner Department of Pediatrics Carmel Medical Center Haifa Israel
Marco Matucci Cerinic Department of Rheumatology University of Florence Florence Italy Gabriela Medina Clinical Research Unit Hospital de Especialidades Centro Mrdico La Raza, IMSS Mexico City Mexico Stephen D. Miller Department of Microbiology-Immunology Feinberg School of Medicine Northwestern University Chicago, IL USA
xi
Gui Milo Department of Medicine E Rabin Medical Center Petah-Tikva Israel
Carmen Navarro Molecular Biology Department Instituto Nacional de Enfermedades Respiratorias Mexico City Mexico
Daniel Mimouni Rabin Medical Center Department of Dermatology Petah-Tiqva Israel
Robert Nussenblatt Laboratory of Immunology National Eye Institute National Institutes of Health Bethesda MD USA
Juan M. Miranda Rheumatology Department Hospital de Especialidades Centro M6dico La Raza, IMSS Mexico City Mexico
Angelina Morand B. Bilate Laboratory of Immunology Heart Institute (Incor) University of S~o Paulo, School of Medicine S~o Paulo Brazil
Kamal D. Moudgil Department of Microbiology and Immunology University of Maryland, School of Medicine Baltimore, MD USA Haralampos M. Moutsopoulos Department of Pathophysiology Medical School National University of Athens Athens Greece Dina C. Naeionales Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA
xii
Christian Pagnoux Department of Internal Medicine HOpital Cochin University of Paris Paris France
Michael P. Pender Department of Medicine Clincial Sciences Building Royal Brisbane and Women's Hospital Herston Queensland Australia
Jaques-Olivier Pers Laboratory of Immunology Brest University Medical School Brest France W-Ludo van der Pol Department of Neurology University Medical Center Utrecht Utrecht The Netherlands Mark E Prummel Department of Endocrinology & Metabolism Academic Medical Center University of Amsterdam The Netherlands
Francisco J. Quintana Department of Immunology The Weizmann Institute of Science Rehovot Israel
B. Rager-Zisman Department of Microbiology and Immunology The University Center for Cancer Research Ben Gurion University of the Negev Beer Sheva Israel
Taha Rashid Division of Life Sciences King's College University of London London UK
Westley H. Reeves Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA
Shimon Reif Division of Pediatric Gastroenterology Dana Children's Hospital Tel Aviv Israel
Jozef Rovensk~ National Institute for Rheumatic Diseases Pie~t' any Slovakia
Minoru Satoh Division of Rheumatology & Clinical Immunology Center for Systemic Autoimmune Diseases University of Florida Gainesville, FL USA
Reinhold E. Schmidt Department of Clinical Immunology Medical School Hannover Hannover Germany
Bruno Seriolo Department of Internal Medicine University of Genova Genova Italy
Marianne C. Severin Center for Autoimmune Diseases Department of Medicine 'B' Sheba Medical Center Tel Hashomer Israel
Yves Renaudineau Laboratory of Immunology Brest University Medical School Brest France
Yehuda Shoenfeld Center for Autoimmune Diseases Department of Medicine 'B' Sheba Medical Center Tel Hashomer Israel
Noel R. Rose Center for Autoimmune Disease Research Department of Pathology The John Hopkins Medical Institutions Baltimore, MD USA
Jean Sibilia Rheumatology department H6pitaux Universitaire de Strasbourg Universit6 Louis Pasteur Strasbourg France
xiii
Lisa A. Snider National Institute of Mental Health Department of Health and Human Services Bethesda, MD USA
Moshe Tishler Department of Medicine 'B' Asaf Harofe Medical Center Zerifin Israel
Samantha S. Soldan Viral Immunology Section NIH/NINDS Bethesda, MD USA
Yaron Tomer Division of Endocrinology Department of Medicine Mount Sinai School of Medicine New York, NY USA
N.M. van Sorge Departments of Neurology and Immunology University Medical Center Utrecht Utrecht The Netherlands
Allen C. Steere Center for Immunology and Inflammatory Diseases Division of Rheumatology, Allergy and Immunology Massachusetts General Hospital Harvard Medical School Boston, MA USA
Matthias Stoll Department of Clinical Immunology Medical School Hannover Hannover Germany
Alberto Sulli Department of Internal Medicine University of Genova Genova Italy Susan E. Swedo National Institute of Mental Health Department of Health and Human Services Bethesda, MD USA
xiv
Alan Tyndall Department of Rheumatology University of Basle Basle Switzerland
Christina M. Vandenbroucke-Grauls Department of Medical Microbiology Vrije Universiteit, Medical School Amsterdam The Netherlands
Carol L. VanderLugt-Castaneda Department of Biology Indiana University Northwest Gray, IN USA
Dimitrios Vassilopoulos Department of Medicine Hippokration General Hospital University of Athens School of Medicine Athens Greece Sandro Vento Section of Infectious Diseases Department of Pathology University of Verona Verona Italy
Olga Vera-Lastra Internal Medicine Department Hospital de Especialidades Centro M6dico La Raza, IMSS Mexico City Mexico
Ronald Villanueva Division of Endocrinology Department of Medicine Mount Sinai School of Medicine New York, NY USA
Dominique Wachsmann Unit6 INSERM U392 "Immunit6-Infection" Universit6 Louis Pasteur Illkirch-Graffenstaden France
Joel V. Weinstock University of Iowa Hospitals and Clinics Division of Gastroenterology Iowa City, IA USA
Georg Wick Institute for Pathophysiology University of Innsbruck Medical School Innsbruck Austria Wilmar M. Wiersinga Department of Endocrinology & Metabolism Academic Medical Center University of Amsterdam The Netherlands Clyde Wilson Division of Life Sciences King's College University of London London UK
Jan G.J. van de Winkel Department of Immunology University Medical Center Utrecht Utrecht The Netherlands
Kai W. Wucherpfennig Department of Cancer Immunology & Aids Dana Farber Cancer Institute Boston, MA USA
Qingbo Xu Department of Cardiological Sciences St. George's Hospital Medical School London UK
Hasan Yazici Department of Internal Medicine Division of Rheumatology Cerrahpasa Medical School Istanbul University Istanbul Turkey
David E. Yocum Arizona Arthritis Center University of Arizona Tucson, AZ USA
Ji-Won Yoon Center for Immunologic Research and Department of Microbiology and Immunology The Chicago Medical School North Chicago, IL USA Pierre Youinou Laboratory of Immunology Brest University Medical School Brest France
XV
Sandra Zampieri Division of Rheumatology Department of Medical and Surgical Science University of Padova Padova Italy
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Gisele Zandman-Goddard Center for Autoimmune Diseases Department of Medicine 'B' Sheba Medical Center Tel Hashomer Israel
9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Introduction: Infection and Autoimmunity Yehuda Shoenfeld ~and Noel R. Rose 2
1Centerfor Autoimmune Diseases, Department of Medicine 'B', Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases, Tel-Aviv University, Israel; 2Centerfor Autoimmune Disease Research, Department of Pathology, and Department of Molecular Microbiology & Immunology, The John Hopkins Medical Institutions, Baltimore, MD, USA
About 80 recognized autoimmune diseases fulfill the Rose-Bona criteria (Table 1). Yet many other conditions are claimed to be of autoimmune origin. While some would say that "everything is autoimmune until proven otherwise", reading the chapters in this book written by world leaders in autoimmunity brings one to the conclusion that everything after all is infectious until proven otherwise (including autoimmune diseases). The story started many years ago with infectious etiology of rheumatic fever (RF) (See Guilherme and Kalil). RF was long known to be induced by infection with beta-hemolytic streptococci. During the last decades the molecular mimicry between the M-protein of the streptococci and its peptides was delineated as a classical mechanism for autoimmunity. The structural similarities between the M-protein on the streptococcal membrane and on the cells of the heart, brain and joint synovium could explain the typical clinical manifestations in patients encountering RF (i.e. migrating arthritis, valve involvement and chorea). Furthermore, a genetic predisposition to RF is implied by the fact that only few infected individuals will be affected. The RF paradigm epitomizes the environmental agent-genetic background interplay in induction of autoimmunity as shown in Fig. 1.
Environmental factor
Autoimmune
Genetic
predisposition
,
I~ disease
Figure 1. The RF paradigm epitomizes the environmental agent-genetic background interplay in induction of autoimmunity.
1. THE OLD AND THE NEW It has taught us many valuable lessons that may be applicable to other autoimmune diseases. Nowadays RF is not only rarely seen in the industrialized countries of Europe and North America, a dramatic change usually attributed to the prompt use of antimicrobials in treating streptococcal infections. This experience suggests that if we can identify microbial agents that trigger an autoimmune disease, it may be possible to prevent the disease, even in genetically susceptible individuals. The classical autoimmune conditions, have been joined by newly described diseases having possible infectious etiology such as the anti-phospholipid syndrome (APS). Characterized originally (1982) by the triad of manifestations i.e. repeated thromboemboric phenomenon, recurrent pregnancy loss and thrombocytopenia [1], this disease became even-
Table 1. The Rose-Bona criteria for recognizing autoimmune diseases (Immunology Today 14: 426-430, 1993) DIRECT EVIDENCE A. Autoantibody-mediated 1. Circulating autoantibodies affecting function a. Destruction or sequestration of a target cell b. Interaction with receptor 1) Stimulated function 2) Impaired function c. Interaction with hormones or enzymes 2. Localized autoantibodies a. Demonstration of immunoglobulin and/or complement components at site of lesion b. Ability to elute antibodies from lesions c. Reproduction of lesions by immunoglobulin eluates 3. Localized immune complexes at site of lesion a. Elution of antibody-antigen complex b. Transfer to experimental animals 4. Reproduction of disease by passive transfer a. Maternal-fetal transfer b. Transfer to experimental animals c. Demonstration of in vitro injury to target cell B. Cell-mediated 1. Transfer of T cells to immunodeficient mice implanted with target organ 2. In vitro cytotoxicity of T cells with cells of target organ II. INDIRECT EVIDENCE A. Reproduction of Autoimmune Disease by Experimental Immunization 1. Identification of initiating antigen 2. Immunization of susceptible experimental animal with analogous antigen 3. Production of characteristic lesions 4. Reaction of antibody or T cells with an analogous antigen or epitope B. Reproduction of Autoimmune Disease through Idiotype Network 1. Identification of disease-associated idiotype 2. Immunization of susceptible host with the idiotype 3. Production of characteristic lesions C. Spontaneous Models in Experimental Animals 1. Identification of disease in an experimental animal 2. Breeding and selection to increase disease frequency 3. Demonstration of self-reactive antibodies/T cells 4. Passive transfer/adoptive transfer of disease to syngeneic recipients 5. Passive transfer/adoptive transfer of disease to syngeneic recipients D. Animal Models Produced by Dysregulation of the Immune System 1. Neonatal thymectomy 2. Irradiation with thymectomy 3. Cytokine-deficient homologous inbred animals 4. Transgenic animals with altered: a. cytokine production b. antigen expression c. co-stimulatory factor expression 11I. CIRCUMSTANTIAL EVIDENCE A. Presence of Autoantibodies B. Association with Other Autoimmune Diseases C. Association with MHC Haplotype D. Lymphocyte Infiltration of Target Organ 1. Presence of germinal centers in lesions 2. Restricted V-gene usage of infiltrating T cells E. Favorable Response to Immunosuppression 1. Nonspecific 2. Specific (including oral tolerance)
tually one of the most common systemic diseases [2] entailing valve involvement (Liebman-Sacks endocarditis) as well as CNS affliction (chorea and cognitive impairment). The APS recently was found to be induced by diverse infectious agents [3]. The syndrome for years was thought to be clinicially "associated" with infections. Yet, only employment of modem techniques such as phage library display, monoclonal autoantibodies and proteinomics led to the conclusive results that APS can be induced by peptides residing on infecting agents' membranes and on the autoantigen ~2-glycoprotein-I (~2GPI) [4]. The genetic predilection for the disease was reported by several groups [5]. Interestingly enough, some kind of cross-reactivity exists between the autoantigens and streptococci of both RF and APS which affects the heart and the brain. This is an example of how the old diseases like RF may relate to the new and modem aspects of others. Despite clinical and epidemiologic evidence that many, if not most, autoimmune diseases are triggered by infection, there are very few instances where the underlying mechanisms have been explored. Autoimmune myocarditis in humans often follows infection by enteroviruses. The disease can be reproduced in genetically susceptible strains of mice by infection with a cardiotropic strain of Coxsackievirus B3 [6]. The antigen in this disease has been identified as cardiac myosin, and the autoimmune disease reproduced without virus in susceptible mice by immunization with purified cardiac myosin heavy chain or its myocarditogenic peptide [7]. The antigen arises from the host itself, and the virus serves both to deliver endogenous intracellular myosin and to provide a favorable inflammatory microenvironment [8]. The book intends to encompass the different mechanisms involved in the infection-autoimmunity association/induction. In addition to molecular mimicry one will find mechanisms such as polyclonal activation, tissue damage and the adjuvant effect of the inflammatory process itself. Kai W. Wucherpfennig discusses the ability of T cells to recognize a number of peptides from different viral and bacterial antigens that are remarkably distinct in their primary sequence, thus indicating the degeneracy of TCR recognition. Kamal D. Moudgil and Malaruithi Du Rai discuss the diversification of
the immune system by an antigen to new T cell and/or antibody specificities during the course of an autoimmune disease known as "epitope spreading". Special attention will be given to HSPs and to trangenic mouse models to better understand infection-induced autoimmunity. Due to the fact that vaccine may contain infectious antigens, the controversial issue of vaccineautoimmunity relationship is critically reviewed.
2. INFECTING AGENTS INDUCING DIVERSE AUTOIMMUNE DISEASES There are notorious infecting agents i.e. EBV, hepatitis-C, parwovirus-19 and others which were reported to be linked to many autoimmune diseases. These are detailed in Section 2. But other infecting agents such as HIV, Theiler's murine encephalomyelitis virus (causing demyelinating disease), endogenous retroviruses (leading to SLE), parasites (i.e. trypanosoma c r u z i - causing Chagas' disease; Yersinia enterocolitica leading to autoimmune thyroid disease and the newly appearing players the Saccharomyces cervisiae), and endogenous retroviruses, will be detailed.
3. AUTOIMMUNE DISEASES INDUCED BY A VARIETY OF INFECTING AGENTS
Another viewpoint of the infection autoimmunity inter-relationship entails the various diseases associated with a variety of infections. Those are summarized in Section 3. Among the diseases having enough data to support an infectious origin, we include rheumatic fever, SLE, APS, Sjrgren's syndrome, polymyositis, IBD, reactive arthritis, autoimmune thyroid diseases, type I diabetes mellitus, autoimmune heart diseases, autoimmune liver diseases, vasculitides and multiple sclerosis. We hope that the readers of this book will gain a deeper insight into these increasingly important etiological aspects of autoimmunity.
REFERENCES 5. 1.
2. 3.
4.
Asherson RA, Cervera R, Piette JC, Shoenfeld Y, eds. The Antiphospholipid Syndrome II: Autoimmune Thrombosis. Amsterdam: Elsevier, 2002; 1457. Shoenfeld Y. Systemic antiphospholipid syndrome. Lupus 2003;12:497--498. Blank M, Krause I, Fridkin M, Keller N, Kopolovic J, Goldberg I, Tobar A, Shoenfeld Y. Bacterial induction of autoantibodies to 132-glycoprotein-I accounts for the infectious etiology of antiphospholipid syndrome. J Clin Invest 2002; 109:797-804. Blank M, Shoenfeld Y, Cabilly S, Heldman Y, Fridkin M, Katchalski-Katzir E. Prevention of experimental antiphospholipid syndrome and endothelial cell activation by synthetic peptides. Proc Natl Acad Sci (USA)
6.
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1999;96:5164-5168. Minisola G, Galeazzi M, Sebastiani DS. HLA class 1I alleles and genetic predisposition to the antiphospholipid syndrome. Autoimmun Rev 2003;2:387-394. Rose NR, Wolfgram LJ, Herskowitz A, Beisel KW. Postinfectious autoimmunity: two distinct phases of Coxsackievirus B3-induced myocarditis. Ann NY Acad Sci 1986;475:146-156. Neu N, Rose NR, Beisel KW, Herskowitz A, GurriGlass G, Craig SW. Cardiac myosin induces myocarditis in genetically predisposed mice. J Immunol 1987; 139:3630-363. Rose NR, Afanasyeva M. The inflammatory process in experimental myocarditis. In: Feuerstein GZ, Libby P, Mann DL, eds. Inflammation and Heart Disease. Basel: Birkhiiuser, 2003;325-333.
9 2004 Elsevier B. E All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Implications of T Cell Receptor Crossreactivity for the Pathogenesis of Autoimmune Diseases Kai W. Wucherpfennig
Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute & Department of Neurology, Harvard Medical School, Boston, MA, USA
1. HOW SPECIFIC IS TCR RECOGNITION OF MHC/PEPTIDE COMPLEXES? The experimental practice of isolating "antigenspecific" T cells by in vivo or in vitro selection with a particular antigen led to the widely held notion that TCR recognition is highly specific since T cell clones selected in such a fashion are typically not activated by control antigens. Within this conceptual framework it was thought that rare microbial antigens in which the peptide sequences were closely related to a self-peptide could represent mimics and be responsible for the induction of autoimmune diseases. Since it was assumed that such peptides would have to possess substantial sequence identity with the self-antigen, sequence alignments with self-peptides were used to identify candidate sequences. This approach was used to identify a peptide from the Hepatitis B virus DNA polymerase that induced histological signs of experimental autoimmune encephalomyelitis (EAE) following immunization of rabbits [1]. However, a large number of groups used this method to identify potential mimicry peptides with largely disappointing results since the vast majority of peptides had no biological activity. It thus appeared that TCR crossreactivity might be a very rare event. Given the paucity of definitive experimental data, the biological relevance of TCR crossreactivity was questioned by many basic scientists. However, structural studies on the interaction of MHC molecules with peptides and on TCR recognition of MHC/peptide complexes suggested that this view of TCR recognition might have to be
reconsidered. Peptide elution studies demonstrated that a single MHC molecule could bind a very large and diverse set of peptides since several hundred distinct peptide masses could be defined with mass spectrometry techniques [2, 3]. Investigation of the structural requirements for peptide binding by MHC class II molecules demonstrated that five peptide side chains contributed to binding [4]. However, each of these 'anchor residues' could typically be substituted by a number of other amino acids so that the resulting peptide binding motifs were highly degenerate [5]. For the multiple sclerosis (MS) associated HLA-DR2 molecule (DP~a., DRB 1" 1501) that has been the focus of our studies, three of these five peptide positions (P6, P7 and P9 pockets) could be substituted by many different amino acids. Even though a higher degree of specificity was observed for the P1 and P4 anchor residues, substitutions by structurally related amino acids were permitted [6]. The crystal structure of HLA-DR1 with a bound peptide from influenza hemagglutinin (HA, 306318) elucidated how peptides are bound with high affinity, despite such degenerate sequence motifs: a significant fraction of the binding energy is derived from interactions between the backbone of the peptide and conserved residues of the MHC class II binding cleft. This structure also demonstrated that the peptide is buried in the binding site such that substitutions of peptide side chains located in deep pockets might not interfere with TCR recognition of the MHC/peptide surface [4]. Analysis of TCR recognition of MHC-bound peptides in light of this structural information demonstrated that specificity was typically confined to a
small number of peptide side chains. For the myelin basic protein (MBP) specific T cell clones that we have studied, three peptide side chains in the core of the MBP peptide (P2 His, P3 Phe and P5 Lys) were particularly relevant for TCR recognition [6, 7]. This observation appeared to be general since similar findings were made for murine TCR reactive with microbial or self-antigens. Global amino acid replacements in the moth cytochrome C (93-103) peptide recognized by murine I-E k restricted T cells demonstrated a strong preference for a particular amino acid at three peptide positions [8]. For the Ac(1-11) peptide of MBP that is encephalitogenic in PL/J mice, only four native MBP residues were required for activation of MBP-specific T cells [9]. Based on these considerations, we developed the hypothesis that TCR recognition is characterized by a considerable degree of crossreactivity and that a TCR could recognize a number of different peptides that may be rather distinct in their sequence. This hypothesis was supported by a reported case of crossreactivity where no obvious sequence similarity was present between the two peptides, as well as the observation that many T cell clones recognized alloreactive MHC/peptide complexes [ 10, 11 ].
2. D E V E L O P M E N T OF A STRATEGY TO SYSTEMATICALLY EXAMINE T C R CROSSREACTIVITY The challenge therefore was to develop a systematic approach that would allow us to identify such peptides even though their structural similarity might not be evident by conventional sequence alignments. Formation of the trimolecular complex of MHC, peptide and TCR is based on two independent binding events: high affinity binding of peptide to an MHC molecule and the more shortlived association of TCR with this MHC/peptide surface. We decided to base our strategy on the minimal structural requirements for each of these two binding events. This approach took advantage of the fact that T cell epitopes can be mapped to short peptide segments and that the contribution of individual peptide side chains to MHC binding and TCR recognition can be evaluated with a series of peptide analogs. Experimentally, this work focused on T cell recognition of a peptide from human MBP
(residues 85-99) that is bound with high affinity by the MS-associated HLA-DR2 molecule (DRA, DRB 1"1501) [6]. Analysis of TCR crossreactivity for T cell clones activated by this HLA-DR2/MBP peptide complex could thus provide insights into disease mechanisms in MS. We defined the minimal structural requirements for binding of the MBP peptide to HLA-DR2 and for TCR recognition of this MHC/peptide complex and searched available sequence databases with this motif information. This approach permitted the identification of many examples of TCR crossreactivity, not only for the human MBP specific T cell clones that we have studied but also for CD4 and CD8 T cells of both human and murine origin, as described in some of the other chapters of this book. A more recent variant of this approach has been to determine the search motif with peptide analogs in which all neighboring positions carry mixtures of amino acids (so called combinatorial peptide libraries/positional scanning libraries) [ 12]. These libraries provide less detailed motif information, but can be used on any MHC class II restricted T cell clone.
3. T CELL CLONES CAN R E C O G N I Z E MULTIPLE PEPTIDES W I T H L I M I T E D SEQUENCE SIMILARITY The search criteria focused on the two major HLADR2 anchor residues of the peptide (P1 and P4) and four putative TCR contact residues (at P-1, P2, P3 and P5), all of which were located in a six amino acid core segment of the peptide. In the initial study, we synthesized a panel of 129 microbial peptides that matched these criteria and tested them for their ability to activate human MBP specific T cell clones that had been isolated from the peripheral blood of two patients with MS. Even though we only analyzed T cell clones that recognized a single selfpeptide, we could identify eight microbial peptides that activated MBP reactive T cell clones [7]. These peptides were remarkably distinct in their sequence from each other and the MBP peptide and only one of the eight peptides had obvious sequence similarity with the MBP peptide in the core segment. The motif-based strategy was thus essential for the identification of these peptides. In the initial study, such peptides were identified for three of
the seven T cell clones that we studied. In order to determine whether microbial peptides could activate the majority of these clones, we examined the recognition motif for two of the four remaining clones in detail and synthesized a panel of peptides that included sequences from recently characterized microbial genomes. A total of five bacterial peptides were identified in this set of experiments [13]. Thus, we have identified a total of thirteen microbial peptides that can activate human T cell clones specific for a single myelin peptide. Since we could only synthesize a subset of peptides identified in each search and a number of microbial genomes have not yet been sequenced, it is evident that a substantial number of microbial peptides can activate T cell clones that recognize this MBP peptide. These experiments thus demonstrated that T cell clones could recognize a variety of different peptides with limited sequence similarity. A number of different terms have been used to describe this property of TCR recognition. TCR crossreactivity describes the basic biological observation, while plasticity and degeneracy suggest structural m e c h a n i s m s - mobility of TCR CDR loops (plasticity) or a relatively poor fit of the TCR on the MHC/peptide surface (degeneracy). Molecular mimicry has been widely utilized to describe the specialized case where TCR crossreactivity involves peptides from an infectious agent and a self-antigen.
4. ACTIVATION OF MBP SPECIFIC T CELLS BY NATURALLY PROCESSED VIRAL ANTIGEN One of the viral peptides was derived from the EBV DNA polymerase and we examined whether the viral peptide is presented by infected antigen presenting cells to MBP specific T cells. The EBV DNA polymerase gene is part of the lyric cycle and is therefore not transcribed in EBV transformed B cells. However. the lytic cycle and expression of the EBV DNA polymerase gene can be induced by treatment of EBV transformed B cells with phorbol esters [14]. A HLA-DR2 § B cell line that had been treated with a phorbol ester activated a MBP specific T cell clone that recognized both MBP and EBV DNA polymerase peptides. T cell activa-
tion was blocked by a mAb specific for HLA-DR but not by a control mAb against HLA-DQ, and was not observed when MHC-mismatched EBV transformed B cells were used as antigen presenting cells. These results demonstrated that the MBP specific T cell clone recognized not only the EBV peptide but also antigen presenting cells in which the viral gene was transcribed [7].
5. CRYSTAL STRUCTURE OF THE HLADR2/MBP PEPTIDE COMPLEX Since the peptides that can be recognized by the same TCR are quite distinct in their primary sequence, an important question relates to the structural basis of TCR crossreactivity. Structural information is also required to determine why only a subset of peptides that match the MHC binding/ TCR recognition motif activate the appropriate T cell clones. We determined the crystal structure of HLA-DR2 (DRA, DRBI*I501) with the bound MBP peptide as a step towards defining molecular mimicry at a structural level [ 15]. Fig. I gives an overview of the structure and illustrates features of HLA-DR2 that are important for MBP peptide binding as well as TCR recognition of the HLA-DR2/ MBP peptide complex. The MBP peptide is bound in an extended conformation as a type II polyproline helix and MBP peptide side chains occupy the P I, P4, P6 and Ix) pockets of the binding groove (Fig. l a). The two major anchor residues of the MBP peptide (Val and Phe) occupy the P I and P4 pockets of HLA-DR2. The P4 pocket of HLA-DR2 is distinct from DR molecules associated with other autoimmune diseases. In HLA-DR2, this pocket is large and predominantly hydrophobic due to the presence of a small residue (Aia) at a key polymorphic position (DRI3 71), which permits an aromatic side chain of the MBP peptide to be accommodated (Phe) (Fig. Ic). The peptide residues shown to be important for TCR recognition of the MBP peptide (P2 His, P3 Phe and P5 Lys) are solvent exposed in the structure of the HLA-DR2/MBP peptide complex (Fig. l b, l d: Fig. 2). Comparison with the published high resolution crystal structures of human MHC class I/peptide/TCR complexes [ 16-18] suggests that P5 Lys binds in a TCR pocket formed by the CDR3 loops of the r and [3 chains.
Figure 1. Crystal structure of the complex of HLA-DR2 (DRA, DRB I * 1501 ) and the MBP (85-99) peptide. (A) Overview of the structure. MBP peptide side chains that are located in four pockets of the binding site are indicated, PI Val, P4 Phe, P6 Asn and P9 Thr. (B) Solvent exposed residues that are important for TCR recognition of the MBP (185-99) peptide, IY2 His, P3 Phe and P5 Lys. (C) P4 pocket of the HLA-DR2 binding site. This large and hydrophobic pocket is occupied by P4 Phe of the MBP peptide. The necessary room for this aromatic side chain is created by the presence of a small residue (Ala) at the polymorphic DRI$ 71 position. (D) Close-up view of MBP peptide residues recognized by the TCR of MBP reactive T cell clones, P-1 Val, P2 His. P3 Phe and P5 Lys. Preferences at these positions were considered in the search criteria for the identification of microbial peptides that activate MBP reactive T cell clones. Reprinted from The Journal of Experimental Medicine [151 with permission of the publisher.
6. C O M M O N F E A T U R E S O F M I C R O B I A L P E P T I D E S T H A T A C T I V A T E MBP SPECIFIC T CELLS The structural information on the H L A - D R 2 / M B P peptide complex was used to dissect the crossreactive peptides identified for the MBP specific T cell clone ( O b . l A I 2 ) for which the largest number of stimulatory microbial peptides were identified. The crossreactive peptides were aligned with the MBP
10
peptide in order to determine residues that may be located in pockets of the HLA-DR2 binding site (Table 1). This analysis shows that the HLA-DR2 contact surface of this set of peptides is highly diverse, No sequence identity with the MBP peptide is required on the HLA-DR2 binding surface, as illustrated by comparison of the MBP and Bacillus subtilis peptides. C o m m o n features of putative HLA-DR2 contact residues are the presence of an aliphatic residue in the P I pocket and a large hydro-
Figure 2. Electron density and model of the MBP peptide in the binding site of HLA-DR2. (A) Experimental electron density of the MBP peptide bound to HLA-DR2. (B) Model of the MBP peptide based on the elecLron density. The DR2/MBP peptide complex crystallized as a dimer of dimers, as other HLA-DR molecules [45, 41. and both peptide copies are shown (blue and yellow). The peptide backbones superimpose in the P-I to P4 segment and are more divergent in the C-terminal segment due to different crystal contacts. Reprinted from The Journal of Experimental Medicine [ 15] with permission of the publisher.
phobic residue in the P4 pocket. At P6, a preference for asparagine is observed, while the residues that occupy the P7 and P9 positions are diverse. These data are in agreement with HLA-DR2 binding studies, which indicated that only two positions of the peptide (Pl and P4) were critical for binding and that they could be substituted with other aliphatic residues or phenylalanine (Pl pocket) or other hydrophobic residues (P4 pocket). Analysis of the residues that may be solvent exposed and that could interact with the TCR shows a higher degree of sequence similarity/identity, in particular in the center of the epitope (Table 2). All peptides that activate this T cell clone carry the two primary TCR contact residues of the MBP peptide (His and Phe at P2 and P3). Also, a preference for a positively charged residue (Lys and Arg) is observed at PS. A high degree of sequence diversity
is observed in the N- and C-terminal flanking segments, even though they are required for efficient T cell stimulation. The data also indicate that combinatorial effects shape the peptide surface that can be recognized by a TCR. Analysis of double-amino acid substitutions of the MBP peptide demonstrated that certain combinations of amino acids at TCR contact residues were stimulatory, even though the individual analogs had no activity [19]. This notion is supported by the observation that the majority of microbial peptides that match the MHC binding/TCR recognition motif did not stimulate the MBP specific T cell clones. Identification of a complete set of peptide sequences that represent agonists for a TCR will therefore require analysis of complex peptide libraries. At present, such analyses represent a technical challenge since a large number of peptides may
II
Table 1. Sequences of microbial peptides that activate human MBP specific T cell clones Organism
Source protein
Sequence
Peptides that activate T cell clone Ob.lAl2 (HLA-DR2 restricted)
Homo sapiens Staphylococcus aureus Mycobacterium avium Mycobacterium tuberculosis Bacillus subtilis Haemophilus influenzae/E, coli
Myelin basic protein VgaB Transposase Transposase YqeE HI0136/ORF
ENPVVHFFKNIVTPR VLARLHFYRNDVHItE QRCRVHFLRNVLAQV QRCRVHFMRNLYTAV ALAVLHFYPDKGAKN DFARVHFISALHGSG
Peptides that activate T cell clone Hy. 1B 11 ( HLA-DQ i restricted)
Homo sapiens Herpes simplex virus type 1 Adenovirus type 12 Human papillomavirus type 7 Pseudomonas aeruginosa
Myelin basic protein ULI5 protein ORF L2 protein Phosphomannomutase
ENPVVHFFKNIVTPR FRQLVHFVRDFAQLL DFEVVTFLKDVLPEF IGGRVHFFKDIS P IA DRLLMLFAKDWSRN
Microbial peptides that activate two human T cell clones reactive with the MBP (85-99) peptide are shown. Clone Ob.lAl2 is restricted by HLA-DR2 (DRA, DRB 1" 1501) and five microbial peptides have been identified that activate this T cell clone. Clone Hy.IB 11 is HLA-DQI restricted and activated by four microbial peptides that are quite distinct in their primary sequence. The peptide from human papillomavirus type 7 is the only peptide with obvious sequence similarity to the MBP peptide within the entire set of identified microbial peptides. Residues that are identical between the MBP peptide and microbial peptides are highlighted. The HLA-DQI restricted T cell clone recognizes the same core segment of the MBP peptide as HLA-DR2 restricted clones [7, 6, 13]. ORF, open reading frame.
need to be sequenced from phage display libraries or peptide libraries on beads. However, the complexity of the peptide repertoire that is recognized by an individual TCR may be underestimated unless the combinatorial nature of peptide recognition by the TCR is taken into consideration.
7. STRUCTURAL FEATURES OF AUTOREACTIVE TCR THAT C O N T R I B U T E TO THE DEGREE OF CROSSREACTIVITY Comparison of the MBP (85-99) reactive T cell clones demonstrated obvious differences in the degree of specificity/crossreactivity. For some of the T cell clones, such as Ob.Al2, amino acid identity was required at two TCR contact residues of the MBP peptide while every TCR contact residue of the MBP peptide could be substituted by at least one structurally related amino acid for other T cell clones.
12
Two of the T cell clones (Ob.lAl2 and Ob.2F3) differed only in the CDR3 loops of TCR ct and 13 since they had identical Vo~-Jt~ and V~-J~ rearrangements [20]. Nevertheless, the clones differed in the level of crossreactivity since only three of the five microbial peptides identified for clone Ob. 1A 12 activated the other clone. We examined the basis for the different level of crossreactivity and found that the clones had a very similar fine specificity for the MBP peptide, except for the P5 position of the peptide (P5 Lys). The microbial peptides that activated clone Ob. I A I 2 were characterized by conservative or non-conservative changes at P5 (Lys to Arg, Ser or Pro). In contrast, clone Ob.2F3 was only stimulated by the peptides that had a conservative lysine to arginine substitution. The degree of specificity in recognition of the P5 side chain was the key difference between these TCR since the Haemophilus influenzae/E, coli peptide stimulated both clones when the P5 position was substituted from serine to arginine [ 13]. In the crystal structure of the HLA-DR2/MBP peptide complex, P5 Lys was a
Table 2. Alignment of microbial peptides based on the crystal structure of the HLA-DR2/MBP peptide complex Organism
Residues located in HLA-DR2 binding pockets Homo sapiens Staphylococcus aureus Mycobacterium avium Mycobacterium tuberculosis Bacillus subtilis Haemophilus influenzae/E, coli
Source protein
Sequence
Myelin basic protein VgaB
....
V-
- F-NI-T-
-
....
L- - Y-ND-H-
-
Transposase
....
V-
-L-NV-A--
Transposase
....
V-
-M-NL-
YqeE
....
L- - Y- DK-A-
-
HI0136/ORF
....
V-
-
1
Solvent exposed residues
4 6 7 9
T- -
- I -AL-G-
23
5
8
HF-
K-
- V-
Homo sapiens Staphylococcus aureus
Myelin basic protein
ENPV-
VgaB
VLAR-HF-
Mycobacterium avium Mycobacterium tuberculosis
Transposase Transposase
QRCR-HF-R-
-L-QV
QRCR-HF-R-
-Y-AV
Bacillus subtilis
YqeE
ALAV-
Haemophilus influenzae/E, coli
HI0136/ORF
DFAR-HF-S-
HF
PR
R- -V-KE
- P - - G - KN -H-SG
Peptide sequences were dissected in terms of putative HLA-DR2 anchor residues and solvent accessible residues that are available for interaction with the TCR. The HLA-DR2 contact surface of these peptides is highly diverse, and no sequence identity is observed at these five positions between the MBP and Bacillus subtilis peptides. A higher degree of sequence similarity/identity is observed for peptide residues that are likely to interact with the TCR (P2, P3 and P5). Numbers above the sequences represent positions in a nine-amino acid peptide core, starting with the anchor residue for the P1 pocket of the binding site.
prominent, solvent exposed residue in the center of the DR2/MBP peptide surface (Fig. 1). Structures of MHC class I/peptide complexes have shown that this peptide side chain occupies a central TCR pocket created by the CDR3 loops of TCRt~ and [16, 18]. This suggests that the two MBP specific TCR differ in the size and shape of the TCR pocket that accomodates the central lysine of the MBP peptide. Similar findings were made for MHC class I restricted T cell clones that recognize the HTLV-1 Tax (11-19) peptide bound to HLA-A2. The crystal structure of the MHC/peptide/TCR complex has been determined for both of these TCRs (A6 and B7), which demonstrated major differences in the shape and charge of the TCR pocket for the P5 peptide residue [16, 18]. The B7 TCR was exquisitely specific for P5 tyrosine of Tax (11-19) since only aromatic substitutions were tolerated. In contrast, the A6 TCR was much more degenerate at the P5 position since 10 of 17 analog peptides induced lysis of target cells at low peptide concentrations
[21]. The absolute requirement for an aromatic side chain by the B7 T cell clone could be explained based on the structure of the P5 pocket: the P5 Tyr represented a tight fit for this TCR pocket and a favorable stacking interaction between the aromatic ring of P5 Tyr and an aromatic residue of the CDR3 loop of the TCR 13chain (Y 104 13) was observed. In contrast, the A6 TCR had a larger P5 pocket and the P5 Tyr did not interact with an aromatic TCR residue. These data demonstrate that the TCR CDR3 loops can determine the degree of specificity and degeneracy of the central TCR pocket. Further characterization of the peptide from the EBV DNA polymerase gene demonstrated a second structural mechanism for TCR crossreactivity. The HLA-DR2 haplotype that confers susceptibility to MS encodes two DR~ chain genes (DRB 1"1501 and DRB5*0101), both of which can pair with the non-polymorphic DRot chain to form functional DR heterodimers [22]. Both DR molecules are expressed by antigen presenting cells in subjects with the HLA-DR2 haplotype. The Hy.2E11 clone recog-
13
nized the MBP peptide bound to DRA, DRB 1"1501 molecules, but surprisingly the EBV peptide bound to DRA, DRB5*0101 molecules. Comparison of the two crystal structures demonstrated a striking degree of similarity, in particular for the peptide positions previously shown to be required for TCR recognition [7, 23]. TCR crossreactivity can therefore involve the recognition of different peptides bound to the same MHC molecule, or recognition of different peptides on other self-MHC molecules.
8. TCR CROSSREACTIVITY AND IMMUNOPATHOLOGY Several different experimental autoimmune diseases have been induced by immunization with microbial peptides, indicating that crossreactive T cell populations can be pathogenic. EAE has been induced in different strains of mice and in Lewis rats with mimicry peptides of MBP and myelin oligodendrocyte glycoprotein [24-28]. However, the physiologically more relevant question is whether autoimmune disease can also result from infection with pathogens that carry such T cell epitopes. To address this question, Olson et al [29] generated recombinant Theiler's viruses in which the candidate sequences were placed into the leader segment of the virus. The first recombinant virus carded the sequence of the PLP (139-151) peptide that is immunodominant in SJL mice and infection with this virus resulted in the rapid development of central nervous system (CNS) inflammation and vigorous CD4 T cell responses to the PLP peptide. It is important to note that the wildtype Theiler's virus also induced CNS pathology, but disease onset was significantly later (day 30, rather than day 10) permitting the two disease states to be distinguished. Also, tolerance induction with the PLP (139-151) peptide prevented induction of the early disease process with the PLP (139-151) expressing virus, but not the late disease caused by wild-type Theiler's virus [30]. Importantly, CNS autoimmunity could not only be induced by a virus that carried the self peptide, but also by a recombinant virus that expressed a peptide from Hemophilus influenzae shown to stimulate PLP (139-151) specific T cells [29]. The relationship between autoimmune disease and viral infection has also been examined with
14
natural pathogens, in particular using the Herpes simplex keratitis (HSK) model [31]. Infection of the eye with Herpes simplex virus (HSV-1, KOS strain) triggers a T cell-mediated autoimmune process that persists after the virus has been cleared. In order to rigorously test the role of molecular mimicry in this disease process, a single amino acid substitution was made in the crossreactive T cell epitope of the viral UL-6 protein. The mimicry T cell epitope was found to be important for disease induction in C.AL-20 mice since 1000-fold larger quantifies of the mutant virus were required than of wild-type HSV-1, even though both viruses replicated at the same rate. However, in mice that expressed the C1-6 TCR and therefore harbored large numbers of autoreactive T cells, infection with HSV-1 was not required since scratching of the cornea or local application of LPS were sufficient for the induction of disease. The crossreactive T cell epitope was thus required for the expansion of autoreactive T cells, but activation of the innate immune system was sufficient when large numbers of such T cells were already present. This model therefore clarified the potential contributions of TCR crossreactivity and 'bystander' activation in the induction of autoimmune diseases. The diverse nature of the viral/bacterial peptides that stimulate autoreactive T cell clones suggests that different infectious agents could initiate autoimmunity by molecular mimicry. However, it is important to keep in mind that a number of other mechanisms could also result in the activation of autoreactive T cells. The diverse nature of the mimicry peptides and the ubiquitous presence of some of these pathogens may make it difficult to establish a direct epidemiological link between infectious agents and the occurrence of certain autoimmune diseases. In particular, the temporal relationship between an infection and development of an autoimmune process may in many cases not be clear because of the time that frequently elapses until clinical symptoms become obvious and a diagnosis is made. Such epidemiological relationships may be more readily established for autoimmune disorders with a rapid disease onset since early diagnosis can greatly increase the likelihood of establishing a link with a preceding infection. Recent data have demonstrated a relationship between an inflammatory, demyelinating disease of the peripheral nervous
system (Guillain-Barr6 syndrome) and preceding infections [32]. Patients with this disease acutely develop severe symptoms and rapid diagnosis permitted isolation of Campylobacter jejuni from approximately a third of new cases, compared to 2% of household controls. A better understanding of the epidemiology of infectious agents and autoimmunity could thus help to advance our understanding of the molecular mechanisms that trigger human autoimmune diseases.
9. TCR CROSSREACTIVITY AS A GENERAL PROPERTY OF T CELL RECOGNITION A large number of studies have now demonstrated TCR crossreactivity for a variety of human and murine T cells [10, 12, 25, 33-37]. An interesting example is the melanoma/melanocyte-derived peptide MART- 1 (res. 27-35) since it demonstrates how crossreactivity can shape the T cell repertoire. In normal human donors with the HLA-A2 haplotype, T cells specific for this melanocyte peptide were detected at a surprisingly high frequency, and such T cells could be visualized directly ex vivo with HLA-A2/MART- 1 tetramers (frequency of -0.1%). This suggested that these T cells crossreacted with microbial peptides and a motif search similar to the one that we had performed for human MBP specific T cell clones yielded twelve peptides that were able to sensitize target cells for lysis. One of these peptides was derived from the glycoprotein C of Herpes simplex virus (HSV) and anti-MART effectors lysed cells infected with a recombinant vaccinia virus encoding HSV-1 glycoprotein C [35, 38]. TCR crossreactivity can also have a profound effect on protective immune responses to viral pathogens in vivo, as shown in a murine model where CD8 T cells crossreacted with peptides from two different v i r u s e s - lymphocytic choriomeningitis virus (LCMV) and Pichinde virus (PV) [37]. LCMV and PV are members of the Arenaviridae family, but the two viruses are only distantly related as shown by sequence comparison. Prior infection with either LCMV or PV provided partial protection against the heterologous virus and LCMV-immune mice showed a 97% reduction in viral titer compared to naive mice when challenged with PV. CD8 T cells
from mice infected with either virus crossreacted with a nucleoprotein-derived peptide (NP 205-212) from the other virus; these two nucleoprotein peptides shared six of eight residues and differed at positions 5 (Tyr versus Phe) and 8 (Leu versus Met). In LCMV infected mice the NP 205-212 epitope was subdominant and 3.6% of CD8 T cells responded to this epitope on day 8 following infection. However, CD8 T cells specific for this NP 205-212 peptide became the predominant CD8 T cell population (30% of all CD8 T cells) when mice that had previously encountered PV were infected with LCMV. These experiments demonstrated that TCR crossreactivity influences the hierarchy of CD8 T cell responses and shapes the pool of memory T cells. TCR crossreactivity is also a critical aspect of T cell development in the thymus and weaker TCR signals are required for positive selection in the thymus compared to activation of mature T cells. Positive selection in the thymus is peptidedependent and is affected both by the density of a particular MHC/peptide complex and the affinity of the TCR for this complex. In thymic organ cultures, positive selection was observed with peptides that represented weak agonists or antagonists for the corresponding mature T cells, or with low densities of the agonist peptide [39-41]. The creation of transgenic mice that expressed a single MHC/ peptide ligand in the thymus provided a striking demonstration of crossreactive TCR recognition in thymic development [42]. In this experiment, a peptide was covalently linked to the N-terminus of the MHC class II ~ chain so that all MHC class II molecules were occupied with this peptide. The total numbers of CD4 T cells in these mice were -20% compared to wild-type mice and these CD4 T cells expressed a wide variety of different VI3 segments, indicating that a relatively diverse T cell repertoire could develop in the presence of a single MHC class II/peptide ligand. T cell hybridomas isolated from these mice reacted with peptides that had no primary sequence identity with the selecting peptide [43]. These experiments demonstrated that T cells could be activated by peptides that were unrelated in sequence to their selecting peptide. The examples described above indicate that TCR crossreactivity is common and represents an
15
important aspect of TCR recognition. The balance between specificity and crossreactivity is likely to represent a compromise that permits a sufficient number of T cells to recognize a pathogen novel to the individual's immune system. The potentially negative impact of TCR crossreactivity may be in part balanced by in vivo selection of T cells with high avidity TCR for the relevant MHC/peptide complexes. It has been postulated that a single TCR can recognize 106 different peptide ligands, and this estimate is based on the observation that a subset of T cell clones can be activated by complex peptide mixtures in which only one peptide position is specified [44]. While the number of peptide variants that can be recognized may be very large, the number of natural ligands from microbial and self-antigens is likely to be considerably smaller. Nevertheless, a number of different peptides can act as agonists for a given T cell and a considerably larger number of peptide ligands may induce weak signals, such as those that promote positive selection in the thymus and survival of naive T cells in the periphery. TCR specificity and crossreactivity thus represent important aspects of T cell biology.
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ACKNOWLEDGEMENTS I would like to acknowledge the important contributions that my colleagues and collaborators have made towards this research. In particular, I would like to acknowledge the contributions of Stefan Hausmann, Katherine Smith, Laurent Gauthier, Heiner Appel, Jason Pyrdol, David A. Hailer, Don C. Wiley and Jack L. Strominger. This work was supported by grants from the National Multiple Sclerosis Society and the NIH (RO1 NS39096).
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44. Mason D. A very high level of crossreactivity is an essential feature of the T-cell receptor. Immunol Today 1998; 19:395-404. 45. Brown JH, Jardetzky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993;364:33-9.
9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Epitope Spreading Kamal D. Moudgil and Malarvizhi Durai
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA
1. INTRODUCTION The phenomenon of "epitope spreading" (or "determinant spreading") is characterized by broadening or diversification of the initial immune response induced by immunization with a single peptide antigen or a multi-determinant antigen [1-3]. The new T cell and/or antibody responses are directed to different epitopes either within the same antigen as that used for immunization (intra-molecular spreading) or another antigen (inter-molecular spreading). The spreading of initial immune reactivity has been shown to occur during the course of a variety of experimentally-induced and spontaneously arising autoimmune diseases in animal models (Tables 1 and 2) [2, 3]. Studies in patients with certain autoimmune diseases, in recipients of organ transplants, and in cancer patients given peptide vaccination (described below) (Table 1) have further validated the significance of epitope spreading in disease pathogenesis. Depending on the disease process, epitope spreading can contribute either to the progression or to the control of an autoimmune disease (Fig. 1) [1, 3, 4]. The timing of epitope spreading during the course of disease and its functional attributes are of significance in designing appropriate immunotherapeutic approaches.
2. EPITOPE SPREADING IN AUTOIMMUNE DISEASES 2.1. Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis Multiple sclerosis (MS) is a human autoimmune disease characterized by mononuclear cell infiltration and discrete areas of demyelination (plaques) within the central nervous system (CNS), and neurological dysfunction. Experimental autoimmune encephalomyelitis (EAE) is an experimental model for MS, and it can be induced in different mouse/rat strains by immunization (in adjuvant) with myelin antigens such as myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte protein (MOG) [3, 5, 6] (Table 2). Epitope spreading was first demonstrated by Lehmann et al in the EAE model using (SJL X B 10.PL) F1 mice [1]. It was shown that the initial T cell response of mice with acute EAE was directed to MBP A c l - l l , but spreading of the T cell response to new determinants of MBP, namely 35-47, 81-100, and 121-140, occurred during the chronic stage of EAE [1, 7]. This broadening of the T cell response was attributed to priming of new T cells by determinants within endogenous MBP following initial CNS damage. Miller's group has established the role of epitope spreading in the pathogenesis of relapsing-EAE (R-EAE). R-EAE can be induced in SJL mice by immunization with PLP 139-151 [8, 9]. Using this model, it was observed that the T cell response to the disease-initiating epitope, PLP 139-151, was maintained in SJL mice throughout the course of
19
Table 1. Examples of epitope spreading in animal models and human diseases References
Diseases A. Autoimmune diseases a) Animal models of autoimmune diseases Experimental autoimmune encephalomyelitis (EAE) Diabetes in the non-obese diabetic (NOD) mouse Adjuvant-induced arthritis (AA) Lupus or Systemic lupus erythematosus (SLE) Experimental autoimmune myasthenia gravis (EAMG) Experimental autoimmune neuritis (EAN) Equine recurrent uveitis (ERU) Experimental autoimmune gastritis (EAG) Autoimmune oophoritis
[1, 8, 13, 17, 19, 20, 22, 24, 27]
[35,36] [4, 52, 63-65] [71, 72, 78, 80] [89,91,92] [96] [97] [98] [99, 100]
b) Human autoimmune diseases Multiple sclerosis (MS) Insulin-dependent diabetes mellitus (IDDM) or Type I diabetes Rheumatoid arthritis (RA) Systemic lupus erythematosus (SLE) or Lupus Myasthenia gravis (MG) Pemphigus
[31-33] [39-42] [671 [83-87] [93, 94] [95]
B. Other diseases
a) Organ transplantation / graft rejection
[102-106]
b) Tumors
[107-110]
c) Infection/Vaccination
[111, 112, 115, 116]
EAE. However, spreading of the T cell response to non-crossreactive PLP 178-191 and MBP 84-104 epitopes occurred after the first and second relapses, respectively. The sequential appearance of the T cell response to the 3 epitopes of PLP/MBP tested was hierarchical (PLP 139-151, PLP 178-191, and MBP 84-104, in order of decreasing level of immunodominance). Furthermore, the T cells against the spreading epitope (PLP 178-191) could transfer disease to naive syngeneic recipients [8]. Interest-
ingly, induction of tolerance in SJL mice against relapse-associated epitopes after the acute episode blocked disease progression and decreased the frequency of subsequent relapses [8]. In addition, short-term blockade of either CD28-CD80 (B7.1) costimulation by anti-CDS0 F(ab) fragment [10] or CD40-CD154 (CD40L) interaction using monoclonal anti-CD154 antibody [1 l] during remission from acute disease significantly reduced both the incidence of disease relapse and the T cell response
aUnless specified, all epitopes mentioned in Table 2 refer to T cell responses. The abbreviations are given in the order of their appearance in Table 2. M B P - Myelin basic protein; P L P - Proteolipid protein; T M E V - Theiler's murine encephalomyelitis virus; S E A Staphylococcal enterotoxin A; MOG - Myelin oligodendrocyte protein; GAD65/67 - Glutamic acid decarboxylase 65/67; HSP65 - Heat shock protein 65; Mtb - heat-killed Mycobacterium tuberculosis H37Ra; Bhsp65 - mycobacterial hsp65; A A Adjuvant-induced arthritis; Sm B/B'- Smith Ag B/B'; La & R o - Antigens within the ribonucleoprotein complex; AChRacetylcholine receptor; CTLA-4 - Cytotoxic T-lymphocyte antigen-4; P0 - Peripheral nervous system myelin P0 glycoprotein; IRBP- Interphotoreceptor retinoid binding protein; S-Ag - retinal S-antigen.
20
Table 2. The antigen specificity of epitope spreading in experimental models of autoimmune diseases Disease model
Animals tested
Disease-inducing antigen/agent
Antigen/epitopes targeted during epitope spreading a
Ref.
Experimental autoimmune encephalomyelitis (EAE)
(SJLxB10.PL)F1 mice SJL/J mice (SWR x SJL)F1 mice PL/J B 10.RIII mice Lewis rats SJL/J mice
MBP A c l - l l
[ 1]
Callithrix jacchus (the common marmoset)
MP4 fusion protein (PLP - MBP)
MBP 35-47, MBP 81-100, and MBP 121-140 PLP 178-191 and MBP 84-104 PLP 249-273, MBP 87-99, and PLP 137-198 MBP 81-100 and MBP 120-140 Multiple T cell epitopes within MBP Multiple T cell epitopes within MBP PLP 139-151, PLP 178-191, PLP 5670, and MOG 92-106 Anti-MOG antibodies
NOD mice
Spontaneous
[35]
NOD mice
Spontaneous
T cell response to GAD65, Carboxypeptidase H, Insulin, and HSP65 T cell and antibody response to GAD65/67, Peripherin, Carboxypeptidase H, and HSP60
Lewis rats
Mtb (H37Ra)
Lewis rats
Mtb (H37Ra)
NZW rabbits
Sm B/B' peptide
Mice
La (or Ro) antigen
(SWR x NZB)F1 Mice (NZB/NZW)F1 mice
Spontaneous
Diabetes in the non-obese diabetic (NOD) mouse
Adjuvant-induced arthritis
PLP 139-151 PLP 139-151 MBP 100-120 + SEA MBP 89-101 MBP TMEV
(AA)
[8] [ 13] [17] [ 19] [20] [24] [27]
[36]
417--431,441-455, 465-479, 513-527, [4] and 521-535 of Bhsp65 Multiple B cell epitopes within Bhsp65 [63] after recovery of AA Antibody response to other epitopes of Sm B/B' antigen and other spliceosomal proteins Antibody response to both La/SS-B and Ro/SS-A proteins Antibody response to nucleosomal components Response to epitopes in V. region of anti-DNA antibody
[71]
Human AChR czsubunit peptides
Antibodies to rabbit AChR
[89]
C57BL/6 mice
tz (146-162) of AChR + anti-CTLA-4 antibody
[91 ]
Rats
AChR {x-subunit
Antibody and T cell response to other subdominant epitopes of AChR txsubunit Antibody response to cytoplasmic region of AChR
Experimental autoimmune neuritis (EAN)
Lewis rats
Peptides 56-71 and 180-199 of P0 protein
T cell response to other epitopes of P0 protein
[96]
Equine recurrent uveitis (ERU)
Horse
IRBP
Multiple T cell epitopes within IRBP and S-Ag
[97]
Experimental autoimmune gastritis (EAG)
Mice
H/K ATPase ~l-subunit T cell and antibody response to {xsubunit of H/K ATPase
Systemic lupus erythematosus (SLE)
Experimental autoimmune NZW rabbits myasthenia gravis (EAMG)
Spontaneous
[72] [77a,b] [78]
[92]
[981
21
to relapse-associated epitopes. Unexpectedly, mice treated with intact anti-B7.1 antibody [10] or antiCTLA-4 antibody [12] during ongoing disease suffered from exacerbated relapsing disease and revealed enhanced epitope spreading. However, the above studies have revealed a window of therapeutic intervention in the face of epitope spreading. Tuohy and colleagues have studied the determinant specificities and functional significance of epitope spreading in R-EAE inducible in (SWR X SJL) F1 mice by injection with PLP 139-151 [13]. It was observed that the T cell response to the disease-inducing peptide, PLP 139-151, gradually diminished, whereas spreading of responses to MBP and new epitopes of PLP occurred with progression of EAE in a sequential pattern (PLP 249-273, MBP 87-99, and PLP 137-198, in that order). Interestingly, the T cells specific for the spreading determinant could passively transfer EAE to naive syngeneic recipients, whereas induction of peptide-specific tolerance to spreading epitopes after onset of EAE could prevent progression of EAE [13]. Furthermore, interferon-I] treatment of mice not only reduced the frequency/severity of disease relapses but also suppressed epitope spreading [14]. The results of another study by the same investigators revealed that the T cell response to the spreading-associated determinant (MBP 87-99) in (SWR X SJL) F1 mice had a proinfiammatory Thl cytokine profile, and that splenocytes activated with MBP 87-99 could adoptively transfer acute EAE to na'fve recipients [15]. Using a novel therapeutic approach based on immune deviation, T cells specific for MBP 87-99 were genetically modified to secrete high level of immunoregulatory cytokine, IL-10 and then transferred into (SWR X SJL) F1 mice after onset of EAE [16]. These recipient mice showed a marked inhibition of both disease progression and demyelination. Thus, induction of a disease-regulating T cell response targeted to relapse-associated epitope provided therapeutic benefit. Taken together, these results strongly support the idea that determinant spreading is involved in the progression of EAE. Using another model of EAE, it was shown that staphylococcal enterotoxin superantigen A (SEA) can reactivate EAE in PL/J mice recovered from MBP-induced EAE, and this re-activation of the disease was associated with intramolecular
22
spreading to epitope 100-120 of MBP [17]. No inter-molecular spreading was observed during the course of reactivated disease. In another set of experiments by the same investigators, it was observed that MBP peptide 100-120 per se failed to induce EAE in PL/J mice [ 17]. However, an additional challenge of these mice with SEA rendered MBP 100-120 encephalitogenic, and the diseased mice developed T cell responses to new epitopes namely, 81-100 and 120-140. These results provide insight into reactivation of autoimmunity following exposure to microbial agents containing the relevant superantigen. Another aspect of epitope spreading was highlighted in the SJL model of MBP-induced EAE: epitope spreading was found to occur in active but not passive EAE [ 18]. These results point to the fine differences in the pathogenesis of active vs. passive disease resulting from activation of T cells against the same autoantigen. Diversification of response to MBP epitopes has also been observed during the course of EAE in B10.RIII mice [19] and the Lewis rat [20]. The initial T cell response of B 10.RIII mice with relapsing EAE was focused predominantly on the immunogen (MBP 89-101), but later spreading of the response occurred to other epitopes within MBP [19]. In the case of Lewis rats with EAE, the dominant encephalitogenic T cells in the induction phase of the disease were directed to epitope 71-90, whereas T cell responses to new epitopes within MBP appeared during the recovery phase of the disease [20]. Another study revealed that Wistar Kyoto (WKY) rats having the same MHC haplotype as the Lewis rat were resistant to EAE despite raising potent T cell responses to the dominant encephalitogenic T cell epitope within MBP [21]. However, the antigen-specific T cell response in WKY rats was skewed towards a predominantly Th2 type compared to predominantly Thl type in Lewis rats. These results along with those of another study in AA using the same rat strain [4] demonstrate that a positive T cell response to the pathogenic epitope may not correlate with the presence or absence of clinical signs of the disease. Furthermore, these results point towards the significance of analysis of the cytokine responses of epitope-specific T cells in addition to measuring the proliferative T cell response in evaluation of epitope spreading and related aspects of the disease process.
In a recent study, autoantibody responses of mice with EAE were measured using a large set of autoantigens in a protein microarray [22]. Different patterns of autoantibody reactivity were observed in acute vs. chronic phase of EAE. Chronic EAE was characterized by both intra- and inter-molecular epitope spreading. Furthermore, attenuation of established EAE by a tolerizing DNA vaccination based on defined autoantigens was associated with reduced epitope spreading of autoantibody responses. Epitope spreading has also been implicated in the induction of autoimmunity in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease in SJL/J mice [23-26]. TMEV causes chronic infection of the CNS. The host immune response (predominantly CD4+ T cells) to the virus is responsible for the initial myelin damage [23]. Interestingly, however, the T cell response specific for self myelin antigens appears during the progression of the disease. After the disease onset, response to the dominant epitope of PLP, PLP139-151, is observed followed by that to relatively less dominant epitopes ofPLP (PLP 178-191 and PLP 56-70)and MOG 92-106 [24, 25]. These results demonstrate that epitope spreading targeting myelin antigens contributes to TMEV-induced autoimmunity. This conclusion is further supported by the findings that tolerance induction against defined T cell determinants within myelin antigens in SJL mice with ongoing TMEV-induced disease results in marked reduction in demyelination [26]. However, unlike in PLP 139-151-induced EAE, costimulation blockade in TMEV-infected mice was found to enhance the disease severity instead of reducing it. Like in rodent EAE, epitope spreading has also been observed during the course of EAE in the common marmoset, Callithrix jacchus, and in patients with MS. The Callithrix jacchus marmoset develops chronic relapsing-remitting form of EAE following challenge with myelin antigens, and both T cells and antibodies serve as immune effector mechanisms in the disease process [27, 28]. Interestingly, treatment of these non-human primates with anti-CD40 antibody prevented intramolecular spreading and afforded protection against EAE [29, 30]. In a study based on patients with isolated monosymptomatic demyelinating syndrome (IMDS), the T cell reactivity to PLP epitopes was found to
decrease over time. However, spreading of T cell responses to other PLP epitopes was observed in those IMDS patients who progressed to clinically definite MS (CDMS) [31-33].
2.2. Insulin-Dependent Diabetes Mellitus (IDDM) or Type I Diabetes IDDM is an autoimmune disease involving mononuclear cell infiltration of the pancreatic islets (insulitis), destruction of the 13-islet cells, and insulin deficiency. Spontaneously-developing diabetes in the non-obese diabetic (NOD) mouse serves as a model for human IDDM [34]. Glutamic acid decarboxylase (GAD65) has been invoked as one of the early target antigens in the pathogenesis of autoimmune diabetes in the NOD mouse [35, 36]. With the progression of disease in the NOD mouse, the T cell response spreads to additional epitopes within GAD65 and to other ~-cell antigens (e.g., to carboxypeptidase-H, insulin, and heat shock protein 65 (Hsp65) in one study [35], and to GAD 67, carboxypeptidase-H, peripherin, and Hsp60 in another study [36]). Furthermore, tolerization of GAD65reactive T cells suppressed the development of insulitis, disease progression, and the spreading of T cell responses [35, 36]. In the NOD mouse, Thl spreading leads to disease progression, whereas Th2 spreading is associated with protection from disease [37]. Induction of a Th2 response to a single I]-cell antigen (GAD/Hsp peptide 277/Insulin B chain) or a peptide of GAD (e.g., p35/p6) led to spreading of the Th2 cellular and antibody response to other I]-cell antigens/epitopes along with protection from diabetes [37]. In another study on emergence of T cell responses to GAD during the course of spontaneously developing disease in the NOD mouse, new potential target epitopes within GAD65 and GAD67 were described [38]. The patterns of T cell responses to epitopes within GAD65/GAD67 were found to vary with the age and disease-status of mice. In addition, NOD mice given a disease-protective regimen (e.g., adjuvant challenge) revealed a different pattern of response to GAD65/67 compared to unmanipulated control NOD mice. These results validate the role of T cell responses to GAD in the pathogenesis of autoimmune diabetes. Studies on epitope spreading in human IDDM have revealed the pattern of autoantibody responses
23
to [3-cell antigens in children of diabetic patients [39]. The anti-islet autoantibody response in these subjects was characterized by appearance of an early IgG1 response to one or more islet antigens, particularly insulin. Thereafter, coupled with a decline in the titer of these antibodies, there was a sequential appearance of antibodies against other I]-cell antigens over a period of several years [39]. In other studies in preclinical childhood type I diabetes, it was observed that the initial antibody response to GAD of offspring of diabetic patients was directed primarily to epitopes within the middle portion of GAD65, but later it spread to epitopes in other regions of GAD65 and GAD67 [40, 41]. Similarly, intermolecular spreading of the T cell reactivity and antibody responses to islet antigens was found to have occurred during the pre-clinical phase of type I diabetes in subjects at risk (as defined by positivity for autoantibodies to l-islet antigens) for developing clinical diabetes [42]. Taken together, the above studies lend support to the role of epitope spreading in the pathogenesis of autoimmune diabetes in humans, and provide impetus for development of new therapies for individuals at risk for type I diabetes. The observations that patients with IDDM have activated T cells against GAD65 [43], and that diabetic siblings possess relatively lower frequency of Valpha24JalphaQ+ T cells compared to non-diabetic twins [44], have provided new insights into the autoreactive T cell repertoire and its regulation in this disease. The precise relationship of the activity of these T cell subsets to the levels and epitope specificity of anti-GAD autoantibodies in diabetes remains to be determined. 2.3. Arthritis
Rheumatoid arthritis (RA) is a human autoimmune disease that primarily affects the joints. It is characterized by persistent inflammatory synovitis, generally involving peripheral joints in a symmetrical fashion. The etiology of RA is not known. Adjuvant-induced arthritis (AA) is an experimental model of human RA, and it can be induced in Lewis rats by immunization with heat-killed Mycobacterium tuberculosis H37Ra in mineral oil [45, 46]. The T cell response to the 65-kD mycobacterial heat shock protein (Bhsp65) has been implicated in the pathogenesis of AA as well as RA [47-50]. We have
24
previously shown that there is a shift in the epitope specificity of the T cell response to Bhsp65 during the course of AA in the Lewis rat [4]. In the acute phase of AA, the T cell response of arthritic Lewis rats was focused on peptide 177-191 (which contains the arthritogenic determinant 180-188) and other epitopes in the middle and N-terminal regions of Bhsp65. However, during the recovery phase of AA, appearance of new T cell responses directed to the 5 C-terminal epitopes of Bhsp65 (namely, 417431, 441455, 465-479, 513-527, and 521-535) was observed. Interestingly, pretreatment of na'fve Lewis rats with the synthetic peptides representing these 5 Bhsp65 C-terminal determinants (BCTD) significantly reduced the severity of subsequent AA [4, 51 ]. Furthermore, T cell responses to BCTD were observed early following M. tuberculosis (H37Ra, heat-killed) challenge in the Wistar Kyoto (WKY) rats that possess the same MHC haplotype as the AA-susceptible Lewis rat but are resistant to induction of AA [4]. The simultaneous emergence of T cell responses to the pathogenic (180-188/ 177-191 determinant) and regulatory (BCTD) epitopes could explain, in part, the AA-resistance of WKY rats. The results of our recent study demonstrate that the C-terminal epitopes of self hsp65 are also disease-regulating in nature (Durai, Gupta and Moudgil, manuscript in press [51 a]). The above results suggest that spreading of the T cell responses to BCTD during the course of AA might be involved in natural recovery from acute AA in the Lewis rat. Furthermore, these findings demonstrate that epitope spreading in the course of an autoimmune disease is not always pathogenic; instead, in another situation, it can also be disease-regulating in nature. This is the first study [4] reporting the disease-regulating aspect of epitope spreading in the course of an autoimmune disease. Another aspect of emergence and spreading of T cell responses was revealed in a study on the immunological basis of environmental modulation of AA in the Fischer F344 (F344) rat [52]. We observed that F344 rats kept in a barrier facility (BF-F344) were susceptible to AA, whereas those maintained in a conventional facility (CV-F344) spontaneously acquired protection (or resistance) against induction of AA. Testing of the T cell responses to peptides of Bhsp65 of naive F344 rats showed that CV-F344 but not BF-F344 rats raised T cell response to mul-
tiple epitopes of Bhsp65, including BCTD. In addition, the level of these spontaneously-arising T cell responses gradually increased with the duration and extent of exposure of F344 rats to the conventional environment. The functional significance of these BCTD-directed T cell responses was evident from results of adoptive transfer experiments: BCTD-restimulated (in vitro) splenic cells of naive CV-F344 rats could adoptively transfer protection against AA to naive BF-F344 recipients [52]. The above results present one of the mechanisms underlying the influence of housing environment on protection against an autoimmune disease. The role of conventional environment in facilitating the induction of an autoimmune disease has been observed in various models of autoimmunity (e.g., EAE, thyroiditis, hemolytic anemia, Pristane-induced arthritis (PIA) etc.) [53-58]. In contrast to this, our study described above [52] along with others in animal models of arthritis and diabetes [59-62a] reflect upon the protective effect of environment on autoimmunity. AA is believed to be a T cell-mediated disease. There is meager information about the role of antibodies to Bhsp65 in the pathogenesis of this disease. However, recent studies have shown that in addition to possessing disease-regulating T cell epitopes, Bhsp65 also harbors protective B cell epitopes. During the course of AA, Lewis rats develop antibodies against Bhsp65, and the number of epitopes within Bhsp65 recognized by these antibodies gradually increases during the recovery phase of the disease [63] (Kim and Moudgil, manuscript in preparation). Thus, like spreading of the T cell response to BCTD in AA described above, arthritic Lewis rats also show spreading of anti-Bhsp65 antibody response. Interestingly, challenge of naive Lewis rats with peptides comprising the B cell epitopes (e.g., peptides 31-46 and 37-52) represented in the diversified response afforded protection against subsequent disease [63]. Similarly, passive immunization with the antibodies directed against one of these epitopes (peptide 31-46) also suppressed subsequent AA [63]. Furthermore, the resistance to AA of BN rats correlates with natural antibody response to the same B cell epitopes as those involved in epitope spreading in the susceptible Lewis rats. These results demonstrate the role of spreading of antibody response to Bhsp65 in regulation of AA. Spreading of the tolerogenic effect of disease-
related epitope of Bhsp65, p176-190, and that of the suppressive effect of antigen-specific anergic T cells have been invoked in downmodulation of the course of avridine-induced arthritis and/or AA [64, 65]. It was observed that induction of nasal tolerance against p176-190 provided protection against subsequent AA as well as avridine-induced arthritis [64]. It was proposed that tolerance of T cells recognizing p176-190 or its mimic spread to T cells of other specificities that are involved in induction of arthritis. Similarly, it was suggested that a subset of anergic T cells, in the presence of the specific antigen recognized by these cells, exerted spreading suppressive activity on T cells of other antigen specificities [65]. Furthermore, this amplification of suppressive effect was attributed to modulation of the activity of the antigen presenting cell (APC) by anergic T cells. The influence of a disease-regulating antigenic challenge on diversification of response has been described in the Pristane-induced arthritis (PIA) model [66]. A comparison of the T cell response of arthritic mice compared to mice protected against disease by pre-treatment with Bhsp65 revealed that there was 'repertoire limitation' (the opposite of diversification of response) in Bhsp65 pre-treated mice [66]. The protective effect of Bhsp65 was attributed in part to prevention of diversification of response via induction of a Th2 response. A study of the T cell repertoire in RA patients showed that several dominant T cell clones were found in the synovial membrane but not in the peripheral blood [67]. Analysis of the complementarity-determining region 3 (CDR3) region following sequencing of the T cell receptor (TCR) V~ V-D-J junctional regions showed evidence for antigen-driven selection of the TCR. This TCR selection was attributed to determinant spreading during the course of RA. More similar studies in RA would help define the fine characteristics of the pathogenic T cell repertoire in this disease.
2.4. Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE) is a multisystem human autoimmune disease characterized by development of autoantibodies against a variety of autoantigens: dsDNA, Ro/La ribonucleoprotein complex, histones and other nucleosomal compo-
25
nents, spliceosomal proteins, ribosomal proteins, etc [68-70b]. Antibodies against these autoantigens have also been found to develop during the course of disease in various experimental models of lupus (Table 2). Studies focused on autoantibodies in lupus patients and animal models of lupus have revealed both inter-molecular and intra-molecular epitope spreading involving one or more of the above-mentioned autoantigens. For example, James et al [71] demonstrated that NZW rabbits immunized with an Sm B/B' peptide (representing a C-terminal epitope) developed antibodies directed against the immunogen and other epitopes within the middle and amino-terminal regions of the Sm B/B' antigen. In addition, these animals also raised antibody response to other spliceosomal proteins (e.g., D, 70K, A, and C). This new experimental model of lupus would help define the role of autoantibodies to Sm B/B' in the pathogenesis of the disease in lupus patients [71]. In another study, mice immunized with La protein developed autoantibodies not only to the immunogen but also to 60-kD Ro, whereas mice immunized with 60-kD Ro produced anti-Ro antibodies as well as anti-La antibodies [72]. These results and those of other studies [73-77] demonstrate that development of antibodies to multiple components of the L a ~ o ribonucleoprotein complex occurs after challenge with a single component of the antigenic complex. In addition to experimentally-induced epitope spreading described above, spreading of the T helper and antibody response to components of the nucleosome in (SWR x NZB)F1 mice [77a, 77b], and intramolecular spreading of the T cell response to T helper (Th) epitopes within the V Hregion of a pathogenic anti-DNA antibody in NZB/NZW F1 (BWF1) mice [78] has been observed during spontaneously-arising disease. The patterns of autoantibody responses observed in animal models of lupus have been shown to be influenced by multiple factors including, genetic makeup (strain differences), the MHC class II haplotype of the host, age of the animal, the level of self tolerance to a particular autoantigen, and nature of the immunogen (homology between the corresponding self and foreign antigenic determinants, immunogenicity of endogenous antigens, the level of crossreactivity between different antigens, sharing of crossreactive epitopes between unrelated and physically-separated antigens, molecular mimicry,
26
etc.) [70a, 70b, 72-77]. Interestingly, tolerization of lupus-prone mice against either autoantibodyderived peptides or the protein/peptides (e.g., nucleosomal peptides) representing antigenic determinants involved in epitope spreading can successfully halt the progression of epitope spreading as well as the disease process [77c, 79, 80]. Alternatively, blockade of costimulation by administration of antiB7.1 and anti-B7.2 antibodies into BW F1 mice has also been shown to suppress the development of SLE [81]. Similarly, induction in lupus-susceptible mice of inhibitory CD8+ T cells that are capable of controlling the activity of autoreactive B cells can downmodulate the course of disease and prolong survival of the vaccinated mice [82]. SLE patients develop autoantibodies to a variety of autoantigens described above. Studies on antigen reactivity of sera of lupus patients have demonstrated temporal shifts either in recognition of another antigen (e.g., inter-molecular spreading from Sm antigen to RNP reactivity) or in reactivity to different epitopes within the same antigen (e.g., intra-molecular spreading within a given antigen depending on the model system: Sm B/B', Sm D, ribosomal protein L7, caspase-8, etc.) [83-87].
2.5. Myasthenia Gravis Myasthenia gravis (MG) is an autoimmune disease characterized by weakness and fatigability of skeletal muscles owing to impaired neuromuscular transmission resulting from antibody-mediated autoimmune attack against the nicotinic acetylcholine receptor (AChR). Experimental autoimmune myasthenia gravis (EAMG) is inducible in animals following immunization with either AChR or its peptide along with an immunomodulator (e.g., antiCTLA-4 antibody that can enhance T cell activation by interfering with inhibitory signal via CTLA-4 molecule), and it serves as a model for human MG [88]. Epitope spreading has been reported in different models of EAMG. NZW rabbits challenged with heterologous (human) AChR t~-subunit peptides developed clinical MG and antibody response to self (rabbit) AChR [88, 89]. These antibodies reacted strongly to rabbit AChR but only weakly to human AChR. These results show that the observed spreading of antibody response to self AChR in rabbits challenged with human AChR is attributable to
processing and presentation of endogenous AChR. EAMG can also be induced in mice. C57BL/6 (B6) mice immunized with AChR and boosted with ~146-162 peptide of AChR develop EAMG [90]. In another study, it was observed that B6 mice challenged with the immunodominant peptide o~146-162 of AChR and given anti-CTLA-4 antibody developed clinical signs of MG [91]. These mice developed both antibody and T cell response against the peptide immunogen (~ 146-162) and other epitopes of AChR cx-subunit [91]. In another recent study, it has been shown that rats immunized with extracellular region of self (rat) AChR ~-subunit initially developed antibodies to the immunogen followed by antibody response to the intracellular cytoplasmic part of AChR [92]. There is some suggestive evidence for epitope spreading in patients with MG. The T cell clones generated from patients with MG were specific for peptide epsilon 201-219 and epsilon subunit of adult AChR, but did not show any reactivity to fetal AChR [93]. However, the serum antibodies of these patients showed higher reactivity with fetal AChR compared to adult AChR. These results suggest that anti-AChR antibodies in MG patients might have been generated following epitope spreading triggered by T cells specific for adult AChR. In this context, it has recently been described that primary human myoblasts treated with IFN-~, for induction of MHC class II expression can present endogenous AChR epitope to an AChR-specific CD4+ T cell clone [94]. These results suggest that during the course of clinical MG, myoblasts can serve as APC and amplify the ongoing T cell response. Furthermore, these myoblasts may also become targets of cytotoxic immune attack, and the resulting release of self antigens can in turn contribute to epitope spreading.
2.6. Other Autoimmune Diseases Pemphigus is an autoimmune disease affecting the skin. Epitope spreading has been observed in pemphigus vulgaris, pemphigus foliaceus, and other cutaneous autoimmune disorders (reviewed in Ref. [95]). Similarly, diversification of the autoimmune response has also been reported in animal models of neuritis [96], uveitis [97], gastritis [98], and oophoritis [99, 100] (Tables 1 and 2).
3. EPITOPE SPREADING IN OTHER IMMUNE-MEDIATED DISEASES 3.1. Organ Transplant/Graft Rejection Alloreactive T cells recognize foreign (allogeneic) MHC molecules either as intact antigens expressed by the donor APC (direct allorecognition) or as epitopes derived from donor MHC but presented by the recipient APC (indirect allorecognition) [ 101 ]. In a study on heart allograft recipients [ 102], the epitope specificity of self-restricted alloreactive T cells (indirect allorecognition) was examined. Sequential blood samples collected from these patients over a 3 year period were tested for allopeptide reactivity using a panel of peptides representing sequences derived from 32 HLA-DR alleles. The higher incidence of complications (in this case, coronary artery vasculopathy; CAV) in these patients directly correlated with persistent alloreactivity as well as epitope spreading (both intra- and inter-molecular). These results demonstrate the role of epitope spreading in chronic graft rejection. In other studies on heart as well as liver transplant recipients [ 103, 104], a differential T cell response to the alloantigens within the graft was observed in recipients undergoing primary acute rejection compared to those having either recurring episodes of rejection or a chronic rejection. The T cell responses in the former were directed to a single dominant epitope in one of the two mismatched HLA-DR antigens, whereas those in the latter were found to spread to both HLA-DR antigens as well as to other alloantigens of the transplanted tissue. Thus, spreading of the alloresponses apparently was involved in the pathogenesis of graft rejection. We have described above that both intra-molecular and inter-molecular epitopes spreading occur during the course of spontaneously developing autoimmune diabetes in the NOD mouse [35, 36]. Interestingly, deliberate deviation (from Thl to Th2 type) of the GAD-specific T cell response in young NOD mice was successful in inhibiting the progression of the disease process [37]. Furthermore, a similar experimental manipulation also prolonged survival of syngeneic islet grafts in NOD mice with diabetes [105]. These results show that tolerance induction to the key autoantigen could also benefit the outcome of organ graft. Similarly, in another
27
study on skin graft tolerance, it was shown that tolerance induction (by donor-specific transfusion; DST) against one MHC molecule (La)-mismatch spread to skin grafts with more than one MHC-mismatch [106]. Thus, tolerance to allogeneic MHC showed spreading that was beneficial for graft survival. 3.2. Tumors
The study of anti-tumor immunity in experimental models as well as patients vaccinated with defined peptide antigens has revealed the occurrence of epitope spreading involving CD8+ cytolytic T cell (CTL) and/or CD4+ T cell responses [107-110]. In a study using ovalbumin (OVA) as a model tumor antigen and EL4 thymoma cells with or without OVA expression (the former is named EG.7OVA) as the tumor-inducing cell line, it was observed that B6 mice immunized with OVA or EG.7OVA raised CTL response directed to the immunodominant epitope 257-264 of OVA [ 107]. Furthermore, CTL response induced by OVA afforded protection against tumor induction by challenge with EG.7OVA cell line. Interestingly, mice that survived EG.7OVA challenge raised additional CTL responses to two other epitopes within OVA, namely 55-62 and 176-183, and to other endogenous antigens within EL4 cells. The emergence of new CTL responses was attributed to release of tumor antigens following tumor rejection and subsequent cross-presentation by the APC of cryptic epitopes within these tumor antigens. Similarly, epitope spreading involving CTL response to tumor antigens was observed in another study based on P815 tumor model [108]. Immunization of DBA/2J mice with a single P815-derived tumor peptide, P1A, induced potent CTL response that could successfully induce rejection of P1A § P511 tumor. Interestingly, mice undergoing tumor rejection developed new CTL responses directed to another P815-derived peptide antigen, P1E, and also could reject P1A- P1.204 tumor. The induction of new CTL response was attributed to processing and presentation of released tumor antigen by the host APC. These results demonstrate that diversification of the CTL response could be beneficial in control or elimination of antigen-loss tumor cell variants that arise during the course of tumor progression and tumor therapy. Diversification of anti-tumor CTL/CD4+
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immune response has also been observed in patients undergoing peptide-based immunotherapy [109, 110]. In one study, induction of immunity against peptides derived from HER-2/neu (a self tumor antigen that is quantitatively over-expressed in certain adenocarcinomas) was tested in patients with breast and ovarian cancer [ 109]. Immunization of patients with HER-2/neu peptides was performed i.d. using GM-CSF as the adjuvant. Peptide-specific CD4+ T cell responses were observed in all patients, and in most of them, the peptide-primed T cells were crossreactive with the native HER-2/neu protein. The T cell response against the HER-2/neu protein included reactivity against new epitopes within the protein (epitope spreading). Induction of these new T cell specificities was attributed to processing and presentation of endogenous HER-2/neu protein. In another study, induction of CTL response against HER-2/neu and MUCl-derived peptides was tested in patients with advanced breast and ovarian cancer [ 110]. Vaccination of patients was performed using autologous dendritic cells pulsed with the appropriate peptides. One of the patients immunized with HER-2/neu peptides raised T cell response directed against MUC-1 protein, whereas another patient vaccinated with MUC-1 peptides raised T cell response against CEA and MAGE-3 peptides. These studies demonstrate that epitope spreading invariably accompanies anti-tumor vaccination in both tumor models and cancer patients, and therefore, it is of significance in determining the outcome of the vaccination regimen. 3.3. Infection/Vaccination
(i) Lymphocytic choriomeningitis virus (LCMV) infectio.n. It has been observed that the cytolytic T cell (CTL) response in mice infected with LCMV is focused on the immunodominant epitope of the viral nucleoprotein during the acute phase of the infection, but it diversifies to sub-dominant epitopes of the viral glycoprotein in the chronic phase of the disease [111]. Furthermore, CTL response to the immunodominant epitopes apparently plays a critical role in induction of acute disease, whereas responses to sub-dominant epitopes are involved in inducing protective immunity and clearing of the infection [ 111, 112]. The density of MHC-peptide complexes on the APC surface and the composi-
tion of the T cell repertoire are among the major factors determining the hierarchy of CTL response [112, 113]; (ii) Lyme disease is caused by infection by Borrelia burgdorferi (Bb), and some of the clinical features of the disease are believed to be of autoimmune origin [114]. For example, molecular mimicry between epitopes within the outer surface protein A (Osp A) of Borrelia burgdorferi and leukocyte function-associated antigen- 1 (LFA-1) has been invoked in the pathogenesis of Lyme arthritis [114]. In one study, diversification of the antibody response to different antigens of the pathogen was observed with progression of Lyme disease [115]. However, the precise significance of this antibody spreading in pathogenesis of the disease has not yet been fully defined; and (iii) DNA vaccination
using human immunodeficiency virus-1 (HIV-1) r~ulatory genes: Both protein and DNA encoding the regulatory genes nef, rev, and tat of HIV-1 were found to be immunogenic in mice, and immunization with DNA plasmids induced higher magnitude of antibody response (compared to protein antigens) along with epitope spreading [116]. However, the protective efficacy against HIV- 1 of these antibodies remains to be determined.
4. M E C H A N I S M S UNDERLYING E P I T O P E SPREADING DURING T H E COURSE OF AN A U T O I M M U N E DISEASE Considering diverse experimental models of autoimmune diseases involving different target organs and predominantly either T cell (CD4+/CD8+)- or antibody-response to one or more disease related antigens as the pathogenic effector mediators (Table 2), various mechanisms have been proposed to explain the phenomenon of epitope spreading observed during the course of different autoimmune diseases (Table 3). These include inter-related factors or conditions that operate in concert in different combinations, depending on the disease process, to induce epitope spreading (Fig. 1, Table 3). These are described below.
1. Upregulation of the display of cryptic~subdominant epitopes within a self antigen under inflammatory conditions. Native self and foreign antigens possess potential T cell epitopes that are
Table 3. Proposed mechanisms underlying the phenomenon of epitope spreading 1. Upregulationof the display of cryptic/sub-dominant epitopes within a self antigen under inflammatory conditions 2. Releaseof self antigens and their processing and presentation following tissue damage in the course of an autoimmune disease or a chronic microbial infection 3. The frequency and avidity of epitope-specific precursor T cells within the mature T cell repertoire favoring responsiveness to certain antigenic determinants over others 4. Presentationof neo-epitopes within a particular self antigen by the B cells specific for that antigen 5. The influence of antigen-bound antibodies on processing and presentation of T cell epitopes within that antigen
processed and presented either efficiently (dominant determinants) or poorly (cryptic/sub-dominant determinants) by the antigen presenting cells (APC) [117]. However, both sets of determinants are immunogenic in the peptide form. In the case of a self antigen, tolerance is readily induced to its dominant but not cryptic/subdominant epitopes [118-121]. For this reason, unlike a foreign dominant epitope that is generally immunogenic, a self dominant determinant generally fails to induce a response owing to self tolerance. However, the T cells against cryptic/sub-dominant epitopes escape tolerance induction in the thymus and therefore, are available in the mature T cell repertoire. These T cells can be activated provided the otherwise poorly processed cryptic/sub-dominant epitopes within the native self antigen are efficiently presented to the T cells by professional APC. This could happen under conditions of upregulated antigen processing and presentation events as in the case of inflammation and/or infection [3, 122-125]. The T ceils specific for cryptic/sub-dominant epitopes of an endogenous self antigen thus activated (constituting epitope spreading) can participate in further propagation of the ongoing disease process (Fig. 1). In this manner, the inflammatory and cytokine milieu created during the initial phase of the disease induced by T cells specific for one self antigen or one of its epitopes can facilitate induction of new T cell responses
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Experimentally-induced autoimmune disease
Spontaneously-developing autoimmune disease
Initiation of the autoimmune process
Tissue damage ~
Inflammation
Upregulation of antigen ~ processing and presentation
Microbial infection
Release of self antigens
1
Display of previously cryptic self epitopes
Priming of new subsets of self-reactive T cells
Propagation of autoimmunity
1 Worsened and chronic disease (Pathogenic epitope spreading)
Control of activity of pathogenic T cells
l
Remission from acute phase of the disease (Regulatory epitope spreading)
Figure 1. Epitope spreading: the underlying mechanisms and role in the disease process. Initiation of an autoimmune disease, either spontaneously or following an antigenic challenge, creates a local inflammatory milieu that is conducive to upregulation of antigen processing and presentation. In addition, the inflammatory events, either of autoimmune origin or induced by a microbial infection, cause tissue damage leading to the release of self antigens. Under these circumstances, self antigens are processed efficiently by the antigen presenting cells (APC) revealing previously cryptic epitopes to potentially self-reactive T cells available in the mature T cell repertoire. The activation of new subsets of autoreactive T cells enhances the ongoing inflammatory events and further amplifies the processes described above. The outcome of priming of the self-reactive T cells depends on multiple factors: the antigen/epitopes targeted during epitope spreading, the nature of the T cell response (e.g., Thl/Th2), the genetic make up of the individual, etc. In this context, epitope spreading could either further perpetuate (pathogenic epitope spreading) or attenuate (protective epitope spreading) the ongoing disease process. The above scheme depicts only induction of the T cell response. However, the activated T cells may provide help to multiple autoreactive B cells displaying one of the epitopes of the antigen recognized by T cells and thereby, lead to spreading of antibody responses. Similarly, activated B cells displaying epitopes of an autoantibody can prime the appropriate T cells, which upon activation help other B cells to effect spreading. In addition, the processing and presentation of certain T cell epitopes within the native self antigen can be modulated by antigen-bound autoantibodies. These processes are described in detail under Section 4.
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directed to other epitopes within the same antigen (intra-molecular spread) and/or another endogenous self antigen (inter-molecular spread). This also is one of the mechanisms by which self antigens released following tissue damage (described below) could contribute to epitope spreading. 2. Release of self antigens and their processing and presentation following tissue damage in the course of a microbial infection or an autoimmune disease. The etiology of most of the human autoimmune diseases is not known. However, one important factor that is believed to constitute a trigger or precipitating factor for induction of autoimmunity is microbial infection. However, the precise role of microbial agents in induction or perpetuation of autoimmunity is not yet fully defined. Some of the mechanisms proposed to explain this association include - a) molecular mimicry: a microbial antigen/epitope structurally mimicking a self antigen/epitope such that the T cells primed following microbial infection can be re-stimulated by the endogenous self antigen, and thereby, can target cells/tissue expressing that self antigen leading to tissue damage [126-129]; b) bystander activation: stimulation of potentially self-reactive T cells under the immune environment where priming of microbial antigen-specific T cells is taking place; the autoreactive T cells can then cause tissue damage [3]; and c) tissue injury. leading to induction of a_utoimmune response: the tissue damage caused by microbial infection results in the release of endogenous self antigens that can then be processed and presented by local as well as peripheral APC leading to priming of self-reactive T cells [3, 130, 131] (Fig. 1). The T cells activated locally can then induce autoimmune damage within the target organ [130, 131], whereas the T cells activated in the periphery can re-enter the target tissue and damage the cells expressing a particular self antigen [3]. The role of virus-mediated tissue damage in induction of autoimmunity has been best demonstrated by Rose and colleagues in the model of autoimmune myocarditis [132, 133], by Sarvetnick and colleagues in the NOD model of type I diabetes [134], and by Miller and colleagues in the TMEV-induced EAE model [23-26]. Autoimmune myocarditis induced by coxsackievirus B3 has been shown to be a biphasic disease: an early 'infection' phase and a subsequent 'autoimmune'
phase characterized by T cell and antibody response to cardiac myosin [133]. In TMEV-induced EAE, the induction of autoimmunity has in addition been linked to epitope spreading resulting from release of self antigens [23-26]. (In a viewpoint different from release of antigens within the target organ, it has been suggested that epitope spreading in EAE might be initiated in the peripheral lymphoid tissues instead of the CNS; this proposition is based on the observation that myelin antigens are expressed in the lymph node, spleen and thymus of SJL mice [135].) An additional aspect relevant to release of tissue antigens is the relative susceptibility/resistance to acute infection of the target organ [136]; for example, the cytokines IFN-~ and IFN-7 produced within the pancreatic islets can significantly influence the susceptibility to coxsackievirus B4 infection of the cells. Another mechanism for the release of intracellular self antigens has been proposed by Rosen and colleagues [137, 138]; in this process, apoptotic cells serve as an important source of self antigens, and the novel antigenic fragments produced in apoptotic surface blebs are implicated in reversal of self tolerance leading to induction of autoimmunity. Taken together, irrespective of the mechanism causing tissue damage (whether the initial phase of an autoimmune damage or a microbial infection), the resulting release of self antigens and their processing and presentation leads to activation of new subsets of self-reactive T cells (Fig. 1) constituting epitope spreading. 3. The frequency and avidity of epitope-specific precursor T cells within the mature T cell repertoire favoring responsiveness to certain antigenic determinants over others. The T cell responses to various epitopes within a native antigen, or to individual antigens within a mixture of antigens, are hierarchical, and accordingly, th.e corresponding antigen/ epitope can be categorized as immunodominant, sub-dominant, or cryptic [ 117]. A similar hierarchy is also observed during the induction of epitopespecific T cell tolerance [139, 140]. The observed hierarchy of T cell response to the immunogen/ tolerogen is influenced by multiple factors operating at the level of the APC (described above) as well as those relating to the composition of the mature T cell repertoire [6, 111-113, 140]. Both the size (frequency) and composition (e.g., the relative levels of
31
high avidity vs. low avidity T cells) of the T cell repertoire can significantly influence the magnitude as well as the timing of appearance of response to an antigenic determinant following challenge with the native antigen. In the context of epitope spreading, the above-mentioned characteristics of the T cell repertoire have been invoked, in part, in explaining the hierarchy as well as ordered sequential appearance of response to different antigens/epitopes involved in inter- or intra-molecular epitope spreading [3, 8, 9, 13, 141].
4. Presentation of neo-epitopes within a particular self antigen by the B cells specific for that antigen. Most of the above discussion on priming of the T cells is based on APC implying primarily dendritic cells and macrophages. However, activated B cells serving as potent APC also can participate in induction and propagation of epitope spreading. Mamula and Janeway proposed an interesting model based on the role of B cells as APC in the diversification of the T cell and antibody response [ 142]. According to this model, the initial T cell priming to self epitopes is done by APC like dendritic cells, and these activated T cells then provide help to the appropriate B cells. The activated B cells in turn can take up antigen by specific interaction between the antigenic determinants and the B cell receptors, and then process and present that antigen to the T cells. The newly displayed antigenic determinants by B cells (as APC) can now activate new subsets of T cells, which then can provide help to new population of B cells. These T-B interactions thus lead to diversification of both T cell and antibody responses. In a parallel situation, B cells could present epitopes of an autoantibody to the T cells, and these T cells could then render help to multiple clones of B cells, each displaying an epitope crossreactive with the original determinant (reciprocal T-B determinant spreading) [78]. The role of B cells as APC in induction of selfdirected antibody response in the setting of epitope spreading has been demonstrated in experimental models of lupus [143, 144].
5. The influence of antigen-bound antibodies on processing and presentation of T cell epitopes within that antigen. We have described above the role of B cells in diversification of the T cell/antibody response. Another mechanism by which
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components of humoral immunity can influence the induction of epitope-specific T cell response is through antigen-specific antibodies. Through an interesting series of experiments, it has been demonstrated that antigen-bound antibodies can significantly influence the processing and presentation of T cell epitopes within that antigen [122, 145]. Depending on the nature and site (in context of the antigenic structure) of interaction between the antigenic determinant and the antibody, antibodies bound to specific epitopes of an antigen can either enhance or suppress the T cell response to the epitope involved. These results provide one of the mechanisms explaining a shift in the epitope specificity of T cell response (as observed in epitope spreading) during the course of an immune response directed towards a microbial or self antigen.
5. THE E F F E C T O R MECHANISMS INVOLVED IN MEDIATING THE FUNCTIONAL O U T C O M E OF EPITOPE SPREADING: THE COMPLEX INTERPLAY BETWEEN TH1-TH2 CYTOKINES We have described above that epitope spreading can be disease-propagating in some diseases (e.g., EAE and diabetes in the NOD mouse) but disease-regulating in others (e.g., AA). In a simplistic model, diseases that are believed to be primarily Th 1-mediated can be perpetuated by new Thl responses arising during the course of disease, whereas those dependent on a Th2 response for disease initiation can be further propagated by new Th2 responses. Similarly, considering both the regulation of an ongoing immune response and the protective aspect of epitope spreading, Thl-mediated diseases can be regulated by a Th2 response, whereas diseases characterized by a predominantly Th2 response can be controlled by a Thl response. However, in recent years, it has increasingly been realized that comprehension of the pathogenesis of autoimmune diseases on the above-mentioned paradigm of Thl-Th2 cross-regulation might be an over-simplification [146]. For example, certain cytokines that are secreted by Thl (e.g., IFN-T and TNF-ct) or Th2 (e.g., IL-4 and IL-10) cells may have dual and opposing function. Taking the example of IFN- 7 in
more detail, in a particular animal model of autoimmunity, IFN-y may be disease-inducing in one set of conditions but disease-regulating in others. It is known that IFN-y plays a critical role in induction of Thl-mediated diseases. However, the results of studies in various experimental models of autoimmunity (e.g., AA [147], EAE [148-152], collageninduced arthritis (CIA) [153, 154], diabetes in the NOD mouse [155, 156], autoimmune myocarditis [157, 158], autoimmune uveitis [159, 160], and glomerulonephritis [ 161 ]) using diverse experimental approaches (e.g., use of IFN- 7 -/- mice, IFN-TR -/- mice, and challenge of mice/rats either with IFN- 7 or with neutralizing anti-IFN-y antibodies) have revealed that IFN-y plays a regulatory role in Thl/IL-12-mediated diseases. It has been suggested that control of the activity of pathogenic T cells by IFN-y might involve one or more of the following mechanisms [156, 160, 162, 163]: suppression of proliferation of target pathogenic T cells, induction of apoptosis in pathogenic T cells, a change in the activity of APC, influence on migration of T cells into the target organ, etc. In addition to the abovementioned information from animal models, limited clinical trials conducted in patients with RA several years ago also pointed towards a beneficial effect of IFN- 7 [ 164-166]. In a contrary situation, it has been suggested that IFN-yplays an important role in mediating the tissue damage in murine SLE [ 167]; lupus is characterized by development of autoantibodies and a predominance of Th2 cytokine response. In view of the above information, both induction and regulation of autoimmune disorders would need a fresh look beyond the simple cross-regulatory function of Thl and Th2 cytokines [ 146].
6. P H Y S I O L O G I C A L SIGNIFICANCE OF EPITOPE SPREADING: I N V O L V E M E N T OF EPITOPE S P R E A D I N G IN THE PATHOGENESIS OF AN A U T O I M M U N E DISEASE.
Experimental evidence from studies in different models of autoimmune diseases supports the role of epitope spreading in the pathogenesis of the disease process. The results of these studies can be categorized into 3 functional outcomes- (i) Pathogenic epitope spreading" most of the published studies
summarized above (Table 2) describe that the new T cell responses emerging via epitope spreading are involved in progression of the initial autoimmune process, and thereby, in perpetuation and chronicity of the disease process. Some of the supportive evidence establishing the pathogenic role of epitope spreading consists of the following observations - a) the intensity and extent of epitope spreading correlates with the severity and/or duration of the disease process, and that the diseased vs. non-diseased animals differ in the pattern of responses to epitopes involved in diversification of response [3, 8, 13, 35, 36]; b) sirrfilarly, the induction of epitope spreading is associated with the frequency and/or chronicity of graft rejection [103, 104], and with complications of organ transplantation [102]; c) tolerization of the T cells specific for the antigenic determinants involved in epitope spreading and relapse of disease, and cytokine modulation can limit the progression of the disease and prevent clinical relapses [3, 8, 13, 16, 35, 36]; and d) blockade of co-stimulation at the appropriate time before or after disease induction prevents disease progression [3, 10, 11]; (ii) Protective epitope spreading: in contrast to the above, other studies provide evidence favoring a regulatory or protective role for T cell/ antibody responses comprising diversification of the initial immune response [4, 63]. The protective role of epitope spreading is evident from the findings showing that- a) pre-treatment of animals with the antigenic determinants involved in epitope spreading using an immunogenic regimen (leading to priming and expansion of antigen-specific T cells) instead of a tolerogenic regimen affords protection from disease [4]; b) passive transfer of antibodies from an animal in recovery phase of the disease into a naive syngeneic recipient leads to suppression of subsequently induced disease in the recipient [63]; and c) epitope spreading occurring during the course of tumor rejection affords protection against subsequent challenge with the same tumorigenic cell line or its antigen negative variant [107, 108]; and (iii) Epitope spreading unrelated to the dis.ease process or no spreading at all: in a couple of studies in EAE, epitope spreading either was evident but without any functional relationship with the disease process [168] or did not occur at all [169]. In another study, a clinically relapsing disease was observed in a single-TCR-transgenic mice that lack
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all T cell specificities except the one required for initiation of the disease process [ 170]. Taken together, the above-mentioned results suggest that there is no single functional outcome that can a priori be assigned to epitope spreading, and therefore, each disease and the antigenic response associated with it needs to be examined objectively and without any preformed notion or bias. In this regard, any prediction regarding the contribution of epitope spreading to the disease process in vastly heterogeneous human population poses an important challenge for both clinical prognosis and custom designing of the therapeutic regimens.
7. IMPLICATIONS OF EPITOPE SPREADING IN IMMUNOTHERAPY OF AUTOIMMUNE DISEASES: HINDRANCE VS. FACILITATION OF THE CONTROL OF THE AUTOIMMUNE PROCESS. A great deal of effort that has been invested in developing immunotherapeutic approaches for autoimmune diseases has centered on inactivation of the potentially pathogenic T cells. It is evident from extensive studies in animal models that it is relatively easier to modulate the antigen-specific immune response for preventing the development of autoimmunity than for controlling the ongoing disease process. However, from the viewpoint of treatment of patients with autoimmune diseases, the development of effective 'therapeutic' approaches has a much higher priority over devising 'preventive' approaches. In the long run, of course, preventive approaches targeted to a potentially susceptible human population would also be very rewarding. In regard to treatment of ongoing disease, epitope spreading that is disease-propagating in nature poses a major hurdle [ 1, 3, 14]. For a successful treatment, the patient would have to be treated very early in the course of disease prior to occurrence of epitope spreading. However, it might be a rather daunting task to make a prediction about the timing of epitope spreading during the natural course of disease in individual members within a patient population. Many more clinical studies are needed to determine the timing of onset of epitope spreading during the course of MS or IDDM, for example. On the other hand, epitope spreading that is disease-regulating in
34
nature can readily be exploited for therapeutic purposes [4, 51 ]. In this situation, therapeutic regimens aimed at priming and expanding the T cells specific for the new epitopes arising during the course of disease could help expedite recovery from acute phase of the disease. In this situation, the precise timing of onset of epitope spreading might not be that much of a concern. However, in either situation, carefully planned clinical trials would be warranted to insure that the programmed modulation of the immune response delivers the expected outcome. Otherwise, strategies aimed at suppression of the disease might rather exacerbate the disease. This has been exemplified in animal models. For example, the use of two versions of the same immunomodulator (in this case, anti-B7.1 antibody) [10] or administration of the same antibody (e.g., anti-CTLA-4 antibody) at different time during the disease course [ 12] had an opposite outcome in mice with EAE.
8. CONCLUDING REMARKS Epitope spreading represents a dynamic quantitative/qualitative change in the T cell and/or antibody specificities evolving during the course of an immune response initiated by a dominant antigen/epitope associated with a pathological condition. The primary event may either be triggered experimentally or arise spontaneously. The subsequently developing new T cell and/or antibody responses then participate in perpetuation of the initial pathological damage leading to chronicity of the disease. However, depending on the disease process, spreading of response to potentially disease-regulating antigens/epitopes can be protective in nature, and therefore, epitope spreading also represents a mechanism by which initial pathological immune responses are limited to effect natural recovery from acute phase of the disease. We suggest that like many other physiological processes in the body, epitope spreading represents a snapshot of dynamic events attempting to strike a balance between the pathogenic and regulatory components of the antigen-specific T cell responses, and that the picture obtained would vary depending on the time that the responses are sampled and tested during the disease process. Study of the temporal pattern of appearance of pathogenic vs. regulatory T cell and/
or antibody responses in relation to epitope spreading would significantly advance our understanding of the pathogenesis of a variety of immune disorders, particularly autoimmune diseases. Such studies would be facilitated by application of new tools like MHC-peptide tetramers [171, 172], MHC-Ig dimers [173] and autoantibody profiling using protein microarrays [22, 174], etc. As evident from results of the studies discussed above, the implications of the phenomenon of epitope spreading cannot be generalized; instead, these need to be evaluated individually in the context of a particular disease, genetic make up of the individual, and the antigen involved. Awareness of these aspects is critical for developing appropriate immunotherapeutic approaches for immune-mediated disorders. At first, the idea of treatment of an ongoing disease in the face of epitope spreading might seem discouraging. This issue is further compounded by the observations that modulation of immune responses associated with epitope spreading could, under some circumstances, exacerbate the disease process instead of suppressing it. However, identification of the 'window' of therapeutic opportunity in terms of selecting the fight target antigen and the timing of intervention in animal models has offered hope for developing better approaches for treatment of human diseases [3, 175]. In addition, the regulatory aspects of epitope spreading could be reinforced for therapeutic advantage. We have described above examples of epitope spreading in a wide spectrum of diseases ranging from highly prevalent to relatively less prevalent diseases. In the near future, more information regarding the nature and function of epitope spreading is expected to be obtained from several infectious diseases and from diseases that are believed to possess an autoimmune component (e.g., atherosclerosis, inflammatory bowel disease, acquired immunodeficiency syndrome, etc.) [176-178]. In addition, at least three aspects of immune response are expected to shed more light on the mechanisms underlying epitope spreading. These include immune regulation, modulation of adaptive immunity by components of the innate immune response, and the host-environment interplay. At this time, there is far more information regarding pathogenic immune responses in epitope spreading than that for the regulatory aspects of the process. Bystander sup-
pression has been suggested to be the counterpart of pathogenic spreading [179]. Further integration of bystander suppression and the participation of CD4+CD25+ T cells and other regulatory T cells in the control of disease-propagating epitope spreading would significantly advance our understanding of the pathogenesis of autoimmune diseases. In regard to the role of components of innate immunity in autoimmunity, it has recently been shown that the complement and complement receptors [ 180] play a critical role in mediating effector arthritogenic response in KXBN transgenic mice [181], and in modulation of the induction of autoimmune myocarditis [182]. It is conceivable that the complement system and other mediators of innate immunity might have a significant effect on both pathogenic and regulatory epitope spreading. Finally, studies in animal models of autoimmunity [52-62] and those on the prevalence of autoimmune disorders in human populations living in different geographical regions of the world [183-188] have further validated the importance of the association between environment/infection and autoimmunity. Study of the interaction between immune response to subclinical/overt infections and autoimmune processes, and vice versa, would provide new insights into the influence of anti-microbial immunity on the induction and regulation of autoimmunity.
ACKNOWLEDGEMENTS We gratefully acknowledge the grant support from the National Institutes of Health (Bethesda, MD), the Arthritis Foundation (Atlanta, GA), the Maryland Chapter of Arthritis Foundation (Baltimore, MD), and the Maryland Arthritis Research Center (MARRC) (Baltimore, MD).
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133. Hill SL, Rose NR. The transition from viral to autoimmune myocarditis. Autoimmunity 2001 ;34(3): 169-76. 134. Horwitz MS, Bradley LM, Harbertson J, Krahl T, Lee J, Sarvetnick N. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat Med 1998;4(7):781-5. 135. Voskuhl RR. Myelin protein expression in lymphoid tissues: implications for peripheral tolerance. Immunol Rev 1998;164:81-92. 136. Flodstrom M, Maday A, Balakrishna D, Cleary MM, Yoshimura A, Sarvetnick N. Target cell defense prevents the development of diabetes after viral infection. Nat Immunol 2002;3(4):373-82. 137. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes. J Exp Med 1994; 179(4): 1317-30. 138. Casciola-Rosen L, Rosen A. Ultraviolet light-induced keratinocyte apoptosis: a potential mechanism for the induction of skin lesions and autoantibody production in LE. Lupus 1997;6(2):175-80. 139. Anderton SM, Wraith DC. Hierarchy in the ability of T cell epitopes to induce peripheral tolerance to antigens from myelin. Eur J Immunol 1998;28(4): 1251--61. 140. Harrington CJ, Paez A, Hunkapiller T, Mannikko V, Brabb T, Aheam M et al. Differential tolerance is induced in T cells recognizing distinct epitopes of myelin basic protein. Immunity 1998;8(5):571-80. 141. Tian J, Gregori S, Adorini L, Kaufman DL. The frequency of high avidity T cells determines the hierarchy of determinant spreading. J Immunol 2001; 166(12): 7144--50. 142. Mamula MJ, Janeway CA Jr. Do B cells drive the diversification of immune responses? Immunol Today 1993;14(4):151-2; discussion 153-4. 143. Mamula MJ, Fatenejad S, Craft J. B cells process and present lupus autoantigens that initiate autoimmune T cell responses. J Immunol 1994;152(3): 1453--61. 144. Shlomchik MJ, Craft JE, Mamula MJ. From T to B and back again: positive feedback in systemic autoimmune disease. Nat Rev Immunol 2001;1(2):147-53. 145. Simitsek PD, Campbell DG, Lanzavecchia A, Fairweather N, Watts C. Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell determinants. J Exp Med 1995;181(6): 1957-63. 146. Gor DO, Rose NR, Greenspan NS. TH1-TH2: a procrustean paradigm. Nat Immuno12003;4(6):503-5. 147. Jacob CO, Holoshitz J, Van der Meide P, Strober S, McDevitt HO. Heterogeneous effects of IFN-gamma in adjuvant arthritis. J Immunol 1989; 142(5): 1500-5. 148. Billiau A, Heremans H, Vandekerckhove F, Dijkmans
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R, Sobis H, Meulepas E et al. Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-gamma. J Immunol 1988;140(5):1506-10. 149. Ferber IA, Brocke S, Taylor-Edwards C, Ridgway W, Dinisco C, Steinman L et al. Mice with a disrupted IFN-gamma gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J Immunol 1996;156(1):5-7. 150. Willenborg DO, Fordham S, Bernard CC, Cowden WB, Ramshaw IA. IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J Immunol 1996; 157(8):3223-7. 151. Krakowski M, Owens T. Interferon-gamma confers resistance to experimental allergic encephalomyelitis. Eur J Immunol 1996;26(7): 1641-6. 152. Kumar V, Sercarz E. Induction or protection from experimental autoimmune encephalomyelitis depends on the cytokine secretion profile of TCR peptide-specific regulatory CD4 T cells. J Immunol 1998;161(12): 6585-91. 153. Vermeire K, Heremans H, Vandeputte M, Huang S, Billiau A, Matthys P. Accelerated collagen-induced arthritis in IFN-gamma receptor-deficient mice. J Immunol 1997; 158( 11 ):5507-13. 154. Manoury-Schwartz B, Chiocchia G, Bessis N, Abehsira-Amar O, Batteux F, Muller Set al. High susceptibility to collagen-induced arthritis in mice lacking IFNgamma receptors. J Immunol 1997;158(11):5501-6. 155. Quinn A, Mclnerney B, Reich EP, Kim O, Jensen KP, Sercarz EE. Regulatory and effector CD4 T cells in nonobese diabetic mice recognize overlapping determinants on glutamic acid decarboxylase and use distinct V beta genes. J Immuno12001 ;166(5):2982-91. 156. Trembleau S, Penna G, Gregori S, Giarratana N, Adorini L. IL-12 Administration accelerates autoimmune diabetes in both wild-type and IFN-gammadeficient nonobese diabetic mice, revealing pathogenic and protective effects of IL-12-induced IFN-gamma. J Immunol 2003; 170(11):5491-501. 157. Eriksson U, Kurrer MO, Bingisser R, Eugster HP, Saremaslani P, Follath F et al. Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction. Circulation 2001 ;103(1): 18-21. 158. Afanasyeva M, Wang Y, Kaya Z, Stafford EA, Dohmen KM, Sadighi Akha AA et al. Interleukin-12 receptor/STAT4 signaling is required for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway. Circulation 2001; 104(25):3145-51. 159. Caspi RR, Chan CC, Grubbs BG, Silver PB, Wiggert B,
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Parsa CF et al. Endogenous systemic IFN-gamma has a protective role against ocular autoimmunity in mice. J Immunol 1994;152(2):890-9. 160. Tarrant TK, Silver PB, Wahlsten JL, Rizzo LV, Chan CC, Wiggert B et al. Intefleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon gamma, nitric oxide, and apoptosis. J Exp Med 1999;189(2):219-30. 161. Ring GH, Dai Z, Saleem S, Baddoura FK, Lakkis FG. Increased susceptibility to immunologically mediated glomerulonephritis in IFN-gamma-deficient mice. J Immunol 1999; 163(4):2243-8. 162. Furlan R, Brambilla E, Ruffini F, Poliani PL, Bergami A, Marconi PC et al. Intrathecal delivery of IFNgamma protects C57BL/6 mice from chronic-progressive experimental autoimmune encephalomyelitis by increasing apoptosis of central nervous system-infiltrating lymphocytes. J Immuno12001;167(3):1821-9. 163. Madakamutil LT, Maricic I, Sercarz E, Kumar V. Regulatory T cells control autoirranunity in vivo by inducing apoptotic depletion of activated pathogenic lymphocytes. J Immunol 2003; 170(6):2985-92. 164. Veys EM, Mielants H, Verbruggen G, Grosclaude JP, Meyer W, Galazka A e t al. Interferon gamma in rheumatoid arthritis - a double blind study comparing human recombinant interferon gamma with placebo. J Rheumatol 1988; 15(4):570-4. 165. Lemmel EM, Brackertz D, Franke M, Gaus W, Hartl PW, Machalke K et al. Results of a multicenter placebocontrolled double-blind randomized phase HI clinical study of treatment of rheumatoid arthritis with recombinant interferon-gamma. Rheumatol Int 1988;8(2): 87-93. 166. Cannon GW, Pincus SH, Emkey RD, Denes A, Cohen SA, Wolfe F et al. Double-blind trial of recombinant gamma-interferon versus placebo in the treatment of rheumatoid arthritis. Arthritis Rheum 1989;32(8): 964-73. 167. Peng SL, Moslehi J, Craft J. Roles of interferongamma and interleukin-4 in murine lupus. J Clin Invest 1997;99(8): 1936--46. 168. Kumar V. Determinant spreading during experimental autoimmune encephalomyelitis: is it potentiating, protecting or participating in the disease? Immunol Rev 1998;164:73-80. 169. Takacs K, Altmann DM. The case against epitope spread in experimental allergic encephalomyelitis. Immunol Rev 1998;164:101-10. 170. Jones RE, Bourdette D, Moes N, Vandenbark A, Zamora A, Offner H. Epitope spreading is not required for relapses in experimental autoimmune encephalomyelitis. J Immuno12003;170(4):1690-8.
171. Kotzin BL, Falta MT, Crawford F, Rosloniec EE Bill J, Marrack Pet al. Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis. Proc Natl Acad Sci USA 2000;97(1):291--6. 172. Trollmo C, Meyer AL, Steere AC, Hailer DA, Huber BT. Molecular mimicry in Lyme arthritis demonstrated at the single cell level: LFA-1 alpha L is a partial agonist for outer surface protein A-reactive T cellS. J Immunol 2001 ;166(8):5286-91. 173. Fahmy TM, Bieler JG, Schneck JP. Probing T cell membrane organization using dimeric MHC-Ig complexes. J Immunol Methods 2002;268(1):93-106. 174. Hueber W, Utz PJ, Steinman L, Robinson WH. Autoantibody profiling for the study and treatment of autoimmune disease. Arthritis Res 2002;4(5):290-5. 175. Steinman L. Despite epitope spreading in the pathogenesis of autoimmune disease, highly restricted approaches to immune therapy may still succeed (with a hedge on this bet). J Autoimmun 2000; 14(4):278-82. 176. Gordon PA, George J, Khamashta MA, Harats D, Hughes G, Shoenfeld Y. Atherosclerosis and autoimmunity. Lupus 2001 ;10(4):249-52. 177. Bouma G, Strober W. The immunological and genetic basis of inflammatory bowel disease. Nat Rev Immunol 2003;3(7):521-33. 178. Zandman-Goddard G, Shoenfeld Y. HIV and autoimmunity. Autoimmun Rev 2002; 1(6):329-37. 179. Bach JF, Koutouzov S, van Endert PM. Are there unique autoantigens triggering autoimmune diseases? Immunol Rev 1998;164:139-55.
180. Carroll MC. The role of complement and complement receptors in induction and regulation of immunity. Annu Rev Immunol 1998;16:545-68. 181. Ji H, Ohmura K, Mahmood U, Lee DM, Hofhuis FM, Boackle SA et al. Arthritis critically dependent on innate immune system players. Immunity 2002;16(2): 157-68. 182. Kaya Z, Afanasyeva M, Wang Y, Dohmen KM, Schlichting J, Tretter T et al. Contribution of the innate immune system to autoimmune myocarditis: a role for complement. Nat Immuno12001;2(8):739-45. 183. Bulman DE, Sadovnick AD, Ebers GC. Age of onset in siblings concordant for multiple sclerosis. Brain 1991;114(Pt 2):937-50. 184. Ewing C, Bernard CC. Insights into the aetiology and pathogenesis of multiple sclerosis. Immunol Cell Biol 1998;76(1):47-54. 185. Rouse BT. Virus-induced immunopathology. Adv Virus Res 1996;47:353-76. 186. Wilson C, Tiwana H, Ebringer A. Molecular mimicry between HLA-DR alleles associated with rheumatoid arthritis and Proteus mirabilis as the Aetiological basis for autoimmunity. Microbes Infect 2000;2(12): 1489-96. 187. Singh B, Rabinovitch A. Influence of microbial agents on the development and prevention of autoimmune diabetes. Autoimmunity 1993; 15(3):209-13. 188. Rose NR. The role of infection in the pathogenesis of autoimmune disease. Semin Immunol 1998;10(1): 5-13.
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9 2004 Elsevier B. V All rights resen,ed. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Molecular Mimicry in Multiple Sclerosis: Role of MHC-Altered Peptide Ligands (MAPL) Dong-Gyun Lim and David A. Hater
Laboratory of Molecular Immunology, Centerfor Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School Boston, MA, USA
Multiple sclerosis (MS) is a chronic inflammatory illness affecting the CNS white matter that can lead to progressive neurologic dysfunction. Together with other organ-specific autoimmune diseases such as type 1 diabetes mellitus and rheumatoid arthritis, MS is thought to be mediated by autoreactive T cells that recognize CNS self-antigens. Supporting evidence for the autoimmune basis of MS includes the inflammatory nature of the CNS lesions, the genetic linkage to the MHC region, similarities to the animal model experimental autoimmune encephalomyelitis (EAE), and the therapeutic effects of immunomodulatory drugs [1]. Data from animal studies in the EAE model established that CD4 +Thl cells specific to myelin antigens can play a central role in the induction and progression of autoimmune demyelinating disease [2, 3]. This finding has fueled efforts to dissect the antigenic specificity and functional characteristics of myelin-reactive CD4 + T cells in the peripheral blood and cerebrospinal fluid of patients with MS. Several peptide epitopes derived from myelin proteins have been found to activate CD4 + T cells in the circulation of patients with MS. Among them, MBP (myelin basic protein) 84-102 and MBP148-162 have been classified as immunodominant epitopes in the context of HLA-DR2 haplotype [4-6]. However, CD4 + T cells reactive to these self-peptides have also been detected in healthy persons, suggesting that the presence of myelinreactive T cells is not sufficient for the development of MS. While resting autoreactive T cells are a part of normal T cell repertoire, the activation status of these cells is different in patients than in healthy
individuals. Consistent with the properties of preactivated T cells, myelin-reactive T cells recovered from patients are less dependent on costimulation for activation and express activation markers, such as IL-2R, on their cell surfaces [7-9]. These findings lead to the question of what induces the activation of myelin-reactive T lymphocytes in patients with MS. One attractive hypothesis is based on the role of microbial infections in the activation of selfreactive T cells. If invading microorganisms contain protein antigens with sufficient structural homology to human proteins, infection itself may be sufficient to activate pre-existing self-reactive T cells. This is the basic concept of molecular mimicry. Several clinical and epidemiological studies support the hypothesis of mimicry as a mechanism of autoimmunity. According to the original observation by Fujinami and Oldstone [10], rabbits immunized with a hepatitis B polymerase peptide that shared six amino acids with the MBP sequence developed CNS lesions reminiscent of EAE. In humans, upper respiratory infections often precede MS exacerbations. The original concept of molecular mimicry evolved from the recent understanding of the degenerative recognition of antigens by the T cell receptor (TCR). In examining the immune response to MBP, we found that complementary mutations in an antigenic peptide allow for cross-reactivity of autoreacfive T cell clones that may be related to shifts of the TCR structure itself [11]. Using combinatorial libraries, Hemmer and co-workers [12] demonstrated that totally unrelated peptides also activate autoreactive T cells. Furthermore, recent data suggest that even different MHC molecules complexed
45
with diverse peptides can activate MBP-reactive T lymphocytes in an agonistic or antagonistic fashion. In the following sections, we will describe examples of this broader concept of molecular mimicry and its clinical relevance in MS.
reveal signs of superantigenic activation of myelinreactive T cells in MS. Thus, molecular mimicry is still a very attractive theory to explain the frequent association of microbial infection with the development or exacerbation of MS.
1. RELATIONSHIP BETWEEN MICROBIAL INFECTIONS AND MS
2. M O L E C U L A R MIMICRY ASSOCIATED WITH SEQUENCE H O M O L O G Y
Since 1894, when Pierre Marie proposed that infection is the cause of MS, many reports have supported the possible involvement of infectious pathogens. Epidemiological studies have formulated a list of suspicious microbes, including Borrelia burgdorferi, Chlamydia pneumoniae, measles virus, rabies virus, paramyxovirus, coronavirus, EpsteinBarr virus, cytomegalovirus, varicella-zoster virus, herpes simplex virus, human herpes virus 6, rubella virus, mumps virus, Marek's disease virus, Semiliki Forest virus, human retroviruses, and human lymphoma virus type I [13]. So far, none of these infectious agents have been found to be specific for MS, although an MS-specific agent may yet be discovered. The apparent absence of an MS-specific infectious agent and the autoimmune nature of MS suggest another role of infection in the development of this disease. Three mechanisms have been proposed to explain the association of microbial infection and MS. One is the molecular mimicry theory, which has received a great deal of attention and will be discussed in detail in the following sections. Another is bystander activation, including epitope spreading [ 14]. Infections can activate autoreactive T cells through the release of sequestered myelin proteins as a result of infection-related tissue damage, activation of antigen-presenting cells (APCs), and induction of secretion of inflammatory cytokines and chemokines, irrespective of the particular microbial determinants. The third is superantigenic T cell activation. Several bacterial and viral products are able to cross-link TCR and MHC molecules independent of specific antigen recognition through the TCR. Cross-linking leads to activation of T cells with particular V[3 families of TCR. Myelin-reactive T cells with a particular TCR VI3 chain can be activated after infection with microbes whose superantigen recognizes this specific VI3 chain. Again, many studies have failed to
Molecular mimicry describes a situation whereby a foreign antigen can initiate an immune response in which a T or B cell component cross-recognizes self. The previous concept for the antigen specificity of T cells predicted that the presence of strict sequence homology between the microbial antigens and self-peptide was necessary to induce autoimmunity (Fig. 1, top A). There are two approaches to determining the foreign microbial antigens that are cross-reactive to self-antigens. One is the initial identification of causative microbes and their major antigenic epitopes and subsequent demonstration of their cross-reactivity to self-antigens. This approach was applied successfully to reveal that two autoimmune diseases are caused by a molecular mimicry mechanism. Autoimmune Lyme arthritis is preceded by infection with B. burgdorferi and is associated with hLFA-1-reactive T cells that were initially primed and expanded by the OspA(165-173) peptide of this spirochete [15]. Herpetic stromal keratitis (HSK) follows infection of the eye with herpes simplex virus 1 and is caused by the activation of autoreactive cells by viral UL6(299-314) peptide [16]. However, it is difficult to apply this strategy for the study of molecular mimicry in MS because, as mentioned previously, no microbial agent has been directly associated with the disease. The other approach is to first identify T cell determinants capable of inducing autoimmunity and then search for the microbial antigens with sequences homologous to those determinants. An initial study adapting this latter strategy successfully demonstrated that a peptide from hepatitis B virus polymerase (HBVP) with six consecutive amino acids in common with an encephalitogenic determinant of MBP (ICGYGSLPQE in HBVP vs TTHYGSLPQK in MBP) could induce subclinical EAE in rabbits [10]. However, proteins sharing a sequence of more than six amino acids are not
46
Molecular mimicry
Cognate MHC/peptide ligand
A. Extensive sequence homology
I
L
l
1
i
!
TCR
1,
I
B. Minimal sequence homology
I
L
1 L,I
C. Entirely unrelated peptide
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Variant molecular mimicry A. Different MHC/same peptide
B. Different MHC/different peptide
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Figure 1. Schematic representation of molecular mimicry. Top: Classical molecular mimicry occurs by the sequence homology between self and microbial peptide (A). Expanded molecular mimicry can occur either by peptides with minimal sequence homology (B) or even by entirely unrelated peptides complexed with cognate MHC molecule (C). Bottom: Variant molecular mimicry means the cross-recognition of a different MHC/same (A) or different peptide (B) by the identical TCR. common in nature, and this type of homologous foreign peptides has not yet been identified in human MS. Extensive biochemical and structural characterization of the recognition of MHC/peptide ligand by TCR in humans has greatly influenced
the identification of cross-reactive epitopes for the given TCR. While a few amino acid residues of a peptide are important for binding to the MHC molecule, the other one or two amino acid residues serve as critical residues for the recognition by the TCR (Fig. 1, top B). Specifically, in the immunodo-
47
minant peptide ligand (MBP85-99) presented by DRB* 1501, two hydrophobic residues (Val-89 and Phe-92) serve as the primary anchors to the HLADR2 molecule, while Phe-91 and Lys-93 have been defined as the primary TCR contact residues for a MBP85-99-specific T cell clone. Mutations in other amino acid residues are tolerated with respect to recognition by the TCR [17]. On the basis of this information, Wucherpfennig and Strominger [18] developed an efficient strategy to search for peptides that share these critical contact motives rather than sequence homology. This search yielded seven viral and one bacterial peptides derived from herpes simplex virus, adenovirus, human papillomavirus, Epstein-Barr virus, influenza type A virus, reovirus type 3, and Pseudomonas that efficiently activated T cell clones. Interestingly, only one of them has been identified as a molecular mimic by sequence alignment. These data clearly indicated that more foreign peptides may act as molecular mimics than was previously thought.
3. M O L E C U L A R MIMICRY W I T H O U T SEQUENCE H O M O L O G Y Conservation of critical MHC and TCR contact residues appears to be a general rule for activation by the peptide of a specific T cell. However, several peptide sequences with no sequence homology have been shown to be able to activate identical T cells (Fig. 1, top C) [19, 20]. In addition, the use of synthetic peptide combinatorial libraries has clearly illustrated the extreme degeneracy of TCR recognition of antigen. Hemmer et al [12] examined the response of CD4 + T cell clones specific for MBP86-96 to a set of 220 11-mer peptide sublibraries, each containing 10 degenerated amino acids and one defined amino acid in the positional scanning format. The new knowledge obtained from this study was 1) that of highly degenerative recognition of peptides by autoreactive CD4 § T cells, including identification of stimulatory ligands not sharing a single amino acid in corresponding positions with the antigen used to establish the T cell clone and 2) the identification of more potent agonistic peptides than cognate self-peptide. From the database search for natural proteins with deduced peptide sequences, they found four self and three microbial proteins
48
as stimulatory ligands to this T cell clone. Most interestingly, one self-peptide (protein-glutamine gamma-glutamyltransferase, 675-685) and two microbial peptides (human CMV UL71, 166-176; UDP-N-acetylenolpyruvoyl-glucosamine reductase of Salmonella typhimurium, 227-237) were defined as even more potent agonists than MBP86-96. This strategy opened the way for the identification of molecular mimics if relevant autoreactive T cells are defined in MS.
4. VARIANT M O L E C U L A R MIMICRY: MHC-ALTERED PEPTIDE LIGAND The studies discussed above focused on the molecular mimics with respect to the peptide portion of TCR ligand. Since TCRs recognize MHC/peptide as a single unit and exhibit greatly degenerative recognition of their ligands, they might recognize a different MHC combined with a cognate or even a different peptide as an agonist (Fig. 1, bottom). Since most people are heterozygous for the HLADR locus, this type of molecular mimicry is likely to occur in physiological situations. It has been shown that the human MBP peptide 84-102 binds to several different DR molecules and that T cells recognize this peptide presented by these different HLADR2 molecules [21]. We systematically exarpdned this type of cross-reaction using a panel of CD8 § T cell clones specific to Taxi 1-19 peptide of HTLV-1 in the context of HLA-A*0201 [22]. When CD8 § T cells were stimulated with cognate Taxi 1-19 peptide presented by different HLA-A2 subtype alleles, which have one to four amino acid differences at specific positions (Table 1), they showed a diverse pattern of T cell function, encompassing agonistic, weak agonistic, or partial agonistic, depending on the individual T cell clones (Fig. 2). This is similar to the effects induced by antigenic altered peptide ligands (APL). In addition, atypical partial agonistic T cell function was observed; i.e., a number of CD8 + T cell clones proliferated in response to Taxi 1-19 ~ presented by the HLA-A*0205 subtype even though they did not exhibit any cytotoxic activity. The analysis of the structural interaction between the TCR and MHC/peptide complex indicated that polymorphic amino acids in the HLA-A2 peptide-binding groove, especially the D-pocket, rather than the dif-
Table 1. Summary of amino acid sequences at polymor-
phic HLA-a2 positionsa HLA-A2 subtype
Amino acid sequence at specific position .
o~1 domain
.
.
.
.
.
t~2 domain
9
43
95
149
152
156
A*0201
F
Q
V
A
V
L
A*0202
F
R
L
A
V
W
A*0203
F
Q
V
T
E
W
A*0205
Y
R
L
A
V
W
A*0206
Y
Q
V
A
V
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aReproduced from Ref. [22] by copyright permission of the Rockefeller University Press. ferences on the MHC residues in direct contact with the TCR, were responsible for this partial agonism (Fig. 3A). This was supported by the finding that reciprocal mutations of the Tax peptide side chain that engaged the D-pocket restored the agonist functions of the MHC/peptide complex (Fig. 3B). Thus, our study clearly demonstrated that MHC molecules play an important role in T cell activation not only by restricting antigen element but also by altering the antigenic nature of peptide. We termed this type of TCR ligand MHC-altered peptide ligand (MAPL). CD4 § T cell can also exhibit MAPL effects on the recognition of variant MHC class II molecules. Germain and colleagues [23] previously suggested that a peptide presented on a mutant MHC class II molecule induces a response different from the response induced with the native MHC molecule. We also recently examined the modulation of T cell responses induced by stimulation with different MHC class II/peptide complexes, demonstrating the existence of significant cross-reactivity of autoreactive CD4 + T cells in the context of the distinct MHC class II molecules. The Ob 1A12 T cell clone is reactive to the immunodominant MBP peptide, MBP85-99, presented by HLA-DRA/DRB*1501 and was generated from peripheral blood mononuclear cells of a patient with MS [24]. Characterization of this T cell clone showed that it recognizes several single amino acid substitutes of MBP85-99 presented by DRB 1" 1501 in a degenerative fashion.
Moreover, when the responses of this T cell to APLs in the context of the other self-DR allele (DRM DRBI*0401) were examined, the OblA12 T cell clone responded to the MBP85-99 88V--->K APL, even though it did not recognize the MBP85-99 presented by this allele (Fig. 4, [25]). These data indicate that APL-induced cross-reactivity provides a further degree of T cell receptor degeneracy among different DR molecules. It should be noted that the functional outcome of this type of crossreaction could be agonistic, partial agonistic, or even antagonistic depending on the specific combination of MHC molecules and peptides. The cross-reaction induced by MAPLs containing cognate, altered, or even irrelevant peptides has not been well appreciated as a potential mechanism of molecular mimicry. However, our data generated from the systemic analyses in vitro, as mentioned above, and the increasing awareness of the highly degenerative nature of TCR recognition strongly predict that this type of molecular mimicry might effect the activation of autoreactive T cells. In fact, a recent report provides a good example for this type of molecular mimicry by showing that T cells derived from a patients with MS recognized an Epstein-Barr virus DNA polymerase peptide in the context of DRB5*0101 as well as the immunodominant MBP85-99 epitope in the context of DRB 1" 1501 [26]. The structural similarity in TCR contact surfaces between these two MHC/peptide complexes explains this cross-reactivity. If this type of molecular mimicry is frequent, the chances for autoreactive T cell activation would be higher than previously thought, while the identification of mimicry peptides would be more complicated. This means that we need to consider both the peptide epitopes and MHC class II alleles to carry out an exhaustive study of the molecular mimics.
5. THE RELEVANCE OF M O L E C U L A R MIMICRY IN MS It is important to define whether molecular mimicry induces the initiation of autoimmune responses or contributes only to the exacerbation of existing autoimmune responses. Unfortunately, no convincing evidence is currently available for a role of molecular mimicry in the initiation of any autoim-
49
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Figure 2. Summary of CD8§ cell clone functions with Taxll-19 presentation by different HLA-A2 subtype molecules. Lymphoblastoid cell lines expressing different HLA-A2 subtypes were loaded with 50 I.tM Taxi 1-19 and used as target cells or APCs. Cytotoxicity was tested by 5~Crreleases assay at an E/T ratio of 10:1. Interferon-q, was measured in culture supernatants by ELISA after 48 h of incubation. Proliferation was determined by an 18 h-[3H]thymidine incorporation assay at the end of a 72-h culture and expressed as the stimulation index (S.I.). N.D., not determined. Individual T cells can recognize the different MHC/same peptide ligand as an agonist, weak agonist, or partial agonist. Reproduced from Ref. [22] by copyright permission of the Rockefeller University Press.
50
Figure 3. Polymorphic amino acids located in the HLA-A2 peptide-binding groove alter peptide ligands to induce atypical partial agonistic T cell function. (A) Location of HLA-A2 subtype polymorphic residues. The HLA-A2/Taxl 1-19 complex viewed from above the peptide-binding groove. Residue 156 is located in D-pocket of the groove and is within the Van der Waals radius of the P3 side chain of the Tax peptide. Thus, the substitution of Leu for a bulky Trp in HLA-A*0205 is most likely to affect TCR recognition by the structural change in peptide bound in the MHC molecule. (B) Cytotoxicity and proliferative responses of T cell clone to Taxi 1-19 peptide analogues with single amino acid substitutions at P3 position presented by HLA-A*0201 or A*0205. Reciprocal mutation (Phe to Asn) of the Tax peptide side chain engaging the D-pocket restores the agonist function of MHC/peptide complex. Reproduced from Ref. [22] by copyright permission of the Rockefeller University Press.
51
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Figure 4. Cross-reaction of human autoreactive T-cell receptor with APLs in the absence of reaction to cognate peptide presented by different MHC class II molecules. (A) The MBP85-99 peptide was substituted in the core recognition region and presented by either DRBI*1501 or DRBI*0401. The responses of OblA12.TCR hybridoma to these ligands were analyzed by HT.2 assay measuring the interleukin-2 production. (B) As the MBP 88K peptide was recognized in the context of DRBI*0401, a series of MBP85-99 peptide with substitutions at position 88 were synthesized and tested for their ability to activate Ob 1A12.TCR hybridoma.
mune disease. It should be emphasized that all the cross-reactive foreign peptides have been defined by their stimulatory capacity and selected from protein databases or screening based on the role of individual amino acid residues. Therefore, it is not clear whether these peptides can be processed by and presented on APCs after microbial infection. If they can be, it still remains to be defined whether or not cross-reactive T cells can be successfully activated and expanded sufficiently to attack self. Even though epiderniological studies suggested that sev-
52
eral viral and bacterial infections often precede the exacerbation of MS [27], it is difficult to discriminate between two potential mechanisms for this effect: the molecular mimicry described here and the bystander activation of autoreactive T cells. The latter includes the effect of inflammation, epitope spreading, or a superantigenic effect of invading microbes. In addition, a chronologic relationship is difficult to establish because the starting point of the disease is usually unknown and most suspicious microbes induce persistent infection. The lack of an
animal model of spontaneous development of MS is another obstacle to finding answers to these questions. Thus, although a great deal of fragmentary evidence is available, a definitive role for molecular mimicry in the pathogenesis of MS has not been proven. Potential evidence for the role of molecular mimicry in human MS has recently emerged from a clinical trial of altered peptide ligand therapy [28, 29]. In vitro and animal studies found that APLs can inhibit the response of T cells to agonistic autoantigen and can block EAE [30, 31]. Furthermore, APLs could induce a novel, APL-reactive T cell population that had a Th2 phenotype and cross-reacted with native MBP antigens [32]. These data supported the use of APLs as a therapeutic agent for MS. Unfortunately, unexpected detrimental side effects, including a hypersensitivity reaction to APLs and exacerbation of disease, necessitated the premature termination of all the trials before completion. An immunological study suggested that the latter side effect came from the expansion of APL-specific T cells with pro-inflammatory phenotype, which cross-reacted with MBP [30]. This finding is perhaps the strongest argument that, in some situations, molecular mimicry indeed can play a role in the pathogenesis of MS. Considering the highly degenerative nature of T cell recognition, it can be proposed that APLs may be presented by any of the HLA class II molecules and activate the pro-inflammatory T cells, which cross-react with myelin antigens presented either by APL-restricting or another class II molecule. However, the previous studies employed a single HLADR molecule for the selection of the APLs. Since the majority of patients are heterozygous for HLADR locus, the effect of APLs should be considered in conjunction with the individual's entire HLA class II haplotype. Actually, we recently observed that one APL (88V---)K) of MBP85-99 could stimulate the OblA12 TCR in the context of both DRB*1501 and DRBI*0401, whereas the original MBP85-99 peptide could not be recognized in the context of DRB*0401 [25]. Therefore, stricter criteria, considering all the self-MHC class II molecules as potential restriction elements, should be applied for the selection of APLs.
6. CONCLUDING R E M A R K S Molecular mimicry still remains an attractive hypothesis to explain the initiation and maintenance of MS lesions. Epidemiological and immunological studies support this theory. Indeed, several microbial peptides that cross-react with self myelin proteins have been identified. In addition, more microbial peptides will be identified in the near future if the MAPL concept is considered during screening for cross-reacting antigens. However, as mentioned previously, not all the available data can be taken as direct evidence that this molecular mechanism is at work in the pathogenesis of MS. Considering the highly degenerative nature of TCR recognition and the presence of self-reactive T cells in a normal repertoire, why do only a small percentage of people suffer from MS after microbial infections? One possible explanation is the multifactorial origin of MS. A recent twin study convincingly showed the importance of genetic traits as a risk factor [33]. If genetically predisposed individuals suffer from infections, they may develop MS due to molecular mimicry. Recently, there has been a conceptual movement in the MS field that MS is not a single disease entity but rather a syndrome composed of different disorders with different causes [34]. In addition, major pathogenic mechanisms might differ depending on the disease stage. Therefore, the molecular mimicry hypothesis should be re-evaluated according to the new concept of the complexity of T cell cross-reactivity as well as the disease entity.
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c, Ito K, Nagy ZA, Field JA, Steere AC, Huber BT. Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 1998;281: 703-706. Zhao Z-S, Granucci F, Yeh L, Schaffer PA, Cantor H. Molecular mimicry by herpes simplex virus-type I: autoimmune disease after viral infection. Science 1998;279:1344-1347. Wucherpfennig KW, Sette A, Southwood S, Oseroff C, Matsui M, Strominger JL, Hailer DA. Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones. J Exp Med 1994;179:279-290. 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. Bhardwaj V, Kumar V, Geysen HM, Sercarz EE. Degenerate recognition of a dissimilar antigenic peptide by myelin basic protein-reactive T cells. Implications for thymic education and autoimmunity. J Immunol 1993;151:5000-5010. Hagerty DT, Allen PM. Intramolecular mimicry. Identification and analysis of two cross-reactive T cell epitopes within a single protein. J Immunol 1995;155: 2993-3001. Martin R, Howell MD, Jaraquemada D, Flerlage M, Richert J, Brostoff S, Long EO, MaFarlin DE, McFarland HE A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J Exp Med 1991;173:19-24. Lim D-G, Slavik JM, Bourcier K, Smith ICJ, Hailer DA. Allelic variation of MHC structure alters peptide ligands to induce atypical partial agonistic CD8 § T cell function. J Exp Med 2003;198:99-109. Racit~ppi L, Ronchese F, Matis LA, Germain RN. Peptide-major histocompatibility complex class II complexes with mixed agonist/antagonist properties provide evidence for ligand-related differences in T cell receptor-dependent intracellular signaling. J Exp Med 1993:177; 1047-1060. Ota K, Matsui M, Milford EL, Mackin GA, Weiner HL, Hailer DA. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990:346;183-187. Mycko MP, Waldner H, Bourcier KD, Wucherpfennig K, Kuchroo VK, Hailer DA. Cross-reactive human autoreactive T-ceU receptor responses to altered peptide ligands presented by different MHC class II molecules. (Submitted). Lang HLE, Jacobsen H, Ikemizu S, Andersson C,
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30. De Magistris MT, Alexander J, Coggeshall M, Altman A, Gaeta FC, Grey HM, Sette A. Antigen analog-major histocompatibility complexes act as antagonists of the T cell receptor. Cell 1992;68:625-634. 31. Karin N, Mitchell DJ, Brocke S, Ling N, Steinman L. Reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: T cell receptor antagonism and reduction of interferon ~/and tumor necrosis factor ct production. J Exp Med 1994;180:2227-2237. 32. Nicholson LB, Greer JM, Sobel RA, Lees MB, Kuchroo VK. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity 1995;3:397-405. 33. Ebers GC, Sadovnick AD, Risch NJ. A genetic basis for familial aggregation in multiple sclerosis. Canadian collaborative study group. Nature 1995;377:150-151. 34. Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. Heterogeneity of multiple sclerosis lesions: Implications for the pathogenesis of demyelination. Ann Neurol 2000;47:707-717.
55
9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Adverse Events of Desirable Gain in Immunocompetence: The Immune Restoration Inflammatory Syndromes (IRIS) Matthias Stoll and Reinhold E. Schmidt
Department Clinical Immunology, Medical School Hannover, Hannover, Germany
1. INTRODUCTION Sumilar signs of inflammation are common and correspondingly in a broad spectrum of heterogeneous infectious diseases. In infectious diseases inflammation reflects the ability of the adaptive immune system to actively compete with "nonself'. Inflammatory symptoms develop for the first time after an incubation period, in which usually (a) the infectious agent spreads inapparently in the host and (b) primary or secondary specific adaptive immune responses develop. Beginning from that point declining extent of inflammation reflects increasing immunological control of infection in an immunocompetent host. Therefore intensity of inflammation and extent of infection are sometimes equated improper. In contrast, in the immunodeficient host inflammation will not occur or to less extent - even in presence of latent infection. Therefore in case of (re-)emerging immunocompetence in an immunodeficienct state an increase of "paradoxical" inflammatory response may develop (Fig. 1). The delayed acute onset of "paradoxical" inflammation resembles a recently acquired intercurrent or acute disease. Therefore IRIS rather reflects an uncommon pathophysiologic mechanism or special kind of inflammation than the (retrospective) recognition of a - previously incompetent - reconstituted immune system. This may justify the assumption of a "paradoxical" inflammatory response. However, both underlying possible scenarios require specific clinical management: Spread of infection which escapes immunosurveillance demands an intensification or change of antinfective
treatment. But prolonged inflammation as sign of improving immunologic control might need predominantly antiinflammatory treatment [ 1]. A consent for the definition of IRIS has still to be found. Until than it could be characterized: (i) by the description of predispopsing risks for certain forms of IRIS; (ii) by its particular characteristics and clinical appearance; (iii) by its specific therapeutical requirements; (iv) and by the identification of distinct immunopathologic features in IRIS.
2. STATE OF IMMUNOCOMPETENCE The term IRIS might suggest restriction to one particular state of immunity. Indeed it may develop under different clinical conditions. IRIS may occur in individuals with defects in the specific or the innate immune system respectively. Beyond immunodeficiencies IRIS has been described as well in cases with extensive infections presenting without immunodeficiency.
2.1. IRIS Without Apparent Immunodeficiency Any whitespread infection by itself may overwhelm the capacity of the immune control in a non-immunocompromized host. In that case effective antimicrobial treatment allows the host's immune system to generate more potent inflammatory response at the site of remaining infection by lowering the high burden of infectious antigen. The historically classical example for IRIS is called "reversal reaction"
57
Figure 1. Model illustrating balance of driving forces in inflammatory reactions during immune restoration. and occurs commonly during the treatment of multibacillary "lepromatous" leprosy. Multibacillary forms of leprosy reflect a lower extent of specific immunity with predominant Th2-immune response than Thl dominated paucibacillary "tuberculoid" leprosy [2]. Therefore certain antiinflammatory or
58
immunosuppressive drugs are added to polychemotherapy of lepromatous leprosy. Correspondingly in disseminated tuberculosis IRIS with development of cerebral tuberculoma after initiation of tuberculostatic treatment may cause fatal outcome [3]. Reversion from negative tuberculin skin test in
disseminated tuberculosis to positive reaction after chemotherapy is of additional evidence for specific immune reconstitution in such scenario [4]. The addition of immunosuppressive corticosteroids is established and improves the outcome of certain forms of tuberculosis [5] or bacterial meningitis [6] by controlling the overshoot of inflammation under antimicrobial chemotherapy, indicating clinical significance of IRIS.
frequently. Commonly these cases suffer previously from fever of 'unknown' origin (FUO) during neutropenia. FUO is characterized by the lack of any infectious focus. The subsequent development of such foci - often after complete resolution of fever - c a n be explained by reconstitution of elements of the innate immune system in neutropenia [10, 13].
3. CLINICAL CHARACTERISTICS
2.2. Cellular Immunodeficiency Until the pandemic spread of the human immunodeficiency virus (HIV) severe defects of the cellular immune system were uncommon in adults and up to the introduction of highly active antiretroviral treatment (HAART) there was hardly any option to enhance immunity in immunodeficient patients [7]. I ~ S as an apparent new entity of disease have been described in cases of virologic and immunologic successful HAART [8-10]. IRIS occurs predominantly in patients who are severely immunocompromised in a narrow timely correlation to the initiation of HAART. The synchronous and widespread use of HAART in a large number of HIV infected patients with severe immunodeficiencies focusses in a considerable number of cases presenting with IRIS within a short period. Because of the individually proven immunological effectiveness of HAART in these cases IRIS imposes as unexpected and atypical event of inflammation. In most cases it is due to HIV-associated or AIDS-defining infectious disease, but IRIS presents often with unusual clinical features as compared to the corresponding classical opportunistic diseases. A couple of studies give evidence for the hypothesis that IRIS can be explained as increased inflammatory response during the restoration of the previously incompetent specific immunity [ 11, 12].
2.3. Defects of Innate Immunity Paradoxical inflammatory responses are not restricted to the reconstitution of specific adaptive immunity. Extensive and acutely developing pulmonary infiltrates or spread of focal hepatic lesions during the hematopoietic reconstitution after high dose cytostatic chemotherapy in patients with hematological malignancies have been described
IRIS imposes as unexpected event or paradoxical deterioration, when increasing inflammatory potency in a reconvalescent host either unmasks subclinical disease or worsens intensity of an already apparent inflammation. By certain clinical characteristics progress of classical overt disease can be distinguished from complementary IRIS. 3.1. Mycobacteria Tuberculous IRIS occurs commonly in (a) a narrow timely correlation to the initiation of antimycobacterial treatment or the initiation of HAART, (b) conversion of specific skin tests in some cases, and (c) the lack of increase of acid fast stained rods [8], which reflects increase of inflammation without increase of the underlying pathogen. IRIS reveals emerging and long lasting pulmonary infiltrates, lymphadenitis or tuberculoma [3, 14]. Focal lymphadenitis as a typical presentation of IRIS due to nontuberculous mycobacteria [15] clearly differs from the classical disseminated multiorgan disease in advanced immunodeficiency.
3.2. Herpesviridae Relapses of Cytomegalovirus (CMV)-retinitis under maintenance CMV-therapy were seen in up to half of all HIV-infected individuals at risk after the initiation of HAART [8, 9]. In contrast to AIDSdefining CMV-retinitis, IRIS may occur in patients with CD4+ T cells within normal range [8], without markers of CMV replication, and with atypical clinical manifestations including uveitis, vitritis, macular edema, epiretinal membranes and cataract [16]. A more pronounced increase of specific anti-CMV-IgG antibodies in these patients might serve as evidence for a specific pathogenetic role of
59
immune reconstitution in CMV-IRIS [ 11 ]. Repeated reactivation of dermatomal zoster developed up to more than half a year after HAART has been started [9, 17]. VZV-IRIS has been described in cases with higher increase of CD4+ T cells during the first weeks of HAART [9] and with more elevated CD8+ T cell proportions [ 17]. Herpes simplex (HSV) reactivation with unusually localized erosive lesions has been reported as IRIS in cases, who shared certain HLA Class I and HLA Class II antigens [18]. Transient reactivation of EBV replication occurs after initiation of HAART more often in patients with good immunological responses, especially in those, who respond immunologically well, present with increased immunoglobulin levels but do not reach complete control of retroviral replication [121. IL-6, a growth factor for HHV-8 was found to be elevated in patients with IRIS [19]. In this context two HHV-8 associated diseases presented atypically during immune restoration: Castleman's disease and Kaposi's sarcoma [20]. 3.3. Noninfectious
Disease
Increase of inflammatory response during immune restoration in a couple of noninfectious diseases provides additional evidence for the pathophysiological concept of IRIS: Descriptions include cases of autoimmune diseases, like systemic lupus erythematosous [21], rheumatoid arthritis [22], polymyositis [231, autoimmune thyreopathy [24], alopecia universalis [25], allergic reaction e.g. against prexisiting tattoos [26], disease of unknown etiology, like sarcoidosis [27], and the induction of atherogenic chronic inflammation respectively [28-30]. Additional clinical aspects of IRIS are summarized in detail elsewhere [ 1, 8-10, 31 ]
4. I M M U N O P A T H O L O G I C F E A T U R E S Certain correlations could be demonstrated between immunological markers or genetic characteristics and IRIS. As different opportunistic diseases require particular mechanisms of immunologic control disease specific "risk factors" could be
60
Table 1. Putative risk factors for development of IRIS after initiation of HAART in HIV-infection with severe immunodeficiency 9 9 9 9
Duration of immunodeficiency Extent of immunodeficiency Velocity and (relative) extent of immune reconstitution Specific pattern of immune reconstitution under HAART - immune reconstitution without complete suppression of HIV replication - high levels of CD8+ T lymphocytes - high levels of IL-6 and soluble IL-6 receptor - increase in (CMV-) specific IgG antibodies - high levels of soluble CD30 and soluble CD26 (dipeptidyl peptidase IV) activity - high levels of IFN gamma producing cells - Increased expression of CCR3 and CCR5 on monocytes and/or granulocytes - persisting polyclonal hypergammaglobulinemia - development of specific delayed type hypersensitivity 9 Genetic susceptibility - Distinct HLA haplotypes (e.g.: HLA B72, Cw0202, DRB4; HLA A2, B44 and HLA A1, B8, DR3 in conjunction with TNF-alpha polymorphism) - Polymorphisms in cytokine genes 9 TNF-alpha (in conjunction with certain HLAhaplotypes) 9 IL-6 9 IL-12
identified for IRIS (Table 1). The risk increases with speed and strength of immune restoration. In the majority of mycobacterial IRIS increase of CD4+ cells remained suboptimal whereas a more pronounced rise of CD4+ and/or CD8+ T cells were found in Herpesvirus IRIS [8, 9, 11, 17]. In CMVand EBV-IRIS predominance of Th2- over T h l immune response develops [ 11, 12, 32, 33] and correspondingly IL-6 and soluble IL-6 receptor were found elevated [19]. Increased chemokine receptor expression may result in persistence of irranunostimulation as one additional risk factor for the onset of IRIS [33] and usually succesful HAART leads to a decrease of immunostimulation [34]. Evidence for the individually genetic predisposition for certain manifestations of IRIS is based on the association of IRIS with certain histocompafibility antigens [18, 35] and with gene polymorphisms of cytokines like IL-6 and TNF-alpha [19, 36].
Table 2. Proposal for diagnostic criteria for IRIS within a setting of immune reconstitution Clinical criteria
Immunological criteria Major
Minor
Unexpected onset or paradoxical deterioration
Demonstrable immune reconstitution
Specific and predisposing pattern of immune stimulation or immune restoration a
Specific symptoms, which should allow distinction from "regular" opportunistic disease
Specific immune reconstitution against the presumed pathogen a
Predisposing genetic factors (e.g. histocompatibility antigens, cytokine gene polymorphisms)a
Rapid onset and close correlation to restoration of the immune system Proof of underlying pathogen a Routine tests for these criteria are either not available or these criteria are preliminary and therefore restricted to scientific investigation.
5. DIAGNOSIS
Consensus for diagnostic criteria and treatment is not yet defined. One proposal for diagnostic criteria for IRIS within a setting of immune reconstitution is given [ 1] in Table 2.
6. TREATMENT Until treatment guidelines will be defined and evaluated in clinical studies an empirical proposal for treatment of IRIS should consider [ 1]: 9 Treatment of underlying disease by antiinfective chemotherapy. 9 Temporarily suppression of inflammation by suitable interventions, e.g. with antiphlogistic or immunesuppressive drugs. 9 Surgical or symptomatic treatment of certain manifestations of I ~ S (e.g.: iritis bombata, necrotizing lymphadenitis).
and can mimic opportonistic disease, allergic reaction or features of autoimmunity respectively. 9 Timely correlation to immune restoration, distinct clinical appearance, particular immunologic and genetic characteristics, and different therapeutic requirements justify the assumption of an indepent entity for IRIS. 9 Differential therapeutic approaches require to separate IRIS from corresponding opportunistic disease: Antiinflammatory or immunosuppressive drugs are cornerstones of treatment in IRIS. 9 Certain patterns of immune response, different levels and pathways of immune stimulation and genetic factors have been identified in IRIS. In future these factors may allow an individual risk assessment and development of rational therapeutic approaches.
REFERENCES 1.
7. TAKE HOME MESSAGES
9 Immune restoration inflammatory syndromes (IRIS) present as unexpected event or paradoxical deterioration of inflammation. 9 IRIS is correlated to a variety of infectious diseases or noninfectious immunogenic antigens
2.
3.
Stoll M, Schmidt RE. Immune restoration inflammatory syndromes: The dark side of successful antiretroviral treatment. Curr Infect Dis Rep 2003;5:266-276. Moubasher AD, Kamel NA, Zedan H, Raheem DD. Cytokines in leprosy, II. Effect of treatment on serum cytokines in leprosy. Int J Dermatol 1998;37(10): 741-746. Lees AJ, MacLeod AF, Marshall J. Cerebral tuberculo-
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mas developing during treatment of tuberculous meningitis. Lancet 1980; 1(8180): 1208-1211. Rooney JJ, Crocco JA, Kramer S, Lyons HA. Further observations on tuberculin reactions in active tuberculosis. Am J Med 1976;60(4):517-522. Dooley DE Carpenter JL, Rademacher S. Adjunctive corticosteroid therapy for tuberculosis: a critical reappraisal of the literature. Clin Infect Dis 1997;25(4): 872-887. De Gans J, van de Beek D, European Dexamethasone in Adulthood Bacterial Meningitis Study Group. Dexamethasone in adults with bacterial meningitis. N Engl J Med 2002;347(20): 1549-1556. Egger M, May M, Chene G, Phillips AN, Ledergerber B, Dabis F et al. Prognosis of HIV-l-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies. Lancet 2002;360(9327): 119-129. Behrens G, Meyer D, Stoll M, Schmidt RE. Immune reconstitution syndromes in human immunodeficiency virus infection following effective antiretroviral therapy. Immunobio12000;202:186-193. French MA, Lenzo N, John M, Mallal SA, McKinnon EJ, James IR et al. Immune restoration disease after the treatment of immunodeficient HIV-infected patients with highly active antiretroviral therapy. HIV Med 2000; 1(2): 107-115. Cheng VC, Yuen KY, Chan WM, Wong SS, Ma ES, Chan RM. Immunorestitution disease involving the innate and adaptive response. Clin Infect Dis 2000;30( 6):882-892. Stone SE Price E Tay K, French MA. Cytomegalovirus (CMV) retinitis immune restoration disease occurs during highly active antiretroviral therapy-induced restoration of CMV-specific immune responses within a predominant Th2 cytokine environment. J Infect Dis 2002; 185(12): 1813-1817. Righetti E, Ballon G, Ometto L, Cattelan AM, Menin C, Zanchetta Met al. Dynamics of Epstein-Barr virus in HIV-l-infected subjects on highly active antiretroviral therapy. AIDS 2002; 16(1):63-73. Heussel CP, Kauczor HU, Heussel G, Fischer B, Mildenberger E Thelen M. Early detection of pneumonia in febrile neutropenic patients: use of thin-section CT. AJR Am J Roentgenol 1997;169(5):1347-1353. Narita M, Ashkin D, Hollender ES, Pitchenik AE. Paradoxical worsening of tuberculosis following antiretroviral therapy in patients with AIDS. Am J Respir Crit Care Med 1998;158(1):157-161. Race EM, Adelson-Mitty J, Kriegel GR, Barlam TF, Reimann KA, Letvin NL et al. Focal mycobacterial lymphadenitis following initiation of protease-inhibitot therapy in patients with advanced HIV-1 disease.
Lancet 1998;351:252-255. 16. Whitcup SM. Cytomegalovirus retinitis in the era of highly active antiretroviral therapy. JAMA 2000;283(5): 653-657. 17. Domingo P, Torres OH, Ris J, Vazquez G. Herpes zoster as an immune reconstitution disease after initiation of combination antiretroviral therapy in patients with human immunodeficiency virus type-1 infection. Am J Med 2001;110(8):605-609. 18. Fox PA, Barton SE, Francis N, Youle M, Henderson DC, Pillay D et al. Chronic erosive herpes simplex virus infection of the penis, a possible immune reconstitution disease. HIV Med 1999;1(1):10-18. 19. Stone SF, Price P, Keane NM, Murray RJ, French MA. Levels of IL-6 and soluble IL-6 receptor are increased in HIV patients with a history of immune restoration disease after HAART. HIV Med 2002;3(1):21-27. 20. Zietz C, Bogner JR, Goebel FD, Lohrs U. An unusual cluster of cases of Castleman's disease during highly active antiretroviral therapy for AIDS. N Engl J Med 1999;340(24): 1923-1924. 21. Behrens G, Knuth C, Schedel I, Mendila M, Schmidt RE. Highly active antiretroviral therapy. Lancet 1998;351:1057-1058. 22. Bell C, Nelson M, Kaye S. A case of immune reconstitution rheumatoid arthritis. Int J STD AIDS 2002; 13(8): 580-581. 23. Sellier P, Monsuez JJ, Evans J, Minozzi C, Passeron J, Vittecoq D et al. Human immunodeficiency virusassociated polymyositis during immune restoration with combination antiretroviral therapy. Am J Med 2000; 109(6):510-512. 24. Jubault V, Penfornis A, Schillo F, Hoen B, Izembart M, Timsit J et al. Sequential occurrence of thyroid autoantibodies and Graves' disease after immune restoration in severely immunocompromised human immunodeficiency virus-l-infected patients. J Clin Endocrinol Metab 2000;85( 11):4254--4257. 25. Sereti I, Sarlis NJ, Arioglu E, Turner ML, Mican JM. Alopecia universalis and Graves' disease in the setting of immune restoration after highly active antiretroviral therapy. AIDS 2001;15(1):138-140. 26. Silvestre JF, Albares MP, Ramon R, Botella R. Cutaneous intolerance to tattoos in a patient with human immunodeficiency virus: a manifestation of the immune restoration syndrome. Arch Dermatol 2001;137(5): 669-670. 27. Wittram C, Fogg J, Farber H. Immune restoration syndrome manifested by pulmonary sarcoidosis. AJR/san J Roentgenol 2001;177(6):1427. 28. Behrens G, Stoll M, Schmidt RE. Lipodystrophy syndrome with protease inhibitors: what is it, what causes it and how can it be managed? Drug Saf 2000;23:
57-76. 29. Grahame C, Alber DG, Lucas SB, Miller R, Vallance P. Association between Kaposi's sarcoma and atherosclerosis: implications for gammaherpesviruses and vascular disease. AIDS 2001; 15(14): 1902-1904. 30. Lewis W. Atherosclerosis in AIDS: potential pathogenetic roles of antiretroviral therapy and HIV. J Mol Cell Cardio12000;32(12):2115-2129. 31. Shelburne SA, Hamill RJ. The immune reconstitution inflammatory syndrome. AIDS Rev 5(2):67-79. 32. Johnson SC, Benson CA, Johnson DW, Weinberg A. Recurrences of cytomegalovirus retinitis in a human immunodeficiency virus-infected patient, despite potent antiretroviral therapy and apparent immune reconstitution. Clin Infect Dis 2001;32(5):815-819. 33. Keane NM, Price P, Lee S, Stone SF, French MA. An evaluation of serum soluble CD30 levels and serum CD26 (DPPIV) enzyme activity as markers of type
2 and type 1 cytokines in HIV patients receiving highly active antiretroviral therapy. Clin Exp Immunol 2001 ;126(1): 111-116. 34. Behbahani H, Landay A, Patterson BK, Jones P, Pottage J, Agnoli M et al. Normalization of immune activation in lymphoid tissue following highly active antiretroviral therapy. J Acquir Immune Defic Syndr 2000;25(2): 150-156. 35. Price P, Mathiot N, Krueger R, Stone S, Keane NM, French MA. Immune dysfunction and immune restoration disease in HIV patients given highly active antiretroviral therapy. J Clin Virol 2001 ;22(3):279-287. 36. Price P, Morahan G, Huang D, Stone E, Cheong KY, Castley A et al. Polymorphisms in cytokine genes define subpopulations of HIV-1 patients who experienced immune restoration diseases. AIDS 2002; 16(15): 2043-2047.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
CD5-Expressing B Cells and Infection Y. Renaudineau, J.O. Pers and P. Youinou
Laboratory of Immunology, Brest University Medical School, Brest, France
The 67 kDa T cell marker CD5 was originally identified on malignant human B cells [1], and subsequently shown to act as a coreceptor on a proportion of normal B lymphocytes in humans [2] and mice [3]. These have been classified into B-2, representing the conventional cells, and B-l, predominating in serous cavities [4]. The latter population comprises B-la which express CD5 (Fig. 1), and B-lb subpopulations which do not, but share all the other attributes of B-1 cells [5], such as the presence of mRNA for CD5, the expression of the myelomonoytic marker Mac-1, and the reduced density of the high molecular weight isoform of the common leukocyte antigen (Ag) CD45RA. Over the past decade, evidence has been accumulating to suggest that such B-1 lymphocytes are key in the defense from infectious agents. For example, they produce much of the immunoglobulin (Ig), almost all natural antibody (Ab) reactive with lipopolysaccharide [6], and most of the innate Ab in serum [7], although constituting only a minor fraction of the B compartment. Furthermore, they contribute significantly to the IgA-producing plasma cells in the lamina propria of the gut [8]. Indeed, in germ-free conditions, few peritoneal B-1 cells are detected in the mouse, while a number of these exist in specific pathogen-free conditions, indicating that bacterial infections are necessary for their generation [9]. The finding that numerous autoimmune conditions are associated with elevated levels of circulating B-1 cells, and the demonstration that, due to their resistance to apoptosis, such lymphocytes accumulate in chronic lymphocytic leukemia (CLL) and other B cell malignancies have sparked off a great deal of interest in the possibility of a role
of B-1 cells in the pathophysiology of a number of diseases, including infectious conditions. The general conclusions regarding B-1 lymphocytes are that only in some cases has this subset been demonstrated to be responsible for the production of Ab to infectious agents as well as autoAb. In general, however, the function of these cells in different disease states remains unclear.
1. C H A R A C T E R I Z A T I O N OF CD5+ B CELLS
1.1. Origins of the Cells Still it is unclear whether CD5 signifies a different lineage or is induced by activation. On the one hand, there are a number of evidence in support of a different lineage. Early experiments showed that irradiated mice could be reconstituted with CD5+ B cells if the graft contained bone marrow stem cells together with peritoneal cells [10]. Additional support came from studies on the severe combined immunodeficiency mouse (SCID). The animals fail to develop either T or B cells due to a genetic deficiency in the enzyme required for rearrangements of Ag receptor genes. Immunological reconstitution with CD5+ B cells can be achieved by injection of fetal liver but not adult bone marrow. In similar experiments with severe common immune deficiency (SC1D) mice, repopulation of CD5+ B cells, but not conventional B cells, B2, was obtained by injection of fetal omental cells. Levels of serum IgM and IgG3 Ab became detectable in these mice. Although the SCID mice experiments seem definitive, it has been argued that these animals might provide an alien environment for the develop-
65
Figure 1. On the left, double-staining of peripheral blood lymphocyte using a B cell marker which is CD19 and a T cell marker which is CD5 permits identification of B cells which express CD5, i.e., B-1 cells (arrows), as opposed to conventional B-2 cells. On the fight, polymerase chain reaction of the transcripts for CD5 followed by electrophoresis and blots show the message in T cells, and, to a lesser degree, in B cells.
ment of B cells along the conventional pathway from a common B cell precursor. Also consistent with the lineage paradigm, is the striking similarity in the relative proportion of circulating CD5+ B cells in monozygotic twins [11 ] and family members [12] of patients with rheumatoid arthritis (RA). On the other hand, arguments against the notion that CD5+ B cells are a separate lineage support the general view that such cells are just an activated subpopulation. One argument is that human CD5B cells can be induced to become CD5+ in vitro by activation with phorbol myristic acetate (PMA) [13] and several cytokines modulate the expression of this molecule [ 14]. Similar data is now available in the mouse showing that anti-IgM and interleukin (IL)-6 treatment increased CD5 expression on splenic B cells. To account for these opposite viewpoints, we
66
have proposed a reconciliation of the divergent theses [15] by postulating two different classes of CD5+ B cells (Fig. 2): those where CD5 expression is constitutive ("classical" CD5+B cells) particularly in the fetal liver and in the cord blood (CB), and conventional B-2 cells induced to express CD5 on appropriate activation ("induced" CD5+B cells) usually located in the germinal center of any secondary lymphoid organ.
1.2. Functions of CD5+ B Cells This question has been approached using immortalized CB clones. There appeared that B-1 lymphocytes have a propensity to produce low-affinity polyreactive Ab binding to self, as well as to exogenous Ag including several bacteria [16]. Some positive clones were specific for the carbohydrate
Figure 2. Innate or "classical" CD5+B cells in the cord blood are distinct from acquired or "induced" CD5+ B cells in the germinal center of a secondary lymphoid organ. B-1a refers to CD5-positive and B-lb to CD5-negative B-1 cells, as opposed to conventional B-2 cells.
erythrocyte iI [ 17], which is accessible to the B lymphocytes in the CB. This observation suggests that the CD5+ clones have been driven by such autoAg and, therefore, selected in vivo, and agrees with the previous finding that, at least in the Hemophilus influenza model, innate (B-1 cells) and acquired (B2 cells) humoral immunities are mediated by distinct arms of the system [7]. Most of these B-1 cells, however, use germ-line genes [18] and express cross-reactive idiotypes [ 16].
1.3. Control of the CD5+ B Cell Population As suggested by the data, already mentioned, on monozygotic twins [11] and family members of RA patients [12], the size of B cell subsets seems to be under the control of the major histocompatibility complex, as established in the mouse [19]. The distribution of B cells into B-1 a, B-lb and B-2 would thus be genetically regulated, while IL-10
rescues B-1 a cells from apoptosis and encourages B-lb cell proliferation [20]. Interestingly, an Igindependent regulating feedback mechanism of the B cell compartments has also been described in the mouse [21 ].
2. CD5+ B CELLS AND DISEASE
2.1. Connective Tissue Diseases Several groups have reported that the CD5+ B cell subset may be expanded in patients with RA. Using double-fluorochrome ultraviolet light rvficroscopy, this was originally found to comprise an average of 20% of the B cells of 16 RA patients, compared to a maximum of 3% in eight normal controls, though the average absolute numbers of circulating B cells were comparable in these two groups of subjects [12]. Although CD5 molecules are present at low
67
Autoimmune disease
Control
3% I
Chronic lymphocytic leukemia
22%
91%
I
CD 19-FITC F i g u r e 3. The level of CD5+ B cells is increased in some nonorgan-specific diseases as well as infectious states. These cells accumulate in chronic lymphocytic leukemia.
density on B lymphocytes, this is increased [13] following treatment of B-lymphocyte-enriched cell suspensions with PMA. These results were subsequently confirmed by flow cytometry analysis (Fig. 3). It was thus possible to detect coexpression of CD5 on a larger population of B cells from RA patients and controls than earlier studies, but the mean proportions of B cells that express CD5 were still greater in patients than in controls. These data conflict with some reports showing no significant differences in percentages of circulating CD5+ B cells in RA patients compared with normal individuals. In fact, they fall into two categories, twothirds with CD5+B cell levels within the normal range, and a third with elevated levels. Clearly, the elevation do CD5+ B cells p e r se is insufficient to give rise to RA. The corollary is that high levels of this B-cell subpopulation is not a prerequisite for developing the disease. The number of circulating CD5+ B cells does, however, correlate with the titer of rheumatoid factor (RF). Whilst there were reports of significantly elevated frequencies of these cells in RA patients with extremely high titers of RF, other studies have claimed that increased levels of CD5+B cells were associated with RF and antinuclear antibody in such patients. The level of CD5+B cells was also elevated in patients with primary Sjrgren's
68
syndrome [22] particularly in those patients with associated monoclonal Ig. Surprisingly, in most of the cases of systemic lupus erythematosus (SLE), there are not elevated numbers of CD5+B cells. Increased numbers have, however, been described in some of them [23], suggesting that polyclonal activation might also affect this B cell subset in a proportion of SLE patients.
2.2. Lymphoid Malignancies CLL comprise a heterogeneous group of disorders, in which three main conditions have emerged: CLL, pro-lymphocytic leukaemia (PLL) and hairy cell leukaemia (HCL). The malignant cells from approximately 95% of the CLL patients co-express CD5 and other B-cell surface markers [24]. Thus, in most cases of CLL there is a proliferation of a B cell clone characterized by low amounts of surface Ig and increased expression of the CD5 Ag. Different subtypes of CLL have, however, been delineated, and CD5 shown to be expressed on the leukemic cells, not only from patients with genuine CLL, but also from those with cleaved cell lymphocytic leukaemia, and with lymphoplasmocytoid leukaemia. The B cells of CLL with a particularly indolent character have been claimed to be more frequently CD5+ than those B cells in patients with more
aggressive CLL [25], but no correlation was definitively established between the expression of CD5 and surface Ig class or type, clinical stage, disease activity or age at diagnosis by other investigators. B cell markers are present on the leukemic cells in all cases of PLL, but in some cases they do not express the CD5 marker, and only weakly to moderately in others. In contrast, PLL have been found to carry molecules recognized by anti-CD5 monoclonal Ab. Typically, malignant cells forming HCL do not express CD5, although is expression has been reported occasionally [26]. Expression of CD5 is not a trait shared by immature B cell malignancies, such as pre-B acute lymphoblastic leukaemia, or by endstage differentiated B cell malignancies, e.g., multiple myeloma or Waldenstrrm's macroglobulinemia. Nonetheless, there are no clear differences between the latter disease and an otherwise typical CLL associated with monoclonal Ig in the serum and urine or cases terminating in lymphoplasmocytoid leukaemia or lymphoma. Less than half of the B cell-derived non-Hodgkin's lymphomas are CD5+ [27]. The marker is mainly found on B cell lymphomas composed predominantly of small lymphocytes, such as diffuse well-differentiated lymphocytic lymphomas, or intermediate lymphocytic lymphomas. Malignant cells are prone to express CD5, when solid tumors are associated with lymphocytosis. In fact, the immunophenotype of these lymphoma or leukaemia cells is reminiscent of that of lymphocytes in normal primary follicles and the mantle zone of secondary follicles of secondary lymphoid organs. It is well documented that surface Ig receptors on malignant B cells exhibit specificity for a variety of self Ag. This concept has been extended [28] by studies of IgM Ab secreted by leukemic B cells, after stimulation with PMA, which results in the production of low-affinity polyreactive autoAb. 2.3. Infections States
Elevated levels of circulating CD5+ B cells have also been reported in a great number of infectious diseases, especially those from viral origin. Surprisingly, this has been described in infectious mononucleosis [29], but never confirmed because Epstein-Barr virus reduces the expression of CD5. On the other hand, CD5+ B cells have repeatedly
been found enhanced in chronic hepatitis C virus infection (HCV), as compared with the patients with resolved infection [30]. In fact, chronic infection with HCV is associated with disturbance of B lymphocyte activation and function, leading to serological abnormalities, such as autoAb production, mixed cryoglobulinemia and B cell lymphomas [31]. The possibility exists that they reflect chronic Ag stimulation or aberrant signaling through the B cell CD81 glycoprotein. Alternatively, CD81 which is a putative HCV receptor is upregulated in CD5+ cells, compared with conventional B cells [32]. Are these unique cells involved in the defense from more common infections? The major target Ag of B-1 lymphocyte-derived IgA are normal intestinal bacteria [33]. Their coating with IgA results in immune exclusion, as established for pathogenic microbes, although the bacterial microflora of the gut is an extremely stable ecosystem. Furthermore, several parasites have been associated with an expansion of CD5+ B cells. Included are Toxoplasma gondii [34], Trypanosoma evansi [35] and Schistosiamis mansoni [36]. In the latest parasitemia, whether or not B-1 a cells are responsible for Ab against egg Ag polylactosamine sugars, as described for a mouse model previously, has not yet been determined.
3. FUNCTION OF THE CD5 M O L E C U L E CD5 is physically [37] and functionally [38] associated with the B cell receptor (BCR). Increased numbers of CD5+ B cells might thus reflect defective regulation of B cell function through CD5 itself (Fig. 4). There is now a growing body of evidence that CD5 is essential in modulating signals downstream of the BCR. In this respect, we have shown that ligation of CD5 or IgM on tonsillar B but not blood T cells resulted in apoptosis [39]. This observation has since been confirmed in a group of patients with CLL [40], and shown to take the BCR pathway [41]. In addition, anti-CD5 sustains the proliferation of tonsillar B cells pre-activated with anti-IgM Ab and IL-2 [42]. This was in contrast to CB CD5+ B cells which do not apoptose in response to anti-CD5, but might rather reflect the fact these CB B cells are continuously exposed to autoAg in vivo. It is important to note that the src-homolgy
69
(~ P-Tyrosine @ Serine
Figure 4. The B cell receptor (BCR) comprises membrane IgM with Igct/Ig[3as transducing molecules. CD5 is made up of three extra-cytoplasmic domains (D l-D3) and associated with the BCR and brings about the src-homology 2 domaincontaining phosphatase (SHP-1) to dampen down the transducing cascade. The tyrosine residues are phorphorylated (P) by phosphorylases, but not the serine(s) residues.
2 domain-containing protein tyrosine phosphatase (SHP-1) is constitutively linked with the Ig~/Ig]] chains of the BCR through the immunoregulatory tyrosine based inhibitory motif of CD5 [43]. The tyorisine residues are phosphorylated by phosphorylases (P), but not the serine(s) residues. It has thus been suggested that such an interaction with CD5 "sequesters" the SHP-1, and limits its role with important molecules in positive signaling through the BCR [44]. Furthermore, the role of CD5 in the maintenance of clonal anergy has recently been addressed by the elegant experiments of Hippen et al using the hen egg lysosyme (HEL)-Ig transgenic (Tg) mouse [45]. In this model, mice Tg for HEL-Ig and the membrane-bound form of the self Ag HEL produce apoptosis of anti-HEL B cells, while those Tg for HEL-Ig and the soluble form of HEL initiate anergy through the SHP-1. Breeding of the latter Tg mice onto a CD5-/- background results in loss of tolerance. These data indicate that the presence of CD5 raises the threshold required for activation of self-reactive B cells, in such a way that it determines their ultimate face. Consistent with this role for CD5 is a more recent model in which CD5- spleen cells from mice made Tg for anti-ribonucleo-protein
70
(RNP), a common autoAb in SLE and other connective tissue diseases, were injected into irradiated naive mice. They migrated to the peritoneal cavity where most of the CD5+ B cells are found, and began to express CD5 which prevented their production of anti-RNP autoAb [46]. As well as CD5 being important in this negative regulation of autoreactive B cells, other molecules have been shown to play a role. For example, CD 19 amplifies BCR signaling by favoring the activity of phosphorylases, such that a modest 10-20% increase in CD 19 expression may be sufficient to shift the balance between tolerance and immunity to autoimmunity [47]. In contrast, CD22, dampens down the signals by recruiting SHP-1, so that deficiency in CD22 encourages the development of autoimmunity [48]. Furthermore, defective signaling through the BCR has already been demonstrated for B cells from patients with SLE [49].
4. REGULATION OF THE CD5 E X P R E S S I O N Several lines of evidence indicate that the expression of CD5 is tightly regulated. Thus, membrane density of CD5 is --30-folds higher in T than in B cells, and the expression of CD5 developmentally regulated, since the membrane density of CD5 is higher on mature T cells than on thymocytes. Moreover, CD5-expressing B cells represent the majority of B lineage cells during fetal and neonatal fife, but the number of CD5+ B cells declines in relative number with age [50]. Further evidence in support of tight CD5 regulation comes from experiments showing that ex vivo B cells downregulate their membrane CD5 expression when cultured in the presence of IL-4 [14], but upregulate CD5 following activation with PMA [ 13, 51 ], or when their membrane IgM is cross-linked in the presence of IL-6 [52]. Consistent with this view are findings that, despite their loss of membrane expression of CD5-, B-lb cells retain CD5 mRNA, albeit at lower levels than B-la cells [5]. Finally, a feedback regulation of murine B-la cells has also been advocated [21]. In an apparent contrast to these findings, a recent report suggested that all B cells constitutively express CD5, but the level of expression varies considerably, from B l a cells at one end of the spectrum to B2 cells at the other [53]. All in all, these observations imply that
Figure 5. The CD5 gene is made up of 12 exons. Exon 1 associates the classical exon 1, termed 1A and the alternative exon 1, designated lB. The former is expressed in B and T cells, and the latter present exclusively in B cells. When exon 1A splices to exon 2, the initiation site AUG is located within the exon 1 and the resulting CD5 molecule is full-length, whereas when exon 1B splices out exon 1A and binds to AUG-free exon 2, the first initiation site AUG is located within exon 3 and the resulting CD5 molecule truncated, because the 5' segment of exon 3 is not transcribed into mRNA.
multiple regulatory mechanisms for CD5 expression exist.
4.1. Shedding of CD5 At the posttranslational level, shedding of the molecule has been described, and suggested to be exaggerated in nonorgan-specific autoimmune and infectious diseases [54]. Cell-free CD5 could even bind to cells endowed with the related receptors, leading to an over-estimation of CD5+ B cells.
4.2. Internalization of the Membrane Molecule Interplay between several mechanisms is likely to be involved in the accurate regulation of CD5 expression at the membrane. CD5 internalization which is enhanced in T cells, but inhibited in B cells, upon Ag receptor crosslinking is another mechanism known to be involved in CD5 regulation [55], even though the spontaneous turn-over of this molecule is rather low.
pre-mRNA splicing, message stability and translation in different lymphocyte populations, are totally unknown. These 11 exons are, however, conserved in size and number in the mouse, as well as most of their transcription regulatory elements. A novel exon 1 that is exclusively transcribed in B cells has just been discovered [57]. Intriguingly, the existence of this new exon is due to a defective human endogenous retrovirus (HERV). The data also provides attractive evidence for a reciprocal expression of this alternative exon 1, designated exon 1B, with the conventional exon 1, hereafter referred to as exon 1A. Exon 1B-type transcripts are translated into a truncated variant of the CD5 molecule devoid of leader peptide. Consequently, whereas exon 1A promotes expression of membrane CD5 protein in T and a subset of B cells (Fig. 5), exon 1B acts to reduce CD5 protein expression in BL and, therefore, possibly, reduce the signaling functions of CD5, such as the production of Ab against infectious agents and auto-Ab. This balance between the two exons 1 might be important in the regulation of membrane expression of CD5.
4.3. Alternative Splicing of the Gene The CD5 protein is encoded by a single gene in both T and B cells, mapping to chromosome 11q12.2, and consisting of at least 11 exons [56]. The precise stages of regulation, i.e., transcription, alternative
To conclude, the Bar Mitzvah is being celebrated for B-1 cells [58]. We are indeed close to understand the way they operate in autoimmunity and infection. Paradoxically, in the light of recent findings of the modulation of B cell signaling by CD5, this
71
and other molecules play a crucial role in preventing autoimmunity. Aberrations of the transduction through CD5 are thought to exist. They could lead to autoimmune disorders. Hence, the present views on the potential functions of CD5+ B cells in autoimmunity are quite different from the previous and rather naive interpretation that the increased levels of CD5 B cells in patients with nonorganspecific autoimmune diseases represented a direct source of autoAb leading to pathogenesis.
ACKNOWLEDGEMENTS
8.
9.
10.
11.
Studies mentioned in this review were supported by the Acad6mie Nationale Franqaise de M6decine, the Conseil R6gional de Bretagne and the C o m m u naut6 Urbaine de Brest. The secretarial assistance of S imone Forest is appreciated.
12.
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45. Hippen K, Tze LE, Behrens TW. CD5 maintains tolerance in anergic B cells. J Exp Med 2000;191:883-889. 46. Qiou Y, Sartiago C, Borrero M, Tedder TF, Clarke SH. Lupus-specific antiribonucleoprotein B cell tolerance in nonautoimmune mice is maintained by differentiation of B-1 and governed by B cell receptor signaling thresholds. J Immuno12001 ;166:2412-2419. 47. Sato S, Hasegawa M, Fujimoto M, Tedder TF, Takehara K. Quantitative genetic variation in CD19 expression correlates with autoimmunity. J Immunol 2000;165: 6635-6643. 48. Smith KG, Tarlington DM, Doody GM, Hibbs ML, Fearon DT. Inhibition of the B cell by CD22: a requirement for Lyn. J Exp Med 1998;187:807-811. 49. Liossis SN, Kovacs B, Dennis G, Kammer GM, Tsokos GC. B cells from patients with systemic lupus erythematosus display abnormal antigen receptor-mediated early signal transduction events. J Clin Invest 1996;98: 2549-2557. 50. Bergler W, Adam S, Gross HJ, HiSrmann K, SchwartzAlbiez R. Age-dependent altered populations of tonsillar lymphocytes. Clin Exp Immunol 1999; 116:9-18. 51. Miller RA, Gralow J. The induction of Leu-1 antigen expression in human malignant and normal B cells by phorbol myristis acetate. J Immunol 1984;133: 3408-3414.
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52. Ying-zi C, Rabin E, Wortis HH. Treatment of murine CD5-B cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways. Int Immunol 1991;3:467-476. 53. Kaplan D, Smith D, Meyerson H, Pecora N, Lewandowska K. CD5 expression by B lymphocytes and its regulation upon Epstein-Barr virus transformation. Proc Natl Acad Sci USA 2001;98:13850-13853. 54. Jamin C, Magadur G, Lamour A, MacKenzie LE, Lydyard PM, Katsikis PD, Youinou P. Cell-free CD5 in patients with rheumatoid diseases. Immunol Letter 1991;31:79-84. 55. Lu X, Axtell RC, Collawn JF, Gibson A, Justement LB, Raman C. AP2 adaptator complex-dependent internalization of CD5: differential regulation in T and B ceils. J Immuno12002; 168:5612-5620. 56. Padilla O, Calvo J, Vilh JM, Arman M, Gimferrer I, Places L, Arias MT, Pujana MA, Vives J, Lozano E Genomic organization on the human CD5 gene. Immunogenetics 2000;51:993-1001. 57. Renaudineau Y, Mageed RA, Youinou P. An alternative exon 1 of human CD5 gene regulates CD5 expression. Submitted. 58. Tarakhovsky A. Bar Mitzvah for B-1 cells: how will they grow up ? J Exp Med 1997; 185:981-984.
9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Endothelial Cell Autoreactivity and Infection C. Dugu6, Y. Renaudineau and E Youinou
Laboratory of Immunology, Brest University Medical School Brest, France
Owing to their permanent contact with circulating immune effectors, endothelial cells (EC) have long been suspected of being a target for antibody (Ab)-mediated assaut. In spite of incredibly wide variations in the results [ 1], it is, therefore, not surprising that anti-EC Ab (AECA) have been reported in a variety of clinical settings having in common to be accompanied by vascular changes [2]. These include not only most of the nonorgan-specific auto-immune diseases, but also numerous infectious states. AECA were first detected by indirect immunofluorescence (IIF) analysis [3, 4], and subsequently characterized using purified IgG and F(ab') 2 fragments [5]. The disorders associated with AECA has become impressively diverse [6], and sera apparently negative for this autoAb on a given cell type may turn positive if, instead, appropriate substrate cells are used [7]. Thus, AECA certainly represent a heterogeneous family of autoAb. As a corollary, the antigens (Ag) recognized by AECA may be infered to be multiple, though we have hitherto been unable to identify any of these EC Ag [8]. Furthermore, the presence of such autoAb does not imply causation, since it may follow, rather than precede EC damage. There is, nonetheless, compelling evidence that they are pathogenic. At this time, the most persuasive argument for this interpretation has come from the development of an idiotypic experimental model of systemic vasculitis [9]. A number of recent findings have indeed kindled a new debate on their pathogenicity. Concomitantly, the interest for further analysis of infection-induced AECA has been revived by the finding that infectious agents, such as Mycobacterium leprae, cytomegalovirus (CMV) and
dengue virus [10-12] colonize EC and contribute to the pathophysiology of vasculitis. Evidence has also been presented that some AECA recognize the membrane, while others react with components of their cytosol [13]. The former may be involved in the pathogenesis, but the latter would merely constitute a disease marker. Such findings support the view that AECA are also functionally heterogeneous, depending on their specificity.
1. PITFALLS IN AECA DETECTION The detection methods of AECA can be classified into those requiring fixation of the cells, and those using suspensions of EC. The group with fixation includes IIF on tissue sections, cytotoxicity of EC labelled with Cr51 or I111, radio-immunoassay and cell enzyme-linked immunosorbent assay (ELISA). There are, in fact, some major pitfalls in this method developed by Hashemi et al [14]. We have indeed reported that heterophile Ab against fetal calf serum (FCS) may be mistaken for AECA [15]. Such an interference can be eliminated simply by absorption with FCS-containing dilution buffer. Given that antinuclear Ab and rheumatoid factor may interfere with AECA, these may also be detected by an ELISA using Ab from EC lysate [16]. In addition, since false-negative AECA may serult from lack of expression of specific Ag, analysis of infectious or other AECA [ 17] dictates the use of several EC types, including microvascular EC. However, because fixation with glutaraldehyde permeabilizes the cells, autoAb to non-EC-specific cytosolic components can been detected in these assays, as reported in malaria [13].
75
Table 1. Prevalence of antiendothelial cell antibodies in infectious disease Infectious diseases
Antiendothelial cell antibodies [No positive/No tested (% positive)]
Reference
Year
13/16 (81)
[24]
1986
3/17 (18)
[28]
1992
13/72 (18)
[27] [30] [29]
1993 1997 1999
35/68 (51)
[33] [131
2001 2003
30/34 (88)
[ 13 ]
2003
Viral infections 9 Kawasaki disease 9 Puumala virus o Behqet's disease 9 Cytomegalovirus
10/23 (43)
9 Hepatitis C virus
28/69 (41)
Bacterial infections 9 Infective endocarditis 9 Leprosy
7/15 (47)
Parasitic infections 9 Malaria 9 Toxocara infection
3/5 (60)
unpublished
2003
9 Amebiasis
4/5 (80)
unpublished
2003
9 Echinococcis
4/6 (67)
unpublished
2003
9 Schistosomiasis
5/5 (100)
unpublished
2003
EC may rather be used as a suspension. Fluorescence-activated cell sorting (FACS) analysis, immunoprecipitation (IP) and Western blotting (WB) have thus been developed [18]. Human umbilical vein endothelial cells (HUVEC) remain the most widely used substrate cells. Hybrid cell lines, such as EAhy-926 and various cell lines, are occasionally employed [19]. However, needless to stress that HUVEC have a very limited use on a routine basis, due to the fact that their number is so limited (approximately 106 cells are eluted from a cord) that the procedure becomes extremely tedious. In addition, the phenotype of the cells is not stable, and EC die at the third or fourth passage. EA.hy926 cells have the unvaluable advantage of consisting of a non-limited number of cells, of which the phenotype remains the same until 50 passages or more. It may be necessary to absorb the sera with the mother epithelial cells A-549 before use. However, though "AECA" has long been coined as a designation for these autoAb, this does not necessarily mean that they recognize exquisitely EC. Numerous other human, bovine, and murine cell lines are available. With regard to the practical development of the AECA binding, the most accurate method is the
76
cell-ELISA [ 18]. It is quantitative since EC are used only once confluent. It is therefore possible to test a large number of sera at the same time. l i e a semiquantitative test, is not recommended, given that it does not permit adequate follow-up of the AECA levels. Cytotoxicity tests and radioimmunoassay are not in use any more. FACS is a technique where the cells do not adhere to a matrix, but are suspended in a buffer [ 18, 19]. It has been claimed to be the most suitable method to measure membrane-specific AECA [20], and we have shown that such autoAb detected that way predominate in leprosy [13]. Yet, its standardization has not been achieved and creating suspension of adherent cells may underestimate the expression of certain Ag [21]. The results of the ongoing programs could standardize the test and perhaps bring about more insights into the understanding of these autoAb. At present, they have to be compared with those obtained by ELISA. EC display different Ag distributions on their surface, depending on whether or not they are in contact with the solid support (subendothelial matrix or plastic), or flooding in the supernatant. AutoAb against the extracellular matrix can maskarade AECA [22], and have patho-
logical significance in vascular damage. IP and WB are too complicated to be applied on a routine basis. An additional problem is that cytoplasmic proteins can be precipitated, along with surface glycoproteins. Due to the uncertainty of such results, we have evaluated the same sera using in-house ceI1-ELISA, FACS analysis and WB completed by densitometric quantitation, and identified AECA using these three different methods [18]. We came to the conclusion that, ideally, the three methods should be applied. It is equally important to agree on the mean to express of results [23]. Given the variations in the number of cells in a well, they should not be expressed in optical densities, but in binding index, using the formula: 100 x (S - A) / (B - A), where S is the result of the disease, A the negative value and B the positive value.
2. DETECTION OF AECA
2.1. Viral Infections Despite obvious differences in their pathophysiology, similar AECA have been reported in a vast array of diseases (Table 1). For example, IgG and IgM AECA have long been described [24] in Kawasaki syndrome (KS), and claimed to be involved in the development of its acute phases [25]. Such a statement has been subsequently challenged [26], so that still very little is known about the etiology of the syndrome. In Behqet disease which is also of unknown origin but possibly triggered by a virus, AECA have been detected in 18% of the cases, found to be associated with thrombotic events, and therefore suspected to be pathogenic [27]. There is increasing evidence that AECA are associated with various viral infections. The autoAb have thus been reported in nephropathia epidemica, one of the milder forms of haemorrhagic fever caused by Puumala virus, and in three of 17 patients, as well as in four of nine and two of 19 sera from patients with influenza A and influenza B, respectively [28]. Interestingly, AECA is a common finding in hepatitis C virus (HCV) infection, but not in non-HCV chronic liver diseases [29]. It might be a risk factor for vascular rejection in CMV-infected recipients of cardiac or renal [30] or liver [31 ] allograft. In this respect, the dengue virus is another
particular case, because EC are permissive to this virus and AECA generated in the majority of the patients [32].
2.2. Other Infections AECA have also been found in under half of the sera from infective endocarditis [33], and over half of those from leprosy [13]. In the latter disease, more of the patients with the lepromatous and the borderline lepromatous that of the patients with the tuberculoid and the borderline tuberculoid forms scored positive, and the truly specific autoAbs to the membrane of EC were preferentially associated with multibacillary than with paucibacillary leprosy. Other bacteria, including Chlamydia pneumoniae and Helicobacter pylori activate EC, though, for unknown reasons, they do not promote the synthesis of AECA [34]. Finally, the cell-ELISA for these autoAb was recorded positive in 30 of 34 patients with malaria, compared with 17 of 50 local controls [13]. This baseline production in healthy African individuals is relatively high, as would be expected in an area where parasitic infection is endemic. Consistent with this view is the high level of AECA in toxocara infection (60%), amebiasis (80%), echinococas (67%) and schistosomiasis (100%).
2.3. Pathogenic Effects of AECA The pathogenicity of AECA remains uncertain. The likelihood of such an effect was first suggested by the observation that the autoAb levels fluctuate with disease activity in patients with systemic lupus erythematosus (SLE), Wegener granulomatosis (WG) and KS. The AECA test can even identify subsets of systemic sclerosis (SSc) [35], vasculitides [36] or inflammatory myopathies [37] with differing prognoses. Interestingly, the production of AECA is complicated by renal failure in SLE [38], vasculitis in rheumatoid arthritis (RA) [39] and lung fibrosis in dermatomyositis [40]. Some of them cause complement-mediated killing of EC in SLE [5], KS [25] and hemolytic-uremic syndrome [41], or induce Ab-dependent cellular cytotoxicity in WG [42]. Thrombomodulin, an EC-specific glycoprotein, is released by damage to these cells in WG and other systemic vasculitides [43]. Plasma from patients with thrombotic thrombo-
77
Figure 1. On the left, endothelial cells (EC) stained with May-Griinwald-Giemsa exhibit a typical morphological aspect of apoptosis (arrows). On the right, agarose gel electrophoresis shows EC incubated with control IgG or with apoptosis-inducing autoantibodies. These generate fragmentation of DNA. cytopenic purpura and sporadic hemolytic-uremic syndrome induces apoptosis in restricted lineages of human microsvascular EC, although the agents responsible for initiating EC injury and the exact role played by AECA is elusive [44]. Also supporting an apoptotic process in these thrombotic microangiopathies are the EC detachment from affected vessels, their appearance in the periphery [45], and the clear absence of inflammatory changes. The recent finding that one of the earliest events of a chicken model of SSc is EC apoptosis [46] may be highly relevant to this problem. Similar endothelial changes are also found in the initial phase of human generalized and local scleroderma. In addition, speculation about the mechanism of vascular conditions associated with AECA has focused on raised expression of adhesion molecules, such as E-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1, by EC [47]. This enhancement, together with the production of chemotactic cytokines, e.g., intefleukin (IL)- 1~, IL6, IL-8 and monocyte chemotatic protein 1, would facilitate adhesion of leukocytes to the inflamed vessel walls, followed by their extravascular migra-
78
tion and granuloma formation. A different group of AECA has been shown to induce tissue factor in EC [48]. An other appealing possibility is that EC apoptosis is initiated by AECA. In this study, incubation of EC with AECA derived from patients with vasculitis or mouse monoclonal AECA resulted in the expression of phosphatidylserine (PS) on the surface of the cells, as established through the binding of cationic annexin V [49]. Hypoploid cell enumeration, DNA fragmentation study and optical, immunofluorescence, confocal and electron microscopy analysis confirmed apoptosis of EC (Fig. 1). In some but not all sera, a subgroup of AECA may thus be pathogenic that way. Such a complication has been described in leprosy [13] and denguevirus infection [50]. We have, therefore, addressed the issue of whether activation is a prerequisite for AECA-mediated apoptosis of EC [51 ], shown that the ability of some AECA to activate the cells was irrelevant to the nature of the underlying disorder, and established that activation does not play a role in the advent of apoptosis. We have since extended these studies and found
that AECA binding to HUVEC makes anionic phospholipids (PL) accessible to anti-[32 glycoprotein I (I3zGPI) Ab (52]. A mechanism by which some antiPL Ab (aPL) bind to EC has thus been proposed. Should PS become available, following the binding of AECA, circulating ~2GPI would attach to EC and thereby, allow the recognition of the 132GPI~L complex by autoimmune aPL. It is not yet known whether anti- I3zGPI Ab from patients with primary aPL syndrome recognize new epitopes formed after binding of the molecule to anionic structures displayed by native ~2GPI when available at increased density, as one would expect for low-affinity Ab [53]. In line with the first interpretation is the recent report by Pittoni et al that a monoclonal from an SLE patient reacts with a cryptic epitope on I]2GPI, following binding to apoptotic cells [54]. Thus, not only AECA encourage the binding of pre-existing aPL to apoptotic EC, but the PS exposure might result in de novo production of aPL. It may be argued that if AECA were essential to the production of aPL, they should be present in all patients with aPL. However, a proportion of sera contain aPL but not AECA. One possibility is that, by the time a patient is investigated for autoAb, AECA may have already disappeared, so that the serum, while aPL-positive, has become AECAnegative. As suggested by Shoenfeld [55], aPL may be infectiously-induced. Inasmuch as the infectious diseases to be associated with AECA are plenty, it is tempting to speculate on a role for these infectionrelated autoAb in the production of aPL.
4. MECHANISMS OF AECA PRODUCTION IN INFECTIOUS DISEASE 4.1. Direct Involvement of EC
Three mechanisms deserve to be considered. The first refers to molecular mimicry, as described in dengue virus infection where Ab cross-react with EC, and their binding inhibited by pretreatment of the cells with nonstructural protein 1 from the virus [32]. Human heat shock protein (HSP)-70, which is a chaperone molecule, is recognized by some autoAb from some lepromatous sera [13]. This target Ag of AECA reproduces the C-terminal half of the M. leprae HSP-70 [56]. Accordingly, it
may initiate cross-reactive autoAb, either singularly or through interaction with any chaperoned autoAg. Alternatively, AECA may represent one of many serological hallmarks of polyclonal B cell activation. This second mechanism, well established in SLE, has been demonstrated in the production of malaria AECA [13, 57]. The third possibility is the induction of cell proliferation and morphological changes through colonization of EC. One example is M. leprae, most notably in those lining epineurial and perineurial blood vessels [10]. Dengue virus-EC interaction as also been studied in depth by using differential display reverse transcriptionpolymerase chain reaction (PCR), real time PCR, and Affymetrix oligonucleotide microarrays [58]. Stricking changes in gene expression were seen after infection of HUVEC with the virus. 4.2. Indirect Involvement of EC
Several indirect mechanisms have been suspected to initiate the production of AECA. These include activation of EC by IL-lct released by epithelial cells infected with respiratory syncytial virus [59], and upregulation of CD40 expression on EC infected with CMV [60].
5. THE TARGET AG OF AECA 5.1. Cell Membrane Specificity
Early studies have excluded anti-ABO and antiHLA Ab from the AECA [61]. Specificity for membrane, that cannot be absorbed with cytosolic lysates, predominates over that for cytosolic components in leprosy [13]. WB analysis, expression bank evaluation and two-dimensional electrophoresis have revealed that calreticulin, vimentin, tubulin and HSP-70 are recognized by AECA from patients with leprosy, while numerous proteins remain unidentified. (Table 2). In SLE, which is the prototype vasculitis-associated disease, 19 bands ranging from 15 to 200 kDa were identified by van der Zee et al [62] using WB, and Ab against 38, 41 and 150 kDa proteins shown to be tightly associated with lupus nephritis. Li et al [63] reported, however, that SLE patients with nephritis, vasculitis and hypocomplement raise IgG-AECA against a 66 kDa membrane
79
Table 2. Membrane components recognized by antiendo-
thelial cell antibodies Disease Leprosy
Calreticulin, vimentin, tubulin, Heat-Shock Protein (HSP)-70
Systemic lupus erythematosus
Ribosomal P-protein
Systemic vasculitides
Triose phosphate isomerase
Systemic sclerosis
Heparan sulfate
Wegener granulomatosis 70 kDa protein (HSP-70?)
Ag, whereas a 55 kDa Ag would be the specific target for AECA in patients with thrombocytopenia, and another 18 kDa component in those with pleuritis. Other groups have demonstrated that ribosomal P protein is an EC target for autoAb. It may be involved in the pathogenesis of lupus nephritis [64]. Although the AECA epitopes vary from a given patient to another, a subgroup can be IP only by SLE sera, suggesting that the way AECA react might be specific for each disease [65]. It has also been established that in renal diseases and kidney transplantation, AECA from patients with systemic vasculitis recognize 30-35 kDa Ag. In contrast, a 28 kDa Ag has been claimed to be specific for vasculitis and to share 93% amino acid with triose phosphate isomerase [66]. Interestingly, Wheeler et al [67] have demonstrated the association of these anti-triose phosphate isomerase Ab with IgM anti-vimentin Ab in human transplantassociated coronary artery disease. The latter observation might be a clue to the concept that a fraction of AECAs enter the cells. In RA, 12 proteins, ranging from 16 to 48 kDa, have been identified by WB and IP. In those patients with RA vasculitis, IgG-AECA were as directed towards a 44 kDa EC membrane Ag [68]. This is reminiscent of the intriguing finding that a 44 kDa protein was targeted by AECA from patients with Behqet's disease when human dermal microvascular EC were used [69]. Del Papa et al [65] found that any one of five proteins in WG reacts with AECA (180, 155, 125, 38 and 25 kDa). Ab binding to a
80
43 kDa as yet unknown component in the cytosol and the nucleus of human microvascular renal EC have also been identified in hemolytic uremic syndrome and thrombotic thrombocytopenic purpura [70]. In heparin-induced thrombocytopenia, some circulating Ab react with platelet factor 4 complexed with heparin, and others with heparan sulfate incorporated into the membrane of EC. Thus, it is not unreasonable to assume that they may play an active role in the development of thrombosis [71]. Similar reactions have been reported to occur [72] in connective tissue diseases associated with vasculitis. Finally, a 18 kDa EC membrane antigen was shown important for autoAb from patients with SSc. It should be stressed that the related AECA are associated with the CREST variant of this disorder. Finally, the possibility exists that murine monoclonal AECA produced by idiotypic manipulation with human Ab recognizes HSP-70 [73].
6. OTHER AG POSSIBLY R E C O G N I Z E D BY AECA
There appears that most of the AECA-positive malarian sera react with the cytosol but not with the membrane of EC [13]. This is substantiated (Fig. 2) by our finding that sera negative in the FACS analysis turn positive, once EC have been permeabilized with saponin. Clearly the target Ag of these pseudoAECA are specific neither for malaria nor for EC. The majority of AECA that bind to Ag seem to be EC membrane proteins. Yet the demonstration that extensive washes of radiolabeled preparations with high molar buffers result in the reduction of AECA from SLE sera indicates that some of these Ab are also able to recognize non-constitutive proteins. This is further supported by the description of monoclonal and polyclonal anti-DNA Ab binding in vitro to EC through DNA or DNA/histone complexes attached to the cell membrane [74]. To conclude, there is compelling evidence that more and more specific proteins and epitopes recognized by those apoptosis-inducing AECA are identified. Still, clarification of the function of target Ag is required to achieve a better understanding of the effects of AECA on associated infectious diseases.
Leprosy After
}, |l
i
s
Malaria '
il
After
Figure 2. Detection of antiendothelial cell (EC) antibodies (Ab) from lepromatous (top) and malarian (bottom) sera using flow cytometry analysis. The serum from the patient with leprosy is already positive before permeabilization of the cells since Ab bind to the membrane, whereas that from the patient with malaria needs incubation of EC with saponin to encounter cytosolic antigens.
ACKNOWLEDGEMENTS The studies m e n t i o n e d in this review were supported by the Conseil R6gional de Bretagne and the C o m m u n a u t 6 Urbaine de Brest. The secretarial assistance of S imone Forest is appreciated.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Induction of Autoimmunity by Adjuvant Hydrocarbons Kindra M. Kelly, Yoshiki Kuroda, Dina C. Nacionales, Jun Akaogi, Minoru Satoh and Westley H. Reeves
Division of Rheumatology & Clinical Immunology, Center for Systemic Autoimmune Diseases, University of Florida, Gainesville, FL, USA
Although susceptibility to systemic lupus erythematosus (SLE) is genetically determined [1 ], environmental factors such as ultraviolet radiation, chemicals, or infections are likely to play an important role in triggering the disease in susceptible individuals. In view of the diversity of environmental exposures in human populations, animal models afford the best opportunity to identify exogenous triggers of lupus. We have reported that a lupus-like disease with disease-specific autoantibodies and nephritis develops in non-autoimmune prone mice treated with pristane, a hydrocarbon derived from the metabolism of chlorophyll [2, 3]. Some strains also develop an erosive and destructive arthritis reminiscent of rheumatoid arthritis [4]. BALB/c, C57BL/6, and nearly all other immunocompetent mice are susceptible to pristane-induced lupus, but autoantibody production, the severity of renal disease, and the development of arthritis exhibit strain-to-strain variation [5]. More recently, it has become clear that other hydrocarbons, notably the mineral oil Bayol F and the endogenous hydrocarbon squalene, also can induce lupus-like disease in mice [6]. These substances share the capacity to serve as immunological adjuvants, defined as "substances used in combination with a specific antigen that produce more immunity than the antigen alone" [7]. The induction of murine lupus by immunological adjuvants is significant for two reasons. First, it provides a model for the interaction of environmental triggers with the genetic background in systemic autoimmunity and secondly, it raises the possibility that adjuvant hydrocarbons might trigger autoimmune disease in susceptible humans. Consistent with that possibility, mineral oil and hydrocarbon adjuvants are known to
induce inflammatory disease in humans, including lipoid pneumonia, granulomas, and synovitis [8-10]. This review summarizes the current state of understanding about the immunological effects of adjuvants and our recent work on the induction of autoimmunity by these materials.
1. IMMUNOLOGICAL BASIS OF ADJUVANTICITY
Although many early vaccines consisted of live, attenuated intact microorganisms or heat-killed intact organisms, the use of these vaccines is limited by difficulties culturing certain organisms and by adverse effects, such as the induction of disease in immunocompromised hosts or unacceptable inflammatory reactions. This has led to the development of more antigenically restricted vaccines, such as viral subunit vaccines. However, it has been long recognized that many protein antigens are, by themselves, poorly immunogenic in comparison with intact microorganisms, e.g. viruses or bacteria. Accordingly, adjuvants have been added to these antigens to boost the immune responses. Adjuvants used in humans must be selected carefully so as to enhance immune responsiveness sufficiently without causing undue toxicity. At present, few effective adjuvants are considered safe for use in humans. 1.1. Mineral Salts
Alum, the only adjuvant currently approved in the United States for human vaccines, is a relatively weak adjuvant and a poor inducer of cell-mediated
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Table 1. Some adjuvants used in human or veterinary vaccines Adjuvant
Potency
Status
Advantages/disadvantages
Aluminum-based mineral salts (alum)~
Weak
FDA approved for human use.
Safe; poor enhancement of cell-mediated immunity; can induce IgE responses and allergic reactions
MF59 (Squalene)b
Moderate Licensed in Europe. In clinical trials for influenza, HIV, herpes simplex, CMV, and hepatitis B vaccines.
More potent than alum in enhancing cell mediated immunity. Safe and well tolerated in humans.
Incomplete Freund' s adjuvant (mineral oil)b
Moderate Used in human influenza vaccine in the 1940s. No longer used in human vaccines, but used commonly in veterinary vaccines,
More potent than alum. Human use discontinued due to formation of granulomas and abscesses and because of induction of plasmacytomas in mice.
Complete Freund' s High adjuvant (mineral oil plus heat killed mycobacteria)b'~
Limited use in animals. Not approved in humans
Too toxic to use in humans (causes severe granulomatous inflammation, sterile abscesses, pain and fever)
Muramyl dipeptide~
Moderate Limited use in animals
Good inducer of both humoral and cellmediated immunity. Unacceptable for human use due to fever.
CpG DNA~
Moderate In clinical trials
Good inducer of both humoral and cellmediated immunity. So far appears to be safe and well tolerated.
Lipopolysaccharide c
Moderate Not approved for human use (but whole organism vaccines for typhoid, cholera, and pertussis contain substantial amounts of LPS)
Good inducer of both humoral and cellmediated immunity. Too toxic for human use (fever).
aMineral salt. bOil emulsion. Clmmunostimulatory microbial product (Toll receptor ligand).
immunity (Table 1). Alum and other mineral salts are thought to enhance immune responses mainly by binding antigen and acting as a slow-release depot for the antigen, thus prolonging antigen presentation [ 11].
1.2. Oil Emulsions In contrast to the mineral salts, oil adjuvants stimulate cellular as well as humoral immunity. A variety of hydrocarbon oils exhibit adjuvant properties [ 11, 12] (Table 1). Le Moignic and Pinoy discovered the adjuvant effect of mineral oil in 1916. Freund subsequently discovered that the potency could be enhanced significantly by adding heat-killed mycobacteria. Incomplete Freund's adjuvant (IFA) consists of the mineral oil Bayol F plus an emulsifier
88
(Arlacel A), whereas complete Freund's adjuvant (CFA) contains heat-killed mycobacteria in IFA [13]. IFA was used in human influenza vaccines up until the early 1960s when its use was discontinued due to the occurrence of granulomatous inflammatory reactions and sterile abscesses in some individuals, and in light of evidence that mineral oil can induce plasmacytomas in mice [14]. Despite these concerns, there generally were few side effects and in one 16-18-year follow-up study of 18,000 military recruits who received influenza vaccine in the mineral oil Draceol, there was no evidence of an increased incidence of either neoplasia or systemic autoimmune disease [ 15]. Besides mineral oil, a number of other hydrocarbon oils, including squalene (MF59) and pristane (2,6,10,14-tetramethylpentadecane), have adjuvant
effects (Table 1). Straight chain hydrocarbons containing 15-20 carbons (C15-20) can substitute effectively for standard mineral oil (a complex mixture of hydrocarbons) in Freund's adjuvants, at least for inducing experimental autoimmune encephalomyelitis, whereas longer and shorter carbon chains are ineffective [ 16]. The threshold for adjuvanticity is a chain length of 12 carbons (C 12) [ 17]. The adjuvant effect appears to be related in some manner to inflammation. Inflammatory responses to hydrocarbon oils have been studied in experimental models employing turpentine, alkanes, and a variety of other hydrocarbons [ 16, 18, 19]. Nevertheless, although all commonly used adjuvant oils cause inflammation, short-chain alkanes are intensely inflammatory but are poor adjuvants, suggesting that there is not a simple relationship between adjuvanticity and the strength of the inflammatory response. Recent studies have shed some light on the question of how hydrocarbon oils exert their adjuvant effects [20]. Squalene (MF59) does not appear to facilitate the transport of antigen nor does it have a depot effect, as previously supposed [21]. Rather, the oil appears to be internalized by macrophages, which are transported to regional lymph nodes where they undergo apoptotic cell death and are taken up by dendritic cells [20, 22]. These or other antigen-presenting cells exposed to the oil probably are responsible for the subsequent T and B cell activation seen in squalene-treated mice. Despite these conceptual advances, it remains uncertain precisely how hydrocarbon oils cause inflammation.
1.3. lmmunostimulatory Adjuvants Innate immunity mediated by antigen presenting cells tightly controls the activation of antigen-specific T and B-lymphocytes. A number of immunostimulatory molecules of microbial origin have been used as vaccine adjuvants (Table 1). Some of these substances, such as lipopolysaccharide (LPS), bacterial DNA containing unmethylated CpG motifs, and muramyl dipeptide, are ligands for Toll-like receptors (TLRs) (Table 2), whereas others are the cytokines (e.g. IL-12, GM-CSF) produced upon engagement of TLRs. Like oil emulsion adjuvants, immunostimulatory adjuvants effectively stimulate both cellular and humoral immune responses. Most of these substances are too toxic to use as human
Table 2. Stimulation of IFNot/~ by Toll-like receptors Toll-like Ligands receptor
MyD88 MyD88 dependent independent pathway pathway
TLR3
Doublestranded RNA
Yes
Yes
TLR4
Lipopolysaccharide, Yes Lipoteichoic acid, Taxol, RSV F protein, HSP60
Yes
TLR7
Imidazoquinoline Yes compounds (imiquimod; R-848)
No
TLR9
CpG DNA
No
Yes
vaccine adjuvants, though it should be noted that heat-killed intact organisms are replete with TLR ligands. CpG DNA is perhaps the most promising of the immunostimulatory adjuvants, and currently is undergoing human trials [23, 24]. Antigen presenting cells, natural killer cells, complement, and type I interferons (IFNs) constitute the innate immune system [25, 26]. Dendritic cells (DCs) link innate immunity with the adaptive immune response [25, 27]. Antigens picked up by DCs from apoptotic cells are transported to the lymph nodes [25, 28, 29]. When antigens are presented to T cells by immature DCs, the outcome is usually tolerance [30, 31 ]. In contrast, when DCs are confronted with a "dangerous" foreign antigen [32], they mature and express higher levels of costimulatory molecules (e.g. CD86) and MHC class II [25]. Antigen presenting cells sense "danger" via pattern receptors such as the TLRs. The immunostimulatory adjuvants LPS, lipoarabinomannan (muramyl dipeptide), and CpG DNA signal via TLR 4, TLR2, and TLR9, respectively [33]. Moreover, whole organism vaccines for typhoid, cholera, and pertussis contain substantial amounts of the TLR4 ligand LPS. The adapter protein MyD88 is a key element of the signaling pathway for many of the TLRs, including TLR4 and TLR9 [33, 34]. Signal transduction through MyD88 recruits the IL-1 receptor associated kinase (IRAK) and leads to activation of the transcription factors NFw_B and MAP kinase. TLR4 also signals via a MyD88 independ-
89
ent pathway, which activates the transcription factor interferon regulatory factor 3 (IRF-3) [35]. NFrd3 and IRF-3, in turn, activate several "primary viral response genes" including IFN~I, IP-10, RANTES and others [36]. The interaction of IFNI~ with the type I IFN receptor activates the transcription of an additional set of "secondary viral response genes", further enhancing cellular defenses. TLRs that efficiently stimulate type I IFN production include TLR3, TLR4, TLR7, and TLR9 [37]. Recent studies strongly suggest that type I IFNs (IFNo~, ~, to) are important mediators of the "adjuvant effect" [38].
2. IMPORTANCE OF TYPE I INTERFERONS FOR ADJUVANTICITY
Type I IFNs were found in 1957 to possess potent anti-viral activity. Since then, much has been leamed about their regulation and mode of action. Type I IFNs include up to 18 IFNct genes and pseudogenes, one IFN~ and one IFNto gene, all of which are located in the IFN gene cluster on the short arm of human chromosome 9 [39, 40]. The Type I IFNs are active as monomers and bind a specific receptor complex composed of two subunits, IFNAR1 and IFNAR2 [41]. The Jak non-receptor tyrosine kinases Tyk2 and Jakl associate with IFNAR1 and IFNAR2. IFN-receptor interactions cause reciprocal transphosphorylation of Jaks leading to receptor phosphorylation and recruitment and phosphorylation of STAT1 and STAT2. This causes activation of IFN regulatory factors (IRFs), transcription factors that induce the expression of IFN regulated proteins. Type I IFNs are a key component of the cellular response to viral infection. Working in part through the extracellular TLR3 molecule, viral double stranded (ds) RNA activates several intracellular kinases including the dsRNA-dependent protein kinase (PKR), culminating in IFN expression [42]. IFNs can activate PKR in a positive feedback loop, and induce other antiviral proteins that amplify the antiviral response. A variety of other stimuli are now known to induce IFN~I5 production including bacterial lipopolysaccharide (LPS), and bacterial CpG DNA working through TLR4 and TLR9 respectively. IFN inducible genes mediate various effector
90
pathways, including PKR, 2'5' oligoadenylate synthase (OAS), the Mx proteins, TRAIL, caspases, IRFs, and other proteins [43]. PKR can inhibit translation by phosphorylating Eif-2t~ and activates inflammation by causing nuclear translocation of NFrd3. OAS induces mRNA degradation, the Mx proteins inhibit viral replication, and TRAIL inhibits viral infectivity. IRFs and IRSs are involved in transduction of IFN signals. 2.1. Adjuvanticity is Associated with IFN Production
Type I IFNs have immunomodulatory effects, including enhancement of class II MHC expression on APCs and promotion of DC maturation and survival [38, 44]. IFNt~ stimulation increases the expression of BLyS and APRIL, promoting CD40-independent immunoglobulin class switching as well as plasma cell differentiation [45]. IFNtx also promotes TH1 responses [44] and the survival of activated T cells [46]. Its effects on B cells are more diverse. B cell lymphopoiesis is inhibited by Type I IFNs through a mechanism involving down-regulation of Bcl-2 and apoptosis [47], whereas in other situations, B cell survival is prolonged [48] and the maturation of B cells into plasmablasts facilitated [49]. The production of polyclonal IgG in response to influenza virus is abrogated when plasmacytoid dendritic cells, the primary Type I IFN producing cells, are depleted. Plasmacytoid dendritic cells drive CD40L activated B cells to mature into plasmablasts, which subsequently undergo IL-6-mediated differentiation into antibody secreting cells (see below). Moreover, Type I IFNs influence the balance of immunoglobulin isotypes produced in response to polyclonal [50] or antigen-specific [38] immune stimulation. Production of Type I IFNs is stimulated by several TLR ligands, including double stranded RNA, LPS, and CpG DNA [36, 51, 52]. Since these molecules also are potent adjuvants, there is considerable interest in the immunomodulatory effects of Type I IFNs. It has been shown that Type I IFNs augment antigen-specific immunoglobulin production following immunization with soluble antigen and are required for memory B cell responses [38]. IFNtx is as potent an adjuvant as IFA, an effect mediated by its action on dendritic cell maturation. In conclusion, there is increasing evidence that Type I IFNs are critical
mediators of adjuvanticity, a fact that may have considerable implications for the induction of autoimmunity by hydrocarbon adjuvants.
Table 3. Susceptibility to pristane-induced lupus BALB/c DBA/1 C57BL/6 B10.S Anti-RNP/Sm
55%
83%
24%
5%
3. INDUCTION OF LUPUS AUTOANTIBODIES BY HYDROCARBON ADJUVANTS
Anti-ribosomal P
0
0
16%
62%
Anti-dsDNA
38%
N/A
0
0
Anti-NF90/NF45
0
0
26%
33%
During the course of generating monoclonal antibody-enriched ascitic fluid, we found unexpectedly that the intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane, 0.5 ml i.p.) in BALB/c and other non-autoimmune strains of mice results in the production of autoantibodies characteristic of SLE as well as immune complex-mediated glomerulonephritis resembling lupus nephritis [2, 3]. More recently, it has become apparent that certain other hydrocarbons have the same effect, and that the ability to induce lupus autoantibodies correlates to some degree with adjuvanticity and cytokine production [6]. Remarkably, although various hydrocarbons may be more or less likely to induce lupus like disease, they all induce a similar spectrum of lupus-associated autoantibodies suggesting that their mechanisms of action are similar. Following intraperitoneal injection of pristane, IFA, squalene, or hexadecane, BALB/c and most other immunocompetent mice develop high levels of antinuclear antibodies [2, 3, 6] (Y Kuroda et al, unpublished data). The specificities include autoantibodies thought to be pathognomonic of SLE, such as anti-Sm, anti-dsDNA, and anti-ribosomal P [5, 53, 54] (Table 3). Other specificities include antinRNP, anti-Su, anti-chromatin, anti-ssDNA, and anti-NF90/NF45 [55] as well as myositis-specific anti-OJ autoantibodies [56]. The pattern of autoantibody production is remarkably similar to that seen in SLE (Fig. 1), and titers are comparable to those found in lupus-prone strains, such as MRL, or in humans with SLE. A puzzling aspect of this and other murine models is the complete absence of responses to the Ro (SS-A) and La (SS-B) antigens. We have examined many strains bearing different H-2 haplotypes, and although there is significant inter-strain variability in the frequency of standard autoantibodies induced by pristane [5], anti-Ro and La are never seen. Following intraperitoneal injection of pristane
Anti-OJ
0
0
0
5%
Anti-Ro (SSA) or La (SSB)
0
0
0
0
Anti-Scl70, fibrillarin, RNA polymerase I/III
0
0
0
0
Glomerular IC
S
S
S
S
Proteinuria
S
S
R
N/A
Arthritis
S
S
R
R
S, susceptible; R, resistant; N/A, not available.
or other active hydrocarbons, the earliest autoantibodies to appear are IgM anti-ssDNA mad IgM anti-chromatin antibodies, which appear after about 2 weeks [3]. Anti-Su antibodies are detected at 2-3 months followed by anti-nRNP/Sm at 3-4 months. Anti-dsDNA antibodies appear much later (6-10 months). Interestingly, this is well after the onset of nephritis. Unlike the early IgM anti-ssDNA response, these late-appearing autoantibodies are primarily of T cell dependent isotypes: IgG2a and IgG2b in the case of anti-Su, and IgG2a in the case of anti-nRNP/Sm and anti-dsDNA. Autoantibody production varies somewhat from strain to strain, but only within the rather limited repertoire mentioned above. Thus, pristane treatment causes BALB/c mice to produce anti-nRNP/ Sm (55%) but not anti-ribosomal P (Table 3). In contrast, SJL and B 10.S mice produce anti-ribosomal P frequently but not anti-Sm [5, 54]. Other strains fall somewhere between these extremes. There also are some differences depending on the hydrocarbon used to induce peritoneal inflammation. Pristane more potently induces anti-nRNP/Sm, whereas medicinal mineral oils induce mainly antissDNA and anti-chromatin. It is important to note that other than the major specificities (anti-nRNP/
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Figure 1. Spectrum of autoantibodies induced in mice by pristane is similar to that seen spontaneously in SLE. K562 cells were labeled with [35S]methionine and cell extracts were immunoprecipitated using sera from patients with SLE, serum from a healthy control subject (NHS), or with sera from pristane-treated or medicinal mineral oil-treated BALB/c mice. Immunoprecipitated proteins characteristic of SLE include the Sm (U5-200, Sm-B, Sm-D, Sm-E/F, and Sm-G) and RNP (U1-A, U1-C) proteins, the 100 kDa Su antigen, and the ribosomal P (rP) proteins P0, P1, and P2.
Sm, Su, ribosomal P, dsDNA, ssDNA, chromatin, and to a lesser degree NF90/NF45 and anti-OJ), additional specificities are unusual regardless of which hydrocarbon is used to induce autoantibody production. Following intraperitoneal injection of pristane, large amounts of IL-6, IL-12, and/or TNFa are produced locally in BALB/c and many other strains of mice [6]. There is inter-strain variability, however. For instance, C57BL/6 mice produce little IL-6 [57]. We investigated the effects of these cytokines on pristane-induced lupus in cytokine knockout mice. In view of the absence of autoantibodies in T cell deficient nude mice [58], we also examined the effects of T cell cytokines (IL-4 and IFN7) in cytokine knockout mice (Table 4).
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Table 4. Cytokine dependence of pristane-induced lupus in BALB/c mice a
Manifestation
IL-6 -/-
IL-12 -/-
IFN)'-/-
IL-4 -/-
Anfi-dsDNA
,H,
No A
,l,,I,
No A
Anti-chromatin
,1,,I,
$,1,
,1,$
No A
Anti-nRNP/Sm
No a b
S J,
$,1,
No a
Nephritis
,H,
$,1,
$,],
No h
aData from Refs. [53, 58, 75, 76]. bDecreased levels but comparable prevalence.
3.1. IL-6 Deficient Mice IL-6 is a pleiotropic cytokine thought to play a pivotal role in immune regulation through its effects on B and T cells [59]. It acts primarily on the late phase of B cell differentiation, consistent with the observation that IL-6R is expressed on mitogenactivated, but not resting, B cells [60]. Although Type I IFN is a key mediator of B cell differentiation into plasmablasts, their further maturation into antibody secreting cells requires IL-6 [49]. Moreover, IL-6 promotes the development of plasmacytomas in pristane and mineral oil treated mice [61-63]. Polyclonal activation and hypergammaglobulinemia in lupus may reflect both the over-expression of IL-6 receptors on B cells [64, 65] and increased IL-6 levels [66]. IL-6 overproduction in disorders such as atrial myxomas and Castleman's disease is associated with hypergammaglobulinemia and autoimmune phenomena [59, 67, 68]. In view of the strong indirect evidence linking IL-6 with autoantibody formation, we examined whether IL-6 deficient mice were susceptible to pristane-induced lupus [53]. Pristane induces high levels of IgG anti-ssDNA and chromatin antibodies as well as anti-dsDNA in wild type BALB/c mice, but IL-6 deficient BALB/c mice do not produce these autoantibodies [53]. Interestingly, wild type mice spontaneously develop low levels of anti-chromatin autoantibodies in an age-dependent manner, and effect abrogated in IL-6 -/- mice [53]. In contrast to anti-DNA, the frequencies of anti-nRNP/Sm an anti-Su antibodies are similar in pristane-treated IL-6-/- and IL-6 +/+ mice, although levels were lower in the knockout mice, consistent with the known effects of this cytokine on B cell maturation. We concluded from analyzing IL-6 deficient mice that anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6dependent, whereas induction of anti-nRNP/Sm and Su autoantibodies is relatively IL-6-independent. 3.2. IL-12 and IFNy Deficient Mice The potent adjuvant CFA is derived from IFA by adding heat-killed mycobacteria, which stimulate TLRs and enhance the production of IL-12 by maturing DCs [29, 69, 70]. For example, CpG oligonucleotides stimulate IL-12 production through
binding to TLR9, polarizing the immune response to TH1 [71, 72]. Analysis of the isotypes of polyclonal IgG and specific autoantibodies induced by pristane, especially anti-nRNP/Sm and anti-dsDNA, indicated a strong IgG2a response [73]. Since IgG2a is an IFNy [74] and IFNt~ [50] dependent isotype, we hypothesized that pristane-induced lupus may be a TH1 mediated disease. This possibility was examined using IL-12 and IFN 7 knockout mice. IgG anti-chromatin autoantibodies are absent following pristane treatment of IFNy deficient BALB/c mice, whereas their frequency and level are similar in IL-4 deficient mice vs. wild type controls [75]. The frequency of IgG anti-nRNP/Sm autoantibodies was reduced markedly in pristane-treated IFNy 4- mice compared with +/+ controls (22% vs. 77%), but in the few IFNy-/- mice producing these autoantibodies, levels were comparable to those in wild type controls. Likewise, the frequency of anti-Su antibodies was reduced (55% vs. 17%), but not the level. IL-12 and IL-18 are key cytokines produced by antigen presenting cells (APCs) that regulate the production of IFNy. As noted above, local (intraperitoneal) IL-12 production is greatly enhanced by pristane treatment. IL-12 deficient mice exhibit a similar, though not identical, autoantibody phenotype to that of IFNy-/- mice [76]. The major difference is that anti-dsDNA autoantibody production following pristane treatment is not substantially reduced in the IL-12-/- mice vs. controls, despite the fact that glomerulonephritis is nearly completely abrogated [76].
4. INDUCTION OF POLYCLONAL HYPERGAMMAGLOBULINEMIA BY HYDROCARBON ADJUVANTS One of the first changes noted in BALB/c mice given an intraperitoneal injection of pristane is a striking increase in total serum IgM [3]. This is followed by a rise in IgG1, IgG2a and IgG2b [77]. In most strains, IgG2a increases out of proportion to IgG1. Despite the role of IL-6 in hypergammaglobulinemia in other situations, an increased total IgM level is apparent as early as 2 weeks after pristane treatment in BALB/c IL-6 +/+ as well as IL-6 -/- mice, but not in PBS-treated controls. IgG2a, IgG2b, and
93
IgG3 levels also increase markedly from 1 to 3 months after pristane treatment both in IL-6 +/+ and -/- mice, suggesting that other cytokines contribute. Not surprisingly, a substantial reduction in polyclonal IgG2a (an IFN~, dependent isotype) inducible by pristane is seen in BALB/c IFNT 4- mice [75]. Likewise, IgG1 (IL-4 dependent isotype) polyclonal hypergammaglobulinemia following pristane treatment is substantially reduced in IL-4 deficient mice [75]. Different hydrocarbons can yield quite different effects on the total levels of IgM vs. IgG isotypes. Thus, whereas pristane and to a lesser extent squalene and IFA promote IgG2a hypergammaglobulinemia, medicinal mineral oils tend to stimulate the T cell independent isotypes IgM and IgG3 out of proportion to IgG2a. These data suggest that the polyclonal hypergammaglobulinemia induced by pristane, mineral oil, squalene [6, 75], and silicone oil [78] is mediated at least in part through the stimulation of cytokine production. Consistent with that notion, intraperitoneal injection of lupus-inducing hydrocarbons (pristane, squalene, and IFA) causes the production of IL-6, IL-12, and sometimes TNFcz [6]. The production of these cytokines is substantially lower when nonlupus inducing hydrocarbons, such as medicinal mineral oils, are injected. IL-6 production also is likely to contribute to the growth of plasma cell neoplasms arising in the peritoneal cavity of BALB/ cPtAn mice treated with pristane [14, 61]. The cytokine-dependent induction of specific autoantibodies thus occurs in the setting of polyclonal hypergammaglobulinemia, the nature of which is influenced by the type of hydrocarbon injected and the cytokine response to it.
5. INDUCTION OF GLOMERULONEPHRITIS AND ARTHRITIS BY HYDROCARBON ADJUVANTS
complexes, but not renal disease [80]. The role of inflammatory cells in nephritis depends largely on the site of immune complex deposition: subendothelial and mesangial deposits are associated with inflammatory lesions, whereas subepithelial lesions (characteristic of membranous nephropathy) are not [81 ]. The predominantly mesangial and subendothelial immune deposits characteristic of human lupus nephritis and pristane-induced lupus are consistent with the inflammatory nature of the renal lesion in SLE. Renal lesions in pristane treated mice are initially mesangial, but subendothelial lesions reminiscent of diffuse proliferative lupus nephritis develop later on [3]. The mesangial expansion in pristane-induced lupus [3, 54] is consistent with the appearance of lupus nephritis [82]. IL-6 stimulates mesangial cell proliferation [83, 84] and may exacerbate lupus nephritis [85]. IFN~/also has been implicated in lupus nephritis [86; 87]. The light microscopic changes and severe proteinuria in pristane treated BALB/c mice are nearly abrogated by IFNT or IL- 12 deficiency and there is a significant reduction in immune complex deposition [75, 76]. In contrast, IL-4 deficiency has little effect or actually increases the severity of nephritis. IL-6 deficient BALB/cAn mice also are highly resistant to the induction of lupus nephritis by pristane. Light microscopic changes and proteinuria are eliminated and glomerular immune complex deposition is reduced dramatically [53]. Taken together, these data strongly suggest that the proinflammatory cytokines IFN),, IL-12, and IL-6 are critical mediators of pristane-induced lupus nephritis. It is possible that the striking reduction in glomerular immune complex deposits and the lower levels of immune complexes containing IgG2a in particular decreases the inflammatory response mediated by FcTRI and III. Alternatively, cytokine deficiency could decrease the recruitment of phagocytes and/or T cells into the glomerular lesions [88].
5.1. Nephritis 5.2. Arthritis
The interaction of immune complexes with Fc receptors (FqRI or Fc~,RIII) on phagocytes causes production of proinflammatory cytokines and other mediators of glomerulonephritis [79]. Interestingly, NZB/W mice lacking the common T chain shared by FcTR I and III exhibit glomerular immune
94
Several hydrocarbon oils induce arthritis in rodents [4]. BALB/cJ, DBA/1 and several other strains of mice develop synovial hyperplasia, periostitis, and marginal erosions reminiscent of rheumatoid arthritis following intraperitoneal pristane treatment
[4, 89]. Serological abnormalities consistent with rheumatoid arthritis also develop, including autoantibodies against type II collagen and rheumatoid factor [4]. Susceptibility to pristane-induced arthritis is associated with the IF-1 locus, which regulates circulating IFNct/~, TNFo~, and IL-6 levels induced by Newcastle disease virus [4]. BALB/c and DBA/1 mice (susceptible to arthritis) have the IF-1 ~allele, whereas most resistant strains, such as C57BL/6 and DBA/2, have the IF-lh allele and express high levels of I F N ~ , TNFct, and IL-6 when infected [90]. This is unexpected in view of the importance of TNFot and IL-6 in the pathogenesis of rheumatoid arthritis and collagen-induced arthritis [91]. It is all the more surprising in view of a report indicating that pristane-induced arthritis is ameliorated by TNFt~ inhibition [92]. Other hydrocarbon oils also can induce arthritis in rats. The adjuvanticity of a hydrocarbon corresponds to its arthritogenicity. Normal (unbranched) alkanes with 12 or more carbons are arthritogenic, whereas C12-C16 alkenes (olefins) consistently induce less severe disease than corresponding saturated hydrocarbons [17]. Squalene is arthritogenic in arthritis-prone DA rats following intradermal injection, raising the possibility that this endogenous cholesterol precursor has the potential to trigger autoimmune disease [93]. The arthritis induced by squalene, like pristane-induced arthritis in mice, is erosive and T cell-mediated, but is not accompanied by anti-collagen autoantibodies. The pathogenesis of erosive arthritis in rodents exposed to hydrocarbons is not well understood. TNFt~ has been implicated as well as monocyte/macrophages, suggesting that the pathogenesis may be similar in some respects to that of rheumatoid arthritis.
6. ACCELERATION OF SPONTANEOUS LUPUS BY PRISTANE Besides triggering the onset of autoantibody production and lupus-like autoimmune disease in non-lupus prone mice, pristane can accelerate spontaneous (genetically mediated) lupus-like disease in (NZB X NZW) F1 and MRL +/+ mice (Table 5). This is a useful model for studying the interaction between the environment and the genetic background in lupus.
6.1. (NZB X NZW) F1 Mice
In addition to anti-chromatin/DNA responses, NZB/W mice spontaneously produce autoantibodies against the double-stranded RNA binding protein RNA helicase A (RHA). In contrast, this strain does not produce autoantibodies against the nRNP, Sm, Ro, and La antigens. Pristane exposure greatly accelerates the production of anti-chromatin and anti-DNA antibodies and dramatically accelerates renal disease. Production of anti-nRNP/Sm and Su autoantibodies also is induced, indicating that the unresponsiveness of NZB/W mice to these antigens can be overcome. Unexpectedly, pristane treatment does not enhance the production of anti-RHA and may actually inhibit it, suggesting that these autoantibodies are regulated differently. 6.2. MRL Mice and Effect of the
lpr Defect
Similarly, the onset of spontaneous anti-nRNP/Sm and anti-Su autoantibody production and the development of lupus nephritis were accelerated greatly in MRL +/+ mice by pristane treatment (A Mizutani et al, submitted for publication) (Table 5). However, a major surprise is that the Ipr and gld mutations eliminate susceptibility to pristane-induced lupus nearly completely [57]. The TNF/TNFR family members Fas and FasL signal apoptosis and act as lupus susceptibility genes [94]. In lupus-prone mice, deficiency of Fas or FasL greatly accelerates the onset and severity of lupus. Even in non-autoimmune B6 mice, Fas Jp~promotes the development of mild autoimmunity, with anti-chromatin autoantibody production. How Fas deficiency promotes autoimmunity is unclear, but it has been suggested that autoreactive B and/or T cells are deleted in the periphery by Fas-FasL signaling [95, 96]. In view of the consistent acceleration of spontaneous lupus-like disease in mice by lpr (Fas mutation) or gld (FasL mutation) the finding that these mutations have just the opposite effect on autoantibody formation in B6 mice with pristaneinduced lupus was unexpected [57]. B6/lpr and B6/gld mice are highly resistant to the induction of autoantibodies by pristane. Pristane induces IgM anti-ssDNA at 2 weeks and IgG anti-nRNP/Sm/Su/ribosomal P autoantibodies at 6 months in wild type B6 mice, but this is abrogated
95
Table 5. Effects of pristane on spontaneous autoimmunity Autoimmune strain
Manifestation
Effect of pristane
Reference
(NZB X NZW)F1
Anti-dsDNA Anti-nRNP/Sm Anti-Su Anti-RNA helicase A Glomerulonephritis
Acceleration Induction Induction Inhibition Acceleration
[ 103]
MRL +/+
Anti-dsDNA Anti-nRNP/S m Anti-Su Glomerulonephritis
Induction/Acceleration Acceleration No effect Acceleration
Unpublished data
MRL/Ipr
Anti-dsDNA Anti-nRNP/Sm Anti-Su Glomerulonephritis
No effect No effect No effect Inhibition
Unpublished data
B6/lpr
Anti-chromatin
No effect
[57]
CBA/N
Anti-RNA helicase A
Inhibition
[ 101 ]
in lpr or gld mice, suggesting that intact Fas signaling is necessary for autoantibody induction. Pristane also does not enhance IgG anti-chromatin antibody production in B6/lpr or B6/gld mice, suggesting that it does not influence spontaneous autoantibody production in Fas deficient mice. Similarly, although autoantibody production and nephritis in MRL +/+ mice are accelerated by pristane treatment, MRL lpr/lpr mice are completely refractory and the onset of renal disease is delayed by pristane treatment. This paradoxical effect of Fas deficiency might be explained in several ways. Cells undergoing Fas-mediated cell death may provide a source of antigens driving autoantibody formation in pristaneinduced lupus, consistent with suggestions that the abnormal clearance of apoptotic material induces autoantibody formation [97, 98]. Alternatively, pristane might cause Fas-mediated cell death of a population of cells that normally prevents autoimmunity. Another possibility is that pristane and Fas stimulate autoimmunity through mutually antagonistic pathways and that pre-existing Fas or FasL deficiency precludes the induction of autoimmunity by pristane. Recent studies in our laboratory raise the possibility that pristane exposure may promote the maturation of myeloid DCs, an important producer of IL-12. It has been proposed that whereas immature DCs tolerize autoreactive T cells, mature DCs
96
are activators [30, 99]. Since DCs capture apoptotic cells via interactions with surface receptors such as the integrin avl] 5 [99, 100], perhaps the uptake of apoptotic cells by a subset of pristane-activated DCs could help trigger autoimmunity. 6.3. CBA/N (x/d) Mice Although generally considered to be an "immunocompromised" strain, CBA/N mice, which have a genetic defect in the Bruton's tyrosine kinase (btk) gene, have recently be shown to produce autoantibodies against RNA helicase A (RHA) [ 101 ]. AntiRHA autoantibodies are associated with spontaneous lupus in humans [ 102] and mice [ 103]. Pristane treatment antagonizes the spontaneous production of anti-RHA autoantibodies in both CBA/N and NZB/W mice (Table 6) [101,103]. In contrast to the striking predominance of IgG2a class anti-nRNP/ Sm autoantibodies in pristane treated mice and in MRL mice with spontaneous lupus, IgG1 antiRHA autoantibodies are produced at high levels. We hypothesize that by enhancing the production of IFNy and IFNr pristane may inhibit the production of IgG1 anti-RHA antibodies. However, it remains to be determined why anti-nRNP/Sm is so strongly skewed toward IgG2a whereas anti-RHA responses are skewed more toward IgG1.
7. HOW DO HYDROCARBONS CAUSE AUTOANTIBODY PRODUCTION?
Just as the mechanisms responsible for adjuvanticity remain incompletely understood, we do not know precisely why certain hydrocarbons promote autoimmunity while others do not. Interestingly, there is a rough correlation between the two phenomena: hydrocarbons that are good adjuvants tend to promote lupus whereas those that are weak adjuvants, such as medicinal mineral oils, do not [6]. Lupus-inducing hydrocarbons tend to be more potent than inactive hydrocarbons at stimulating early IL-12 production [6], consistent with its role in linking innate with adaptive immunity. However, there are many other unanswered questions that are only now being addressed. 7.1. Is There a Receptor for Pristane?
Hydrocarbons could bind to a specific receptor or receptors that transmit a "danger signal" [32] analogous to the binding of LPS to TLR4 or glycolipids to CD1 [104]. The fact that CDld deficient mice remain sensitive to pristane-induced lupus argues that CD1 is not a receptor for pristane or other hydrocarbons [56]. The role of Toll-like receptors in the recognition of hydrocarbons is not known and under investigation. An alternative possibility is that pristane becomes incorporated into the plasma membrane [105-107], altering inflammatory signaling pathways or that pristane gains access to the inside of the cell by dissolving in the plasma membrane followed either by binding to an intracellular receptor or modification of a subset of intracellular proteins, as has been proposed for urushiol, the inflammatory oil that causes poison ivy [108, 109]. 7.2. Is the Site of Pristane Exposure Important?
Thus far, studies of hydrocarbon-induced lupus all have employed intraperitoneal injection of the oil. There is little information about whether intraperitoneal injection is necessary or if other sites work equally well. Experiments to address this question are underway in our laboratory. There are significant differences in secondary lymphoid tissues located at different sites, such as lymph nodes, Peyer's patches, or the peritoneal cavity. The peritoneal
cavity of mice, in particular, is highly enriched in the B-I subset of B-lymphocytes [110]. However, we have shown that these cells are rapidly depleted upon injection of pristane [111]. We do not know at present whether the B-1 cells undergo apoptosis or become sequestered at some other site, such as in "granulomas" forming in response to intraperitoneal pristane injection. 7.3. What is the Role of "Oil Granulomas" Induced by Pristane?
Potter et al were the first to record the development of inflammatory nodules in the peritoneal cavities of mice following the injection of mineral oil or pristane [112-114]. They termed these structures "granulomas" in view of the fact that they contained inflammatory cells and numerous oil droplets. These later become organized into polypoid structures. Plasmacytomas develop within the granulomas of certain strains of mice, notably BALB/c, starting -10 months after pristane injection. The evolution of granulomas may begin with so-called "milky spots" [115]. More recently, we have found that the term "granuloma" is not entirely accurate. These structures closely resemble ectopic lymphoid tissue (tertiary lymphoid tissue, lymphoid neogenesis), since they contain collections of B and T lymphocytes, dendritic cells, and macrophages and have a variety of other features consistent with lymphoid tissue (D Nacionales et al, manuscript in preparation). The role of this ectopic lymphoid tissue in the pathogenesis of autoantibodies and lupus-like disease is incompletely defined. However, ectopic lymphoid tissue in a variety of other situations is associated with autoantibody-mediated autoimmune diseases [116]. The thyroid gland in Hashimoto's thyroiditis, the thymus of some patients with myasthenia gravis, CNS lesions in multiple sclerosis, the salivary glands in Sj6gren's syndrome, and the synovium in rheumatoid arthritis all have morphological and functional features of lymphoid neogenesis, including the presence of high endothelial venules, DCs and follicular dendritic cells, antigen-driven clonal proliferation of B-cells, and lymphoid tbllicles with clonally expanded lymphocytes [ 117-120]. Ectopic lymphoid tissue may provide a focal milieu where interactions between lymphocytes and APCs occur in the partial absence of normal censoring mecha-
97
nisms. 7.4. What is the Role of Microbial Exposure as a Co-factor?
Autoantibody production induced by pristane is attenuated in mice housed under specific pathogen free conditions in comparison with mice housed under standard conditions [77]. In contrast, autoimmune diabetes is milder in NOD mice housed under standard conditions than in SPF mice [121]. These and other observations suggest that the microbial environment can modulate autoimmune disease. Studies of pristane-induced lupus in SPF mouse raised the possibility that pristane might increase the exposure to microbial substances such as LPS, which stimulate innate immunity. For instance, inflammation of the bowel has been shown to increase bowel permeability to bacteria [122]. To address this question, we recently completed studies of pristane-induced lupus in germfree mice (A Mizutani et al, submitted for publication). Germfree mice were susceptible to pristane-induced lupus, and made autoantibodies at frequencies comparable to those in SPF mice. Thus, it seems likely that pristane has other actions besides just increasing microbial stimulation. We have found that the peritoneal exudate cells from pristane treated mice are hyper-responsive to stimulation by LPS, suggesting that pristane may act synergistically with certain TLR ligands. 7.5. What is the Role of Cytokines in the Pathogenesis of Pristane Induced Lupus?
As described above, IL-6, IL-12, and IFNy appear to be directly involved in the pathogenesis of pristane-induced lupus. Anti-nRNP,-Sm, and-Su, and -dsDNA autoantibody production is greatly reduced in IL-12 or IFNy deficient mice, and glomerulonephritis is much milder. In contrast, whereas antidsDNA antibody production is nearly eliminated in IL-6 deficient mice, the prevalence of anti-nRNE Sm, and Su autoantibodies is not altered substantially. More recently, we have shown that "granulomas" induced by pristane have high type I IFN activity in comparison with those induced by medicinal mineral oil (DC Nacionales et al, unpublished data), suggesting that type I IFNs are involved in the
98
pathogenesis of pristane induced lupus. IL-6, IL-12, IFNT, and I F N ~ have important effects on the generation of autoreactive T and B cells that may help explain their role in the pathogenesis of hydrocarbon-induced lupus in mice (Fig. 2). DCs, which undergo maturation in response to IFN 7, IFN~, or LPS, regulate T cell activation [25, 30]. Immature dendritic cells (iDC) are tolerogenic whereas mature DCs are sfimulatory. IFNot promotes the differentiation of monocytes into DC-like cells, which can capture antigens from dying cells and present them to CD4+ T cells and also enhances both DC and T cell survival [38, 44, 46, 123]. Mature DCs produce IL-12, which along with IFN 7 polarizes the CD4+ T cells toward the TH1 phenotype. B cell activation and isotype switching are influenced by signals delivered by CD4+ T cells, including CD40L and cytokines. Switching to IgG2a, the predominant isotype of autoantibodies induced by pristane, is enhanced by IFNy and IFNc~ [50, 74]. The maturation of B cells into plasmablasts and plasma cells is driven by IFN~ and IL-6 [49]. Thus, the major cytokines implicated in pristane-induced lupus may promote autoimmunity at multiple levels, including DC maturation, autoreactive T cell activation and survival, and activation, isotype switching, and maturation of B cells. 7.6. Are the Cytokine Abnormalities in PristaneInduced Lupus Relevant to Human SLE?
The cytokines involved in pristane-induced lupus, IL-6, IFN~I3, and IFN 7, have been implicated in the pathogenesis of human SLE. For instance, IL6 levels have been reported to be elevated in SLE [124] and both IL-6 and IFNy are thought to play a role in the pathogenesis of lupus nephritis in humans [ 125, 126]. Both Castleman's disease [ 127] and atrial myxoma [59] are associated with the production of high levels of IL-6 as well as the production of lupus autoantibodies and the development of autoimmune disease. Therapeutic use of IFNy has been associated with the development of lupuslike disease [ 128]. The same is true of IFNc~, which when used to treat hepatitis C infection, malignant carcinoid syndrome, or chronic myelogenous leukemia is sometimes associated with autoimmune phenomena, including sarcoidosis [129], autoimmune
Figure 2. Cytokine effects on autoreactive T and B cells. Cytokines implicated in the pathogenesis of hydrocarbon-induced lupus and their possible mechanisms of action. Dendritic cell (DC) maturation is promoted by IFNtx and IFN~,, as well as by TLR ligands, such as lipopolysaccharide (LPS). Whereas immature dendritic cells (iDC) promote T cell tolerance, mature DCs promote T cell activation. IL-12 and IFN~,drive the differentiation of type I (Tal) T cells, and IFNtx permits activated T cells to survive. B cell development also is cytokine dependent. IFN~,produced by TH1 cells promotes isotype switching to IgG1, the predominant isotype of autoantibodies produced in hydrocarbon-induced lupus. IFNct and IL-6 stimulate B cell differentiation into plasmablasts and plasma cells, respectively, and type I IFNs are necessary for the generation of memory B cells.
thyroiditis, and autoimmune hepatitis [130]. The induction of antinuclear antibodies and anti-dsDNA antibodies as well as overt lupus has been reported, as well [131-133]. Moreover, the serum level of IFNct correlates with anti-dsDNA antibody levels and disease activity in SLE [134-136]. Finally, recent studies suggest the existence of a type I IFN gene expression "signature" that is associated with active SLE [137, 138]. Together, these data suggest that the cytokine abnormalities identified in pristane-induced lupus are relevant to human SLE. A major challenge for the future will be to understand how non-specific inflammation and cytokine production resulting from hydrocarbon exposure leads to the production of a highly restricted subset of autoantibodies that is pathognomonic of SLE. A second challenge will be to determine whether environmental triggers of lupus, such as viral infections or exposure to chemicals, promote disease by stimulating increased production of IL-6, IFN~,, and type I IFNs.
ACKNOWLEDGEMENTS We gratefully acknowledge the technical assistance of Ms. Minna Honkanen-Scott. This work was supported by research grants R01-AR44731 and R01-AI44074 from the United States Public Health Service, by training grant T32-AR07603, and by research support from Lupus Link.
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129. Fiorani C, Sacchi S, Bonacorsi G, Cosenza M. Systemic sarcoidosis associated with interferon-alpha treatment for chronic myelogenous leukemia. Haematologica 2000;85(9): 1006-1007. 130. Dumoulin FL, Leifeld L, Sauerbruch T, Spengler U. Autoimmunity induced by interferon-alpha therapy for chronic viral hepatitis. B iomed Pharmacother 1999;53(5-6):242-254. 131. Ioannou Y, Isenberg DA. Current evidence for the induction of autoimmune rheumatic manifestations by cytokine therapy. Arthritis Rheum 2000;43:14311442. 132. Kalkner KM, Ronnblom L, Karlsson Parra AK, Bengtsson M, Olsson Y, Oberg K. Antibodies against doublestranded DNA and development of polymyositis during treatment with interferon. Q J Med 1998;91(6): 393-399. 133. Ronnblom LE, Aim GV, Oberg KE. Autoimmunity after alpha-interferon therapy for malignant carcinoid tumors (see comments). Ann Intern Med 1991;115(3): 178-183. 134. Kim T, Kanayama Y, Negoro N, Okamura M, Takeda T, Inoue T. Serum levels of interferons in patients with systemic lupus erythematosus. Clin Exp Immunol 1987 ;70(3 ):562-569. 135. Hooks JJ, Moutsopoulos HM, Geis SA, Stahl NI, Decker JL, Notkins AL. Immune interferon in the circulation of patients with autoimmune disease. N Engl J Med 1979;301 (1):5-8. 136. Preble OT, Black RJ, Friedman RM, Klippel JH, Vilcek J. Systemic lupus erythematosus: presence in human serum of an unusual acid-labile leukocyte interferon. Science (Wash, DC) 1982;216(4544):429--431. 137. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ et al. Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc Natl Acad Sci USA 2003; 100(5):2610-2615. 138. Bennett L, Palucka AK, Arce E, Cantrell V, Borvak J, Banchereau J e t al. Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J Exp Med 2003;197(6):711-723.
9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Vaccination and Autoimmunity Anabel Aron-Maor ~and Yehuda Shoenfeld ~,2
;Department of Medicine 'B' & Center for Autoimmune Diseases, Chaim Sheba Medical Center (affiliated to Tel-Aviv University), Tel-Hashomer 52621, Israel; 2Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases, Tel-Aviv University
Starting in the third decade of the 20th century vaccination against some of the most common infectious diseases (measles, mumps, diphtheria, rubella, polio) was introduced, reducing the morbidity of these diseases by close to 100% by the end of the century [ 1]. Thus, the benefits of immunization are irrefutable. However, there have been over the last 15 years or so several reports of adverse autoimmune reactions to various vaccines. Mostly the connection between the vaccination and the autoimmune reaction was temporal and not causal. This nevertheless did not prevent such concern (to put it mildly) among the medical and general community that certain childhood vaccinations all but stopped being administered to large populations (such as the MMR vaccine in Britain in the 80's). There are two kinds of vaccination: 9 Active vaccination: when a live, generally attenuated infectious agent (microbe or virus) is used, or an inactivated infectious agent (or constituents thereof), or products obtained by genetic recombination. Active vaccination may also be achieved when injecting a toxoid. 9 Passive vaccination: usually provides temporary immunity and consists of immune globulin preparations or antitoxins. The vaccines usually contain not just the specific antigens but also adjutants (such as aluminum salts or carder proteins). These are introduced in order to potentiate, or boost, the immune response to some antigens. Their action is non-specific. The purpose of vaccination is to induce immunization a reaction of the immune system that will provide the organism with protection against -
disease. As any other medical treatment, vaccines also have side effects, from local reactions, to systemic side effects such as flu-like or hypersensitivity reactions. For more than 15 years reports have been accumulating of autoimmune reactions to various vaccines. Mostly case reports but also some case series of patients who developed autoimmune signs and syndromes. The aim of this chapter is to surmnarize the autoimmune manifestations that have been reported in connection with various vaccines, as well as the possible explanations to these occurrences. As already mentioned, it is imperative to emphasize that so far no causal connection has been demonstrated between any one vaccine and an autoimmune syndrome (even though strong evidence exists to suggest such a connection in regard to reactive arthritis following rubella vaccine [2, 3]). All reported cases of autoimmune manifestations have been only temporally related to the respective vaccines.
1. VACCINES AND ARTHRITIS The occurrence of arthritis has been described following administration of several vaccines (Table 1) and can be divided into isolated or reactive arthritis (poly or monoarticular) and arthritis as a symptom of a systemic autoimmune disease (such as Systemic Lupus Erythematosus (SLE) or rheumatoid arthritis (RA)). Some vaccines have been implicated more often than others.
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Table 1. Vaccines associated with post-vaccination reactive arthritis and RA Disease
Vaccine
Reactive arthritis
HBV
Rheumatoid arthritis
SLE
Rubella Mumps and measles Influenza DPT Typhoid HBV
Tetanus HBV Polyvaccine: mumps, measles, tetanus, meningococcal, hepatitis A, polio
1.1. Arthritis and Hepatitis B Vaccine Over the past 10-15 years more than 30 cases of arthritis following vaccination with the HBV recombinant vaccine have been reported. Some were cases of isolated inflammation of the joints, others turned out to be harbingers of frank RA. In 1990 two cases of arthritis were reported shortly after the patients had received HBV vaccines [4, 5]. One of the patients developed poly-arthritis and also erythema nodosum and the other patient reactive arthritis only. The symptoms receded in both patients and there was no evidence of a systemic autoimmune illness later on. During the next nine years more reports were published of people developing arthritis after HBV vaccination [6-12]. Some of the patients described were found to have high titers of rheumatoid factor (RF) in their sera without fulfilling other ACR criteria for RA [7]. Others were carders of genetic markers predisposing to autoimmune disease [12]. In 1998 eleven patients
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No. of cases
1 4 3 1 >10 >100 1 1 2 2 15 1 6 5 13 7 5
Additional symptoms Erythema nodosum Migratory arthritis, urticaria, oedema of the glottis Hypercalcemia, lytic bone lesions Myalgia Vasculitis Adult-onset Still's disease Reactive arthritis alone
Cutaneous, renal, hematological Cutaneous, renal, hematological
were reported who developed arthritis after receiving HBV recombinant vaccine [12]. Ten of these patients fulfilled the ACR criteria for RA, nine of those required disease modifying drugs. Five of the subjects were carriers of the HLA-DR4 haplotype. Nine of the eleven patients genotyped for HLA-DR and DQ expressed the RA shared motif in their HLA class II genes. The findings from this report suggested that HBV recombinant vaccine may trigger RA in genetically prone individuals. An additional case report supports to a certain extent this hypothesis [9]. This is the case of a 44 year old man who had had myasthenia gravis 20 years earlier and had developed arthritis shortly after administration of HBV vaccine. Overall the occurrence of arthritis, and especially RA after anti-HBV immunization is a rare one [13]. Moreover, in studies that examined the response of known RA patients to HBV vaccination it was shown that the administration of the vaccine was not associated with an appreciable deterioration of any laboratory or clinical parameters of
the disease [ 14], and that 68% of patients produced antibodies. Older age and higher scores of daytime pain were associated with a lower rate of antibody production after vaccination. To summarize there are three explanations suggested [15] to the apparent association between immunization and arthritis: - it represents the chance occurrence of two common phenomena. - immunization precipitates a specific form of arthritis that is distinct from RA and that is usually self-limited (post-immunization arthritis). - immunization is one of the factors that can trigger the manifestation of R A - as can infection.
1.2. Arthritis and Rubella Vaccine Joint manifestations have been documented often in connection with rubella vaccine, as well as with the wild virus itself [16, 17]. In 1991 the Institute of Medicine released a report in JAMA examining adverse effects of the DPT (diphtheria-pertussis-tetanus) vaccine and the rubella vaccine (strain 27/3). The report concluded that the evidence suggests a causal relation between rubella vaccine and acute arthritis in adult women [18, 19]. No animal studies were available to support or disprove this conclusion. In a large study [20] that included 2658 immunized and 2359 non-immunized children, the incidence of joint manifestations was assessed six weeks after immunization with the MMR (measlesmumps-rubella) vaccine. There was an increased risk of arthralgia or arthritis in the immunized children six weeks after immunization. The risk for frank arthritis was less than after wild rubella infection. As with the HBV vaccine a genetic predisposition to develop autoimmune disease may play a role in the manifestation of acute reactive arthritis in proximity to the administration of the rubella vaccine. In 1998 [21] a group of scientists examined the frequency of HLA-DR in relation to the incidence of acute joint manifestations in 283 white women who had received rubella vaccination post-partum. The conclusion, based on statistical analysis (after adjustment for age, treatment and time post-partum) was that the risk for developing arthritis was 1.9 times greater after rubella vaccination that after placebo. The risk for arthropathy was also influenced by DR interactions - odds to develop post-vaccination arthropathies were 8 times greater in individu-
als with both DR1 and DR4, and 7.6 times greater with both DR4 and DR6 present. An additional risk factor to the development of post-vaccine arthropathy [22] apparently is the titer of pre-vaccine rubella antibodies: the lower the titer the higher the risk of post-vaccination arthropathy. In conclusion, there seems to be a causal relation between the rubella vaccine and post-vaccination arthropathies (arthralgia and arthritis) with an increased risk for individuals with genetic predisposition (HLA-DR4) and for individuals with low pre-vaccination titers of anti-rubella antibodies.
1.3. Arthritis and BCG Vaccine (see separate chapter) Oligo- and poly-articular arthritis has been reported in approximately 3% of patients treated with intravesicular BCG (for bladder carcinoma) 1-3 months after start of treatment [23]. The arthritis is sterile and HLA-B27 has been demonstrated in several of these patients suggesting a resemblance to reactive arthritis [24].
2. VACCINES AND SLE Systemic lupus erythematosus (SLE) is an autoimmune illness involving multiple organs. Its etiology is believed to be multifactorial since presentation (as well as flare-up) of the disease has been observed after exposure to infectious agents(see chapter), ultra-violet light, drugs and various chemicals. Genetic factors also, inevitably, play a significant role in determining who will develop SLE and when. Viral infections have specifically been causally associated with SLE [25-31] and there are documented cases of SLE presenting after vaccinations.
2.1. SLE and HBV Vaccine The HBV vaccine has been relatively frequently associated with manifestations of SLE in both sexes and all age groups [32-37]. It is interesting to note familial "clustering" of cases of post-vaccination lupus. Such as the case of a 24 year old woman and her 7 year old daughter who both developed autoimmune disease (the mother SLE and the daughter
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ITP-idiopathic thrombocytopenic purpura) 4 and 10 months (respectively) after vaccination against HBV [33]. The issue of safety of HBV immunization of SLE patients has also been addressed so far only in retrospective studies. The safety of such immunization has yet to be definitely determined and prospective studies are needed. So far it seems that most patients mount an adequate immune response to vaccination, even though it may be quantitatively and qualitatively less than in healthy controls [37]. It is recommended that individuals at risk of exposure to hepatitis B, be immunized.
3. VACCINATION AND N E U R O L O G I C A L AUTOIMMUNE MANIFESTATIONS
Table 2. Vaccines associated with GBS Tetanus toxoid [48, 49] Bacille Calmette-Guerin [48] Rabies [48] Smallpox [48] Mumps [48] Rubella [48] Hepatitis B [48] Diphtheria [49] Poliovirus [48, 49]
Three major neurological autoimmune manifestations have been addressed in conjunction with vaccination: the Guillain- Barre syndrome (GBS), multiple sclerosis and autism.
lion adults received influenza ("swine-flu") virus vaccine [48] the incidence of GBS increased by a factor of four to eight. Additional vaccines have been associated with the occurrence of GBS and Table 2 summarizes the vaccines that have been related with GBS.
3.1. Vaccination and GBS
3.2. Vaccination and Multiple Sclerosis
GBS is a transient neurological disorder characterized by areflexic motor paralysis with mild sensory disturbances. In the patients' cerebrospinal fluid there is an acellular rise of total protein associated with inflammatory demyelination of the peripheral nerves [38]. The exact etiology of the syndrome remains unclear, however there is increasing evidence to suggest an autoimmune etiology [39]. Autoantibodies to various myelin-associated glycoconjugates are described in GBS patients [40] and prior viral infections are often associated with the onset of GBS [41-45]. Approximately 30% of GBS cases are preceded by Campylobacterjejuni infections [46] as detected by serologic tests. It can be concluded that GBS is probably both a humoral and a cellular autoimmune disease induced by infection with multiple microorganisms. The presence of microbe-specific antibodies and T-cells with cross-reactivity to various nerve-sheath components initiates inflammatory demyelination and shedding of peripheral nerve auto-antigens [47]. Presumably by a similar mechanism vaccines can induce an autoimmune reaction. In the autumn of 1976 after a government-sponsored mass-inoculation program in which 45 mil-
Multiple sclerosis (MS) is a disease characterized by central nervous system demyelination and progressive paralysis. It is considered an autoimmune disease of unknown etiology in which the pathologic process is caused by a cell-mediated autoimmune process directed against nerve-sheath myelin. Autoantibodies specific for the central nervous system/oligodendrocyte glycoprotein were identified [50]. These autoantibodies were specifically bound to disintegrating myelin around axons in lesions of acute MS. MS has been connected to hepatitis B vaccine in one of the largest and most heated debates and law suites in France and the US [51]. More than 600 cases of illnesses, many with MS-like symptoms have been reported in France among people who have received recombinant HBV vaccine. The temporal association between MS and HBV vaccination has been reported on few occasions [52-53]: neurological symptoms and signs as well as magnetic resonance imaging documenting CNS demyelinization have been documented days to weeks after BV vaccination. On the other hand, a French government sponsored study in 1997 revealed that vaccinated individuals were less likely to have MS [52].
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The measles vaccine also has been investigated in conjunction with MS. There are several lines of evidence that support the possibility that MS may be an age dependent host-response to measles. MS patients have higher titers of measles antibodies than do healthy controls and paramyxovirus inclusions have been found in brain cells of MS patients [54]. A hypothesis has been raised that measles might be one cause of MS, based on the finding that in areas where measles occurs at a later age in the majority of population (such as in Scandinavia) the incidence of MS is higher. Whereas in areas where measles occurs at a generally younger age the incidence of MS is significantly lower [54]. However, even though the incidence of measles dropped precipitously after measles vaccination began in the US in 1963, no effect was seen approximately 30 years later on the incidence of MS [55].
3.3. Vaccination and Autism The behavioural syndrome of autism in children is considered to be a neuro-developmental disorder identified by neuro-psychiatric manifestations that include few or no imaginative and language skills, repetitive rocking and self-injurious behavior, and abnormal responses to sensations, people, events and objects. The cause of the syndrome is not known but the etiology may be multifactorial, including environmental, genetic, immunological and as yet undiscovered biochemical and neuropathological factors. An immune hypothesis involving autoimmunity as one possible pathogenetic mechanism in autism has been suggested [56] based on a family study of infantile autism in the presence of autoimmune disease. The vaccine most commonly associated with autism was the measles vaccine. The hypothesis suggested was that were an immunological assault to occur prenatally or post-nataly (during infancy or early childhood) it could possibly result in poor myelination or abnormal function of the axon myelin. The hypothesis was supported by an association found in autistic children, between anti-viral and brain autoantibodies [57]. Slightly higher titers of measles IgG were found in autistic children compared to normal controis and the higher the measles antibodies' titer the greater the likelihood of brain autoantibodies [58]. An additional theory has been suggested connect-
ing autism, the measles-mumps-rubella vaccine and a new pathological entity - a form of chronic inflammatory bowel syndrome - lymphoid nodular hyperplasia (LNH) [59]. It is a different entity from Crohn's disease or ulcerative colitis - this is a reactive swelling of the lymphoid (immune) tissue of the ileal and colonic lining. Symptoms include abdominal pain and change of bowel habits and it may transient or persistent. Autistic children with this finding were also found to be immune deficient [60] lacking in one or more lymphocyte subsets or in immunoglobulin IgG subclasses - findings consistent with an acquired immunodeficiency. The measles component of the MMR vaccine has been implicated in the etiology of this syndrome. The connection between the gastrointestinal findings and autism was hypothetically explained by the possibility that the primary site of damage in autism is outside the brain causing an arrest in the normal development of the brain and its function. Other studies, however, failed to demonstrate the presence of measles virus in lesions of inflamed bowel from these children. So far data is conflicting and there has been no consistent scientific support to the alleged connection between the measles vaccine (or wild virus) and autism [61 ]. An epidemiological study reassessing the association between measles and autism was published in 1999 [62]. It transpired from this study that since 1979 there was a steady increase in the number of cases of autistic children by year of birth with no sudden increase after the introduction of the MMR vaccine. Also there was no difference in age of diagnosis between children vaccinated before or after 18 months of age and those who were never vaccinated. An increased potential risk for neurodevelopmental disorders might related to increased doses of thimerosal- an organic mercury compound that is metabolized to ethyl-mercury and thiosalycylate and that has been used as a preservative in some vaccines since the 1930's [63]. Other, systemic, autoimmune phenomena have been sometimes described in relation to several vaccines. Especially renal involvement has been documented on several occasions [64-66]. The renal disorder developed after receiving a variety of vaccines, among which polio vaccine [67], smallpox, tetanus toxoid and influenza [68]. Rarely vasculitis has been described in conjunction with vaccination
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with HBV recombinant vaccine [69].
4. VACCINATION OF PATIENTS W I T H KNOWN A U T O I M M U N E DISEASE The question was addressed in a number of studies over the years and several vaccines have been examined in this context. The swine-flu influenza vaccination was well tolerated by patients with MS [70--71] without any influence on the course of the disease (more exacerbations). Likewise, the widely used influenza vaccine has been well tolerated by MS patients [72] with similar rates of systemic reactions as the general population. Influenza vaccine did not cause any worsening of the disease in SLE patients either [73-74]. Response to immunization however (as measured by antibody titers), was lower in patients with lupus than in healthy controls [74-75]. A similar kind of reduced response to vaccination was observed in RA patients as well [76]. Nevertheless the vaccine does not have any noxious effects on RA Patients and does not cause an increased frequency of flares of the disease [76-77]. Since the danger to these patients with systemic autoimmune diseases is great in case of influenza infection they should receive the vaccine. The same is true for other vaccines where the infection itself poses a real and significant danger to these patients.
5. POSSIBLE M E C H A N I S M S OF VACCINERELATED A U T O I M M U N I T Y Many common infections can induce a transient rise in autoantibody production. A similar rise in autoantibody production has been observed after various vaccinations. Such autoantibodies usually resolve within a period of two months [78] but can persist in rare cases. Several studies indicate that stimulation of autoantibody production has become one of the criteria of establishing the safety of vaccines. It is to be remembered however that although autoantibodies are a characteristic of autoimmune disease it is often unclear whether they are an epiphenomena or represent the causal agents of the illness. The human immune system is highly complex, it displays both specificity and memory and
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is designed to provide protection against almost all infections. The drawback of such a complex and broadly responding immune system is that in responding to infection, the immune system of a few individuals will "turn against" the self and cause autoimmunity [79]. An infection (or vaccine for that matter) can induce autoimmunity via two mechanisms: antigen-specific or antigen-non-specific. An autoimmune condition will arise, however, only if the individual is genetically predisposed to that condition. A common explanation of how an infectious agent can cause autoimmunity via an antigen-specific mechanism is the molecular mimicry theory. Antigenic determinants of the infectious can thus be recognized by the host immune system as being similar to antigenic determinants of the host itself [79]. The situation is more complex for molecular mimicry that involves T lymphocytes. These cells recognize their antigen as short peptides bound to MHC molecules. To serve as a molecular mimic an infectious agent's antigen must copy the shape of a self-antigentic epitope bound to the appropriate MHC molecule. Experimental findings have shown that a single T-cell receptor can recognize a broad range of sequences [80-81 ] including peptides with totally different sequences. It has been calculated that each individual T-cell should be able to recognize more than one million distinct peptide epitopes [81]. Thus, the probability of T-cell cross-reactivity is so high that one wonders why all infection do not induce severe autoimmune disease?! Another mechanism whereby microorganisms (or vaccines) may cause autoimmunity involves bystander activation. This is an antigen-non-specific mechanism. In this instance the infecting agent causes release of previously sequestered self-antigens or stimulates the innate immune response, resulting in activation of self-antigen-expressing antigen presenting cells. Evidence for this mechanism has come from several studies on transgenic mice [82-83]. Autoimmune disease is most likely to be induced in the infected organ. For example mice that harbour high numbers of islet antigen-specific T cells developed diabetes only when infected with an islet-cell tropic virus [84]. The effect of this virus has been reproduced by an islet-cell damaging drug but not by non-specific T-cell activation [85]. These findings imply that viruses can precipitate disease by damaging tissue and causing the release and presentation of previ-
ously sequestered self-antigens. So far we can conclude that there is a high probability for infectiousagents' antigen to cross-react with self-antigens and also that autoimmune disorders can be triggered by the innate immune response to microorganisms. The fact that autoimmune disease does not occur more frequently is probably due to a "fail-safe" mechanism that the immune system has evolved to prevent extensive tissue damage in response to infection. The immune system is controlled by homeostatic mechanisms [86]. Lymphocytes have to compete with each other for antigen and growth factors. Furthrmore, T cell reaction to antigen is limited by activation-induced cell-death [87]. These mechanisms are designed to keep the lymphocyte population at an optimal predetermined level thus limiting the expansion of self-reactive lymphocytes [86]. In a lymphopenic setting, self-reactive lymphocytes undergo homeostatic proliferation and are released from peripheral tolerance' thus causing autoimmune disease [88-91]. The immune system is equipped with a wide variety of lymphocytes bearing receptors with varying affinity to antigen [79]. The immune response to a given antigen selects only a strictly limited set of these lymphocytes. The selection depends on several mechanisms" a) the role of antigen processing and MHC-peptide complex formation; b) selective binding of antigenic epitopes to specific MHC molecules; and c) selective depletion of specific lymphocytes by overstimulation (clonal exhaustion or deletion) [92]. Also, the fact that the threshold for activation of T cells is close to the threshold for activation-induced cell death results in a highly focused reaction of the immune system to any antigen [93, 94]. These mechanisms limit the immune response to antigen and prevent activation of cells beating high-affinity receptors. Since high-affinity receptors are more likely to be crossreactive it is likely this mechanism has evolved to prevent collateral tissue damage, which occurs during the immune response to infection, and to limit the likelihood of self-reactive lymphocyte activation during infection [79]. An additional control mechanism is imposed on the immune system by "regulatory T cells". The best characterized subset pf T cells is the CD4+CD25+ cell [95, 96]. These cells arise in the thymus where they are positively selected by recognition of self antigen [97]. Unlike the majority of the T cell population which leave the
thymus as naive lymphocytes, the CD25+ cells emigrate the thymus but do not proliferate in response to antigen. They are capable of suppressing the response to self antigens. These cells were first described by Sakaguchi et al [98], who noted that thymectomy of young mice prevented their generation and resulted in widespread autoimmune disease in adult animals. Additional examples exist to the role and importance of these cells [99]. The physiological role of T-regulatory type I cells is probably to moderate the immune response to infection and thereby limit the collateral damage that results from the immune response to an infectious agent [100]. These combined homeostatic and regulatory mechanisms have evolved to ensure that the immune response is focused and controlled, and they prevent the individual from developing autoimmune disease during the course of infection [79]. These mechanisms also apply to the host response to vaccination. It is probable that a killed vaccine would be less likely to activate the innate response to infection and to cause tissue disruption, that a live-attenuated one, thereby reducing the risk of autoimmune disease. Nevertheless the degree of activation achieved by an attenuated organism will be much less than that induced by the wild strain. Every new vaccine should therefore be assessed on a case-by-case basis giving extreme consideration to the potential benefit, in terms of public health provision.
6. VACCINATION AND DIABETES Over the past few decades there has been a steady increase in the incidence of type I diabetes in most countries in the world. It is not surprising therefore that childhood vaccinations have been deemed suspect as a potential trigger for this disease. This possibility has been assessed in several epidemiological studies. Results of a case-control study done in Sweden in the 1980's has shown no significant influence of several vaccines (anti tuberculosis, smallpox, tetanus, pertussis and rubella) on the incidence of type I diabetes [101 ]. One vaccine in particular has been suggested to be related with an increased risk for diabetes - the Haemophilus influenza type b (Hib) vaccine [102, 103] especially if given at age two months or older. This theory however, was not confirmed in a 10-year follow-
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up study that included more than 100,000 Finnish children [104]. Results of this study showed no increased risk of diabetes when children who had received four doses of vaccine at ages 3, 4, 6 and 14-18 months were compared with those who had received only one dose at age 2 years. Additionally, findings of a study undertaken in four large health-maintenance organizations in the USA did not suggest an association between administration of routine childhood vaccines and an increased risk for diabetes, irrespective of the timing of Hib or hepatitis B vaccination [105]. Therefore, at the present time there is no conclusive evidence of any great effect of childhood vaccines on the occurrence of diabetes type I.
7. CONCLUSION Vaccination has been perhaps the greatest medical discovery of the 20th century with the greatest impact on public health. Thanks to this treatment some infectious diseases have been virtually eradicated since the beginning of the century. Over the last two decades there has been increasing concern about possible effects of vaccines on the occurrence of autoimmune diseases, based mainly on case reports connecting the onset of autoimmune phenomena to vaccination. In this chapter we have reviewed the main data available on vaccines and autoimmune diseases. There exist no criteria for diagnosing vaccine-related autoimmune disease. Epidemiological studies so far have not shown conclusively that there exists a causal relation between any one vaccine and any autoimmune disease. Appropriate epidemiological studies should be done before a particular autoimmune clinical condition is associated with a given vaccination. A possible increased risk for the development of autoimmune conditions has been suggested by findings in several case reports and series, where familial or genetic risk factors for autoimmune conditions has been found in many of the patients who had developed autoimmune disease (or phenomena) shortly after vaccinations. It is interesting to note that autoimmune phenomena related to vaccination occur equally in males and females, unlike "regular" autoimmune diseases which are prevalent mainly in women). Based on the above, vaccination of any person with known
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such risk factors should be carefully considered. However, the degree of vaccine-related risk should always be compared with that associated with the corresponding natural infection, either for the whole population or for a specific subgroup. It is important to mention that vaccination of patients with known autoimmune diseases (such as RA or SLE) has not caused exacerbation of their condition and that most of these patients have mounted a good antibody response to the vaccine (even though attenuated as compared to healthy controls). Criteria for the assessment of adverse effects of vaccines have been established by the World Health Organization (WHO). The four basic principles that apply to autoimmune disease are: the consistency, strength and specificity of the association between the administration of a vaccine and an adverse event, and the temporal association. 1) Consistency and strength- the findings should be the same if the vaccine is given to a different group of people, by different investigators and irrespective of the method of investigation. 2) Specificity - the association should be distinctive and the adverse event linked uniquely or specifically to the vaccine concerned. An adverse event could be caused by a vaccine adjuvant or additive, rather than by its active component. 3) Temporal relation - receipt of the vaccine should precede the earliest manifestation of the event or a clear exacerbation of a continuing condition. A clear distinction should be made between autoimmunity and autoimmune disease. Autoimmunity is a feature of the healthy immune system. Laboratory measurable signs of autoimmunity can associate with infection and occasionally with vaccination. Fortunately, the immune system has evolved sufficient fail-safe mechanisms to prevent these sign from developing into clinical autoimmune disease in the majority of instances.
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9-12. 66. Bishop WB, Carlton RF, Fanders LL. Diffuse vasculitis and death after hyperimmunization with pertussis vaccine. Report of a case. New Engl J Med 1966;274: 661-669. 67. Izumi AK, Matsunaga J. BCG vaccine-induced lupus vulgaris. Arch Dermatol 1982; 118:171-172. 68. Baxter AG, Horsfall AC, Healey D, Ozegbe P, Day S, Wiliams DJ, Cooke A. Mycobacteria precipitate an SLlike syndrome in diabetes-prone NOD mice. Immunology 1994;83:227-231. 69. Le Hello C, Cohen P. Boousser MG, Letellier P. Suspected hepatitis B vaccination-related vasculitis. J Rheumatol 1999;26:191-192. 70. Bamford CR, Sibley WA, Laguna JE Swine influenza vaccination in patients with multiple sclerosis. Arch Neurol 1978;35:242-243. 71. Kurland LT, Molgaard CA, Kurland EM, Wiederholt WC, Kirkpatrick JW. Swine flu influenza vaccine and multiple sclerosis. JAMA 1984;251:2672-2675. 72. Salvetti M, Pisani A, Bastianello S, Millefiorini E, Buttinelli C, Pozzilli C. Clinical and MRI assessment of disease activity in patients with multiple sclerosis after influenza vacciniation. J Neurol 1995;242:143-146. 73. Brodman R, Gilfillan R, Glass D, Schur PH. Influenzal vaccine response in systemic lupus erythematosus. Ann Intern Med 1976;88:735-740. 74. Williams GW, Steinberg AD, Reinertsen JL, Klassen LW, Decker JL, Dolin R. Influenza immunization in systemic lupus erythematosus. A double blind trial. Ann Intern Med 1978;88:729-734. 75. Ristow SC, Douglas RG, Condemi JJ. Influenza vaccination of patients with systemic lupus erythematosus. Ann Intern Med 1978;88:786-789. 76. Cimmino MA, Seriolo B, Accardo S. Influenza vaccination in rheumatoid arthritis. J Rheumatol 1995;22: 1802. 77. Heron A, Dettleff G, Hixon B, Brandwin L, Ortbals D, Hornick R, Hahn B. Influenza vaccination in patients with rheumatic diseases. Safety and efficacy. JAMA 1979;242:53-56. 78. Borchers AT, Keen CL, Shoenfeld Y, Silva J, Gershwin ME. Vaccines, viruses and voodoo. J Invest Allergol Clin Immuno12002;12:155-168. 79. Wraith DC, Goldman M, Lambert PH. Vaccination and autoimmune disease: what is the evidence? Lancet (published online on June 3, 2003 on http: //image.thelancet.corn/extras/02art9340web.pdf). 80. Hemmer B, Jacobsen M, Somner M. Degeneracy in T-cell antigen recognition: implications for the pathogenesis of autoimmune diseases. J Neuroimmunol 2000; 107:148-153. 81. Mason D. A very high level of crossreactivity is an
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
BCG Vaccination Moshe Tishler
Department of Medicine 'B', Assaf Harofe Medical Center, Zerifin, Israel; Tel Aviv University Sackler School of Medicine, Israel
1. INTRODUCTION
2. BACKGROUND PATHOPHYSIOLOGY
The issue of autoimmune manifestations or diseases following administration of various vaccines has been discussed extensively in the literature, both in case reports and reviews. Even though vaccinations have been found to be one of the greatest achievements of modem medicine, a growing number of reports have raised the question of the causal relationship between them and autoimmune phenomena. The relationship between vaccinations and autoimmunity is bi-directional. On one hand, vaccinations prevent infectious diseases and thus can prevent autoimmune diseases that might be triggered by infectious agents. On the other hand, case reports and series that describe post-vaccination autoimmune phenomena give rise to the suspicion that they can also trigger some autoimmune diseases in a similar way to the infectious agents from which we try to protect. Bacillus Calmette-Gu6rin (BCG) vaccine was derived from an attenuated strain of M. bovis and was first administered to humans in 1921. This vaccine is recommended for routine use at birth in countries with high tuberculosis prevalence and for health care employees in high risk areas. Another use for this vaccine is as an adjuvant treatment for superficial bladder cancer where it is given intravesically. In this chapter the link between mycobacterial infection, BCG immunotherapy and autoimmunity will be discussed.
The BCG vaccine acts mainly by an immunological mechanism. Mycobacteria have been found to be immunogenic [ 1] and antibodies to phosphoglycolipids extracted from mycobacterial cell wall have been suggested as a diagnostic tool for detecting active tuberculosis. Furthermore, many autoantibodies can be detected with high frequency in infected patients with mycobacteria [2]. It has been postulated that mycobacteria share antigens with human tissue. Monoclonal anti-DNA antibodies derived from patients and mice with systemic lupus erythematosus (SLE) were found to bind to three glycoproteins derived from mycobacterial cell wall [3]. This binding could be inhibited by prior incubation of the antibodies with glycolipid antigens and with anti ss-DNA, thus indicating that mycobacterial and human tissue share common antigens. On the other hand, autoantibodies detected in chromic relapsing experimental autoimmune encephalomyelitis have been shown to react as well with mycobacteria [5]. The similarity between human and mycobacterial antigens is not limited to the humoral levels and extends to cellular mechanisms also. Studies done in 1984/5 in a model or of rat with adjuvant arthritis succeeded in establishing a Tcell clone specific for M. tuberculosis which was strongly arthritogenic [6]. This clone has been able to recognize, in addition to M. tuberculosis antigens present in human synovial fluid, progeoglycans purified from human cartilage and chondrocyte culture medium [7]. The molecular mimicry between mycobacteria and human tissues has been based on studies of
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adjuvant arthritis in a rat model. In this model cross antigenicity between M. tuberculosis and proteoglycans purified from human cartilage has been demonstrated [7]. Furthermore, T-lymphocytes taken from patient with rheumatoid arthritis (RA) have shown augmented reactivity to a fraction of mycobacteria cross-reactive with human cartilage [8]. The systemic immune effect of mycobacteria is not limited to this model and has also been assessed in autoimmune-prone mice. In an animal SLE model a single dose of M. bovis injected to prediabetic NOD mice resulted in prevention of type I diabetes and onset of a systemic autoimmune disease similar to SLE [9]. This disease was characterized by hemolytic anemia antinuclear antibodies (ANA), severe sialadenitis and glomerulonephritis. Characterization of the B-cell responses in these mice have shown that they were directed against ds DNA and the Sm ribonucleoprotein complex [10]. The antids DNA and anti-Sm antibodies were not a direct result of polyclonal stimulation although it was most likely occurring [11]. This molecular similarity between mycobacterial and host antigens, which has repeatedly been determined, was the conceptual basis for the usage of mycobacteria as a therapeutic agent.
only as case reports with no more than 30 cases reported thus far [14-17]. Usually it is manifested by symmetric polyarthritis affecting the large joints of the lower limbs, associated also with low back pain. Arthritis can spread to other joints after recurrent instillations, and severe polyarthritis has been described even after the eighth BCG administration [17]. Radiographic evaluation usually reveals no abnormal findings and laboratory tests show only non-specific signs of inflammation. In all cases reported the synovial fluid culture was negative for mycobacterial and negative PCR tests argue against the possibility of active mycobacterial infection. Review of the literature discloses that in approximately 50% of the cases HLA B27 was positive and in fewer cases HLA DR4 was demonstrated [18]. Most cases resolve completely with NSAID treatment without further joint sequelae. Another very rare autoimmune phenomenon induced by BCG vaccination given as TB prophylaxis is dermatomyositis. Only three case reports could be detected in the literature, describing three adolescents developing dermatomyositis [ 19-21 ].
4. BCG AND ARTHRITIS- MECHANISM OF ACTION 3. BCG IN CLINICAL PRACTICE Treatment of urinary bladder carcinoma by intravesical installation of Bacillus Calmette-Gu6rin has been used successfully since 1976. The BCG given for superficial bladder carcinoma does not destroy the tumor cells directly but rather increases the local immune response, eventually eliminating the tumor cells [ 12]. The great majority of patients (- 95%) tolerate treatment without any serious side effects. The most common side effects are malaise, low-grade fever, cystitis and hematuria, all of which are short term and resolve spontaneously. Intravesical BCG can also result in more severe side effects with systemic phenomena such as rash (0.5%), renal abscess (0.1%), epididymitis (8.4%), sepsis (8.4%), pneumonitis and hepatitis (0.7%), cytopenia (0.7%) and arthritis or arthralgia (0.5%) [13]. Arthritis secondary to intravesical BCG administration is a rare systemic side effect poorly documented in the literature. BCG induced arthritis has been reported
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The mechanism of action of BcG induced arthritis is not clear. Animal studies have shown that an intact host immune system is required for the antitumoral effect of BCG [15], which hints us that the probable mechanism involved in the therapeutic activity of the vaccine is immunologic. Furthermore, clinical and laboratory evidence suggests that the anti-tumor activity is concentrated at the site of BCG administration, thus supporting the local immune mechanism as an important factor for its therapeutic effect [22]. It has been shown that following repeated instillations of BCG organisms into the bladder, large quantities of various cytokines can be detected in the urine. Although the responses among patients were heterogeneous, concentration of all cytokines detected (IL-1, IL-2, IL-6, IL-8, IL-10, TNF alpha, IFN gamma and soluble ICAM-1) were increased following BCG intravesical therapy [24]. The most likely explanation is based on the concept of molecular mimicry which has developed following the
model of adjuvant arthritis. In this model performed in rats cross antigenicity was demonstrated between M. tuberculosis and proteoglycans purified from cartilage [7]. Moreover, T-cell fines grown from patients shortly after intravesical BCG treatment showed a strong expression of HLA-DR on their surface which persisted several months afterwards [24]. The results of these studies suggest that proliferation of a CD4 T-cell clone might take place. This clone might have specificity to a common antigen shared by both cartilage proteoglycans and the mycobacterial cell wall [25]. Penetration of the bacterial or bacterial antigens through the wall of the vascularized tumor in the bladder to the circulation can generate a systemic immune response. Therefore, the T-cell clone that has been suggested to proliferate can now attack the joints. Such attack can take place in genetically susceptible individuals such as those having specific HLA antigens. Indeed, more than 50% of patients with BCG induced arthritis were carrying the HLA B27 antigen. Genetic susceptibility has been demonstrated by in vitro studies as well. It has been shown that peripheral blood lymphocytes from patients with RA responded vigorously to specifically to PPD but not to other polyclonal mitogens [26]. Enhanced T-cell responses to a fraction of mycobacteria have also been shown in RA patients. This T-cell response was more pronounced in these patients compared to patients with degenerative joint disease and to healthy controls. This effect of generating an attack on a host organism from within has been given the name "Trojan Horse" [27] and is not unique just to BCG vaccination but rather to numerous autoimmune phenomena following vaccination.
important role as well.
5. C O N C L U S I O N
9.
BCG immunization, especially by intravesical instillation, can act as "a double edged sword". Although it proved to be a powerful tool in the treatment of superficial bladder cancer, it can trigger autoimmune phenomena and even full blown autoimmune disease, which are fortunately rare. The link between mycobacteria and autoimmunity is probably a consequence of molecular mimicry, although genetic and environmental factors play an
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21. Kass E, Straume S, Mellbye OJ, Munthe E, Salheim BG. Dermatomyositis associated with BCG vaccination. Scand J Rheum 1979;8:187-191. 22. Prescott S, Jackson AM, Hawkyard SJ, Alexandroff AB, James K. Mechanisms of action of intravesical BCG: local immune mechanisms. Clin Infect Dis 2000;31:591-593. 23. Jackson AM, Alexandroff AB, Kelly RW, Skibinska A, Esuvaranathan K, Prescott S et al. Changes in urinary cytokines and soluble intracellular adhesion molecule-1 (ICAM-1) in bladder cancer patients after BCG immunotherapy. Clin Exp Immunol 1995;99:369-375. 24. Prescott S, James K, Busuttil A, Hargreave TB, Chisholm GD, Smyth JF. HLA-DR expression by high grade superficial bladder cancer treated with BCG. Br J Urol 1989;63:264-269. 25. Holoshitz J, Naparstek Y, Ben Nun A, Cohen IR. Lines of T lymphocytes induce or vaccinate against autoimmune arthritis. Science 1983;219:56-58. 26. Abrahamson G, Froland SS, Matvig JB. In vitro mitogen stimulation of synovial fluid lymphocytes from rheumatoid arthritis and juvenile rheumatoid arthritis patients: dissociation between the response to antigens and polyclonal mitogens. Scand J Immunol 1978;7: 81-90. 27. Aron-Maor A, Shoenfeld Y. BCG immunisation and the 'qu Horse" phenomenon of vaccination. Clin Rheumato12003;22:6-7.
9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Viruses: The Culprits of Autoimmune Diseases? A.M. Denman ~and B. Rager-Zisman 2
Worthwick Park Hospital Harrow, Harrow, UK; 2Dept of Microbiology and Immunology, The University Center for Cancer Research, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel
1. INTRODUCTION One of the first principles in the organisation of the immune system is the need to avoid autoimmune diseases resulting from immune reactions to self antigens (Ehrlich's "horror autotoxicus"). The classical explanation for the prevention of autoimmunity was given by Burnet in his clonal selection theory which postulated that clones with the potential to produce autoantibodies are selectively eliminated during the development of immune responses in normal individuals. Breakdown of tolerance to serf-antigens was viewed as a general mechanism leading to autoimmune diseases [1]. Subsequent discoveries have revealed the complexities of the immune system and its regulation. Consequently this theory is now seen by many observers as an over simplification since autoimmune responses may have a physiological role in tissue repair and maintenance [2]. Furthermore it is increasingly evident that autoantibodies in human autoimmune disease are directed at a very limited number of self-proteins, perhaps 2% of the estimated 20,000-60,000 of the potential total [3]. Indeed the same limited repertoire of auto-antigens is the target in mouse strains developing spontaneous autoimmune disease. Although this observation does not negate classical theories based on broken tolerance, it suggests that changes in the milieu in which auto-antigens are presented to the immune system must be at least as important as universal defects in tolerance maintenance. In recent years the idea has been prominent that autoimmune diseases result from a failure of regulatory mechanisms. Particular emphasis has been placed on the regulatory
role of the T cell suppressor population identifiable by their specific CD25 + CD4 § phenotype [4]. The basic issue of tolerance maintenance has important implications for those seeking an infectious aetiology for autoimmune diseases. If the immune system has evolved to meet pathogens, it is likely that these disorders arise as part of the anti-microbial response and not as the result of the random emergence of auto-reactive clones unrelated to host defence [5]. Furthermore, the pathogenesis of human autoimmune diseases is unlikely to have a single cause since experimental models point to aberrations in a wide range of immune responses which may lead to autoimmune diseases [6]. Tissue damage, irrespective of the cause, may induce ephemeral autoimmune reactions but these rarely persist as autoimmune diseases. Viral infections are an attractive explanation for human autoimmune diseases because of the many potential ways in which these may perpetuate local inflammation and also subvert local and systemic immune responses. In classical terminology, proponents of the viral theory of autoimmune diseases must find persuasive evidence to back the many explanations based on the theoretical ability of these agents to break tolerance. This task has at least been eased by the recognition that auto-reactive T and B cell clones are part of the normal immune repertoire. The issue can be more precisely defined as ways in which virus infection may subvert the mechanisms which normally limit this reactivity. There are many excellent recent reviews summarising the evidence for a viral aetiology of autoimmune diseases [7, 8]. However fresh insights into
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the complex mechanisms of anti-viral host defence and its interactions with different viruses continue to provide new insights and experimental evidence for this hypothesis. The object of this review is to examine the validity of the viral hypothesis in the light of this evidence. In a broader perspective there is increasing acceptance that infections contribute to chronic diseases of unknown aetiology [9]. Given the wealth of information on this issue, our approach is largely dictated by the need to devise acceptable criteria for linking infection by a given virus to a specific autoimmune disease. For this purpose we have attempted to adapt Koch's classical postulates [10] for a bacterial aetiology of disease in a way which takes account of the complex interactions between viruses and the host immune response. However we recognise that almost certainly autoimmune diseases do not arise through a single mechanism. Furthermore autoimmune diseases can be induced experimentally which are clearly independent of viral or indeed any obvious antigenic stimulation. A cogent example is myopathy with autoantibodies to histidyl-transfer RNA synthetase (anti-Jo-1) in mice with transgenic overexpression of MHC class 1 molecules [ 11 ].
2. HISTORY AND EVOLUTION OF K O C H ' S POSTULATES There were many doubts in Koch's times about the relevance of bacterial infection to the pathogenesis of disease even in pulmonary tuberculosis when the characteristic symptoms and signs of infection formed a reasonably consistent picture [10]. His postulates provided a satisfactory basis for experimental validation because isolating tubercle bacilli correlated reasonably well with disease activity. Furthermore there was an available laboratory model of tuberculosis induced by the bacillus. Yet even in this situation there are recognised difficulties. The presence of m. tuberculosis in tissues does not necessarily imply tuberculous disease. The outcome of infection is largely determined by host immunity. Perhaps the greatest benefit derived from the postulates was to establish verifiable rules in contrast with the unbridled, often philosophical speculation which characterised so much contemporary discussion on the aetiology of tuberculosis
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and indeed most diseases. Any attempt to link viral infections with human autoimmune diseases must depend on comparable rules adapted to test this hypothesis. However the difficulties are formidable. Viruses have more complex life histories with a wider range of possible outcomes than bacterial infections. The range of anti-viral host responses is immense and limiting or eradicating viral infection necessitates an attack on the cells in which viruses complete their replication cycle or persist in defective or latent form. Furthermore, viruses have evolved strategies for circumventing or subverting these responses in order to enhance their survival and replication. Thus there is an almost unlimited number of plausible mechanisms by which viral infection could initiate autoimmune diseases. A basic problem is our ignorance of many fundamental issues concerning the natural history of bacterial and viral infections alike and their interactions with host defence mechanisms. To compound these difficulties, there is still controversy over the extent to which autoimmunity contributes to many chronic inflammatory human diseases even when these are associated with autoimmune features such as circulating autoantibodies. There is a vast body of information about the immunopathological features of these diseases but these have thrown rather limited light on their origin. Equally, there is no lack of contrived or spontaneous experimental models of diseases whose features resemble human autoimmune disease. A major difficulty is to determine the relevance of these models to the aetiology operating in human autoimmune disease. Perhaps the most reassuring feature of these models is the almost identical yet limited repertoire of auto-antigens recognized by the immune system in human a autoimmune diseases models [3]. This review attempts to expand and adapt Koch's criteria to the problem of linking viral infection with autoimmune disease.
3. GENERAL T H E O R I E S OF AUTOIMMUNE DISEASES There are many excellent general reviews on the general subject of autoimmune diseases. However our ideas on the origin of these diseases general are rapidly changing. Even the traditional distinction between conventional and autoimmune reactions
appears less absolute [12]. The molecular mechanisms of organ-specific autoimmune disease such as myasthenia gravis [ 13] have been elucidated in fine detail. The precise role of autoimmunity is more difficult to unravel in multi-system diseases such as the connective tissue diseases and rheumatoid arthritis where autoantibody may be secondary to other proinflammatory mechanisms. Its contribution to other diseases of unknown aetiology such as multiple sclerosis is even more controversial. We consider only those aspects which are crucial to the issue of viral infection and autoimmune disease. Auto-immune reactivity has been shown to form part of the normal T and B cell repertoire. Indeed self recognition is likely to prove an essential step in the generating an effective response to foreign antigens including microbial infection [14]. For example T cells from normal donors proliferate in response to in vitro stimulation with histones and nucleosomes and clonally expanded T cells reactive to these auto-antigens circulate in similar numbers in normal control subjects and SLE patients [15]. Even persistent, circulating autoantibodies are not necessarily associated with disease; organ-specific autoantibodies, rheumatoid factor, and anti-nuclear antibodies are present in some 2-5% of females over the age of 40 without any apparent immunopathological consequences. Classical immunological theory rigidly distinguished between non-specific, innate immunity and specific immunity mediated by T and B cells. The origin of autoimmune diseases has long been considered to arise from anomalous T or B lymphocyte function. It is now evident that this rigid distinction is incorrect; since all modalities of nonspecific and specific immunity have been variously implicated even immediate type hypersensitivity, an area not usually considered relevant in the context of autoimmune diseases, can not be ignored. Histamine release in immediate type reactions enhances Tn 1 responses through its activating effect on their type 1 histamine receptors [16]. As a result histamine secretion by mast cells contributes to the inflammatory reaction in experimental autoimmune diseases such as experimental allergic encephalomyelitis and to human autoimmune diseases such as rheumatoid arthritis and bullous pemphigus [ 17]. In keeping with these observations, mice strains which are genetically deficient in mast cells are resistant
to an experimental model of inflammatory arthritis
[~8]. The wider spectrum of responses potentially contributing to autoimmune disease is highly relevant to theories of initiation by viral or indeed other forms of infection because the many it increases the potential points at which excessive inflammation could initiate autoimmune disease. Nevertheless the dogma that autoimmune diseases result from a breakdown of tolerance to self antigens remains the key issue [19] even if classical theory needs extensive revision. There is still a consensus view that central tolerance depends on the elimination in the thymus of potentially autoreactive T cells with high affinity receptors for self-antigens. This form of tolerance is difficult to break. Peripheral tolerance is defined as the process by which T cells migrating from the thymus to the periphery express low affinity receptors for autoantigens and normally remain tolerant. However peripheral tolerance is readily broken, usually as the result of inflammation or tissue damage. Indeed, peripheral autoreactivity is not necessarily pathological but can reasonably be considered a physiological process. Furthermore, the ability to mount reactions against self-components is an integral part of the process of counteracting the destructive effects of inflammatory reactions. This role is amplified in the host response to infections associated with tissue damage to an extent dependent on the duration and extent of the infection. It is a situation especially inherent in viral infections which are commonly characterised by indefinite viral persistence in a wide range of host cells. Transient autoimmune phenomena commonly accompany infectious diseases but rarely lead to overt autoimmune disease. Peripheral events affecting tolerance are complex because of the vast range of interactions between different cell populations and their products. For example experimental manipulation indicates that a given cytokine for example will break or enhance tolerance depending on the experimental conditions. Transgenic models show that constitutive over production of selected cytokines induces autoimmune diseases and this over-expression is reproduced in the resulting inflammatory lesions. Nevertheless some general principles have emerged. Disease induction depends critically on
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the nature of auto-antigen presentation and abrogation of the many regulatory steps which limit the proliferation of T cells with autoreactive receptors [6]. Transgenic models emphasise the many safeguards against the prolonged breakdown of peripheral tolerance and the emergence of autoimmune disease. There is good experimental evidence that the induction of tolerance in T cells exposed to novel virus-related antigens is also regulated during the maturation of these cells [20]. There are other mechanisms for controlling potential aggressiveness by autoreactive T cells which have escaped elimination in the thymus, a process which has been termed "fine tuning" [21]. It is not surprising therefore that persistent autoimmunity represents a rare escape from a network of restraining factors. The factors governing B cell tolerance are similarly elaborate. Germ line encoded autoreactivity is a normal feature of B cell development and maturation before eventual IgV gene diversification through hypermutation [22]. Experiments involving autoreactivity to snRNPs and ss DNA indicate that tolerance induction in B cells partly depends on encountering auto-antigens during the transition from immature to mature B cells [23]. B cell maturation depends on interactions with other cell populations and cytokines. In addition it is possible that an accumulation of immune complexes interferes with B cell tolerance to auto-antigens; an inability to clear immune complexes and autoantibody production are characteristic findings in experimentally induced complement deficiency and genetically determined complement deficient in man. There are also intriguing observations that malignant lymphoproliferation, an unequivocal example of failed B cell regulation, is associated with a wide spectrum of organ specific and systemic autoimmune diseases [24].
4. GENETIC SUSCEPTIBILITY TO AUTOIMMUNE DISEASE Any hypothesis invoking viral infection in the pathogenesis of autoimmune disease must satisfactorily explain the overwhelming evidence for genetic factors in determining susceptibility to these disorders. Indeed there are animal models of autoimmune disease in which at first sight it appears
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superfluous to invoke any factors other than single inherited errors in the immune system. In support of classical; theories, genetic abnormalities in T and B cell regulation lead to autoimmune disorders with features resembling human disease. For example mice lacking protein kinase C lambda develop hypergammaglobulinaemia and glomerulonephritis which are characteristic of systemic lupus erythematosus (SLE) [25]. Regulatory defects confined to B cell activation such as kinase deficiency also lead to autoimmune disease [26]. Inherited defects in apoptosis resulting from a single gene defect also induces autoimmune disease as, for example, in mice lacking the membrane tyrosine kinase c-mer [27]. However even autoimmune diseases induced by single gene manipulation result in secondary abnormalities and are thereby realistic models of what may occur in diseases where there are no such clear leads to the underlying error or errors in immune regulation [6]. These abnormalities could well lead to an unusual outcome of infections. Indeed as yet unidentified environmental factors are important in determining the outcome in individuals with familial susceptibility to organ-specific immune diseases who inherit a mutated immune regulatory gene (AIRE) on chromosome 21q22.3 and also HLA class II genes linked to susceptibility to these diseases [28, 29]. A mouse model of this mutation suggests that the proliferation of antigen responsive T cells may also be exaggerated [30] and this could also affect the response to infection. Models in which a combination of genetic factors unequivocally determines the onset of autoimmune disease more closely reproduce the multigenic susceptibility characteristic of human autoimmune disorders such as type I diabetes and SLE. Thus Fas (CD95) deficient mice develop more severe autoimmune haemolytic anaemia and thrombocytopenia when IL-10 levels are constitutively high [31]. We can postulate that human autoimmune diseases arise when infection perturbs the cytokine network and acts in combination with other genetic determined events. Certainly studies of disease incidence in identical twins who are genetically susceptible to autoimmune diseases emphasise the importance of as yet undefined environmental factors. Particular attention has been given to the well documented association between susceptibility or resistance to autoimmune diseases and the inherit-
ance of certain class II HLA genes in man and their MHC counterpart in mice. There is good evidence that these genes control antigen presentation and hence the character of the resulting anti-viral T cell reposnse. This response largely determines the outcome of viral infections. For example HLA antigens determine susceptibility to HIV infection [32] and a vigorous response to this virus anti-viral is associated with the expression of HLA A*02 [33]. HIV infected individuals inheriting HLA B'5701 or 5703 are more resistant to HIV infection because they develop a broad range of CD8 T cell responses variants of the conserved viral p24 epitope [34]. Indeed the evolution of HIV-1 infection is largely determined by HLA restricted anti-viral immune responses [35]. Similarly the HLA class II haplotype DQB 1"0301 protects against hepatitis C virus infection because it enables infected individuals to maintain an effective CD4 T cell response [36]. It is also apparent that more subtle genetic variation determines the risk of post-infectious autoimmune disease. Chagas disease is caused by Trypanosoma cruzi infection and the most serious disease feature is cardiomyopathy. In a mouse model of the disease the myocarditis is directly related to the genetically controlled levels of anti-myosin cell mediated and humoral response [37]. In the human disease there is good evidence that genetically controlled T cell recognition of myosin in an inflammatory milieu contributes to cardiomyopathy [38]. In other situations selective experimental manipulation of the cytokine network affects the pattern of postinfectious immunopathology. Borrelia burgdorferei infection in man usually causes acute disease manifestations including arthritis and myocarditis (Lyme disease). Chronic arthritis with a persistent acute phase response and rheumatoid factor production has been linked to autoimmunity. In experimental Borrelia burgdorferei infection of mice, passively administered antibody to IL-12 increases the severity of the arthritis [39]. This observation suggests that genetically determined differences in cytokine production determine the outcome of human infection by this agent. Linkage studies in human autoimmune diseases emphasise the polygenic risk factors. It is conceivable that these could operate through host resistance to infections including viral infection. Genes predisposing to autoimmune disease control many other features of innate and specific
immunity including cytokine production, complement, and apoptosis. Increasing the complexities, genetic mapping shows that susceptibility genes increase the risk of developing several, different autoimmune diseases and not just a single disease. For example there is an association within the same families between increased susceptibility to organ specific autoimmune diseases and SLE. Genetic predisposition to organ specific autoimmune disease within the same family extends to type I diabetes and thyroiditis. Disease severity is also genetically controlled. The increased susceptibility could also be explained by polygenic influence on the outcome of infections. However the polygenic control of disease manifestation and severity further complicates the exploration of a viral aetiology. The perturbations induced by viral infections could influence the immune response might be secondary effects stages which are not confined to the initial interactions between viral antigens, specialised antigen presenting cells, and responding T cells. In support of this view, studies in a wide range of animal models and clinical situations indicate that genetically determined susceptibility to autoimmune diseases involves many different mechanisms [40, 41].
5. IDENTIFICATION OF HUMAN AUTOIMMUNE DISEASES Autoimmune diseases are defined as diseases arising from autoimmune attack on target organs, tissues, or cells. Autoimmune diseases affect 3-5% of most populations in developed countries although there is some indication that this figure may be lower in undeveloped regions of Africa and Asia. The prevalence of these diseases has been estimated to be as high as 20% [42] but this is probably an exaggeration. Nevertheless the number of autoimmune diseases continues to increase with investigative advances and diagnostic ascertainment is also improving. Conventionally, autoimmune diseases are classified as organ specific or systemic. Organ specific autoimmune diseases are associated with autoantibodies to antigens usually specific for the target organ (Table 1) and the distribution of multiorgan autoimmune disease correlates with the distribution of these antigens. It is reasonable to conclude that these anti-
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Table 1. Classification of autoimmune diseases Autoimmune pathogenesis
Disease
Established Organ specific
Endocrine
Thyroiditis Type 1 (insulin dependent) diabetes Adrenal insufficiency (Addison's disease) (uncommon) Hypoparathyroid Ovarian disease
Gastro-intestinal
Gastritis and pernicious anemia Hepatitis (some forms)
Haematological
Haemolytic anemia Trombocytopenia Neutropenia
Nervous
Myasthenia gravis Peripheral neuropathy (some forms) Para-neoplastic neuro-muscular diseases Rare disorders (e.g. Stiff-man syndrome)
Renal
Goodpasture's syndrome Nephritis (some forms)
Skin
Pemphigus Pemphigoid Chronic urticaria
Ocular
Sympathetic ophthalmia
Possible Organ specific
Coeliac disease Ulcerative colitis Fibrosing alveolitis Uveitis (some forms) Multiple sclerosis Myocarditis Rheumatic fever
Established Systemic
Systemic lupus erythematosus SjiSgren's disease Vasculitis and polyarthritis (some forms)
Possible Systemic
Rheumatoid arthritis Polymyositis; dermatomyositis Scleroderma
gens are the targets for the immunopathological events which eventually destroy the target organs. However it is more difficult to be sure that these antigens are the same as those which initiated the autoimmune reaction. It should also be admitted that the true contribution of autoimmunity to many of the diseases listed in Table 1 remains controversial. Longitudinal studies of first degree relatives of patients with autoimmune diseases who develop autoantibodies before the advent of clini-
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cally detectable disease suggest that the number of initiating antigens is very limited. Subsequently self antigens may become the target for autoimmune reactions because the autoirranune response becomes more widespread as the result of epitope spreading or bystander effects. The delayed appearance of antibodies to other auto-antigens may have important clinical consequences even though these appear as secondary events. Thus autoantibodies to the beta 1-adrenoreceptor contribute to the circula-
tory problems of patients with idiopathic dilated cardiomyopathy [43] but this potential consequence of myocardial infection is not considered in experimental models of virus induced myocarditis. This issue is relevant to the viral hypothesis because analysis of the initiating auto-antigens is likely to provide the best clues to the responsible viruses. As an additional misleading factor, an initiating infection in the mother may be responsible for autoimmune disease in her children. Maternal antibodies may well stimulate the production of autoreactive T cells [44, 45]. In support of this idea, eliminating the transfer of maternal autoantibodies to pancreatic islet cells prevents diabetes in NOD mice [46]. The attribution of pathogenic effects to autoantibodies is well attested in myaesthenia gravis and other neurological diseases in which autoantibodies to acetylcholine receptors, voltage-gated calcium channels, and voltage-gated potassium channels account for muscle weakness and related symptoms. Reasonably selective removal of these autoantibodies leads to clinical improvement [13]. Nevertheless, while autoantibodies are often good predictors of organ specific autoimmune disease, circulating autoantibodies are not necessarily associated with clinically significant disease [47]. Nevertheless, these may be provoked by the same factors which initiate autoimmune disease. For example rheumatoid factors, so long regarded as harmful contributors to the immunopathogenesis of rheumatoid arthritis, will probably prove to be part of the normal host defence against infection [48]. Autoantibodies in general may only become harmful when they achieve high affinity. There are other difficulties in defining the contribution of autoimmunity in experimental models and in human disease. T cells isolated from target organs and clones derived from these cells may be demonstrably autoreactive for target organ cells or their antigens judged by in vitro proliferative or cytotoxic assays. However the interpretation of these findings is not straightforward. In vitro experiments do not necessarily reflect the in vivo behaviour of effector populations subject to regulatory constraints. Furthermore tests selected to demonstrate destructive autoimmunity may overlook T cell populations with protective properties; thus autoreactive T cells protect vulnerable neurones from damage in rats with experimentally damaged optic nerves [49].
Similarly, a population of CD4 alpha/beta cells in non-obese diabetic (NOD) mice limits the damage to pancreatic islet cells induced by autoreactive T cells [50]. Such observations have been incorporated in a more general scheme which assigns a possibly regulatory role to autoreactive T cells in response to infection [51 ].
6. PREVALENCE OF AUTOIMMUNE DISEASES The epidemiology of autoimmune diseases could in principle give clues to an infectious aetiology for these disorders although to date such hopes have not materialised. There is much speculation that the incidence of allergic and autoimmune diseases is increasing in developed societies because innate and specific immunity have been blunted by immunisation strategies, improved hygiene, and antibiotics. A decline in the incidence of infectious diseases has been considered a factor in the seeming increasing in the incidence of allergic and autoimmune diseases possibly because of resulting distortions in the relative numbers of T cell populations; decreased infection might lead to a reduction in Th2 cell numbers and of immunoregulatory IL-10 [52]. There is little evidence that immunisation directly induces autoimmune disease [53, 54]. However a longer period of observation and detailed analysis of immunological memory for the immunising virus may be needed, to take full account of immunisation's effects on the long term accuracy of immunological memory for the immunising viruses. T cell memory may be directed at unrelated viruses and may extend to inappropriate reactivity against self antigens. It is also possible that low level virus persistence in target organs following immunization may induce autoimmune rather than effective anti-viral responses [44]. Although there is little doubt that the prevalence of autoimmune diseases is higher in developed than in third world countries, there is no firm evidence that this is related to immunisation.
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7. ANTI-VIRAL HOST DEFENCES AND THEIR SUBVERSION
Viruses have evolved many strategies to avoid elimination by host defences [55, 56]. LCM virus is a striking example of a virus which can persist in low levels despite a seemingly efficient immune response [57]. Viral persistence increases the risks of autoimmune disease in two general ways. Firstly, a continuing anti-viral response creates an inflammatory environment in which peripheral tolerance is more likely to be broken. Secondly, the strategies by which virus infections persist are likely to disrupt other regulatory mechanisms which normally prevent autoimmunity. A related issue is the duration of virus infection's effects on the regulation of immunity. If disruption continues after the initiating virus has been eliminated or it can no longer replicate, it will be more difficult to link remote infection with persistent immunopathology. Indeed long term disruption of regulatory mechanisms could theoretically act in the same way as inherited abnormalities conferring susceptibility to autoimmune disease. Traditionally, anti-viral immunity has been analysed in rigid compartments, namely innate immunity, and specific humoral and cell mediated immunity. However it is now evident that there are multiple pathways whereby these systems interact in order to mount effective inflammatory and immune reactions to viruses and other pathogens whilst limiting the damage these responses may inflict on infected tissues [58]. The complexities of virus-host interactions are further increased by genetically determined variations in the control of each component of the host response. These topics are the subject of comprehensive reviews and we only consider the implications for theories of virus-induced autoimmunity (Table 2). It is also important to emphasise an emerging theme that defects in innate or specific immunity predispose to inappropriate B cell activation. Defects in the acute phase protein SAP, the complement system, and NK cells predispose to this problem [59]. Viral infection leads to apoptotic or immune mediated host cell death associated with non-specific local inflammation and often a specific autoreactive response to the antigens released by cell death. Delayed clearance of cell debris secondary to viral infection and the resulting immune complexes accentuate the risk.
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Table 2. Major contributors to innate anti-viral immunity
Reference NK cells
[60]
Complement
[64]
Dendritic cells
[65]
Defensins
[68]
Chemokines
[66, 67]
Cytokines
[69]
Innate T and B lymphocytes
[70]
Gamma/delta T cells
[63]
Inherited defects are extreme examples of situations which may also be acquired as the result of viral infection. For example herpeseviruses in general and CMV in particular down regulate the expression of adhesion molecules and class I MHC molecules as a strategies for evading the host immune response [60]. Similarly some viruses have evolved a strategy for blocking antigen presentation by antigen presenting cells to naive CD8 T cells by encoding "viral proteins interfering with antigen presentation ("VIPRs") [61]. Indeed the range of strategies for viral persistence continues to grow. For example human and murine CMV encodes proteins which have been termed immunoevasins. These interfere with viral antigen processing and presentation by class II MHC antigens [62]. 7.1. Toll Receptors and the Defensin System
In common with other pathogens, viruses are recognised by germline encoded receptors termed pattern recognition receptors which include Tolllike receptors [71, 72] and the defensin system. In mice with defective expression of the Toll receptor TLR-4 deficiency respiratory syncytial virus infection (RSV) induces severe lung disease with immunopathological features resembling fibrosing alveolitis in man [73]. Toll-like receptors expressed in high levels on microglia and astrocytes in the central nervous system present auto-antigens to the immune system and this may contribute to chronic immunopathology [74].
7.2. Cytokines and Chemokines Interactions with inflammatory and specific immune cell populations are mediated mainly by chemokines and cytokines. Genetically determined polymorphism in the chemokine and cytokine systems largely determines the pattern and intensity of innate and specific responses to microbial infection. Inherited patterns of chemokine receptors govern cell activation in response to external stimuli including microbial products [75]. In common with other complex mediator systems, there are also regulatory proteins. The IRAK family of kinases down regulate the pro-inflammatory signals transmitted to monocytes and macrophages thereby reducing cytokine release [72]. It is now apparent that these systems contribute to autoimmune disease. Rheumatoid arthritis is an interesting example of the likely interplay of innate and specific immunity in the pathogenesis of a suspected autoimmune disease. Innate immunity is activated in the rheumatoid synovial membrane. Inappropriate macrophage stimulation could lead to excessive cytokine production including IL-12 initiated by CpG sequences in DNA [76]. Chemokines belong to a superfamily of chemoattractants which attract leukocytes to inflammatory lesions. This superfamily comprises at least 50 structurally related peptide agonists and 20 G protein-coupled receptors. So far more than 30 virally encoded mimics of chemokines and chemokine receptors have been identified [67]. These viruses are mainly herpesviruses, poxviruses, and lentiviruses. The net effect of any given viral infection in vivo is unpredictable and could in theory enhance or suppress local inflammation. The potential of this mechanism to influence the outcome of infection is increased by the discovery that chemokines also influence lymphocyte traffic [77]. Furthermore, T cell subsets with the chemokine receptors CXCR3 and CCR5 are specifically associated with inflammatory reactions [78]. Recent evidence suggests that in a transgenic model of type I diabetes, T cells may be attracted to pancreatic islet beta cells because the latter express chemokine CXC ligand 9 [79]. The distinction between innate and specific immunity has become increasingly blurred in terms of host defence against microbial infections and the risk of developing autoimmune diseases. The vast field of cytokine physiology began with
the discovery of the anti-viral effects of the interferons. The complexities even of the interferon system in isolation are daunting. While there are only fourteen Interferon-alpha, one interferon-beta, and one interferon-gamma gene, DNA microarray technology has revealed that there are hundreds of genes whose expression is influenced by the interferon family [69]. The regulation of other cytokine is also complex. There are many ways in which virus infection could initiate imbalances in the cytokine network leading to autoimmune disease. However the pleomorphic effects of individual cytokines and the complexities of the cytokine network make it difficult to predict the outcome of such infections [80]. The relevance of this field to theories of autoirnmune disease is well illustrated by the variable outcome of viral infections in hosts with genetically determined differences in their interferon response. A crucial phase in the susceptibility of mice to diabetes following coxsackie B4 virus infection is the extent to which the virus grows in host cells. Interferon alpha and beta production in pancreatic islet beta cells is genetically controlled. Low levels of production predispose to cell mediated destruction of infected cells initially mediated by NK cells [81]. However the interferon system illustrates a major dilemma; it is very difficult to disentangle viral virulence enhanced by genetically determined deficiencies from suppression mediated by viral genes [69]. For example mutations in the non-structural genes of the highly virulent influenza virus strain H5N1 confer resistance to interferons and TNF alpha [82].
7.3. Non-Specific Inflammatory Cell Populations The activation of B cells and non-specific inflammatory cell populations also depends on Fc gamma receptors. Deficient expression of the neutrophil specific Fc gamma receptor Fc gamma RIIIB is a risk factor for developing SLE and deletions in the promoter region for an inhibitory Fc gamma receptor are found in some lupus prone mouse strains [83]. Viral infection of dendritic cells is a potent strategy for suppressing specific anti-viral immunity or even inducing long term tolerance. Human cytomegalovirus is especially adept at influencing dendritic cells to reduce viral antigen presentation by class II
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MHC molecules and deleting potential anti-CMV T cells [84]. Measles virus has similar capacities [85]. Dendritic cells also determine whether immunity or tolerance follows antigen presentation to T cells. This issue is complicated by the heterogeneity of dendritic cells and the nature of cytokine exposure during the process of antigen presentation [65]. Furthermore some dendritic cells have the unusual capacity to perform a process termed cross presentation. Usually MHC class II antigens present peptides derived from microbial antigens which enter the cell by endocytosis. These peptides are recognised by helper CD4 T cells. In contrast MHC class I molecules present peptides primarily derived from endogenously synthesised self or microbial proteins which have been degraded by the proteasome and then transported by TAP molecules of antigen presenting cells. These peptides are presented to cytotoxic CD8 T cells. This distinction ensures that CD8 cells only attack virus infected host cells thereby reducing reduces the risk that uninfected cells will be the target of an autoimmune response. Crosspresentation permits peptides derived from exogenous viral antigens processed by dendritic cells to be presented by class I MHC antigens to cytotoxic T cells. [86]. Probably many viral antigens including influenza, vaccinia, polio, and LCMV are processed and presented in this manner. Cross-presentation implies that self antigens associated with viral peptides can potentially be presented to cytotoxic T cells thereby inducing an autoimmune response. Indeed there is experimental evidence which supports this conjecture. Mice expressing influenza haemagglutinin as a transgenically coded develop diabetes after subsequent infection with influenza virus because anti-viral T cells destroy the islet cells [20]. However it is impossible to generalise as the effects of viral infection on dendritic cells depends on the infecting agent; many viruses including herpesvirus type I, measles, retroviruses, vaccinia, and LCM suppress antigen presentation by these cells. There are other mechanisms by which inflammatory responses to virus infections are expedited with the potential risk of provoking autoimmune reactions. Innate T and B lymphocytes are especially interesting because they express a restricted set of germ-line encoded receptors which recognise conserved structures including self-antigens that are
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encountered in inflammatory situations including infections [70]. Similarly the activation of NK cells depends on a balance between stimulating signals transmitted by activating receptors and inhibitory signals dependent on contact with histocompatibility antigens. NK cells are activated by contact with virus infected cells and are especially important in host defence against herpesvirus infections. The outcome is determined partly by the nature of the viral infection and partly by host genetic factors. Herpesviruses in general and cytomegalovirus (CMV) in particular depress NK cell activation by simulating the normal inhibitory signal transmitted by histocompatibility antigens [60]. Genetic control of NK cell activation has been shown to affect virus growth. For example C3H/HeN mice are relatively resistant and CBA/J mice are relatively susceptible to Polyomavirus. The virus grows to high titres in the latter strain because the inhibitory molecule CD94-NKG2A is related to weak T cell cytotoxicity for Polyoma virus infected cells [87, 88]. Memour T cells in CBA/J mice are also impaired. In fact viruses have evolved a wide range of mechanisms for suppressing anti-viral defence by NK cells. These involve modulating the expression of receptors on virus infected cells which normally activate NK cells or on NK cells themselves. Furthermore viruses can increase the production of cytokines which inhibit NK cell responses [89]. Other virusinduced proteins inhibit NK destruction of HIV and EBV infected cells. Conversely, NK cells may be non-specifically activated through recognition of viral antigens on infected cells by V gamma 9 / V lambda 2 receptors on NK cells respond to antigens expressed by virus infected cells. These receptors also engage cell surface lipids on infected cells undergoing apoptosis [70]. These interactions are potent routes for activating auto-reactive T cells.
7.4. Specific Immunity The interactions between innate and specific immunity and the many viral factors which influence innate immunity complicate former, relatively simple assumptions about the maintenance of tolerance in the face of acute or persistent viral infections. Clearly, acquired or induced defects in innate
immunity predispose to loss of both T and B cell tolerance. In addition defective T cell function encourages virus virulence and persistence. In particular, infection is aided by major genetic defects which negate normal CD8 T cell function or genetic variation which reduces the efficiency of the response to certain infections. In addition some viruses affect T and B cell function as a survival strategy. Conversely, the host response is a balance between efficiently killing infected cells and the risks of excessive cell death mediated in part at least by autoimmunity. CD8 T cell mediated killing is regulated in large part by CD4 § CD25 § T cells. Appropriate regulation is crucial to the outcome of persistent infections by all pathogens; excessive regulation favours persistence and re-infection while deficient regulation encourages cell death [90]. Another problem arises from the nature of T cell anti-viral memory. This must be sufficiently specific for the host to respond to further infections by the same agent yet sufficiently diverse to respond appropriately to other pathogens [91]. However increasing diverse memory increases the risk of potentially harmful autoreactivity. T cell anti-viral memory becomes less virus specific with the passing of time. For example CD8 memory T cells sensitised by an initial LCM virus infection produce interferon gamma after in vivo exposure to vaccinia virus infection [92]. Furthermore the cross-reactivity with epitopes expressed by a different virus has immunopathological consequences since it induces different forms of lung inflammation including bronchiolitis and destructive changes. The issue of anti-viral memory specificity may be important if T cell memory remains latent after an initial infection but can be reactivated by subsequent infections by the same or different viruses against residual viral antigens from the first infection. This possibility is foreshadowed by experiments in which T cell tolerance to influenza haemagglutin in the islet cells of transgenic mice is broken by subsequent influenza virus infection [20]. There is limited information concerning the duration and nature of anti-viral T cell memory. Antibody levels may be a poor guide to previous infection. Thus specific CD4 and CD8 T cell responses to hepatitis C virus infection are often detectable when anti-viral antibodies are no longer detectable [93]. The duration of memory in different T cell popu-
lations also varies. CD8 T cell memory for LCM virus infection is more stable than CD4 cell memory [94]. The phenotype of memory T cells is not the same in different herpesvirus infections [95]. Viral persistence in different lymphocyte populations has long been recognised and has been explored using both in vitro and in vivo systems. The usual outcome is immunosuppression. Recently many mechanisms have been described by which virus infection aids its persistence by inhibiting specific immunity to the infecting agent. Thus many viruses including respiratory syncytial virus infection suppress CD8 T cell memory [96]. Direct infection is not a prerequisite for selective immunosuppression since there are inhibitory receptors on T cells which can be exploited by viruses such as cytomegalovims [87]. One issue of potential importance is the possibility that viral infections initiate autoimmune reactions by their effects on the repertoire of receptors expressed by T cells and antibodies secreted by B cells. The T cell immunodeficiency characteristic of HIV infection is associated with autoimmune thrombocytopenia and connective tissue diseases resembling SjiSgren's syndrome. However it is not clear whether these complications are related to the accompanying polyclonal proliferation of B cells or to more subtle defects involving altered antigen recognition by surviving T cells. Autoantibody production in vitro by EBV infected B cells is a well documented consequence of the accompanying polyclonal stimulation and ephemeral autoantibody production is a recognised feature of infectious mononucleosis. However the relevance of these observations to the pathogenesis of autoimmune disease remains doubtful. Indeed the short duration and usually inconsequential nature of in vivo autoimmune phenomena after primary EBV infection is more a tribute to the robust nature of regulatory mechanisms than evidence of EBV aetiology for autoimmune diseases. Nevertheless the concept that viruses may stimulate autoantibody production through a mitogenic effect is interesting because other ubiquitously encountered viral antigens such as influenza haemagglutinin have this property, at least in vitro [97].
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8. PROPOSED MECHANISMS FOR VIRAL INDUCTION OF AUTOIMMUNE DISEASES 8.1. General Theories Many general theories have been advanced to account for virus-induced autoimmune diseases (Table 3). However this classification is artificial since many mechanisms may operate simultaneously. In particular T and B cells may be activated relatively specifically because of defects in the immune system or as a secondary event in an inflammatory environment. It is especially difficult to determine what determines the transition form post-viral immunopathology to a sustained autoimmune response which is more than a passing phenomenon [98]. It is also necessary to consider ways in which viruses might cause organ specific or systemic autoimmune diseases in different circumstances [99]. If theories for a viral aetiology of autoimmune diseases are to prove useful, they must satisfy two sets of conditions. Firstly, they must fit what is already known about the disease in question. Above all, any postulated mechanism must account for an autoimmune attack on a dominant auto-antigen even if this is not confined to a single organ [19]. Indeed the theory must account for the limited number of autoantigens in general which are the targets for autoimmune responses [3]. Clearly it is essential to account for genetic susceptibility [ 112]. Since virus infections are common and autoimmune diseases are relatively uncommon. It is also essential to identify the factors which might lead to this outcome. The second condition is the possibility of testing the proposed contribution of the infection by a set of tests equivalent to Koch's postulates. In general terms there is a wide range of theoretical possibilities for viral infections to transform normally ephemeral autoimmune responses into persistent autoimmune diseases. As shown in the schematic diagram in Fig. 1 the risk of autoimmune disease is likely to increase in line with viral persistence. This conclusion is based on two considerations. Firstly, many studies have failed to show any link between acute virus infections and the onset of autoimmune diseases. Secondly, proposed mecha-
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Table 3. Proposed general mechanisms for virus-induced autoimmunity Mechanism
A) Non-specific hostfactors Cytokine dysregulation Complement deficiency Disturbed apoptosis Mitogenic effects ("superstimulation") Disturbed lymphocyte traffic Lymphocyte killing Disturbed antigen presentation B) Specific hostfactors Dysregulated autoantigen expression Neo-antigen production
C) Immunological recognition Mimicry Cross-presentation Epitope spreading Bystander involvement Immune deviation Polyclonal stimulation Superantigen Adjuvant effects D) Viralfactors Persistence - complete or defective? Virulent or attenuated Coding potential Susceptibility to environmental factors
Reference
[69] [64] [ 100] speculative [77] [ 101] [61] [ 102] mainly speculative [103] [ 104] [86] [ 105] [ 106] [ 107] [ 108] [ 109] [97] [ 110] (The EBV model) [55] [ 111] [52]
nisms such as auto-reactive T cell responses are unlikely to arise after acute infections. In the following sections we consider the most plausible of the proposed mechanisms.
8.2. Altered Autoantigens Theories based on a viral aetiology must take detailed account of the natural history of viral infections of postulated relevance. Persistent infection with the continuous production of viral encoded proteins is more likely to elicit conventional T cell or antibody responses to self antigens. Latent infection may not induce the production of any viral antigens but could still disrupt the regulation of immune responses to unrelated antigens. Indeed the most significant pathological consequences of
graduated evolution of autoimmunity after a viral infection
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Figure 1. In this schema autoimmune disease after viral infection is envisaged as a late complication of immunopathology generated by persistent infection. persistent herpesvirus infections concern gamma herpesviruses which, alone of this group, are able to persist in latent form. Another central issue is the starting point of proposed virus-induced autoimmune disease. One set of theories propose that the autoimmune response is driven by a normal immune response to autoantigens whose expression is qualitatively or quantitatively abnormal. According to the alternative, classical view the primary abnormality lies in the immune system. Indeed the most frequently advocated theories of virus induced autoimmune disease concentrate in altered patterns of antigen recognition by T cells secondary to continued immune stimulation by unaltered auto-antigens. Many mechanisms have been suggested by which auto-antigen expression might be affected by viral infection but there is tittle experimental information bearing on this issue. Viral infection might increase auto-antigen expression through its effects on the genetic regulation of this process. No structural changes in these antigens need be postulated and viral infection would operate in a manner analogous with autoimmune disease resulting from transgenic manipulation of antigen expression or experimentally induced exposure to an abnormal cytokine environment. This remains speculative. Altemafively, auto-antigens might be physically altered by direct association with viral antigens or by other
effects of contiguous viral replication. This hypothesis would be more persuasive if viruses implicated in the pathogenesis of autoimmune diseases used common target auto-antigens in their replication cycle. There is some evidence that this may occur. Antinuclear autoantibody characteristic of many systemic autoimmune diseases react with aminoacyl-tRNA synthetases which are also used in virus replication [ 113] thereby stimulating the notion that the autoreactivity might be primarily anti-viral. In contrast there is limited information concerning the strategies evolved by different viruses which allow the viral genome to obtain access to intra-nuclear replication sites. When it is forthcoming, the information is very surprising and encourages the idea that associations through this mechanism may stimulate autoreactivity. Intact adenovirus 2 is too large to penetrate pores in the nuclear membrane. The virus docks with the nuclear pore complex receptor CAN/Nup214, slowly disassembles, and transfers its genetic information through the nuclear pore while still attached to the nuclear pore receptor. The passage of viral genetic information to the cell nucleus is further assisted by the linker histone HI protein which normally travels backwards and forwards between the nucleoplasm and cytoplasm and acts as a transport vehicle in infected cells [ 102, 111]. Histones are the target for autoantibodies in many general autoimmune diseases and an asso-
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ciation with viral components could explain these responses. Indeed there is experimental evidence that viral antigens can act as haptens allowing T cells to initiate autoantibody responses to histones and nucleosomes by B cells encoding these autoantibodies as part of their normal repertoire [15]. These experiments suggest that T cells reactive with histone or the polyoma T antigen provide help for autoreactive B cells. Interestingly the frequency of reactive T cells and the complementarity determining sequences in the T cell receptors were similar in normal individuals and SLE patients. However this kind of speculation raises further issues such as the manner in which these antigens would become accessible to T cell recognition and the threshold necessary to elicit such a response. There is no evidence that any of the many virus infections implicated in the immunopathogenesis of type I diabetes disrupt the genes encoding target auto-antigens for the immune destruction of pancreatic islet beta cells. However targeted disruption of the gene encoding the protein tyrosine phosphataselike molecule IA-2 induces hyperglycaemia and impaired glucose tolerance in mice [ 114]. Anti-IA-2 autoantibodies may appear years before human type I diabetes becomes clinically manifest. It is intriguing to speculate that virus infections might produce similar disruption and insidious consequences. Classical immunological theory proposed that the release of sequestered antigens was a plausible mechanism for inducing autoimmune disease. This notion was upset by the realisation that tissue damage commonly results in temporary loss of peripheral tolerance. It has been partially revived by the discovery that autoimmune disease accompanies inherited or acquired defects in immune complex clearance including complexes between auto-antigens and autoantibodies. It is reasonable to propose that the destruction of virus infected cells either secondary to immune destruction or apoptosis might overwhelm clearance mechanisms especially in individuals with defective clearance. However there is little supporting evidence in general or in viral infections. There is some evidence that granzyme B secreted by cytotoxic lymphocytes cleaves auto-antigens into novel fragments during the process of cell death. Furthermore, these fragments are not generated during apoptosis initiated by other mechanisms. The inflammatory infiltrate in
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Table 4. Proposed "Koch's" postulates for autoimmune diseases 1) Sequencehomology (molecular mimicry) 2) Epitopepresented in immuogenic form after pathogen processing by antigen-presenting cells 3) Immunogenicconfiguration in vivo 4) T cell and/or B cell activation demonstrable in vitro 5) T cell and/or B cell activation demonstrable in vivo 6) Experimentalmodel shows that inappropriate recognition and activation produces an autoimmune disease 7) Evidencein a human autoimmune disease that cross reactivity contributes to the disease process.
salivary glands affected by Sjrgren's syndrome generates novel fragments of La, alpha-fodrin, and type 3 muscarinic actylcholine receptor. Presentation of alpha-fodrin and possibly other auto-antigens by class II molecules to lymphocytes can be blocked by inhibitors of cathepsin S leading to reduced salivary gland inflammation in a murine model of Sj/Sgren's disease [ll5]. These observations are of general interest but there is no information on the extent to which the presentation of intact or cleaved autoantigens in persistent viral infection contributes to autoimmune-mediated inflammation.
8.3. Molecular Mimicry Molecular mimicry is the process by which T cells recognise identical sequences in antigens encoded in viral infections and self antigens thereby providing help for autoreactive B cells. This mechanism is commonly proposed as mechanism for post-viral autoimmune disease but the available evidence remains fragmentary and unsatisfying. Much of it relies on sequence homologies derived from computer based searches. It is possible, indeed essential to devise "Koch's postulates" for attributing autoimmune diseases to this mechanism (Table 4). Some of these criteria can be satisfied fairly easily. However it is very difficult to validate in vitro findings either in experimental models or in human disease. Thus glutamic acid decarboxylase (GAD65) is a major autoantigen in type I diabetes and cross reacts with a peptide in human cytome-
galovirus DNA-binding protein. Furthermore the cytomegalovirus-derived peptide is processed by antigen presenting cells and stimulates T cells [116]. However the other criteria are notoriously difficult to satisfy.
8.4. Epitope Spreading Epitope spreading is defined as the process in which specific anti-viral responses in the early stages of infection becomes less sharply defined and extend to self antigens. Most of the evidence for this theory is derived from experimental models of virus-induced disease. There are many difficulties in applying the findings in experimental systems to clinical situations. Autoantibody specificity remains constant in organ-specific and generalised autoimmune diseases. This is not what one would predict from the theory of epitope spreading. Nor does it account for the restricted range of auto-antigens which are the target for immune attack, a point consistently and correctly emphasised. Credibility depends on demonstrating a peculiar vulnerability of these auto-antigens to immune attention in virus-induced immunopathology and so far there is little evidence to this effect.
8.5. Bystander Effect The bystander effect is defined as a process whereby the continued immune response to infection and attendant inflammation allows exposure of normally sequestered auto-antigens to the immune response. Theoretically this could operate by T cell recognition resulting in help for potentially auto-reactive B cells. Alternatively auto-antigens could bypass T cell help by stimulating B cells responding polyclonally to mitogens generated secondary to tissue damage or virus infection. The distinction between this mechanism and epitope spreading is a fine one and adds little to the difficulties of dissecting the pathogenesis of human autoimmune diseases.
8.6. Virus Induced Apoptosis Genetically determined defects in apoptosis clearly account for immunoproliferative B cell diseases leading to autoimmunity in mice and similar defects
may operate in human autoimmune diseases. In contrast novel treatment which augments B cell apoptosis through Fas-and TNF receptor-independent mechanisms suppresses autoimmunity [ 117]. In addition, defective removal of apoptotic cells predisposes to autoimmunity as occurs in Ciq defective mice and humans. Accumulating complexes trigger autoantibody production by B cells which form immune complexes and also convert anti-inflammatory reactions by macrophages into pro-inflammatory ones [118]. The situation in organ specific autoimmune diseases is still more complicated. One interpretation of thyroid autoimmune disease proposes that increased thyrocyte apoptosis contributes to this process. However this might be the primary event or secondary to an autoimmune attack. It has also been proposed that up-regulation of the TNF receptor superfamily is the primary event which attracts pro-apoptotic T cell ligands [119]. Furthermore thyrocytes themselves may be able to kill other thyrocytes through caspase mediated apoptosis independent of lymphocyte-mediated apoptosis [120]. Alternatively ganzyme B released by infiltrating T cells might increase apoptosis[121]. Furthermore Fas-mediated apoptosis may also lead to cell death. However the pattern of Fas and FasL expression by infiltrating lymphocytes and thyrocytes is different in Hashimoto's thyroiditis and hyperthyroidism suggesting more complex explanations [ 119, 122]. One can argue simplistically that viruses interfere with apoptosis as a strategy for survival because prolonging host cell life favours viral persistence [ 100]. Viruses modulate both the apoptotic machinery of the cell and also the extrinsic pathway mediated by TNF-alpha. However some viral infections increase apoptosis through their direct effects on infected cells. Interestingly too, they may provoke apoptosis through indirect mechanisms which may be relevant to human autoimmune disease. Scleroderma is marked by major micro-vascular damage, endothelial cell apoptosis, and fibroblastic proliferation. Sera from scleroderma patients contain an autoantibody against the surface integrin-NAG-2 complex on endothelial cells which induces their apoptosis. This antibody also reacts with the homologous cytomegalovirus late protein UL94 suggesting virus mimicry secondary to infection by this virus [123]. Viruses also abet their persistence by
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killing lymphocytes through activating apoptosis. The lymphopenia accompanying many infections not only blunts specific immunity to the invading virus but also disrupts other cell populations with unpredictable consequences [ 101]. In contrast, killing virus-infected cells enables the host to eliminate infection at the cost of losing that cell's normal function. Currently one can only speculate about the possibility that increased apoptosis secondary to viral infection contributes to autoimmune disease in individuals with overtly or subtly defective clearance mechanisms. 8.7. Other Mechanisms
In considering currently popular theories we should not lose sight of other possibilities. One interesting requirement for autoreactive T cells to induce disease is their ability to home to the target organ. Lymphocyte homing through interactions with endothelial cells is a major area of study. Less considered is the need for virus specific CD8 T cells to home accurately to infected tissues as part of an efficient host response. For example herpesvirus type 2 specific T cells have been shown to express cutaneous lymphocyte-associated antigen (CLA) thereby suggesting a means by which they can home to cells infected by this virus [124]. The intriguing possibility that deviant homing may also occur is unexplored.
9. MODELS OF VIRUS INDUCED AUTOIMMUNE DISEASE
Relatively few animal models have been used to explore virus-induced autoimmune disease. Even in these models there are difficulties in distinguishing in ascribing a primary role to autoimmunity. Indeed the complexities perversely reproduce the difficulties in dissecting the human diseases they are intended to help clarify. 9.1. Viral Myocarditis
Coxsackie B3 virus induces myocarditis in mice which closely resembles the human disease but the contribution of autoimmunity is difficult to delineate. The model is potentially useful in some respects
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since the pathogenesis in mouse and man have features in common. CB3 damages cardiac muscle in the majority of infected strains irrespective of their genetic background yet chronic myocarditis develops only in a minority of strains. The outcome of the infection is dependent on host anti-viral irmnunity and manipulating this response reveals some of the factors predisposing to chronic inflammation. For example transgenic mice expressing interferon gamma coding genes in their islet cells have raised circulating interferon levels and are protected from myocarditis [125]. Although it is not clear which of the proposed mechanisms for virus-induced myocarditis are operating, it seems most likely that autoimmunity is induced by antigens released from muscle fibres damaged by viral infection rather than mimicry [106]. Nevertheless these experiments do not exclude the possibility that autoimmunity is a secondary response proportional to the extent of viral; replication and the resulting inflammatory reaction. 9.2. Theiler's Virus
Theiler's murine encephalomyelitis agent, generally termed Theiler's virus, induces chronic inflammation and demyelination of the spinal cord in mice [ 126, 127]. It provides an attractive model for those investigators who believe that multiple sclerosis and other human demyelinating diseases result from virus-induced autoimmunity. Virus persistence is a crucial feature essential as the agent can readily be isolated. The innate and specific immune response to the virus are genetically controlled and determine the levels of viral replication. T cell responses to basic myelin protein antigens can readily be demonstrated. Epitope spreading has been invoked as the most likely mechanism operating in this model by analogy with experimental autoimmune encephalomyelitis induced by immunisation with the dominant epitope of myelin [105]. The T cell responses to myelin antigens seems to progress in a regular order determined by the efficiency with which these antigens are processed and presented to autoreactive T cells [128]. Furthermore the disease can be adoptively transferred to normal mice by autoreactive T cells. Unfortunately, the absolute requirement for viral persistence makes it difficult to decide whether anti-myelin autoreactivity contributes to
the lesions or is a secondary phenomenon irrespective of the mechanism or mechanisms responsible for its induction. Certainly, adoptive transfer shows that the sensitised T cells damage myelin in the new host but it is difficult to extrapolate too uncritically about their role in the original host.
9.3. Herpesvirus Keratitis Herpesvirus 1 (HV-1) induces stromal keratitis in the eyes of infected mice [8, 104, 129]. Not all mouse strains are susceptible. Genetic factors determine both the level of viral replication and the nature of the host immune response; both these factors determine disease susceptibility. In this model viral persistence is also indispensable. Although HV-1 infection initiates the disease, corneal inflammation is delayed for 1-2 weeks after the infection by which time persistent local infection is difficult to demonstrate. However this is not surprising in the face of a fully established host anti-viral immune response. Autoimmune reactions also appear at this stage of inflammation. Some T cells in the lesions react with a corneal antigen and others with an IgG2a sequence. Furthermore T cell clones stimulated in vitro with HV-1 infected cells recognise a viral peptide identical with this IgG sequence. Infected cells infected with an HV-1 variant which do not encode this viral peptide fail to induce T cell reactivity with the IgG determinant, suggesting that the viral peptide is essential for inducing the inflammatory disease. An additional argument in favour of the pathogenic importance of this viral peptide is the inability of HV-1 infected immunodeficient mice to develop herpetic stromal keratitits unless immunised with the same peptide. At first sight these observations seem to indicate that the inflammation is mediated by virus-reactive T cells which recognise an auto-antigen through molecular mimicry. However other observations make this argument less compelling. Firstly, viral infection of the host or stimulating cell lines is indispensable. Secondly, the target corneal antigen for the postulated autoimmune attack has not been identified. Finally there is other evidence that herpes stromal keratitis can be induced in mice which are unable to mount a T cell response to the allegedly crucial HV-1 or IgG antigens. Other evidence suggests that quantitative factors also determine susceptibility to
the disease. Specific HV-1 infection is essential for disease induction in mice with low numbers of autoreactive T cells. Furthermore, non-specific stimuli suffice to induce disease in mice with high numbers of autoreactive T cells [99, 130]. Cross reaction between the retinal S antigen and viral pepetides has also been proposed mainly on the basis of T cell proliferative responses [131] but the evidence remains fragmentary.
9.4. Murine Cytomegalovirus Infection and SjSgren's Syndrome Cytomegalovirus infection in mice (MCMV) provides an interesting model of the systemic autoimmune disease Sj6gren's syndrome [132]. The human disease is characterised by chronic destructive inflammation of the salivary and lacrimal glands, and B cell proliferation with autoantibody production notably to the Ro/SSA and La/SSB auto-antigens. Four different mouse strains infected with MCMV developed acute sialadenitis. However chronic sialadenitits with the characteristic autoantibodies developed only in the B6-1pr/lpr strain with a genetic background of defective Fas mediated apoptosis. Infectious MCMV could not be detected after 100 days post-infection. This group reported a similar outcome in MCMV infected B6-gld/gld mice which carry a defective ligand (FasL) and hence have impaired Fas-mediated apoptosis [ 133]. The chronic sialadenitis was partially reversed by local FasL treatment. This model is of particular interest because, although MCMV persistence may be a prerequisite for disease development, this postulate is difficult to prove. Furthermore the pattern of inflammatory disease in target tissues and the autoantibody profile closely resemble the spontaneous human disease.
10. GENERAL EVIDENCE FOR A VIRAL A E T I O L O G Y IN HUMAN AUTOIMMUNE DISEASES There have been many attempts to identify viruses contributing in whole or in part to the pathogenesis of human autoimmune disease [8]. The stimulus for a continued search for a viral aetiology of human autoimmune diseases rests largely on theoretical
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speculation and clinical observation of post-viral autoimmune phenomena. Acute viral infections commonly induce transient autoimmune responses usually directed at circulating bone marrow progeny namely platelets, neutrophils, and red cells.There is also a high incidence of transient autoantibodies and disease manifestations such as arthritis after viral infection or immunisation [134] but these rarely progress into established autoimmune disease. For example the polyarthritis provoked by parvovirus B 19 or rubella infection rarely progresses to chronic arthritis such as rheumatoid arthritis. These almost invariably transient events are conventionally attributed to polyclonal B cell activation but this is likely to prove an over-simplification. If viral infections do contribute in whole or in part to these diseases, we must postulate delayed mechanisms which only become evident a long time after the initial infection. The most plausible proposition is viral persistence. If the viral genome persists in latent form without any transcription of viral proteins, there might be an indefinite silent period without any disease manifestations. However even in these circumstances there is the theoretical possibility that the viral genome may alter the expression of host antigens. Indeed it is theoretically possible that transient infection may alter the regulation and expression of host genes encoding autoantigens without the need for persistence of any viral genes. However the lessons learnt from viral oncology make it more likely that viruses would need to persist in some form to elicit autoimmune diseases. In the current state of knowledge it seems more likely that some expression of virus-coded proteins would be needed to induce an autoimmune response. Many human viruses persist indefinitely which are transcribed continually or intermittently. Most prominent among these agents are herpes viruses, measles virus, and retroviruses. There have been many attempts to link viral infections with autoimmune diseases. Many studies have used classical, indirect techniques, notably antibody titres. To date these studies have been negative or inconclusive. Early studies compared anti-viral antibody titres in patients and controls but the results were often difficult to interpret. Thus the polyclonal hypergammaglobulinaemia of systemic lupus erythematosus (SLE) generates a non-specific increase in anti-viral antibody titres which reflect accumulated immunological memory but
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not necessarily infections relevant to the aetiology of the disease. Antibody titres to viral antigens are often a poor guide to long term immunopathological consequences even when the link with disease is indisputable. For example there is a poor correlation between antibody titres and recurrent "cold sores" induced by herpes virus type 1 or Epstein-Barr (EBV) infection and Burkitt's lymphoma. Both viruses persist life long in most individuals yet cold sores affect only 10% of the population and the incidence of Burkitt's lymphoma is low even in susceptible populations. Yet EBV infection admirably illustrates the difficulties in relying on conventional markers of viral persistence as evidence for the role of a given virus in disease pathogenesis. The association between EBV infection and Burkitt's lymphoma was established mainly on epidemiological grounds. There is now direct evidence that selective EBV gene latency in B cells provokes proliferation of the infected B memory cells and evasion of the cytotoxic T cell response lymphoma cells thereby allowing the emergence of Burkitt's lymphoma clones [ 135]. Whether EBV acts in a similar manner to generate conventionally benign but autonomous autoantibody producing B cell clones is speculative. Almost universal infection EBV makes it very difficult to correlate classical markers of infection such as anti-viral antibody titres with autoimmune diseases which almost certainly develop many years after primary infection. Attempts to link EBV infection with multiple sclerosis illustrate this dilemma [136, 137]. These studies established a strong association between high titres of anti-VCA and EBNA2 antibodies encoded by EBV and multiple sclerosis. However it is difficult to determine whether this increase results from an increased virulence of common virus infections in patients destined to develop the disease, a direct aetiological relationship, or the role of EBV as an adjuvant in an unrelated pathological process. Similarly the detection of the EBV genome in cells involved in an autoimmune disease does not establish an aetiological role for the virus. The same difficulties arise from attempts to implicate EBV infection in the pathogenesis of systemic lupus erythematosus (SLE). These studies have been encouraged by other evidence such as sequence homology between the EBV peptide PPPGRRP and the peptide PPPGMRPP of the SM B'B antigen of the human spliceosome. Cross
reactivity to these sequences might account for the Sm reactive autoantibody encountered in SLE patients. A higher percentage of peripheral blood B cells from SLE patients than normal controls carry the EBV genome [138], but it is quite possible that this is secondary to the polyclonal B cell activation characteristic of this disease. In addition anti-EBV antibodies are detectable in a higher percentage of SLE patients than controls [139], but this could easily be a secondary event. There is evidence from other situations that viruses may act as adjuvants rather than prime causes of immune-mediated disease. An analysis of heart transplant rejection illustrates this dilemma. Although this process is not strictly analogous with autoimmune myocarditis, the initiating immune disorder at least has a known starting point. Heart transplant rejection in children is increased 6.5 fold if the transplant is infected by viruses, notably adenoviruses [ 140]. The evidence from studies using other techniques is still more fragmentary. The results of attempted viral isolation from target tissues and organs involved in autoimmune diseases are contentious and also difficult to interpret because any immunoproliferative disorder is likely to reactivate latent virus infections. Many studies have sought evidence based on molecular mimicry and other mechanisms invoked in experimental models of virus-induced autoimmune diseases. The same problems of interpretation arise in clinical studies. Attempts to incriminate specific viral infections in human autoimmune disease by invoking molecular mimicry are not persuasive if the claims are based solely on sequence homology. It is relatively easy to obtain these data from sequence banks. It is far more difficult to establish that the viral sequence induces a cross reactive T cell response in vivo. Many other considerations come into play including tertiary structure, accessibility to T cells, and evidence that the sequence is presented in immunogenic form. Nevertheless viral infections which indubitably provoke systemic diseases would be considered primary autoimmune diseases in the absence of this information. Hepatitis C virus infection induces a disease with many features resembling idiopathic SLE. Furthermore, a possible mechanism involving molecular mimicry has been identified [ 141 ]. CD8 T cells reactive with liver cytochrome P450 peptide
Table 5. Human autoimmune diseases investigated for a viral aetiology Disease
Reference
A) Organ or system specific
Type I diabetes Thyroiditis Myocarditis Multiple sclerosis Peripheral neuropathy Polymyositis
[143] [144] [106] [127] [145] [ 146]
B) Systemic
Rheumatoid arthritis Systemic lupus erythematosus Sj6gren' s syndrome
[147] [ 148] [149]
sequences also react to homologous sequences encoded by hepatitis C virus. The reactivity is HLA class II restricted. Although the features of hepatitis C virus associated SLE differ in some respects from those encountered in idiopathic SLE, there are many common features [ 142].
11. EVIDENCE FROM SPECIFIC HUMAN AUTOIMMUNE DISEASES The autoimmune diseases which have been most intensively investigated for a viral aetiology are listed in Table 5. A review of the evidence for a viral aetiology in some of these diseases illustrates the difficulties. 11.1. Type 1 Diabetes Virus-induced experimental models of type I diabetes highlight the difficulties in investigating the human disease [150]. The M variant of the picornavirus encephalomyocarditis virus (EMCM) induces a syndrome resembling human type I diabetes in genetically susceptible mice. The small nucleotide differences distinguishing the diabetogenic from other strains have been characterised and centre on the crucial position of an alanine in a highly conserved, strongly hydrophilic part of the sequence. This site governs viral attachment to pancreatic islet beta cells and subsequent infection. Heavily infected islet cells are rapidly destroyed by
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viral replication and innate immune mechanisms, notably macrophages and TNF-alpha. Low titre systemic and islet cell infection by virus results in their more protracted destruction by macrophages activated through Src kinases. The Kilham rat (KRV) parvovirus causes diabetes in rats which are genetically resistant to the spontaneous disease. However, in contrast with the EMC-M model, KRV does not infect islet cells but initiates autoimmune destruction of these cells through macrophage activation and the preferential activation of cytotoxic T cells. These observations indicate that selected virus strains can induce diabetes in genetically susceptible hosts through mechanisms which do not invariably involve T cell destruction of infected islet cells. There is no guarantee that the model has any relevance to the human disease. If they are relevant, the challenges for investigators are formidable. Routine antibody screening is unlikely to detect infection by diabetogenic strains. Islet cells from diabetic patients are hardly ever available for analysis. Even if material was available, the KRV model indicates that even the most detailed virological analysis might be unrewarding. It is therefore not surprising that the search for a viral aetiology in the human disorder has been at best inconclusive [143]. The polygenic susceptibility of patients to autoimmune destruction of islet beta cells has been established beyond reasonable doubt. However no specific infection has been identified which might be a plausible primary cause of the disease or might exacerbate a primary autoimmune process unrelated to infection. The hope of progress depends on the development of culture systems with tissues from genetically well characterised donors which permit the study of interactions between islet cells, potentially diabetogenic virus strains, and the immune system.
11.2. Autoimmune Thyroid Diseases Autoimmune thyroid diseases are important for many reasons. They are the commonest diseases in which autoimmunity is unequivocally the most likely key to their aetiology. The incidence of autoimmune thyroiditis in middle age women, the most susceptible group, is around 2%. Intriguingly, the average incidence of thyroid autoantibodies in different populations in this group is about 15%; in
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the majority of these individuals anti-thyroid autoantibodies have no apparent clinical significance but they may nevertheless be attributable to thyroiditis. Despite formidable technical difficulties, the antigen-autoantibody systems in autoimmune thyroid disease have been largely characterised [144]. The main target autoantigen in autoimmune hypothyroidism is the cell surface protein thyroid peroxidase (TPO). The main target autoantigen in hyperthyroidism is the thyrotrophin receptor (TSHR), a G protein with seven membrane-spanning segments. In cultured cells, the extra-cellular subunit A is shed from the cell surface during cleavage. TPO is the target of T-cell mediated autoimunity as well as autoantibodies. TPO-reactive autoantibodies have been isolated from immunoglobulin gene recombinatorial libraries constructed from thyroid infiltrating B lymphocytes. The germline H and L genes are similar to those in many other antibodies. However the H chain genes show a high degree of somatic mutation characteristic of antigen driven maturation and consistent with their high affinity. Defective receptor editing may also contribute to the generation of TPO-reactive autoantibodies [151]. Four largely overlapping epitopic domains have been identified. Interestingly, the epitope specificity of TPO autoantibodies from individual patients remains constant for many years with no evidence of epitope spreading. Because of technical difficulties, TSHR antibodies and their epitope specificities have been less fully characterised. TPO-reactive autoantibodies from patients with hyperthyrodism preferentially select different VH domains from those associated with hypothyroidism [151]. There is little information on the epitopes recognised by infiltrating T cells. No convincing link with viral infection has been established by indirect means. There is meagre information about the long term natural history of patients with acute, self-limited thyroiditis. A detailed survey showed no association with common viral infections of childhood or immunisation against these infections [152]. In thyroid disease, this failure can not be attributed to lack of material which has been readily available from surgical or biopsy material. Thyroiditis may be associated with hepatitis C virus infection even before interferonalpha treatment is started [ 153]. However, although
the incidence of thyroid autoantibodies [12.1%] was higher than in controls [4.0%], the prevalence of autoantibodies to TPO in infected individuals was the same. Nor was the increase associated with detectable thyroid dysfunction. In general, detailed knowledge of the auto-antigens and autoantibodies contributing to thyroid autoimmune disease has not resulted in any substantial clues to a viral aetiology.
11.3. Post-Infectious Polyneuropathy and Related Neurological Syndromes Campylobacter jejuni is the commonest identifiable cause of post-infective polyneuropathy (Guillain Barre syndrome) but viral infection is implicated in some 20% of patients. In theory nerve damage following Campylobacter jejuni infection may result from autoantibodies to about 20 distinct gangliosides provoked by molecular mimicry with similar carbohydrate sequences in bacterial lipopolysaccharides. The fine specificity of these autoantibodies is probably crucial. Anti-GQlb ganglioside autoantibodies demonstrably damage the motor nerve terminal through a complement dependent mechanism. A particularly striking association between these autoantibodies and neuropathy is seen in Miller-Fisher syndrome in which patients develop ataxia, areflexia, and ophthalmoplegia [154]. In contrast, anti-GM2 ganglioside antibodies are less specifically associated with neuropathy and their neurotoxic potential is more dubious [145]. These observations point to the possibility that similar mimicries might account for post-viral peripheral neuropathy but emphasise the difficulties in ascribing pathological significance to post-infectious autoantibodies even when these are detected.
11.4. Polymyositis, Myocarditis and Related Diseases The search for a viral aetiology for polymyositis, muocarditis, and related diseases has produced tantalising clues but no convincing solution. Coxsackieviruses have received particular attention because some strains induce myocarditis in mice and myalgia is a prominent symptom of myalgia in human coxsackievirus infections. However it is fair to conclude that the search for persistent or latent virus in affected striated or heart muscle, serological
studies, and assays of anti-viral T cell reactions have produced conflicting and often controversial results. Coxsackievirus infections are common and 60-80% of a given population have antibodies against the prevalent strains. In contrast polymyositis is uncommon with a prevalence ranging from 2.4-10.7 per 100,000 [146]. Clearly, if an association between coxsackievirus infection and polymyositis exists, it results from the interaction of a peculiar strain with an unusual host. This requirement is underscored by early attempts to establish a viral model of polymyositis which was eventually achieved by infecting CD1 Swiss mice less than 48 hours old with the Tucson strain of coxsackie B 1 virus [155]. Interesting features of the model were the disappearance of detectable virus despite persistent myositis and the difficulties in distinguishing myopathic from nonmyopathic virus strains by their virological properties. Subsequent studies have shown that myotropic, myopathic clones of coxsackie B 1 virus differ from myotropic but non-myopathic clones by 20 nucleotides. Only myopathic clones induced anti-muscle and anti-nuclear antibodies [ 156]. Non-viral models of polymyositis introduce the important concept that virus infections may initiate an inflammatory response which is perpetuated by autoimmunity unrelated to classical therories. For example an initial increased expression of class I HLA antigens might be the critical event [11]. Intriguingly, auto-antigenic aminoacyl-tRNA synthetases released from damaged muscles may be chemotactic for inflammatory cells thereby stimulating chronic autoreactive inflammation [157]. These observations are important because they emphasise the potential contribution of immune mechanisms to autoimmune disease which are not mediated by auto-reactive T and B cell clones. Further grist for the idea of viral initiation without persistence comes from the observation that adeno-associated virus, a plausible cause of inflammatory myositis, is less likely to be found in myositic muscle than in muscles from normal individuals or patients with non-inflammatory myopathies [158].
11.5. Anti-Phospholipid Syndrome The anti-phosphoplipid syndrome (APS) causes thrombotic episodes with a protean range of clinical problems including cerebral ischaemic
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episodes, early foetal loss, and pulmonary infarction. Although its florid clinical presentations are well recognised, it may be involved in many other disorders whose pathology involves endothelial cell damage and small vessel occlusion. The responsible autoantibody is directed at beta2-glycoprotein-1. Many microbial infections have been implicated in this syndrome including hepatitis C virus and EBV and there is strong evidence that these infections induce antibodies to microbial peptides which cross-react with anti-beta2-glycoprotein- 1 [ 159].
11.6. Chronic Urticaria Justifiable preoccupation with severe, often life threatening autoimmune diseases is liable to distract attention from other problems which, although less devastating, are nevertheless common, chronic, and often very distressing. The rash and angioedema of chronic urticaria come into this category and can be so severe as to necessitate treatment with immunosuppressive drugs or plasma exchange. Specific causes such as drug allergy can be identified in many patients but in some 70% there is no obvious cause. However some 20% of patients give a history of a preceding infection which is usually upper respiratory and has features suggesting a viral infection. In one third of patients chronic urticaria results from histamine release mediated by autoantibodies to the high affinity IgE receptor FcepsilonR1 or IgE itself [160]. As interest in the pathogenesis of chronic urticaria shifts from a preoccupation with food intolerance and "pseudoallergy", observations of this kind rightly draw attention to common mechanisms which may underlie allergic and autoimmune diseases [ 17].
12. EVIDENCE THAT THE PRIMARY EVENT IN ORGAN SPECIFIC AUTOIMMUNITY MAY NOT BE IMMUNOLOGICALLY MEDIATED A major problem in autoimmune diseases is to determine the extent to which autoimmunity is the primary event in diseases undoubtedly accompanied by autoimmune reactions. This remains an issue even in many virus-induced models of autoimmune diseases where viral growth and per-
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sistence are exacerbated by hosts with defective innate or specific immunity. It can be argued that autoimmunity contributes to tissue damage but only as a secondar3, event. There are also situations in which autoimmune disease is provoked by viral infections because of pre-existing or virus-induced defects which are unrelated to host immunity. For example abnormalities in the dystrophin coding gene predispose to cardiomyopathy. Coxcsackie B3 virus (CVB3) encodes a protease which cleaves dystrophin and disrupts the dystrophin-glycoprotein complex with consequences resembling those encountered in hereditary disease. CVB3 causes more severe cardiomyopathy in dystrophin deficient than in wild mice [ 161 ]. There are similar situations in human disease. Thrombotic thrombocytopenic purpura is a disease characterised by intra-vascular destruction of red cells and platelets which results in the formation of platelet micro-thrombi and small vessel obstruction. It follows viral infections in seemingly normal individuals or in those already affected by chronic inflammatory diseases such as juvenile chronic arthritis. Susceptibility to this chain of events results from an inherited defect in the proteolytic breakdown of the clotting factor von Willebrand factor so that the formation of microthrombi is encouraged [162]. Interactions between virus infection, apoptosis, and neuro-degenerative disease are a still more subtle illustration of degenerative disease which can later be interpreted as autoimmune in origin. A provirus insertion in apoptosis-inducing factor (Aif) in Harlequin mice interferes with the apoptosis of neurones damaged by oxidative stress. Damaged cells which would normally be removed by apoptosis re-enter the cell cycle and their exposure to oxidative stress results in neurodegeneration [163]. Observations of this kind enjoin caution about necessarily accepting that autoimmune aggression is the primary cause of common diseases associated with autoimmunity. The association between thyroiditis, the commonest organ specific autoimmune disease, and Down's syndrome, the commonest genetic disorder is well established but the implications of a seemingly nonimmunological genetic disorder for later autoimmunity are unexplained [ 164, 165].
13. RETROVIRUSES Retroviruses in many different species are recognised causes of degenerative and immunodeficiency diseases accompanied by some autoimmune features, especially directed at red cells and platelets. HIV infection in man exacerbates some inflammatory disorders with a possible autoimmune component such as psoriasis and Reiter's syndrome. The variable temporal relationship between autoimmune platelet destruction and other manifestations of infection indicates that autoimmune mechanisms may be subtle [166]. There have been suggestions that the characteristic depletion of CD4 T cells is mediated at least in part through autoimmunity induced by viral mimicry [167, 168], and not exclusively or even predominantly through direct viral infection of susceptible cells. However the view that HIV is irrelevant to the pathogenesis of AIDS has lost whatever credence it first enjoyed. An infectious retrovirus designated HRV-5 has been detected in synovial tissue from rheumatoid arthritis patients and blood mononuclear cells of patients with this disease or SLE [ 169]. However in general attempts to attribute human autoimmune diseases such as rheumatoid arthritis and SLE to retroviruses transmitted by conventional infection have been unsuccessful [ 148, 170]. A more difficult subject to address is the possibility that inherited or acquired endogenous retroviruses are involved in the pathogenesis of autoimmune diseases [171]. Theoretically, these sequences could disrupt the regulation of the myriad of the components of innate and acquired immunity which contribute to these diseases. Endogenous retroviral sequences have been detected in normal and rheumatoid synovial membranes. However there is evidence that these sequences may be selectively expressed in rheumatoid arthritis. This expression could be linked to the intriguing histological observation that synovial cell proliferation in early rheumatoid arthritis appears to precede the immunopathological events which later dominate the histological picture. However, as always in inflammatory lesions a major problem is to determine whether the altered expression of endogenous retroviral gene sequences is a primary event or the consequence of immunoproliferative disease. Retrotransposons have also been linked with
autoimmune diseases. These genetic elements resemble retroviruses but lack the env gene. They can be transmitted between genes. A retrotransposon inserted in thefas gene of MRL lpr/Ipr accounts for the destructive synovial hyperplasia accompanying the lymphoproliferative disease characteristic of this strain. Retrotransposons have been consistently detected in rheumatoid synovial memebranes. Their RNA sequences are similar to ORF2/L1 and THE1 retrotransposons, human endogenous retrovirus (ERV)-E, and other elements [ 147]. In theory, these sequences could up-regulate the genes controlling the cytokines and kinases responsible for synovial inflammation. However these changes could also be secondary to hyperplasia induced by totally unrelated events. Cytokine activation of retroviral superantigens secondary to conventional virus infection has also been postulated as the mechanism responsible for pancreatic beta cell destruction in diabetes [109]. As yet these issues remain unresolved and there is no conclusive evidence that retroviruses in any form cause human autoimmune diseases.
14. IMMUNOLOGICAL SURVEILLANCE Apoptosis is a proven strategy by which the host eliminates virus-infected cells. This is achieved by activating intrinsic pathways or by extrinsic inflammatory cells and their products. It is also possible that apoptosis is a mechanism for removing cells damaged more subtly by viral infection which do not display any obvious evidence of viral infection. Retroviruses are often cited as persistent agents with the capacity to damage cells directly as well as to induce immunopathological mechanisms. Indeed the concept of immunological surveillance of endogenous retroviral sequences has been made the basis of a general theory for autoimmune diseases [172]. Autoimmune diseases are thereby considered the price of controlling DNA damage. The indirect or delayed consequences of viral infection in general might not be revealed by conventional studies of primary infections and their outcome. For example, although B19 parvovirus infection does not induce chronic destructive arthritis, there is some evidence that it induces an invasive phenotype in normal human synovial fibroblasts [173].
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Apoptosis secondary to immune mediated inflammarion and cell lysis by traditional immunological mechanisms could be perceived as part of a general defence strategy. However these ideas are currently mainly speculative.
15. E X T E N D E D KOCH'S POSTULATES
The complexities of host defence mechanisms and the many viral strategies for persistence make any attempt to adapt Koch's postulates to this field extremely difficult. Attempts to devise credible presuppose that the disease in question presuppose that its autoimmune nature has been unequivocally established. We suggest the following scheme: 1) The virus or its products can be consistently identified in patients with a given autoimmune disease. 2) The virus persists in a form in which it is able to initiate the autoimmune mechanisms responsible for the disease. 3) There are demonstrable mechanisms by which the virus induces the autoimmune disease. 4) The autoimmune reactions must be related to viral infection. 5) There are host features in patients with autoimmune disease attributed to the virus which confer susceptibility to the disease and distinguish them from other individuals infected by the same virus who do not develop the disease. 6) The virus induces comparable disease in an animal model.
16. CONCLUSIONS There are obvious difficulties in se problems in attempting to ascribe a viral aetiology to autoimmune diseases [7]. Nevertheless there are certain points relating to this issue which are generally recognised as fundamental, even if these are hard to resolve [ 19]. Any theory must account for autoantigenic specificity and the polygenic factors conferring susceptibility to disease. The classical dogmas about loss of T or B cell tolerance or a combination of these cell populations are still valid but the factors which lead to the breakdown of tolerance are
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more complex than was formerly envisaged. The complexities of host defence against viral infections are increasingly apparent. Furthermore the classical, sharp distinction between innate, non-specific immunity and specific immunity is no longer tenable since these contribute to host defence in an integrated manner. Auto-reactivity is part of the normal T and B cell repertoire of responses and contributes to anti-viral immunity. In a sense, peripheral tolerance is regularly broken as part of the host strategy for destroying infected cells. Nevertheless the central tenet of classical tolerance remains unshaken; autoimmunity may be common but persistent autoimmune diseases are seemingly rare after viral infection. Autoimmune disease results from a wide range of very different defects. Some are entirely genetic and distort the proliferation of potentially reactive immune cells. Others are only apparent after immune stimulation including microbial infection. Although the general principle of abnormal immune regulation has been validated, early monotheistic views attributing autoimmune diseases to a failure of suppressor T cells have been abandoned now that so many pathways for excessive inflammation have been discovered. To add to the possibilities, viral strategies for persistence further disrupt the control of inflammation with varying risks for abetting sustained autoimmune response. There is little evidence that viruses are the primary cause of most organ specific and general autoimmune diseases. Experimental models of virus-induced autoimmune diseases have served mainly to uncover new complexities of observation and interpretation. A major difficulty is to discern whether autoimmunity in these models is the initiating event or secondary to inflammation. Classical techniques for implicating viruses in human disease have produced hints but no solutions. One difficulty is the likely heterogeneity of many human diseases which have been given a single label. The immune complex disease and autoimmune processes produced by hepatitis C virus, for example, may satisfy rather arbitrary criteria for the diagnosis of SLE. However clearly SLE does not have a single cause. It is more likely that a common immune defect confers susceptibility to this disease from a variety of causes including viruses. Nevertheless the slow progress in this field is insufficient reason for abandoning an investigative route which remains
plausible and suggestive evidence is still forthcoming. Put pithily, persistent inflammation is caused by infection when we know the agent and attributed to autoimmunity when we do not [ 174]. The prospects for progress depend on advances in specific areas. As discussed, we need a modem version of Koch's postulates which not only identifies the suspected agent but also indicates why ubiquitous viruses might cause autoimmune disease only in a susceptible individuals. It is unlikely than novel observations will disclose a simple cause and effect relationship between agent and disease. Indeed this has not proved to be the case in the classic example of tuberculosis. Analysis of viral and host gene expression and the products they encode is crucial to this process. Otherwise issues such as molecular mimicry and linked immunogenic expression of host and viral encoded genes will remain unsolved. Furthermore the chain of events which lead from initial infection to sustained autoimmunity is likely to depend on quantitative factors which can never be spelt out adequately in descriptive terms. New technologies giving new data and insights into this question. In particular mapping the human genome and the science of proteomics are key scientific developments which will enable us to determine the genetic coding and degree of expression of self antigens already recognised as targets for autoimmune attack. Furthermore microarray technologies may well identify new targets for autoimmune attack and should help to distinguish between auto-antigens encoded by host genes and viral genes. Moreover these auto-antigens may prove to be encoded by viral genes incorporated into the genome following conventional infection or transmitted vertically in the germ line. The enormous mass of information on viral life span, viral interactions with the many components of the host response, the cross-talk between these components, and the genetic control of each step of this process continues to generate a wealth of defensible hypotheses but few certainties. Only an integrated, computed model of the interactions between virus and host responses will suffice [175]. From a different perspective, traditional ideas about viruses as infective agents may have underestimated the vast range of outcomes of virus infection. The intellectual separation of "viruses" and "genes" which has characterised so much of our
thinking about autoimmune diseases may be largely illusory. "The virus, instead of being single-minded agents of disease and death, now begin to look more like mobile genes. Evolution is still an infinitely long and tedious biological game, with only the winners staying at the table, but the rules beginning to look more flexible. We live in a dancing matrix of viruses; they dart, rather like bees, from organism to organism, from plant to insect to mammal to me and back again, and into the sea, tugging along pieces of this genome, strings of genes from that, transplanting grafts of DNA. passing around heredity as though at a great party. They may be a mechanism for keeping new, mutant kinds of DNA in the widest circulation amongst us. If this true, the odd virus disease, on which we all focus so much of our attention in medicine, may be looked on as an accident, something dropped" [ 176].
ACKNOWLEDGEMENTS The authors would like to thank Dr. Evelyn Denman and Dr. Tatiana Dvorkin for their invaluable help in preparing the manuscript. This work was supported in part by a grant (BRZ) from the Center for the Study of Emerging Diseases and the Israel Science Foundation.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
How Transgenic Mouse Models Contribute to a Better Understanding of Virus-Induced Autoimmunity /
Philippe K r e b s 1,2 and Burkhard Ludewig ~
IKantonal Hospital St. Gallen, Research Department, St. Gallen, Switzerland; 2Institute of Experimental Immunology, Department of Pathology, University Hospital Ziirich, Ziirich, Switzerland
Abbreviations: Ad-LacZ: adenovirus recombinant for [3-galactosidase, CNS: central nervous system, CTL: cytotoxic T lymphocyte, CTLP: precursor CTL, HA: influenza virus hemagglutinin protein, HBV: hepatits B virus, IDDM: insulin-dependant diabetes mellitus, LCMV: lymphocytic choriomeningitis virus, LCMV-GP: glycoprotein of LCMV, LCMV-NP: nucleoprotein of LCMV, MBP: myelin basic protein, MS: multiple sclerosis, OVA: ovalbumin, RIP: rat insulin promoter, SM: smooth muscle promoter, TCR: T cell receptor, VSV." vesicular stomatitis virus; VE." vaccinia virus.
1. INTRODUCTION The prevalence of autoimmune diseases in the Western World is high with approximately 3-5% of the general population [1, 2] and the incidence of major autoimmune disorders such as multiple sclerosis, systemic lupus erythematosus, myasthenia gravis and primary biliary cirrhosis has been steadily increasing over recent years [1]. The frequent association of autoimmune reaction with viral and bacterial infections suggests a causative link between infection and autoimmunity [3]. However, attempts to establish a direct linkage between viral infections and autoimmune diseases have been impeded by the fact that patients usually have gone through infections with several pathogens before an autoimmune disease is finally diagnosed. Furthermore, antimicrobial immune responses are detectable at the time of onset of the autoimmune disease; viral or bacterial antigens, however, are often barely
detectable. It is therefore important to delineate the infection-associated initiating events that lead to the break of tolerance and eventually provoke autoimmune diseases. The majority of self-reactive T cells is eliminated in the thymus through clonal deletion of highaffinity autoreactive T cells (Fig. 1A). However, T cells with specificity for autoantigens exclusively expressed in the periphery and low-affinity T cells may escape thymic negative selection and therefore peripheral tolerance mechanisms are required to control potential autoimmune disease-mediating T cells. The simplest scenario of peripheral tolerance is that self-reactive T cells remain quiescent because the antigen is not presented in secondary lymphoid organs in sufficient amounts, a process that has been termed immunological ignorance. A further mechanism of peripheral tolerance is the induction of T cell anergy where functional inactivation of T cells is usually induced by TCR triggering in the absence of costimulation [4, 5] (Fig. 1B). In addition, longlasting presence of antigen in the periphery and lymphoid organs may result in the physical deletion of antigen-specific T cells, most likely after induction of initial functional impairment [6, 7]. Autoimmunity is most likely initiated in the course of an infection when target tissue inflammation is provoked either by direct cytopathic effects or by immunopathological reactions against a persisting microbial agent. The consequence may be the initiation of a dominant immune response against a single self-epitope that may broaden and thereby spread to other regions of the molecule and to other target molecules of the same tissue,
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Figure 1. Viruses break peripheral tolerance. (A) T cells that bind to self antigens with high affinity are negatively selected in the thymus. Autoreactive T cells specific for sequestered peripheral tissue antigens may escape clonal deletion and migrate towards the periphery. (B) Peripheral auto-reactive T lymphocytes either immunologicallyignore their cognate self antigens or become anergized due to the lack of efficient costimulation. (C) Following viral infection, professional antigen-presenting cells (APC) may display viral determinants that share homology with self antigens and hence activate autoreactive T cells (molecular mimicry). Alternatively, the inflammatory environment elicited by the infection may produce cytokines and chemokines that activate self-reactive bystander T cells. Due to tissue damage caused by the virus itself or by virus-specific immune cells, self antigens are released into the inflammatory milieu that trigger autoreactive T lymphocytes (epitope spreading). i.e. "epitope spreading" [8]. Infection-associated inflammation, for example, involves the release of cytokines and chemokines which attract antiviral effector cells and lymphocytes of other specificities. This "bystander effect" can be sufficient to activate lymphocytes directed against self antigens [9] leading to initiation and/or exacerbation of autoimmune disease. A third mechanism that may lead to the initiation of autoimmunity in the course of an infection is the activation of T or B cells via antigenic determinants shared between the pathogen and the host which has been termed "molecular mimicry" [10] (Fig. 1C). Cross-reactivities between pathogen-derived and self antigens have been described for human autoimmune diseases such as insulindependent diabetes, multiple sclerosis, and Guillain-Barre syndrome.
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Transgenic technology enabled establishment of mouse models with microbial antigens present in potential autoimmune target organs with clinical manifestations that resemble the phenotype of human autoimmune diseases [11]. These models have the advantage that the onset and progression of disease can be usually controlled. Furthermore, transgenic mice with viral or bacterial antigen expression in peripheral tissues are useful to address fundamental questions on the pathogenesis of autoimmune diseases such as the contribution of the genetic background, the nature of immunogenic self-antigens, or the role of immunoregulatory molecules and cells (Table 1). In this article, we will discuss how genetically engineered transgenic mice with defined tissue expression of viral antigens have contributed to our understanding of human
Table 1. Advantages of transgenic animals models for virus-induced autoimmunity
9 Focusingof autoimmunity to a single organ through tissue-specific transgene expression 9 Use of well-characterized antigens facilitates tracking of autoreactive T and B cells 9 Elucidation of epitope spreading from the known initiating antigenic determinants 9 Analysisof the single gene effects and their combination by crossing with other transgenic or knock-out mice 9 Valuablefor the design and the assessment of new potential therapies
autoimmune disease. The different models will be described and discussed according to the human disease they mimic.
2. TRANSGENIC MODELS FOR VIRUSINDUCED AUTOIMMUNE INSULINDEPENDENT DIABETES MELLITUS
A diabetes model of virus-induced autoimmunity illustrates the phenomenon of immunological ignorance of an extrathymically displayed neo-self antigen [12, 13]. The transgenic mouse lines express the viral glycoprotein (GP) of the leukocytic choriomeningitis virus (LCMV) under the control of the rat insulin promoter (RIP) exclusively in pancreatic islet cells (see Table 2). RIP-GP mice did neither spontaneously develop insulin-dependent diabetes mellitus (IDDM) nor did they delete potentially autoreactive GP-specific T cells. The latter was demonstrated by the fact that GP-specific immune responses can be induced by LCMV infection leading to autoimmune destruction of GP-expressing pancreatic islet cells and autoimmune diabetes in only 8 to 14 days. In a second transgenic mouse model expressing the LCMV nucleoprotein (RIPNP mice) both in I~ islet cells and thymus, diabetes developed slowly within three to six months after LCMV infection thus modeling slow onset autoimmune diabetes that is mediated mainly by low avidity T cells [14]. Interestingly, a less immunogenic LCMV-GP recombinant vaccinia virus (VV) elicited only a mild insulitis in RIP-GP mice without signifi-
cant elevation of blood glucose levels [ 15]. In addition, important observations on quantitative aspects of autoimmunity have been made in RIP-GP mice. Elevation of CTLp frequencies in double transgenic mice expressing the LCMV-GP in the pancreas and a specific TCR on their CTL (RIP-GPxTCR) strongly accelerated disease and compensated for the weak CTL induction by LCMV-GP recombinant VV [ 12, 15]. VV-GP-induced autoimmune diabetes could also be generated when the B7 costimulatory molecule was locally expressed in the 13-islets of RIP-GP mice (RIP-GPxRIP-B7) [ 16, 17], or when TNF-t~ was expressed in pancreatic islets (RIPGPxRIP-TNF-t~) [15, 18]. These mouse models present not only the characteristic hallmarks of IDDM in humans, namely hyperglycemia, hypoinsulinemia, and mononuclear cell infiltration in the ~l islets, but can also be used for the assessment of novel immunotherapeutical approaches [ 19-21 ] or the effect of regulatory T cells in the pathogenesis of autoimmune diabetes [22]. Transgenic mice expressing the influenza virus hemagglutinin (HA) as a neo-self antigen in the pancreatic islet [~ cells (Ins-HA mice) revealed that peripheral antigen may efficiently anergize self-reactive T cells [23]. Since thymocyte development is not impaired in Ins-HA mice [24], the absence of autoimmune destruction in pancreatic islets after influenza virus infection suggested efficient peripheral tolerization of self-reactive T cells. Indeed, HA-specific TCR-transgenic CD8 § T cells were activated and proliferated exclusively in the draining lymph nodes of the pancreas [25] and were subsequently functionally deleted [26]. It is most likely that HA is cross-presented by bone marrow-derived antigen presenting cells in the local lymphoid tissue in Ins-HA mice. Interestingly, not only naive but also memory CD8 § T cells may be tolerized under these conditions by the peripherally expressed antigen [27].
3. MODELING VIRUS-INDUCED LIVER DISEASE
Infection with the hepatitis B virus (HBV) or hepatitis C virus (HCV) causes severe inflammatory liver disease of variable duration and severity. Persistently infected patients with ongoing liver disease
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Table 2. Transgenic models Disease modeled
Transgene construct (promoter and antigen)
Infectious agent
Findings
References
IDDM
(RIP)-LCMV-GP
LCMV
Peripherally expressed self-antigen may be immunologically ignored
[12, 13]
IDDM
(RIP)-LCMV-GP
LCMV-GP recombinant VV
Amount of self-reactive T cells and inflammatory milieu in the target organs determine the extent of autoimmune disease
[15-18]
IDDM
(RIP)-LCMV-NP
LCMV
Low affinity CTL mediate slow-onset IDDM
[14]
IDDM
(RIP)-Influenza HA
Influenza and HA-VV
Peripheral antigen may anergize self-reactive CTL in the local lymph node; memory CTL may be tolerized
[23, 25-27]
Hepatitis
(Albumin)-HBV envelope proteins
HBV recombinant VV
Induction of autoantibodies, but absence of HBV-specific CTL and liver disease
[29, 30]
Hepatitis
(Albumin)-LCMV-GP
LCMV
Break of tolerance and self-limited hepatitis after LCMV infection only if TCR transgenic CTL had been adoptively transferred
[31]
Multiple sclerosis
(MBP)-LCMV-GP and -NP
LCMV
Induction of CNS inflammatory and demyelinating disease through repeated infection
[32]
VSV-OVA
Adoptive transfer of IL-12-treated effector CTL augmented myocarditis
[37]
Virus-induced tolerization of self-reactive CTL through functional paralysis and/or exhaustion.
Krebs & Ludewig (in preparation)
Myocarditis (Murine cardiac ormyosin heavy-chain)OVA
Myocarditis (SM-22)-~l-galactosidase Ad-LacZ
Abbreviations: Ad-LacZ, adenovirus recombinant for ~-galactosidase; CNS, central nervous system; CTL, cytotoxic T lymphocyte; HA, influenza virus hemagglutinin protein; HBV, hepatits B virus; IDDM, insulin-dependant diabetes mellitus; LCMV, lymphocytic choriomeningitis virus; LCMV-GP, glycoprotein of LCMV; LCMV-NP, nucleoprotein of LCMV; MBP, myelin basic protein; MS, multiple sclerosis; OT-I, ovalbumin-specific transgenic CTL; OVA, ovalburnin; RIP, rat insulin promoter; SM, smooth muscle promoter; TCR, T cell receptor; VSV, vesicular stomatitis virus; VV, vaccinia virus.
may develop cirrhosis, hepatocellular carcinoma, and, in the case of chronic HCV infection, may also develop autoimmune liver disease. Transgenic mice constitutively expressing the HBV envelope proteins containing hepatitis B surface antigen (HBsAg) in the liver under the transcriptional control of the mouse albumin promoter represent a well-established model for virus-induced immunopathological liver damage [28]. In these mice, HBsAg was detected in virtually all hepatocytes and was also secreted into the blood. Repetitive infection with HBV envelope-recombinant VV leads to production of low titers of T cell-dependent antiHBV IgG autoantibodies that clear HBsAg from the
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blood, but not to activation of HBsAg-specific CTL [29]. In this model, immunization of the transgenic animals failed to induce ongoing autoimrnune liver disease, whereas adoptive transfer of effector CTL elicited fulminant and resolving hepatitis [30]. A similar model has been established by Voehringer et al [31] who expressed the GP33 peptide of the LCMV-GP under the control of the albumin promoter (ALB 1-GP33 mice). Partial thymic deletion of GP33-specific T cells resulted in reduced GP33-specific CTL responses after LCMV infection and most likely helped to avoid LCMV-induced liver damage in ALB 1-GP33 mice [31]. It is interesting to note that adoptively transferred TCR trans-
genic CTL recognizing GP33 ignored the peripheral antigen expressed in hepatocytes, whereas adoptive transfer of activated GP33-specific CTL elicited a significant fiver disease. Virus-induced autoimmune hepatitis could be induced by adoptive transfer of naive TCR-transgenic CTL followed by LCMV infection [31]. Overall, these studies indicate that the liver may serve as a target organ for virusinduced autoimmune disease.
4. VIRUS-INDUCED AUTOIMMUNITY IN THE CENTRAL NERVOUS SYSTEM Human autoimmune demyelinating diseases such as multiple sclerosis is most likely a CD4 + T-ceU mediated disease that is associated with viral infections [8]. Virus-induced autoimmunity in the central nervous system has been studied by Evans et al. [32] who expressed LCMV viral antigens (glycoprotein and nucleoprotein) as transgenes in oligodendrocytes of the central nervous system (CNS) under the control of the myelin basic protein (MBP) (MBP-GP and MBP-NP mice). Similar to RIP-GP mice, autoreactive lymphocytes that escaped thymic negative selection were present in the periphery and were activated by LCMV infection. Virally activated autoreactive CTL were able to cross the blood-brain barrier, migrated into the CNS and lysed transgeneexpressing oligodentrocytes. Following a second LCMV infection, the amount of infiltrating cells massively increased, leading to significant motor dysfunction in infected transgenic animals. The experimental disorder in MBP-GP and MBP-NP mice disorder resembles some characteristics of human demyelinating disease [33] suggesting that relapses in multiple sclerosis that often occur after viral infections could be caused by a reactivation of oligodendrocyte-specific T cells that were initially generated through molecular mimicry.
5. VIRAL INFECTION AND AUTOIMMUNE MECHANISMS OF MYOCARDITIS Dilated cardiomyopathy is one of the leading causes of heart failure and is most likely a sequel of myocarditis induced by infectious agents such as Coxsackievirus B (CVB) or cytomegalovirus [34]. Dis-
ease in mice induced by serotype 3 CVB resembles the human situation because infection of susceptible mouse strains elicits first an acute myocarditis that resolves around day 14 post infection, followed by a chronic phase with persistent low level inflammation of the cardiac muscle. The immune system not only plays an important protective role against the infection and subsequent heart disease induced by CVB3, but may also contribute to cardiac damage by attacking heart cells. For example, cardiac damage is dramatically reduced in mice lacking a functional T cell response [35], indicating that immunopathological damage contributes crucially to myocardial injury during CVB infection. It has been suggested that molecular mimicry between viral pathogens and myocardial proteins leads to induction of cross-reactive T cells directed against heart antigens [36]. However, other studies indicate that CVB-induced "bystander activation" of selfreactive T cells is the major immunopathological mechanism in insulin-dependent diabetes [9]. It is thus still an open question to which extent the different immunopathological mechanisms contribute to virus-mediated acute and chronic heart disease. Recently, a mouse line has been developed that expresses cardiac myocyte-restricted membranebound ovalbumin (CMy-mOVA) [37]. Despite no detectable transgene expression the thymus, these transgenic mice were tolerant to OVA as shown by the lack or OVA-specific immune responses following infection with OVA-expressing vesicular stomatitis virus (VSV-OVA). However, adoptive transfer of naive OVA-specific TCR transgenic CTL and subsequent infection with VSV-OVA induced myocarditis in CMy-mOVA mice. Adoptive transfer experiments revealed that OVA-specific effector CTL require IL-12 during their in vitro stimulation to acquire full pathogenic potential. A transgenic mouse model with defined T cellmediated cardiovascular immunopathology has been recently established by our group [38]. SMLacZ mice express the bacterial 13-galactosidase (I]-gal) antigen in cardiomyocytes of the fight heart and in arterial smooth muscle cells [39]. The [~-gal transgene is immunologically ignored in these mice, despite widespread expression in the vascular system. Repetitive priming of SM-LacZ mice with dendritic cells (DC) presenting [3-gal peptide caused acute vascular immunopathology with
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strong lymphocytic infiltration in lung arteries and aorta (arteritis) and in the fight heart (myocarditis). In the chronic phase, despite ceasing immunization with 13-gal peptide-loaded DC, SM-LacZ mice show a severe loss of functional heart tissue and fibrosis that eventually leads to dilated cardiomyopathy [40]. This transgenic model is therefore well-suited for the characterization of pathological mechanisms in cardiovascular diseases [41]. Interestingly, following intravenous administration of replicationdeficient [~-gal-recombinant adenovirus (Ad-LacZ), only limited cellular infiltrations were observed in lungs and myocardium of SM-LacZ mice (Krebs and Ludewig, manuscript in preparation). Moreover, SM-LacZ mice displayed only weak [3galactosidase-specific CTL response compared to wild type C57BL/6 mice. In this particular model, peripheral tolerance is thus most likely established by anergization and/or clonal deletion of specific CTL because high amounts of Ad-LacZ-encoded [3galactosidase antigen are presented for too long in peripheral and lymphoid organs, leading to exhaustive activation of 13-galactosidase-specific CTL.
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5. 6.
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10. 6. C O N C L U S I O N Viruses may disturb the fine-tuned balance of the immune system by acting as "adjuvant" and/or by stimulating cross-reactive T and B cells with specifity for both viral and self antigens. Transgenic mouse models have helped to uncover the basic rules how virus interfere with self tolerance. Furthermore, the different transgenic mouse models of virus-induced autoimmunity described here represent valuable tools to delineate basic pathogenic mechanisms and to evaluate therapeutical strategies to intervene with early detrimental processes that lead to manifest autoimmune disease.
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Benoist C, Mathis D. Autoimmunity provoked by infection: how good is the case for T cell epitope mimicry? Nat Immunol 2001 ;2(9):797-801. Jenkins MK, Schwartz RH. Antigen presentation by chemically modified splenocytes induces antigen-specific T cell unresponsiveness in vitro and in vivo. J Exp Med 1987;165(2):302-319. SchwartzRH. A cell culture model for T lymphocyte clonal anergy. Science 1990;248(4961): 1349-1356. Kyburz D, Aichele P, Speiser DE, Hengartner H, Zinkemagel RM, Pircher H. T cell immunity after a viral infection versus T cell tolerance induced by soluble viral peptides. Eur J Immunol 1993;23(8): 1956-1962. Wherry EJ, Blattman JN, Murali-Krishna K, van der MR, Ahmed R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J Virol 2003;77(8):4911-4927. Vanderlugt CL, Miller SD. Epitope spreading in immune-mediated diseases: implications for imnmnotherapy. Nat Rev Immunol 2002;2(2):85-95. Horwitz MS, Bradley LM, Harbertson J, Krahl T, Lee J, Sarvetnick N. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat Med 1998;4(7):781-785. Oldstone MB. Molecular mimicry and autoimmune disease. Cell 1987;50(6):819-820. Boyton RJ, Altmann DM. Transgenic models of autoimmune disease. Clin Exp Immunol 2002;127(1): 4-11. Ohashi PS, Oehen S, Buerki K, Pircher H, Ohashi CT, Odermatt Bet al. Ablation of "tolerance" and induction of diabetes by virus infection in viral antigen transgenic mice. Cell 1991;65(2):305-317. Oldstone MB, Nerenberg M, Southern P, Price J, Lewicki H. Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response. Cell 1991;65(2):319-331. Von Herrath MG, Dockter J, Oldstone MB. How virus induces a rapid or slow onset insulin-dependent diabetes mellitus in a transgenic model. Immunity 1994;1(3): 231-242. Ohashi PS, Oehen S, Aichele P, Pircher H, Odermatt B, Herrera Pet al. Induction of diabetes is influenced by the infectious virus and local expression of MHC class I and tumor necrosis factor-or. J Immunol 1993;150(11): 5185-5194. Harlan DM, Hengartner H, Huang ML, Kang YH, Abe R, Moreadith RW et al. Mice expressing both B7-1 and viral glycoprotein on pancreatic beta cells along with glycoprotein-specific transgenic T cells develop diabetes due to a breakdown of T-lymphocyte unresponsive-
ness. Proc Natl Acad Sci USA 1994;91(8):3137-3141. 17. Von Herrath MG, Guerder S, Lewicki H, Flavell RA, Oldstone MB. Coexpression of B7-1 and viral ("self") transgenes in pancreatic beta cells can break peripheral ignorance and lead to spontaneous autoimmune diabetes. Immunity 1995;3(6):727-738. 18. Higuchi Y, Herrera P, Muniesa P, Huarte J, Belin D, Ohashi P e t al. Expression of a tumor necrosis factor alpha transgene in murine pancreatic beta cells results in severe and permanent insulitis without evolution towards diabetes. J Exp Med 1992; 176(6): 1719-1731. 19. Aichele P, Kyburz D, Ohashi PS, Odermatt B, Zinkemagel RM, Hengartner H et al. Peptide-induced T-cell tolerance to prevent autoimmune diabetes in a transgenic mouse model. Proc Natl Acad Sci USA 1994;91(2): 444--448. 20. Bot A, Smith D, Bot S, Hughes A, Wolfe T, Wang Let al. Plasmid vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice. J Immuno12001;167(5):2950-2955. 21. Wolfe T, Bot A, Hughes A, Mohrle U, Rodrigo E, Jaume JC et al. Endogenous expression levels of autoantigens influence success or failure of DNA immunizations to prevent type 1 diabetes: addition of IL-4 increases safety. Eur J Immuno12002;32(1): 113-121. 22. Homann D, Jahreis A, Wolfe T, Hughes A, Coon B, van Stipdonk MJ et al. CD40L blockade prevents autoimmune diabetes by induction of bitypic NK/DC regulatory cells. Immunity 2002; 16(3):403-415. 23. Lo D, Freedman J, Hesse S, Palmiter RD, Brinster RL, Sherman LA. Peripheral tolerance to an islet cell-specific hemagglutinin transgene affects both CD4+ and CD8+ T cells. Eur J Immunol 1992;22(4):1013-1022. 24. Morgan DJ, Liblau R, Scott B, Fleck S, McDevitt HO, Sarvetnick N et al. CD8(+) T cell-mediated spontaneous diabetes in neonatal mice. J Immunol 1996;157(3): 978-983. 25. Morgan DJ, Kurts C, Kreuwel HT, Hoist KL, Heath WR, Sherman LA. Ontogeny of T cell tolerance to peripherally expressed antigens. Proc Natl Acad Sci USA 1999;96(7):3854-3858. 26. Morgan DJ, Kreuwel HT, Sherman LA. Antigen concentration and precursor frequency determine the rate of CD8+ T cell tolerance to peripherally expressed antigens. J Immunol 1999; 163(2):723-727. 27. Kreuwel HT, Aung S, Silao C, Sherman LA. Memory CD8(+) T cells undergo peripheral tolerance. Immunity 2002;17(1):73-81. 28. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol 1995; 13:29-60:29-60. 29. Wirth S, Guidotti LG, Ando K, Schlicht HJ, Chisari bag". Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmurfity Y. Shoenfeld and N.R. Rose, editors
Epstein-Barr Virus and Autoimmunity Michael P. Pender
Neuroimmunology Research Centre, School of Medicine, The University of Queensland, and Department of Neurology, Royal Brisbane and Women's Hospital Brisbane, Queensland, Australia
1. I N T R O D U C T I O N There is a large body of evidence that infection with the Epstein-Barr virus (EBV), the aetiological agent of infectious mononucleosis, has a role in the pathogenesis of many human chronic autoimmune diseases. This chapter will review the evidence for the role of EBV in each of these diseases and also focus on the features that are common to the different human chronic autoimmune diseases, with the aim of providing an explanation for what appears to be a unique role for EBV in the pathogenesis of these diseases.
2. G E N E R A L ASPECTS OF HUMAN C H R O N I C A U T O I M M U N E DISEASES Human chronic autoimmune diseases share a number of common features. The various autoimmune diseases have similarities in their patterns of genetic susceptibility. The major histocompatibility complex (MHC) class II region contributes to this genetic susceptibility, and each autoimmune disease is associated with particular MHC class II genes [1]. However, there is increasing evidence that another important genetic component is susceptibility to 'autoimmunity-in-general'. People with one particular autoimmune disease such as multiple sclerosis (MS) have an increased risk of developing other autoimmune diseases, and their first-degree relatives also have an increased risk of developing other autoimmune diseases [2]. Studies on autoimmune family pedigrees have led to the proposal that autoimmunity is an autosomal dominant trait with
penetrance (disease expression) in -92% of females and 49% of males carrying the abnormal gene [3, 4]. Furthermore, people with organ-specific autoimmune diseases, such as insulin-dependent diabetes mellitus [5], autoimmune thyroid disease [6], MS [7] and inflammatory bowel disease [8] have an increased incidence of antinuclear antibodies. I have recently proposed that the genetic susceptibility to 'autoimmunity-in-general' is mediated by susceptibility to the effects of B-cell infection by EBV [9]. Human autoimmune diseases are generally more common in females than males and tend to be exacerbated in the post-partum period. Many chronic autoimmune diseases have a relapsing-remitting course, for example rheumatoid arthritis (RA), ulcerative colitis and MS, suggesting fluctuations in the autoimmune attack. Other autoimmune diseases, such as insulin-dependent diabetes mellitus and autoimmune hypothyroidism, do not become clinically apparent until much of the target organ has been destroyed; fluctuating autoimmune attack might also be occurring in these diseases but would not be clinically evident. Some chronic autoimmune diseases are manifested clinically by a primary progressive course, such as primary progressive MS, where there is progressive clinical deterioration without clear relapses or remissions. In such diseases there still could be fluctuations in the level of autoimmune attack but these could be masked by a lack of target organ repair and a subsequent lack of any periods of clinical improvement. There is also evidence of similarities in the environmental factors that predispose to or exacerbate different chronic autoimmune diseases; for example, exacerbations can be triggered by a variety of infections.
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3. GENERAL ASPECTS OF EBV INFECTION
EBV has the unique ability to infect, activate and latently persist in B lymphocytes. When EBV infects resting B cells in vitro, it drives them into activation and proliferation independently of T-cell help. Infection of B cells from normal individuals in vitro results in the production of monoclonal autoantibodies reacting with antigens in multiple organs [10]. This accounts for the transient appearance of autoantibodies during the course of infectious mononucleosis [11]. Usually, the proliferating infected B cells are eventually eliminated by EBV-specific cytotoxic CD8+ T cells, but latently infected non-proliferating memory B cells persist in the individual for life [12]. Antigen-driven differentiation of latently infected memory B cells into plasma cells might trigger entry into the lytic cycle with the production of infectious virus [ 12].
4. POSSIBLE MECHANISMS BY WHICH EBV INFECTION COULD PROMOTE AUTOIMMUNE DISEASE
EBV infection could promote autoimmune disease by: inducing cross-reactive immune responses against self antigens; infection of organs with resultant tissue damage and release of antigens and secondary immune sensitization; non-specific general upregulation of the immune system; infection of autoreactive B cells which could produce autoantibodies and act as professional antigen-presenting cells in the target organ. There is evidence for T-cell or antibody cross-reactivity between EBV antigens and self antigens, for example myelin basic protein in MS [13, 14], La antigen in Sjrgren's syndrome [15], SmD in systemic lupus erythematosus (SLE) [16] and self MHC-derived peptides in oligoarticular juvenile idiopathic arthritis [17]. However, cross-reactivity between self antigens and viral antigens is a phenomenon applicable to all infectious agents and is therefore unlikely to be the primary mechanism for the unique role that EBV appears to have in the pathogenesis of autoimmune diseases such as MS and SLE. Infection of organs with resultant tissue damage, release of antigens and secondary immune sensitization is also a mechanism that potentially could occur following infections
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with many different agents. Similarly, non-specific general upregulation of the immune system, for example through upregulation of cytokines and adhesion molecules, could also occur following any infection. In contrast, the ability of EBV to infect and immortalize B cells, including autoreactive B cells, is unique and therefore a likely explanation for a unique pathogenic role of EBV in human chronic autoimmune diseases [9]. EBV-infected autoreactive B cells could produce pathogenic autoantibodies. They could also act as professional antigen-presenting cells in the target organ where they could provide a costimulatory survival signal to autoreactive T cells that have been activated in peripheral lymphoid organs by cross-reactivity with infectious agents and that would otherwise undergo activation-induced apoptosis when they enter the target organ [ 18-20]. On receiving a costimulatory survival signal from the EBV-infected B cells, the autoreactive T cells could instead proliferate and produce cytokines, which recruit other inflammatory cells, with resultant target organ damage and chronic autoimmune disease [9].
5. RELATIONSHIPS BETWEEN EBV INFECTION AND PARTICULAR AUTOIMMUNE DISEASES 5.1. Multiple Sclerosis (MS)
In 1980 Sumaya et al [21] reported a higher frequency of EBV seropositivity and a higher prevalence of high anti-EBV antibody titres in patients with MS compared to controls. Subsequent studies have shown that patients with MS are almost universally seropositive for EBV, raising the possibility that EBV infection might be a prerequisite for the development of MS. A review of eight published case-control studies comparing EBV serology in MS patients and controls revealed that 99% of MS patients were EBV-seropositive compared to 90% of controls; the summary odds ratio of MS comparing EBV-seropositive individuals with EBV-seronegative individuals was 13.5 (95% confidence interval = 6.3-31.4) [22]. This difference does not apply to other herpes viruses [23]. Furthermore, a definite clinical history of infectious mononucleosis, which indicates primary infection with EBV with a high
frequency of infected B cells [ 11] further increases the risk of MS in EBV-seropositive subjects (eightfold, if infection occurs before the age of 18 years) [24]. Levin et al [25], in a study of blood samples collected from US military personnel before the onset of MS, have shown that the presence of high titres of antibodies to EBV increases the risk 34-fold for developing MS. In some cases the first attack of MS has occurred at the time of primary EBV infection [26]. Interestingly, elevated anti-EBV antibody levels were found in a child who developed MS at the age of 10 months [27]. Anti-EBV antibodies occur more often in the cerebrospinal fluid (CSF) of MS patients than controls [28], but MS patients exhibit local central nervous system (CNS) production of antibodies to various viruses [29]. Some patients have CSF oligoclonal bands of IgG reacting with EBV nuclear antigen-1 (EBNA-1) [30]. In 1979 Fraser et al [31] reported that patients with clinically active MS had an increased tendency to spontaneous in-vitro B-lymphocyte transformation compared to healthy subjects and patients with clinically quiescent MS. This could result from an increased frequency of circulating EBV-infected B cells or from defective control of outgrowth of EBV-transformed B cells in vitro by EBV-specific cytotoxic T cells. Wandinger et al [23] found EBV DNA in the sera of patients with clinically active MS but not in those with clinically stable disease. They interpreted this as evidence of an association between disease activity and EBV replication, which was supported by the finding of increased IgM and IgA responses to EBV early antigens in the patients with clinically active disease. Analysis of the CSF from MS patients using the polymerase chain reaction has not detected EBV DNA [32]; this makes it unlikely that EBV is a major target for immune attack in the CNS but does not exclude the presence of EBV-infected B cells that could act as professional antigen-presenting cells in the CNS. I have suggested [9] that EBV-infected B cells could be the source of the monoclonally expanded B cells present in the CSF of MS patients [33] and be responsible for the development of primary B-cell lymphoma in the CNS in MS [34]. Patients with MS have defective T-cell control of EBV-infected B cells [35]. One possible mechanism for this is decreased MHC class I expression on B cells, which has been reported to occur in patients
with MS [36] and other autoimmune diseases [37], although it remains unclear whether the reported decrease is sufficient to cause decreased EBV-specific CD8+ T-cell cytotoxicity. A recent study found an increased frequency of CD8+ T cells responding to two immunodominant EBV epitopes in MS patients but it was not determined whether these T cells were cytotoxic [38]. EBV-specific CD8+ T cells are enriched in MS brain lesions compared to the peripheral blood, but such enrichment is also found for EBV-specific and cytomegalovirus-specific CD8+ T cells in other inflammatory lesions of the brain and other organs, including non-autoimmune inflammatory lesions [39]. This might simply reflect the accumulation of activated T cells in any chronic inflarmnatory lesion and does not necessarily imply that the virus-specific T cells are recognizing viral antigen or cross-reacting self antigen in the inflamed organ. There is evidence of T-cell cross-reactivity between EBV antigens and the myelin antigen, myelin basic protein [13]. A CD4+ T-cell clone from an MS patient has been found to react with both a DPO35*0101-restricted EBV peptide and a DRB 1" 1501-restricted myelin basic protein peptide [14]. Furthermore, EBV infection induces the B-cell expression of ctB-crystallin, a small heat-shock protein [40], which has been reported to be present in MS lesions and to be an immunodominant myelin antigen for T cells from healthy subjects and MS patients [41]. These findings have been interpreted as evidence that T cells generated in response to t~B-crystallin expressed and presented by EBVinfected B cells might be pathogenic for CNS myelin expressing the same stress-induced protein [40].
5.2. Systemic Lupus Erythematosus (SLE) In 1971 Evans et al [42] reported elevated levels of anti-EBV antibodies in the sera of patients with SLE. Subsequent studies have shown that 99% of SLE patients are seropositive for EBV [43, 44]. The association of EBV-seropositivity with SLE is particularly striking in young patients, 99% of whom are seropositive compared to 70% of agematched controls (odds ratio 49.9, 95% confidence interval 9.3-1025, P < 0.00000000001) [43]. Seroconversion rates for other herpes viruses do not
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differ between SLE patients and controls [43, 44]. SLE can develop immediately after EBV-induced infectious mononucleosis [45]. T cells from patients with SLE cannot control the numbers of EBV-infected B cells from SLE patients or normal subjects but T cells from normal EBV-seropositive subjects can control infected B cells from SLE patients [46]; this indicates impaired T-cell control of EBV-infected B cells in SLE. This might be explained by the reported decrease in MHC class I expression on B cells in patients with SLE [37]. Patients with SLE have autoantibodies that bind an amino acid sequence which is shared between SmD, a small nuclear ribonucleoprotein, and EBNA-1 [16].
5.3. Rheumatoid Arthritis (RA) Patients with RA have increased anti-EBV antibody levels in their sera compared to healthy subjects [47]. They also have an increased frequency of circulating EBV-infected B cells, as determined by the frequency of spontaneously transforming B cells [48]. A recent study using real-time polymerase chain reaction has demonstrated a 10-fold increase in the EBV DNA load in the peripheral blood mononuclear cells of patients with RA compared to normal controls [49]. The high frequency of EBV-infected B cells in patients with RA is not due to increased uptake of the virus by B cells [48] but might be explained by the defective control of infected B cells by EBV-specific T cells [50, 51]. This might be explained by the reported decrease in MHC class I expression on B cells in patients with RA [37]. A study using a highly sensitive in-situ hybridization technique to detect EBV-encoded small nuclear RNAs (EBERs) in synovial membrane biopsy samples of patients with RA concluded that there was a lack of evidence for involvement of EBV [52]. Yet, the study actually found EBERs in seven (19%) of 37 patients with RA and in zero of 51 patients with other joint diseases; cells expressing EBERs were B cells and plasma cells. These results could also be interpreted as supporting a role for EBV infection of B cells in the pathogenesis of RA if the negative results in the other patients with RA were due to the limitations imposed by sampling. EBV-specific CD8+ T cells are enriched in the inflamed joints of patients with RA compared to the peripheral blood,
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but such enrichment is also found for cytomegalovirus-specific CD8+ T cells in the inflamed joints and for EBV-specific and cytomegalovirus-specific CD8+ T cells in autoimmune and non-autoimmune inflammatory lesions in other organs [39].
5.4. Sjiigren's Syndrome Patients with Sj6gren's syndrome have increased levels of anti-EBV antibodies in their sera [53, 54], an increased tendency to spontaneous in-vitro B-lymphocyte transformation from the peripheral blood [54] and an increased frequency of shedding of EBV from the oropharynx [54]. They also have decreased EBV-specific T-cell cytotoxicity [55] which accounts for the impaired ability to abort in-vitro outgrowth in regression assays of EBVinduced B-cell transformation [54, 55]. Decreased EBV-specific T-cell cytotoxicity might be explained by the reported decrease in MHC class I expression on B cells of patients with Sj6gren's syndrome [37]. EBV-infected B cells could be the source of the monoclonally expanded B cells in the salivary glands in Sj6gren's syndrome [56] and be responsible for the increased risk of the development of B-cell lymphoma in the salivary glands in Sj6gren's syndrome [57]. Moreover, antibodies to the La autoantigen of Sj6gren's syndrome also react with EBERs complexed with protein [15].
5.5. Autoimmune Thyroid Disease Patients with autoimmune thyroiditis have increased titres of anti-EBV antibodies in their sera compared to healthy subjects [58]. Thyrotoxicosis can develop immediately after infectious mononucleosis due to primary EBV infection, and autoimmune hypothyroidism can develop in association with acute EBV infection [59]. Intrathyroidal EBV-infected B cells could be the source of the monoclonally expanded B cells in the thyroid gland in autoimmune thyroiditis [60] and might be responsible for the increased risk of development of B-cell lymphoma in the thyroid gland in patients with autoimmune thyroiditis [61 ].
5.6. Scleroderma Patients with scleroderma have defective T-cell control of EBV-infected B cells [62]. Progressive
systemic sclerosis has developed in an infant five months after infectious mononucleosis [63].
5.7. Autoimmune Liver Disease There is evidence for a role of EBV in both primary biliary cirrhosis and autoimmune hepatitis. Patients with primary biliary cirrhosis have increased levels of EBV DNA in their peripheral blood mononuclear cells, fiver and saliva compared to controls [64]. They also have defective T-cell control of EBVinfected B cells [65]. Autoimmune hepatitis can develop soon after infectious mononucleosis due to primary EBV infection [66]. 5.8. Inflammatory Bowel Disease Latently and productively EBV-infected B cells are present at a higher frequency in the colonic mucosa of patients with ulcerative colitis than controls [67, 68]. Patients with Crohn's disease also have a higher frequency of EBV-infected B cells in the colonic mucosa than controls [67].
organs; an increased risk of developing B-cell lymphoma in the target organs of chronic autoimmune disease; and T-cell and antibody cross-reactivity between EBV antigens and self antigens. These findings can be explained by the hypothesis that chronic autoimmune diseases occur in individuals genetically susceptible to the effects of B-cell infection by EBV, resulting in an increased frequency of latently EBV-infected autoreactive B cells. EBV-infected autoreactive B cells could produce pathogenic autoantibodies; they could also act as professional antigen-presenting cells in the target organ where they could provide a costimulatory survival signal to autoreactive T cells that have been activated in peripheral lymphoid organs by cross-reactivity with infectious agents and that would otherwise undergo activation-induced apoptosis in the target organ. On receiving a costimulatory survival signal from the EBV-infected B cells, the autoreactive T cells could proliferate and produce cytokines, which recruit other inflammatory cells, with resultant target organ damage and chronic autoimmune disease.
5.9. Cryptogenic Fibrosing Alveolitis
REFERENCES
Patients with cryptogenic fibrosing alveolitis have increased serum levels of antibodies against EBV, but not against herpes simplex virus or cytomegalovirus, compared to controls [69]. Furthermore, EBV DNA is detected in lung tissue more frequently in patients with cryptogenic fibrosing alveolitis than in controls [70].
1.
6. CONCLUSION
There is a large body of evidence indicating that EBV infection has a major role in the pathogenesis of organ-specific and non-organ-specific human chronic autoimmune diseases. This evidence includes: a high frequency and high levels of circulating anti-EBV antibodies; triggering of the first attack of autoimmune disease by infectious mononucleosis due to primary EBV infection; an increased frequency of circulating EBV-infected B cells; defective T-cell control of EBV-infected B cells; an increased level of EBV DNA in target tissues; monoclonal B-cell expansion in the target
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
HIV and Autoimmunity Gisele Zandman-Goddard ~and Yehuda Shoenfeld ~,2
;Center for Autoimmune Diseases, Department of Medicine 'B', Sheba Medical Center, Tel-Hashomer; Sackler Faculty of Medicine, Tel-Aviv University, Israel; 2Incumbent of the Laura Schwartz-Kipp Research Chair in Autoimmune Diseases, Tel Aviv University, Israel
1. INTRODUCTION The combination of immune dysfunction in patients with HIV infection and AIDS and the development of autoimmune diseases is intriguing. Yet, the spectrum of reported autoimmune phenomena in these patients is increasing [1, 2]. This wide range of reported autoimmune diseases is due to different patient selection, and the association with the development of AIDS. An infectious trigger for immune activation is one of the postulated mechanisms in autoimmunity and derives from molecular mimicry [3, 4]. During frank loss of immunocompetence, autoimmune diseases that are predominantly T cell subtype CD8 driven may predominate. Multiple anti-retroviral drug therapy for patients with AIDS provides prolonged survival and immune restoration, a setting where autoimmune diseases develop. We propose a staging of autoimmune manifestations related to HIV/AIDS manifestations and CD4 count that may be beneficial in identifying the type of autoimmune disease and establishing the proper therapy (Table 1). During Stage I there is the acute HIV infection, and the immune system is intact. In this stage, autoimmune diseases may present. While Stage II is a quiescent period without overt manifestations of AIDS, there is a declining CD4 count indicative of some immunosuppression. Autoimmune diseases are not found. During Stage III further immunosuppression is encountered manifested by a low CD4 count. However, diseases where T cell subtype CD8 predominant such as psoriasis and diffuse immune lymphocytic syndrome (Sjtigren's-
like syndrome) may present or even be the initial manifestation of AIDS. Autoimmune diseases are not found. In Stage IV there is restoration of immune competence following highly active antiretroviral therapy (HAART). In this setting, there may be a resurgence of autoimmune diseases. This review describes the various autoimmune diseases that develop in HIV/AIDS patients through possible mechanisms related to immune activation.
2. AUTOIMMUNE DISEASES IN HIV INFECTION The frequency of rheumatological syndromes in HIV patients varies from less than 1% to 60% [2, 5-7]. The list of reported autoimmune diseases in HIV/AIDS is found in Table 2.
2.1. Systemic Lupus Erythematosus (SLE) The unrestrained state of immune activation may contribute to chronic inflarmnatory and autoimmune sequelae in HIV-infected individuals. Several rheumatic entities, such as Reiter's syndrome, psoriatic arthritis, SjSgren's-like syndrome, myopathy and HIV-related vasculitis are often correlated with the severity of the HIV infection and improve with anti-retroviral therapy. However, other entities, such as SLE and sarcoidosis [8, 9], have a decreased incidence in the HIV infected population than would be expected in the general population. This inconsistency suggests that the immunosuppressive effect of HIV may inhibit the development of autoimmune
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Table
1. HIV and autoimmunity
Stage
Stagedescription
CD4 count
Viral load
AIDS
Autoimmunity
I II III IV
Clinical latency Cellularresponse Immunedeficiency Immunerestoration
High (> 500) Normal/Low(20(0-499) Low (< 200) High (> 500)
High High High Low
No No Yes Controlled
Autoimmune disease Immune-complex, vasculitis Spondyloarthropathy Autoimmune disease
Autoimmune disease can occur with a preserved immune system requiting B and T cell interactions (normal CD4 count). Therefore, autoimmunity is possible in Stages I, II, and IV. With profound immunodeficiency (low CD4 count) autoimmune diseases are not found. Stage IV (high CD4 count) describes HIV-infected patients with immune restoration but possibly altered immunoregulation enabling the resurgence of autoimmune diseases.
Table 2. Autoimmune diseases in HIV patients Autoimmune disease
References
SLE Antiphospholipid syndrome Autoimmune thrombocytopenia Vasculitis Polymyositis Graves' disease Primary biliary cirrhosis Raynaud' s phenomena, Behqet's disease
[8-14] [17-29] [33] [2, 34-36] [37-39] [40] [41] [42, 43]
diathesis. However, HIV infection that is controlled by protease inhibitors and other anti-retroviral agents renders the immune system no longer immunodeficient. There is immune restoration with normalization of the CD4 count and functional T cell reconstitution [ 10], so that a genetically predisposed host can develop autoimmunity. This has been postulated in the coexistence of HIV with SLE [ 11 ]. Systemic lupus erythematosus (SLE) may be influenced by human immunodeficiency virus type-1 (HIV) infection. It has been suggested that the immunosuppression resulting from HIV infection can prevent the emergence of SLE. There appear to be fewer cases of SLE in the HIV infected population than would be predicted, based on the overall incidence of SLE. One case report described a female patient with systemic lupus erythematosus (SLE) who was infected with HIV; using stored serum, the precise timing of HIV seroconversion was determined and the early effects of HIV infection on SLE examined.
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The infection resulted in clinical improvement and the disappearance of autoantibody production [ 12]. Another case report reported a patient with HIV infection who developed SLE after the initiation of highly active antiretroviral therapy [ 13]. To date, 29 cases of association between the two diseases have been reported, but the diagnosis was simultaneous in just two of these and only 18 fulfilled the ACR criteria for the diagnosis of SLE. Most patients experienced an improvement in their SLE after development of their HIV associated immunosuppression and a reactivation of lupus manifestations also were noted after immunological recovery secondary to antiretroviral therapy [ 14]. A number of clinical and laboratory features of HIV infection are found in systemic lupus erythematosus (SLE). The presence of circulating antibodies to small nuclear ribonucleoproteins (snRNP) in both diseases was analyzed [15]. Sera from 44 HIV-infected children, from 22 patients with childhood-onset SLE, and from 50 healthy children were studied. Results included the detection of anti-snRNP antibodies by ELISA in 30 HIV-infected patients (68.1%) and 19 SLE patients (86.3%). These antibodies were directed against U1-RNP (61.3% and 77.2%, respectively), Sm (29.5% and 54.5%, respectively), 60 kDa Ro/SSA (47.7% and 50%, respectively), and La/SS-B proteins (18.1% and 9%, respectively). None of the HIV-infected children and 11 SLE patients (50%) showed anti-snRNP antibodies by counter immunoelectrophoresis (CIE). None of the HIV-infected patients showed anti-70 kDa U1-RNP or anti-DSm antibodies by immunoblotting. No differences between the two groups were noted on the presence
of nonprecipitating anti-snRNP antibodies. No such reactivities were observed among the normal sera tested. The authors concluded that non-precipitating anti-snRNP antibodies in HIV-infected children are as frequent as in childhood-onset SLE. The significance of these antibodies is not clear at present. Although polyreactive and low-affinity antibodies and a mechanism of molecular mimicry may explain these results, a specific stimulation of B cells by nuclear antigens could not be excluded. SLE patients produce high titer antibodies to various retroviral proteins, including Gag, Env, and Nef of HIV and HTLV, in the absence of overt retroviral infection. In particular, the role of HTLV1-related endogenous sequence (HRES-1) should be considered in SLE. Molecular mimicry may be a mechanism between HRES-1 and the small ribonucleoprotein complex that initiate the production of autoantibodies, leading to immune complex formation, complement fixation, and pathological tissue deposition [16].
2.2. Antiphospholipid Syndrome (APS)/ Anti-Cardiolipin Antibodies/ Anti-[i2 GPI Antibodies In 1992, the association of aCL antibodies with HIV infection in male homosexuals was reported [17]. Since then, many studies have alluded to this specific combination [18-21]. We described an unusual presentation of APS associated with acute HIV infection. The APS in this patient was characterized by elevated titers of aCL antibodies and anti-132GPI, necrotic lesions in the lower extremities and testicular necrosis requiting orchiectomy. The patient had no history of AIDS, no previous opportunistic infections, and was not on any retroviral medications. The CD4 count was only minimally decreased (CD4-322) indicating that the patient had an acute infection and was not immunosuppressed [ 18]. In another case, a 42 year old woman with a 12 year history of HIV infection developed gangrene of both forefeet. A skin biopsy revealed intracapillary thrombi and severe necrosis of the hypodermis with no evidence of vasculitis. Elevated titers of IgA antibodies were detected [22]. Anti-cardiolipin antibodies and stroke was reported in 2 HIV infected patients [23, 24]. A 33-year-old female with AIDS, a prior small cer-
ebrovascular accident, thrombocytopenia, and a coagulopathy suddenly developed left upper quadrant pain and tendemess due to splenic infarction associated with a high titer of anticardiolipin antibodies. Possible clinical manifestations of anticardiolipin antibodies in this patient include recurrent thromboembolism, coagulopathy, and thrombocytopenia. This case report suggests that anticardiolipin antibodies are associated with splenic infarction and that anticardiolipin antibodies associated with AIDS may sometimes be clinically significant [25]. Another study reported four cases with acute livedo reticularis, avascular necrosis of the femoral head, thrombosis of the inferior vena cava and pulmonary embolus, and a major pulmonary embolus [26]. Avascular necrosis (AVN) in HIV infection associated with aCL was reported in 3 cases. While no other risk factor for thromboembolic event was known, hyperlipidemia (associated with antiretroviral therapy) may have been an additional risk factor for AVN [27, 28]. Other manifestations of APS found in HIV infected patients that are yet to be elucidated are thrombotic microangiopathy (TMA) and pulmonary hypertension [29]. The aCL described in HIV patients are of both the pathogenic one (~I2GPI cofactor dependent) and the infectious type (non-132GPI dependent). It seems that following infections, one may see both types of aCL as well as all isotypes and diversity of aPL including anti-PS [30]. Antiphospholipid antibodies have previously been detected in HIV patients. The presence of lupus anticoagulant (LAC), aCL antibodies, anti-prothrombin antibodies, and anti~12 glycoprotein I antibodies were investigated in 61 HIV patients and 45 patients with APS. LAC was present in 72% of HIV patients and 81% of APS patients. Anticardiolipin antibodies were detected in 67% of the HIV patients and 84% of APS patients. The detection of anti-prothrombin and anti-132 GPI antibodies was significantly less in HIV patients [31]. A recent study demonstrated a high frequency of anti-prothrombin antibodies in a group of 100 HIV infected black patients in South Africa. These variations may be due to the study population due to a different strain of HIV encountered and predominating in infected South African patients [31]. In another study, the phospholipid specificity, avidity, and reactivity with ~2 GPI in 44 patients
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with HIV infection and compared to the results in 6 SLE patients with secondary APS, 30 SLE patients without APS, and 11 patients with primary APS was investigated [20]. Interestingly, the prevalence of aCL, anti-phosphotidyl serine, anti-phosphotidyl inositol, and anti-phosphotidyl choline (36%, 56%, 34%, and 43% respectively) was similar to that found in the SLE/APS and primary APS patients. The prevalence of these antibodies was significantly higher than that observed in SLE/non-APS patients. Anti-132 GPI antibodies occurred in only 5% of HIV-1 infected patients. A significant decrease of aPL binding after treatment with urea and NaC1 was observed in the sera of HIV- 1 infected patients when compared to APS patients, indicating that aPL antibodies from HIV patients have low resistance to dissociating agents. Anti-132 GPI antibody isotype and IgG subclass in APS patients and a variety of other thrombotic and non-thrombotic disorders including infections was studied [21 ]. Elevated levels of IgM anti-~2 GPI antibodies were observed in 65% of patients with APS and 27% of patients with HIV infection. In another study, the distribution of aCL isotypes and requirement of protein cofactor in viral infections including HIV was investigated. The isotype distribution of anti-cardiolipin antibodies in the sera from 40 patients, with infection caused by HIV-1, was studied by ELISA in the presence and absence of protein cofactor (mainly [32-GPI). The prevalence of one or more aCL antibody isotypes in serum of patients with HIV-1 infection was 47%. Most of these antibodies were mainly cofactor independent [32].
2.3. Autoimmune Thrombocytopenia Immune thrombocytopenic purpura (ITP)occurs in as many as 40% of patients infected with the human immunodeficiency virus (HIV). The evaluation of the effect of highly active antiretroviral therapy (HAART) on platelet counts in 11 homosexual men with HIV-associated ITP patients was sought. At initial evaluation, 7 patients were antiretroviral naive, 2 were taking zidovudine alone, and 2 were receiving combination antiretroviral therapy for known HIV infection. For 6 patients with < 30 • 109 platelets, prednisone was initially co-administered with HAART. The primary outcome measure was
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the platelet count response to HAART, which was measured weekly until counts had normalized on 3 consecutive occasions, then every 3 months while on HAART. Secondary outcome measures were HIVviral RNA levels and CD4+ cell counts. The results were that one month after the initiation of HAART, 10 patients had an increase in mean platelet count. This statistically significant improvement was sustained at 6 and 12 months' follow-up for 9 of 10 patients. There were no thrombocytopenic relapses at a median follow-up of 30 months. The 9 longterm platelet responders maintained on HAART, at 12 months, had a mean reduction of > 1.5 log 10 in HIV viral RNA serum levels and a marked improvement in CD4+ T-lymphocyte cell count. HAART was effective in improving platelet counts in the setting of HIV-associated ITP, enhanced CD4+ cell counts, and reduced HIV viral loads [33].
2.4. Vasculitis Different types of vasculitis are associated with HIV infection. Co-infections inducing vasculitis have been reported, including hepatitis B and C. Systemic necrotizing vasculitis, leukocytoclastic vasculitis, cryoglobulinemia, and CNS vasculitis have been reported [2, 34, 35]. Panarteritis nodosum more frequently affects the neuromuscular system and skin. Antineutrophilic cytoplasmic antibodies are found less commonly. Vasculitis of the peripheral nerve may cause mononeuritis multiplex or polyneuropathy, sometimes the presenting symptom of HIV infection or after the development of AIDS [35]. HIV antigens and HIV-particles can be identified by electron microscope and positive in situ hybridization studies for HIV have been reported in perivascular cells [2]. Evidence of HIV pathogenicity was described in a 32 year old HIV patient without coronary heart disease risk factors who developed acute coronary vasculitis resulting in a fatal myocardial infarction. Histological analysis of two coronary arteries on autopsy showed a dense infiltration of lymphocytes with necrosis of the intima. In sire hybridization showed sparse intense staining indicating the presence of HIV-1 sequences within the arterial wall [36].
2.5. Polymyositis and Dermatomyositis
in HIV-1 patients without hyperthyroidism.
HIV-associated polymyositis was first described in 1983, and many reports in the past several years confirm this association [2, 37]. Dermatomyositis is also seen in HIV infection [38]. The clinical course, laboratory and electromyography findings are similar to the idiopathic form [2]. Polymyositis in 64 HIV/AIDS patients referred for the presence of elevated creatine kinase (CK) levels or muscle weakness was evaluated. Patients underwent neurologic and rheumatologic evaluation, electromyography, and muscle biopsy after exclusion for recreational drug or alcohol use, metabolic/endocrine disorders, zidovudine therapy, and other infections. Thirteen patients (20%) had biopsy-proven myositis. The median duration of HIV infection prior to diagnosis of myositis was 4.3 years. Six patients had concomitant diffuse infiltrative lymphocytosis syndrome. There was no correlation of severity of weakness, stage of HIV infection, or retroviral treatment with the CK level at diagnosis. Eight patients received prednisone (60 mg/day) with 5 attaining complete resolution of myositis. The remaining 3 patients received immunosuppressive therapy (azathioprine or methotrexate and intravenous immunoglobulin) and had normalization of strength and CK. Four patients had spontaneous resolution of their myositis without treatment. In this study, HIV-associated myositis occurred at any stage of HIV infection, had a relatively good prognosis, and responded well to immunosuppressive therapy [39].
2.7. Primary Biliary Cirrhosis
2.6. Thyroid Disease/Graves' Disease/ Anti-Thyroglobulin Antibodies/ Anti-Thyroid Peroxidase Antibodies The kinetics of CD4 cells, HIV viral load, and autoantibodies in AIDS patients with Graves' disease after immune restoration on (HAART) was investigated [40]. Five patients were diagnosed with Graves' disease after 20 months on HAART, several months after the plasma HIV viral load was undetectable, and when the CD4 count had risen from 14 to 340x 106 cells/L. Anti-thyroid peroxidase (anti-TPO) and anti-TSHR antibodies appeared 14 months after starting HAART and 12 months after the rise in the CD4 count. No other autoantibodies were detected. The autoantibodies were not detected
The role of retroviruses in the development of primary biliary cirrhosis was sought by utilization of immunoblots [41]. Western blot tests were performed for HIV-1 and the human intracisternal A-type particle (HIAP), on serum samples from 77 patients with primary biliary cirrhosis, 126 patients with chronic liver disease, 48 patients with systemic lupus erythematosus, and 25 healthy volunteers. HIV-1 p24 gag seroreactivity was found in 35% patients with primary biliary cirrhosis, 29% patients with systemic lupus erythematosus, 50% of patients with chronic viral hepatitis, and 39% patients with either primary sclerosing cholangitis or biliary atresia, compared with only 4% of 24 patients with alcohol-related liver disease or alphal-antitrypsindeficiency liver disease, and 4% healthy volunteers (p = 0.003). Western blot reactivity to more than two HIAP proteins was found in 51% of patients with primary biliary cirrhosis, in 58% of patients with SLE, in 20% of patients with chronic viral hepatitis, and in 17% of those with other biliary diseases. None of the 23 patients with either alcohol-related liver disease or alphal-antitrypsin deficiency, and only one of the healthy controls showed the same reactivity to HIAP proteins (p <0.0001). The results showed a strong association between HIAP seroreactivity and the detection of autoantibodies to double-stranded DNA. HIAP seroreactivity was also strongly associated with the detection of mitochondrial, nuclear, and extractable nuclear antigens. The HIV-1 and HIAP antibody reactivity found in patients with primary biliary cirrhosis and other biliary disorders may be attributable either to an autoimmune response to antigenically related cellular proteins or to an immune response to uncharacterised viral proteins that share antigenic determinants with these retroviruses.
2.8. Other Autoimmune Diseases Other reported autoimmune diseases in HIVinfected patients include Raynaud's phenomenon, and Behqet's disease. The prevalence of these disorders is not known. [42, 43].
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Table 3. Autoantibodiesin HIV Autoantibody
Disease
Study group
Anti-alpha myosin Anti-EPO Anti-TPO Anti-TSHR Anti-cardiolipin Anti-PS Anti-PI Anti-PC Anti-132GPI Anti-prothrombin Anti-DNA Anti-snRNP
Left ventricular dysfunction 43% Anemia 23.5% Grave' s disease 5 patients Grave' s disease 5 patients APS 1 patient APS 56% APS 34% APS 43% APS 5%-27% ND ND ND ND ND 68.1%
Reference [45] [46] [40] [40] [ 17, 22-27, 29] [20] [20] [20] [301
[171 [44] [20]
APS - Antiphospholipid syndrome; EPO - erythropoietin; ND - not described.
3. AUTOANTIBODIES IN HIV I N F E C T I O N The array of autoantibodies reported in HIV/AIDS patients is found in Table 3. In a prospective study of 100 sequentially selected HIV-infected patients, the frequency and specificity of autoantibodies in HIV-infected subjects and their association with rheumatic manifestations, immunodeficiency, total CD4+ cell count and prognosis was evaluated. Patients were followed for 2 years. HIVinfected patients presented high overall frequency of autoantibodies. No difference was observed between immunodeficient and asymptomatic HIVinfected patients. The most frequent specificities were antibodies to cardiolipin and to denatured DNA. Immunolobulin serum levels did not correlate with the occurrence of autoantibodies. The presence of autoantibodies was associated with lower CD4+ cell counts and with higher mortality within 2 years. Rheumatic manifestations were observed in 35/100 HIV-infected patients and were not associated with the occurrence of autoantibodies or the presence of immunodeficiency. The presence of autoantibodies was significantly associated with lower CD4+ lymphocyte counts and increased mortality, which implies prognostic significance to this phenomenon in the context of HIV infection [44].
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3.1. Cardiac Autoimmunity/Anti-myosin Autoantibodies The frequency of circulating specific autoantibodies in 74 HIV positive patients with and without echocardiographic evidence of left ventricular failure was investigated. Abnormal anti-alpha myosin autoantibody concentrations were found more often in HIV patients with heart disease (43%) than in HIV positive patients without heart disease (19%) or in HIV negative controls (3%). The data support a role for cardiac autoimmunity in the pathogenesis of HIV related heart muscle disease [45]. 3.2. Anemia/Anti-erythropoietin Antibodies The association of circulating autoantibodies to endogenous erythropoietin (EPO) with HIV-1 related anemia was studied in a cohort of 204 unselected consecutive HIV-1 infected patients. Circulating autoantibodies to EPO were present in 23.5% of the patients. The circulating autoantibodies were an independent predictor of anemia. Autoimmunity may contribute to the pathogenesis of HIV-1 related anemia [46].
Autoimmune response
Total CD4 count
I I
HIV
I ...
t
II
Latency
IV HAART
III
AIDS
Stage of autoimmune response Figure 1. Autoimmune disease may develop in HIV-infected patients parallel to normal CD4 count (Stage I,II). Once the CD4 count decreases past a threshold, autoimmune disease is not present (Stage III). Following HAART, a rise in the CD4 count above the threshold enables autoimmune disease to emerge (Stage IV). 4. M E C H A N I S M S
The possible mechanisms for autoimmune manifestations of HIV infection include the direct effect of HIV on endothelial, synovial, and other hematopoietic cells resulting in destruction of CD4 cells, increased cytotoxic cell activity, and increased expression of autoantigens. Molecular mimicry is one of the proposed mechanism in the development of autoimmune disease. The exogenous infectious agent may have molecular similarity to a self-antigen and may therefore induce an autoimmune response. The basis for this mechanism has been substantiated in several studies. A previous study demonstrated that about one-third of patients with either Sj/Sgren's syndrome (SS) or SLE react to human immunodeficiency virus (HIV) p24 core protein antigen without any evidence of exposure to, or infection with, HIV itself [47]. They further characterized the specificity of this reaction using enzyme-linked immunosorbent assay to peptides representing fragments of p24. Characteristic epitope-specific profiles were seen for SS and SLE patients. All sera-reactive peptides from regions of knownstructure of HIV p24 were located in the apex of the p24 molecule. Thus, the specificity of the peptide reactivities described indicates a specific pattern of a nonrandom cross-reactivity between
HIV type 1 p24 and autoimmune sera that may be partially syndrome specific. Autoimmunity during HIV-1 infection may contribute to the immunopathogenesis of AIDS. Titers of autoantibodies to HLA molecules and other surface markers of CD4+ T cells appear to increase with the progression of disease and may correlate with lymphopenia. Other autoantibodies are directed at a number of regulatory molecules of the immune system. Genesis of autoreactivity may be related to structural homologies of HIV-1 env-products to such functional molecules involved in the control of self-tolerance. The most impressive similarities include the HLA-DR4 and DR2, the variable regions of TCR alpha-, beta-, and gamma-chain, the Fas protein, and several functional domains of IgG and IgA. Thus, HIV-1 infection may induce dysregulation leading to autoimmune response, through a number of molecular mimicry mechanisms. Pathogenicity of antibodies to T cells could also include the activation of membrane-to-nucleus signal transducers resulting in increased apoptosis. The evolution of autoimmune mechanisms during HIV1 infection cannot exclude, however, progression to immunoproliferative malignancy, since aspects of oligoclonal immune response to HIV-1 components may occur in several autoimmune diseases [48].
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In summary, autoimmune diseases and autoantibodies are present in HIV infection. Autoimmune diseases may develop during acute viral infection (Stage I), with normal to low CD4 counts (Stage II). However, past a threshold where the CD4 count is profoundly low, autoimmune disease cannot develop (Stage III). Following HAART, immune restoration (normal CD4 count) with possible altered immune regulation may lead to the emergence of autoimmune diseases (Stage IV) (Fig. 1). More studies are necessary to identify the subgroups of HIV-infected patients that may be prone to develop autoimmune diseases and autoantibodies.
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1019-20. 24. Keeling DM, Birley H, Machin SJ. Multiple transient ischaemic attacks and a mild thrombotic stroke in a HIV-positive patient with anticardiolipin antibodies. Blood Coagul Fibrinolysis. 1990;1:333-35. 25. Cappell MS, Simon T, Tiku M. Splenic infarction associated with anticardiolipin antibodies with acquired immunodeficiency syndrome. Dig Dis Sci 1993;38: 1153-55. 26. Turhal NS, Peters VB, Rand JH. Antiphospholipid syndrome in HIV infection-report on four cases and review of the literature. ACI Int 2001;13:268-71. 27. Belmonte MA, Garcia-Portales R, Domenech I, Fernandez-Nebro A, Camps MT, De Ramon E. Avascular necrosis of bone in human immunodeficiency virus infection and antiphospholipid antibodies. J Rheumatol 1993 ;20:1425-28. 28. Monier P, McKown K, Bronze MS. Osteonecrosis complicating highly active antiretroviral therapy in patients infected with human immunodeficiency virus. Clin Infect Dis 2000;31:1488-92. 29. Asherson RA, Shoenfeld Y. Human immunodeficiency virus infection, antiphospholipid antibodies, and the antiphospholipid syndrome. J Rheumatol 2003;30: 214-19. 30. Guerin J, Casey E, Feighery et al. Anti-beta-2-glycoprtein I antibody isotype and IgG subclass in antiphospholipid syndrome patients. Autoimmunity 1999;31: 10%16. 31. Loizou S, Singh S, Wypkema E, Asherson RA. IgG, IgH and IgA aPL in black South African infectious disease patients (abstract). Lupus 2002; 11:570. 32. Guglielmone H, Vitozzi S, Elbarcha O, Fernandez E. Cofactor dependence and isotype distribution of anticardiolipin antibodies in viral infections. Ann Rheum Dis 2001 ;60:500-04. 33. Aboulafia DM, Bundow D, Waide S, Bennet C, Kerr D. Initial observations on the efficacy of highly active antiretroviral therapy in the treatment of HIV-associated autoimmune thrombocytopenia. Am J Med Sci 2000;320:117-23, 34. Calabrese LH, Esyes M, Yen-Liebermann B e t al. Systemic vasculitis in association with human immunodeficiency virus infection. Arthritis Rheum 1989;32:569. 35. Brannagan TH. Retroviral-associated vasculitis of the nervous system. Neurologic Clinics 1997;15:927-44. 36. Barbaro G, Barbarini G, Pellicelli AM. HIV-associated coronary arteritis in a patient with fatal myocardial infarction. New Eng J Med 2001 ;23:1799. 37. Dalakas MC, Pezeshkpour GH. Neuromuscular diseases associated with human immunodeficiency virus
infection. Ann Neurol 1988;16:1397. 38. Gresh JP, Aguilar JL, Espinoza LR. Human immunodeficiency virus infection-associated dermatomyositis. J Rheumatol 1989; 16:1397. 39. Johnson RW, Williams FM, Kazi S, Dimachkie MM, Reveille JD. Human immunodeficiency virus-associated polymyositis: a longitudinal study of outcome. Arthritis Rheum 2003;49:172-78. 40. Jubault V, Penfornis A, Schillo F, Hoen B, Izembart M, Timsit J, Kazatchkine MD, Gilquin J, Viard JP. Sequential occurrence of thyroid autoantibodies and Grave's disease after immune restoration in severely immunocompromised human immunodeficiency virus1-infected patients. J Clin Endocrinol Metab 2000;85: 4254-57. 41. Mason AL, Xu L, Guo L, Munoz S, Jaspan JB, BryerAsh M, Cao Y, Sander DM, Shoenfeld Y, Ahmed A, Van de Water J, Gershwin ME, Garry RF. Detection of retroviral antibodies in primary biliary cirrhosis and other idiopathic biliary disorders. Lancet 1998;351: 1620-24. 42. Munoz-Fernandez S, Cardenal A, Balsa A e t al. Rheumatic manifestations in 556 patients with human immunodeficiency virus infection. Semin Arthritis Rheum 1991;21:30. 43. Routy JP, Blanc AP, Viallet C et al. Cause rare d'arthrite, la maladie de Behqet chez un sujet VIH positif, auge 69 ans. Presse Med 1989;18:525. 44. Massabki PS, Accetturi C, Nishie IA, da Silva NP, Sato EI, Andrade LE. Clinical implications of autoantibodies in HIV infection. AIDS 1997;11:1845-50. 45. Currie PF, Goldman JH, Caforio AL, Jacob AJ, Baig MK, Brettle RP, Haven AJ, Boon NA, McKenna WJ. Cardiac autoimmunity in HIV related heart muscle disease. Heart 1998;79:599-604. 46. Sipsas NV, Kokori SI, Ioannidis JP, Kyriaki D, Tzioufas AG, Kordossis T. Circulating autoantibodies to erythropoietin are associated with human immunodeficiency virus type 1-related anemia. J Infect Dis 1999;180: 2044--47. 47. Deas JE, Liu LG, Thompson JJ, Sander DM, Soble SS, Garry RF, Gallaher WR. Reactivity of sera from systemic lupus erythematosus and Sj6gren's syndrome patients with peptides derived from human immunodeficiency virus p24 capsid antigen. Clin Diagn Lab Immunol 1998;5:181-85. 48. Silvestris, Williams RC Jr, Dammacco E Children with HIV infection: a comparative study with childhoodonset systemic lupus erythematosus. Clinical Immunology Immunopathology 1995;75:197-205.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Parvovirus B19 Infection and its Association to Autoimmune Disease Marianne C. Severin 1 and Yehuda Shoenfeld ~,2
tCenter for Autoimmune Diseases, Department of Medicine 'B', Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel; 2Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases
1. PARVOVIRUS
Human parvovirus B 19 is a relatively new virus, discovered in 1975. It belongs to the genus Erythrovirus and has a tropism for erythroid precursors. Parvovirus B 19 does not need a helper virus, that is, an additional virus during time of infection to replicate, and it will only replicate productively in human cells. Parvo B 19 is a small, icosahedral, nonenveloped single stranded DNA virus with a genome of 5600 nucleotides. The cellular receptor is the P antigen, expressed on most erythroid progenitor cells, erythrocytes, megakaryocytes, endothelial cells, placental cells, and fetal liver and heart. Those who lack the P antigen are not at risk for parvo B 19 infections [3]. Since it lacks a lipid envelope, it is heat and solvent stable, resisting temperatures up to 56 ~ C at pH 3 for an hour and stable when dissolved in ether or chloroform. Parvo B19 encodes three major proteins, VP1 and VP2 of 84,000 and 58,000 d respectively, which are structural proteins, NS1 and two small amino acids which are nonstructural proteins. VP1 and VP2 are identical except for an amino-terminal end of 227 amino acids located on the VP1 protein and expressed on the particle surface, therefore being exposed to neutralizing antibodies. VP2 accounts for 96% of the total capsid proteins. NS1 (77,000 d) is the major nonstructural protein and its functions include having specific DNA-binding and helicase activities, acting as a cellular promoter, and involved in ATPase acitivity and cytotoxicity of the protein. Replication and assembly occurs in the cell nucleus. The incubation time ranges from 6 to up to
28 days. Generally, most parvovirus B 19 infections pass unnoticed without any clinical symptoms. The infection is biphasic, consisting of a viremic phase and an antibody response phase. During the viremic phase, symptoms range from being asymptomatic to having flu-like symptoms. The second phase of the infection is associated with rash, arthalgias and arthritis. The virus resides in nasal and oropharyngeal tissue, and during viremia, the person is infectious via nasal and oral secretions. No viral DNA is detected in urine or feces. After initial infection, the body first responds to the major capsid antigen VP2 with both IgM and IgG, the IgM phase appearing on days 10-12 after inoculation, and lasting one to three months afterwards. Peak viremia occurs on days 8, 9 after inoculation and clearance is proportional to high titer anti-B 19 IgM antibodies. IgG antibodies appear several days after the IgM response and are believed to persist throughout one's lifetime. During peak viremia, there is an absolute areticulocytosis until the appearance of antibodies followed by rebound reticulocytosis [4]. There is a fall in hemoglobin as well as neutropenia, lymphopenia, and thrombocytopenia. During convalescence, antibodies to another capsid protein, VP1 was formed. A prolonged viral presence also results in antibodies against the nonstructural protein NS 1 [5]. Diagnosis of an acute parvo infection is made by IgM serology. There is a brief window of opportunity in which the diagnosis can be determined since it has been estimated that 40%-80% of the adult population has already been exposed. IgG antibodies to parvo B 19 begins appearing and is most common after the age of five suggesting that they
181
are infected after entering school. In North America, most outbreaks occur during the late winter and early summer months.
2. CLINICAL MANIFESTATIONS
2.1. Transient Aplastic Crisis Transient aplastic crisis is defined as "An acute fall in hemoglobin associated with cessation of reticulocyte production in the setting of chronic hemolytic anemia" [7]. Seven to ten days afterwards, there is reticulocytosis and a return to previous hemoglobin levels. Infection is suspected when it occurs in chronic anemic populations and does not recur implying some sort of immunity. The association of B19 with other hemolytic anemias was later confirmed.
2.2. Erythema Infectiosum Once thought to be one of the six major childhood diseases, erythema infectiosum or fifth disease does indeed primarily occur in the pediatric population, resulting in a characteristic rash of the face and torso. It may be macular, maculopapular, occasionally vesicular or hemorrhagic, and pruritus occurs in about half. The rash occurs after virus resolution, usually after sun exposure, hot bath, or physical activity. Relapses occur, but are not associated with new onset viremia or viral shedding. Many are asymptomatic, and when symptoms do occur, they are usually mild. Adults lack the usual "slapped cheek" appearance as seen in children, however tend to have more joint involvement such as arthritis, arthalgia, joint swelling, and severe flu-like symptoms. In a report describing an outbreak of erythema infectiosum from 1961-1962, 77% of the adults were reported to have had joint pain, and 60% joint swelling as opposed to children under 10 yrs who had had 5% and 3% respectively [8-10]. Later, it was demonstrated that parvo B 19 could cause a "chronic rheumatoid-like arthropathy", showing a female preponderance. Patients presented with acute, moderately severe, symmetric polyarthritis which usually began in the hands or knees. Within 24--48 hours there was other joint involvement such as the wrists, ankles,
182
feet, elbows, and shoulders. All patients suffered from joint pain and stiffness, with varying degrees of swelling, and most experienced an improvement within two weeks. Only two of the 153 patients had a complete resolution. More than half had general symptoms of malaise, fever, gastrointestinal disturbances, or rash, and two thirds had recurrences with disease free intervals. There were however no erosions or joint destruction.
2.3. Hydrops Fetalis Infection with parvo B 19 will cause fetal infection in approximately 30% of the fetuses and of those, 9% will have adverse effects such as an aplastic crisis, edema, ascites, pleural effusions, viral cardiomyopathy, and polyhydramnions. Congenitally, it is characterized by anemia, thrombocytopenia, cardiac and hepatic dysfunction. Fetuses known to be infected have also been successfully treated by transfusion in utero showing the importance about B 19 awareness.
2.4. Immunocompromised Patients Patients with inadequate immune responses, be it congenital or acquired, may be unable to clear parvo viremia. There are reported cases of various immunocompromised patients such as AIDS, cancer patients, etc. who have had chronic B 19 infections and persistent bone marrow suppression. Unlike immune competent patients, these patients fail to produce IgG antibodies which normally neutralize B 19, and prevent future infections.
2.5. Additional Clinical Manifestations of Parvo B19 Infections Parvo infections can present with a palette of other manifestations which are however less typical. Atypical skin manifestations include a combination of morbilliform and vesiculopustular lesions, extravasation of erythrocytes into the dermis, purpura or petechiae without thrombocytopenia, Henoch-Schoenlein purpura appearance and gloves and socks syndrome, which can also be caused by other viral infections. Atypical neurological presentations include peripheral neuropathies, encephalopathy, aseptic meningitis, and a possible trigger for
fibromyalgia. Parvo B 19 can cause isolated neutropenia, thrombocytopenia, or anemia. Cases of ITP have been reported as well as hemophagocytic syndrome [ 11]. In addition, patients have had transient elevations in liver enzymes, hepatitis, myositas, and neurological disease [ 12, 13]. Persistent infections have been reported in both immunocompromised patients and non-immunocompromised patients.
3. PARVOVIRUS B19 INFECTION AND LINKS TO A U T O I M M U N I T Y Human parvovirus B19 has recently been associated with various rheumatic disorders and other autoimmune diseases. Acute parvovirus B 19 can present with symptoms resembling rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), vasculitis, and various hematological disorders and similarities encompass not only clinical symptoms, but also serology, histology, and laboratory abnormalities. 3.1. Rheumatoid Arthritis (RA) Various studies have proposed an infectious etiology to RA such as parvovirus B 19, although this is still controversial [14, 15]. Rheumatoid arthritis is an autoimmune disease of unknown origin characterized by chronic inflammation of the synovia, infiltration of lymphocytes, inflammatory cells, fibroblasts, cytokines, and proteolytic enzymes. The disease is usually progressive and results in joint deformation, destruction, and impaired function. There are reported cases of patients who, after having had an acute parvovirus B 19 infection developed active RA including joint destruction, rheumatoid nodules, and rheumatoid factor [16]. Some patients demonstrated persistence of B 19 DNA and VP-1 antigen for more than 7 years [ 17]. Further analysis revealed that B 19 DNA was rarely found in the blood with the exception of initial episodes of RA. In the majority of the cases, (almost 75%) parvovirus B19 were found in the synovial tissue, and a third of the cases were found in the bone marrow [18]. VP-1 protein was found exclusively in synovial cells. It should also be noted that B 19 DNA was found in non-arthropathy patients, however B 19 RNA and VP-1 antigen in the synovia were specific to RA [19].
Table 1. Similarities between RA and parvo B 19 infections Morning stiffness Joint deformation Joint destruction Rheumatoid nodules Rheumatoid factor Chronicity Production of lymphocytes Induction of IL-6 and TNF tx .
.
.
.
.
.
.
.
Parvovirus B19 is thought to be involved as a causative agent of RA since patients who were treated with IVIG therapy demonstrated a decrease of VP-1 protein in the bone marrow and a clinical improvement. Another important association involves the cells it affected. As mentioned earlier, parvovirus B19 targets erythroblasts, erythroid progenitor cells, and megakaryocytes due to the P antigen, which is located on the red cell membrane. However, VP-1 as well as parvovirus B 19 were also found in follicular dendritic cells, macrophages, T and B cells in the synovia of RA patients [20]. Normally in humans, after parvovirus B 19 infects its target cell, apoptosis is shortly after induced. However, in RA associated B 19 infections, parvovirus, which also infects lymphocytes, macrophages, and dendritic cells, may be non-cytotoxic in these cells. When these cells are infected, they may in turn influence various cytokines such as IL-6 and tumor necrosis factor cz (TNF cz). It is hypothesized that B 19 positive T cells and macrophages infiltrate the synovia, which then recruit immunocytes as well as activating various cytokines [21, 22]. Another mechanism proposed is due exclusively to its tie to IL-6. In an experiment by Moffatt et al [23], it was shown that transfected human hematopoietic cell lines, which produced NS 1 also secreted IL-6 in response. No other cytokines such as IL-1 beta, IL-8 or TNF cz were produced. Additionally, IL-6 was transiently induced in human endothelial cells when primed with NS1. IL-6 production due to NS 1 occurs not only in erythroid cells, but also in lymphoid cells, monocytes, and endothelial cells and is dependent on the intracellular environment. It is thought that the virus possesses a factor,
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which induces IL-6 that can possibly lead to RA. RA patients have huge amounts of IL-6 in inflamed joints and administration of anti IL-6 was associated with inhibition of major clinical manifestations, acute phase proteins and IL-6 quantifies [24]. Evaluating the relationship between IL-6 and RA from another perspective, deregulation of IL-6 has been strongly implicated in polyclonal B cell activation [25] and synovial cell proliferation in RA patients [26]. This is suggestive that abnormal IL-6 production may contribute to autoantibodies, rheumatoid factor, or generalized autoimmune diseases. In conclusion, NS 1 may be involved in the pathogenesis of parvovirus B 19 related diseases.
3.2. Systemic Lupus Erythematosus (SLE) SLE, a multi-systemic, chronic, inflammatory autoimmune disease of unknown origin is characterized by the production of autoreactive cells and antibodies. Today, it is considered to be the prototype of autoimmune diseases and can potentially involve all organ systems unlike other autoimmune diseases, which are more organ-specific. There are many reported cases of an association between parvovirus B 19 and SLE. Patients with known SLE have had disease exacerbations after being infected with parvo B 19 [27]. Hsu et al [28] postulate that SLE patients are in general more apt to be infected and re-infected by parvovirus B 19 due to lack of antibodies, either due to natural immune deficiencies or immunosuppressive drugs. Producing antibodies to the capsid protein is essential in stopping the parvo infection and those with prolonged disease suggest a quantitative defect [29]. Persistent infections may indicate an inability to handle the viral load or inability to produce the necessary antibodies [30]. Clinicians recount of previously healthy patients who presented with rash, malar rash, arthalgia, arthritis, fever, fatigue renal involvement, and varying serolgies as listed in the table, such that 3-5/11 of the criteria for the American College of Rheumatology were fulfilled. In the majority of the cases, the disease was self-limited, causing clinicians to re-evaluate their original diagnosis. In all cases, there were significant IgM titers to parvovirus B 19. In these cases, it would appear that parvovirus mimicked SLE. However, the remainder of the cases progressed to frank SLE implying that parvovirus
184
Table 2. Similarities between SLE and parvovirus B19 infections Clinical features Malar rash Fever Photosensitivity Non-destructive arthritis/arthalgia Symmetric, polyarticular (adults) PIP, MCP involvement Knee involvement (children) Inflammation Morning stiffness Pain Fatigue Lymphadenopathy Hepatitis (rare) Hepatomegaly Abortion [57] Headache GI symptoms: nausea, vomiting, abdominal pain Pleural effusion Pericardial effusion [58] Proteinuria [59] Myalgia Female predisposition Vasculitis/Angioedema [60, 61] Encephalopathy/Aseptic meningitis [62, 63]
B 19 may have acted as a trigger. Reasons may be due to a particular HLA predisposition [31 ].
3.3. Antiphospholipid Syndrome (APS) Antiphospholipid syndrome (APS) is a disorders characterized by one or more thrombolic/thromboembolic events, multiple abortions and the presence of antiphospholipid antibodies. Increased antiphospholipid antibodieS (aPL) were first described in patients with autoimmune connective tissue disorders. Later, it was also recognized that aPL also appeared after certain infections such as chicken pox, hepatitis A, infectious mononucleosis, rubella, adenovirus, mumps, HIV, syphilis, parvovims, and others [32]. In a study by Loizou et al [33], antiphospholipid antibody specifity from SLE and
APS patients were compared to that of parvovirus B19 infected patients. Parvovirus was shown to bind to negatively charged phospholipids and [32 glycoprotein-I ([32 GP-I), both typical of APS and SLE as well as similar phospholipid specifity and isotype distribution. ~I2GP-I, a plasma protein and cofactor enhancing binding to the antiphospolipid antigen showed enhanced binding to parvovirus, unlike other viral infections [34]. Despite these similarities, it is still unclear why this occurs. Possible explanations include cross reactivity, faulty regulation and/or molecular mimicry. One of the proposed mechanisms involves production of anti-phospholipid antibodies due to phospholipase-A2-1ike activity found in the structural protein VP1 of parvovirus [35]. This in tum may conduce the production of leukotrienes and prostaglandins as well as lead to cleaved cellular phospholipid products. In this setting, including a gentetic predisposition may result in a parvovirus induced antiphospholipid-like syndrome.
3.4. Vasculitis Parvovirus B 19 has also been implicated in various vasculitis such as polyarteritis nodosa, temporal arteritis, Wegener's granulomatosis, and Kawasaki's disease [36]. This theory was put fourth based on sporadic reports of new onset vasculitis supported by serological proof of an acute parvovirus B 19 infection. In certain cases, patients who were treated with IVIG therapy, had remissions without relapses, supporting the idea that parvovirus B 19 may have induced the vasculitis [37]. In the case of temporal arteritis, parvo B 19 was actually detected in temporal artery tissue. The question was raised whether parvo B19 infection is purely circumstantial and not etiological. However, when these vasculitis cases were treated with conventional therapy such as corticosteroids or cyclophosphamide, there was no clinical improvement. Only after IVIG administration did the symptoms improve and the virus cleared without recurrence, implying a causative role. The fact the IVIG therapy was comerstone might suggest that patients had defective humoral immune responses and were possibly unable to clear the virus alone. Normally in healthy individuals, IgG to parvovirus is demonstrable in blood years after the initial infec-
tion. However in some of these "vasculitis" patients, there were no detectable IgG antibodies after having received IVIG therapy [38]. Parvovirus B 19 was once thought to cause vasculifts by deposits of circulating immune complexes in the vascular endothelium, however due to low circulating complexes and a poor antibody response, this no longer holds true. Instead, it is thought that the P antigen located on erythroid cells and endothelial cells, is also the receptor for parvovirus B 19, and vasculitis is thought to arise from direct injury and direct infection by the virus [39]. In a recent case-control study Eden et al [40] compared the prevalence of parvovirus and another Erythrovirus, V9 among patients with antineutrophil cytoplasmic antibodies (ANCA) vasculitis, and healthy controls. ANCA is usually present in various vasculitides such as Wegener's granulomatosis (88%) [41], microscopic polyangiitis (75%) [42], and Churg-Strauss syndrome (48%) [43]. In this case report thirteen patients with one of the above mentioned vasculitides were compared to three age and sex matched controls. They found that 77% of the vasculitis patients were infected with either parvovirus B 19 or V9 while 79% of the healthy controls were infected. Both groups demonstrated only IgG antibodies in the sera, none had IgM. Eden et al in turn conclude that neither parvovirus nor V9 play an etiological role in ANCA associated vasculitides.
3.5. Autoimmune Cytopenia Autoimmune cytopenias are disorders, which usually occur in childhood and can affect any of the bone marrow precursor cells resulting in anemia, neutropenia, or thrombocytopenia. Usually one cell line is affected although various combinations do exist such as Evan's Syndrome, (autoimmune hemolytic anemia and thrombocytopenia), simultaneous neutropenia and thrombocytopenia. Chronic neutropenia of childhood is defined as neutropenia in children occurring for three or more months, however clear criteria for diagnosis have not been established. Autoantibodies are not always detected and are not necessary for the diagnosis [44]. The neutropenia usually runs a benign course and remits spontaneously. Possible causes include viral infections, drugs, autoimmune diseases, and
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glycogen storage diseases. Parvovirus B 19 infections have been linked to various cytopenias however reports are somewhat conflicting. There are reports of anemia, thrombocytopenia and associated neutropenia after parvovirus infection [45]. One study by Bux et al [46] suggested a parvovirus etiology after demonstration of anti-B 19 IgM in five of eleven cases. Another study by Koch et al [47] refuted this after examining 110 serum samples from infants with primary autoimmune neutropenia (AIN). Approximately only one third of the cases which demonstrated anti-B 19 IgG/IgM or virus specific DNA, and had at best, only threshold values [48]. Another group [49] reported of an 11 year old boy who presented with thrombocytopenia, neutropenia and splenomegaly without specific neutrophil antibodies. Polymerase chain reaction (PCR) revealed parvovirus B-19 specific antibodies in the bone marrow but not the serum including antibodies to VP1, VP2, and NS1. In addition, he had persistant evidence of parvo B 19 infection in the bone marrow. Although this condition is rare, it is believed that parvo B 19 acted as a permanent trigger [50]. The mechanism for this condition is not yet established, however there exist a number of hypotheses. Cytopenias may be central, due to bone marrow suppression, or peripheral, due to antibody formation to the particular cell line. Central cytopenia is thought to be caused by the NS 1 protein which can suppress megakaryocyte formation [51], lower myeloid precursor cells or neutrophils [52]. As outlined earlier, after an acute parvovirus infection, IgM antibodies against VP1 and VP2 begin to rise followed by IgG two weeks later which persist throughout one's lifetime. Six weeks after the initial infection, antibodies to NS1 form. Persistence of NS specific antibodies has been associated with delayed virus elimination and delayed antibody formation[53] as well as various autoimmune diseases such as arthritis, chronic anemia, SLE, thrombocytopenia and others [54]. After viral infections, autoantibodies may emerge. Disease may be a result of molecular mimicry, implying common antigenic epitopes, although to date none have yet been identified. It is therefore proposed that parvo B19 may trigger and sustain an autoimmune reaction similar to cases reported of SLE [55]. Disease may also result from cross reaction of antibodies to self such as keratin, col-
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lagen type II, thryeoglobulin, or antinuclear bodies and has been demonstrated in patients with chronic parvovirus B 19 infection [56].
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16. Takahashi, Y, Murai C, Shibata S, Munakata Y, Ishii, T, Ishii K, Saitoh T, Sawai T, Sugamura K, Sasaki T. Human parvovirus B 19 as a causative agent for rheumatoid arthritis. Proc Natl Acad Sci 1998;95:8227-8232. 17. Sasaki T, Takahashi Y, Murai C, Munakata Y, Ishii K, Sugamura K. Arthritis Rheum 1996;9:147. 18. Takahashi, Y, Murai C, Shibata S, Munakata Y, Ishii, T, Ishii K, Saitoh T, Sawai T, Sugamura K, Sasaki T. Human parvovirus B 19 as a causative agent for rheumatoid arthritis. Proc Natl Acad Sci 1998;95:8227-8232. 19. Soderlund M, von Essen R, Haapasaari J, Kustala U, Kiviluoto O, Hedman K. Lancet 1997;349:1063-1065. 20. Takahashi, Y, Murai C, Shibata S, Munakata Y, Ishii, T, Ishii K, Saitoh T, Sawai T, Sugamura K, Sasaki T. Human parvovirus B 19 as a causative agent for rheumatoid arthritis. Proc Natl Acad Sci 1998;95:8227-8232. 21. Moffatt S, Tanaka N, Tada K, Nose M, Nakamura M, Muralka O, Hirano T, Sugamura K. A cytotoxic nonstructural protein NS 1, of human parvovirus B 19 induces activation of interleukin-6 gene expression. J Virol
1996;70:8485-8491. 22. Tak PP, Smeets TJ, Daha MR, Kluin PM, Meijers, KA, Brand R, Meinder AE, Liew FY. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 1997 ;40:217-225. 23. Moffatt S, Tanaka N, Tada K, Nose M, Nakamura M, Muralka O, Hirano T, Sugamura K. A cytotoxic nonstructural protein NS1, of human parvovirus B19 induces activation of interleukin-6 gene expression. J Virol 1996;70:8485-8491. 24. Bataille R, Barlogie B, Lu ZY, Rossi JF, LavabreBertrand T, Beck J, Wijdeness J, Brochier J, Klein B. Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood 1995 ;68:685-691. 25. Hirano T, Akira T, Taga T, Kishimoto T. Biological and clinical aspects of interleukin 6. Immunol Today 1990;11:443-449. 26. Jorgensen C, Angel J, Fournier C. Regulation of synovial cell proliferation and prostaglandin E2 production by combined action of cytokines. Eur Cytokine Netw 1991;2:207-215. 27. Hemauer A, Bechenlehrer K, Wolf H, Bernhard L, Modrow S. Acute Parvo B 19 infection in connection with a flare of systemic lupus erythematosus in a female patient. J Clin Virol 1999;14:73-77. 28. Hsu TC, Tsay GJ. Human parvovirus B 19 infection in patients with systemic lupus erythematosus. Rheumatology 2001 ;40:152-157. 29. Kurtzman GJ, Cohen BJ, Field AM et al. Immune response to B19 parvovirus and an antibody defect in persistent viral infection. J Clin Invest 1989;84:
1114--1123. 30. Moore TL, Bandlamudi R, Alam SM, Nesher G. Parvoviris infection mimicking systemic lupus erythematosus in a pediatric population. Semin Arthritis Rheumat 1999;28:314-318. 31. Tovari E, Mezey I, Hedman K, Czirjak L. Self limiting lupus-like symptoms in patients with parvovirus B19 infection. Ann Rheumat Dis 2002;61:662-3. 32. Loizou S, Cazabon J, Walport M, Tait D, So A. Similarities of Specifity and Cofactor Dependence in Serum Antiphospholipid Antibodies from Patients with Human Parvovirus B 19 Infection and From Those With Systemic Lupus Erythmatosus. Arthritis & Rheum 1997;40:103-108. 33. Vaarala O, Palosuo T, Marjaana K, Aho K. Anticardiolipin response in acute infections. Clin Immunol Immunopathol 1986;41:8-15. 34. Loizou S, Cazabon J, Walport M, Tait D, So A. Similarities of specifity and cofactor dependence in serum antiphospholipid antibodies from patients with human parvovirus B 19 infection and from those with systemic lupus erythmatosus. Arthritis & Rheumat 1997;40: 103-108. 35. Dorsch S, Liebisch G, Kaufmann B, von Landenberg P, Hoffman JH, Drobnik W, Modrow S. The VP1 unique region of parvovirus B 19 and its constituent phospholipase A2-1ike activity. J Viro12002;76:2014-2018. 36. Magro CM, Crowson AN, Davood M, Nuovo GJ. Parvoviral infection of endothelial cells and its possible role in vasculitis and autoimmune diseases. J Rheumato12002;29:1227-1235. 37. Viguier M, Guillevin L, Laroche L. Treatment of parvovirus B19-associated polyartertitis nodosa with intravenous immune globulin. N Engl J Med 2001 ;344: 1481-2. 38. Gabriel SE, Epsy M, Erdman DD, Bjomsson J, Smith TF, Hunder GG. The role of parvovirus B 19 in the pathogenesis of giant cell arteritis. Arthritis & Rheumat 1999;42:155-1258. 39. Finkel TH, Torok TJ, Ferguson J, Durigon EL, Zaki SR. Leung DY, Harbeck RJ, Gelfand EW, Saulsbury FT, Hollister JR, Anderson LJ. Chronic parvovirus B 19 infection and systemic necrotizing vasculitis: opportunistic infection or aetiological agent'?, Lancet 1994;343: 1255-1258. 40. Eden A, Mahr A, Servant A, Radjef N, Amard S, Mouthon L, Garbarg-Chenon A, Guillevin L. Lack of association between B 19 or V9 erythrovirus infection and ANCA-positive vasculitides: a case-control study. Rheumato12003 ;42:660-664. 41. Hoffman GS, Kerr GS, Leavitt RY et al. Wegener granulomatosis: an analysis of 158 patients. Ann Intern Med, 1992;116:488-498.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity
Y. Shoenfeld and N.R. Rose, editors
Hepatitis C Infection and Vasculitis Dimitrios Vassilopoulos
University of Athens School of Medicine, Department of Medicine, Hippokration General Hospital, Athens, Greece
Vasculitis in the setting of chronic hepatitis C virus (HCV) infection occurs in different forms (Table 1) [1]. The most common form is a small vessel vasculitis due to the deposition of immune complexes containing cryoglobulins i.e. HCV-associated cryoglobulinemic vasculitis (CV). Involvement of medium-size vessels is an uncommon finding in HCV-associated CV whereas there are few reports of medium size vasculitis in the setting of chronic HCV infection, in the absence of circulating cryoglobulins (polyarteritis nodosa/PAN-like). There are also rare case reports of large vessel vasculitis (giant cell arteritis) or central nervous system (CNS) vasculitis in patients with hepatitis C, but a clear association between the two entities has not been clearly established. In this chapter, the epidemiological, pathogenetic, clinical and laboratory findings in HCVassociated vasculitis will be reviewed as well as the currently available therapeutic approaches.
1. H C V - A S S O C I A T E D C R Y O G L O B U L I N E M I C VASCULITIS 1.1. Definition
HCV-associated CV is a clinical syndrome that develops in a subset of patients with HCV-associated mixed cryoglobulinemia (MC). There are currently no widely accepted diagnostic or classification criteria for HCV-associated CV or CV in general. The definition that was adopted by the Chapel Hill Consensus Conference on the Nomenclature of Systemic Vasculitides, defined essential CV as
Table 1. Types of HCV-associated vasculitis 9
HCV-associated cryoglobulinemic vasculitis a (mainly affecting small vessels, rarely medium size vessels)
Medium-sizevasculitis (systemic PAN-like, cutaneous PAN) 9 Largevessel vasculitisb 9 CNS vasculitisb
9
aMost common type. bUncertain association.
Abbreviations: HCV = Hepatitis C virus, PAN = polyarteritis nodosa, CNS = central nervous system.
a "vasculitis with cryoglobulin immune deposits affecting small vessels (i.e. capillaries, venules or arterioles) and associated cryoglobulins in serum" [2]. It was noted that the skin and glomeruli are frequently involved. Based on the criteria proposed by the GISC (Italian Group for the Study of Cryoglobulinemias) [3], Lamprecht et al utilized a similar set of classification criteria that included the presence of symptomatic cryoglobulinemia for > 6 months, > 2 symptoms of the Meltzer's triad (purpura, arthralgias, weakness), detection of high rheumatoid factor (RF) activity and/or low C4 levels, in the absence of coexistent diseases (autoimmune, infectious, lymphoproliferative) that may account for the cryoglobulinemia [4]. Recently, Ferri and his colleagues have also proposed a set of classification criteria for MC [5]. None of these criteria though have been validated in a standardized fashion, thus the need for a consensus in formulating classification criteria becomes
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evident. Despite the absence of accepted criteria for HCVassociated CV, most authors agree that for an accurate diagnosis a number of serologic, pathologic and clinical findings should be present [5-8]: 9 Active chronic HCV infection should be estabfished by the presence of anti HCV antibodies and HCV RNA in the serum by established methods [8]. 9 Circulating mixed cryoglobulins (type II or III, according to the classification proposed by Brouet) [9] measured by an appropriate method must be present in the serum of infected patients (cryoglobulinemia). Since cryoglobulinemia is a common laboratory finding of patients with chronic HCV infection (35-55%), clinical and pathological findings strongly suggestive of vasculitic involvement should be also present. 9 Clinical findings suggestive of vasculitis including purpura, neuropathy (symmetric distal polyneuropathy or mononeuritis multiplex), membranoproliferative glomerulonephritis (MPGN) and skin ulcerations or digital necrosis. Rarely, clinical findings suggestive of gastrointestinal, cardiac or CNS vasculitic involvement may be present. 9 Pathological findings of small-vessel vasculitis affecting the skin (leukocytoclastic purpura), nerves, muscles or other involved organs are extremely helpful in making the correct diagnosis. The documentation of the presence of immune complexes in affected vessels either by immunofluorescence or electron microscopy (kidneys) is an additional important diagnostic tool.
1.2. Epidemiology The frequency of HCV-associated CV has not been investigated in large epidemiological studies. The worldwide prevalence of HCV infection has been estimated to be approximately 3% [10]. Although, as mentioned earlier, the prevalence of cryoglobulinemia is reported consistently in the range between 35 to 55% in HCV infected individuals, the frequency of HCV-associated CV is significantly lower [8]. Although earlier studies, have indicated a high frequency of HCV-associated CV in HCV
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patients with cryoglobulinemia [11], recent studies including large number of patients have reported a much lower frequency of vasculitis [ 12]. Cacoub et al in a prospective study of 1614 patients with HCV infection found an overall frequency of vasculitis of 1% while in patients with cryoglobulinemia the frequency was 2-3% [12]. Similar low rates of HCV-associated CV (0-11%) have been reported in recent studies [13-15]. Extrapolating from these data, one can assume that the prevalence of HCV-associated CV in the general population should range between 0.010.3% (based on the prevalence of HCV infection in the studied population). This estimate though is probably an overestimation, since most studies examining the frequency of cryoglobulinemia and HCV-associated CV in HCV patients are biased (referral and patient selection bias) [16]. Moreover, there is clearly a shortage of well designed population-based studies on the frequency of this type of vasculitis. In a recent retrospective study of a well-defined population of Northwestern Spain (--250,000 people), Gonzalez-Gay et al found only one case of HCV-associated CV over a 10-year period [ 17]. Geographical variation exists with the disease being more common in Southern Europe compared to Northern Europe and America [6]. The mean age of patients with HCV-associated CV is approximately 50 years (range 40-60 years) while there appears to be a female predominance (female" male ratio = 3:1) [5, 18, 19].
1.3. Clinical Characteristics 1.3.1. Skin manifestations Deposition of cryoglobulins (with or without associated HCV) in skin vessels leads to a localized inflammatory reaction that is manifested clinically by a number of skin findings including purpura, leg ulcers and more rarely digital necrosis [5, 18-21]. The hallmark of HCV-associated CV is the appearance of purpuric lesions in the lower extremities. Purpura has been reported in 65-90% of patients and in most cases represents the presenting manifestation of the disease [5, 18-21]. Typically its appearance follows an intermittent pattern while the lesions tend to be non-pruritic with a lower extrem-
Table 2. Demographic and clinical characteristics of patients with HCV-associated mixed cryoglobulinemia Characteristic Mean age (range, years) Female : male ratio Purpura Weakness (asthenia) Arthralgia Leg ulcers Peripheral neuropathy Renal involvement " Sicca syndrome Raynaud's phenomenon
Table3. Laboratory findings of patients with HCVassociated mixed cryoglobulinemia Characteristic
50 (40-60) 3:1
Rheumatoid factor (RF)
68-75 %
Low C4
50-85%
65-90% 45-90% 40-80% 30-40% 8-55 % 20-35% 6-36% 3-40%
ANA
12-32%
SMA
-23%
AMA
-10%
Anti-thyroid antibodies
- 10
Data from Refs. [5, 18-21].
ity predilection [22]. Purpuric lesions are found more commonly in limbs with venous insufficiency and their disappearance is followed by residual skin hyperpigmentation which can last for prolonged period of time [23]. Biopsies of the purpuric lesions show the typical findings of leukocytoclastic vasculitis with a predominance of mononuclear cells and neutrophils [19, 24]. Immunofluorescent studies reveal the deposition of IgM and C3 in the vessel walls in approximately 80% of the cases [ 19, 25]. Attempts to detect HCV RNA in skin biopsies from patients with HCV-associated CV has given conflicting results, with some studies showing the presence of HCV virions in endothelial cells [24, 26] or vessel walls [26] whereas other studies failed to reproduce these findings [27]. In the study by Agnello et al, HCV RNA was detected in most cases in complexes with IgM and/or IgG antibodies [26]. The appearance of leg ulcers is another common skin manifestation of HCV-associated CV (30-40%, Table 1) [ 19, 20, 22]. The ulcers typically are localized in the lower extremities (above the malleoli) in association with purpuric lesions. Other less common skin manifestations of HCVassociated CV include digital necrosis, nodules and urticarial lesions [24] with variable histopathologic findings [24].
Data from Refs. [5, 7, 19, 21].
1.3.2. Nerve involvement
The predominant form of nerve involvement in HCV-associated CV is peripheral neuropathy that is detected in 8-55% of HCV patients with cryoglobulinemia (Table 2) [5, 18-21]. Peripheral nerve involvement occurs either as a symmetric distal polyneuropathy (--80%) or as mononeuritis multiplex (--10%) [28]. The role of mixed cryoglobulins in the pathogenesis of peripheral neuropathy appears crucial, since the development of neuropathy in non-cryoglobulinemic HCV infected patients is an uncommon event [28, 29]. Patients with distal polyneuropathy present with a painful symmetric neuropathy with predominant sensory findings (paresthesias) [28]. Electromyographic studies reveal an axonal sensory neuropathic process while nerve or muscle biopsies from the affected areas show inflammatory vascular lesions in the majority of cases (-83%) [28]. These inflammatory lesions take the form of vasculitis of the small and/or medium-size vessels or infiltration of vessel wall by mononuclear cells without necrosis [29]. A direct pathogenetic role for HCV has been postulated based on the detection of HCV RNA by sensitive assays in biopsied material [28, 30, 31]. HCV RNA has been found in endothelial cells, infiltrating mononuclear cells or in immune complexes deposited in the arterial wall [28]. Despite its presence though, localized viral replication as evidenced by the detection of its replicative (negative) strand has not been documented so far [28]. Mononeuritis multiplex is another less common
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neurological manifestation with prominent inflammatory vascular lesions in pathological specimens [28] and IgM deposition by immunofluorescent studies [32]. In a recent study by Authier et al, HCV RNA was not present in muscle or nerve biopsies of three patients with mononeuritis multiplex [28]. CNS involvement has been rarely reported in patients with HCV-associated cryoglobulinemia [19, 33-35]. A number of clinical manifestations have been observed including cerebrovascular accidents, seizures, encephalopathy, dizziness and dementia [19, 33-35]. Interpretation of these limited data is problematic since detailed analysis including angiography and/or brain biopsy has not been performed in each case. 1.3.3. Renal involvement
Kidney involvement is present in 20-35% of HCV patients with mixed cryoglobulinemia (Table 2) [5, 18-21]. The most common form of renal involvement is that of MPGN (55-80%) [36--38]. Less common forms include messangial proliferative glomerulopathy, membranous nephropathy and focal segmental glomerulosclerosis [37]. Patients with MPGN typically present with hypertension (~80%), proteinuria (--55%, usually in the nephrotic range), hypoalbuminemia and mild to moderate renal insufficiency [37, 38]. Typically, renal involvement develops during the evolution of HCV-associated systemic MC [36, 38] with only 15% of the cases displaying a concomitant renal and extrarenal involvement at presentation [39]. HCV-associated MPGN usually follows a fluctuating clinical course with frequent episodes of exacerbation [39]. The true incidence of end-stage renal disease in these patients is unknown. In the largest study in the literature, Tarantino et al reported that approximately 15% of 105 patients developed end-stage renal disease requiting dialysis during a 10 year follow-up [40]. This group of patients though had a high mortality rate (40%) during the same follow-up period, indicating a possible patient selection bias (inclusion of referred patients with more severe renal disease). Although data on the clinical course and prognosis of non-MPGN forms of HCV-associated renal disease are limited [36, 40], no significant differences with MPGN have been observed.
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Renal biopsies in patients with MPGN reveal the typical histological findings of an immune-complex mediated glomerulonephritis characterized by hypercellular glomeruli (mainly by infiltrating monocytes/macrophages), subendothelial and endocapillary deposits, and IgM/IgG and C3 glomerular deposition [36, 39]. In some cases, characteristic intraluminal thrombi composed of deposited immune complexes are noted [36, 39]. Vasculitis of small and medium size vessels is present in one third of cases [39]. As is the case with peripheral neuropathy in patients with HCV associated CV, HCV RNA has been detected in kidney tissues in a number of studies but its direct pathogenetic role has not been proven [37, 41]. Search for HCV-encoded proteins in kidney biopsies has given inconsistent results so far [37, 42]. 1.3.4. Other clinical findings
A number of other clinical manifestations have been described in patients with HCV-associated MC including arthralgias (16-83%), arthritis (10%), sicca syndrome (6-36%) and Raynaud's phenomenon (3--40%) [5, 18-21]. Since some of these manifestations occur also in cryoglobulin-negative patients with chronic HCV infection, its true association with HCV-associated CV is unknown.
1.4. Laboratory Findings The hallmark of HCV-associated CV is the presence of mixed cryoglobulins in the serum. Cryoglobulins are immunoglobulins with distinct physicochemical characteristics illustrated by their tendency to precipitate at temperatures below 37 ~ (see recent reviews, [5, 20]). Cryoglobulins in chronic hepatitis C are immune complexes composed of IgM with RF activity either monoclonal (type II) or polyclonal (type HI) directed against polyclonal IgG immunoglobulins. Intermediate forms of mixed cryoglobulins composed of oligoclonal IgM-RF have been also observed [5, 20]. Special attention to blood draw and sample handling for the accurate measurement of circulating cryoglobulins has been emphasized [5, 20]. Standardized assays for the qualitative measurements of cryoglobulins would assist in better characterizing HCV patients with
cryoglobulinemia. A number of studies have examined the frequency of type II or HI cryoglobulins in patients with chronic HCV infection [43]. The majority of patients with predominant liver disease without associated extrahepatic diseases, demonstrate more commonly type III cryoglobulins [43]. In contrast, patients with symptomatic HCV-associated CV are more comlnonly positive for type II cryoglobulins [5, 15, 19]. Similarly, in a study by Donada et al patients with chronic HCV infection and type II circulating cryoglobulins, were older and more likely to develop manifestations of HCV-associated CV such as purpura, neuropathy and nephropathy compared to patients with type III cryoglobulins [ 15]. Patients with HCV-associated CV display a number of autoimmune laboratory findings (see Table 2). Among them the detection of elevated titers of RF (68-75%) is the most prevalent [5, 7, 19, 21]. Other laboratory findings include low levels of C4 indicating immune-complex formation and tissue deposition, presence of autoantibodies such as ANA, SMA and more rarely AMA or anti-thyroid antibodies (Table 2). It should be mentioned that patients with chronic hepatitis C demonstrate a similar array of autoantibodies, indicating a chronic polyclonal activation of B lymphocytes in these patients. Patients with HCV-associated CV and cryoglobulinemia in general display much higher titers or percentage of positive RF tests, compared to HCV patients without circulating cryoglobulins [43, 44] (Vassilopoulos D/Calabrese LH, unpublished data). The activity and chronicity of the underlying liver disease in patients with circulating cryoglobulins or HCV-associated CV is a debatable issue. In a recent meta-analysis, Kayali et al reported an overall incidence of cirrhosis of 40% in patients with cryoglobulinemia compared to 17% in patients without cryoglobulins [43]. The difference remained significant even after adjustment for age, gender and disease duration [43, 44].
1.5. Pathogenesis The precise pathogenetic mechanisms that lead to the production of cryoglobulins and furthermore, HCV-associated CV during chronic HCV infection are unknown. A number of epidemiological, clinical
and laboratory observations combined with recent demonstrations of specific gene rearrangements in patients with HCV-associated CV have provided more insight in this complex process. The prevailing theory is that the development of HCV-associated CV is a sequential process (Fig. 1) [7, 45]. Chronic B cell stimulation by HCV or its antigens, leads initially to polyclonal B cell proliferation and production of type HI cryoglobulins characterized by the presence of IgM RF with polyclonal activity. In a certain subset of patients, years after the initial exposure to the virus, a monoclonal or oligoclonal B cell subpopulation arises. These B cells are located preferentially in the liver or bone marrow and produce monoclonal or oligoclonal RF (type II or intermediate typelI/I/I). A number of recent studies have shown that in such patients there is an enrichment in B cells bearing the t(14;18) translocation associated with an overexpression of the Bcl-2 anti-apoptotic protein [46-48]. These long-lived B cells may play a significant role in the production of mono- or oligo-clonal IgM RF that constitute the predominant autoantibody in type II or type IIBII cryoglobulins (Fig. 1). Deposition of monoclonal IgM RF (type II) with or without complexed IgG molecules and HCV RNA in different vascular beds leads to local complement activation and chemoattraction of neutrophils and/or monocytes/macrophages. The firing of this inflammatory cascade is responsible for the various clinical manifestations of HCV-associated CV. Although this theory is based on solid epidemiological, clinical and experimental data, a number of unanswered questions remain. It is unclear why the syndrome of HCV-associated CV is so uncommon despite the frequent presence of circulating cryoglobulins (~50%) in the large HCV infected population worldwide. Additional genetic, immunologic and viral factors have been implicated as additional necessary co-factors but none of them seems to play an exceptional role. Second, there are no well performed prospective studies with long term follow-up that validate these laboratory and experimental findings. In a study by Donada et al, among 102 patients with type III cryoglobulins only 4 developed type II cryoglobulins over a 2 year follow-up period [ 15]. During the same period, none of the cryoglobulin negative patients devel-
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Figure 1. Pathogenesis of HCV-associated cryoglobulinemic vasculitis (CV). The prevailing theory of cryoglobulin formation and vasculitis during the course of chronic HCV infection is illustrated. Chronic stimulation of B cells by HCV (directly or indirectly) leads to their polyclonal proliferation and production of IgM rheumatoid factor (RF) with polyclonal activity (type III cryoglobulins). During the chronic evolution of the disease, a number of monoclonal or oligoclonal RF arise (mRF) and expand (type II or II/lII cryoglobulins). The role of Bcl-2 translocation may be critical in that direction. Immune complexes (IC) containing mRF, polyclonal IgG _+HCV are deposited in vessel walls (mainly skin, nerves) followed by a localized immune response leading to the development of vasculitis. Similarly, deposition of mRF in certain tissues (mainly glomeruli) leads to in situ formation of IC, chemoattraction of mononuclear cells (MNC) and polymoprhonuclear neutrophils (PMNs) that cause tissue damage (glomerulonephritis). oped cryoglobulins. Furthermore, in a recent study by Persico et al, none of a small number of patients with circulating cryoglobulins developed HCVassociated CV over a 7 year follow-up period [ 14]. Third, a direct role for HCV in this process, beyond its well-accepted participation in the generation of cryoglobulins, has not been proven. Detailed comparison of the clinical and laboratory findings of HCV positive vs. HCV negative patients with symptomatic cryoglobulinemia, shows only minor differences in a recent study by Rieu et al [19]. These findings indicate that deposition of circulating cryoglobulins is the main pathogenetic factor in the development of symptomatic disease, regardless of the presence of HCV. The absence of replicating HCV RNA in the majority of tissue specimens from
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patients with HCV-associated CV further support these clinical and laboratory findings.
1.6. Therapy The therapy of patients with HCV-associated CV is a challenging task for the involved physicians [8]. The goals of therapy are clear: eradication of the responsible causative agent (HCV) and suppression of the vasculitic inflammatory process [49]. The recent advances in the antiviral treatment of chronic hepatitis C offer new therapeutic options for patients with HCV-associated CV.
1.6.1. Antiviral therapy Interferon-a. Interferon-a (IFN-a) remains the most important agent in the treatment of chronic HCV infection [8]. Bonomo et al used IFN-a for the treatment of HCV-associated CV, even before the discovery of HCV [50]. Following the discovery of HCV and its clear association with MC [51, 52], a number of small randomized and open trials have examined the role of standard IFNa therapy in patients with HCV-associated CV [8]. About 75% of the patients demonstrated partial or complete clinical response at the end of therapy (6-12 months), but approximately 70% of these patients relapsed after treatment discontinuation [8]. Furthermore, the clinical improvement was noted predominantly in skin lesions (purpura) and less so in renal and neurological manifestations [8, 53]. The inability of standard IFN-a to provide a sustained clinical response is directly related to the low sustained virological response rate achieved in these chronically infected HCV patients (-15%) [8].
Combination therapy (interferon a and ribavirin). Multicenter randomized clinical trials at the end of last decade confirmed the superiority of a combination scheme consisting of standard IFN-a and ribavirin over standard IFN-a monotherapy in patients with chronic hepatitis C [54]. As expected though, in these large randomized studies patients with HCV-associated vasculitis were excluded. Data on the efficacy of combination antiviral therapy in patients with HCV-associated CV are derived from single case reports or small uncontrolled studies [37, 55-61]. In the largest study of 27 patients with HCV-associated vasculitis by Cacoub et al, a complete or partial clinical response was seen in 85% of patients treated with the combination of IFN-a (mean duration = 20 months) and ribavirin (mean duration = 14 months) [60]. The major determinant of sustained clinical response to combination therapy was the rate of sustained virological response (only 1 patient with persistent viremia showed clinical remission) [60]. It should be noted that approximately 40% of patients received corticosteroids and plasmapheresis as part of their initial therapeutic scheme [60]. The introduction of pegylated interferons instead of standard IFN-a has increased the efficacy of the
combination scheme in patients with chronic hepatitis C. Currently, the combination of a pegylated IFN-a and ribavirin for 6-12 months is the optimal therapeutic approach for naive patients with chronic hepatitis C [62]. With this regimen 50 to 80% of patients (determined mainly by the HCV genotype) clear the virus [62]. There are currently no short or long-term data on the safety and efficacy of pegylated IFN-a treatment in patients with HCVassociated CV. Antiviral treatment (mono- or combination therapy) in patients with an underlying vasculitis should be administered with great caution and knowledge of its potential for severe side effects. Apart from the known contraindications and side-effects of IFN-a and ribavirin treatment [8], IFN-a has also the potential to exacerbate underlying skin [63], nerve [64] or renal [65] lesions in patients with HCVassociated CV. In a recent study, 22% of patients with HCV-associated vasculitis, discontinued IFN-a treatment due to side effects [60]. Ribavirin is contraindicated in patients with moderate to severe renal dysfunction (creatinine clearance < 50 ml/min) while reduction in its dose is frequently needed due to hemolytic anemia [49]. About 1/3 of patients with HCV-associated vasculitis had to reduce their ribavirin dose due to hemolytic anemia in a recent study by Cacoub et al [60].
1.6.2. Immunosuppressive therapy The goals of immunosuppressive therapy are to suppress the production of the pathogenic cryoglobulins by B cells and to downregulate the host immune response that is responsible for the localized vascular inflammatory process [8]. Prior to the discovery of HCV, patients with severe "essential" MC were frequently treated with a combination of corticosteroids, cyclophosphamide and plasmapheresis in an uncontrolled fashion with mixed results [23]. The administration of immunosuppressive therapy in patients with a chronic viral infection raises reasonable concerns about their short and long-term side effects [8]. Short term corticosteroid use in patients with chronic hepatitis C is associated with transient increase in HCV RNA levels but acute deterioration of liver function during or after therapy is rare [8, 60]. Similarly, short courses of cyclophosphamide therapy have not
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been linked to acute liver failure in a large study of Italian patients with HCV-associated MPGN [39]. On the other hand, there is accumulating evidence that long-term immunosuppressive therapy leads to accelerated rates of cirrhosis in HCV infected patients [8]. Collectively, these limited data suggest that short term immunosuppressive therapy is not associated with acute liver toxicity in patients with HCV-associated vasculitis. Long-term continuous immunosuppressive therapy though may enhance chronic liver damage leading to cirrhosis.
1.6.3. Apheresis Plasmapheresis is used temporally in patients with severe/life-threatening manifestations of HCV-associated CV including rapidly deteriorating glomerulonephritis, skin necrosis, CNS or motor neuropathy and hyperviscosity syndrome [6, 39]. It is usually administered in combination with corticosteroids and/or cyclophosphamide. Controlled data on its efficacy are not available but anecdotal evidence supports its use in patients with life-threatening disease.
1.6.4. New immunosuppressive agents Biologic agents that specifically target elements of the immune system that participate in the pathogenesis of HCV-associated CV (see Fig. 1) are currently under investigation for the treatment of this disorder. So far, published data are available only for rituximab [66, 67]. Rituximab is a chimeric monoclonal antibody (anti-CD20) that specifically targets and depletes B cells from the circulation. This agent is already being used in clinical practice for the treatment of B-cell Non-Hodgkin's lymphomas and autoimmune cytopenias (autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura) [68, 69]. Recently, two studies reporting on the efficacy of rituximab in Italian patients with MC were published [66, 67]. In both studies, patients with treatment resistant HCV-associated MC (12 and 20 patients respectively) were treated with 4 weekly intravenous infusions of standard dose Rituximab (375 mg/m 2) [66, 67]. The majority of patients had mild to moderate disease activity at baseline
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(purpura = 75-80%, neuropathy = 33--60%, renal disease = 5-17%). This initial course of therapy was associated with significant clinical response mainly in the skin (-75%) and nerve (50-100%) manifestations [66, 67]. The medication was well tolerated without significant side effects. Relapses were noted in 25% of patients in the study by Sansonno et al [66] while 1/3 of patients in the study by Zaja et al [67] had to be retreated. There were no significant changes in ALT levels during therapy in both studies but increases in HCV RNA levels were noted in one study (mainly in clinically responding patients) [66]. More studies with particular emphasis on the long term safety of this promising agent are needed.
1.6.5. Treatment guidelines for HCV-associated CV Management of HCVoassociated CV should be designed on an individual basis. Careful initial assessment of the severity of vasculitic involvement, the status of the underlying liver disease and the virological characteristics of each patient are necessary prior to the initial decision making. In nah've patients with mild to moderate disease activity (purpura, arthralgias/arthritis, mild sensory neuropathy, mild proteinuria/hematuria with normal creatinine values), combination therapy with IFN-a and ribavirin should be offered in addition to symptomatic therapy [5, 7, 8, 53]. Given the latest data on the enhanced efficacy of pegylated interferons, these agents should be tried first in combination with ribavirin. Regular follow-up of the patients with sensitive measurements of HCV RNA levels is required as well as increased vigilance for therapy related side effects. The optimal duration of treatment should be based on the viral genotype and the clinical/virological response to therapy. In patients with severe or life-threatening disease (rapidly progressive glomerulonephritis, motor neuropathy, CNS, gastrointestinal or myocardial involvement, digital necrosis), combination therapy with immunosuppressive and antiviral therapy should be tried. Immunosuppressive therapy should be given for a short period of time (2-4 weeks) followed by antiviral treatment. Corticosteroids and/or cyclophosphamide (IV or per os) therapy are the agents most commonly used in this setting. In
patients with life-threatening disease, plasmapheresis can be also offered in combination with the immunosuppressive therapies. In resistant cases, inclusion of patients in carefully designed study protocols in referral centers is strongly recommended. In patients with relapsing disease that show clinical response to a second course of antiviral treatment, long term antiviral treatment (> 1 year) may be necessary (preferably with pegylated interferons).
3. H C V - A S S O C I A T E D L A R G E V E S S E L VASCULITIS Symptoms suggestive of giant cell arteritis in HCV infected patients have been noted in two settings. Rarely, in patients with HCV-associated CV involvement of the small vessels around the temporal artery occurs [77, 78]. This small vessel vasculitis manifests as typical giant cell arteritis. Direct involvement of the temporal artery in a patient with classical symptoms of giant cell arteritis and chronic HCV infection has been documented by Ferracioli et al [79].
2. HCV-ASSOCIATED MEDIUM S I Z E VASCULITIS ACKNOWLEDGEMENTS Medium-size vasculitis is uncommon in patients with chronic hepatitis C [12]. In most cases, cryoglobulins are also present in the circulation, so a clear distinction from HCV-associated CV with medium size vessel involvement can not be made [70]. Cacoub et al reported that patients with such characteristics present with more severe systemic disease manifestations that resemble polyarteritis nodosa (PAN) [70]. Biopsies of involved tissues demonstrate a necrotizing medium size vasculitis with mononuclear and polymorphonuclear cell infiltration. In a subset of patients, characteristic intrarenal microaneurysms were noted by angiographic studies [70, 71 ]. In unselected populations of patients with features of classic PAN, the frequency of HCV infection is < 10% [72-74]. Thus, a clear causative association between chronic HCV infection and PAN can not be made. A small number of patients with predominant skin manifestations (nodules, livedo reticularis) in the absence of systemic disease and histological findings suggestive of cutaneous PAN have been also described [75, 76]. The therapy of patients with systemic PAN-like disease does not differ from the suggested therapy for severe HCV-associated CV.
I would like to thank Dr Calabrese for his continuous support and critical review of the manuscript.
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vasculitis. Rheumatology (Oxf) 2002;41:708-710. 72. Cacoub P, Lunel-Fabiani F, Du LT. Polyarteritis nodosa and hepatitis C virus infection [letter]. Ann Intern Med 1992; 116:605-606. 73. Carson CW, Conn DL, Czaja AJ, Wright TL, Brecher ME. Frequency and significance of antibodies to hepatitis C virus in polyarteritis nodosa. J Rheumatol 1993;20:304-309. 74. Quint L, Deny P, Guillevin L, Granger B, Jarrousse B, Lhote F et al. Hepatitis C virus in patients with polyarteritis nodosa. Prevalence in 38 patients. Clin Exp Rheumatol 1991 ;9:253-257. 75. Vitali C, Galluzzo E, Ciancia EM, Moretti A, Marchi S. Giant cell arteritis of the leg in a patient with hepatitis C virus infection. Ann Rheum Dis 1997;56:697-698. 76. Soufir N, Descamps V, Crickx B, Thibault V, Cosnes A, Becherel PA et al. Hepatitis C virus infection in cutaneous polyarteritis nodosa: a retrospective study of 16 cases. Arch Dermatol 1999; 135:1001-1002. 77. Disdier P, Pellissier JF, Harle JR, Figarella-Branger D, Bolla G, Weiller PJ. Significance of isolated vasculitis of the vasa vasorum on temporal artery biopsy. J Rheumatol 1994;21:258-260. 78. Genereau T, Martin A, Lortholary O, Noel V, Guillevin L. Temporal arteritis symptoms in a patient with hepatitis C virus associated type II cryoglobulinemia and small vessel vasculitis. J Rheumatol 1998;25:183-185. 79. Ferraccioli GF, Mariuzzi L, Damato R, Rocco M, Pirisi M, Beltrami CA. Jaw and leg claudication in a patient with temporal arteritis, chronic sialoadenitis and previous hepatitis C virus infection. Clin Exp Rheumatol 1998;16:463-468.
9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
HCV and Cryoglobulinemia Clodoveo Ferri I and Stefano Bombardieri 2
1Rheumatology Unit, Department of lnternal Medicine, University of Modena, Medical School Modena, Italy; 2Rheumatology Unit, Department of Internal Medicine, University of Pisa, Medical School Pisa, Italy
1. C R Y O G L O B U L I N E M I A
Cryoglobulinemia is defined as the presence of circulating immunoglobulins (Ig) that precipitate at temperatures < 37 ~ C and redissolve on re-worming [1]. Such an in vitro phenomenon is detectable in a wide number of chronic infectious and immunological disorders, as well as in some hematological malignancies [ 1-3]. The real mechanism(s) of cryoprecipitation that remains largely unknown; it could be secondary to intrinsic characteristics of both mono- and polyclonal immunoglobulin components and/or to the interaction among single components of the cryoprecipitate [ 1-5]. Cryoglobulinemia is usually classified into three subgroups according to Brouet et al [4]: type I, composed by single monoclonal Ig; type II and HI, which contained a mixture of polyclonal IgG and mono- (type II) or polyclonal (type III) IgM rheumatoid factor (RF). Cryoglobulinemia type I or monoclonal cryoimmunoglobulinemia is frequently associated to a well known hematological disorders; in particular, lymphoid tumors such as Waldenstrom's macroglobulinaemia, multiple myeloma, and immunocytoma. Monoclonal cryoimmunoglobulinemia is generally asymptomatic, in only few cases it can be complicated by hyperviscosity syndrome. Type II and III mixed cryoglobulinemia (MC) are often responsible for a clinical syndrome characterized by leucocytoclastic vasculitis of small and medium sized vessels, and multiple organ involvement [1-5]. MC is classified as essential or secondary in the absence/presence of other well defined- infectious, immunological or neoplastic - d i s e a s e s [2-5]. Table 1 shows the main biologi-
cal and clinico-pathological characteristics of cryoglobulinemia subgroups, including a newly proposed serological variant the type II-III MC [4, 6]. The analysis of cryoprecipitates is generally carded out by means of immunoelectrophoresis or immunofixation. With more sensitive methodologies, i.e. immunoblotting or 2-dimensional polyacrylamide gel electrophoresis, type II MC frequently shows a microheterogeneous composition; in particular, oligoclonal IgM or a mixture of polyclonal and monoclonal IgM can be detected [6]. Type II-III MC could fit together the most recent molecular studies showing the presence of oligoclonal B-lymphocyte proliferation's in liver and bone marrow biopsies from MC patients [7, 8]. In two third of type II MC a cross-idiotype WA monoclonal RF has been demonstrated [3, 9]. This WA (after the patient in whom it was first detected) autoantibody almost invariably express a Vk light chain derived from a single germinal gene, the human KV 325. The same WA monoclonal IgMk RF has also been detected in type II MC secondary to lymphoid malignancies, probably expression of an antigen-independent clonal B-cell lymphoproliferation.
2. M I X E D C R Y O G L O B U L I N E M I A
The so-called 'essential' MC was first described in 1966; this term refers to a distinct clinical syndrome in the absence of other well known systemic or neoplastic disorders [5]. Clinically, MC is characterized by a typical t r i a d - purpura, weakness, arthralgias - and by multisystem organ involvement including chronic hepatitis, membranoproliferative glomeru-
201
Table 1. Classification and clinico-pathological characteristics of different cryoglobulinemias Composition
Pathological findings
Clinical associations
Type I cryoglobulinaemia
monoclonal Ig, mainly IgG, or IgM, or IgA self-aggregation through Fc fragment of Ig
tissue histological alterations of underlying disorder
lymphoproliferative dis.: MM, WM, CLL, B-cell NHL
Type II mixed cryogl.
monoclonal IgM (or IgG, or IgA) with RF activity (often crossidiotype WA-mRF) and polyclonal Ig (mainly IgG)
leukocytoclastic vasculitis B-lymphocyte expansion with tissue infiltrates
infections (mainly HCV) autoimmune/lymphoproliferative dis. rarely 'essential'
Type H-Ill mixed cryogl.
oligoclonal IgM RF or mixture of poly/monoclonal IgM (often crossidiotype WA-mRF)
leukocytoclastic vasculitis B-lymphocyte expansion with tissue infiltrates
infections (mainly HCV) autoimmune/lymphoproliferative dis. rarely 'essential'
Type III mixed cryogl.
polyclonal mixed Ig (all isotypes) with RF activity of one polyclonal component (usually IgM)
leukocytoclastic vasculitis B-lymphocyte expansion with tissue infiltrates
infections (mainly HCV) more often autoimmune disorders rarely 'essential'
Lymphoproliferative disorders: MM (multiple myeloma), WM (Waldenstrom's macroglobulinemia), CLL (chronic lymphocytic leukemia), B-cell non-Hodgkin's lymphoma; Ig: immunoglobulin; RF: rheumatoid factor; HCV: hepatitis C virus.
lonephritis (MPGN), peripheral neuropathy, skin ulcers, widespread vasculitis, and less frequently lymphatic and hepatic malignancies [2-5, 10] (Table 2). A variety of circulating immune-complexes, mainly mixed cryoglobulins with RF activity, a low hemolytic complement activity, and markedly low C4 are the typical serological findings of MC [2-5, 10]. MC is considered to be a relatively rare disorder; however, its prevalence among different countries shows a great geographical etherogeneity, being the disease more frequent in Southern Europe than in Northern Europe or Northern America. Because its clinical polymorphism, MC patients are often referred to different specialties according to the main symptom(s), i.e. skin vasculitis, hepatitis, nephritis, peripheral neuropathy, etc. Consequently, the actual prevalence of the MC is probably underestimated; moreover, in some patients a correct diagnosis can be delayed or overlooked entirely. There are not available classification/diagnostic criteria for MC. In the clinical practice, the main diagnostic parameters are serum mixed cryoglobulins with RF activity, frequently associated to low C4, orthostatic skin purpura due to leukocytoclastic vasculitis of small/medium-sized blood vessels [25, 10]. This latter is an immune-complex-mediated vasculitis, with the possible contribution of hemorheological and/or local factors [2, 10]. A polyclo-
202
Table 2. Clinico-epidemiological and laboratory features of 200 MC patients .
.
.
.
.
.
.
.
Age, mean +_SD years (range)* Female/male ratio Disease duration, mean _+SD years (range)
52+_12 (29-75) 2.6 12_+6(1-35)
Purpura Weakness Arthralgias Arthritis (non-erosive) Raynaud's phenomenon Sicca syndrome Peripheral neuropathy Renal involvement Liver involvement B-cell non-Hodgkin' s lymphoma Hepatocellular carcinoma
92% 90% 81% 10% 35% 36% 36% 29% 68% 7.5% 2.5%
Cryocrit, mean __SD % 4_+8 Type IFtype Ill mixed cryoglobulins 2/1 C3, mean _+SD mg/dl (normal 60-130) 72_+30 C4, mean _+SD mg/dl (normal 20-55) 9_+15 Autoantibodies 34% Anti-HCV antibodies HCV RNA Anti-HBV antibodies HBsAg
92% 85% 38% 3%
nal or mono-oligoclonal B-lymphocyte expansion represents the underlying pathological alteration detectable in the large majority of MC patients in the serum and/or in different tissues as lymphoid aggregates, with diffuse or nodular pattern [2, 3, 7, 8, 10]. Besides the above clinico-serological and pathological findings, the presence of one or more organ involvement can be useful for a correct classification of MC syndrome [ 10]. Both type II and type III MC are characterized by large amounts of circulating immune-complexes; the fraction of cryoprecipitable immune-complexes varies greatly among MC patients and in the same patient during the course of the disease [ 10, 11 ]. In some subjects with overt MC syndrome but without detectable serum cryoglobulins it is necessary to repeat at intervals the cryocrit determination. Cryoglobulin detection and characterization are necessary for a correct classification and diagnosis; however, the amount of serum cryoglobulins generally does not correlate with the severity and prognosis of the disease [ 10, 11].
30 and 54% of MC patients, respectively [17, 18]. One year later, HCV viremia was first reported in a large series of MC patients along with a striking correlation between HCV seropositivity and viremia (91% vs 86%) [19]. A subsequent study demonstrated that HCV RNA was markedly more concentrated (1000-fold) in the cryoprecipitate than in the supernatants [20]. The prevalent role of HCV in MC has been definitely established on the basis of epidemiological, pathological, and laboratory studies [ 10, 21]. In particular, immunohistochemical and molecular biology studies, including HCV RNA detection by in situ hybridisation, have reinforced the hypothesis of a direct involvement of HCV antigens in the immune-complex-mediated cryoglobulinemic vasculitis [21-24]. Being HCV the main triggering factor of MC the term 'essential' is no longer appropriate for the majority of cases [10,21-24].
3. HCV INFECTION AND MIXED CRYOGLOBULINEMIA
The association between HCV and MC [21] together with other immune-system alterations observed in chronically HCV-infected individuals [25-34] suggested that the same virus could be the triggering factor of other extrahepatic immunological disorders. Table 3 summarizes the main organ or systemic diseases that may be related to HCV infection according to the strength of association. Besides the well-established association with the MC syndrome, HCV can be detected in a significantly high percentage of patients with autoimmune or neoplastic diseases when compared to HCV prevalence in the general population. In particular, porphyria cutanea tarda (PCT), glomerulonephritis, diabetes, thyroid disorders, and B-cell neoplasias have been widely investigated for this purpose [27-35, 10]. In patients with the sporadic variant of PCT, a metabolic disorder characterized by reduced hepatic activity of uroporphyrinogen decarboxylase, a clear-cut association with HCV infection has been demonstrated [27, 28]. The majority of patients with PCT present chronic liver involvement along with some HCV-driven clinico-serological autoimmune phenomena [28]. Renal involvement complicating HCV infection is generally represented by type I
Being liver involvement one of the most frequent clinical features of the MC (Table 2), a causative role of hepatotropic viruses in MC has long been hypothesized during the seventies [2, 10, 12, 13]. Following the demonstration of a significant association between hepatitis B virus (HBV) and another systemic vasculitis - the polyarteritis nodosa- [ 14], a possible role of HBV was suggested also for MC [12, 13]. However, the presence of HBV antigenemia is seldom recorded, while anti-HBV antibodies largely varied among different MC patients' series [12, 15]. Thereafter, it can be estimated that HBV can represent an etiological factor in a minority of individuals, generally less than 5% of MC [10] (Table 2). In 1989 hepatitis C virus (HCV) has been identified as the major etiologic agent of post-transfusion and sporadic parentally-transmitted non-A-non-B hepatitis [16]. A role of HCV in MC was initially suggested in 1990 by two distinct studies reporting the presence of antibodies against HCV (anti-HCV; first generation ELISA, Chiron, Emeryville CA) in
4. HCV-ASSOCIATED AUTOIMMUNE AND LYMPHOPROLIFERATIVE DISORDERS
203
Table 3. Hepatitis C virus infection and extrahepatic disorders Established associationa mixedcryoglobulinemia Significant associationb B-cell NHL monoclonal gammopathies porphyria cutanea tarda diabetes mellitus thyroid disorders glomerulonephriitis Possible associationc
chronic polyarthritis sicca syndrome/Sj6gren's s. lung fibrosis polyarteritis nodosa poly/dermatomyositis gonadal (erectile) dysfunction lichen planus, other skin dis. Mooren comeal ulcers
aHCV infection in the large majority of patients. bHCV in a significant percentage of patients compared to general population. cSuggested but unproven association. MPGN, more often as visceral complication of MC syndrome. Type I MPGN alone, and less frequently milder glomerulonephritis patterns, can be also observed in HCV-positive individuals [36]. With regard to some endocrinological manifestations a number of epidemiological studies have reported a significantly higher risk to develop diabetes mellitus and thyroid disorders in patients with HCV infection compared to general population [10, 37-39]. Moreover, gonadal involvement responsible for erectile dysfunction in HCV-positive males has been recently reported [40]. Patients with type II MC can develop a B-cell lymphoma, usually after a long-term follow up [3, 10, 22, 41-43]. It was shownthat these patients frequently carry lymphoid infiltrates in the liver and bone marrow characterized by peculiar clinicopathologic characteristics [10, 22]. These infiltrates have been regarded as "early lymphomas", since they are sustained by lymphoid components indistinguishable from those of B-cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (B-CLL) and immunocytoma (Ic) [10, 22]. However, conversely to overt lymphomas, they tend to remain unmodified for years or even decades and are followed by malignant lymphoid tumor in about 10% of cases [41, 43]. These characteristics justify
204
the recently proposed term of "monotypic lymphoproliferative disorder of undetermined significance (MLDUS)" [10, 22]. This condition includes two main pathological patterns; namely, the B-CLLlike and the Ic-like. Since MC may be regarded as potential pre-lymphomatous disorder and HCV represents its triggering factor, a possible role of the same virus also in 'idiopathic' B-cell NHL had been suggested [44]. In 1994, HCV infection was first demonstrated in a significant percentage of Italian patients with unselected B-cell non-Hodgkin's lymphomas (B-NHLs), regardless of the histotype [45]. This association was then confirmed by several studies on various B-cell NHL patient populations from Italy and other Countries [ 10, 22, 46, 47]. An increasing number of clinico-epiderniological and immunopathological studies seems to support a causative role of HCV in the above immune system disorders [10, 48]. The actual strength of these associations is controversial; however, HCV might play a pathogenetic role for at least certain patients' subsets and in some geographical areas. Of interest, different HCV-related diseases show an intriguing clinico-serological overlap [10]. In this scenario, MC can be represent a crossing road between some classical autoimmune disorders (autoimmune hepatitis, sicca syndrome, glomerulonephritis, thyroiditis, etc.) and malignancies (B-cell lymphomas, hepatocellular carcinoma) [ 10, 49] (Fig. 1). Because of possible methodological bias it is difficult to verify whether other suggested but unproven associations between HCV and some immunological disorders (Table 3c) is coincidental or a pathogenetic link actually exists. HCV infection is rarely complicated by classic rheumatoid arthritis; whereas an intermittent, non-erosive olygoarthritis of large-medium sized joints, is often observed in a significant number of HCV-infected individuals, more often without overt MC syndrome [10, 34, 50, 51]. Generally, polyarthritis in HCV-positive patients shows a more benign clinical course and good response to low steroid dosage and hydroxychlorochine treatment [50]. In some instances, some symptoms such as polyarthritis, glomerulonephritis, or neuropathy may present as apparently isolated manifestation of HCV infection. In these patients a concomitant, subclinical hepatitis, cryoglobulinemic syndrome, or B-cell lymphoma should be carefully investigated.
Figure 1. Possible etiopathogenesis of mixed cryoglobulinaemia (MC) and other HCV-related disorders. HCV infection may exert a chronic stimulus on the immune-system; in particular, various pathogenetic mechanisms can be taken in account: a) the interaction between HCV envelope protein E2 and CD81 on both hepatocytes and lymphocytes; b) a molecular mimicry mechanism involving HCV antigens and possible autoantigens; and c) T(14;18) translocation commonly found in HCV-infected individuals, particularly in MC patients; the consequent activation of Bcl2 proto-oncogene may lead to prolonged B-cell survival. B-lymphocyte expansion may be responsible for various autoantibodies production, including rheumatoid factor and cryo- and non-cryoprecipitable immune-complexes (CIC). Consequently, various auto'immune disorders and cryoglobulinemic vasculitis may develop. The indolent B-cell proliferation underlying MC may be complicated by framkmalignant lymphoma in about 10% of patients. Moreover, HCV is the major causative factor of hepatocellular carcinoma; finally, a possible link between HCV and thyroid cancer has been also suggested. There is a clinico-serologic and pathologic overlap among different HCV-related diseases; mixed cryoglobulinaemia syndrome represents a crossroads between these autoimmune and neoplastic disorders.
As observed for MC, the prevalence of different HCV-related autoimmune-lymphoproliferative diseases shows a geographically heterogeneous distribution [10, 27-31, 36], suggesting a role of important co-factors also in these HCV-related disorders.
5. P A T H O G E N E S I S O F H C V - A S S O C I A T E D MIXED CRYOGLOBULINEMIA Following its identification, HCV has been recognized to be both hepato- and lymphotropic virus, as firstly demonstrated by the presence of active or latent viral replication in the peripheral lymphocytes of patients with type C hepatitis [52]. Of great interest, the infection of lymphoid tissue may explain the appearance of a constellation of autoimmune
205
Table 4. Treatmentof HCV-associated mixed cryoglobulinaemia proposed treatments
none
asymptomatic mild-moderate manifestations
purpura, weakness arthralgias, arthritis, peripheral sensory neuropathy
~
low dosage of steroids and/or LAC-diet other symptomatics
severe manifestations
nephropathy, skin ulcers sensory-motor neuropathy widespread vasculitis active hepatitis
attempt at HCV eradication"
~ steroids and/or plasma exchange and/or cyclophosphamide rituximab interferon + ribavirin
cancer
B-cell NHL, HCC
chemotherapy, surgery
NHL: non-Hodgkin's lymphoma; HCC: hepatocellular carcinoma; *alpha-interferon+ ribavirin; LAC-diet: low antigen content diet
and lymphoproliferative disorders in HCV-infected individuals [10, 44, 52]. HCV-related types HI and II MC are comparable with regard their organ involvement and clinical course, with the exception of their potential evolution to malignancy. Although not definitely demonstrated, they might represent two different steps of the same disorder: MC type III may evolve to benign linfoproliferative disorder, the mono-oligoclonal B-cell proliferation of MC type II, which in some individuals can be complicated by frank B-cell non-Hodgkin's lymphoma (NHL), usually after a long-term follow-up period [ 10, 22]. Circulating mixed cryoglobulins are frequently detectable in HCV-infected individuals (50%); whereas, overt cryoglobulinemic syndrome develops in only a minority of cases (5%) [10]. HCV infection presents a homogeneous diffusion worldwide, which contrasts with the geographical etherogeneity in the prevalence of HCV-related MC as well as other immune-system disorders. The involvement of particular HCV genotypes, environmental and/or host genetic factors should contribute to the pathogenesis of MC; however, the actual role of the above co-factors remains still to be demonstrated [ 10, 22,
53, 54]. The above considerations suggest that HCV p e r se might be insufficient to drive the autoim-
mune-lymphoproliferative phenomena observed in
206
a limited but significant proportion of infected individuals. HCV is a positive, single-stranded RNA virus without a DNA intermediate in its replicative cycle, so that viral genomic sequences cannot be integrated into the host genome. Therefore, it has been proposed that HCV infection exerts a chronic stimulus to the immune system, which facilitates the polyclonal B-lymphocyte expansion and the selection in some subjects of malignant B-cell clones [10, 22]. In spite of morphology and monotypic Ig light chain expression, the lymphoproliferation occurring in HCV-positive patients with type II MC should not be regarded as a real lymphomatous situation, since: 1) it is usually characterized by oligoclonality, and 2) the overt malignant lymphoma which eventually develops during follow-up, more often stems from a B-cell clone other than the ones sustaining MLDUS as suggested by the molecular analysis of liver and bone marrow lymphoid infiltrates [7, 8, 22]. More interestingly, the presence of t(14;18) translocation leading to Bcl-2 activation has been demonstrated in a significant percentage of peripheral blood lymphocytes in HCV-infected individuals, particularly in those with MC [55-57]. Besides, the recent identification of HCV envelop protein E2 able to bind CD81 molecule expressed on both hepatocytes and B-lymphocytes [58] could help to clarify the pathogenesis of HCV-related autoim-
mune and neoplastic diseases (Fig. 1). In fact, CD81 is a cell-surface protein that, on B-cell, is part of a complex with CD21, CD 19, and Leu 13. This complex reduces the threshold for B-cell activation by bridging antigen specific recognition and CD21-mediated complement recognition. It can be hypothesized that the interaction between HCV-E2 and CD81 may increase the frequency of VDJ rearrangement in antigen-reactive B-cell. One possible consequence could be the above mentioned bcl-2 activation observed in HCV-related diseases, mainly MC [55-57]. This proto-oncogene is able to inhibit the apoptosis leading to extended cell survival [59]. The aberration of bcl-2 can explain, at least in part, the B-lymphocyte expansion and the wide autoantibody production observed in HCV-infected individuals [ 10, 22, 28, 29, 31 ]. Other mechanisms such as molecular mimicry can be involved in B lymphocyte activation responsible for different hepatic and extrahepatic autoimmune disorders. On the other hand, the prolonged B-cell survival can expose these cells to other genetic aberrations leading to overt malignant lymphoma (Fig. 2). HCV exerts a well-known oncogenic potential as definitely demonstrated for hepatocellular carcinoma; the same virus seems to be also involved in the lymphomagenesis and, possibly, in other malignancies such as thyroid cancer [33, 37].
6. MANAGEMENT OF HCV-ASSOCIATED MIXED CRYOGLOBULINEMIA The clinical symptoms of MC largely vary among patients and in the same patient during the followup. Usually, MC shows a relatively benign clinical course; the disease is often oligosymptomatic for long time intervals characterized by mild weakness, arthralgias, and sporadic flares of purpura on the legs. In other cases MC syndrome may start with or may be studded by one or more severe symptoms such as renal, neurological, and/or liver involvement, widespread vasculitis, and/or neoplastic complications [ 10]. The cumulative survival of MC shows a significantly poor prognosis if compared to general population [60]. Overall, the treatment of MC syndrome is particularly challenging because of its complex etiopathogenesis [10]. For a correct therapeutic approach to HCV-related MC
HCVeradication immunosuppressors LAC-dietPlasma'exchange steroids
HCV infection I
ill
Benign B-cell expansion autoantibodies and cryoglobulin production
.........
l
~1Cryoglobulinemic vasculitis /
chemotherapy
_
" ]B-cell
,~,
_ _
_
lymphomaI
Figure 2. Mixed cryoglobulinaemia is a combination of three main clinico-pathological alterations: chronic HCV infection, B-cell lymphoproliferation, and immune-complex vasculitis. We can treat the disease at different levels by means of combined - etiologic, pathogenetic, and symptomatic - therapies. LAC-diet: low antigen content diet.
we must deal with the concomitance of conflicting conditions: HCV infection, autoimmune, and lymphoproliferative alterations. According to the cascade of events leading from HCV infection to cryoglobulinemic vasculitis (Figs. 1, 2) we can treat the disease at three different levels by means of etiologic, pathogenetic, and/or symptomatic therapies. Since HCV represents the triggering factor of the disease and probably exerts a chronic stimulus on the immune system (Fig. 1), an attempt at HCV eradication should be done in all cases of HCV-associated MC. In this respect encouraging data came from some preliminary observations: in MC patients with MLDUS repeated bone-marrow biopsies, before/after interferon therapy, showed a regression of lymphoid infiltrates along with HCV clearance [61 ]; moreover, the antiviral therapy may induce the regression of T(14; 18) beating B-cell clones in HCV-positive patients [62]. On these basis, we can hypothesize that antiviral therapy (interferon + ribavirin) may improve or treat the lymphoproliferative disorder underlying the MC. Unfortunately, HCV eradication is obtained in a small percentage of cases, while the beneficial effect observed with interferon treatment is often transient and not rarely associated with important immune-mediated complications, in particular, the peripheral sensory-motor neuropathy [63-68]. There are no parameters available for predicting
207
this hamafial complication; thus, alpha-interferon therapy should be avoided at least in those patients with clinically evident peripheral neuropathy. Similarly, in patients treated by alpha-interferon for type C hepatitis without MC syndrome, it is not rare to encounter complications such as peripheral neuropathy, thyroiditis, and rheumatoid-like polyarthritis. Probably, in predisposed subjects, alpha-interferon, both an antiviral and immunomodulating agent, can trigger or exacerbate some pre-existing, often subclinical, symptoms [66-69]. On the whole, the usefulness of alpha-interferon treatment in MC patients is limited by the low rate of responders and frequent side effects. The association of pegylated interferon and ribavirin might achieve the eradication of HCV infection in a rather significant number of treated subjects, as recently demonstrated in patients with type C chronic hepatitis [70-72]. Controlled clinical trials are necessary to definitely evaluate the usefulness of such combined antiviral therapy in HCV-related MC patients. With the rapid growth of molecular biology a vaccine against HCV might be available in the near future. The identification of the interaction between HCV envelope protein E2 and CD81 on both hepatocytes and lymphocytes [58] suggests the possibility of interfering with HCV binding to target cells. In HCV-infected individuals a vaccine-based therapy with recombinant HCV proteins [58, 73] could be able to prevent the evolution from HCV infection to both severe hepatic and extra-hepatic complications, and could possibly interrupt the selfperpetuating autoimmune mechanism underlying HCV-related disorders. Immunosuppressive treatment is still the firstline intervention in rare cases of 'essential' MC. In the setting of HCV-related MC the immunosuppressive treatment should be considered mainly in those patients who have failed to respond to alpha-interferon. An immunosuppressive treatment with cyclophosphamide often in association with steroids, and/or plasma exchange may be able to treat some severe MC complications such as nephropathy, sensory-motor neuropathy, or widespread vasculitis [2, 10, 74, 75]. Both traditional and double-filtration plasma exchange are able to achieve a dramatic reduction of circulating immune-complex levels, including the cryoglobu-
208
lins, as well as the viral loading [ 10, 76]. The beneficial effect of such 'symptomatic' treatment can be reinforced by means of oral cyclophosphamide during the tapering of apheretic sessions (50--100 mg/day for 4-8 weeks). In particular, it can prevent the rebound phenomena that may be observed after the discontinuation of apheresis. Plasma exchange is useful in severe MC complications, and particularly in active cryoglobulinemic nephropathy. Low-antigen-content diet (LAC-diet) has been employed in some immune-complex-mediated disorders, namely MC and IgA-nephropathy [77, 78]. In MC patients, this particular dietetic treatment can improve the serum clearance of immune-complexes by restoring the activity of the reticulo-endothelial system, overloaded by large amounts of circulating cryoglobulins [77]. LAC-diet and/or low dosage of steroids may be sufficient to improve mild-moderate manifestations of MC, i.e. purpura, arthralgias, peripheral sensory neuropathy, etc. [ 10]. More recently, a pathogenetic treatment with rituximab, a monoclonal chimeric antibody that binds to the B-cell surface antigen CD20, has been proposed in HCV-positive patients with type II MC [79, 80]. The selective B-cell blockade leads to the improvement of MC manifestations, including skin vasculitis, peripheral neuropathy, and lowgrade B-cell lymphoma along with a significant reduction of serum RF and cryoglobulin levels. Of interest, it has been noticed that serum HCV RNA increased approximately twice the baseline levels in the responders [80]. The impact of rituximab on HCV viremia suggests the possible use of combined therapy with this monoclonal antibody and other (antiviral?) agents. Finally, the long-term efficacy and safety of rituximab need to be investigated by controlled clinical trials. On the whole, MC treatment should be tailored for the single patient according to the severity of clinical symptoms. While asymptomatic patients usually do not need any treatment, even in the presence of high levels of cryocrit, patients with mildmoderate symptoms, such as palpable purpura, are particularly sensitive to the smallest variations of daily steroid dosage (1-2 mg). On the contrary, severe, life-threatening vasculitic manifestations must be promptly treated with a combined therapy based on plasma exchange, high doses of steroids, and/or immunosuppressors. A careful clinical moni-
toting of the disease is mandatory in all cases, with particular attention to neoplastic complications.
6.
ACKNOWLEDGEMENTS 7. We thank all the following people who actively contributed to our studies: L. La Civita, MD, G Longombardo, BS, G Porciello, MD, P Fadda, MD, M Sebastiani, D Giuggioli, M Cazzato, R. Cecchetti, G Pasero, MD: Rheumatology Unit,
Department of Internal Medicine, University of Pisa, Pisa, Italy; A.L. Zignego: Istituto Medicina Interna, University of Florence, Italy; S. Pileri: Pathologic Anatomy and Haematopathology Unit, University of Bologna, Italy; E Caracciolo, MD, M. Petrini: Cattedra di Ematologia; University of Pisa; Pisa, Italy; E Greco, MD, A. Mazzoni, MD: Blood Center, Ospedale S. Chiara, Pisa, Italy; L. Moriconi, R Puccini: Nephrology Unit, Ospedale S. Chiara, Pisa, Italy; S. Marchi, MD, and F. Costa, MD: Clinica Medica I, University of Pisa; P. Highfield, Ph, and T. Corbishley, Ph: Wellcome Diagnostic, Beckenham, UK; M.P. Manns, MD: Department of Gastroenterology and Hepatology, Zentrum Innere Medizin, Medizinische Hochschule, Hannover, Germany.
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45. Ferri C, Caracciolo F, Zignego AL, La Civita L, Monti M, Longobardo G et al. Hepatitis C virus infection in patients with non-Hodgkin's lymphoma. Br J Haematol 1994;88:392-394. 46. Luppi M, Grazia FM, Bonaccorsi G, Longo G, Nami F, Barozzi Pet al. Hepatitis C virus infection in a subset of neoplastic lymphoproliferations not associated with cryoglobulinemia. Leukemia 1996; 10:351-355. 47. Zuckerman E, Zuckerman T, Levine AM, Douer D, Gutekunst K, Mizokami M, Qian DG, Velankar M, Nathwani BN, Fong TL. Hepatitis C virus infection in patients with B-cell non-Hodgkin's lymphoma. Ann Intern Med 1997;127:423-428. 48. Dammacco F, Sansonno D, Piccoli C, Racanelli V, D'Amore FP, Lauletta G. The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinemia, and overt B-cell malignancy. Semin Liver Dis 2000;20:143-57. Review. 49. Ferri C, La Civita L, Longombardo G, Zignego AL, Pasero G. Mixed cryoglobulinemia: a cross-road between autoimmune and lymphoproliferative disorders. Lupus 1998;7:275-279. 50. Fadda P, La Civita L, Zignego AL, Ferri C. Hepatitis C virus infection and arthritis. A clinico-serological investigation of arthritis in patients with or without cryoglobulinemic syndrome. Reumatismo 2002;54: 316-23. 51. Buskila D. Hepatitis C-associated arthritis. Curr Opin Rheumato12000;12:295-9. Review. 52. Zignego AL, Macchia D, Monti M, Thiers V, Mazzetti M, Foschi Met al. Infection of peripheral mononuclear blood cells by hepatitis C virus. J Hepatol 1992;15: 382-386. 53. Zignego AL, Ferri C, Giannini C, Monti M, La Civita L, Careccia G, Longombardo G, Lombardini F, Bombardier S, Gentilini P. Hepatitis C virus genotype analysis in patients with type II mixed cryoglobulinemia. Ann Intern Med 1996;124:31-34. 54. Lenzi M, Frisoni M, Mantovani V, Ricci P, Muratori L, Francesconi R, Cuccia M, Ferri S, Bianchi FB. Haplotype HLA-B8-DR3 confers susceptibility to hepatitis C virus-related mixed cryoglobulinemia. Blood 1998;91: 2062-6. 55. Zignego AL, Giannelli F, Marrocchi ME, Mazzocca A, Ferri C, Giannini C, Monti M, Caini P, Villa GL, Laffi G, Gentilini P. T(14;18) translocation in chronic hepatitis C virus infection. Hepatology 2000;31:474-9. 56. Zignego AL, Ferri C, Giannelli F, Giannini C, Caini P, Monti M, Marrocchi ME, Di Pietro E, La Villa G, Laffi G, Gentilini P. Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas. Ann Intern Med 2002; 137:571-80.
57. Kitay-Cohen Y, Amiel A, Hilzenrat N, Buskila D, Fejgin M, Gaber E, Safdi R, Tur-kaspa R, Lishner M. Bcl-2 rearrangement in patients with chronic hepatitis C associated with essential mixed cryoglobulinemia type II. Blood 2000;96:2910-2. 58. Pileri P, Uematsu Y, Campagnoli S, Galli G, Falugi F, Petracca R et al. Binding of hepatitis C virus to CD81. Science 1998;282:938-941. 59. Korsmeyer SJ. Bcl-2: a repressor of lymphocyte death. Immunol Today 1992; 13:285-287. 60. Ferri C, Sebastiani M, Giuggioli D, Cazzato M, Longombardo G, Alessandro Antonelli A, Rodolfo Puccini R, Claudio Michelassi C, Zignego AL. Mixed cryoglobulinemia: demographic, clinical, and serological features, and survival in 231 patients. Sem Arthritis Rheum 2004;33: in press. 61. Mazzaro C, Franzin F, Tulissi P, Pussini E, Crovatto M, Carniello GS et al. Regression of monoclonal B-cell expansion in patients affected by mixed cryoglobulinemia responsive to a-interferon therapy. Cancer 1996;77: 2604-2613. 62. Giannelli F, Moscarella S, Giannini C, Caini P, Monti M, Gragnani L, Romanelli RG, Solazzo V, Laffi G, La Villa G, Gentilini P, Zignego AL. Effect of antiviral treatment in patients with chronic HCV infection and t(14; 18) translocation. Blood 2003. 63. Ferri C, Marzo E, Longombardo G, Lombardini E La Civita L, Vanacore R, Liberati AM, Gedi R, Greco F, Moretti A, Monti M, Gentilini P, Bombardieri S, Zignego AL. Alpha-Interferon in Mixed Cryoglobulinemia patients: a randomized crossover controlled trial. Blood 81:1132-1136;1993. 64. Misiani R, Bellavita P, Fenili D, Vicari O, Marchesi D, Sironi PL, Zilio P, Vernocchi A, Massazza M, Vendramin G. Interferon alfa-2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 1994;330:751-6. 65. Casato M, Lagana B, Antonelli G, Dianzani F, Bonomo L Long-term results of therapy with interferon-alpha for type II essential mixed cryoglobulinemia. Blood 1991 ;78(12):3142-7. 66. Di Lullo L, De Rosa FG, Coviello R, Sorgi ML, Coen G, Zorzin LR, Casato M. Interferon toxicity in hepatitis C virus-associated type II cryoglobulinemia. Clin Exp Rheumatol 1998;16:506. 67. La Civita L, Zignego AL, Lombardini F, Monti M, Longombardo G, Pasero G, Ferri C. Exacerbation of peripheral neuropathy during alpha-interferon therapy in a patient with mixed cryoglobulinemia and hepatitis B virus infection. J Rheumatol 1996;23:1641-1643. 68. Lidove O, Cacoub P, Hausfater P, Wechsler B, Frances C, Leger JM, Piette JC. Cryoglobulinemia and hepatitis C. Worsening of peripheral neuropathy after interferon
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Virus-Induced Systemic Vasculitides Lo'ic Guillevin, Pascal Cohen and Christian Pagnoux
Department of Internal Medicine, HOpital Cochin, University of Paris, Paris, France
1. I N T R O D U C T I O N Viruses have been demonstrated to be the etiological agent responsible for several vasculitides, which can affect vessels of various calibers and which are usually not associated with antineutrophil cytoplasmic antibodies (ANCA). Two major vasculitides can occur as a consequence of viral infection: classic polyarteritis nodosa (PAN), as a result of hepatitis B virus (HBV) infection [1], and mixed cryoglobulinemia, in patients infected with hepatitis C virus (HCV) [2]. Other viruses can also be associated, albeit less frequently, to the occurrence of vasculitis, for example human immunodeficiency virus (HIV) [3] and parvovirus B19 [4], among others. The demonstration of a close relationship between viral infection and vasculitis justified the original therapeutic approach, avoiding prolonged administration of steroids and cytotoxic agents and based on the combination of antiviral agents and plasma exchanges (PE). Furthermore, the prospective trials organized by the French Vasculitis Study Group (FVSG) validated this therapeutic strategy [5]. A specific antiviral strategy is also recommended for HCV-related cryoglobulinemia vasculitis, despite the less favorable results.
2. POLYARTERITIS NODOSA Since the first reports on HBV-related PAN (HBVPAN) [1, 6], this causal relationship has been largely confirmed based on clinical, epidemiological and therapeutic data [5]. During the 1970s, the rate of HBV infection in patients with classic PAN reached 50%. However, over the past few years,
the frequency of HBV-PAN has declined to less than 5% [7]. In France, the incidence of HBV-PAN has decreased dramatically since blood testing and donor selection have been reinforced, and large vaccination campaigns have been organized in teenagers and people at risk. Intravenous drug use is now becoming the major cause of HBV-PAN. Since 2002, we have seen very few new cases of HBVPAN in the French population but also fewer new PAN cases, observations that indirectly support the hypothesis of a viral cause of PAN. Some other viruses have been associated with PAN but could only explain the occurrence of a few cases per year. HCV is not a major etiological factor for PAN and its responsibility as such was advanced in only a few publications [8, 9]. Less than 5% of our patients are infected with HCV, which confirms our previous findings [ 10]. GB virus-C, when sought in patients with PAN, has not been found to be an agent responsible for the disease [ 11 ]. When present, HCV was often observed in association with other viruses, HBV or HIV, and also with mixed cryoglobulinemia. Several concomitant parvovirus B 19 infections have been described too [4, 12] but a systematic survey of PAN patients did not show them to have a higher frequency of parvovirus B 19 than the control population [12]. Other viruses have been incriminated in the development of PAN, including anecdotal cases of HIV infection [3, 13, 141. The immunological process responsible for HBV-PAN mainly in patients under 40 years of age usually becomes manifest less than 12 months after infection. Hepatitis is rarely diagnosed, as it remains silent before the occurrence of PAN. Clinical manifestations are of acute onset and are roughly
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the same as those commonly observed in PAN [ 15]. HBV-PAN is certainly the purest form of PAN and no overlap with other vasculitides, especially microscopic polyangiitis, has been observed in our experience. HBe antigen (Ag) to anti-HBe antibody (Ab) seroconversion usually leads to recovery. The major sequelae are the consequence of vascular nephropathy and peripheral neuropathy but, even in patients who initially develop renal insufficiency, it is possible to cure PAN with little residual impairment of renal function.
2.1. Relapses HBV-PAN tends not to recur once remission is induced. In our series, only 6% of the patients relapsed [15]. At present, it is still not possible to identify the subgroup of patients who will relapse. The clinical pattern of relapse does not necessarily mimic the original presentation, in that previously unaffected organs can be involved at relapse. Due to the low frequency of relapses, maintenance treatment is not necessary and short-term treatment can be envisaged.
2.2. Deaths The causes of death can be divided into three categories: related to vasculitis manifestations, attributed to treatment side effects and miscellaneous causes, usually independent of the vasculitis. 2.2.1. Deaths related to vasculitis
In all vasculitides, involvement of major organs can have lethal issue. A few patients die early from multivisceral involvement, often gastrointestinal [ 16], that cannot be controlled by treatment. In such cases, the course of the disease is generally characterized by fever, rapid weight loss, diffuse pain and involvement of one or several major organs. 2.2.2. Deaths attributed to treatment side effects
Conventional treatment with steroids and cyclophosphamide jeopardizes the patient's outcome by allowing the virus to persist, stimulating its replication and thereby facilitating evolution towards chronic hepatitis and liver cirrhosis. Thus, cyclo-
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phosphamide, like prolonged steroid treatment, is contraindicated. In addition to these long-term side effects, infections are more frequent when immunosuppressants are prescribed. Steroids are also responsible for side effects, which are not detailed here [ 16]. PE can also favor the occurrence of infections when a central venous access is necessary.
3. HCV-RELATED CRYOGLOBULINEMIA Mixed cryoglobulinemias of type ii and, more rarely, type III are the consequence of HCV infection in more than 80% of the patients [2]. Cryoglobulinemia is asymptomatic in most patients but persists for decades and the disease duration might be a factor associated with the occurrence of clinical symptoms of vasculitis. When symptoms are present, the most frequent are purpura, peripheral neuropathy, glomerulonephritis, leg ulcers, arthritis and sicca syndrome. Cryoglobulinemia vasculitis is a small vessel vasculitis, as defined by the Chapel Hill nomenclature [17]. The clinical symptoms [ 18, 19] may develop progressively and are often of moderate intensity at their onset. Neuropathy can be symmetric and limited to sensory signs, including hypoesthesia and pain. This distal neuropathy is more frequently present in the lower than upper limbs. Neuropathy can also be mononeuritis multiplex, as described in PAN. The outcome of neuropathy is chronic and, although the motor symptoms can regress, the sensory symptoms can remain definitively. Kidney involvement, when present, is glomerulonephritis but, unlike ANCA-associated vasculitides, pauci-immune glomerulonephritis is not found. Few patients progress to end-stage renal failure. Sicca syndrome is present in 20% of the patients but without the immunological features of Sj6gren's syndrome. The presence of the cryoglobulins, usually type II, IgM kappa, is characteristic of the disease. Complement, especially the C4 component is low and a rheumatoid factor may be found, and, because it is sometimes difficult to detect a cryoprecipitate, this association is highly suggestive of the diagnosis. Autoantibodies are absent, especially ANCA. The outcome of mixed cryoglobulinemia vasculitis is characterized by chronicity and relapses, even under treatment.
4. HIV-ASSOCIATED VASCULITIS
5. T R E A T M E N T
Vasculitides occurring during the course of HIV infection have been reported [3, 14]. Most of them involved skin, peripheral neuropathy or the central nervous system. The clinical spectrum and histological findings of HIV-associated vasculitis vary widely. Large-, medium- and small-sized arteries can be affected. Necrotizing arteritis, non-necrotizing arteritis, giant-cell arteritis and eosinophil arteritis have been observed [14]. According to Calabrese [ 14], the frequency is low (1%), and most of the reported cases were identified at autopsy. In our experience [3], HIV-associated vasculitis is an extremely rare entity and we have seen only a few cases in our vasculitis reference center, which is networked to several large centers specializing in the management of HIV infection. Vasculitis can develop in adults and children at any stage of HIV infection as defined by the Centers for Disease Control classification. Some cases seem to be directly caused by opportunistic infections, such as Pneumocystis carinii, cytomegalovirus or Toxoplasma gondii, non-opportunistic infectious agents or drug-induced hypersensitivity. HIV was thought to be the etiological agent in a few patients because of the in situ localization of the virus and the absence of evidence suggesting other mechanisms. However, the etiology remains unknown in most cases. The pathogenesis of HIV-associated vasculitis is heterogeneous, but at least two general mechanisms have been hypothesized: first, virus replication might induce direct injury of the vessel wall or vascular damage might be the result of an immune mechanism. These mechanisms may be cellular and/or humoral and include deposition of immune complexes (IC) and/or their in situ formation [20]. IC are frequently detected in AIDS patients and their frequency increases with advancing stages of infection [3]. Some authors have analyzed their composition and found them to contain both antibodies specific to HIV and HIV antigens. However, their role in the vasculitic process remains to be demonstrated.
5.1. Treatment of HBV-Related PAN
For many years, HBV-PAN was treated in the same way as non-vir~s-related PAN and patients received steroids, sometimes combined with cytotoxic agents, mainly cyclophosphamide. This treatment was often effective in the short-term but careful analysis of long-term results showed that relapses and complications (chronic hepatitis or liver cirrhosis) occurred because of virus persistence. According to McMahon et al [21 ], who followed Eskimos with PAN, 4 (31%) patients died during the course of PAN. In our first randomized study [22] in which patients were not selected according to their virus status, 14/71 were HBV-positive; 84% of them recovered from PAN but 2 subsequently died of liver cirrhosis. The rationale for combining PE and antiviral treatment was to obtain the following effects" initial corticosteroids to rapidly control the most severe life-threatening manifestations of PAN which are common during the first weeks of the disease, and abrupt stoppage of corticosteroids to enhance immunological clearance of HBV-infected hepatocytes and favor HBe seroconversion. PE can almost always control the course of these PAN without the addition of steroids or cyclophosphamide. An alternative therapy was also needed to lower PAN mortality and improve prognosis. In a retrospective study, we showed that, when steroids and immunosuppressants were prescribed to treat HBVPAN, the outcome was poorer than for non-viralPAN [15]. Therefore, based on the efficacies of antiviral agents against chronic hepatitis and of PE in PAN, together with Trrpo [ 1], who described the responsibility of HBV in the development of PAN, we combined the two therapies to treat HBV-PAN [23, 24].
Vidarabine. When this therapeutic strategy was first applied, the only available antiviral agent was vidarabine. After a 3-week course of vidarabine, administered after 1 week of steroids (1 mg/kg/d) and combined with PE, a full clinical recovery was obtained in three-quarters of the patients and HBe seroconversion was observed in nearly half of the patients.
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IFN~ IFNet has replaced vidarabine and gives better results. In a series of patients, HBe seroconversion was obtained in two-thirds of the patients and HBsAg-to-anti-HBsAb seroconversion in half of them. The dose of 3 millions units, injected subcutaneously 3 times a week is recommended. Pegylated IFNct can also be prescribed. In HBV-PAN, the combination of antiviral agents (vidarabine or interferon-alpha-2a or 2-b (IFNot)) gave excellent overall therapeutic results [5] and should be preferred to conventional regimens that jeopardize the outcome, as described above. The efficacy of this strategy was confirmed in a series of 41 patients [5]. Twenty-three (56.1%) no longer exhibit serological evidence of replication and 80.5% recovered. Lamivudine. Lamivudine is an antiviral agent specifically designed for the treatment of HBV and HIV infections. In a small series of patients (personal data), we prescribed lamivudine (100 mg/day) in combination with PE, after a few days of steroids. Because lamivudine is eliminated by the kidney, its dose should be adapted to renal function and lower for patients with renal insufficiency. In that study [25], 9/10 patients recovered and 6/9 HBe seroconverted. One patient died. Plasma exchanges. In a few cases [26], the antiviral agent was prescribed alone. In our opinion, even if it is possible to obtain good clinical results in some patients, the severity of the disease in most patients requires therapy able to control immediately the severe or life-threatening manifestations of PAN. PE are able to rapidly clear the IC responsible for the disease. This rapid intervention is the most appropriate to control the disease. In our protocol, steroids are also prescribed for a few days to control as quickly as possible the clinical manifestations while waiting for IFNot or other antiviral agent efficacy to kick in. In our opinion, PE are not indicated because of their superiority to other medications but because they are, in combination with antiviral drugs, able to replace the deleterious therapies commonly used in virus-associated vasculitides with equivalent efficacy. The optimal schedule is as follows: 4 sessions/ week for 3 weeks, then 3 sessions/week for 2 to 3 weeks, followed by progressive lengthening of the
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intervals between sessions. One plasma volume (60 ml/kg) is usually exchanged using 4% albumin as the replacement fluid. The circuit can be primed with starch. During the first weeks of treatment, the high number of PE can decrease the level of clotting factors and thereby lead to bleeding. Should bleeding occur, fresh frozen plasma can be used instead of albumin. Usually, the tolerance of PE is excellent.
Outcome and follow-up. The previously described short- and long-term outcomes of the patients showed the progressive improvement of seroconversion rates for patients receiving IFNtx. One of the major advances obtained under our antiviral strategy was the very rapid cure of HBV-PAN, even in its most severe forms. The majority of patients received the antiviral drug for a few weeks or months but PE, which were specifically given to control the acute manifestations of the disease, were stopped after 2 months. All signs of vasculitis were sometimes eliminated more quickly, with some of our patients recovering within 3 weeks. In the days following treatment onset, transaminase levels decrease. They usually return to normal within a few days or weeks. For patients who received vidarabine, a second increase of transaminase levels was observed prior to seroconversion. This usually mild immunological response was considered normal, as it attested the patient's ability to reject the virus via the hepatocytes. Nevertheless, transaminase levels can rise sharply and fulminant hepatitis can coincide with HBe seroconversion, as for one of our patients [27] who died of fulminant hepatitis several days after seroconversion. The response observed under IFNt~ is markedly different: transaminases normalize progressively and their levels do not rise after stopping the treatment, even when seroconversion has not been obtained. When HbeAb are detected, PE should be stopped to avoid the clearance of the newly synthesized antibodies. In a few cases, the antibody levels fluctuated, sometimes being present or absent. This Ag-Ab equilibrium can be very unstable and treatment should be continued. In such cases, it is more reliable to monitor virus activity by quantitative measurements of viral DNA. After recovery from the symptoms of the vasculitis, the clinician potentially faces two different
virological situations. First, replication continues, as demonstrated by the absence of HBeAb and the positivity of viral DNA, and PAN remission has been obtained but relapses may still occur. We therefore recommend prolonging IFNt~ administration for a total of 6 to 12 months, according to the viral response measured by quantitative DNA, and focusing on the treatment of viral hepatitis and not PAN, which has been cured. Second, Ab to HBe, at least, or to HBs, at best, are present, the patient can be considered cured and relapses will never occur. If, despite the presence of HBsAb, new manifestations of PAN appear, the clinician should consider the possibility of the vasculitis occurring coincidentally with virus infection but not linked to it. For patients who do not respond to one of the antiviral drugs, a combination of IFNtx and lamivudine could be tested.
5.2. Treatment of HCV-Associated Cryoglobulinemic Vasculitis Only a quarter of the patients with chronic hepatitis C achieved a sustained virological response [28]. A higher response rate was obtained with pegylated IFNtx. In the most recent study [29], 69% of the patients had responded at 48 weeks and achieved clinical recovery. Combining IFNt~ and ribavirin also increased the seroconversion rate in hepatitis C. No treatment is able to cure the majority of mixed cryoglobulinemias definitively and an optimal therapeutic strategy has not yet been clearly defined. Steroids and immunosuppressive drugs are commonly used to treat severe forms, but they have the same noxious effects as stated above. As we did for HBV-PAN, we devised a strategy associating antiviral drugs and PE for some patients [30]. For asymptomatic patients, there is no argument to treat, and monitoring could be sufficient. For patients with moderate symptoms of cryoglobulinemia vasculitis (e.g., arthralgias, purpura, sensory peripheral neuropathy), combining IFNtz and ribavirin is indicated. Ribavirin alone is not able to completely suppress viral replication but, in conjunction with IFNtx, viral replication was no longer detectable in 48% of the patients receiving the combination for 12 months [28]. We can expect that virus suppression will also be obtained in cryoglobulinemia.
Although the majority of the patients seen for symptomatic cryoglobulinemia have virus-positive polymerase chain reaction (PCR) assays, reflecting virus replication, a few of them remain serologically positive but become PCR-negative, reflecting past contamination. We also observed, in 2 of our patients with very severe vasculitis, the disappearance of the virus under antiviral treatment and PE but the persistence of clinical symptoms, which necessitated prolonged symptomatic treatment with PE.
Plasma exchanges. The indications of PE in HCVrelated cryoglobulinemia are controversial. Based on the effectiveness observed in our patients who failed to respond to other treatments and several reported failures of IFNt~, we recommend combining PE and antiviral drugs. PE should not be prescribed systematically for every newly diagnosed case of cryoglobulinemia because the majority of patients present no or very few symptoms, and we do not know, at present, whether or not treatment is indeed indicated in these pauci- or asymptomatic forms. PE are indicated for patients with symptoms requiting medical intervention. Purpura and sicca syndrome do not constitute such indications: the former regresses spontaneously and the latter is refractory to this treatment. In the case of glomerulonephritis due to cryoglobulinemia, PE combined with IFNtx can be effective but randomized controlled trials are needed to assess their contribution. PE are mainly indicated to treat rapidly progressing peripheral neuropathy and leg ulcers. The latter manifestation is often very severe and accompanied with pain that can require intensive therapy, including morphine. Under PE, arteriolar ulcers regress quickly and complete healing can be obtained in a few weeks. PE should be tapered progressively to avoid a rebound phenomenon due to the increased production of cryoglobulins as a consequence of the stimulation of the B-cell clones responsible for their production. Some of our patients remain PEdependent: clinical symptoms recur or worsen while tapering or after abrupt discontinuation of the sessions. Maintenance treatment should therefore be prescribed and the clinician has to try to determine the minimal number of sessions able to control the disease. When indicated, the number of sessions is not
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clearly established. We propose the following schedule: 3 sessions a week for 3 weeks, then 2 sessions a week for 2 to 3 weeks, then 1 session every week or every 10 days until clinical symptoms disappear or the optimal clinical result is obtained.
term results are better and relapses are rare. This therapeutic approach should be applied and results will surely improve with the greater efficacy of new antiviral drugs.
5.3. Treatment of HIV-Associated Vasculitis
REFERENCES
Treatment of HIV-associated vasculitis has not yet been well defined, and steroids and immunosuppressants should be used cautiously, as they could favor the development of opportunistic infections and other clinical manifestations of AIDS. Again, the dual objective is to cure the vasculitis and to control HIV infection, and thus to avoid steroids and cytotoxic agents. The first objective is HIV-replication suppression, which is more easily obtained with the combination of 2, 3 or 4 antiviral agents. Nucleotide, non-nucleotide and protease inhibitors are the most frequently used families of drugs. Based on the presence of IC, we have proposed, as for other virus-associated vasculitides, to treat the patients with PE, using the same scheme as that for HBV-PAN. PE can clear IC and cytokines involved in the vasculitic process. In our clinical experience, this regimen was successful [3] and the patients we treated improved and vasculitis remissions were obtained. This strategy was also effective for patients with HCV and HIV or HBV and HIV coinfections. Some patients with cryoglobulinemia responded very quickly to this therapy. HCV cryoglobulinemia usually recurs after stopping PE and, unfortunately, anti-HIV drugs are not able to suppress cryoglobulin production. We also observed anti-cardiolipin Ab in a patient with HIV- and HCVrelated vasculitis. HIV-associated vasculitides appear to be a oneshot disease and do not recur and one to three months of therapy are usually sufficient to cure them.
1.
6. C O N C L U S I O N Virus-associated vasculitides are not uncommon and require a specific therapeutic strategy. The combination of antiviral agent(s) and PE is effective in the majority of patients and, because this strategy is adapted to the pathogenesis of the disease, long-
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Tr6po C, Thivolet J. Hepatitis associated antigen and periarteritis nodosa (PAN). Vox Sang 1970;19:410-1. 2. Agnello V, Chung RT, Kaplan LM. A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 1992;327:1490-5. 3. Gisselbrecht M, Cohen P, Lortholary O, Jarrousse B, Gayraud M, Lecompte I e t al. Human immunodeficiency virus-related vasculitis. Clinical presentation of and therapeutic approach to eight cases. Ann M6d Interne (Paris) 1998;149:398-405. 4. Corman LC, Dolson DJ. Polyarteritis nodosa and parvovirus B 19 infection. Lancet 1992;339:491. 5. Guillevin L, Lhote F, Cohen P, Sauvaget F, Jarrousse B, Lortholary O et al. Polyarteritis nodosa related to hepatitis B virus. A prospective study with long-term observation of 41 patients. Medicine (Baltimore) 1995;74: 238-53. 6. Prince AM, Tr6po C. Role of immune complexes involving SH antigen in pathogenesis of chronic active hepatitis and polyarteritis nodosa. Lancet 1971;1: 1309-12. 7. Mahr A, Guillevin L, Poissonnet M, Aym6 S. Prevalence of polyarteritis nodosa, microscopic polyangiitis, Wegener's granulomatosis and Churg-Strauss syndrome in a French urban population in 2000: a capturerecapture estimate [abstract]. Cleve Clin J Med 2002;69 (Suppl 2):170-1. 8. Cacoub P, Lunel-Fabiani F, Du LT. Polyarteritis nodosa and hepatitis C virus infection. Ann Intern Med 1992; 116:605-6. 9. Cacoub P, Maisonobe T, Thibault V, Gatel A, Servan J, Musset L et al. Systemic vasculitis in patients with hepatitis C. J Rheumatol 2001;28:109-18. 10. Quint L, D6ny P, Guillevin L, Granger B, Jarrousse B, Lhote F et al. Hepatitis C virus in patients with polyarteritis nodosa. Prevalence in 38 patients. Clin Exp Rheumatol 1991;9:253-7. 11. Servant A, Bogard M, Delaugerre C, Cohen P, D6ny P, Guillevin L. GB virus C in systemic medium- and small-vessel necrotizing vasculitides. Br J Rheumatol 1998;37:1292-4. 12. Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003;300:455.
13. Calabrese L. The rheumatic manifestations of infection with the human imunodeficiency virus. Semin Arthritis Rheum 1989; 18:225-9. 14. Calabrese LH. Vasculitis and infection with the human immunodeficiency virus. Rheum Dis Clin North Am 1991;17:131-47. 15. Guillevin L, Lhote F, Jarrousse B, Bironne P, Barrier J, D6ny Pet al. Polyarteritis nodosa related to hepatitis B virus. A retrospective study of 66 patients. Ann M6d Interne (Paris) 1992;143:63-74. 16. Gayraud M, Guillevin L, le Toumelin P, Cohen P, Lhote F, Casassus Pet al. Long-term followup of polyarteritis nodosa, microscopic polyangiitis, and Churg-Strauss syndrome: analysis of four prospective trials including 278 patients. French Vasculitis Study Group. Arthritis Rheum 2001 ;44:666-75. 17. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL et al. Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum 1994;37:187-192. 18. Cacoub P, Hausfater P, Musset L, Piette JC. Mixed cryoglobulinemia in hepatitis C patients. GERMIVIC. Ann M6d Interne (Paris) 2000; 151:20-9. 19. Rieu V, Cohen P, Andr6 MH, Mouthon L, Jarrousse B et al. Characteristics and outcome of 49 patients with symptomatic cryoglobulinaemia. Rheumatology (Oxf) 2002;41:290-300. 20. Gherardi R, Lebargy F, Gaulard P, Mhiri C, Bernaudin J, Gray F. Necrotizing vasculitis and HIV replication in peripheral nerves (letter). N Engl J Med 1989;321: 685-686. 21. McMahon BJ, Heyward WL, Templin DW, Clement D, Lanier AP. Hepatitis B-associated polyarteritis nodosa in Alaskan Eskimos: clinical and epidemiologic features and long-term follow-up. Hepatology 1989;9: 97-101. 22. Guillevin L, Jarrousse B, Lok C, Lhote F, Jais JP, Le THDD et al. Longterm followup after treatment of polyarteritis nodosa and Churg-Strauss angiitis with comparison of steroids, plasma exchange and cyclophosphamide to steroids and plasma exchange. A prospective randomized trial of 71 patients. The Coopera-
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tive Study Group for Polyarteritis Nodosa. J Rheumatol 1991 ;18:567-74. Guillevin L, Merrouche Y, Gayraud M, Jarrousse B, Royer I, L6on A et al. P6riart6rite noueuse due au virus de l'h6patite B. D6termination d'une nouvelle strat6gie th6rapeutique chez 13 patients. Presse M6d 1988;17: 1522-6. Tr6po C, Ouzan D, Delmont J, Tremisi J. Sup6riorit6 d'un nouveau traitement 6tiopathog6nique gu6rissant la p6riart6rite noueuse due au virus de l'h6patite B par la combinaison d'une br~ve corticoth6rapie, de vidarabine et d'6changes plasmatiques. Presse M6d 1988;17: 1527-31. Guillevin L, Mahr A, Cohen P, Larroche L, Queyrel V, Loustaud-Ratti V e t al. Short-term corticosteroids then lamivudine and plasma exchanges to treat hepatitis B virus-related polyarteritis nodosa. Arthritis Rheum (in press) 2003. Avsar E, Savas B, T6ztin N, Ulusoy NB, Kalayci C. Successful treatment of polyarteritis nodosa related to hepatitis B virus with interferon alpha as first-line therapy [letter]. J Hepatol 1998;28:525-6. Guillevin L, Lhote F, L6on A, Fauvelle F, Vivitski L, Tr6po C. Treatment of polyarteritis nodosa related to hepatitis B virus with short term steroid therapy associated with antiviral agents and plasma exchanges. A prospective trial in 33 patients. J Rheumatol 1993;20: 289-98. Poynard T, Marcellin P, Lee S et al. Randomized trial of interferon alpha 2b plus ribavirin for 48 weeks or for 24 weeks versus alpha 2b plus placebo for 48 weeks for treatment of chronic hepatitis C virus. Lancet 1998;352: 1426--32. Zeuzem S, Feinman S, Rasenack Jet al. Peginterferon alpha-2a in patients with chronic hepatitis C. N Engl J Med 2000;343:1666-72. Cohen P, Nguyen QT, D6ny P, Ferri~re F, Roulot D, Lortholary O et al. Treatment of mixed cryoglobulinemia with recombinant interferon alpha and adjuvant therapies. A prospective study on 20 patients. Ann M6d Interne 1996;147:81-6.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Viral Infections and Autoimmune Hepatitis Sandro Vento ~and Francesca Cainelli
1Section of lnfectious Diseases, Department of Pathology, University of Verona, Verona, Italy
Abbrevations: AH: autoimmune hepatitis, ANA: antinuclear antibodies, ASMA: anti-smooth muscle antibodies, LKMI: liver-kidney microsomal type 1 antibodies, ASGPR: asialoglycoprotein receptor, HAV: hepatitis A virus, HCV: hepatitis C virus, CYP2D6: cytochrome P450IID6, HSV: herpes simplex virus, HBV: hepatitis B virus, CMV." cytomegalovirus, EBV: Epstein-Barr virus, TTV."TT virus.
1. INTRODUCTION Autoimmune hepatitis (AH) is (similarly to the other autoimmune diseases, with the exceptions of rheumatoid arthritis and thyroiditis) a rare disease; the etiology is unknown, the female predominance is strong and the prevalence in the population is around 0.01-0.02%. The disease is characterised serologically by a striking increase in serum IgG [1]. The natural course is marked by recurrent necro-inflammatory episodes within the liver lobules and at the interface with the portal tracts (piecemeal necrosis) eventually leading to cirrhosis and possibly liver failure, despite a responsiveness to corticosteroids (approximately 85%) which is among highest of all autoimmune diseases. Autoimmune hepatitis is divided into two main forms, types 1 and 2; the former is characterized by high titer (> 1:80) autoantibodies to nuclei (ANA, homogeneous or speckled pattern) reactive with chromatin and occasionally dsDNA and/or antibodies to substrates of smooth muscle (ASMA) reactive with F-actin microfilaments. Type 2 is overall rare (20 times less frequent than type 1) but usually occurs in younger patients and with typical type 1 antibodies to liver/ kidney microsomes (LKM1) directed against the
cytochrome P450 isoform 2D6. Despite attempts over the latest 20 years at establishing a reliable experimental animal model by Australian, Japanese and German research groups, this is still lacking. A genetic predisposition for the type 1 form of the disease is suggested by descriptions of impah'ed suppressor T cell function in patients and first degree relatives [2, 3], enhancing reactivity to autoantigens and favoring an antibody-dependent cellular form of cytotoxicity against self antigens expressed on the hepatocyte surface and by the association with HLA haplotypes, which varies however among different ethnic groups. DRB 1*0301 (DR3) and DRB 1*0401 (DR4) are associated with type 1 AH in white European and North American populations; DRB 1"0405 (DR4) is the principal association in Japanese and adult Argentine patients, whereas DRBl*0404 (DR4) is the main susceptibility allele in Mestizo Mexicans [4, 5]. Furthermore, in patients of northern European origin an association with the extended HLA haplotype A1, B8, DR3 is notable [6].
2. PROPOSED MECHANISMS OF LIVER CELL INJURY AND UNDERLYI-NG IMMUNE DEFECTS
The detection of IgG on hepatocytes isolated from liver biopsy specimens, the linear pattern of immunofluorescence [7], suggestive of a reaction against antigens diffusely distributed on the cell membrane [8], the predominance of B cells and T lymphocytes of helper phenotype in the mononuclear cell infiltrate in the liver [8] and the finding of antigen-presenting dendritic cells in the periportal areas of the lobule [9] all point to a role for antibodies synthe-
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sized within the fiver and directed against surface membrane antigens expressed on hepatocytes in the establishment and persistence of liver cell injury in autoimmune hepatitis. In addition, antibodydependent cell-mediated cytotoxicity is the major mechanism of cytotoxicity in vitro in AH [ 10]. Although a number of autoantibodies reacting with antigens thought to be expressed on the liver cell surface have been described, especially in patients with active disease, the strongest candidate as a substantial contributor to hepatocellular damage in adult AH type 1 is directed against the asialoglycoprotein receptor (ASGPR), the liver cell-specific receptor for desialylated glycoproteins inserted on the hepatocyte sinusoidal plasma cell membrane [ 11 ]. T cells reactive to ASGPR are also detectable in patients with AH [2, 3] and T lymphocytes from healthy, unrelated subjects, added in a low ratio in culture, can specifically suppress the in vitro response to ASGPR of T cells from patients with autoimmune hepatitis type 1 [2, 3]. CD4+ve T cell inducers (specific for the asialoglycoprotein receptor) of CD8+ve suppressor T lymphocytes are present in the circulation of normal healthy subjects and defective in the patient population [12], appear to be activated in vivo in normal subjects and may play an essential role in controlling liver-directed autoreactivity. This liverspecific suppressor/inducer T cell defect might be genetically determined, as it has also been found in a high proportion of healthy relatives of patients with AH [3]. The hypothesis has been put forward [12] that the disease (in its far most frequent form, namely type 1) occurs only in those individuals who, in addition to having an ASGPR-specific suppressor/inducer defect, develop (owing to environmental triggers) T lymphocyte reactivity to the same autoantigen.
3. VIRUSES AS TRIGGERS IN ! AUTOIMMUNE HEPATITIS TYPE 1
If indeed a trigger is required to set off a sequence of events leading to autoimmune hepatitis in a predisposed individual, viruses are among the most likely candidates.
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3.1. Measles Virus
The first candidate virus put forward was the measles virus, IgM antibodies to which were found to be raised in one study in patients with AH [ 13] and the genome of which reportedly persisted in patients' lymphocytes, suggesting an etiologic relationship [14]; however, the results reported by Robertson and colleagues were not reproduced by others [ 15] and the subsequent finding by a different group of a similar proportion of anti-measles antibody-positive individuals among patients with AH and the general population of comparable age did not support the suggestion [16]. Although measles virus appears overall not to be a frequent trigger of AH, it may occasionally act as such, and the report of a 4-yearold girl with a strong genetic predisposition who developed AH type 1 in strict temporal relation to this viral infection [ 17] supports this possibility. 3.2. Hepatitis A Virus
In 1991, following a 4-years prospective study, two cases of AH type 1 were described in association with hepatitis A virus (HAV) infection in patients who had first-degree relatives with the disease [18]. In that study two of the three patients with subclinical acute hepatitis A from a group of 58 healthy relatives of 13 patients with AH developed AH within 5 months. These 2 patients had the above mentioned suppressor/inducer T cell defect specific for the ASGPR, and antibodies to this autoantigen appeared during acute HAV infection and increased during follow-up. Anti-ASGPR antibodies are indeed commonly found transiently in acute hepatitis A [ 19] and appear before the detection of T cell immunity to the same antigen. This humoral autoimmune response is therefore unlikely to be driven by autoreactive T cells recognizing ASGPR. As immune reactions against viral antigens expressed on cell surfaces are present in patients with hepatitis A [20], it might be that helper T cells reactive to HAV antigens exposed on the surface of infected liver cells provide help for autoreactive B cells specific for an antigen (the ASGPR) coexpressed on the hepatocyte membrane. The results of the study published in 1991 also showed that T cell immunity to the ASGPR is detectable, during HAV infection, after the peak
in aminotransferase concentrations, suggesting that massive antigen release on hepatocyte damage is necessary to activate specific T helper lymphocytes. In the presence of functional ASGPR-specific suppressor/inducer T cells, this activation is transient and both specific T helper cells and the corresponding antibodies rapidly disappear. In contrast, in subjects with defective suppressor/inducer T cell control of immune responses to the ASGPR, T helper cell activation and antibody production continue and increase, and appears to be responsible for an autoimmune liver damage. Subsequently Rahaman and coworkers [21] described a middle-aged woman in whom serologically defined acute hepatitis A also triggered the onset of AH, and Huppertz and colleagues [22] reported a 7-year-old patient who developed the disease in association with preceding HAV infection and in the absence of a family history of AH. In a study reporting the clinical profile of 59 patients presenting with acute HAV infection, two women presented with concomitant AH type 1 [23]. Other similar cases have been reported in adults [24] and children [25]. Taken together, the above observations indicate that in the rare patients in whom chronic liver disease follows acute hepatitis A, the disease is AH type 1 and demonstrate conclusively that HAV infection can act, although in few cases, as a trigger for this organ-specific autoimmune disease in predisposed individuals. Which peculiar characteristics may underline the role of HAV as a trigger for an autoimmune liver disease? HAV is a non-cytopathic virus (either in vitro or in vivo), and elicits powerful cellular immune reactions against viral antigens expressed on the cell surface of infected hepatocytes. Although these reactions are considered to be responsible for the associated hepatocyte necrosis [20], it must be outlined that the liver damage resembles histologically an autoimmune damage, as even an experienced pathologist would have difficulties in distinguishing the histological appearance of the liver during acute hepatitis A from that of the liver of a patient with AH. Indeed the portal inflammatory infiltrate is similarly rich in plasma cells, piecemeal necrosis is frequently observed during acute hepatitis A (but not in other virally-induced acute liver disease) [26] and the main lymphoid subsets present in portal
areas are helper/inducer T cells and B cells [27]. Occurrence of autoantibodies typical of AH is also quite frequent during acute hepatitis A: anti-actin antibodies were first reported in 1983 and antinuclear, anti-smooth muscle and anti-ASGPR antibodies [19] are also transiently produced after the peak in aminotransferase concentrations. Moreover, in children from Argentina and Brazil (two countries with a still high prevalence of H~V infection) AH type 1 is associated with a unique HLA allele, DRBI*1301 (a particular HLA DR13 allele); the very same allele is associated with protracted liver damage and persistent high titers of anti-smooth muscle/actin antibodies following HAV infection [28]. Anti-smooth muscle/actin antibodies are not associated with HLA-DRB 1" 1301 if the infection is not prolonged, and it seems therefore that their long-lasting presence only in the protracted forms is associated with the sustained release of a self antigen related to a particular genetic background. As these children do not however progress to AH, it may be that in areas of high endemicity some unknown factors protect most individuals from progression to overt AH.
3.3. Epstein-Barr Virus In 1995, the extended follow-up of the very same cohort of relatives of patients with autoimmune hepatitis which had led to the identification of HAV as a possible trigger allowed the discovery of a second trigger, namely Epstein-Barr virus (EBV). Two women, aged 24 and 15 years, developed AH type 1 in strict temporal relation to EBV-induced infectious mononucleosis; in both cases, a defect in suppressor/inducer T cells controlling the response to the ASGPR had been identified prior to the viral infection, and anti-ASGPR antibodies persisted and increased after the viral illness [29]. As EBV is a polyclonal B lymphocyte activator and induces proliferation of specific B-cell clones [30], it may trigger AH through continuining proliferation and antibody production of ASGPR-specific B lymphocytes unchecked by defective suppressor/inducer T cells of the same specificity. Three further cases of AH type 1 following EBV infection have been described: one in a young Italian woman [31], one in an old Japanese man [32], and the latest in a 5year-old Italian girl [33]. The above reports clearly
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define EBV as a trigger for AH type 1, albeit rare.
4. CAN A ROLE FOR VIRUSES BE ENVISAGED IN AUTOIMMUNE HEPATITIS TYPE 2?
The target of the hallmark of this form of autoimmune liver disease, i.e. liver-kidney microsomal type 1 antibody, is cytochrome P450IID6, a member of the hepatic P450 enzyme family. Manns and coauthors suggested that reactivity to the major epitope of CYP2D6 recognized by LKM1 antibodies may arise through a crossreactive response to hepatitis C virus (HCV) or herpes simplex virus type 1 (HSV1), as the aminoacids 310-324 of the envelope region E1 HCV and aminoacids 156-170 of immediate early protein IE175 of HSV1 share sequence homology with the immunodominant region, aminoacids 254-271, of CYP2D6, recognised by 85% of patients with AH type 2 [34]. Klein and coworkers demonstrated however that reactivity to the linear B-cell epitope of cytochrome P2D6 CYP2D6196_218is found in as many as 68% of patients with autoimmune hepatitis type 2 but in only 18% of LKMI+ve HCV-infected patients [35]. The complexity of the situation is further illustrated by the observation of crossreactive antibody recognition of homologous regions of HCV (NS5B HCW2985_2990)and cytomegalovirus (CMV) (EXON CMV130_135)antigens in LKMI+ve HCV-infected patients recognizing CYP2D6204_209 [36], and by the homologies between CYP2D6239_271and proteins from Salmonella typhimurium and human T lymphotropic viruses 1 and 2 [37]. Are all these crossreactivities of any importance in clinical practice, i.e. do they lead to autoimmune liver disease? Lenzi and coauthors first reported an impressively high occurrence of HCV infection in their cases of anti-LKMl-positive individuals with chronic liver disease [38], and suggested a role for HCV in the induction of AH type 2, but failed to provide direct evidence; a case report from Germany described two identical twins (HLA A1,B8,DR3), only one of whom was affected by AH and had LKM1 autoantibodies and anti-HSV 1 antibodies [39], suggesting a role for this virus in the development of disease. Dalekos and coworkers [40], while studying antibody titers and per-
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forming epitope mapping of LKMl-positive sera from patients with chronic hepatitis C, found one young female patient with a very high LKM1 titer and autoantibodies directed against the epitope of amino acids 257-269 of CYP2D6 (preferentially recognized by patients with AH type 2) [35], who showed exacerbation of the disease under interferon treatment and was switched successfully to immunosuppressive therapy. This latter patient suffered from AH type 2 as the dominant cause for liver damage, but no proof was provided that HCV induced the appearance of the autoimmune disease. Furthermore, a survey conducted in 25 LKMI+ve patients in United Kingdom failed to demonstrate any association between AH type 2 and either antibodies to HSV- 1 or to HCV [41 ]. The most striking case of viral-induced AH type 2 is the case of a 29-year-old nurse who developed the disease following acquisition and rapid clearance of HCV [42]. The finding of IgM anti-LKM1 antibodies during acute HCV infection, the subsequent switch to IgG anti-LKM1 and the reactivity of these antibodies against amino acids 257-269 of CYP2D6 all point to a real (but unique) case of HCV-induced AH type 2. Overall, paradoxically (taking into account the immunological crossreactivities observed between CYP2D6 and a variety of microorganisms sequences) evidence for a causal role of viruses in triggering AH type 2 in humans is quite lacking, especially if one considers that the LKM1 antibodypositive cases of chronic HCV infection are generally middle aged men, whereas the true AH type 2 cases typically occur in young girls.
5. CAN OTHER VIRUSES TRIGGER, OR BE ASSOCIATED WITH, AUTOIMMUNE HEPATITIS?
Two case reports have linked hepatitis B virus infection to AH type 1: the first case followed acute HBV infection in a young woman who had eliminated the virus [43], the second developed in a chronic HBV cartier concurrently with the emergence of mutant, HBeAg-negative virus and was directly related to viral multiplication, as the disease went in remission following inhibition of HBV replication [44]. TT virus, a DNA virus first isolated in Japan from serum
of a patient with post-transfusion non-A-G hepatitis, does not appear to be related to AH in German or Japanese [45] patients. Contrasting reports refer to GB-virus/hepatitis G virus, a flavivirus unable to act alone as a cause of liver damage; while an Austrian study found a significantly increased prevalence of ongoing infection in patients with all types of AH [46], German [47] and Japanese authors [45] failed to find the same. SEN virus, a DNA virus related to the TTV family, has also been investigated and ruled out as a possible cause of AH [48]. Finally, retroviruses have been proposed as potential triggers of autoimmune diseases, including type 1 diabetes, Sj6gren's syndrome and primary biliary cirrhosis. Their role has not been investigated in AH, but it is not likely, as it is still unclear whether endogenous retroviruses can play a pathogenic role in autoimmune diseases in humans and they are more likely to be activated by the chronic inflammation associated with these diseases, the production of retroviral particles being in this latter case a mere epiphenomenon.
6. CONCLUSIONS Despite decades of complex studies employing sophisticated techniques, the obscure origins of autoimmunity and the even more obscure factors leading to overt autoimmune diseases are far from being uncovered, and genetic background, hormones and environmental agents are persistently listed as contributing factors. Viruses have been proposed as triggers for several autoimmune diseases, the most cited examples being B3 Coxsackie virus for myocarditis and B4 Coxsackie virus, cytomegalovirus and rubella for type 1 diabetes mellitus. Autoimmune hepatitis is no exception, but a role for a few viruses has been convincingly shown only in rare cases of AH type 1, and is unconvincing in AH type 2, the very form where one of the most held views in modem immunology, i.e. molecular mimicry, has been repeatedly invoked in the latest 14 years. Viruses can not act alone: even the association with the putative defect in ASGPR-specific suppressor/ inducer T cells does not necessarily induce AH, as one of the patients (an 18-year-old man) of the Italian cohort who was affected by AH following HAV infection had previously acquired EBV infection
without developing the autoimmune liver disease. It can therefore be argued that, even in patients with a genetic predisposition, a trigger must intervene at the "fight" time for the disease to develop; perhaps this "fight" time has to do with sexual hormones fluctuations or with concurrent drug administration, but again no clues are available in this respect. Immunologists need to go back to the innovative, provocative, simple and challenging experiments which used to nourish the specialty long ago and led to unexpected results, and which have now been replaced by extremely complicated laboratory investigation aiming almost exclusively at confirming trendy theories; provocative results such as those reported by Burns and coworkers [49] are the kind of nowadays rare, unexpected data which can really advance our understanding. In our opinion, the most important lesson to be learned form the studies that we described is that careful observation of single cases at the onset of viral infections and prospective follow-up of cohort of individuals at possible risk for autoimmune diseases, although painstaking, are the most reasonable ways of proceeding if a role for viruses in human autoimmune diseases (including autoimmune hepatitis) has to be uncovered. Laboratory investigation conducted in patients with established disease or only aiming (as usual) at confirming trendy theories (however attractive these may appear) will never give chances of significant breakthroughs.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Viral Infections and Type 1 Diabetes Hee-Sook Jun and Ji-Won Yoon
Center for Immunologic Research and Department of Microbiology and Immunology, The Chicago Medical School North Chicago, IL, USA
1. INTRODUCTION Type 1 (insulin-dependent) diabetes mellitus results from the destruction of insulin-producing [~ cells in the pancreatic islets [1-8]. Although strong genetic predisposition is associated with the development of type 1 diabetes [7, 9-12], there is considerable evidence that environmental factors play an important role in the etiology of type 1 diabetes [13-16]. The concordance rate for type 1 diabetes in identical twins is only 25-60% [17-19]. Epidemiological studies also indicate a role for environmental factors in the development of type 1 diabetes. Variations in the incidence of type 1 diabetes have been observed among populations with similar genetic backgrounds but from different geographical areas, and between migrant populations and their indigenous population [20]. Environmental factors that are suspected to be involved in the initiation and/or progression of [3 cell destruction leading to type 1 diabetes include dietary composition, [3 cell toxins, and viruses. Viruses have long been suspected to contribute to the development of human type 1 diabetes, largely by temporal and geographic association between the disease and viral infection, serological evidence of infection in patients recently diagnosed with type 1 diabetes, and isolation of viruses from the pancreas of diabetic patients in a few cases [21]. As well, some viruses have been reported to be associated with the development of type 1 diabetes in animals. Viruses can induce type 1 diabetes either by direct infection and cytolytic killing of [~ cells or by triggering [3 cell autoimmunity with or without direct infection of the [~ cells.
In this review, we will discuss viruses that are considered to be associated with the pathogenesis of type 1 diabetes in humans and animals (Table 1) and the possible mechanisms by which they induce this disease.
2. VIRUSES AND TYPE 1 DIABETES IN HUMANS 2.1. Coxsackie B Virus and Enterovirus
Considerable evidence indicates that Coxsackie B virus, especially the B4 serotype, is associated with the development of type 1 diabetes. Several epidemiological studies have shown high frequencies of anti-Coxsackie B viral antibodies in children newly diagnosed with type 1 diabetes as compared to non-diabetic subjects [22-31]. In addition, T cell responses against a Coxsackie B viral protein were observed in new-onset type 1 diabetic patients [32-34]. While these studies support the involvement of Coxsackie B virus in the development of human type 1 diabetes, other epidemiological studies have come to the opposite conclusion. Several studies found no evidence for a correlation between the onset of type 1 diabetes and Coxsackie B viral infections [35-38], and other studies found higher levels of anti-Coxsackie B virus-specific antibodies in non-diabetic control subjects than in recent-onset type 1 diabetic patients [39, 40]. The controversy may arise from the nature of the virus and genetically determined host factors. There are different variants of virus within each serotype. For example, Prabhakar et al [41] isolated thirteen variants of
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Table 1. Viruses associated with the development of type 1 diabetes Virus RNA VIRUSES: Picornaviridae Coxsackie B virus Encephalomyocarditis virus Mengovirus Foot-and-mouth disease virus Ljungan virus Retroviridae Retrovirus Togaviridae Rubella virus
Bovine viral diarrhoea-mucosal disease virus ParamyxovilTdae Mumps virus Reoviridae Rotavirus Reovirus DNA VIRUSES: Parvoviridae Kilham rat virus Herpesviridae Cytomegalovirus Epstein-Barr virus
Coxsackie B4 virus. In another study, four variants of Coxsackie B4 virus were tested and only one was found to be diabetogenic, while the remaining three were not [42]. This is an indication of the possible rarity of diabetogenic variants of Coxsackie B4 virus. Also, it is difficult to distinguish between diabetogenic and non-diabetogenic variants using routine neutralizing antibody or ELISA assays, since antibodies against one variant cross-react with other variants. Therefore, if a person is exposed to a more common variant of Coxsackie B4 virus before exposure to a more rare diabetogenic variant of the same serotype, that person will have already developed antibodies against the non-diabetogenic variant, which will neutralize the diabetogenic variant during a subsequent infection; thus, the person will
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Host
Involvement of genetic factors
Humans Mice Non-human primates Mice Hamster Mice Pigs, cattle Bank voles
Not determined Yes Yes Yes Yes Yes Not determined Not determined
Humans
Mice
Not determined Yes
Humans Hamsters Rabbits Cattle
Not determined Not determined Not determined Not determined
Humans
Yes
Humans Mice
Not determined Yes
Rats
Yes
Humans Degu Humans
Not determined Not determined Not determined
not become diabetic, even if he or she is genetically predisposed to the disease. If this person is a subject in an epidemiological study, then the results will be misleading, as the lack of diabetes will not be a result of lack of exposure to a diabetogenic Coxsackie B virus, and no correlation between Coxsackie B viral infection and incidence of diabetes will be found. In contrast, outbreaks of diabetogenic virus in certain areas before outbreaks of non-diabetogenic virus will result in a high correlation between Coxsackie B viral infection and the development of diabetes. In addition, there are genetically determined differences in susceptibility to virus-induced diabetes, as has been shown in experiments using different strains of mice infected with Coxsackie B4 virus [43]. It is thought that humans, as well, will not
become diabetic when infected with diabetogenic Coxsackie B4 virus unless they are genetically predisposed to developing the disease. Thus, the correlation between Coxsackie B viral infection and the development of diabetes seen in some epidemiological studies but not in others may depend on the genetic makeup of the virus and the genetic background of the patients. In addition to these epidemiological studies, there are many anecdotal reports describing the development of type 1 diabetes in patients with recent or concurrent Coxsackie B viral infections [42, 44-52]. Direct evidence supporting the involvement of Coxsackie B viral infection in the development of type 1 diabetes comes from studies in which Coxsackie B4 and B5 viruses have been isolated from the pancreata of patients with acute-onset type 1 diabetes and the isolates have been shown to induce diabetes in susceptible strains of mice [53, 54]. A patient who died of diabetes after Coxsackie viral infection showed lymphocytic infiltration of the islets and ~ cell necrosis at autopsy (Fig. 1) and the isolated virus was able to induce diabetes in SJL mice but not in C57BL6, Balb/c or CBA mice [53]. Additional evidence includes a study in which Coxsackie B virus-specific antigens were detected in islets showing marked 13cell damage in children who developed diabetes following severe infections by these viruses and died [55]. As well, in vitro studies have shown that Coxsackie B viral infection can impair human islet cell metabolism. Infection of human 13cells with Coxsackie B3 and B4 viruses decreased insulin content beginning at 24 h after infection and the decrease in insulin roughly paralleled the increase in viral titer [53, 56, 57]. Although the exact mechanisms for Coxsackie virus-induced type 1 diabetes are not known, direct destruction of 13cells by cytolytic infection, molecular mimicry, and bystander activation of pre-existing autoreactive T cells have been suggested. Coxsackie B virus may directly infect pancreatic ~ cells and destroy them by cytolysis, as has been found in mice [43, 58]. Other studies have suggested that molecular mimicry could underlie autoimmune responses that result in [3 cell damage after Coxsackie viral infection. P-2C, a non-capsid protein of Coxsackie B4 virus, has sequence homology with glutamic acid decarboxylase (GAD), a putative autoantigen expressed by [~ cells [59]. Moreover, infection with
the virus increases expression of GAD by [~ cells [60]. Antibodies that react with both P2-C and GAD have been detected in type 1 diabetic patients [61]. However, this hypothesis is not supported by studies of antibodies produced by lymphocytes isolated from a newly diagnosed type 1 diabetic patient. Four of six antibodies studied recognized and bound to the region of GAD65 that is homologous to P2C, but none cross-reacted with P2-C itself or with any other Coxsackie B4 viral proteins. The lack of cross-reactivity between these two proteins may be due to differences in secondary or tertiary structure [62]. On the other hand, the capacity of murine T lymphocytes to cross-react with P2-C and GAD is associated with a diabetes susceptibility allele; cross-reactive T-cell recognition of GAD65 could therefore contribute to the initiation or amplification of autoimmune responses against the ~l cell, and perhaps contribute to the association of type 1 diabetes with certain human leukocyte antigen (HLA) alleles [63]. Another hypothesis is that Coxsackie B virus induces diabetes via "bystander" activation of autoreactive T cells against islet antigens. In mice with diabetes-susceptible major histocompatibility complex (MHC) alleles, these viruses did not accelerate the development of diabetes, whereas transgenic mice carrying a T cell receptor specific for an islet autoantigen rapidly became diabetic. This suggests that Coxsackie B virus induces diabetes by direct local infection, leading to inflammation, tissue damage, and the release of sequestered islet antigens that results in the re-stimulation of resting autoreactive T cells [64]. A further possibility is that a defective Coxsackie B virus, lacking the usual high lytic activity, could cause persistent infection of [3 cells, resulting in bystander activation of autoreactive T cells [65]. This hypothesis would be consistent with evidence of continuing Coxsackie B viral infection in other diseases. Interferon (IFN)-t~ production associated with hyperexpression of HLA type I antigens, was identified in the 13cells of three out of four children who died from Coxsackie B viral pancreatitis [66]. In addition, it was reported that plasma IFN-ct levels were elevated in some type 1 diabetic patients, and that this was associated with Coxsackie B viral infection [67]. Persistent Coxsackie B viral infection of the 13 cells might stimulate them to synthesis and release IFN-ct,
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Figure 1. Pancreatic sections from a non-diabetic subject and a diabetic, Coxsackie B4 virus-infected patient. (A) Section of a pancreas from a non-diabetic subject, showing a single intact islet of Langerhans surrounded by acinar cells (x 160). (B-F) Sections from different locations of a pancreas from a Coxsackie B4 virus-infected 10 year-old boy who died after acute onset of type 1 diabetes. (B) Islet with moderate insulitis (x 230). (C) Atrophied islet with severe insulitis (x 160). (D) Lymphocytic infiltration in the periphery of the islet. (E) Islet with extensive inflammatory infiltrate, loss of islet architecture and severe islet destruction. (F) Islet with severe necrosis of the 13cells and only a few lymphocytes remaining in the islet. which in turn could induce hyperexpression of HLA class I antigens and the production of chemokines that recruit and activate macrophages and autoreactive T cells. These activated immune cells could then kill the 13 cells, resulting in type 1 diabetes (Fig. 2). Finally, Coxsackie viral infections may be
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involved in the pathogenesis of type 1 diabetes by acting as the terminal insult in individuals who have already lost substantial [3-cell mass through ongoing autoimmune damage. Destruction of a critical number of residual cells would result in the abrupt onset of type 1 diabetes.
Figure 2. Hypothetical scheme of a possible mechanism of Coxsackie B4 virus-induced diabetes by persistent infection. Coxsackie B4 virus may persistently infect pancreatic 13cells and induce the expression of IFN-~. In turn, IFN-~ may induce the expression of chemokines and MHC-I on the 13cells. These chemokines may recruit macrophages and T cells to the pancreatic islets, which then are activated and may kill 13cells in conjunction with the hyperexpressed MHC class I molecules, resulting in the development of type 1 diabetes.
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There is considerable information that type 1 diabetes may be associated with infection with other enteroviruses in addition to Coxsackie B virus. Epidemiological studies show a strong association between the development of type 1 diabetes and enteroviral infection [25, 68-70], and antibodies against enterovirus [26-31, 71 ] or T cell responses to enterovirus [32-34, 72] have been detected in new-onset diabetic patients. In addition, enteroviral RNA was detected with high frequency in the serum or lymphocytes of type 1 diabetes patients [44, 45, 73-75]. Some case studies have reported that type 1 diabetes developed after enteroviral infection [76-78]; however the role of enteroviruses in type 1 diabetes is still poorly understood [79]. There is also some indirect evidence that Coxsackie A virus may also be associated with type 1 diabetes [80, 81 ].
2.2. Mumps Virus Mumps virus was one of the first viruses reported to be associated with human type 1 diabetes. Several cases were reported in which mumps infection appeared to precede the onset of type 1 diabetes [82, 83]. Mumps-related type 1 diabetes may have an autoimmune basis. Some children with mumps parotitis develop islet-cell antibodies [84], and there is evidence that the virus may induce an autoimmune response against ~ cells, or might intensify a pre-existing autoimmune attack. Mumps infection of a human l-cell line (insulinoma) in vitro induced the release of interleukin (IL)-I and IL-6 and also up-regulated the expression of HLA class I and II antigens [85, 86]. The production of interleukins and increased or aberrant expression of HLA antigens by 13 cells may be crucial steps in provoking autoimmune ~ cell damage. However, studies on the impact of mumps vaccination on the development of type 1 diabetes reported that there is no association with childhood mumps vaccinations and the development of islet autoimmunity [87] or type 1 diabetes [88], suggesting that mumps virus may not be a good candidate virus for the induction of diabetes. In contrast, one study reported that the elimination of mumps infections by vaccination may have been responsible for the decreased risk of developing type 1 diabetes over the time period studied [89], suggesting that infection with mumps virus may be associated with the development of type 1 diabetes.
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Further studies are required to determine whether mumps virus is definitely involved in the development of type 1 diabetes.
2.3. Rubella Virus Rubella virus has been implicated in type 1 diabetes, because approximately 12-20% of patients with congenital rubella syndrome (CRS) develop diabetes by 5-20 years of age [90-98]. Islet cell and anti-insulin antibodies were found in 50-80% of diabetic patients with CRS, whereas these antibodies were present in only 20% of nondiabetic CRS patients, suggesting an underlying autoimmune disorder. There was also an increased frequency of HLA-DR3 in patients with CRS, suggesting some genetic susceptibility might be involved in the development of type 1 diabetes in CRS patients [99]. While rubella virus appears to be involved in the development of type 1 diabetes in patients with CRS, more research is required to discover if infection by rubella virus after birth plays any role in the induction of type 1 diabetes. In vitro studies have shown that human islets are susceptible to rubella infection. Human fetal islets exposed to rubella virus showed rubella viral antigens in both 13and non-[~ cells and had lowered levels of insulin production [ 100], although without any observable cytopathology [ 101 ]. Several mechanisms have been suggested by which rubella viral infection may induce type 1 diabetes. Rubella viral infection may alter antigens in the plasma membrane of infected 13 cells that may be perceived as foreign by the host's immune system, resulting in the induction of ~ cell-specific autoimmunity. Altematively, rubella viral infection may induce autoimmune type 1 diabetes by molecular mimicry. It was found that a monoclonal antibody directed against a rubella capsid protein crossreacted with extracts from rat and human islets and rat insulinoma cells, and the shared epitope was shown to be part of a unidentified 52 kD [3 cell protein [ 102]. It has also been shown that T cells from type 1 diabetic patients cross-react with epitopes in rubella viral proteins and the ~ cell isoform of GAD [ 103]. These results suggest that rubella viral infection may lead to the generation of viral antigenspecific cytotoxic T cells that also recognize 13 cell antigens in susceptible individuals.
2.4. Cytomegalovirus (CMV) Case reports have described type 1 diabetes developing in CMV-infected individuals, including a child with congenital CMV infection [104] and an adult with severe CMV infection that caused acute pancreatitis and rhabdomyolysis [ 105]. In addition, CMV infection can cause [~ cell damage under certain circumstances and characteristic inclusion bodies have been found in 13 cells in children who died with disseminated CMV [ 106]. A study showed that 20% of type 1 diabetic patients had CMV genomic DNA in their lymphocytes, compared to only 2% of normal controls. Furthermore, 80% of patients who had both antiCMV antibodies and the CMV genome in their lymphocytes also had islet cell autoantibodies [107]. Another study found that non-diabetic siblings of type 1 diabetes patients had a significant association between high titres of anti-CMV antibodies and islet cell autoantibodies, but no correlation between anti-CMV antibodies and HLA-DR antigens [ 108]. Although these results are largely circumstantial, they suggest that chronic CMV infection may be associated with islet cell autoantibody production, but that other factors may be needed for the development of clinical type 1 diabetes. Evidence for molecular mimicry is the finding that human CMV can induce an islet cell antibody that reacts with a 38 kD autoantigen expressed in human pancreatic islets [ 109]. Also, a study showed that a CD4 § T cell clone reactive to GAD65 isolated from a prediabetic Stiffman syndrome patient cross-reacted with a peptide of human CMV major DNA binding protein, suggesting that human CMV may be involved in the induction of autoimmunity by molecular mimicry of the 13 cell autoantigen, GAD65 [110]. However, further investigation is required to determine whether CMV is truly involved in the development of type 1 diabetes.
2.5. Retrovirus Many studies have suggested that retroviruses may be implicated in autoimmune disease, including type 1 diabetes [ 111, 112]. Retroviral-like particles were found in patients with multiple sclerosis and Sj6gren's syndrome [ 113, 114]. Nucleotide sequence homology was found between human retrovirus and
self-antigens, in particular between ribonucleoproteins and the p30 C-type retroviral gag gene product [115-117]. Anti-insulin autoantibodies from type 1 diabetic patients and their non-diabetic, first-degree relatives have been found to cross-react with retroviral p73 antigen, suggesting that endogenous retroviruses may be involved in the pathogenesis of type 1 diabetes [ 118]. A novel human endogenous retroviral gene, designated IDDMK1.222, was reported to be expressed in the plasma of recent-onset type 1 diabetes patients but not in non-diabetic control subjects [ 119]. This virus was thought to belong to the mouse mammary tumor virus-related family of human endogenous retroviruses (HERV)-K. However, careful studies have shown that the IDDMK1.222 sequence was not present in either the plasma or peripheral lymphocytes from either diabetic or control subjects [120-122]. Instead, a related human endogenous retrovirus with 90-93% sequence homology with IDDMK1.222 was present with equal frequency in both diabetic and nondiabetic subjects [120]. These human endogenous retroviruses are therefore unlikely to play a role in the development of autoimmune type 1 diabetes in humans [120-122]. Even though it appears that the endogenous retroviral gene homologous with IDDMK1.222 is not associated with type 1 diabetes, it does not necessarily exclude the involvement of other human retroviruses or endogenous retrovims genes in the pathogenesis of autoimmune diabetes. An interesting report showed that the expression of the defective retroviral gene, the HERV-K18 provirus encoding superantigen, is induced by IFN-~ and subsequently stimulates V137 T cells [123], which was correlated with the onset of type 1 diabetes. Whether the HERV-K18 provirus is truly involved in the development of autoimmune diabetes remains to be determined.
2.6. Epstein-Barr Virus (EBV) EBV has been implicated in the etiology of several autoimmune diseases [124], and a few cases have been reported to be linked with the onset of type 1 diabetes [125]. It is suggested that EBV may be potentially capable of triggering autoimmune type 1 diabetes by molecular mimicry, since an eleven amino acid sequence of the EBV protein, BOLF1, was found to be homologous with residues in the
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Asp-57 region of the HLA-DQW8 13 chain peptide [ 126]. It was also found that a pentapeptide sequence in the Asp-57 region of the HLA-DQI] chain is successively repeated six times in the EBV-BERF4 epitope [127, 128]. Two patients who produced antibodies against this epitope during acute EBV infection developed type 1 diabetes soon thereafter, while five individuals also acutely infected but not producing antibodies against this epitope did not develop type 1 diabetes [ 128]. Further investigation is needed to find the relationship between EBV and type 1 diabetes. 2.7. Other Viruses There is circumstantial evidence that hepatitis A virus [129], varicella zoster virus [130], measles virus [ 130], polio virus [ 130], influenza virus [ 131 ], and rotavirus [132] may be associated with the development of type 1 diabetes in humans. However, whether these viruses are truly associated with the development of type 1 diabetes remains to be determined.
3. VIRUSES AND TYPE 1 DIABETES IN ANIMALS 3.1. Encephalomyocarditis (EMC) Virus EMC virus selectively infects pancreatic 13 cells [133] and induces diabetes in genetically predisposed mice by the destruction of pancreatic [3 cells [134, 135]. This virus has been the most thoroughly studied of the diabetogenic viruses in animals [ 136]. The EMC virus has two antigenically indistinguishable variants: EMC-D virus causes diabetes by direct cytolytic destruction of 13 cells in over 90% of the animals it infects, whereas EMC-B virus is completely non-diabetogenic [137]. However, diabetes only develops after EMC-D viral infection in some strains of mice such as SJL/J, SWR/J, DBA/1J, and DBAJ2J, whereas C57BL/6J, CBA/J, and AKR/J strains are resistant. Susceptibility to EMC-D virus-induced diabetes is determined by a single autosomal recessive gene [138], which may modulate the expression of viral receptors on [3 cells [135, 139-141]. Examination of the complete nucleotide
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sequence of the genomes of the EMC-D and EMCB variants showed that they were different in only 14 nucleotide positions [142, 143]. Further studies using several mutant viruses showed that only one amino acid, alanine at the 152nd amino acid residue of the major capsid protein VP1, is critical for diabetogenicity of the EMC virus [ 144, 145]. Threedimensional molecular modeling of the VP 1 protein showed that the van der Waals interactions are greater and the residues surrounding position 152 are more closely packed in recombinant chimeric viruses containing Thr, Ser, Pro, Asp, or Val in this position than in recombinant chimeric viruses containing Ala in the same position. The surface area surrounding Ala at position 152 of VP1 is more accessible, thus increasing the availability of the binding sites for attachment to 13 cell receptors and resulting in viral infection and the development of diabetes [ 146]. Two different animals models have been established with respect to pathogenic mechanisms for EMC virus-induced diabetes. The first model involves animals infected with a high dose (105 plaque-forming units [PFU]/mouse) of EMC virus, in which replication of the virus within the 13 cells plays a major role and recruitment of macrophages plays a minor role in 1~cell destruction. In contrast, the second model involves animals infected with a low dose (< 102 PFU/mouse) of EMC virus, in which activated macrophages that are recruited to the [3 cells play a major role and replication of virus within the [3 cells plays a minor role in [3 cell destruction [136]. In low-dose EMC virus-infected mice, it was found that macrophage-derived soluble mediators play a critical role in the destruction of pancreatic I] cells. Further study revealed that EMCD virus infects and activates macrophages, but does not replicate within them [147]. The expression of macrophage-derived soluble mediators such as IL-113, tumor necrosis factor (TNF)-c~, and inducible nitric oxide synthase (iNOS) was selectively detected in the pancreatic islets of mice infected with a low dose of EMC-D virus. In addition, treatment of EMC-D virus-infected mice with antibody against IL-1 [~ or TNF-c~ or with the iNOS inhibitor, aminoguanidine, exhibited a significant decrease in the incidence of diabetes [148]. These results suggest that macrophage-derived soluble mediators play a critical role in the destruction of pancreatic
[3 cells resulting in the development of diabetes in mice infected with a low dose of EMC-D virus. Studies have shown that tyrosine kinase signaling pathways are involved in the activation of macrophages by EMC-D virus infection. Extracellular signal-regulated kinases (ERK)I/2, p38 mitogenactivated protein kinase (MAPK), and c-Jun-terminal activation kinase (JNK) were activated in macrophages after EMC-D viral infection. Treatment of mice infected with a low dose EMC-D virus with a tyrosine kinase inhibitor, AG126, decreased the incidence of diabetes and suppressed the production of IL- 1[3, TNF-ot, and iNOS in the pancreata of these mice as compared with vehicle-treated control mice [147]. In addition, it was found that hematopoietic cell kinase (hck), a Src family kinase, is involved in the activation of macrophages by EMC-D viral infection and that treatment of EMC-D virusinfected mice with a Src kinase inhibitor prevented the development of diabetes [ 149].
3.2. Mengovirus Mengovirus induces fatal encephalitis in mice similar to EMC virus and is antigenically similar to EMC virus. Plaque purification of Mengovirus resulted in the isolated clone, Mengovirus-2T, that could cause diabetes in strains of mice resistant to EMC-D viral infection [150]. Marked ~ cell necrosis, severe inflammatory infiltration of the islets and decreased insulin content without evidence of autoimmune responses were observed in Mengovirus-2T-infected mice [ 150]. It appears that Mengovirus-2T causes diabetes by directly infecting [~ cells.
3.3. Coxsackie B4 Virus Coxsackie B virus causes diabetes in susceptible strains of mice, such as SJL/J and SWR/J mice. Coxsackie B4 virus isolated from a patient with acute-onset diabetes or patients with Coxsackie viral infection induced abnormal glucose tolerance and transient hyperglycemia in infected mice [151]. A diabetogenic Coxsackie B4 virus generated by repeated passaging of the virus in 1~ cell cultures, which enhanced its 13 cell tropism [43], resulted in lymphocytic infiltration of the islets and [3 cell destruction in infected mice. During the acute
phase of Coxsackie B4 viral infection, antigens from this virus were observed in the pancreatic islets [43]. Some clinical isolates of Coxsackie B4 virus produced 13cell damage in vivo in mice [152] or in vitro [153]. The E2 variant of Coxsackie B4 virus isolated from a child who died from systemic infection induced a diabetes-like syndrome in infected mice, such as abnormal glucose tolerance and transient hyperglycemia [ 154]. Hyperglycemia developed between 6-8 weeks after the viral infection and CD4 § T cells predominated in the pancreatic infiltrates of the infected mice [155]. However, the role of these CD4 + T cells in the development of diabetes in E2 variant-infected mice remains to be determined. Interestingly, it was reported that the expression of GAD65, which has a sequence homology with a non-structural protein (P2-C) of Coxsackie B4 virus, is increased in the islets at 72 h after E2 variant-infection [60], suggesting that molecular mimicry may play a role in the development of Coxsackie B4 virus-induced diabetes. Studies on the Coxsackie B4 viral genome have identified the amino acid residues responsible for the virulence of the virus [156]; however only preliminary work has been done on identifying the residues responsible for diabetogenicity. Sequence comparison data between the diabetogenic E2 variant of Coxsackie B4 virus and the prototype nondiabetogenic JVB strain of virus [157] revealed 111 amino acid differences [158]. Another group sequenced the genome of a ~ cell-tropic variant of Coxsackie B4 virus JVB and compared it with the sequence of the prototype strain and found only seven amino acids that were different [159]. The identification of the specific amino acids responsible for the diabetogenicity of Coxsackie B4 virus remain to be determined. In addition to causing diabetes in susceptible mouse strains, Coxsackie B4 virus can also cause a diabetes-like syndrome in certain species of monkeys. Monkey [3 cell passaged Coxsackie B4 virus was shown to impair insulin secretion and glucose tolerance in the Patas monkey, but had no apparent diabetogenic effects in other primates such as Cebus, Cynomotgus, and Rhesus monkeys [160], suggesting that genetic factors are involved in susceptibility to Coxsackie B4 virus-induced diabetes in monkeys. From the results of the above research on humans
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and animals, it is speculated that Coxsackie B virus, especially the B4 serotype, may play a role in the development of type 1 diabetes, either by initiating the development of the disease or by operating as the final insult to [3 cells in individuals where ongoing autoimmune [3 cell destruction has already been taking place. Whatever the mechanism, evidence from studies on mice, non-human primates, and humans indicates that Coxsackie B virus affects glucose homeostasis. Research on Coxsackie B4 virus has demonstrated that antigenic changes at the epitope level occur at a frequency greater than 1/100 [41, 161 ]. This suggests that even within the same virus pool, there may be many antigenic variants with different tissue tropisms and different physiological properties, which would account for the wide spectrum of clinical disease produced by the Coxsackie B virus. Only rare variants may be diabetogenic, explaining why type 1 diabetes appears to be associated with Coxsackie B viral infection in infrequent isolated cases [68].
3.4. Kilham Rat Virus (KRV) KRV induces diabetes by autoimmune responses against [3 cells rather than by direct [3 cell cytolysis in diabetes-resistant BioBreeding (DR-BB) rats [162, 163], which are derived from diabetes-prone BB (DP-BB) rats, but do not normally develop the disease. When DR-BB rats were infected with KRV at 3 weeks of age, about 30% developed autoimmune diabetes within 2-4 weeks after infection and a further 30% showed insulitis without diabetes [ 162]. The incidence of KRV-induced diabetes could be increased to 80--100% by injection of polyI:C for three consecutive days after KRV infection [ 162]. KRV infects lymphoid organs such as the spleen, thymus, and lymph nodes, but not [3 cells. The precise mechanisms by which KRV induces autoimmune type 1 diabetes without infection of ~ cells are not clearly understood; however it is known that macrophages play an important role. Inactivation of macrophages with liposomal dichoromethylene diphosphonate results in the near complete prevention of insulitis and diabetes in KRV-infected DR-BB rats [164]. In addition, it was found that splenocytes from macrophage-depleted, KRVinfected DR-BB rats treated with polyI:C did not transfer diabetes to young DP-BB recipient rats,
238
whereas splenocytes from macrophage-containing, KRV-infected DR-BB rats treated with polyI:C transferred diabetes to 80% of the recipients [ 164]. The expression of macrophage-derived cytokines such as IL-12, TNF-o~, and IL-I~ in pancreatic islets and splenic lymphocytes was increased after KRV infection, and the increased expression of Thl cytokines such as IL-2 and IFN-y was closely correlated with an elevation in IL-12, suggesting that macrophage-derived cytokines may play a critical role in the cascade of events leading to the destruction of pancreatic 13 cells in KRV-infected DR-BB rats [ 164]. Molecular mimicry has been proposed for KRVinduced diabetes. KRV infection could generate viral peptide-specific T lymphocytes, which might cross-react with epitopes on pancreatic ~ cells and attack them, resulting in the development of diabetes. However, experimental data showed that infection of DR-BB rats with recombinant vaccinia viruses expressing various KRV peptides (VP1, VP2, NS 1, or NS2) did not cause insulitis or diabetes, even though viral peptide-specific T cells and antibodies were generated [165]. This result suggests that molecular mimicry between KRV peptides and [~ cell-specific autoantigens is unlikely to be a mechanism by which KRV induces [3 cell-specific autoimmune type 1 diabetes in DR-BB rats. An alternative hypothesis is that KRV infection of DR-BB rats might disturb the immune balance and activate 1~ cell-specific autoreactive effector T cells [ 166], which are normally held silent by immunoregulatory control involving the RT6.1 subset of T cells [ 167, 168], thus resulting in the destruction of 13 cells. Several studies have focused on identifying the population that contains the autoreactive effector T cells involved in I] cell destruction. It was found that KRV infection resulted in an increase in the percentage of CD8 + T cells, whereas the percentage of CD4 + T cells decreased. In addition, CD8 § T cells preferentially proliferated as compared with CD4 + T cells, and treatment of with OX8 monoclonal antibody, which inactivates CD8 § T cells, significantly decreased the incidence of KRVinduced diabetes. These results indicate that CD8 + T cells may play an important role in KRV-induced autoimmune diabetes. Further studies showed that the number of Th2-1ike CD45RC-CD4 + T cells was significantly reduced and the number of Thl-like
Figure 3. Schematic model of KRV-induced diabetes in DR-BB rats. KRV infection of DR-BB rats activates macrophages, resulting in the production of proinflammatory cytokines such as IL-12, IL-113, TNF-tz, and IFN-~,. These cytokines activate 13cell-specific CD8§ T cells and differentiate CD4§ T cells into Thl-like CD4§ T cells. KRV can also replicate within amplified CD4§ and CD8§ T cell populations. Ultimately, the CD8§ T cell-rnediated cytotoxic response (CTL response) and Thl-type CD4§ T cell-mediated Thl response contribute to pancreatic l~cell death, leading to diabetes in KRV-infected DR-BB rats. CD45RC§ § T cells was significantly increased in the splenocytes of KRV-infected DR-BB rats as compared with controls [165]. Adoptive transfer of Thl-like CD45RC§ § T cells and CD8 § T cells from KRV-infected DR-BB rats resulted in the development of diabetes in 88% of the recipients, suggesting that these two cell populations are major effector T cells that can induce diabetes. As well, it was recently reported that KRV infection regulated the CD25§ § regulatory T cell population [169]. Taken together, it is suggested that KRV infection activates macrophages, which then produce inflammatory cytokines. These cytokines disturb the finely tuned immune balance, resulting in the upregulation of pre-existing [3 cell-specific autoreactive CD8 § T
cells and Thl-type CD4 § T cells. The toxicity of macrophage-derived cytokines and Thl-type CD4 § T cell-derived cytokines to 13 cells, together with the damage incurred by KRV [~ cell-specific CD8 § cytotoxic T cells, may lead to 13cell destruction and autoimmune type 1 diabetes in KRV-infected DRBB rats (Fig. 3).
3.5. Retrovirus Endogenous retroviruses have been implicated in the pathogenesis of type 1 diabetes in NOD mice [170-172], but the evidence is largely circumstantial. It was found that islet cells of NOD mice express various retroviral messenger RNAs
239
(mRNAs) encoded by the gag, pol and env genes, and 1~ cells in particular express the group-specific antigen p73 of the A-type retrovirus [ 173]. In addition, the presence of both A-type and C-type retroviral particles was found in pancreatic 13 cells of NOD mice [ 171, 172, 174] and was correlated with the development of autoimmune type 1 diabetes in these animals. It is not certain how retroviruses might be involved in the pathogenesis of autoimmune type 1 diabetes in NOD mice. It is possible that a retroviral antigen expressed on the 13 cells might be recognized as foreign and presented by antigen-presenting cells, such as macrophages and dendritic cells, resulting in the development of effector T cells that can destroy the 13 cells. Another related mechanism might be the alteration of the expression of cellular genes by the retroviral genomes in the [~ cells, resulting in a ~ cell-specific altered antigen(s). An altered antigen might be recognized as foreign by immunocytes, leading to I] cell-specific autoimmunity. In addition, it is possible that cellular proteins from 13 cells taken up in the retroviral envelope may elicit an autoimmune response or that IFN-y induced as a result of retroviral infection may subsequently induce the expression of HLA-II and trigger autoimmunity through CD4 § T lymphocytes.
3.6. Reovirus Reovirus has been associated with type 1 diabetes in animals, but its mode of action is not known. Direct infection of the ~ cells has been suggested by studies in which mice infected with [~ cell-passaged reovirus type 3 showed specific viral antigens and viral particles in some ~l cells. These mice had abnormal glucose tolerance tests within 10 days after infection, but glucose tolerance returned to normal after 3 weeks [175]. Alterations in the immune system, such as induction of autoimmune mechanisms or a shift to a Th2 response, have been suggested as a mechanisms by which reoviruses might induce type 1 diabetes. It was found that mice infected with [I cell-passaged reovirus type 1 developed transient diabetes, and their sera contained autoantibodies that reacted with cytoplasmic antigens from the islets of Langerhans, the anterior pituitary, and the gastric mucosa of uninfected mice [176]. As well, administration of immunosuppressive drugs to reovirus-
240
infected SJL and NFS mice reduced or prevented the development of reovirus-induced diabetes and mortality [177], suggesting the involvement of an autoimmune response. Recent studies suggest that a Thl response induced by the increased expression of IL-12 may be responsible for the development of diabetes in newborn DBA/1 mice infected with reovirus [ 178].
3.7. Ljungan Virus It was recently found that 33% of wild bank voles (Clethrionomys glareolus), which were trapped and kept in the laboratory for one month, developed diabetes due to [3 cell lysis [179]. These diabetic animals had increased levels of GAD65, IA-2, and insulin autoantibodies. The islets of these mice stained positively for Ljungan virus, a novel picornavirus found in bank voles. When non-diabetic wild bank voles were infected with Ljungan virus in the laboratory, 15 cell lysis was induced. These results show that the development of type 1 diabetes in bank voles is associated with Ljungan virus infection.
3.8. Rubella Virus Rubella virus has been shown to induce type 1 diabetes in hamsters, apparently by direct infection of the [3 cells. Neonatal golden Syrian hamsters infected with [3 cell-passaged rubella virus developed hyperglycemia and hypoinsulinemia between 7 and 10 days of age, and their 13cells were positive for rubella virus antigen. An autoimmune process may be involved, as 40% of infected animals had cytoplasmic islet cell antibodies and 34.5% had insulitis [ 180].
3.9. Bovine Viral Diarrhoea-Mucosal Disease (BVD-MD) Virus B VD-MD virus has been reported to be associated with type 1 diabetes in cattle, however not all animals with B VD-MD viral infection develop diabetes [181]. This may be attributable to the existence of different variants of the virus or to genetic differences among the hosts. It appears that the diabetogenic effect of B VD-MD virus is not a direct effect of the virus on [I cells. Infected cattle with
Table 2. Viruses associated with the prevention of type 1 diabetes Virus
Animal model
Possible mechanism
Lymphocytic choriomeningitis virus
NOD mice BB rats
Depletion of CD4§T cell subpopulation
Mouse hepatitis virus
NOD mice
Induction of the Th2 immune response
Coxsackie B virus
NOD mice
Induction of immunoregulatory response
Encephalomyocarditis virus
NOD mice
Induction of immunoregulatory response
type 1 diabetes showed the presence of B VD-MD viral genes in the pancreas, but not in the islet cells; many of these cattle also had islet cell autoantibodies [182]. More research is needed to determine if B VD-MD virus truly induces autoimmune responses that result in type 1 diabetes in genetically susceptible animals.
4. PREVENTION OF TYPE 1 DIABETES BY VIRUSES In addition to the diabetogenic viruses described above, some viruses can prevent the development of type 1 diabetes under some circumstances (Table 2). Lymphocytic choriomeningitis virus (LCMV) [ 183, 184] and mouse hepatitis virus (MHV) [185] protected against the development of autoimmune type 1 diabetes in spontaneously diabetic DP-BB rats and nonobese diabetic (NOD) mice. Recently, it was reported that infection of NOD mice with Coxsackie B virus significantly reduced the incidence of diabetes as compared with that in mock-infected control mice [186]. Interestingly, EMC-D virus, which is diabetogenic in some strains of mice, could prevent autoimmune diabetes in NOD mice [ 187]. Possible mechanisms have been proposed for the preventive effects of these viruses: 1) viral infection may affect the immune system, resulting in the induction of immunoregulatory cells or the Th2 immune response, or 2) viral infection may deplete a CD4 § T cell subpopulation, which could interfere with the general immune response [ 188].
5. CONCLUSIONS Viruses have been considered to be an important environmental factor in the etiology of type 1 diabetes. Coxsackie virus/enterovirus, mumps virus, CMV, rubella virus, retrovirus, EBV, hepatitis A virus, varicella zoster virus, measles virus, polio virus, influenza virus, and rotavirus have been implicated as potential causal agents for human type 1 diabetes. In addition, EMC virus, KRV, Coxsackie virus, retrovirus, rubella virus, BVD-MD virus, Mengovirus, foot and mouth disease virus, and CMV are known to be associated with the development of type 1 diabetes in animals. However, the precise etiology for the involvement of viruses and their pathogenic mechanisms, particularly in human type 1 diabetes, are poorly understood. Viruses may cause type 1 diabetes by directly infecting and destroying pancreatic [3 cells. For example, susceptible strains of mice infected with a high dose of EMC-D virus develop diabetes within 3 days of infection as a result of pancreatic l] cell destruction mainly caused by replication of the virus within the [3 cells. In contrast to direct cytolytic infection of [3 cells, many viruses appear to cause type 1 diabetes by contributing to [3 cell-specific autoimmunity by a variety of mechanisms, with or without [3 cell infection. Infection of susceptible strains of mice with a low dose EMC-D virus results in the development of diabetes mainly as a result of the activation of macrophages and subsequent induction of [3 celltoxic, macrophage-derived soluble mediators. Other viruses such as retroviruses may infect [3 cells and change existing antigens into immunogenic forms or may induce new antigens, which are recognized as foreign by the immune system and lead to ~ cell-
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specific autoimmunity. Viruses such as Coxsackie virus may cause diabetes by bystander activation of pre-existing 1~cell-specific immunocytes. In this case, it is thought that Coxsackie viral infection of 1~ cells may induce the expression of I F N - a and subsequently chemokines and cytokines, which recruits cytotoxic immunocytes to the ~ cells and results in their destruction. Viruses such as KRV may also activate pre-existing 13 cell-specific autoreactive immunocytes in genetically susceptible animals, but unlike Coxsackie virus, direct infection of the [3 cells does not occur. Instead, it is thought that KRV activates Thl-like immunocytes that disrupt the immune balance and result in the activation of I] cell-specific immunocytes that are usually held silent. Viruses such as rubella virus and CMV may exert their diabetogenic effects by molecular mimicry; effector T cells generated against viral epitopes may cross-react with homologous epitopes on pancreatic 13 cells. Lastly, several viruses such as retroviruses and mumps virus may act by inducing the expression of IFN-y in infected [3 cells, which may upregulate MHC class I and II molecules, leading to the initiation of [~ cell-specific autoimmunity. Viruses not only cause diabetes, but may also prevent the disease in autoimmune diabetes-prone animals. Infection of young NOD mice or DP-BB rats with LCMV and NOD mice with MHV, Coxsackie virus, or EMC virus prevents diabetes, probably by affecting the immune system, such as by the induction of Th2 immune responses or deletion of effector cells (i.e. Thl CD4 § T cells, CD8 § T cells, or macrophages). The identification of causative viruses in human type 1 diabetes is extremely difficult. The acute phase of viral infection may be already passed by the time that diabetes symptoms are shown. In addition, it is difficult to distinguish between diabetogenic and non-diabetogenic variants of the same virus by serological tests. A large prospective cohort study in prediabetic or genetically susceptible individuals as well as newly diabetic patients may help to understand the viral etiology of type 1 diabetes in humans. The identification of causative viruses will help to develop a preventive strategy for human type 1 diabetes.
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L. Persistent MHV (mouse hepatitis virus) infection reduces the incidence of diabetes mellitus in non-obese diabetic mice. Diabetologia 1991 ;34:2-5. 186. Tracy S, Drescher KM, Chapman NM et al. Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence. J Viro12002;76:12097-12111. 187. Hermitte L, Vialettes B, Naquet P, Atlan C, Payan MJ, Vague P. Paradoxical lessening of autoimmune processes in non-obese diabetic mice after infection with the diabetogenic variant of encephalomyocarditis virus. Eur J Immunol 1990;20:1297-1303. 188. Oldstone MBA. Viruses as therapeutic agents: I. Treatment of nonobese insulin-dependent diabetes mice with virus prevents insulin-dependent diabetes mellitus while maintaining general immune competence. J Exp Med 1990; 101:2077-2089.
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9 2004 Elsevier B. V All rights reserved. Infection and Aumimmunity Y. Shoenfeld and N.R. Rose, editors
Theiler's Murine Encephalomyelitis Virus-Induced Demyelinating Disease (TMEu and Autoimmunity Stephen D. Miller ~and Carol L. VanderLugt-Castaneda 2
1Department of Microbiology-Immunology and Interdepartmental Immunobiology Center, Feinberg School of Medicine, Northwestern University, Chicago, IL USA; 2Department of Biology, Indiana University Northwest, Gary, IN, USA 1. TMEV-IDD AND MS
Theiler's murine encephalomyelitis virus (TMEV) belongs to the Picornaviridae family and is a natural enteric pathogen of mice [1]. Neurovirulence upon experimental intracerebral injection varies depending on the strain of TMEV ranging from a rapidly fatal encephalitis in which grey matter neurons are infected and lysed upon infection with the GDVII strain to an initial acute phase of grey matter involvement followed by a chronic phase of viral persistence, inflammation and demyelination in the white matter of the spinal cord following infection with the BeAn or DA strains [2, 3]. The extent of acute phase grey matter involvement and mechanisms of chronic demyelination differ in BeAn and DA infection. The acute phase in DA infection of SJL mice is characterized by microglial proliferation and necrosis of neuronal motor neurons which results in flaccid paralysis (i.e., poliomyelitis) [4]. Surviving mice develop TMEV-induced demyelinating disease (TMEV-IDD). In contrast, BeAn infection results in a very limited early acute phase grey matter disease, with no clinical signs. Chronic demyelinating disease in these mice appears later (d30) and is due to the immune response itself, not to direct lysis of virally infected oligodendrocytes
[5, 6]. Intracerebral injection of the BeAn strain of TMEV into SJL mice results in a chronic demyelinating disease, TMEV-IDD, which resembles multiple sclerosis (MS) in many ways. In addition to the epidemiological data suggesting an infectious etiology of MS, histopathology, consisting of inflamma-
tory infiltrate and areas of demyelination, and clinical disease bear many similarities to MS. Although no one virus has been shown to be consistently associated with MS, early infection may trigger events, through molecular mimicry [7] or epitope spreading [8], that eventually result in autoimmune disease. Like TMEV-IDD, MS is a immune-mediated demyelinating disease characterized by perivascular CD4 § T cell and mononuclear cell infiltration, with subsequent primary demyelination of axonal tracts, leading to progressive paralysis [9]. MS is generally considered to have an autoimmune component, however a direct cause-effect relationship between myelin reactivity and disease has not been established. Interestingly, although TMEV-IDD is due initially to a persistent viral infection of the central nervous system (CNS), autoimmune anti-myelin Thl responses are seen during the chronic phase of disease [8, 10, 11].
2. PERSISTENT INFECTION AND CHRONIC DISEASE Demyelination and the resulting clinical disease symptoms are not the consequence of viral lysis of oligodendrocytes which construct the myelin sheath [12], but are immune mediated, due to a CD4 § T cell inflammatory response in the CNS [5, 13]. Initial observations using immunohistochemistry demonstrated that viral antigens could be found in abundance in the spinal cord of TMEV (BeAn) infected mice in macrophage/microglial cells and in astrocytes but not in oligodendrocytes [12, 14]. In
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contrast to the abundance of viral antigen, infections virus in the CNS of infected mice has been consistently shown to be low. Historically, macrophage/ microglia have been thought to be the major reservoir of infectious virus. However, it had also been demonstrated that viral replication is blocked in these cells [12]. Analysis of the copy number of TMEV genomes, plus- to minus-strand ratios, and full-length detects large numbers of viral genomes [ 15]. Therefore, during the chronic phase of this disease, there is an abundance of viral antigen and viral genomes combined with very low amounts of infectious virus. This can be explained to some degree by its restricted growth in macrophages, blocked after viral RNA replication. There is some evidence that astrocytes may also serve as a reservoir of infectious virus [ 16]. Supporting the major role of the immune response itself in this chronic phase of disease, nonspecific immunosuppression with cyclophosphamide, anti-thymocyte serum, or CD4 § T cell (not CD8 § depletion after the initial viremia, reduces the inflammatory mononuclear cell infiltration into the CNS and the subsequent demyelination [ 17-20]. In fact, in vivo depletion of CD4 + T cells in SJL mice infected with the BeAn strain of TMEV results in a decreased incidence and slower onset of disease [20]. Susceptibility to TMEV-IDD correlates with chronic high levels of TMEV-specific delayed-typehypersensitivity (DTH) [6, 21] along with the presence of predominately Thl-derived cytokines [20] and transfer of TMEV-specific CD4 § Thl blasts into suboptimally infected SJL mice results in an increase in disease incidence and severity. Although it is clear that the initial anti-virus immune response is responsible for the clinical symptoms of TMEVIDD, viral persistence is required for chronic disease, since mouse strains which clear the virus do not go on to develop the chronic demyelination that characterizes TMEV-IDD [21].
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3. FROM VIRAL INFECTION TO AUTOIMMUNITY
3.1. Virus-Specific CD4 § T Cell Responses Initiate Disease, Leading to Myelin Damage The adaptive immune response is exquisitely specific in that T and B cell responses initiated against one pathogen usually do not target other pathogens or self tissue. Two points at which some "non-specificity" can be introduced are 1) T or B cell receptor (T/BCR) interaction with their antigen and 2) the use of innate mechanisms as the effector phase of the immune response. First, T/BCRs are not as rigidly specific as once thought. Both B and T cell receptors can bind and respond to epitopes on molecules other than the original stimulant (molecular mimicry) if these epitopes include the important amino acids for TCR: peptide/MHC binding or if these epitopes fold into a similar shape with certain chemical characteristics recognized by the BCR. This mechanism (degeneracy) suggests that infectious viruses may encode within their sequence, epitopes or peptides that share homology with self-antigens. T cell activation during viral infection may thus produce T cells that cross-react with self peptides. These self-reactive T cells can then lead to self-tissue destruction and perpetuate an autoimmune response. Degeneracy in the TCR allows for the recognition of peptides with varying sequences by the same T cell clone. In fact, degeneracy in MHC class II peptide binding and TCR recognition of self-myelin peptide myelin basic protein (MBP)85-99 in the context of the human MHC class II HLA-DR2 haplotype, which is associated with MS patients, has been demonstrated [22]. Secondly, while CD8 § cytotoxic lymphocytes kill in a very specific manner, CD4 § T helper 1 (Thl) cells induce the influx and activation of macrophages, which carry out effector functions in a 'non-specific' manner. Macrophage influx and activation leads to bystander tissue destruction. During infection with TMEV, initiation of myelin damage is associated with the activation of monocyte/ macrophages by pro-inflammatory cytokines [2325] from TMEV-specific Thl cells responding to viral epitopes presented by CNS-resident antigenpresenting cells (APCs), which harbor persistent
virus for many months following infection [26]. Initially, time-course studies comparing the development of T cell responses to both virus and myelin epitopes during TMEV-IDD showed that autoreactivity to myelin epitopes is not detected prior to disease onset (30-35 days post infection) [27, 28] while immune responses to TMEV epitopes are clearly demonstrable by 5-7 days post infection [29]. Induction of peripheral tolerance to mouse spinal cord homogenate (MSCH), which effectively prevents MSCH-induced experimental autoimmune encephalomyelitis (EAE), at the time of TMEV infection does not affect the clinical onset or the development of virus-specific T cell responses in TMEV-IDD [30]. These results demonstrate that virus-specific CD4 + T cell responses initiate bystander tissue destruction (demyelination) and the clinical signs of TMEV-IDD. Infected animals exhibit a chronic progressive demyelinating disease characterized by low level persistence of TMEV in CNS microglia/ macrophages and/or astrocytes throughout the fifetime of the animals. CNS mononuclear infiltrates can be detected as early as 7 days post-infection [29] and are initially composed of peripheral macrophages and virus-specific T cells [24, 31-33]. In the SJL mouse, CD4 § T cell reactivity to the dominant myelin epitope proteolipid protein (PLP139_151), can be detected in the periphery of these animals beginning 50-55 days post-infection [8] indicating the initiation of an autoimmune component secondary to viral infection. In contrast to molecular mimicry, these data suggest that myelin debris is being processed and presented to T cells specific for autoantigens (epitope spreading).
3.2. Myelin Damage Results In Endogenous Presentation Of Myelin Epitopes And Epitope Spreading Accumulating data demonstrate that chronic immune-mediated tissue damage can lead to de novo activation of autoreactivity via epitope spreading. Epitope spreading is the process whereby epitopes distinct from, and non-cross-reactive with, an inducing epitope become major targets of an ongoing immune response. Two prominent examples of epitope spreading in CD4 + T cell-mediated autoimmune models are diabetes in NOD mice
[34-36] and R-EAE [37-39]. In addition, epitope spreading has been demonstrated following viral infections with picornaviruses, such as TMEV [8] and Coxsackie virus [40]. Initiation of myelin damage in TMEV-infected SJL mice by TMEV-specific CD4 § T cells targeting virus persisting in CNS-resident APCs leads to up-regulation of pro-inflammatory cytokines in the CNS, and is associated with the activation of CD4 § myelin-specific T cells during the chronic phase of disease. These autoreactive T cells appear to be primed via epitope spreading as determined by their late appearance in disease (> 50-60 days PI) and by the fact that there are no apparent viral epitopes that are shared with the major encephalitogenic myelin epitopes on PLP, MBP or MOG, i.e., there is no evidence for molecular mimicry in this system [8]. The spreading process was demonstrated in TMEV-IDD by observing temporal changes in the specificity of delayed-type hypersensitivity (DTH), T cell proliferative, and IFN-~, responses to viral and myelin epitopes [41] in peripheral lymphoid tissues. Anti-viral DTH and in vitro anti-viral T cell responses appear within a few days post-infection, and these responses continue throughout the disease. In contrast, myelin-specific responses can be detected beginning only 50-60 days post infection, i.e. 3-4 wk after clinical disease onset. Most interestingly, T cell responses against myelin epitopes arise in an ordered progression, initially targeting the immunodominant myelin epitope in the SJL mouse, PLP139_ls1. Reactivity toward this peptide then continues throughout disease. As disease progresses, T cell responses to PLP178_191 followed by responses to MBP84_~04arise, paralleling the relative order of their appearance in PLP139_~5~-induced R-EAE in SJL mice [42]. Reactivity toward additional myelin epitopes is also observed. The similarity in the strength and order of epitope spreading in TMEV-IDD and R-EAE suggests a hierarchy in the processing and presentation of these epitopes and/or the precursor frequency of T cells specific for the various myelin epitopes in SJL mice. The T cell precursor frequency of PLPI39_151 > PLP178_191 > MBP84_104 in SJL mice [42] correlates with the order of the appearance of specific T cell responses in the periphery of TMEVinfected SJL mice. However, the dynamics involved in the processing myelin tissue and presentation of
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myelin epitopes by the various types of antigen presenting cells in the CNS may effect this hierarchy and are currently not known. The availability of various myelin epitopes which are targeted in the epitope spreading process in the spinal cord of infected mice was examined by adding antigen presenting cells isolated from the spinal cord (CNS APCs) to myelin- or virus-specific T cell lines or hybridomas. As expected in a chronic CNS infection, endogenous presentation of viral envelope peptides (no exogenous peptide added) was demonstrated on day 35 (the earliest time point at which sufficient CNS APCs could be collected), and endogenous expression of these epitopes on CNS APCs persisted through 150 days post infection. This was expected since the CNS APC population is known to harbor persistent viral antigen, presumably allowing these cells to present this antigen in vivo or in vitro. In contrast, the endogenous presentation of all myelin epitopes assayed could be demonstrated (using 2.5-3.5x104 APC/well) only >80 days post infection. These data suggest that CNS APCs can present myelin epitopes endogenously only after sufficient demyelination by CNS microglia/macrophages has taken place. CNS APCs were also able to endogenously present multiple myelin epitopes around the same time post-infection, which suggests that T cell precursor frequency governs the hierarchy of epitope spreading rather than a peptide hierarchy of processibility in the CNS inflammatory environment or in CNS APCs. Immunohistochemical analysis of spinal cordinfiltrating mononuclear cells reveals that the number of CD4 + T cells and activated F4/80 + macrophages/microglia increase dramatically after TMEV infection. The F4/80 + cells also increase in size and up-regulate the requisite molecules required for activation of naive CD4 + T cells (i.e., MHC class II, B7-1, and B7-2), penetrate into the parenchyma, and accumulate in the CNS. This progressive accumulation correlates with the disease severity and the increasing number of CNS APCs that can be recovered from spinal cords of infected mice. Analyses of the F4/80 + population in the CNS reveals two subpopulations based on levels of expression of CD45 - resident C D 4 5 dim microglia and CD45 bright infiltrating peripheral macrophages. The ability of either of these CNS APCs to activate PLP139_~s~-specific Thl cells can be inhibited by both anti-MHC
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class II and by blocking costimulation with CTLA4 Ig, indicating that the presentation of endogenous myelin epitopes is B7 dependent and MHC class II-restricted [ 10]. Within the normal CNS, a variety of cells are capable of antigen presentation to T cells, including astrocytes, microglia, and macrophages. IFN"t-treated primary astrocytes [43, 44] and microglia [45, 46] cultured from neonatal mouse brain upregulate MHC class II and can present antigens to T cells in vitro, but this may not reflect the in vivo state in adult animals. Microglia directly isolated from adult rats can more efficiently present MBP to T cell lines in vitro compared with neonatally derived microglia [47]. In our hands, CNS mononuclear cells isolated from na'fve mice are inefficient at endogenously activating myelin-specific T cells. They are, however, capable of processing and presenting exogenously added myelin proteins/ peptides, albeit with less efficiency than irradiated splenic APCs [41]. The roles played by each of these CNS APCs in epitope spreading has been difficult to unravel and continues to be investigated. Freshly isolated F4/80 +, I-A s+, CD45 + plasticadherent mononuclear cells from the spinal cord of TMEV-infected mice, which include macrophages, monocytes, and microglia, were able to process and present exogenous TMEV or horse myoglobin epitope to antigen-specific T cell lines [33] and have the ability to endogenously process and present virus epitopes at both acute and chronic stages of the disease [ 10, 41 ]. However, the relative contributions of the resident microglia vs. infiltrating macrophages in antigen presentation during TMEV-IDD had not yet been clearly delineated. Recently, we isolated CNS-resident microglia and CNS-infiltrating macrophages from TMEV-infected mice based on their differential expression levels of CD45 (see above) in order to test the APC capabilities of each cell type at various stages of disease [Mack, C, manuscript submitted]. Microglia from na'fve adult mice are clearly in a "resting state" and are not competent antigen presenting cells as shown by the lack of APC markers such as MHC class II and B7 costimulatory molecules, as well as their inability to stimulate proliferation of an antigen-specific Thl line. In contrast to microglia purified from neonatal brain [48], stimulation of na'fve microglia from adult SJL mice in vitro with pro-inflammatory cytokines,
under defined conditions, resulted in a low expression of B7-2, but no detectable upregulation MHC class ILl, and the persistent inability to stimulate T cell proliferation. The finding that na'fve adult microglia remain incompetent APCs even following stimulation with pro-inflammatory cytokdnes has also been shown by other investigators [47, 49]. In contrast to microglia from naive adult mice, in the inflammatory setting of TMEV-IDD microglia become activated, express the necessary molecular machinery to serve as competent APCs and effectively stimulate T cell proliferation and cytokine production. At the time of clinical disease onset (37 days post-infection), both the CD45 dim microglia and the CD45 b~gh~ infiltrating macrophages express similar levels of APC surface markers and are capable of stimulating proliferation and IFN- 7 production of a PLP139_~51-specific Thl line to a similar degree. The role of the resident microglia at this early point in TMEV-IDD may primarily involve processing and presentation of viral epitopes. In fact, microglia cultured from neonatal SJL mice can be persistently infected with TMEV in vitro and that infection significantly upregulated expression of costimulatory (B7-1, B7-2 and CD40) and MHC class II molecules. Most significantly, TMEV-infected microglia were able to efficiently process and present both endogenous virus epitopes and exogenous myelin epitopes to inflammatory CD4 § Thl cells [48]. Microglia are activated early in response to a number of different infections or injuries to the CNS [50, 51 ]. Interestingly, during the chronic-progressive stage of TMEV-IDD (90 days post-infection), the C D 4 5 b~ight infiltrating macrophages express higher levels of APC markers and spinal cord-infiltrating macrophages are more potent stimulators of T cell proliferation when compared to the CD45 dimmicrogila. Increasing numbers of infiltrating macrophages as disease progresses leads to an increased secretion of TNF-t~ which, along with IFN-), derived from the virus and myelin peptide-specific T cells, may lead to up-regulation of MHC-class II and B7 surface expression on these cells, creating more potent APCs. The amount of TNF-oc mRNA expression in the spinal cords of SJL mice with TMEV-IDD sharply increases as the disease progresses [52]. The resident microglia may not be as responsive as the infiltrating macrophage to TNF-o~-facilitated, IFN-
q-mediated upregulation of APC surface markers and presenting function. Another explanation for the differential APC capability of macrophages versus microglia later in disease may be due to differential inhibition by nitric oxide. In support of this hypothesis, it has been reported that microglia in the setting of MOG35_55 peptide-induced EAE were competent APCs during the peak of disease [53]. However, late in this disease, after mice had partially recovered, there was a reduction in microglial APC capability that was attributable to enhanced production of nitric oxide by infiltrating macrophages. It is of major interest to determine whether T cells involved in the epitope spreading process that are specific for endogenous myelin epitopes become activated in the periphery (draining lymph nodes and spleen) and/orthe CNS. It is possible that following inflammatory disruption of the blood-brain barrier, myelin debris and/or macrophages/microglia that have ingested myelin proteins gain access to the cervical lymph nodes that drain the cerebrospinal fluid [54] or to the spleen, which concentrates blood-borne material. In support of this hypothesis, it has been reported that donor cells from alloantigen-disparate solid CNS grafts placed intracerebrally can be later identified in the host spleen and lymph nodes [55]. We are currently assessing the ability of APCs purified from the spleen and deep cervical lymph nodes of mice with chronic disease to endogenously present self epitopes. In contrast, it is also possible that T cells specific for endogenous myelin epitopes are activated in the local inflammatory environment within the CNS. In TMEVIDD, the inflammatory infiltrate is composed of T and B lymphocytes, activated microglia derived from the CNS-resident pool, and macrophages infiltrating from the peripheral blood [33, 56, 57]. Macrophages/microglia within the demyelinated areas contain phagocytized myelin debris [58] and are capable of processing and presenting myelin epitopes. Therefore, any myelin-specific T cells that enter the CNS during the anti-viral inflammatory response, whether already primed in the periphery or not, could potentially be induced to proliferate and/or to secrete pro-inflammatory cytokines in response to myelin epitopes.
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4. DIFFERENTIAL ABILITIES OF CNS RESIDENT MICROGLIA, ENDOTHELIAL CELLS, AND ASTROCYTES TO SERVE AS INDUCIBLE ANTIGEN-PRESENTING CELLS A variety of cells within the normal CNS are capable of antigen presentation to T cells. MHC class II and costimulatory molecule expressing cells can be found in MS lesions [59-62] and human microglia have been shown to express costimulatory molecules required for activation of T cells [63, 64]. As previously discussed, microglia and macrophages differ in their APC ability depending on the microenvironment. Cerebrovascular endothelial cells (CVEs) upregulate MHC class II and B7-1 costimulatory molecule in response to IFN-y in vitro. However, murine CVEs did not elicit significant MHC class II-restricted T cell responses [65]. IFN-y-treated SJL astrocytes fed intact PLP or MP4 (a fusion protein containing PLP and MBP portions) efficiently activated lines and hybridomas specific for the immunodominant PLP139_151 epitope. However, T cell lines specific for the less immunodominant self encephalitogenic epitopes (PLP56_7o, PLP~04_~7, and PLP178_191) were not activated by IFN-y-treated astrocytes fed intact PLP, but were activated by astrocytes pulsed with the relevant autologous peptide [44]. Similar results were seen with multiple independently derived PLP peptide-specific T cell lines and hybridomas specific for the less dominant epitopes and when astrocytes were activated with a combination of TNF-cx and IFN-y, which enhances MHC class II and Ii expression above the levels seen for astrocytes stimulated with IFN-y alone. Under the pro-inflammatory conditions examined it appears that astrocytes may not play a major role in the phenomenon of epitope spreading. However, it is also possible that in the local milieu of the CNS, additional cytokines may play a role in activating astrocytes to more effectively process and present the subdominant PLP epitopes. Significantly, IFNy-treated SJL/J astrocytes pulsed with either intact MP4 or PLP139_151 were also capable of activating PLP~39_~5~-specific T cells for the adoptive transfer of R-EAE, indicating that they can induce the upregulation of the appropriate integrins and cytokines necessary for CD4 § T cells to home to the CNS and initiate the demyelinating process.
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In addition to the inflammatory environment in which these CNS APCs reside, TMEV infection itself may also play a role in upregulation of APC function. As previously detailed, microglia from SJL mice can be persistently infected in vitro with TMEV and, as a result of this infection, these cells are activated to function as competent APCs with the ability to process and present both virus and myelin epitopes to memory CD4 § Thl cells [48]. Concomitant with the acquisition of this functional antigen .presentation capacity, TMEV infection induced the upregulation of cytokines involved in innate immune responses and of cytokdnes and costimulatory molecules required for the activation and differentiation Th 1 effector cells. Most significantly, direct TMEV infection of microglia was nearly as effective as stimulation with high levels of IFN-y in conferring APC function.
5. USING TMEV TO INVESTIGATE MOLECULAR MIMICRY, AN ALTERNATE MECHANISM FOR INDUCTION OF AUTOIMMUNITY DURING INFECTION The mechanism(s) underlying the initiation and progression of multiple sclerosis and other autoimmune diseases are not well understood, but epidemiologic studies have provided strong suggestive evidence for a role of virus infection(s) in the development and/or exacerbations of MS. The possible mechanisms by which virus infection can trigger an autoimmune response include molecular mimicry, bystander activation, and epitope spreading. In the TMEV-IDD model of MS, we have demonstrated bystander activation, the non-specific activation of autoreactive T cells resulting from the virus-specific CD4 + Thl inflammatory response itself on tissue in the target organ, followed by epitope spreading, the activation of autoreactive T cells due to the tissue damage following release of self epitopes during that immune response. Molecular mimicry, on the other hand, theoretically results following infection with a virus expressing a peptide determinant(s) that shares homology with a self peptide, resulting in activation of T cells that can crossreact with the self epitope. The discovery of TCR degeneracy, the TCR's abil-
ity to recognize multiple peptides with only a few key amino acid positions in common, has led to the widespread belief that some microbial proteins probably contain peptide sequences that are able to activate self-reactive T cells. Recent studies have shown degeneracy in the TCR specific for the human myelin basic protein MBP85_99peptide, with the TCR requiting only a few critical residues for recognition [22]. T cell clones specific for MBP 85-99 established from MS patients were shown to crossreact with viral peptides expressed by a number of viruses, including HSV, adenovirus, reovirus, and human papillomavirus [22]. Likewise, a few critical residues were shown to be necessary for recognition of PLP139_151by its TCR [66]. PLPx39_lsl-specific T cell hybridomas derived from SJL mice were also shown to cross-react with peptides expressed by various mouse pathogens, demonstrating degeneracy in the PLP139_151TCR [66]. Therefore, myelin-specific T cells have been shown by in vitro studies to have the potential to cross-react with viral epitopes, supporting the molecular mimicry model described in these studies. In order to directly investigate molecular mimicry as a potential mechanism of CD4 § T cellmediated autoimmunity, we developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant PLP~39_~5~epitope into the coding region of a nonpathogenic TMEV variant (PLP~39-TMEV) [67, 68]. PLP139-TMEV-infected mice developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLPa39_~5~-specific CD4+Th 1 responses within 10-14 days post-infection. These data demonstrate that the early onset demyelinating disease induced by PLPI39-TMEV is the direct result of autoreactive PLP~39_~5~-specific CD4 § T cell responses. PLP139_~5~-specific CD4 § T cells from PLP~39-TMEV-infected mice transferred demyelinating disease to naive recipients and infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139_xs~-Specific CD4 § T cells could be activated in the periphery and traffic to the CNS. Importantly, PLP139_~5~ epitope-specific tolerance before infection with PLP~39-TMEV resulted in the specific reduction of PLP139_x51-specific CD4 § Thl responses that directly correlated with a significant reduction in the incidence and severity of the early
onset demyelinating disease. In addition, mimic PLP139_151 sequences were constructed in which amino acid substitutions were made at the primary (amino acid 144) or secondary (amino acid 147) TCR contact residues [67, 68]. Infection with the virus carrying a substitution in the secondary TCR contact residue induced early-onset demyelinating disease and activated cross-reactive PLPi39_lsl-specific CD4+T cells. In contrast, infection with the virus substituted at the primary TCR contact residue (position 144) failed to induce early demyelinating disease or activation of cross-reactive PLP139_151-specificCD4§ cells. An additional mimic virus was constructed by inserting a sequence from H. influenzae that shared only 6 of 13 amino acids with the core PLP139_151epitope [67]. More significant to a role for molecular mimicry in induction of autoimmune disease, infection with this mimic virus resulted in early onset demyelinating disease and activation of Thl cells cross-reactive with the native PLP139_151determinant. This model is the first to directly demonstrate that a virus encoding a mimic of an encephalitogenic self myelin epitope could induce an autoreactive CD4§ cell response leading to a CNS demyelinating disease. The ability of any microbial peptide mimicking self to be processed is required for APC presentation to T cells. Ongoing work inserting 30mers into the virus coding region will determine if the induction of the autoimmune disease requires that the mimic epitope be processed from its native flanking regions in addition to the requirement that the core epitope be presented in an appropriate fashion to activate the self-reactive Thl response.
6. SUMMARY
The epidemiology of MS strongly suggests a role for an infectious agent, most likely a virus. Presentation of viral antigens within the CNS (leading to bystander demyelination), of neuroantigens crossreactive with viral antigens (molecular mimicry), or of neuroantigens liberated by immune or virusinduced CNS damage (epitope spreading) are all possible mechanisms by which pathogenic immune reactions could be initiated by viruses within the CNS (Fig. 1). TMEV-IDD is a well-characterized CD4 § T cell-
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Figure 1. Possible Mechanisms of Virus-Induced T Cell-Mediated Autoimmune Disease. The figure illustrates induction of CD4§ T cell-mediated autoimmune tissue destruction via induction of a self antigen-specific cross-reactive T cell response (i.e., molecular mimicry) following peripheral virus infection (Left Panel) and via epitope spreading to self antigen-reactive T cells secondary to bystander tissue destruction and release of self antigens initiated by a specific T cell response to virus persistent in the target tissue (Right Panel).
mediated model of MS. Life-long persistent viral infection of CNS resident microglia, macrophages, and astrocytes is directly related to the development of the chronic demyelinating disease. Initial myelin damage is mediated by a bystander mechanism wherein the primary effector cells are mononuclear phagocytes (microglia/macrophages) activated by inflammatory cytokines produced from TMEVspecific Thl cells responding to viral epitopes that persist in the CNS. Early myelin destruction leads to the de novo activation of myelin-specific T cells (epitope spreading). The initial myelin response is directed toward the immunodominant PLP 139-151 epitope, and epitope spreading then leads to an ordered progression of T cell responses to multiple myelin autoepitopes which appear to play a significant role in the chronic phase of the disease by escalating the demyelinating process. The continuous presence of the virus within the CNS perpetuates this chronic inflammatory process in which epitope spreading leads to the induction of autoreactive T cells. These findings enhance our understanding of the pathogenesis of human MS. MHC class II-bearing
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macrophages, astrocytes, and endothelial cells have been observed in or near MS lesions, together with expression of B7 costimulatory molecules. Therefore, multiple cells in MS lesions have the potential to fully activate both naive and memory T cells within the CNS. Although not the mechanism of autoimmunity in mice infected with native TMEV, mimic peptide- and natural pathogen peptide-engineered TMEV infection models demonstrate that molecular mimicry can also lead from viral infection to autoimmune disease.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Viruses and Multiple Sclerosis A. Achiron
Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Israel
1. INTRODUCTION
2. MICROBIAL-INDUCED AUTOIMMUNITY
Multiple sclerosis (MS) is a central nervous system (CNS) white-matter demyelinating disease affecting young adults. The characteristic clinical course of MS in 85% of patients is relapsing-remitting, whereas in about 15% of patients the disease presents as a primary progressive course. Within 10 years from onset, 50% of patients with relapsing-remitting disease will advance to the secondary progressive phase, with consequent increases in neurological disability [1]. The disease has an autoimmune component, with the presence of selfreactive lymphocytes targeting myelin peptides such as myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), leading to inflammation and myelin destruction within the brain and spinal cord [2, 3]. The etiology of MS is as yet unknown, and the hypothesis that an infectious agent is responsible for triggering the disease has waxed and waned over the last two centuries (since Pierre Marie first proposed that MS often starts as an infectious process). The possible role of infectious agents has been suggested by the different temporal patterns of the disease in different geographic areas, changes in prevalence due to migration and the effect of age at migration, the relapsing-remitting course of MS, and the induction of demyelination in animal models by various viruses [4-6].
The different mechanisms by which infectious agents might activate autoreactive lymphocytes and lead to an autoimmune disease fall into two major classes: antigen-nonspecific and antigen-specific. 2.1. Antigen-Nonspecific - The 'Innocent Bystander Activation' Theory
This theory is based on nonspecific antigen activation with no particular microbial determinant implicated. Once the immune system becomes primed to attack the infecting pathogen, there is a possibility that the myelin could be inadvertently attacked in the process. The mechanisms suggested to be involved include: (a) Direct inflammatory damage caused by the inflammatory response to the microbial agent, resulting in cell destruction and the subsequent release of different cell ingredients that will be presented to the immune system at the inflammation site. These newly presented self-determinants induce an immune response that will result in an autoimmune disease. In MS, the autoimmune process within the CNS involves activation of microglia by signals originating from either activated monocytes and lymphocytes in the blood stream, or from activated macrophages or astrocytes within the brain. This microglial activation subsequently results in the release of excitotoxins, cytokines and chemokines, with further myelin destruction. (b) A microbial-induced alteration in the phenotype of antigen-presenting cells (APCs). This can result in the enhanced expression of co-stimulatory molecules, increased
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production of inflammatory cytokines such as rumor necrosis factor (TNF)-0~ and interleukin (IL)1 that may promote retroviral replication, and the modification of lymphocyte migration patterns. (c) Provoked T-cell lines/clones induced via either a mitogen or a superantigen effect by the microbial agent. 2.2. Antigen-Specific - 'Epitope Mimicry'
The cornerstone of the antigen-specific theory is epitope mimicry; an antigenic determinant on one of the proteins of the microbe is structurally similar to a determinant of a host protein, although different enough to be recognised as foreign by the host's immune system. For T-cells, the determinants involved would be linear peptide stretches of about 8-15 amino acids long. The immune response to the microbial determinant cross-reacts with host tissue and eventually results in autoimmune destruction
[7, 8]. 3. MICROBIAL-INDUCED AUTOIMMUNITY IN MS It is currently not known whether an organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. Several studies have showed that a naturally infectious virus encoding a myelin epitope can directly initiate organ-specific T-cell-mediated autoimmunity. Lenz et al identified a 20-mer peptide from a protein specific to Chlamydia pneumoniae, which shares a 7-aminoacid motif with a critical epitope of MBP, a major CNS antigen targeted by the immune system in MS [9]. This bacterial peptide induced a Thl response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective in many aspects of MS. In a similar study, Olson et al studied the potential of virus-induced molecular mimicry to initiate autoimmune demyelination, using a nonpathogenic Theiler's murine encephalomyelitis virus (TMEV) variant that was engineered to encode a 30-mer peptide encompassing the immunodominant encephalitogenic myelin PLP(139-151) epitope [ 10]. Within 10-14 days of
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infection with the PLP(139-151)-encoding TMEV, a rapid-onset paralytic demyelinating disease, characterised by PLP(139-151)-specific CD4+ Thl responses, was observed. Mice infected with TMEV encoding a Haemophilus influenzae mimic peptide, sharing only six of the 13 amino acids of PLP(139-151), displayed rapid-onset disease and developed cross-reactive, PLP(139-151)-specific CD4+ Thl responses. These studies suggest that the epitope mimicry mechanism may be involved in triggering MS.
4. INFECTIOUS AGENTS AND MS 4.1. Herpes Viruses
One of the greatest challenges in confirming or refuting a role for herpes viruses (e.g. Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6)) in MS is their ubiquitous nature - they are neurotropic, become latent and persist even with very limited genome expression, can be reactivated with the relapsing-remitting course of MS, and have been shown to induce demyelination. In addition, both herpes reactivations and MS exacerbations can be brought on by infections with other viruses.
4.1.1. Human herpes virus 6 HHV-6 was discovered in 1988 and consists of two subtypes, HHV-6A and HHV-6B, both of which are prevalent in the normal population [ 11]. HHV-6B is the causative agent of exanthem subitum, a common childhood illness, and has been associated with meningitis, myalgic encephalomyelitis and febrile seizures [12]. HHV-6A has not yet been shown to cause human disease. The most consistent neuropathologic changes associated with HHV-6 infections of the CNS have been demyelination, ranging from diffuse and extensive loss of myelin, to sharply circumscribed foci of demyelination [13, 14], combined with the destruction of axons within areas of the most severe pathological changes [12]. As MS is also associated with prominent demyelination combined with axonal destruction, it was suggested that there might be an association between HHV-6 infection and MS. Studies describing associations between HHV-6
and MS are based on either detection of HHV-6 antibodies in serum or cerebral spinal fluid (CSF), or amplification of HHV-6 DNA from serum or CSF of patients with MS but not control subjects [15, 16]. Following this lead, HHV-6 antigen expression was detected in MS brains and shown to be associated specifically with MS plaques [17]. Moore & Wolfson systematically reviewed the published evidence for a relationship between human HHV-6 and MS [ 18]. They searched the medical literature using MEDLINE and the Cochrane database, retrieving 28 studies according to 12 different experimental techniques used. When the technologies could not distinguish between active and latent HHV-6 infections (PCR analysis of blood leukocytes, CSFcontaining cells or CNS tissue), no differences were noted between samples from patients with MS and control subjects. In contrast, when diagnostic technologies were restricted to the detection of active HHV-6 infections (PCR analysis of acellular specimens, detection of HHV-6-specific IgM antibodies or immunohistochemical staining of CNS tissues), evidence for a relationship between HHV-6 and MS was found, but no causative relationship could be demonstrated. Thus, it has been suggested that the finding of a relationship between HHV-6 and MS is merely a result of the immune system activation or blood-brain-barrier breakdown in MS making signs of prior infection with HHV-6 more easily detectable. The mechanisms by which HHV-6 was suggested to cause MS could be related to the ability of the vires to infect and destroy oligodendrocytes [ 19, 20], or to the capability of HHV-6 to induce TNF-c~ production in blood mononuclear cells [21 ], as this pro-inflammatory cytokine is also known to mediate demyelination in MS, and its production by blood mononuclear Cells correlates with disease activity [22]. Interestingly, the antiviral drug acyclovir, which provides effective prophylaxis against HHV6 infections in bone marrow transplant patients, has been shown to significantly reduce the frequency of disease exacerbations in patients with MS [23].
4.1.2. Herpes simplex virus I Herpes simplex virus 1 (HSV-1) DNA has been found in some cases of acute MS but not in stable MS or healthy controls [24]. These data suggest
that HSV-1 reactivates in patients during clinical relapses and may be a trigger of MS relapses.
4.1.3. Human herpes virus 7 Human herpes virus 7 (HHV-7) has been found to be equally prevalent in a latent form in peripheral blood mononuclear cells of both MS patients and healthy controls [25]. Soldan et al measured the lymphoproliferative response to HHV-7-infected cell lysate and found no significant difference between patients with MS and controls [26]. Taus et al also reported no relationship between MS and HHV-7 [27].
4.1.4. Epstein-Barr virus Individual epidemiologic studies assessing the relationship between EBV and MS have been inconclusive, in part because of the high prevalence of previous EBV infection among individuals without MS. The reported prevalence of antiEBV seropositivity in patients with MS is 100%, compared with 80-95% in matched controls [28]. Higher concentrations of serum antibodies against both the EBV viral capsid antigen (VCA) and nuclear antigens (EBNA-1) have been reported in patients with MS [29]. Moreover a more frequent history of infectious mononucleosis, and late age at infection, have been described in patients with MS [30, 31 ]. Ascherio & Munch conducted a systematic review of case-control studies comparing EBV serology in patients with MS and controls [32]. Eight published studies were identified, including a total of 1005 cases and 1060 controls. The summary odds ratio of MS, comparing EBV seropositive individuals with EBV seronegative individuals, was 13.5 (95% CI = 6.3,-31.4). The strength and consistency of this association and the high sensitivity and specificity of EBV serology support a role for EBV in the etiology of MS. However, assuming that 90% of both patients with MS and controls were in fact infected by EBV, the results observed could be obtained if the specificity of the diagnostic test was 10% or less among patients with MS, and close to 100% among controls. Alternatively, under the assumption that both cases and controls were all infected, the results could be obtained if the sensitivity was 90% or less among
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controls and 100% among patients with MS. In a recent large case-control study conducted among more than 3 million US military, Levin et al evaluated whether antibodies to EBV are elevated before the onset of MS [33]. The risk of MS increased monotonically with VCA or the nuclear antigens EBNA complex antibody titers, and a relationship between EBV infection and development of MS was therefore suggested. The ability of EBV to interfere with the normal process of T-cell repertoire [34], and the cross-reactions of anti-EBNA antibodies with epitopes of a neuroglial antigen [35], also support a causal association with MS, mainly related to EBV reactivation and disease activity in patients with MS, suggesting that EBV might trigger an underlying disease process [36].
4.1.5. Cytomegalovirus Cytomegalovirus (CMV) is a prevalent viral pathogen. The majority of patients with acute CMV will experience an inapparent infection. A primary CMV infection will cause up to 7% of cases of mononucleosis syndrome and will manifest symptoms almost indistinguishable from those of EBV-induced mononucleosis. There are no reports demonstrating a convincing link between MS and CMV. Sanders et al used a PCR approach to compare active and inactive plaques from patients with MS [37]. CMV sequences were detected in 9-22% of specimens, irrespective of disease activity. In another large population-based study, no association was found between CMV antibodies and MS [33].
4.1.6. Varicella zoster virus The association between varicella zoster virus (VZV) and MS is limited largely to clinical experience and epidemiological surveys. Both MS and varicella are most prevalent in temperate zones and rare in countries close to the equator. An early serological study found the geometric mean titer of antibodies to VZV to be significantly higher among patients with MS than among patients with other diseases and normal individuals [38]. Recently, Marrie & Wolfson reviewed the epidemiological evidence for an etiological role of VZV infection
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in the development of MS; a MEDLINE search of English language literature published between 1965 and 1999 identified 40 studies that were classified according to strict methodological criteria [39]. Five studies that utilised the best methodology failed to show an increased risk of MS associated with varicelia or zoster infections. At the present time, there is insufficient evidence to support an important etiological role of VZV infection in the development of MS. 4.2. Retroviruses
4.2.1. Human immunodeficiency virus Only a few cases of human immunodeficiency virus (HIV)-positive patients with MS or MS-like lesions have been reported. In four of the seven patients described by Berger et al, the MS preceded the HIV infection by a long period. In the other three patients, HIV seroconversion occurred concomitantly or within 3 months of the onset of the neurological symptoms [40]. Gray et al described two patients with a fulminating demyelinating leukoencephalopathy in the early course of HIV infection [41 ], and Graber et al reported a patient with a relapsing and remitting leukoencephalopathy, who was HIV-positive at the onset of the disease, but HIV-negative 9 months earlier [42].
4.2.2. Multiple sclerosis-associated retrovirus The island of Sardinia has a high and increasing incidence of MS. Serra et al and Dolei et al searched for environmental factors that may account for this anomalously high incidence [43, 44]. They detected MS-associated retrovirus (MSRV), an exogenous member of the human endogenous retrovirus family W (HERV-W) in all patients with MS, in most patients with inflammatory neurologic diseases, and rarely in healthy blood donors. MSRV was found in the plasma and CSF of patients with MS, and was produced in vitro by their cells. Detection of MSRV is not restricted to MS, as the virus has also been found in synovial fluids of rheumatoid arthritis patients [45]. Similar to HSV-1, it has been suggested that MSRV may contribute to MS reactivation in Sardinia.
4.3. Parvovirus
Human parvovirus B19 (PVB19), the etiologic agent of Etythema infectiosum, causes transient and persistent immune derangements. PVB 19 infections have been reported to be associated with chronic immune-mediated disorders, including rheumatoid arthritis and systemic lupus erythematosus. PVB 19 can invade the CNS, possibly resulting in encephalopathy and meningitis. Only one study to date has evaluated the association between PVB 19 and MS [46]. The prevalence of serum anti-PVB 19 IgG was shown to be significantly higher in patients with MS than in healthy subjects. On the other hand, none of the patients developed an E. infectiosum infection nor had serological or molecular evidence of an active PVB 19 infection, such as the presence of anti-PVB 19 IgM or PVB 19 DNA. Furthermore, serum anti-PVB 19 IgM and PVB 19 DNA in CSF were consistently negative in patients during exacerbation of MS [46]. 4.4. TT Virus
TT virus (TTV), identified in 1998, is a widespread infectious agent of humans. In infected individuals, TTV induces persistent viremia. However, the life-cycle and pathogenic potential of TTV are still poorly understood. In only one study, 21 paired samples from CSF and serum from patients with MS were tested for TTV using real-time PCR. The majority of MS serum samples (71%) were TTVpositive as expected based on TTV prevalence and viremia levels in the general population, but none of the CSF samples of patients with MS were TTVpositive, suggesting no role for this virus in MS [47].
5. VIRAL INFECTIONS AND DISEASE ACTIVITY
A possible increase in the risk to develop MS following infections of any kind (upper respiratory tract, gastrointestinal, urinary tract) has been investigated in several studies. Hernan et al investigated these associations in a case-control study that included 301 patients with MS and matched controls [48]. Except for infec-
tious mononucleosis, which was a moderate risk factor, little association was found between the history of common viral diseases or exposure to canine distemper virus and risk of developing MS. However, a relationship between mumps and measles after 15 years of age and MS was found. The question of whether infectious diseases can induce MS disease activity was assessed by Sibley et al [49]. A total of 170 patients with MS were evaluated for a mean of 5.2 years. The results showed a 2.8-fold increase in relapse rate during risk periods associated with infection. Andersen et al reported a relative risk for relapse during infection risk periods of only 1.3 in 60 patients with MS followed for a mean of 31 months [50], and Buljevac et al, in a prospective survey of 73 patients with MS followed for a mean of 1.7 years, found a relative risk of 2.1 during infection-related risk periods [51]. These findings confirm the association between infections and relapses in MS and can be explained by the antigen nonspecific theory where activation of the host immune system by viral superantigens results in activation of autoreactive T-cells, pro-inflammatory cytokine production and activation of the disease process [52]. However, no significant changes in MRI activity related to the infections could be demonstrated.
6. VACCINATION AND MS Several case reports describing the onset or exacerbation of MS shortly after vaccination have suggested that vaccines may increase the risk of the disease. The major question raised was whether vaccine-preventable infectious diseases increase the risk of MS onset or exacerbations. DeStefano et al studied 440 patients with MS or optic neuritis and 950 matched controls for the onset of first symptoms of demyelinating disease at any time after vaccination [53]. The odds ratios of the associations between ever having been vaccinated and risk of demyelinating disease were: 0.9 (0.6-1.5) for hepatitis B vaccine; 0.6 (0.4-0.8) for tetanus vaccination; 0.8 (0.6-1.2) for influenza vaccine; 0.8 (0.5-1.5) for measles-mumps-rubella vaccine; 0.9 (0.5-1.4) for measles vaccine; and 0.7 (0.4-1.0) for rubella vaccine. The study concluded that vaccination against hepatitis B, influenza, tetanus, measles
267
or rubella is not associated with an increased risk of MS or optic neuritis. In the early 1990s, several cases of demyelinating diseases were reported in France in association with the vaccination against hepatitis B. A large scientific, regulatory and public debate took place to reassure the growing concern of the population. Even on the basis of the early findings, which appeared to be compatible with a low increase in the risk associated with the vaccination, it was apparent that the risk-benefit profile was unchanged for newborns, and was essentially unchanged for adolescents and high-risk adults [54]. To further address the safety of immunisation in patients with MS in relation to increased risk of relapses after vaccination, the MS Council for Clinical Practice Guidelines commissioned a systematic review [55]. Upon conducting a meta-analysis of 130 articles, after screening 667 citations and 280 full-text articles, strong evidence was found against an increased risk of MS exacerbation after influenza immunisation. There was no evidence to suggest that hepatitis B, varicella, tetanus or Bacille Calmette-Guerin vaccines increase the risk of MS exacerbations. Insufficient evidence was found for other vaccines.
7. C O N C L U S I O N S The viral hypothesis in MS is hampered by the lack of evidence for a specific agent, in addition to the weakness of the results of analytical studies that have tested the association between MS and previous infections. None of the organisms so far investigated for a role in MS has gained acceptance as the causative agent. Although several studies have implicated the role of viruses in the etiology of MS, with the temporal relationship sometimes being impressive, many of the associations appear less than convincing, and even for those that seem to be on solid footing, there is no real understanding of the underlying mechanism(s). It is essential to rely on well-conducted systematic studies that produce valid and reliable estimates of the risk-associated profile of a causative viral agent, as many commonly circulating viruses may be indirectly activated under the autoimmune circumstances occurring in MS, and not necessary associated to these circumstances.
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In conclusion, the strict criteria of evidencebased medicine that includes the detection of viral DNA/RNA in the brain or spinal cord of patients with MS, confirmation of epidemiological studies in a variety of geographic regions, and validation of viral antibodies by several laboratories and in comparison with the normal population have not come close to be satisfying. Additional studies will be needed to clarify any connection.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Endogenous Retroviruses as Etiological Agents in Systemic Lupus Erythematosus Miranda K. Adelman, David E. Yocum and John J. Marchalonis
College of Medicine, University of Arizona, Tucson, AZ, USA
I. I N T R O D U C T I O N Great insights into the etiopathogenesis of Systemic Lupus Erythematosus (SLE) have been made in recent years and point toward a multi-factorial origin consisting of environmental, genetic and retroviral factors. Environmental factors include UV light, which has been shown to induce skin rashes [1], or certain drugs [2, 3], including hydralazine, procainamide or the use of birth control pills, that are known to induce disease and may be associated with flares. Genetic influences are indicated by sibling and familial studies where 'the concordance rate for monozygotic twins is >20% and for dizygotic twins is 2-3% [4-6]. Moreover, the use of murine markers [7], genome-wide scans of murine and human chromosomes [4], and linkage analyses [8-10] have enabled the identification of numerous genes that predispose to SLE [4, 6], as well as to other autoimmune diseases [7]. The New Zealand B lacUNew Zealand White (NZBAV) mouse model for SLE provided the first suggestion of a retroviral association for SLE by demonstrating the presence of a retroviral envelope (Env) protein related to murine leukemia virus (MuLV), termed gp70, in deposited immune complexes [11]. Hence, the search for a retroviral etiology for SLE was begun. 1.1. Retroviral Elements
Of the 3 billion DNA bases comprising our genome, an estimated 3% code for the 30,000-40,000 genes that are translated into the proteins essential for life [12]. The evolutionary significance of the non-
coding "junk DNA" is not completely understood, although it is believed to function in packaging and gene expression [ 13]. Intriguingly, an estimated 50% and likely more [12, 14, 15] of the human genome is derived from the integration of various retroviral elements, which are divided into four groups based on their genomic organization: exogenous retroviruses, human endogenous retroviruses (HERVS), retrotransposons and retroposons. In contrast to exogenous retroviruses that are passed horizontally and require a replication cycle where proviral DNA is integrated into the host DNA, HERVs are transmitted vertically as stable Mendelian elements [ 16, 17]. Accounting for approximately 8% of the genome [12], it is generally believed that most HERVs integrated into the human lineage as exogenous progenitors prior to the divergence of hominid from Old World Primates [ 17]. The third group of retroelements, the retrotransposons, differs from both exogenous and endogenous retroviruses by lacking the env gene. Additionally, some truncated HERVs are classified as retrotransposons when lacking functional env genes. Although sequence analyses have revealed that the genomes of most HERVs are disrupted by frame shift mutations, termination codons and deletions, some HERVS are transcriptionally active, are expressed in a tissue specific manner and produce functional retroviral proteins [17-19]. The retroposons include the long interspersed elements (LINEs) and the short interspersed elements (SINEs) [17], most of which are present in high copy numbers and are found in species as diverse as sharks and humans [20]. HERVs share similar genomic structures as
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A) Exogenous retroviruses MatrixMA~ Capsid C A Nucleocapsid N C Protease a PR
I
~
Protease a PR Surface glycoproteins SU Reverse transcriptase RT T r a n s m e m b r a n e protein T M Integrase IN
B) Endogenous retroviruses (HERVs)
gagHi pol ~ l l l l k ~ C) Retrotransposons D) Retroposons
Long interspersedelements (LINES) 5 ' ~ O1~ 1[ ~ A A A A - ~ , , Short interspersedelements (SINES)
5'91DqltRNA I-AAAAFigure 1. Genetic organization of retroviral elements. (A) Exogenous retroviruses, (B) human endogenous retroviruses (HERVs), (C) retrotransposons, and (D) retroposons all share similar genetic information. Flanked by direct repeats (I~) and long terminal repeats (LTRs) are the retroviral group specific antigen (gag) gene which encodes MA, CA, NC and sometimes PRa, the polymerase (pol) gene which encodes the RT, IN and sometimes PRa and the envelope (env) gene which encodes SU and TM. Although they share the same genomic organization, HERVs differ from exogenous infectious retroviruses in their inability to bud from the cell membrane and lack of infectivity. Retrotransposons (and some truncated HERVs) do not have the env gene and they too are consequently non-infectious. The retroposons, also called non-LTR retrotransposons, include the SINEs and LINEs. LINEs carry genes for a promotor (P), an open reading frame (ORF1) and pol, and have a polyA tail (AAAA) at their 3'end. SINEs have a promotor region, tRNA and a polyA tail, but do not encode an ORF or pol gene. SINEs consequently are dependent on LINEs for their replicative enzymes.
exogenous retroviruses in that they carry sequences homologous to the retroviral group specific antigen (gag), polymerase (pol) and env genes that are flanked by long terminal repeats (LTRs) (Fig. 1). Briefly, the gag gene codes for the matrix, capsid and structural core proteins of the virus, while the pol gene encodes reverse transcriptase (RT), which copies viral RNA into DNA, and protease and integrase, which facilitate protein cleavage and integration of proviral DNA into the host genome, respectively. Finally, the env gene codes for viral membrane proteins and mediates binding of the virus to its receptor and subsequent entry into the host cell, the necessary first steps in establishing an infection (for an exogenous retrovirus). The LTRs contain inverted repeats, the TATA box, promotors,
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enhancers, polyadenylation signals, trans-activation regions and a tRNA primer binding site [15, 17]. However, in contrast to exogenous retroviruses, HERVs stop short of viral budding and consequently are non-infectious [ 13].
1.2. Discovery of HERV Families The first HERV was identified in 1981 by Martin et al [21]. Over the next 23 years, molecular techniques including screening human genomic libraries under low stringency conditions with DNA probes from animal retroviruses [ 13, 17, 22, 23] and polymerase chain reaction (PCR) using degenerate retroviral primers capable of recognizing all known exogenous retroviruses [24] have facilitated the iden-
Table 1. Classification of human endogenous retroviruses (HERVs) by polymerase (reverse transciptase) gene homologi and tRNA primer binding site~ Class 1:
c:.t~ related Family 1- HERV-HF HERV-H (RTVL-H, RGH) HERV-F Family 2: HERV-RW HERV-W HERV-R (ERV9) HERV-P (I-IuERS-P, HuRRS-P) F_ami!y Y: HERV-ER1 HERV-E (4-1, ERVA0 NP-2) 51-t HERV-R (ERV3) Family 4~ HBRV-T HERV-T (S71, CRTKI, CRTK6) Family 5: HERV-IP HERV-! (RTVL-D HERV-IP-T47D (ERV-FTD) Family 6': ERV-FRD ERV-FRD
Class 2: B- & D-type related, HER V-Kb Family 1: HML-I HERV-K (HML- 1.1) Family 2:HML-2 HERV-K10 HERV-K-HTDV Family 3:HML-3 HERV-K (HML3.1) Family 4:HML-4 HERV-K-T47D Family 5:HML-5 HERV-K-NMWV2 Family 6:HML-6 HERV-K (HML-6p) Family 7:HML-7 HERV-K-NMWV7 Family 8:HML-8 HERV-K-NMWV3 Family 9:HML-9 HERV-K-NMWV9 Family 10: HML- 10 HERV-KC4
"Note that this scheme omits characterized HERVs including HRES-1 and HERV-16. bAll Class 2 HERVs use lysine (K) as their tRNA primer binding site. cGrouped into the ER1 superfamily based on substantial homologies in the polymerase, envelope and group specific antigen genes of murine leukemia virus and baboon endogenous virus.
tification and characterization of over 20 species of HERVs [13] (Table 1). The analysis of human chromosomes and loci, coupled with information and knowledge gained from the human genome project, has further contributed to the identification and study of HERVs. HERVs are classified based on sequence similarity to animal retroviruses [16]. Animal retroviruses, belonging to the virus family Retroviridae, containing subfamilies oncovirinae, lentivirinae and spumavirinae, are classified by morphological and biological features, in addition to the observation of retroviral particles in infected cells, as established by the International Committee for Taxonomy of Viruses [25]. Retroviruses are further divided into A-type retroviruses, which are devoid of an envelope and subsequently are seen only in infected cells, and B-, C- and D-type retroviruses, which are enveloped, produce extracellular particles and consequently are infectious. The classification of both exogenous and endogenous retroviruses, the latter
based on similarity to exogenous retroviruses, has been difficult since many exogenous retroviruses were originally named after multiple investigators/ discoverers, or by the disease they caused or host cells they infected [17], for example, the human T cell leukemia viruses (HTLV-1 and-2). A tentative plan for naming HERVs was based on the single letter amino acid (code) tRNA primer binding site used by the virus (i.e. HERV-K uses AAU, lysine, k) [13, 15, 17]. However, distantly related HERV families use the same tRNA primer binding sites [13] and hence were grouped. Today, HERVs are divided into 3 classes based on RT/pol gene homologies to exogenous animal retroviruses [16]. Class 1 HERVs are related to mammalian C:type retroviruses and are subdivided into 6 families, 3 of which have been grouped into the ER1 superfamily based on homologies to the murine leukemia and baboon endogenous retroviruses in the conserved pol gene, as well as in the gag and env genes [13, 17]. Class 2 HERVs share homologies to mamma-
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lian A-, B- and D-type retroviruses and are divided into 10 families based on pol gene homologies [ 13, 16, 22]. All class 2 HERVs possess a lysine (K) tRNA primer binding site, hence the name HERVK, as do B- and D-type mammalian retroviruses [17]. HERV-K retroelements are termed the most biologically active HERVs [13, 26], have long open reading frames (ORFs) encoding all of the retroviral genes [ 17, 27] and are highly expressed in over 20 human teratocarcinoma cell lines that were derived from teratocarcinomas or embryonic carcinomas [17, 27]. Lastly, class 3 HERVs are related to spumaviruses (foamy virus) and consist of only 1 member, HERV-L [13]. This nomenclature/classification scheme, however, is not without its own limitation, since it omits various HERVs that have already been characterized. In particular, HTLV-1related endogenous sequence (HRES-1) has been implicated as a potential etiological agent in SLE [15, 18, 28, 29], Multiple Sclerosis (MS) [30] and Sjrgren's Syndrome (SJS) [31], and is the source of autoimmunity in MRL/Ipr mice [32-34], as it integrated into the fas gene and thereby prevents expression of Fas protein on various cell types, including activated lymphocytes. HRES-1 is a Class 1 HERV, although it shares only limited homology to the HTLV-1 LTR region [ 13], but is less related to the Class 2 HERV-K endogenous sequences.
CD4 on T cells and macrophages that likely induces the immune abnormalities seen in HIV infection. Gpl20-stimulation of the T cell receptor (TCR) specifically results in tyrosine kinase activation of p561ck, activation of CD4 T cells, internalization (down modulation) of CD4, CD4 T cell anergy and apoptosis, and finally TCR inactivation [19]. In the case of gpl20-stimulation of macrophages, the binding of gpl20 to CD4 mediated Th2-cytokineinduced (IL-6, IL-10, TNFt~) polyclonal B cell activation and increased production of various chemokines, including RANTES and MCP-1, as well as the down modulation of CD4 [19]. Interestingly, IL-16 in SLE patients is thought to act on CD4 in a manner similar to that exerted by gp120 in HIV-infected persons [19]. Again reviewed by Sekigawa et al [19], IL-16 is produced from activated CD8 T cells and serum titers are increased in SLE [35, 36] and HIV infection [37]. Furthermore, increased serum titers of IL-16 are strongly correlated to active disease in some SLE patients [36], and perhaps serve as a marker for these individuals. The IL-16 receptor is located on the CD4 molecule on CD4 T cells and consequent binding by IL-16 may promote T cell activation, anergy [38] and perhaps apoptosis of CD4 cells, resulting in a shift in the CD4/CD8 ratio.
2.2. Low Incidence of HIV Infection in Exposed Individuals with SLE 2. C O R R E L A T I O N S B E T W E E N SLE AND HIV
2.1. Immune Abnormalities There is an impressive array of immune abnormalities in common to patients with HIV and SLE. As reviewed by Sekigawa et al [19], both diseases are characterized by polyclonal B cell activation, a decrease in the CD4/CD8 T cell ratio, T cell anergy, increased expression of major histocompatibility complex (MHC) class II by CD4 and CD8 T cells, defective CD4 and CD8 function and a similar shift in cytokine profile from T helper 1 (Th 1) to T helper 2 (Th2). In both diseases, the increased expression of MHC class II on CD4 and particularly CD8 T cells is associated with disease activity and is indicative of the early activation of T cells, which may lead to anergy [19]. It is the binding of HIV gpl20 to
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Based on prevalence data for both diseases [39], it is estimated that approximately 400 Americans should have both SLE and HIV, as opposed to the reported 20 or so individuals with both diseases [ 19, 40]. Even though there have been numerous cases of HIV transmission with organ transplantation, SLE patients who received unscreened blood from 1978-1983 failed to develop HIV infection [41]. Additionally, in the few patients who have both SLE and HIV, increases in HIV viral load were seen after control of lupus flares with immunosuppressant therapy [42]. On the other hand, flares of SLE were seen following highly active antiretroviral therapy (HAART) for HIV infection in patients with both diseases [40]. Still yet, there have been cases when SLE resolves or improves with HIV infection or progression of HIV-related immunodeficiency [39], most likely as a result of augmentation or depletion
of CD4 T cells. Although the use of immunosuppressant drugs in SLE or HAART therapy for HIV might explain some of these findings in patients with both diseases, the numbers alone raise the possibility that SLE patients produce factors that are protective against infection with HIV since the proportion of people with both diseases is much lower than predicted. Intriguingly, IL-16 in SLE may exert protective effects against infection with HIV. Again, CD8 T cells produce IL-16 and it is thought to act upon its receptor, the CD4 molecule on CD4 T cells, in a manner analogous to that exerted by HIV gpl20. IL-16 inhibits HIV infection in vitro, most likely by repressing the HIV promotor and hence transcription via signaling incurred by the interaction of CD4 and IL-16 [19, 38, 43]. Elevated levels of IL-16 seen in SLE patients [ 19, 36], therefore, may be protective against infection with HIV perhaps by competitive inhibition of binding to CD4 by IL-16, although gpl20 and IL-16 do use different epitopes on the CD4 molecule [19, 43].
2.3. Retroviral-Type Activity in SLE Patients Major lines of evidence in support of a retroviral link to the etiopathogenesis of SLE and other autoimmune diseases include the presence of antiretroviral antibodies and the isolation of retroviral-like particles from autoimmune patients. Patients with SLE [31, 44-47], RA [48], SJS [31, 49, 50] or MS [31] are known to produce IgM and IgG antibodies reactive with various retroviral proteins, including Gag, Env, Nef (negative regulation factor) and the p24 capsid protein. However, in some of these cases, PCR using exogenous retroviral primers failed to amplify sequences related to HIV- 1 or HTLV- 1 [51 ]. Since PCR did not detect the presence of HIV-1 or HTLV-1 in autoimmune patients with measurable antiretroviral antibody titers, scientists were perplexed as to why these patients produced antiretroviral antibodies in the absence of exogenous retroviral infection and questioned what affect, if any, the antiretroviral antibodies had on the SLE disease process. Intriguingly, as many as one third of SLE patients are reported to have antibodies reactive to peptides based on the HIV p24 capsid sequence [44, 45], while up to 52% of SLE patients and 48% of patients with
other autoimmune diseases produce antibodies reactive with the HRES-1 endogenous retrovirus, as compared to approximately 4% of normal individuals [18, 52]. Furthermore, comparative sequence analysis has revealed remarkable sequence similarities between HIV genes, in particular gag, and the genes encoding common nuclear antigens [53]. To be discussed below, molecular mimicry, therefore, between autoantigens and retroviral antigens might explain the presence of antiretroviral antibodies in SLE patients [ 18, 28, 54, 55]. The first reports of retroviral-like particles isolated from the lips or salivary glands of patients with SJS [56, 57], from the synovium of patients with RA [48, 58-60], from the peripheral blood of SLE patients [48, 61] or from cells cultured from patients with MS [62-64] were reported in the 1990s. Additionally, detection of an atypical interferon characteristic of lentiviruses and RT activity in the supernatant was induced when lymphocytes from SLE patients were cultured [65, 66], perhaps indicating that retroviral gene products are involved in the SLE disease process. Thus, first the observation of antiretroviral antibodies in autoimmune patients and second the isolation of retroviral-like particles from autoimmune patients, spurred investigators to search for a retroviral etiology for SLE and other autoimmune diseases.
2.4. Retroviral Influences in SLE Murine Models Two mouse models for SLE, in particular the NZB/ W and MRL/lpr models, point to a strong retroviral influence in the etiopathogenesis of disease. Of interest with regards to both of these models is that they produce pathogenic antibodies to the MuLV-related Env protein, gp70 [ 11, 67, 68], which is involved in immune complex deposition in the kidneys [67, 69]. NZB/W mice exhibit severe proliferative glomerulonephritis resulting in thickening of the basement membrane and obliteration of the capillary lumina as a consequence of immune complex deposition, in addition to other autoimmune phenomena [34, 70]. Furthermore, the isolation of a HERV Env protein related to MuLV in deposited immune complexes of NZB/W kidneys suggests that the protein is significantly involved in pathogenesis, in particular glomerulonephritis [11, 67, 68, 70, 71]. Addition-
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ally, cDNA microarray analyses of renal cortex RNAs taken from NZB/W mice and NZW control mice, identified the most up-regulated gene (5.5fold as compared to NZW mice) as corresponding to the endogenous MuLV-related to the Duplan retrovirus (EDV, L08395) [70]. In addition to microarray analyses, histopathology demonstrated that the increased expression of the EDV transcript occurred by ,4-8 weeks of age, prior tothe onset of inflammation in the kidneys of NZB/W mice [ 11, 70], thereby indicating that increased expression of EDV is not aresult of and:actually proceeds inflammation [70]. Thus, these findings suggest that proteins translated from the MuLV-related EDV are involved in deposited immune complexes that result in glomerulonephritis in NZB/W mice. MRL/lpr mice demonstrate direct evidence of a HERV influence, specifically HRES-1, in the etiopathogenesis of autoimmune disease. The autoimmunity in MRL/lpr mice results from the integration of HRES-I into the fas gene, located on chromosome 1 at positionq42 (Clq42) [32, 33]. Integration of HRES-1 results in decreased expression of Fas protein on cell surfaces and the consequent lack of apoptosis in certain cell populations, including activated T lymphocytes [32, 33]. Activation of Fas by its ligand initiates the necessary signaling for apoptosis of cells expressing Fas, most likely via activation-induced cell death (AICD) [32, 33, 69]. The region at Clq42 in the mouse has been identified by repeat linkage analyses as a region conferring predisposition to SLE-like disease in NZB/W mice [8, 34, 72]. Interestingly, Clq42 has been identified as a susceptibility region for human SLE as well [8-10, 32, 33, 73, 74], although it is not the gene for human fas.
3. MECHANISMS OF AUTOIMMUNITY INDUCED BY HERVS There are a variety of proposed mechanisms by which HERVs may initiate and/or perpetuate autoimmune responses (Fig. 2). Although the major focus here is molecular mimicry between HRES-1 and the common autoantigen the small ribonucleoprotein complex (snRNP) in the etiopathogenesis of SLE, other potential mechanisms and a limited number of examples utilized by HERVs in the
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initiation of various autoimmune or inflammatory processes are explored, briefly. However, a point worth making is that no single HERV is unique to any particular autoimmune disease, but rather different autoimmune diseases share various features, potentially those brought about by HERVs. The concepts of HERV-encoded superantigens and skewing of the V~ T cell repertoire in MS and Insulin-Dependent Diabetes Mellitus (IDDM) are discussed, as are cases of retroviral integrations into genes that are critical in the control and regulation of the immune system. Still yet, the concepts of HERV-encoded cis- or trans-regulatory elements and the immunosuppressive effects of HERV proteins are considered. 3.1. Superantigens Superantigens are non-processed, non-MHC restricted peptides that are produced by many bacteria, mycoplasmas and viruses. They bind to conserved regions of the MHC class II molecule, outside of the classic peptide-binding groove, and specific TCR 13-chain variable (V]3) regions, irrespective of the antigen-specificity of the TCR. The selective expansion or deletion of T cells with specific V~ regions is an inherent feature of superantigens and they are capable of rapidly activating 106 more T cells than would be activated by presentation in the classic peptide-binding groove [75]. Activation in this fashion may lead to oligo- or polyclonal activation of certain VI3 subsets, leading to cytokine and chemokine production, systemic toxicity and suppression of the adaptive immune response. Superantigen-encoded TCR V~l expansion is associated with MS [76], RA, SLE and IDDM [77-80]. An enrichment of V137 T cells was found in pancreatic islet cells from patients with acute-onset IDDM [78]. Interestingly, these authors [78] isolated a novel HERV, (1,2) termed IDDMK(1,2)22, a member of the HERV-K family, from patients with IDDM. Conrad et al [78] hypothesized that the expansion of VI]7 T cells was induced by the IDDMK(1,2)22-encoded superantigen and that these T cells were involved in islet cell destruction. Although other groups have failed to duplicate these findings [81-87], the results are still intriguing. Genetic mapping has identified another HERV, HERV-K18, with 99.5% sequence homology in
Figure 2. The multi-factorial etiopathogenesis of SLE. Environmental, genetic and retroviral factors contribute to the overall SLE phenotype. Genetic factors are indicated by familial association and MHC haplotype, as well as the extreme gender imbalance seen in SLE. Retroviral factors are indicated by the presence of antiretroviral antibodies and retroviral-like particles in SLE patients. Significant regions of protein sequence homology between retroviral proteins and autoantigens exist and explain the presence of antiretroviral antibodies in SLE patients. Antiretroviral antibodies may serve to increase protection against infection with HIV in patients with SLE. Additionally, linkage analyses have identified a SLE susceptibility locus on chromosome 1 at position q42 (Clq42) that contains the well-characterized HRES-1 HERV. Environmental factors such as UV light, physical and/or emotional stress and the use of certain drugs may act upon genetic and retroviral factors by increasing transcription of HERVs. the 3'LTR of the env region to that encoded by IDDMK(1,2)22 [88]. It is located in the first intron of the CD48 gene on chromosome 1 and has three allelic env forms, all of which demonstrate superantigen-type activity by mediating the rapid expansion of V~7 T cells [79, 88]. Interestingly, the chromosomal region containing CD48/HERV-K18 has been identified as a susceptibility region for IDDM [89] and peripheral expansion of V137 T cells occurs prior to the onset of clinical disease [90]. Additionally, since CD48 is one of two ligands for CD2, it is conceivable that HERVs mediate expression of cell surface markers and thus may be intimately involved in the etiopathogenesis of !DDM, perhaps by impaired CD4 T cell activation [88]. Furthermore, IFN-cz was shown to up-regulate transcription of the HERVK18 env gene, resulting in the rapid expansion of
V137 T cells [79]. Since IFN-cz is produced from virus-infected cells, these findings suggest that viral infection may lead to expansion of V137 T cells by increased transcription of the HERV-K18 superantigen. The HERV-KI8 superantigen was also shown to selectively expand V~13 T cells [80], which are also correlated with IDDM [90]. A HERV-encoded superantigen may be associated with T lymphocyte immunopathophysiology in MS. MS associated retrovirus (MSRV), isolated from B lymphocyte cultures derived from MS patients or directly from cerebral spinal fluid (CSF) [62, 91], is a member of the class I HERV-RW family [92]. To investigate the possibility of MSRVmediated immunopathology through a superantigen-type mechanism, Perron et al [76] analyzed in vitro whether infection of PBLs from non-MS
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individuals with MSRV particles or a recombinant MSRV Env protein resulted in the selective expansion or deletion of T cells bearing a particular V[3 chain. It was found that a significant polyclonal expansion or deletion of V~ 16 T cells was induced following inoculation of PBLs with MSRV particles, regardless of MHC class II haplotype [62]. Furthermore, polyclonal modification of V~16 and V[~17 T cell populations was seen after inoculation of PBLs with recombinant MSRV Env protein [62]. Thus, the immune response induced by MSRV is abnormal in that it is characterized by the polyclonal activation of na'fve T cells bearing a particular V~ chain independent of MHC class II haplotype, thereby suggesting that the HERV protein is critical to the superantigen-type activity seen in MS.
3.2. lnsertional Mutagenesis There are several examples where HERVs have integrated into or nearby genes that are critical in the control of the immune system, including fas, complement and the MHC. The MRL/lpr murine model for SLE provides such an example as HRES1 integrated into the murine fas apoptosis-promoting gene, resulting in decreased expression of Fas protein and the consequent failure of apoptosis in activated autoreactive lymphocytes [32, 33, 93]. Hence, the MRL/lpr model provides direct evidence of immunopathology as a consequence of retroviral integration. 3.2.1. MHC class I and H genes
Numerous HERVs, as well as various other retroelements, are found within the classical MHC genes [22, 94-96]. Kulski et al [95] analyzed 16 HERV sequences belonging to the HERV-16 (11 copies), HERV-L (1 copy), HERV-I (2 copies), HERV-K91 (1 copy) or HARLEQUIN (1 copy) families within 656kb of genomic sequence obtained from the ~and [3-blocks of the MHC class I region. The HERV16 copies most likely arose as a result of duplication of genomic sequences containing the human MHC class I and PERBII (MIC) genes, while sequences related to the other HERV families probably arose following duplication after a single insertional event or translocation [96]. Additionally, 4 of the 11 copies of HERV-16 and the single copies of HERV-I
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and HARLEQUIN appear to have receptors facilitating the insertion of other retrotransposons [96], a mechanism to further increase the diversity and polymorphism of the MHC. In addition to providing evolutionary clues, the identification and characterization of HERVs within the MHC class I locus is of particular interest since this region is rich in polymorphic genes that have been associated with various autoimmune or inflammatory diseases, as well as with disease susceptibility following viral infection [97]. Chimpanzees, for example, have a large deletion/transposition in the MHC class I region [98], which probably includes the HERV-L, HERV16 or PERB 11 (MIC) sequences [95]. In contrast to the immune response to HIV in humans, chimpanzees that are actively infected with simian immunodeficiency virus (SIV) are capable of mounting an effective antibody response, preventing progression to AIDS [99]. Therefore, it is possible that the deletion of genes or HERVs in the MHC class I region in chimpanzees may influence susceptibility and progression to AIDS [95, 98]. HERV integrations into the MHC class II region have also been reported and are a factor influencing the polymorphic nature of the various DR haplotypes in particular [22, 94]. Distributed throughout the class II region are numerous (H)ERV9 LTRs that may be transcriptionally active in various cell types [22, 100]. Moreover, the (H)ERV-9 LTRs in the DR region contain regulatory elements capable of mediating retroviral basal and tissue-specific transcription, perhaps by functioning as IFN-y responsive elements [94]. Interestingly, IFN-y is the most potent inducer of gene expression in the DR region and consequently the (H)ERV-9 LTRs might act as IFN-y specific/responsive enhancers for the DR genes [79]. 3.2.2. Complement genes
Inherited deficiencies in components of the classical complement system are associated with SLE, RA and scleroderma [ 101,102]. The ability to clear pathological immune complexes and infectious organisms may occur in a state of complement deficiency, resulting in tissue deposition particularly to the basement membranes of the kidneys, as is seen in lupus nephritis. The most characterized inherited complement deficiency is the partial deficiency of
complement component C4 [ 101, 103,104]. The two isotypes of C4, C4A and C4B, differing by only five nucleotides, are encoded by two very polymorphic genes within the non-classical MHC class HI region [102]. Numerous alleles for both isotypes have been identified, including the null alleles C4AQO and C4BQO. The inheritance of at least one of the C4AQO null alleles occurs in as many as 50% of SLE patients, as compared to 25% of controls [ 101, 103] and is associated with certain ethnic groups [ 105]. Furthermore, a 30kb deletion of most of the C4 gene and the adjacent 5'-21-hydroxylase-A (21OHA) pseudogene is thought to account for twothirds of C4A deficiency in Caucasian SLE patients with the MHC haplotype B8-C4AQO-C4B1-DR3 [102]. Intriguingly, the C4A and C4B genes contain the complete 6.4kb sequence of HERV-K(C4), present in intron nine and absent in the case of the C4A/21-OHA deletion [106]. HERV-K(C4)-related sequences have also been found in the second intron of the C2 complement gene [ 107].
Furthermore, the translated cORF protein accumulates in the nucleolus, indicating that cORF harbors a functional nucleolar localization signal, as do Rev proteins [108]. With respect to functioning as a cisor trans-regulatory element, the HTDVTHERV-K Rev-related cORF protein may exert a pathogenic role by activating or suppressing genes involved in cellular growth and/or immune function. In another study, Horwitz et al [ 109] cloned a family of endogenous HIV-related sequences from the DNA of normal humans, chimpanzees and rhesus monkeys by low-stringency Southern blot hybridization and plaque screening. They identified a protein, termed EHS-2, similar in size, amino acid composition and structure to the arginine-rich RNA binding domain of Rev, complete with nucleolar localization motif [109]. Thus, the findings of HERV encoded Revrelated proteins potentially capable of trans-activating viral gene expression indicates that HERVencoded transcriptional transactivators related to Tat in lentiviruses or Tax in HTLV may exist.
3.3. Cis/Trans Activation
3.4. Immunoregulatory Proteins/Peptides
Although there are no definitive examples of cisor trans-activation of cellular genes by HERVs, there is presumptive evidence in support of such an event. Lower et al [108] explained that the human teratocarcinoma-derived particles (HTDV) present in numerous human teratocarcinoma cells lines are encoded by HERV-K (HTDV/HERV-K). Two forms of HTDV/HERV-K proviral genomes exist, type I and type II, differing by the absence (type I) or presence (type II) of nucleotides encoding the amino-terminus of the env gene and a putative signal sequence that overlaps the carboxyl-terminus of the pol gene. In type I HTDV/HERV-K genomes, the pol and env genes are fused, the proviruses are defective in env splicing and full-length transcripts therefore accumulate. On the other hand, type II transcripts are spliced, resulting in subgenomic env mRNA and a short open reading frame (cORF) of 14KDa with some sequence, structural and functional similarities to the RNA binding and effector domains of the lentivirus rev gene [108]. Like Rev, cORF contains an arginine-rich basic motif at its amino-terminus and a leucine-rich motif at its carboxyl-terminus, homologous to the Rev RNA binding and effector domains, respectively [108].
In addition to directly affecting cellular gene expression by integration, HERV gene products may contribute to autoimmune processes by their actions on cellular genes and physiology. The conserved transmembrane Env protein of C-type mammalian retroviruses and class I HERVs, p l5E, exerts an immunosuppressive effect on monocytes and lymphocytes in vitro [ 110-113] and in vivo [ 114, 115]. Haraguchi et al [ 115] demonstrated that a synthetic peptide corresponding to the p l5E Env protein, CKS-15, suppressed stimulant-induced mRNA expression of the Thl cytokines IL-2, IL-12 and IFN-% but did not suppress the Th2 cytokines IL-4, IL-5, IL-6 and IL-13. Interestingly, densitometric analyses of the RT-PCR products showed that CKS17 peptide up-regulated mRNA accumulation of ILl0, a cytokine capable of inhibiting cell-mediated immunity [116-118], while inhibiting stimulantinduced mRNA expression of IL-12 [115], a critical cytokine that induces Thl responses and inhibits Th2 responses [ 119, 120]. Furthermore, Cianciolo et al [ 121] found that a region of the HIV transmembrane Env protein, gp41, is homologous in sequence to p 15E and exerts inhibitory effects on lymphoproliferative responses when stimulated with anti-CD3
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monoclonal antibody and IL-2. Additionally, they found that human neoplastic effusions contain proteins that are potent inhibitors of monocytes and are detected with anti-pl5E antibodies [121, 122]. These results suggest that pl5E and/or similar HERV-derived peptides may be involved in the immune dysregulation associated with autoimmune disease, HIV infection or cancer.
4. M O L E C U L A R MIMICRY IN THE ETIOPATHOGENESIS OF SLE An additional mechanism by which HERVs or HERV gene products initiate autoimmunity is by mimicry of host structures, sometimes leading to pathological autoantibody production. The literature now supports molecular mimicry between common autoantigens and retroviral proteins as a significant contributor to the overall SLE, MS and SJS autoimmune/inflammatory phenotypes [ 15, 28-31, 53, 54]. For example, database searches have identified a region of the MHC class I E antigen as having extremely high homology to the MuLV-related HERV-E clone 4-1 gag region [54], suggesting that HERV clone 4-1 transposed into the MHC class I E antigen. Interestingly, 48% of Japanese SLE patients have antibodies reactive with the clone 4-1 Gag region, while 11% have antibodies to the Env region [54]. These antibodies were not detected in normal individuals [123, 124]. The class I HERV, HRES-1, is of particular interest to SLE, as well as to MS and SJS, and is proposed to participate in autoimmune processes via its remarkable cross-reactivity with a Gag-related region of the 70KDa (U1) component of snRNP ((U1)snRNP) [18, 53], a common autoantigen associated with SLE, scleroderma and polymyositis [125]. Autoantibodies to snRNP, as well as to other nuclear proteins, are associated with immune complex formation, pathological tissue deposition and the recruitment of inflammatory cells and complement [126, 127]. We [28] and others [18, 53, 128-131] believe that molecular mimicry between HRES- 1 and (U 1)snRNP initiates the production of autoantibodies cross-reactive with both proteins, and that these autoantibodies induce pathology by the formation of immune complexes that subsequently result in tissue deposition and may serve to constantly fix complement.
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4.1. HRES-1
HRES-1 integrated into the human genome during the time of the Old World Primates, most likely as an exogenous and as of yet unidentified retrovirus [31 ]. HRES-1 is transcriptionally active, contains a tRNA primer binding site, a polyadenylation signal, a TATA box, an HIV-1 trans-activation region, inverted repeats and is expressed in a tissue-specific manner [15, 18, 29, 131]. It has been mapped to Clq42, a region identified as a murine [8, 72] and human [8, 10, 74, 132] lupus susceptibility region (discussed below), and is present as a single haploid copy [31]. HRES-1 was (one of) the first HERV(s) identified as having ORFs with the capacity to encode a functional protein [31]. The 28KDa protein encoded by HRES-1 is believed to serve as an autoantigen for Gag-reactive antibodies in patients with autoimmune disease [ 18, 29, 31]. Another relevant feature of HRES-1 that pertains to SLE-like autoimmunity is the consequence of its integration into the murine fas apoptosis-promoting gene in MRL/lpr mice [32, 33, 93]. These mice develop severe lymphoproliferative disease and systemic autoimmune disease similar in features to human SLE [32, 133, 134]. 4.2. Anti-snRNP/HRES-1 Antibodies in SLE
The early 1990s noticed the association of antiretroviral antibodies in autoimmune disease [31, 44, 48, 60, 123, 135-139]. Approximately one-third to one-half of patients with SLE or SJS had circulating antibodies reactive to the HIV-1 p24 capsid protein, as well as to the Gag, Env and Nef proteins of HIV-1 [ 18, 44, 45, 50, 136], in the absence of retroviral infection [ 18, 51 ]. With regard to anti-HRES- 1 antibodies specifically, up to 52% (50/96) of SLE patients had antibodies to HRES-1 [ 18, 31 ], as compared to 3.6% (4/111) of normal donors and none of the 92 patients with either asymptomatic HIV or clinical AIDS [ 18]. It thus appears that anti-HRES- 1 antibodies are present in a majority of patients with SLE, but are not present in patients infected with HIV. Furthermore, Perl et al [18] demonstrated a correlation between the presence of antibodies to HRES-1 and the presence of antibodies to snRNP such that SLE patients with antibodies to HRES-1 were 2.3 times as likely to have clinically active
70KDa snRNPI 5 0 - D P R D A P MoMuLV p30Gag i p30Gag/snRNP~ HRESpl9] 14 HRESp24U
P P T R A E T R ~ E E ' R M ! E R K R R : : E K I,E R R Q Q - 8 0 5 2 3 -ETPEEI~E~R I R.RiE T E E K E - 5 4 0 { - PTRAPSGPRPP24 117 - ~ ~ G
PDRS
PR- 12 7
Figure 3. Sequence alignment of snRNP, p30Gag and HRES-1 peptides. Comparative sequence analyses have revealed regions of amino acid sequence homology between a Gag-related region (p30Gag) of the 70KDa U 1 small ribonucleoprotein complex ((U1)snRNP) (residues 50-80), the Gag region of the Moloney Murine Leukemia virus (MoMuLV) (residues 523-540) [53] and peptides based on the p19 (HRESpl9) and p24 (HRESp24) Gag proteins of HTLV-1 related endogenous sequence (HRES-1) [18, 31]. Residues in agreement between snRNP, p30Gag and HRES-1 are shaded in dark gray boxes and the cross-reactive consensus epitope is listed as the third sequence from the top. The fight gray shaded boxes represent homologous residues between snRNP and the HRESp 19 Gag peptide. Italicized residues in the HRES-1 peptides are residues homologous to the p19 and p24 Gag regions of HTLV-1 [31]. disease [ 18]. The (U1)snRNP protein contains regions with amino acid sequence homology to a portion of the retroviral Gag protein (p30Gag) [53]. Using a synthetic snRNP peptide based on the 70KDa (U1)snRNP protein, Query and Keene [53] demonstrated that when snRNP peptide was pre-incubated with anti-p30Gag serum, the anti-p30Gag serum lost its ability to bind the (U 1)snRNP protein, while reactivity to p30Gag persisted [53]. Additionally, the snRNP peptide blocked binding of affinity purified anti-snRNP antibodies to both p30Gag and the snRNP protein [53]. It therefore appears that the region of homology between (U1)snRNP and p30Gag is responsible for the immunological cross-reactivity between the two proteins (Fig. 3). Comparative sequence analyses have also revealed regions of homology between the 28KDa HRES-1 protein and the Gag-related region of (U1)snRNP [18, 31, 131]. Using synthetic HRES- 1 peptides related to the p 19 and p24 Gag proteins of HTLV-1, Banki's [31] and Perl's [18] research groups have confirmed that the HRES-1 peptide sequences are indeed immunogenic and represent antigenic epitopes cross-reactive with the HTLV-1 Gag protein. In particular, the HRES-1 p24 Gag peptide contains a highly charged RRE-domain (RRE: Arg, Arg, Glu) homologous with snRNP and HTLV-1 p24 Gag [ 18]. Interestingly, a rabbit antibody raised against the HRES-1 p24 Gag peptide recognized a peptide based on the 70KDa (U1)snRNP RREdomain (residues 67-77 of (U 1)snRNP), but did not react with a (U1)snRNP peptide lacking the RREdomain [18]. These results suggest that it is the
RRE-domain of HRES-1 and snRNP that comprise the cross-reactive epitope. In this regard, it is conceivable that molecular mimicry between HRES-1 and snRNP serves as one of the priming mechanisms to initiate an ongoing autoimmune response in SLE. Still yet, molecular mimicry between HRES-1 and snRNP explains both the association of antiretroviral antibodies in SLE patients and a mechanism for their generation.
4.3. HRES-1 and a SLE Susceptibility Region A susceptibility locus for murine and human SLE has been mapped using microsatellite markers to Clq42 [8-10, 72, 74], the integration site for HRES-1. The study of human chromosomal regions syntenic to murine susceptibility loci, coupled with information gained from the genome project and genome-wide scans of both species, has facilitated the study of susceptibility regions associated with human disease [6]. For example, genes associated with glomerulonephritis and antibodies to chromotin, histones and DNA have been mapped to the telomeric end of murine chromosome 1 [140-143]. Since SLE-like susceptibility genes may have been conserved between humans and mice, Tsao et al [8] analyzed the human chromosomal region (Clq31q42) syntenic to the murine lupus susceptibility region in 52 SLE-afflicted sibpairs from 3 ethnic groups and found a 15cM region at Clq41-q42 that was linked to disease. Within the mapped 15cM region at C lq41-q42, an estimated 500 genes are encoded, some of which may have relevance to SLE (TGF-132, ADPRT and HLX1) [8]. Moreover, it is
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highly probable that an unidentified disease-causing gene or genes within this region at C lq41-q42 are associated with SLE pathology. Since HRES-1 is located within the 15cM region at C lq41-q42 identified by Tsao [8] and others [4, 9, 10, 74] as being linked to SLE, perhaps then HRES-1 plays a vital part in contributing to the Clq42 SLE susceptibility region in both mouse and man.
4.4. Polymorphic Genotypes of HRES-1 Correlate with SLE Disease Activity A polymorphic HindlII site defines two alleles of the HRES-1 genomic locus [29, 131]. In order to determine if allelic variation in the HRES-1 locus was associated with SLE, Magistrelli et al [29] used Southern blotting and PCR to characterize the polymorphic HindlII site mapped in the LTR of HRES-1. The probe used differentiated between three HRES1 genotypes: I) 5.5kb fragment only, II) 3.7kb and 1.8kb fragments only and III) all three fragments [29]. Interestingly, the frequency of genotype I with respect to genotype III was 3.1-fold lower in SLE patients, as compared to control donors, while genotype II was the least prevalent in all groups [29]. Likewise, the relative frequency of genotype 11I with respect to genotype I was increased significantly in SLE patients. Additionally, the presence of anti-HRES-1 antibodies was increased in genotype III SLE patients and diminished in genotype I patients [131]. These findings raise the possibility that the genotype I HRES-1 allele is protective against SLE-mediated autoimmunity.
5. CONCLUSIONS From an evolutionary perspective, the finding that upwards of fifty percent of the human genome is encoded by retroelements is extraordinary [12]. More than that bestowed by any other single entity, the conglomeration of genes, polymorphisms, pseudogenes and endless additional features contributed by retroelements have worked to build and shape the human genome. Intriguingly, the human genome can be considered in part a composite of ancient retroelements, most of which inserted prior to the emergence of vertebrates [ 12, 144]. With regard to retroviral mediators of autoimmune/inflammatory
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diseases, coupled with genetic and environmental factors, it is clear that retroelements, in particular HERVs, play a vital and significant role in the etiopathogenesis of such diseases. Due to the genetic diversity of exogenous and endogenous retroviruses, it has been difficult to delineate or identify specific retroviruses associated with specific autoimmune or inflammatory diseases. However, sequencing of the human genome and genome-wide scans of both humans and mice support the hypothesis of a HERV based etiopathogenesis for SLE. Specifically, we propose that molecular mimicry between the Gagrelated region of (U 1)snRNP and HRES-1 initiates the production of cross-reactive autoantibodies and associated immune complexes. In addition to molecular mimicry, other mechanisms of autoimmunity brought about by HERVs include but are not limited to insertional mutagenesis, superantigen-type activity, cis- or trans-regulation of cellular genes and immunomodulation by HERV gene products. Continued research in the field of autoimmune disease and HERVs as etiological factors will certainly facilitate the understanding of the very complex disease, SLE, as well as other autoimmune or inflammatory diseases.
ACKNOWLEDGEMENTS This work was supported in part by NIH grant #AI460 and Arizona Disease Control Research Commission (ADCRC) grant # 5018 to JJM. We are extremely appreciative of the advice and guidance given by Samuel E Schluter, Ph.D., on many areas pertinent to this chapter. Thanks also to Nafees Ahmad, Ph.D., for his expertise in the field of Virology.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Sj/igren's Syndrome - Autoimmune Epithelitis: Role of Coxsacldeviruses in Pathogenesis Dimitrios A Liakos, Efstathia K. Kapsogeorgou and Haralampos M Moutsopoulos
Department of Pathophysiology, Medical School National University of Athens, Athens, Greece
1. INTRODUCTION Sjrgren's syndrome (SS) or autoimmune epithelitis is a chronic autoimmune disorder characterized by inflammation of the salivary and lacrimal glands resulting in xerostomia and keratoconjuctivitis sica [1 ]. The disease can be seen as an entity alone (primary SS) or in association with other rheumatic autoimmune diseases (secondary SS). The prevalence of the syndrome is about 1%-2% in the adult population [2], and primarily affects females (9:1 female to male ratio) in their fourth and fifth decade of fife. Symptoms of the disease may appear six to eight years prior to the full blown clinical development of the syndrome. Progression is slow and usually involves glandular tissues. About one third of the patients can develop some extraglandular manifestation including interstitial renal disease, bronchitis sicca, autoimmune liver disorder and vasculitis. Furthermore, about 5% of patients may develop [3-cell lymphoma. Malignant lymphoma development is a major complication of the disease. The risk of lymphoma development in patients with primary SS is 40 times higher compared to the normal population and is associated with increased mortality [3].
2. CLINICAL PICTURE (REVIEWED IN REF. [21) Sjrgren's syndrome is characterized by dryness of the mouth (xerostomia) caused by decreased production of saliva. Salivary flow rate is used for the evaluation of major salivary gland function as
well as scintigraphy and digital subtraction sialography. Age, gender, medication and psychological factors may influence salivary flow measurements. In patients with xerostomia, the oral mucosa can be sticky, dry and erythematosus. Patients report problems with chewing and swallowing due to the dryness of the mouth. The tongue may be dry, with deep fissures and atrophic papillae. Mouth dryness can lead to fungal infections that can result to angular chelitis and fungal overgrowth on the tongue. Recurrent carries is a problem that is often reported, as well as discomfort with dentures. Major salivary gland enlargement occurs in patients with primary SS and although it may initially appear unilaterally, in most cases it develops bilaterally. Chronic eye dryness leads to irritation and destruction of the corneal and conjuctival epithelium. Tear secretion rate is measured by the Shirmer's test while staining corneal and conjuctival epithelial tissues with Rose Bengal and other stains reveals the extend of epithelial destruction. About one third of Sjrgren's syndrome patients at some point in the course of the disease display some extraglandular systemic manifestation. Raynaud's phenomenon precedes sicca manifestations and is found in 35% of patients. Vasculitis of the skin is presented with palpable purpuric or petechial lesions. Musculoskeletal involvement includes arthralgias, myalgias, fatigue and morning stiffness. Nonerosive arthritis and symmetric polyarthritis can be observed. In patients with primary SS, respiratory tract involvement is mild but frequent. Dry cough is quite common and is caused by xerotrachia or bronchitis sicca. Dysphagia might also occur as a result of dryness of the pharynx and esophagus. Liver
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involvement is rare (5%) in patients with primary SS and presents either as primary billiary cirrhosis or chronic active hepatitis. About 4% of Sjrgren's syndrome patients have clinically significant renal involvement in the form of interstitial nephritis or glomerulonephritis. Peripheral sensory or sensorymotor polyneurpathy and mononeuritis multiplex occurs in 1% to 2% of patients. Furthermore, anxiety, depressed mood and personality structure disorders are frequently observed [3]. In primary Sj/Sgren's syndrome, malignant non-Hodgkin lymphoma occurs in 4% to 6% of patients. Lymphoma usually develops later in the course of the disease. Extranodal localization is quite common and is found in the salivary glands in 55% of lymphoma patients. The presence of parotid gland enlargement, palpable purpura, low C4 levels and mixed monoclonal cryoglobulinemia at the first visit are adverse prognostic factors and adequately distinguish high risk patients for the development of lymphoproliferative disorders. Patients with primary SS and adverse prognostic factors display increased mortality compared to the general population (mortality ratio: 1.15) and one to five deaths of patients with primary SS are attributed to lymphoma. The presence of the adverse prognostic factors is strongly correlated with the increased mortality.
3. IMMUNOPATHOLOGY (REVIEWED IN R E E [4]) The aetiology of Sjtigren's syndrome remains unknown. Even though environmental influences and different genetic factors are related to the disorder, no single factor can be identified as responsible for the pathogenesis of the syndrome. It is considered an autoimmune disease due to the presence of autoantibodies and the focal lymphocytic infiltrations that are observed in the lesions. Furthermore, the association of SS with other autoimmune disorders supports the autoimmune nature of the disease. Sjrgren's syndrome is characterized by B-cell reactivity and destruction of exocrine glands associated with dense lymphocytic infiltrations. B-cell activation is the most prominent immunologic feature of Sjtigren's syndrome. B-cells infiltrating
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Figure 1. Advancedlymphocyticinfiltration in labial minor salivary gland biopsy. The bellow the image of the lesion is a list of the cells that participate in the lesion and their characteristics.
the minor salivary glands are activated since they produce increased amounts of immunoglobulins with autoantibody reactivity. Rheumatoid factor (RF) and antinuclear antibodies (ANA) are found in high frequencies in Sjrgren's syndrome patients. The analysis of specificity of anti nuclear antibodies reveals the presence of antibodies against two ribonucleoproteins, Ro(SSA) and La(SSB). While the function of Ro(SSA) remains unknown, La(SSB) is known to participate in transcription termination of RNA polymerase III and also in the translation of viral RNA. Another autoantigen in Sjrgren's syndrome is a cytoskeletal protein a-fodrin. Oligoclonal B-cell expansion takes place early in the development of the disease. Monoclonal light chains are detected in higher frequencies in patients with systemic involvement compared to patients with glandular disease. Furthermore, about one third of the patients with primary SS have high levels of mixed monoclonal/polyclonal type II cryoglobulins with rheumatoid factor activity [5]. It is possible that B-cell neoplastic transformation takes place in the gland possibly due to the chronic immunologic stimulation. This transformation probably takes place along with immunoglobulin gene rearrangements and mutations in the p53 transcription factor [6]. The lesion that is observed in labial minor salivary gland biopsies of Sjrgren's syndrome patients is characterized by the round cell infiltrates that in
Table 1. Aberrantexpression of molecules implicated in epithelial cell activation in Sjtren's syndrome Minor salivary gland biopsies
Salivary gland cultured epithelial cells
Activation markers
Protooncogenes
Not studied
Immune reactive molecules
MHC I and MHC II B7 costimulatory molecules Adhesion molecules Chemokines CD40 Proinflammatory cytokines
MHC I and MHC II B7 costimulatory molecules Adhesion molecules Chemokines CD40 Proinflammatory cytokines
Apoptosis-related molecules
FAS, FAS Ligand
FAS, FAS Ligand
early lesions surround ductal epithelial cells. As the lesions progress, the infiltrate extends and replaces the functional tissue of the salivary gland (Fig. 1). The majority of the cells are T-cells while B-cells represent about one fourth of the infiltrating cells. The majority of T-cells are CD4 positive, express the memory/inducer marker and the lymphocyte function associated molecule (LFA-1); a cell surface glycoprotein that has been associated with adhesion of lymphocytes and macrophages. Monocytes, macrophages and natural killer cells represent less than 5% of cells in the pathologic lesion. Dendritic cells, the other classical antigen presenting cells, are only found in advanced lesions. These cells express the DRC cell surface marker, are located among B and T lymphocytes and represent about 2% of mononuclear cells that are present in the lesion. This reduced number of professional antigen presenting cells suggests that this role is probably played by some other cell type in the pathologic lesion. Clinical and pathophysiologic findings show that the inflamed tissue in affected organs of Sj6gren's syndrome patients is epithelium. The epithelium plays a central role in the initiation and perpetuation of immune response. This is attested by a series of immunopathologic findings that show aberrant expression of various activation and immune response associated molecules in epithelial cells of minor salivary gland biopsies (Table 1). These cells express molecules implicated in antigen presentation, such as MHC class I (HLA-ABC), MHC class II (HLA-DR) and B7 costimulatory molecules. In addition, there is upregulated expression of mol-
ecules that mediate B and T-cell recruitment as well as molecules that are involved in the expansion of the immune response. These molecules are adhesion molecules, lymphoattractant chemokines and proinflammatory cytokines. Furthermore, the activated state of epithelium is attested by the overexpression of apoptosis related molecules (FAS, FAS ligand). Increased rates of epithelial apoptosis result to the release of intracellular antigens that are recognized by the immune system. These findings were substantiated by the detection of increased constitutive expression of the same molecules in long term cultured non-neoplastic epithelial cell lines established from SjOgren's syndrome patients' salivary glands (Table 1) [2, 7]. The establishment of long-term salivary gland epithelial cell cultures revealed the capacity of these cells to interact with immune cells through the expression of functional costimulatory molecules (B7 and CD40) [7, 8]. Moreover, it has been shown that salivary gland epithelial cells can costimulate the growth of activated T-cells, indicating that epithelial cells are able to participate in the antigen mediated activation and proliferation of the immune response. It is therefore very likely that there are intrinsic activation processes active in affected epithelial cells. The above data indicate that epithelial cells are suitably equipped to act as antigen presenting cells and support their central role in the pathogenesis of Sj6gren's syndrome.
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oo
4. VIRUSES AND SJOGREN'S SYNDROME The implication of viruses in the development of Sj6gren's syndrome has long been suspected. There is no clear evidence so far that proves viral induction of Sj6gren's syndrome. However, there are data that link viruses and the disease. It has been shown by some groups that Sj6gren's syndrome patients express EBV-associated antigens in their salivary glands and also display an increased content of EBV-DNA in their saliva [9]. This coupled to the fact Herpesviruses like cytomegalovirus and EpsteinBarr virus (EBV) can replicate in the salivary glands can be interpreted as an indication of viral involvement in the development of the disease. This hypothesis has been challenged by other groups that have demonstrated that the frequency of EBV detection in Sj6gren's syndrome patients is similar to the frequency observed in normal populations [10, 11 ]. Another link between viruses and the disease comes from the fact that chronic lymphocytic sialadenitis that is linked to viral infection is histologically very similar to Sj6gren's syndrome and can be found in 14% to 50% of hepatitis C virus infected patients [12]. HCV RNA has been detected in salivary glands of patients with chronic HCV infection by in situ hybridization [ 13]. Furthermore, sialadenitis has been reported in transgenic animals carrying the HCV envelope genes [14]. Retroviruses can also cause sialadenitis in patients infected with human immunodeficiency virus (HIV) [15] and human Tlymphotropic virus I (HTLV-I) [16]. Furthermore, serum antibodies to the p24 capsid protein of HIV have been reported in 30% of SS patients compared to 1% to 4% in healthy controls. Although there is great similarity between sialadenitis and Sj6gren's syndrome, the two entities are different. There is a difference in the severity of tissue damage in the salivary gland that separates the two, and more importantly patients with chronic lymphocytic sialadenitis are negative for disease specific autoantibodies in contrast to SS patients. Furthermore, these types of viral infections that can give rise to sialadenitis are very uncommon in patients suffering from Sj6gren's syndrome. It is possible that viruses can act as the initiating factor in the activation of the epithelial cells in the disease. Transient or persistent infection of the epithelial cells by a putative virus may be the
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initiating event that leads to the accumulation of T and B cells. These cells could then prime a local autoimmune response using autoantigens provided by the epithelial cells as a result of the viral infection. Finally, monoclonal expansion of B cells under selective antigenic or T-cell-induced pressure can lead to tissue destruction. To further explore this activation of the epithelial cells, it is essential to identify the factors, possibly of viral origin, that trigger the immune response. The identity of the gene products that play this activating role and their origin are essential information in the understanding of the pathogenesis of the disease. To identify genes that may contribute to primary Sj6gren's syndrome pathogenesis the differential display protocol was applied to minor salivary gland RNA samples of a patient with primary Sj6gren's syndrome and a healthy control individual. After sequencing of several differentially expressed genes a 94 bp-fragment homologous to the VP1 region of coxsackievirus B4 RNA expressed exclusively in the diseased sample was identified. The identification of this viral RNA suggests that this virus could have an active role in the pathogenesis of Sj6gren's syndrome. [Triantafyllopoulou et al, unpublished data.]
5. COXSACKIEVIRUSES Coxsackieviruses belong to the large viral family of picomaviruses. This family includes two major groups of human pathogens, the enteroviruses and rhinoviruses. Coxsackieviruses, as all members of the family of enteroviruses, are small non-enveloped RNA viruses that are classified on the basis of their antigenic response. Coxsackie viruses are divided into two major serotype groups as determined by antibody neutralization tests. The larger group contains 23 viruses (A1-A24, no A23), while the smaller group contains 6 (B l-B6) [17]. Due to the nature of their infection cycle that only utilizes RNA as the genetic material, these viruses have no mechanism that maintains sequence integrity equivalent to the DNA proofreading system. As a result, these viruses mutate very rapidly giving rise to new variants. Mutations in the coding and non-coding regions of the viral sequence can result in viruses with altered virulence [ 18].
Coxsackievirus infection gives rise to a variety of human diseases. Herpangina, acute hemorrhagic conjuctivitis and hand-foot-and-mouth disease are all caused by type A coxsackieviruses. Type B coxsackieviruses are known to cause myocarditis, pericarditis and meningoencephalitis. Both types also give rise to aseptic meningitis, respiratory and undifferentiated febrile illnesses and hepatitis [ 19]. The virion of coxsackieviruses consists of a capsid shell of 60 subunits, arranged in pentamers, each being composed of four proteins (VP1-VP4). The pentamers are arranged to form an icosahedron. These pentamer subunits surround the viral genome that is made up of a single strand of positive sense RNA. The three largest proteins (VP 1, VP2 andVP3) are very similar in structure, with the peptide backbone of the protein looping back to form a barrel of eight strands. The aminoacid chains between this ~-barrel and the ends of the protein form a series of loops that contain the main antigenic sites that are found on the surface of the virion [20]. The five subunits on each pentamer form a cleft or canyon and on the floor of this canyon is the receptor binding site that is used in the attachment of the virion to the host cell. This canyon is too narrow for deep penetration by antibody molecules [21 ]. This should protect this important site from structural variation that could result from antibody selection in hosts. The genome of the virus is an RNA molecule of about 7.4 kb that codes for a single polyprotein that is cleaved to produce the various proteins that are required for virion structure and replication. The RNA is polyadenylated at the 3' end while the 5' end is covalently bound to a small viral protein, VPg. The viral RNA contains 5' and 3' untranslated regions (UTR) flanking the large coding region of the polyprotein. In the 5' UTR the VPg binding site can be found, in addition to other sequences involved in the control of viral RNA translation and replication. This 5' UTR region is highly conserved among enteroviruses indicating that this region contains sequences with common function among this family of viruses. The polyprotein-coding region can be divided into two major regions according to the function of the smaller proteins that result from the cleavage of the single large polyprotein. The polyprotein is cleaved to produce two types of proteins, structural proteins called VP proteins and functional proteins called P proteins. The sequence
for the VP proteins is located in the 5' end of the polyprotein coding region while P protein sequence is on the 3' end [19]. Coxsackie replication takes place in the cytoplasm of the infected host cell. The first step in viral infection involves the attachment of the virion on receptors that are located on the surface of the plasma membrane. Coxsackie viruses gain entrance into cells by binding to two different receptors, coxsackie-adenovirus receptor (CAR) [22] and decayaccelerating factor (DAF) [23]. CAR is a common receptor for coxsackieviruses and adenoviruses. While in coxsackieviruses the receptor is used for both attachment and internalization, in adenoviruses CAR mediates virus attachment and subsequently viral entry is achieved through integrins. CAR is a 46 kDa membrane glycoprotein with two immunoglobulin-like extracellular domains, a transmembrane domain and a long cytoplasmic domain. CAR is expressed in many tissues but its cellular function other than as a virus receptor remains unknown. DAF is a GPI-anchored glycoprotein that functions in protecting cells from lysis by autologous complement. Expression of DAF on the cell surface permits virus attachment but not infection. This suggests that unlike CAR, DAF is incapable of mediating some important post-attachment activity essential for viral entry. CAR appears to bind to the canyon that is formed on the surface of the virus. This binding results in a conformational change of the viral proteins that leads to viral instability and uncoating [21]. This appears to result from the interaction of the receptor with an unknown pocket factor that binds in the base of the canyon and competes with the receptor for the binding site. Because the pocket factor and the receptor have overlaping binding sites, only one can bind to the canyon at a time. The pocket factor stabilizes the virus for transport from cell to cell, but as soon as the receptor competes for the binding site, the pocket factor is removed and viral uncoating can begin. During this uncoating VP4 is lost from the viral structure and the resulting conformational change leads to viral internalization and release of the viral RNA into the host cell. The viral positive strand RNA is translated in the cytoplasm to produce the single polyprotein that following cleavage results to the various structural a n d essential replication proteins. This
293
cleavage is mediated by proteases of viral origin. New viral RNA synthesis begins when the viral replication proteins, including an RNA dependent RNA polymerase, are produced. The infecting plus strand RNA is copied to produce a negative strand RNA that serves as a template for the synthesis of many plus strands. These plus strands can either be translated to form more viral proteins or get packaged into new viruses. Some are also recycled as templates for the production of more negative strand RNA molecules that through the normal replication mechanism generate more plus strands. Plus strands are bound to the VPg protein before they are packaged into virions. Viral synthesis and maturation involves several cleavage reactions. The coat precursor protein p l is cleaved to generate VP0, VP3 and VP1. When an adequate concentration of these is reached, they form pentamers which package VPg attached RNA into provirions. These non-infective provirions become infective virions after VP0 is cleaved to generate VP4 and VP2. There are two types of infections that are observed in coxsackieviral infection. The most common infection leads to cell lysis and release of virus particles through increased cell permeability. By altering cell permeability the virus leaves the cell but at the same time several cellular components escape. In addition to this type of infection, persistent infection has been observed. In this case the virus stays in the cell for long periods of time and remains active in the cell without disrupting normal cell life.
6. COXSACKIEVIRUSES AND S J O G R E N ' S SYNDROME Having detected coxsackieviral RNA in a disease tissue sample, the presence of coxsackieviruses in the minor salivary gland biopsies of a large number of samples had to be evaluated. This was achieved through semi-nested RT-PCT using primers designed to amplify the 5' untranslated region of human enteroviruses [24]. By amplifying this region with this technique it was possible to detect the majority of enteroviruses and identify other viruses of the same family that could be present in the disease tissues. Amplification products of--370
294
bp were detected and the products were sequenced in order to verify their identity. PCR amplification of the ~-actin housekeeping gene was performed for all samples in order to verify the efficiency of the reverse transcription reaction and the integrity of the cDNA [Triantafyllopoulou et al, unpublished data]. Efforts to detect sequences that identify the virus with more accuracy by amplifying regions that code for parts of the protein coat that generate the antigenic response, and characterize the virus serotype were unsuccessful. This can be explained by the fact that these parts of the virus mutate rapidly [18] as a response to antibody selection processes and are difficult to amplify. A question that remained unanswered was whether the viral RNA was present in epithelial cells since the RNA was originally detected in whole minor salivary tissue samples. Furthermore, the presence of the RNA in peripheral blood monocytes could indicate a systemic infection that could present the viral RNA in the glandular tissues through infected lymphocytes. To investigate whether epithelial cells harbour the viral sequences that were detected in the biopsy tissues, cultured salivary gland epithelial cells were used. Cultured salivary gland epithelial cells are non neoplastic cell lines that were generated with the explant outgrowth technique from one lobule of minor salivary gland obtained as a part of Sj6gren's syndrome evaluation [25]. To evaluate the presence of the viral RNA outside the glandular tissues, peripheral blood lymphocytes were isolated at the same time point as the biopsy. RT-PCR utilizing the same primers as for all other cases was used to screen the samples for viral RNA. The results are summarized in Table 2. The viral RNA was detected in minor salivary gland biopsies and long term cultures of epithelial cells originating from salivary glands. The viral RNA was not detected in peripheral blood lymphocytes, indicating that there was no systemic infection at the time of the biopsy (Fig. 2). This suggests that probably the viral RNA that was detected originates from persistent viral infection in the salivary glands of Sj6gren's syndrome patients. [Triantafyllopoulou et al and Liakos et al, unpublished data.] Since the primers that were used were designed to amplify the 5'UTR of a large group of viruses, PCR products had to be sequenced in order to iden-
Figure 2. Agarose gel electrophoresis of RT-PCR products. Lanes 1 and 4 represent control samples, while lanes 2, 3, 5, 6, 7 and 8 represent SS patient samples. 13-actinwas used as a control for the reverse transcription reaction and cDNA integrity. Table 2. Detection of enteroviruses RNA sequences using reverse transcription-PCR (RT-PCR) targeting 5' untranslated region (5'UTR) n
5'UTR RT-PCR Biopsy
Cultured SGEC
Peripheral blood lymphocytes
Primary SS
20
7/8
8/8
0/4
Control
15
0/8
0/5
0/2
Triantaffylopoulou et al and Liakos et al, unpublished data.
tify the virus type. All products were sequenced and two virus types were identified. Ten PCR products were homologous to coxsackievirus type B4, while four were homologous to coxsackievirus type A13. The sequences show 97%-99% homology to the coxsackieviruses. Coxsackie B4 has been linked with autoimmunity and especially with diabetes type I. The first implication of environmental factors in the development of type I diabetes came 70 years ago by the observation that there is seasonal variation in the diagnosis of the disease suggesting that an environmental factor, probably a virus, participates in disease pathogenesis. The first indirect association between coxsackievirus and type I diabetes came from the
examination of the sera of newly diagnosed diabetic patients [26]. These sera contained antibodies against coxsackieviruses in higher frequencies compared to controls. The first direct link came from the isolation of coxsackievirus from the pancreas of a newly diagnosed patient with diabetes ketoacidosis [27]. The advent of molecular techniques enabled direct detection of the virus through polymerase chain reaction, circumventing the indirect detection through antibodies. Furthermore, it has been demonstrated that coxsackfe B virus infection can cause diabetes in two different animal models [27, 28]. The etiology of Sj6gren's syndrome remains obscure despite considerable investigation. For the first time a link between Sj6gren's syndrome
295
Autoimmune Epithelitis
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Development
Figure 3. Proposed role of epithelium in the initiation and perpetuation of immune response. Viral infection leads to activation of the epithelium. Activated epithelia recruit and activate B and T-cells that in turn further enhance epithelial activation. This leads to increased epithelial apoptosis that results in the release of intracellular antigens. Classical antigen presenting cells are recruited and further enhance the immune response. B-cell activation can lead to the development of B-cell lymphoma through mutations in p53.
and viruses has been shown. While the role of the virus in the pathogenesis of the syndrome has not been identified, the presence of viral RNA in salivary glands, and more specifically in salivary gland epithelial cells, makes a strong case for the involvement of the virus in the development of the disease. There are several examples of viral-autoantigen interactions. First of all, one of the functions of La(SSB) is to bind to polioviral RNA and initiate translation of viral RNA [29]. Furthermore it has been shown that infection with Epstein-Barr virus leads to expression of La(SSB) in the cytoplasm instead of the nucleus [30]. Moreover, cleavage of La(SSB) by poliovirus 3C protease leads to the redistribution of La from the nucleus to the cytoplasm [31 ]. The mechanism by which the virus participates in disease pathogenesis could be linked to the interaction of the virus with the La(SSB) autoantigen. Since viral RNA can bind to La(SSB) and modify it by cleavage thus altering its localization pattern, it is possible that viral RNA mediates the transfer of the autoantigen to the surface of the cells and conceivably outside the cell through apoptosis and
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apoptotic blebs [32]. This presentation of the antigen to the cell surface or the surrounding microenvironment could lead to the generation of the immune response. Although the viral sequences that have been detected appear to be of specific origin, they represent a fraction of the viral genome and could be part of some unidentified virus that may be the result of some viral recombination effect. Another question that remains unanswered is whether the virus replicates actively in the cells or the infection is persistent. Virus characterization would give information on the virus type and could provide clues about the life cycle of the virus. The receptor that mediates cell entry for this virus should be also identified. To further characterize the role of the viral sequences in the pathogenesis of SjGgren's syndrome the virus needs better characterization and also more light should be shed to the interaction of the virus with the different cellular components and especially autoantigens. In light of this material we propose the following working hypothesis for the role of coxsackieviruses in the pathogenesis of Sj6gren's syndrome (Fig. 3).
The virus after infection of the epithelium remains latent in some genetically predisposed individuals. Through some hormonal or stress related events the virus can participate in the activation of the infected epithelium by becoming active. These events that trigger the response can be connected either to the virus life cycle or the epithelial cell-virus interactions leading to the activation of the epithelium. This activated state of epithelia initiates and perpetuates the immune response that leads to tissue damage that characterizes Sjrgren's syndrome.
10.
11.
12.
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9 2004 Elsevier B. V. All rights reserved.
Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Viral Infection and Heart Disease: Autoimmune Mechanisms Marina Afanasyeva ~and Noel R.
R o s e 1'2
1Department of Pathology and 2Department of Molecular Microbiology and Immunology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA
1. HUMAN MYOCARDITIS
1.1. Viral Etiology Myocarditis, presently defined by "the presence of an inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes," [ 1] is a major cause of sudden death in young adults [2]. In predisposed individuals, myocarditis may evolve into a chronic inflammatory dilated cardiomyopathy (DCM), which typically progresses to heart failure and death in the absence of cardiac transplantation [3, 4]. The etiology of myocarditis remains unknown in the majority of cases but accumulating evidence supports an association with viral infection [5, 6], most often involving coxsackieviruses [7]. Other viruses reported to cause myocarditis include adenovirus, cytomegalovirus, parvovirus, human immunodeficiency virus, measles virus, mumps virus, hepatitis A and C viruses, herpes simplex virus, and encephalomyocarditis virus among others. Among coxsackieviruses, coxsackievirus B3 (CB3) has been frequently associated with myocarditis in the United States [7]. CB3 is a member of the enterovirus genus within the Picornaviridae family, and is, therefore, a non-enveloped virus with a single-stranded positive-sense RNA. Thus, it is typically a cytolytic virus and synthesis of negative-sense RNA is required for viral replication but not for viral protein synthesis. Most coxsackievirus infections are either subclinical or present with mild upper respiratory or gastrointestinal symptoms [8]. However, a small percentage of infected individuals demonstrate signs and symptoms of acute myocar-
ditis. Evidence supporting the role of coxsackieviruses in the development of myocarditis and DCM comes from epidemiologic studies demonstrating an association between prior coxsackievirus infection and subsequent cardiomyopathy [9]. Furthermore, there is serologic evidence of infection in patients with DCM [10-12] and viral PdqA has been isolated from cardiac tissue of myocarditis and DCM patients [12, 13]. These studies also demonstrated that a proportion of patients with DCM have actively replicating virus in the heart as detected by the presence of minus-strand RNA. A significant proportion of patients, however, have only positive-strand RNA, indicating latent viral persistence without replication.
1.2. Evidence for Autoimmunity Autoimmune features of human myocarditis and DCM include familial aggregation [14, 15], a weak association with human leukocyte antigen (HLA)DR4 [16], upregulated expression of HLA class II on cardiac endothelium [17], increased levels of circulating cytokines [18, 19], and presence of cardiac-specific autoantibodies of the IgG class in the blood [20].
1.2.1. Autoantibodies Cardiac autoantibodies provide the strongest evidence for autoimmunity in myocarditis and include those specific to t~ and [3 isoforms of cardiac myosin (CM) heavy chain [21-23], antibodies against some mitochondrial antigens, such as the adenine nucleotide translocator and the branched-chain or-
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ketoacid dehydrogenase dihydrolipoyl transacylase [24, 25], and antibodies to cardiac receptors, such as [~l-adrenoreceptor and M2 muscarinic receptor [26, 27]. An important question has been whether these cardiac-specific antibodies mediate disease or represent a marker, or epiphenomenon, resulting from damage to cardiomyocytes. It is of interest that autoantibodies have been detected in family members of patients with DCM years before the development of disease [16]. Lauer et al [28] found that the presence of anti-CM IgG in serum of patients with chronic myocarditis was associated with the deterioration of both systolic and diastolic function during a six-month follow-up. It has been suggested that anti-receptor antibodies, particularly anti-J31 adrenoreceptor IgG, could either stimulate or block the receptor thereby affecting myocyte contractility [26, 29]. Muller et al [30] demonstrated beneficial effects of IgG adsorption in DCM patients with high-titer anti-J31 receptor antibody. Clinical improvement upon immunoadsorption treatment correlated with the reduction in anti-131 antibody activity in the serum as assessed by an in vitro bioassay involving cultured neonatal rat cardiomyocytes. Similar results were obtained in a different randomized study by Felix el al [31] where a significant hemodynamic improvement was observed in DCM patients treated with immunoadsorption compared to controls. A later study, however, has demonstrated that the beneficial effects of immunoadsorption could not be attributed to the reduction in anti-131 antibody levels since anti-~l antibody-positive and antibody-negative patients benefited equally from such treatment [32].
0.45 [33]. The drawback of the study was that the patients were not discriminated in terms of the presence of an active viral infection in the heart. Such discrimination might have been important since immunosuppression could improve the harmful autoimmune response but impede the protective anti-viral response. A more recent study by Frustaci et al [34] has demonstrated the importance of viral detection in the heart in selecting patients for immunosuppressive therapy. The authors treated patients with active lymphocytic myocarditis with prednisone and azathioprine in addition to a conventional therapy. Based on the retrospective analysis of the presence of a viral genome (enterovirus, adenovirus, influenza A virus, Epstein-Barr virus, parvovirus B 19, and hepatitis C virus) in biopsy specimens and serum cardiac autoantibody at the onset of treatment, the authors concluded that immunosuppression is beneficial in patients with circulating anticardiac antibody and with no viral genome (except for the presence of hepatitis C virus) in the heart. In another clinical study, Wojnicz el al [35] have demonstrated beneficial effects of immunosuppression in a group of myocarditis patients with immunohistological evidence of HLA upregulation in the myocardium. These studies provide insights into the heterogeneity among the lymphocytic myocarditis cases and underscore the importance of further subclassification of lymphocytic myocarditis in order to optimize therapy.
2. ANIMAL MODELS
2.1. CB3-lnduced Myocarditis 1.2.2. Immunosuppressive therapy Based on the hypothesis that the autoimmune component is significant in the course of myocarditis, immunosuppressive drugs have been used in some situations as part of a treatment regimen for myocarditis patients. A large-scale myocarditis treatment trial, however, did not show any improvement in ejection fraction or mortality upon treatment with prednisone plus cyclosporine or.prednisone plus azathioprine compared to conventional therapy in patients with histopathologic evidence of myocarditis and left-ventricular (LV) ejection fraction below
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To better understand the pathologic mechanisms of infection-triggered autoimmune response, several animal models of myocarditis have been established. CB3-induced myocarditis in mice represents one of these models. Following CB3 inoculation, mice develop an acute, inflammatory focal myocarditis with a mixed cellular infiltrate and cardiomyocyte damage, peaking in about 7 days post-infection [36-38]. The severity of the acute disease varies among strains of mice and is associated with the appearance of neutralizing antibody in the serum [39]. Inflammation in the myocardium
gradually subsides by day 21, when there is typically no histologic evidence of myocarditis. Some strains of mice, however, such as A]J and BALB/c, progress to later chronic, or autoimmune, phase of myocarditis peaking around day 35 post-infection [40--42]. The course of CB3-induced myocarditis resembles human disease since majority of humans recover from the acute viral disease without autoimmune sequelae but a small fraction of predisposed individuals, similar to susceptible strains of mice, develop a late autoimmune disease. Histologically, the late phase of myocarditis in mice differs from the acute disease and is characterized by diffuse, rather than focal, leukocyte infiltration with signs of cardiomyocyte "drop out" and fibrosis. The acute viral phase is characterized by the presence of infectious virus in the heart, whereas during the chronic phase no infectious virus can be found, although viral RNA is often present. The two phases of myocarditis also differ in terms of the associated autoantibody profiles. Early after CB3 infection, there is a moderate increase in the natural IgM antibody that binds CM but cross-reacts with skeletal myosin. Those mice that develop the autoimmune phase also produce cardiac-specific and non-crossreactive IgG antibody, with primary cardiac antigen being ct-isoform of CM heavy chain [43]. This is the predominant CM heavy chain isoform in adult mouse ventricles. Adult human ventricles, on the other hand, predominantly express the ~-isoform. In BALB/c mice, these antibodies are mainly of IgG1 subclass. The presence of CM-specific IgG in the serum represents another common feature of the disease in mice and humans [21-23].
2.2. CM-Induced Experimental Autoimmune Myocarditis (EAM) The predominant antibody reactivity to CM led to a hypothesis that autoimmune response to CM might be responsible for ongoing myocarditis. To test this hypothesis, several strains of mice were injected subcutaneously with either an emulsion of purified mouse CM in complete Freund's adjuvant (CFA), or with mouse skeletal myosin in CFA, or CFA alone [44]. CM, but not skeletal myosin or adjuvant alone, administration produced inflammation in the heart resembling that of the autoimmune phase of CB3-induced myocarditis. Remarkably,
the same strains of mice that developed the autoimmune phase of myocarditis following CB3 infection developed myocarditis following injection with CM and, conversely, the strains of mice that were not susceptible to late phase CB3-induced myocarditis did not develop disease following injection with CM. This finding suggested common genetic predisposing factors in the two models of myocarditis. Hence, CB3 appears to trigger an autoimmune disease, namely ongoing myocarditis, making it the first instance in which the antigen responsible for a postinfection autoimmune disease was identified and the disease was reproduced by injecting genetically susceptible animals with that antigen. Both CB3- and CM-induced models have been extensively used to study the pathogenesis of virusinduced autoimmune myocarditis. CB3 model is irreplaceable for studies of viral virulence, viral entry, and of interaction between the virus on one hand and a cardiomyocyte and/or immune system on the other. CM (or EAM) model is valuable to study the autoimmune phenomena which are sometimes difficult to dissect in a more complex viral model, where anti-viral and anti-immune effects often interact.
2.3. Other Models of Myocarditis Similar to human myocarditis, which can be caused by viruses other than coxsackievirus, murine myocarditis can also be induced by murine cytomegalovirus (MCMV) and encephalomyocarditis virus (EMCV) [6, 41, 45]. AJJ and BALB/c mice, which are susceptible to CB3- and CM-induced autoimmune disease, develop both acute and chronic myocarditis following MCMV infection and C57BL/6 mice, resistant to CB3- and CMinduced autoimmune disease, develop only acute phase of MCMV myocarditis. The chronic phase of MCMV myocarditis is also characterized by the presence of CM-specific IgG in the serum. EMCV infection produces myocarditis in susceptible mice (e.g. BALB/c and DBA/2) with distinct acute and chronic phases [6]. Kodama el al [46] described a CM-induced model of myocarditis in Lewis rats. This model is characterized by the presence of giant cells in the myocardium, resembling human giant-cell myocarditis. Interestingly, A/J mice often develop giant
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Figure 1. Eosinophils and giant cells in severe CM-induced autoimmune myocarditis. A, normal myocardium; B, eosinophils in inflammatory infiltrate; C, eosinophils and giant cells (arrows) in inflammatory infiltrate. Hematoxylin and eosin stain. Original magnifications: xl00 (A), x200 (B), and x400 (C). Reprinted with permission from the American Society for Microbiology [143].
cells in the heart upon CM immunization and the presence of giant cells correlates with disease severity (Fig. 1) [47]. EAM can be reproduced in AJJ mice with porcine CM, which induces disease with the same immunohistopathologic features, antibody and cytokine profiles [48]. In some strains of mice, disease-producing epitopes from CM heavy chain have been successfully used to induce EAM; one of them is a 19 amino-acid long peptide (myhc~(334352), NH2-DSAFDVLSFTAEEKAGVYK-COOH ) which binds to I-A k and produces severe myocarditis in AJJ mice [49].
susceptibility is controlled by multiple genes (our unpublished observations). On the other hand, H-2 modifies the severity of disease since A/J, A.SW and A.CA mice develop moderate to severe disease, whereas A.BY mice, which differ only in their H-2 genes, develop mild or no disease following injection with CM.
3. DISEASE PROGRESSION: FROM VIRAL ENTRY TO HEART FAILURE 3.1. Early Events
2.4. Genetic Susceptibility to Myocarditis Susceptibility to autoimmune myocarditis, whether induced by infecting mice with CB3 or injecting them with CM, appears to be under strict genetic control. A/J mice are classical good responders as are most congenics sharing the A background. BALB/c mice are moderate responders, whereas C57BL/10 and C57BL/6 are generally not susceptible to the autoimmune form of myocarditis [39, 44]. This susceptibility is due primarily to genes that are not part of the major histocompatibility (MHC) complex. For example, A.SW mice are good responders to CM immunization, whereas B10.S mice, which share the same MHC genes (or H-2), fail to respond. Hybrids between these two strains exhibit a wide range of responses, indicating that
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Coxsackieviruses are believed to enter cells via coxsackievirus-adenovirus receptor (CAR). As can be inferred from the name, this receptor is also important for cellular entry by adenoviruses, another group of viruses associated with myocarditis and DCM. It has been shown that the expression of CAR is low in normal hearts but is upregulated in the hearts of DCM patients [50]. Similarly, upregulation of CAR has been observed in rat models of EAM and myocardial infarction [51, 52]. The function of CAR as well as the significance of its upregulation remains unknown. Decay accelerating factor (DAF), or CD55, represents a coreceptor for CB3 entry [53]. Liu et al [54] have demonstrated in a mouse model that the sarcoma family kinase Lck (or p56 ~ck) is required for the effective CB3 replication, persist-
ence, and the ability to cause myocarditis. Interestingly, the presence of p56 ~ckin T cells was sufficient to restore susceptibility to myocarditis in p56 ~ckdeficient mice. This study suggested the importance of T cells for CB3 delivery to the heart. It was also shown by others that enteroviruses can replicate in leukocytes and leukocytes may serve as carders promoting viral spread to different organs [53]. Opavsky et al [55] have shown that activation of extracellular signal-regulated kinases 1 and 2 (ERK-1/2) downstream of p56 ~ck is important for viral replication in both T cells and cardiomyocytes. The authors suggested that ERK-1/2 activation may be linked to disease susceptibility based on the observation that such activation was more pronounced in the hearts of susceptible AJJ mice compared to resistant C57BL/6. Luo et al [56] have also found that ERK-1/2 activation is important for CB3 replication and virulence.
3.2. Innate Immunity Innate immune system serves to protect the host against invading pathogens by prompt recognition of certain pathogen-associated molecular patterns (PAMPs). Sometimes, innate immune response may be sufficient for the elimination of a pathogen but, in most cases, the adaptive immune response is necessary to finish the job initiated by the innate response and clear the infection. Innate immunity sets the stage for and determines the quality of the adaptive response.
3.2.1. Toll-like receptor (TLR) 4 Lipopolysaccharide (LPS) of Gram-negative bacteria represents a classic PAMP which is recognized by CD14 and TLR4, both of which have been shown to be expressed in the myocardium [57, 58]. LPS seems to affect the susceptibility of mice to myocarditis, since co-treatment with LPS makes typically resistant B 10.A mice susceptible to CB3-induced autoimmune myocarditis with associated high titers of CM-specific IgG antibody [59]. Expression of TLR4, the receptor for LPS, in the heart has been shown to correlate with enteroviral replication in human myocarditis [60]. In a mouse model, TLR4 deficiency resulted in reduced myocarditis and reduced viral replication in the heart
on day 12 post-CB3 infection despite significant CB3 replication in the heart on day 2 post-infection, suggesting enhanced viral clearance and/or less pro-inflammatory environment in the absence of TLR4 [61]. In support of the latter view, TLR4deficient mice had significantly suppressed production of IL-113 and IL-18 in their hearts on day 12. C3H/HeJ mice, which lack functional TLR4 due to a single missense mutation within its coding gene, seem to be highly susceptible to CB3 myocarditis [62]. These discrepancies could be accounted for by the interaction of susceptibility/resistance genes which may interfere with the interpretation and comparison of data derived from different genetic backgrounds.
3.2.2. Complement Complement, another major component of the innate immune response, affects the susceptibility of mice to developing myocarditis. Anderson et al [63] have shown that complement component C3 interacts with capsid proteins of CB3 and this interaction triggers the alternative complement pathway. The authors also proposed that C3 interaction with CB3 might be important for limiting viral load by retention of the virus in the spleen in an antibodydependent fashion. Experiments using CB3 model of myocarditis demonstrated a strain difference in response to C3 depletion with cobra venom factor with decreased inflammation in DBA/2 but not BALB/c mice upon cobra venom administration as assessed on day 7 post-infection [64]. Neither strain exhibited changes in viral load in the heart in response to cobra venom treatment. The authors suggested that the treatment mainly affected the humoral (antibody) autoimmune response and therefore had an effect in DBA/2 mice, which have a mainly antibody-mediated disease, but not in BALB/c mice, which demonstrate a more pronounced cellular autoimmunity. C3 has been shown to be critical for the development of autoimmune myocarditis in the CM model. Administration of cobra venom factor to mice that were injected with CM resulted in impaired IgG antibody responses to CM and prevented myocarditis [65]. Depletion of C3 at the time of initiation rather than progression of disease was critical since multiple injections of cobra venom
303
factor between days 1 and 9 after immunization, but not between days 10 and 18, were effective in preventing myocarditis. A major product generated during activation of the complement cascade is C3d, which acts mainly through two complement receptors, CR1 (or CD35) and CR2 (or CD21). The incidence and severity of disease are significantly reduced in CR1/CR2 double knockout (KO) mice compared with wild type mice. Similarly, blockade of CR1 and CR2 during the time of CM immunization with a monoclonal antibody (mAb), which binds to the extracellular domain shared by the two receptors, abrogated disease, dramatically reduced the production of CM-specific IgG, and was associated with decreased production of IL-1 and TNF~ by splenocytes cultured with CM. CR1 and CR2 have been shown to be present on a subset of activated/memory CD44h~ghCD62L~~ T cells and their engagement triggers T cell responses, implicating complement as an important player not only in antibody-mediated but also in T cell-mediated autoimmunity [65].
20%, and in some cases up to 50%, of the acute inflammatory cell infiltrate in the heart of CB3infected mice. Upon CB3 infection of mice, depletion of 78+ T cells resulted in increased viral titers in the heart indicating the importance of these cells in controlling viral replication [71]. y8+ T cells, particularly T4+ T cells, have been shown to be important in susceptibility to myocarditis induced by CB3. These cells can recognize MHC class I-like CDld molecules and this recognition has been proposed to mediate the susceptibility to CB3-induced myocarditis. CD 1d-deficient mice developed minimal myocardial inflammation with no significant changes in the cardiac viral titers upon CB3 infection [72], but this effect could not be explained by the lack of NKT cell response since mice deficient in invariant J~281 gene, which is expressed in NKT cells, were highly susceptible to myocarditis. Therefore, it was suggested that the lack of y4+ T cell response was responsible for the reduction in myocarditis.
3.2.3. Natural killer (NK) cells
Type I IFNs, ~ and ~, are associated with early innate immune responses and represent a part of anti-viral defense system. IFN-[3 treatment of patients with inflammatory cardiomyopathy associated with LV dysfunction and presence of either enteroviral or adenoviral genomes in the myocardium resulted in improvement of LV function and clearance of viral genomes [73]. Miric et al [74] have reported beneficial effects of IFN-~ treatment in patients with idiopathic myocarditis and idiopathic DCM which were observed in a small randomized clinical trial. There have also been anecdotal reports of successful treatment of enterovirus-induced myocarditis with IFN-~ [75]. The importance of type I IFNs has been demonstrated in a murine model of CB3induced myocarditis where mice deficient for type I IFNs showed increased mortality within 2 to 4 days after infection [76]. Oral treatment with type I IFNs suppressed the inflammatory response in MCMVinduced myocarditis in mice [77].
NK cells, another component of the innate immune response, can directly kill their target cells and represent a rich source of cytokines, which in turn can influence the inflammatory milieu and affect the adaptive immune response. Godeny et al [66] have shown that NK cells limit CB3 replication. Depletion of NKI.1 § cells exacerbated acute myocarditis induced in mice with MCMV [41 ]. Interleukin (IL)18 augments NK cell activity and its administration has been shown to improve survival, reduce viral load, and decrease myocarditis in a murine model of EMCV-induced acute myocarditis [67]. The therapeutic effect of IL-18 was associated with increased NK cell activity in the spleen. The role of NK cells in the development of the autoimmune phase of myocarditis remains unclear.
3.2.4. y8 T cells T cells expressing Y and 8 chains of the T cell receptor (TCR) have been shown to accumulate in the myocardium during fulminant myocarditis in humans [68]. Huber et al [69, 70] have demonstrated that ?8 + T cells comprise between 5% and
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3.2.5. Type I interferons (IFNs)
3.3. Adaptive Immunity 3.3.1. T cells Autoimmune myocarditis is believed to be a T cellmediated disease. Endomyocardial biopsies from patients with myocarditis and idiopathic DCM show infiltration with CD4 § and CD8 + T cells. Transfer of peripheral blood leukocytes from patients with myocarditis and impaired LV function to mice with severe combined immunodeficiency (SCID) resulted in myocardial infiltration with human leukocytes and impaired LV function [78, 79]. Omerovic et al [80] have found that transfer of peripheral blood lymphocytes from DCM patients to SCID mice induced myocardial fibrosis and deterioration of LV function, as assessed by increased LV dimensions on echocardiography, 75 days post-transfer. Upon CB3 infection, CD4-deficient mice exhibited reduced myocardial infiltration and necrosis but the same survival as the control mice [81]. In the same study, CD8 deficiency increased the severity of disease in terms of both myocardial pathology and survival. However, CD4/CD8 double-deficient mice as well as TCR~-deficient mice showed improved survival and decreased myocarditis as observed during 28 days after infection. None of these deficiencies affected cardiac viral titers on day 7 post-infection. Other studies have also demonstrated the effects of the absence of either CD4 § or CD8 § T cells on the survival, myocarditis, and viral titers after CB3 infection [82]. Overall, however, the results are rather confusing since it is difficult to dissect the effects on viral replication, viral tropism, and inflammatory response in the heart. The possibility that lymphocytes can deliver CB3 to the heart further complicates the interpretation and underscores the complexity of the viral model. Experiments by Smith and Allen [83] showed that CD4 § T cells play a central role in the pathogenesis of CM-induced EAM. Depletion of CD4 § T cells with mAb prevented the development of disease and the disease could be reproduced in immunologically deficient SCID mice by transfer of CD4 § T cells isolated from CM-immunized mice. The heart infiltrate in EAM has greater numbers of CD4 § T cells compared to CD8 + T cells [48]. The predominance of CD4 § T cells persists to day 60 post-immunization in BALB/c mice but the ratio of
CD4 § to CD8 § T cells in the myocardium decreases over time. During the chronic phase of EAM (around day 60 post-immunization), the proportion of CD4 + T cells within the total infiltrating leukocyte population correlates with systolic dysfunction and the development of large LV volumes, the hallmarks of DCM, suggesting the role of CD4 + T cells in the development of cardiac dysfunction(our unpublished observations). The role of CD8 + T cells in EAM is less well defined. It has been demonstrated that CD8 deficiency in mice leads to exacerbation of CM-induced myocarditis [84], the finding similar to that in the CB3 model [81]. However, depletion of CD8 § T cells with a mAb reduced the severity of EAM [85, 86]. The ability of CD8 + T cells to induce myocarditis was demonstrated in mice transgenically expressing an ovalbumin peptide in the heart under cardiac-specific promoter [87]. These mice developed severe myocarditis upon the transfer of CD8 § T cells from transgenic mice that expressed TCR specific for the ovalbumin peptide. In this system, IL-12 was crucial for pathogenicity of CD8 + T cells.
3.3.2. B cells and antibody The role of antibody in the development of myocarditis is less well characterized compared to the role of T cells. In both AJJ and BALB/c mice, disease severity upon CM immunization correlates with CM-specific IgG1 [47]. IgG1 is deposited in the heart and clusters of IgGl-positive cells, which are most likely plasma cells, are found in the myocardial infiltrate on day 21 post-immunization. While it is likely that antibody contributes to the pathogenesis of myocarditis, its role in disease initiation seems to vary among different strains of mice. Transfer of sera collected on day 21 post-immunization in A.SW mice failed to induce myocarditis in A.SW recipients [88]. Liao et al [89] have demonstrated that the transfer of mAb specific for CM induced myocarditis in DBA/2 but not BALB/c mice. The authors found that DBA/2 but not BALB/c mice expressed myosin or myosin-like molecules in the extracellular matrix in the myocardium and offered this finding as a potential explanation for the strain difference. Furthermore, CM-specific IgM antibody failed to induce myocarditis in DBA/2 mice and
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only CM-specific IgG was able to transfer the disease [90]. In another study using BALB/c mice deficient in B cells due to disruption in the IgM gene, B cells have been shown dispensable for the induction of CM-induced myocarditis [91 ].
3.4. The Role of Cytokines Cytokines are the products of activation of both innate and adaptive immune systems, and, therefore, can act early, during disease initiation, and late, during disease progression. For the purpose of discussion they are classified here as proinflammatory (TNF-tx, IL-113, and IL-6), T helper (Th) 1 (IL-12 and IFN-y), and Th2 (IL-4 and IL-10). IL-10, however, does not quite fit into the Th2 group and should rather be classified as an immunoregulatory cytokine.
3.4.1. Proinflammatory cytokines Experiments using both CB3 model and CM model of myocarditis showed that IL-1 and tumor necrosis factor (TNF) are critical for the development of myocarditis [92-95]. Treatment with either of these cytokines rendered otherwise resistant B 10.A mice susceptible to CB3-induced myocarditis [92, 93]. Blocking TNF with a mAb prevented myocarditis in A/J mice immunized with CM [94]. Furthermore, mice deficient in TNF receptor (R) p55 are resistant to the induction of CM-induced EAM [95]. In EMCV-induced myocarditis, genetic deficiency of TNF-t~ resulted in increased mortality and increased viral load but decreased inflammatory response in the heart. These findings suggest that while promoting an autoimmune response, TNF-o~ inhibits viral replication and is necessary for an effective anti-viral response. It is, therefore, important for an effective intervention to differentiate between viral and virus-triggered autoimmune phases of myocarditis. Blocking IL-1 with IL-1R antagonist reduced CB3-induced autoimmune myocarditis in A/J mice [96]. Similarly, expression of IL-1R antagonist in the mouse heart by plasmid DNA decreased myocardial inflammation in CB3-induced myocarditis [97]. More recently, Eriksson et al [98] have shown an important role for IL-1R signaling in the development of EAM by demonstrating that IL-
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1Rl-deficient BALB/c mice were protected from disease. In the same study, the authors examined the role of dendritic cells (DCs) in antigen presentation to CD4 + T cells and initiation of disease. IL-1R1 signaling was critical for the activation of DCs and the ability of CM peptide-pulsed DCs to induce myocarditis. Transfer of IL-1Rl-sufficient DCs restored disease susceptibility in IL-1Rl-deficient mice indicating the mechanism through which IL-1 triggers an autoimmune response. IL-6, another proinflammatory cytokine, has been shown important for the development of EAM induced by a CM-derived peptide [99]. IL-6 KO mice had significantly reduced prevalence of autoimmune myocarditis; this reduction in prevalence was associated with impaired upregulation of complement. The role of IL-6 in viral myocarditis is less clear. In EMCV-induced myocarditis, administration of IL-6 improved disease outcomes but the transgenic expression of IL-6 exacerbated viral myocarditis [ 100-102].
3.4.2. Thl cytokines Depending on the microenvironment, CD4 + T cell activation can lead to their polarization into either Thl or Th2 type. These two polarized and mutually exclusive states differ mainly in terms of which cytokines are produced by activated CD4 § T cells, with Th 1 cells producing IFN-y and TNF-~ and Th2 cells producing IL-4 and IL-5. The initial cytokine milieu is critical for the Thl/Th2 differentiation of na'fve CD4 + T cells. IL-12 and, more recently, IL-23 have been shown to induce Thl responses [ 103, 104]. IL-4, on the other hand, stimulates Th2 polarization. For some time, a generally believed paradigm was that autoimmune diseases are driven by Thl responses and prevented or ameliorated by Th2 responses. While supported by some studies using animal models, this paradigm does not always explain the effects of individual cytokines on the course of an autoimmune disease [105]. The induction of autoimmune myocarditis seems to be dependent on a Thl-inducing cytokine, IL-12, but its development is suppressed by a classic Thl effector cytokine, IFN-y. This conclusion was based on the studies involving the CM model that demonstrated that BALB/c trice deficient in either IL-12 or IL-12R signaling, IL-12 p40 KO, IL-12RI31 KO
Figure 2. Autoimmunemyocarditisprogresses to DCM in IFN-~/KOmice. Left, normal heart. Right, heart with DCM from an IFN-),knockout mouse on day 23 after CM immunization.Reprinted with permission from the American Society for Microbiology [143].
and signal transducer and activator of transcription (STAT) 4 KO, were resistant to EAM [106, 107]. Furthermore, treatment with exogenous recombinant IL-12 exacerbated EAM in BALB/c mice and in Lewis rats [ 107, 108]. Many actions of IL-12 are believed to be mediated by IFN-y; however, this does not seem to be the case in autoimmune myocarditis. The role of IFN-), in EAM has been investigated using four different approaches: depleting IFN-y with a mAb; using IFN-), KO mice; using WN-yR KO mice; and treating mice with exogenous recombinant IFN-y [47, 106, 107, 109]. All these experiments support the conclusion that IFN-), suppresses the development of autoimmune myocarditis. IFN-3' KO mice develop severe acute myocarditis and pronounced cardiac dysfunction. Those mice that survive the acute stage develop extensive cardiac fibrosis, and many develop DCM and die of congestive heart failure (Figs. 2 and 3). The absence of IFN-), leads to impaired apoptosis of CD4 § T cells and subsequent expansion of activated/memory C D 4 4 high T cells, which might explain the exacerbation of myocardial inflammation (our unpublished observations). Thus, IL-12 exerts its proinflammatory effects in an IFN-~,-independent fashion, but the mechanism remains unclear. Reduced disease in the absence of IL-12R signaling was associated with reduced production of proinflammatory cytokines, IL-1
and IL-6, suggesting that IL-12 promotes disease through upregulation of these cytokines [107]. In a CB3 model, IL-12R[31 deficiency also resulted in reduced inflammation as well as reduced viral replication in the heart on day 12 post-infection, indicating that IL-12R signaling is dispensable for the anti-viral protection and contributes to the initiation of the inflammatory response [61]. In EMCV model, however, recombinant IL-12 treatment reduced mortality and decreased viral replication, whereas neutralization of IL-12 with a mAb resulted in increased mortality [110]. It is plausible that IL-12 is more important in clearing EMCV than CB3 or, alternatively, the acute intervention with a cytokine or anti-cytokine antibody has different effects compared to a genetic deficiency. The effects of IL-12R~I deficiency in a CB3 model also seem to be independent of IFN-y. ILl 2RI] 1 KO mice have reduced viral replication in the heart, whereas IFN- 7 KO mice have increased viral replication on day 12 post-infection [61]. Protective effects of IFN-~, have been shown in a number of virus-induced models. Transgenic expression of WN-y in the pancreas, the initial target of CB3, protects mice from CB3-induced myocarditis [111]. Intranasal administration of IFN-y suppresses viral replication and improves prognosis of EMCVinduced myocarditis [112]. Since IFN-~, is protective during both viral and autoimmune disease, it
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Figure 3. Morphologic and functional presentation of DCM in mice. A and B, heart cross-sections; C and D, hematoxylin and eosin stain of heart sections, original magnification • E and F, LV function assessed by pressure-volume relations obtained by means of in vivo LV catheterization. Panels A, C, and E represent a normal heart, and panels B, D, and F represent a heart with DCM. Note the increase in volume and reduction in systolic pressure characteristic of heart failure due to DCM in E Reprinted with permission from the American Society for Microbiology [ 143].
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may represent a potential therapeutic weapon. 3.4.3. Th2 cytokines In the CM model, the analysis of histopathological (presence of eosinophils and giant cells) (Fig. 1) and immunological (correlation of disease with CM-specific IgG1 and upregulation of total IgE responses) profiles has revealed a Th2-1ike phenotype, suggesting a pathogenic role for IL-4, which is important for eosinophil recruitment and IgG 1 class switch [47]. In support of a disease-promoting role of IL-4, treatment with an anti-IL-4 mAb reduced the severity of EAM in A/J mice and induced a shift from a Th2-1ike to a Thl-like phenotype. Such as shift was demonstrated by suppressed IgE and IgG1 responses; upregulated IgG2a response; suppressed production of IL-4, IL-5, and IL-13; and increased production of IFN-~, by cultured splenocytes in response to in vitro stimulation with CM [47]. At the same time, IL-4Rct KO mice on a BALB/c background do not demonstrate reduced severity of EAM and seem to develop the disease earlier compared to the wild type controls [106]. Since IL-13, another Th2 cytokine, signals through the same receptor subunit [ 113], it will be of interest to explore its role in the development of autoimmune myocarditis. IL-10, which is often classified as a Th2 cytokine, exerts immunoregulatory effects by inhibiting activation and effector functions of T cells and antigenpresenting cells [114]. IL-10 seems to suppresses CM-induced murine myocarditis, since an anti-IL10 mAb treatment enhanced disease [115]. This effect was observed when IL-10 was blocked relatively late (starting on day 10 after immunization) but not early (between days 0 and 12), suggesting that IL-10 is more important during the resolution rather than initiation of disease. IL-10 blockade also prevented suppression of EAM induced by nasal tolerance with intra-nasal administration of CM before immunization, implicating IL-10 as a mediator of mucosal tolerance [115, 116]. Watanabe et al [117] demonstrated the disease-suppressive effect of IL-10 in a rat model of EAM by delivering IL10-expressing plasmid vector via electroporation into the tibialis anterior muscles. Treatment with recombinant IL-10 improved the outcomes in viral myocarditis induced by EMCV without any effects
on the viral load in the myocardium [118]. These results suggest that IL-10, similar to IFN-T, can be beneficial regardless of whether the viral or autoimmune component predominates.
3.5. Myocarditis and Cardiac Dysfunction 3.5.1. Changes in the mechanical properties of the heart Human autoimmune myocarditis often presents with symptoms of cardiac dysfunction and heart failure. Echocardiographic studies in patients with myocarditis have demonstrated reduction in ejection fraction indicating deterioration of systolic function [ 119]. Many patients with myocarditis also present with diastolic dysfunction, demonstrated by altered filling patterns on echocardiography with increased peak early (E) diastolic velocity, reduced late (A) diastolic velocity, consequently increased E/A ratio, and with reduced deceleration time [120]. Reduced deceleration time has been shown to represent an increase in diastolic passive stiffness of the LV [121]. Right heart cardiac catheterization demonstrates increased pulmonary artery systolic pressure indicative of increased pulmonary capillary wedge pressure [34]. In some patients, myocarditis-associated cardiac dysfunction is transient and does not lead to chronic cardiomyopathy. In others, myocarditis can progress to DCM, which is manifested by enlarged LV dimensions on echocardiography, reduced LV wall motion, and pronounced impairment of systolic function. The acute phase of EAM is also characterized by deterioration of both systolic and diastolic function as demonstrated by pressure-volume data obtained by means of in vivo LV catheterization (Fig. 3E, F), the gold standard method for assessing cardiac function first described in mice by Georgakopoulos et al [122]. Higher histologic scores of myocarditis severity correlate with reduced cardiac output, stroke work, ejection fraction, end-systolic pressure, maximal rate of pressure development (dP/dtmax) and power output. Concurrently, diastolic dysfunction is manifested by increased end-diastolic pressures, impaired diastolic relaxation, decreased peak filling rate and, most importantly, increased passive stiffness. Extensive myocardial damage eventually leads to the development of DCM, characterized by
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further suppression of systolic function and associated myocardial remodeling resulting in increased LV volumes. DCM leads to congestive heart failure with markedly increased end-diastolic pressures, suppressed end-systolic pressures and the inability to generate adequate cardiac outputs (Fig. 3). Nishio et al [123] assessed cardiac function using pressure-volume method in DBA/2 mice during the first 14 days after infection with EMCV and observed the most pronounced suppression of both systolic and diastolic function on day 7, which somewhat improved by day 14. LV volumes, however, were largest on day 14, indicating cardiac remodeling consistent with the progression to DCM. In this regard, it would be of interest to assess cardiac function at later time points after infection. In preliminary experiments, we assessed cardiac function during the chronic phase of CB3-induced myocarditis in BALB/c mice (day 35 post-infection) and found a significant reduction in systolic function with less pronounced diastolic dysfunction.
3.5.2. Electrical remodeling Arrhythmias and other electrocardiographic abnormalities are frequent manifestations of myocarditis in humans. Despite some differences in electrophysiologic properties between rodents and humans [124], mouse and rat models of myocarditis also demonstrated pro-arrhythmic electrical remodeling. Less et al [125] studied electrical abnormalities during the acute phase (day 21) of EAM in BALB/c mice using the whole cell patch clamp technique. The authors found prolongation of the action potential duration (APD) with a decrease in repolarizing transient outward current (Ito). Increases in APD have been shown to be arrhythmogenic and are similar to prolongation of Q-T interval in humans, which is associated with life-threatening arrhythmias. Arrhythmogenic prolongation of APD associated with repolarization abnormalities has been observed in heart failure patients [126]. Prolonged action potential was also found during the acute phase of EAM in Lewis rats and was associated with decreased levels of mRNA of Kv4.2, a channel subunit important for transient outward K § currents as a part of Ito current [127]. Patients with heart failure have been shown to exhibit abnormalities in Ca 2§ transients [128]. Cardiomyocytes from
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heart failure patients show reduced peak amplitude of Ca 2+ transients, prolongation of resequestration of cytosolic Ca 2§ into the sarcoplasmic reticulum (SR), and reduced levels of Ca 2§ in the SR [129]. The latter may be explained by reduced levels of SR Ca2+-ATPase, which pumps Ca 2+ into the SR, increased levels of phospholamban, which inhibits SR CaZ§ and increased activity of sodiumcalcium exchanger, which transports Ca 2§ out of the cell and decreases its availability for the SR uptake [128, 130]. Studies measuring cardiomyocyte Ca 2§ transients during the acute phase of EAM failed to detect reduced Ca 2§resequestration rates and even suggested a faster rate of Ca 2§ removal from the cytoplasm in cultured cardiomyocytes [125, 131]. Protein levels of SR Ca2+-ATPase were unchanged and levels of phospholamban and sodium-calcium exchanger were actually reduced on days 18 and 35 post-immunization in EAM [131]. Ca 2§ cycling needs to be further evaluated in myocarditis models including studies of the chronic phase and in mice with signs and symptoms of heart failure. Importantly, changes in ion transients in cardiomyocytes should be compared to the mechanical performance of cardiomyocytes and of the whole LV chamber in the same mouse.
3.5.3. Direct virus-mediated damage The above mentioned functional abnormalities in myocarditis can result from myocardial damage caused either directly by the virus itself or by the virus-triggered immune response. Viral infection can directly cause cardiomyocyte lysis. A more subtle change mediated by the virus was illustrated in the study by Badorff et al [132]. The authors demonstrated the ability of 2A protease of CB3 to cleave dystrophin both in cultured cardiomyocytes and in infected murine hearts. Dystrophin is a cytoskeletal protein, which provides structural support to the cardiomyocyte and links the sarcomeric contractile apparatus to the sarcolemma and extracellular matrix. Its disruption may compromise the force-generating capability of myocytes and result in DCM. It has been suggested in humans that defects in dystrophin predispose to DCM [133].
3.5.4. Immune-mediated damage Examples of the immune-mediated damage are more abundant and include direct effects of inflammarion-associated cytokines on the heart.~The classic example is TNF-ct, which has been shown to exert negative inotropic effects on cardiomyocytes, alter intracellular signaling, and promote cardiomyopathy [ 134]. Transgenic mice with cardiac-specific overexpression of TNF-tx develop cardiac dysfunction and DCM [ 135, 136]. Other cytokines, such as IL-1 and IL-6, have been shown to affect signaling in cardiomyocytes altering their metabolism and triggering hypertrophy and/or apoptosis [137]. In addition to cytokines, certain inflammatory cells can cause direct damage to cardiomyocytes. In vitro studies have demonstrated the ability of neutrophils to cause free radical-mediated injury of cardiomyocytes and impair their shortening [ 138, 139]. Neutrophils represent a significant component of myocardial infiltrate in severe myocarditis and their numbers correlate with the severity of myocarditis (our unpublished observations). Cardiac function can also be affected by the remodeling of the extracellular matrix in response to inflammation. Different inflammatory mediators can either stimulate or inhibit collagen production and, once collagen is produced, can either promote or inhibit its degradation. Some matrix metalloproteinases (MMPs) have been shown to degrade fibrillar collagen and this process is inhibited by tissue inhibitors of metalloproteinases (TIMPs). Li et al [140] have shown the upregulation of MMP3 (a stromelysin) and MMP-9 (a gelatinase) and concomitant downregulation of their inhibitors, TIMP-1 and TIMP-4, in BALB/c mice on day 10 post-infection with CB3. There is still much to learn about the signaling pathways in the heart and how they are affected by different cytokines as well as the effects of cytokines on inotropy, cardiomyocyte hypertrophy, and myocardial remodeling including fibrosis.
4. M O L E C U L A R MIMICRY VERSUS ADJUVANT EFFECT The potential mechanisms of how CB3 or other cardiotropic viruses trigger the autoimmune response
have been extensively reviewed in the literature [42]. Despite numerous attempts to study these mechanisms, they remain unclear. For some time the molecular mimicry hypothesis attracted a lot of attention and investigators have sought evidence in its support. The concept of molecular mimicry implies that the infecting microorganism shares an epitope with the tissues of the host. For example, infections by [3-hemolytic streptococci induce an antibody that cross-reacts with streptococcal M protein and CM [141]. It was shown that an antibody can cross-react with both CM and CB3 [ 142]. However, the comparison of CB3 sequence with that of CM failed to demonstrate any significant sequence identity, suggesting a low likelihood for cross-reactivity at a T-cell level. Autoimmune myocarditis could not be induced by immunizing mice with inactivated CB3 in adjuvant (our unpublished observations), again failing to support the molecular mimicry hypothesis. Horwitz et al [ 111] argued against the molecular mimicry hypothesis and for the role of direct viral damage to the heart in triggering myocarditis. In this study, NOD mice with a pancreas-specific transgenic expression of IFN-~, were infected with CB3. They developed pancreatitis but no myocarditis despite the production of heart-specific IgG. The authors concluded that myocarditis could only occur if the virus infected the heart and the presence of the viral infection elsewhere could not initiate the autoimmune process in the heart through molecular mimicry. While the importance of molecular mimicry remains controversial, recent studies favor the alternative explanation that infecting viruses cause myocardial damage and provide an appropriate inflammatory milieu, so that presentation of CM epitopes is enhanced and such presentation does not lead to tolerance but rather to immune activation. This phenomenon could be termed "an adjuvant effect" of the viral infection [143]. Similar to CFA or any other adjuvant, viral infection produces an appropriate context for the immune system activation leading to recognition of self-epitopes and subsequent autoimmune process. The initial infection with CB3 triggers activation of the innate immune system and signaling through a set of pattern recognition receptors resulting in the production of proinflammatory mediators, such as TNF-t~ and IL-1 [3. These proinflammatory mediators then participate in the
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activation of the adaptive immune system and promote presentation of cardiac antigens by DCs. With the activation of the adaptive immune system and further development of inflammation, the balance between the disease-promoting and disease-suppressing factors determines the outcome. Factors, such IL-12, IL-6, and possibly IL-4, may perpetuate the disease and produce severe cardiomyopathy, whereas IFN- T and IL-10 may suppress the inflammation and limit disease.
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5. C O N C L U S I O N
4.
Despite a great deal of effort to understand the nature of virus-triggered inflammatory heart disease, the processes that underlie the progression from viral infection to an autoimmune disease and finally to cardiomyopathy and heart failure remain poorly understood. The animal models provide an opportunity to study the complex phenomena of viral entry and replication, immune response to the viral infection, autoimmune response to cardiac antigens, the role of individual inflammatory components in disease progression, the nature of cardiac remodeling in response to viral damage and inflammation, and the development of cardiac dysfunction. A better knowledge of each of these stages of disease is needed for the improvement of therapeutic interventions. Translation of the research findings into the clinically meaningful data also requires an understanding of the advantages and limitations of individual animal models, formulation of hypotheses based on the basic research findings, and a careful design of clinical trials to address these hypotheses.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Rheumatic Fever: How Streptococcal Throat Infection Triggers an Autoimmune Disease Luiza Guilherme 1,2 and Jorge KaliP ,z,3
tHeart Institute - lnCor, University of Sao Paulo, School of Medicine; 2iii-lnstitute for Immunology Investigation, Millenium Institute; 3Clinical Immunology and Allergy, Department of Clinical Medicine University of Sao Paulo, School of Medicine, S~to Paulo, Brazil
1. I N T R O D U C T I O N Rheumatic Fever (RF) is a sequel of throat infection by group A streptococci (GAS), affecting 3-4% of untreated children. Rheumatic Heart Disease (RHD) develops 4-8 weeks or later after GAS infection in 30 to 45% of individuals with RF. It remains a major cause of morbidity and mortality in developing countries. Data from World Health Organization showed that 25 to 40% of cardiovascular diseases in these countries are due to RF. In Brazil, the damage to heart valves as a consequence of RF is responsible for 90% of children heart surgeries.
[2]. Schematic representation of S. pyogenes is in Fig. 1. Over 100 different serotypes of group A streptococci have been described [2] and it has been consistently found that some serotypes are more frequently associated with rheumatic fever while others are more often associated with acute glomerulonephritis. These serotypes or strains are called rheumatogenic and nephritogenic, respectively [3, 4]. The M protein is the most important antigenic structure and shares structural homology with alpha helical coiled-coil human proteins like cardiac myosin, tropomyosin, keratin, laminin, vimentin and several valvular proteins [5-9].
2. S T R E P T O C O C C U S PYOGENES Studies done by Rebecca Lancefield [1] in 1941 classified streptococci groups based on the cell wall polysaccharides (groups A, B, C, F and G). The S. Pyogenes (group A streptococcus) is characterized by carbohydrates composed of N-acetyl [3 D-glucosamine and rhamnose. The group A streptococci (GAS) contains the M, T and R surface proteins and the lipoteichoic acid (LTA) involved in the bacterial adherence to the throat epithelial cells. The M protein extends from the cell wall and it is composed by approximately 450 amino acid residues with antigenic variations but high homology on aminoterminal (N-terminal) portion, except for the 11 first amino acid residues that define the different serotypes. The carboxi-terminal (C-terminal) half end contains multiple repeat regions and is conserved
3. GENETIC MARKERS Determination of a genetic pattern of susceptibility to RF and RHD was pointed out by Cheadle more than a century ago [10]. To define the pattern of inheritance of RF some researchers have assumed an autossomic recessive model [11 ], whereas others, a mendelian pattern of inheritance [12]. Observation of RF or RHD in identical twins suggested that if a mendelian pattern is present, penetrance must be incomplete [13]. Correlation with blood groups or secretor status of patients with RF was observed with a higher incidence of a nonsecretor pattern in affected subjects as well as a reduction of blood group O frequency in rheumatic children [14]. Patarroyo described the presence of an alloantigen
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M protein LTA
T,R .'-~ +--... I
/
N-terminal portion
9N . N , I li,.~,-N+~ -+'~+N,.~'..t+N'.'
.Nit_*V.P,i-~/*,'
.+.++,..
+/] i C-terminal portion
Outer hyaloronic acid capsule Figure 1. Schematic representation of S. pyogenes. Group A streptococcal cell is covered by an outer hyalorunic acid capsule and is characterized by the group A carbohydrates composed of N-acetyl [] D-glucosamine and rhamnose. M, T and R are surface proteins; LTA: lipoteichoic acid- involved in the bacterial adherence to the throat epithelial cells; N: amino-terminal portion that contains A and B regions; A region defines the serotypes of streptococci strains; C: carboxi-terminal portion contains C and D regions that are highly conserved among the streptococci strains. on the surface of B cells designated 883 present in more than 70% of RF patients from Bogota and New York [15]. However indirect evidence suggested that 883 alloantigen could be related to the HLAclass II molecules [ 16, 17]. A monoclonal antibody was produced against 883 alloantigen [18] called D8/17 that identifies a B cell antigen with enhanced expression in 90-100% of RF patients [ 17]. No consistent association with HLA class I antigens and RF/RHD was found, however, association with different HLA class II antigens has been indicated in several populations (Table 1). The HLA DR4, DR7 and DR9 antigens are in linkage disequilibrium with HLA-DR53. Interestingly, in American Caucasian and Arabian patients an association with HLA-DR4 and rheumatic fever was found [19-21] whereas in Brazilian and Egyptian patients HLA-DR7 was associated with the disease [23-26] (Table 1). HLA class II antigens play an important role in the antigen presentation to the T cell receptor (TCR). The divergence of HLA class II molecules associated with the disease in different
322
countries is probably due to the capability of these molecules to present strain-specific streptococcal epitopes present in more than 80 streptococcal serotypes [30], some of t h e m - including the rheumatogenic s t r a i n s - with peculiar geographic distribution.
4. PATHOGENESIS The pathogenic mechanisms involved in the development of RF/RHD are not fully understood. It is considered that the molecular mimicry mechanism is responsible for the cross reactive reactions between streptococcal antigens and human tissue proteins, mainly heart tissue proteins in susceptible individuals. Nowadays it is clear that the disease is mediated by both humoral and cellular immune responses and that the cellular branch of the immune response is more involved with the development of rheumatic heart disease (RHD).
Table 1. HLA class II antigens and rheumatic fever HLA
Country
Reference
Population ,,
DR4, DR9 DR2 DR4 DR3 DQW2,D8/17 DR7, DR53 Allogenotope TaqI DRbeta 13.81 kb DR7, DQ2 DR7 DR1, DR6 DR11 DR 1
[19, 20]
Saudi Arabia India
American Caucasian American Black Arabian Indian
Brazil Brazil Egypt Brazil South Africa Turkey Brazil
Mulatto Mulatto Egyptian Caucasian African Turkish Mulatto
[23] [24] [25] [26] [27] [28] [29]
USA
[21] [22]
Several HLA antigens are associated with RF/RHD in different countries. HLA-DR4, DR7 and DR9 were associated with HLA-DR53. HLA-DR4, DR9 were found in american caucasian and arabian patients; DR7 in Brazilian (mulatto) and egyptian patients.
4.1. Humoral Immune Response Streptococcal antibodies react with streptococcal antigens and several human tissues including heart, skin, brain, glomerular basement membrane, striated and smooth muscles [31]. Heart-reactive antibodies were described early [32] and Kaplan's studies demonstrated the presence of rabbit and human heart cross reactive antibodies with components of group A streptococci (GAS) in the sera of animals immunized with streptococcal antigens and sera from RF and RHD patients, as well as bound immunoglobulins and complement in the myocardium of ARF patients [33, 34]. After these works several studies were done analyzing sera from both animals and humans or using monoclonal antibodies demonstrating the presence of cross reactive antibodies to streptococcal and human proteins. Cardiac myosin seems to be one of the major cross reactive antigen (reviewed by M. Cunningham) [35]. Recently, we have analyzed the humoral response against overlapping peptides of N-terminal portion of M5 protein and we could identify several immunodominant epitopes recognized by mild RHD patients, most of them with Sydenham's chorea associated. Antibodies from severe RHD patients recognized few N-terminal epitopes (Fig. 2), (manuscript in preparation).
On the other hand, we have also tested the humoral reactivity of sera from RF and RHD patients against heart tissue proteins isolated by molecular weight and isoelectric point. Using this approach we could identify a very large number of heart tissue proteins (316 proteins derived from myocardium and 78 from mitral valve) recognized by sera from these patients. Interestingly, we did not find reactivity against cardiac myosin, but we could characterize six major proteins by peptide mass analysis, one of them with very high homology with vimentin (manuscript in preparation). Although, the presence of human and animal antibodies against streptococcal antigens and human tissue proteins have been described for more than 50 years, their role in the development of the disease remain unclear. One possibility to explain the presence of antibodies in the heart tissue was suggested by the work done by Roberts et al [36] that showed an increased expression of VCAM-1, an adhesion molecule, in the vascular endothelium that was activated after an inflammatory reaction started by antimyosin and N-acetyl-glucosamine. The VCAM-1 molecules interact with VLA-4, another adhesion molecule expressed on CD4 § T lymphocytes. This could be one way to recruit CD4 +T lymphocytes to the heart valves.
323
Residues
Peptide Sequences
11-25"
QRAKEALDKYELENH
Mild RHD/ chorea __~atients
ELENHDLKTKNEGLKTENEG
21-40 41--60
LKTENEGLKTENEGLKTEKK
81-96"
DKLKQQRDTLSTQKET
81-103" 101-120
LKQQRDTLSTQKE,TLEREVQN NGDLTKELNKTRQELANKQQ
111-130 121-140
TRQELANKQQESKENEKALN ESKENEKALNELLEKTVKDK
131-150
ELLEKTVKDKIAKEQENKET
141-160 163-177"
Severe RHD patients
IAKEQENKETIGTLKKILDE iiiiiiiiiiiii~il-iiii!!'i!!!:;.:i!~iiiiiii~!iiiii~ii~i!iiiiiii~i~i
ETIGTLKKILDETVK
iii!!i!ii!iii!~!iiii!iiiiiiiili!iiii!i!iii!!~ii!!! ~
181-200
KILDETVKDKLAKEQKSKQN
183-201 * 191-210
LDETVKDKLAKEQKSKQNI
iiiiiii!iii
i
iiiiiiiiiii!i i
LAKEQKSKQNIGALKQELAK
Figure 2. Immunodominant epitopes of streptococcal N-terminal region from M5 protein recognized by antibodies of RHD and Sydenham's chorea patients. Humoral reactivity against overlapping peptides was tested by ELISA immunoassay. The immunodominat regions were determined by comparing the reactivity of sera from RHD patients with sera from healthy individuals. P values < 0.05 were considered significant. The peptides preferentially recognized are represented as gray for mild RHD patients with or without Sydenham's chorea and dark gray for severe RHD patients. * The sequences of M5 peptides were based on the sequence of the M5 protein published by Manjula et al [51]. The other M5 peptide sequences were based on the sequence of the M5 protein published by Robinson et al [53]. Overlapping peptides are aligned or underlined.
4.2. Cellular I m m u n e Response The studies of cellular branch of immune response began around 1970. In favor of the important role of T cells in RF, some studies have been performed in tonsils and human peripheral blood showing that CD4 + T cells were increased [37, 38]. It was also demonstrated that T cells were able to recognize streptococcal cell wall and tissue antigens [39-44]. A cytotoxic activity towards immortalized human heart cells was also described [45, 46]. The first evidence that CD4 § T cells were
324
involved in RHD lesions was described 20 years ago [47]. The isolation of T cells from heart valves led Yoshinaga et al [48] to compare the reactivity of PHA stimulated T cell lines derived from heart valves specimens and peripheral blood lymphocytes of RF patients and showed that, although these cells recognized cell wall and membrane streptococcal antigens, they failed to react with M protein, myosin or other mammalian cytoskeletal proteins. The functional activity of heart-infiltrating CD4 § T cell clones was directly demonstrated by our group. We defined, for the first time, the presence
Table 2. Immunodominant T cell epitopes of N-terminal portion of streptococcal M5 protein M5 epitope
Sequence
Nature of T lymphocytes
Ref.
1-25" 81-96 83-103 163-177
TVTRGTISDPQRAKEALDKYELENH DKLKOORDTLSTQKET LKQQRDTLSTQKETLE.REVQN ETIGTLKKILDETVK
Human intralesional T cell clones from RHD patientsb
[9]
Peripheral blood of RF/RHD patients
[50]
40-58 (NT4) 59-76 (NT5) 72-89 (NT6) 137-154 (B 1B2) 150-167 (B2) 163-180 (B2B3A) (B3A)
GLKTENEGLKTENEGLKTE KKEHEAENDKLKQQRDTL QRDTLSTQKETLEREVQN VKDKIAKEQENKETIGTL TIGTLKKILDETVKDKIA KDKIAKEQENKETIGTLK IGTLKKILDETVKDKLAK
Murine Lymph node cells
[49]
The sequence of M5 protein (Refs. [9, 50]) was taken from sequence published by Manjula et al [51] and M5 protein used in Ref. [49] by sequence published by Miller et al [52]. aM5(1-25) align with M5(1-35) described by Robinson et al [53]. Underlined, Bold type- shared sequences of M5 peptides. bPeptides recognized by human T cell clones presented cross reaction with human valvar proteins [9]. of intralesional cross reactive T cell clones and we established the significance ofT-cell molecular mimicry in the pathogenesis of RHD. We mapped the Nterminal reactivity of intralesional T cell clones and this study led us to identify three immunodominant regions: 1-25, 81-103 and 163-177 residues within the streptococcal M protein and cross reactive with several heart tissue protein fractions, mainly those derived from valvular tissue with molecular mass of 95-150 kDa, 43-63 kDa and 30--43 kDa [9]. Myosin / M5 protein cross reactive T cell epitopes were also investigated in mice immunized with intact cardiac myosin [49]. Lymph node T cells were tested against overlapping M5 peptides named NT5/6/7 and B 1B2/B2 and B2B3A/B3A align with the M5 regions identified by us, the M5(81-96) and M5(163-177) respectively (Table 2). Robinson et al [53] obtained lymph node T cell clones from mice immunized with recombinant M5 protein that were able to recognize M5 epitopes. Among the M5 epitopes recognized by mice T cell clones, only the M5(1-35) epitope align with the M5(1-25) region recognized by the human infiltrating T cell clones (Table 2). T cells from peripheral blood of RF/RHD disease patients recognized several M5 peptides. Interestingly, the immunodominant peptide M5(81-96) was
preferentially recognized by DR7 § DR53 § severe RHD patients [50], suggesting that HLA DR7 DR53 molecules could be more involved with the selection of streptococcal peptides and their presentation to the T cell receptor (TCR). Several heart protein fractions were also recognized in the periphery by severe RHD patients. In order to better characterize the heart tissue proteins, we recently identified several heart-derived proteins isolated by molecular weight (MW) and isolelectric point (pI). Several valve-derived proteins were recognized by peripheral blood and intralesional T cell clones from severe RHD patients. Among them, we identified vimentin (MW 53 kDa/pI 5.12) and other cytoskeleton proteins as candidates for being the targets of the valvular lesions in RHD (manuscript in preparation). In line with these results, previous work showed the recognition of 50-54 kDa myocardial derived protein by peripheral T lymphocytes from RHD patients [54]. M protein has an important role in anti-streptococcal immune response of the host and was considered with superantigenic properties by some researchers. Superantigens are proteins that polyclonally activate T cells by an MHC class-II dependent, but haplotype-unresctricted mechanism. Proliferative responses to superantigens are limited to T cells
325
Table 3. Degeneracy of antigen recognition by intralesional CD4§T cell clones T cell Clone Antigens Identification Recognized Lu 3.1.8
BV Family CDR3(N-D-N) Sequences
35 kDa/pI 8.84 BV 13
BJ Family
SGRQGRYEQY BJ 2S7 (10aa)
LMM 28 (1647-1664) LMM 28B (1660-1677) LMM 32 (1699-1716) Lu 3.1.29
56-53 kDa/ pI 6.76
BV 13
SGRQGRYEQY BJ 2S7 (10aa)
AV Family CDR3 (N-D-N) Sequences
AJ Family
AV 2
MRTPVTSSI (9aa)
AJ- NT
AV 3
TDPITGTASKLTAJ 44 (12aa)
AV 2
MRTPVTSSI (9aa)
AV 3
TDPITGTASKLTAJ 44 (12aa)
AJ- NT
NT-not tested; LMM-light meromyosin peptides: LMM28-SLQSLLKDTQIQLDDAVR; LMM28B-DDAVRANDDLKENIAIVE; LMM31-LEELRAVVEQTERSRKL; LMM32-RSRKLAEQELIETSERV. Underlined - shared sequences. Adapted of Fa6 et al, Mol Immunol, 2004 (in press).
expressing a particular TCR-BV gene but independent of antigen specificity. In humans pepM5 preparations (pepsin cleaved fragment) were found to be superantigenic for human T cells expressing TCR-BV2, BV4, and BV8 [55-60], however, using recombinant M5 protein or recombinant pep M5 no evidence of superantigenicity was found [61]. It was also reported that the superantigenicity of pepM 1 and pepM5 were due to contamination with pyrogenic exotoxins that had a potent superantigen effect on BV2-bearing human T cells [62, 63]. Our studies on TCR B V usage in the PBMC of severe RHD patients and infiltrating T cell lines derived from myocardium and/or mitral valve showed expansion of several B V families with oligoclonal profiles mainly in infiltrating T cell lines. These results are in favor of no superantigenicity of M proteins in RHD patients. Some major oligoclonal BV expansions were shared between mitral valve and left atrium T cell lines but an indepth analysis of BJ segments usage in these shared expansions, as well as the nucleotide sequencing of the CDR3 regions suggested that different antigenic peptides could be predominantly recognized in the mitral valve and the myocardium [64]. The high frequency and the persistence of T cell oligoclonal expansions in the damaged heart valves seem to be
326
associated with the progression of the disease [65], probably related to the spreading of autoantigen epitope recognition. In agreement with these data, it has been described that it is possible to detect some T cell expansions in the damaged heart valves even 20 years after acute rheumatic fever episode [66]. The TCR analysis of intralesional T cell clones showed a degenerate pattern of reactivity. Several mitral valve-derived T cell clones recognizing different antigens, presented the same TCR B VBJ and CDR3 sequences. They expressed two alpha chains at the RNA level with same AVAJ segments (Table 3) indicating that intralesional T cell clones with common TCR usage can recognize several epitopes that probably amplify the deleterious immune reaction [67].
4.3. Cytokine Profile The evaluation of proinflammatory cytol,dnes levels produced by peripheral blood and tonsillar mononuclear cells after streptococcal antigen and pokeweed mitogen stimulations from RF/RHD patients without congestive heart failure showed a different pattern on PBMC and tonsillar cells. TNF alpha, IL-1 and IL-2 were overproduced by PBMC and decreased by tonsillar mononuclear cells [68].
In acute rheumatic fever (ARF) patients and patients with active RHD increased production of IL-2 and elevated numbers of CD4 § and CD25 § cells were observed suggesting the expansion of activated T CD4 § cells in the peripheral blood during the active phase of the disease [69]. These results were confirmed by other authors and increased plasma levels of TNF alpha in RF/RHD patients was also showed [70--72]. The Aschoff nodule is considered as the pathognomonic lesion of RF. It is composed of an agglomerate of cells having characteristics of monocytic and macrophage cells [73, 74] and probably function as antigen-presenting cells (APC) to the T cells. In the valve lesions of ARF patients, the production of IL-1, TNF alpha and IL-2 correlated with progression of Aschoff nodules as follows: stages 1 and 2, IL-1 and TNF alpha were secreted by monocytes/macrophages and stage 3, IL-2 by T lymphocytes [75]. We analyzed the cytokine pattern of infiltrating mononuclear cells in the mitral valve and myocardium tissue of ARF and chronic RHD patients (manuscript submitted). Our results showed a predominantly T1 type of cytokine produced mainly by CD4 § T cells that could mediate Rheumatic Heart Disease heart lesions.
5. ANIMAL MODEL Different protocols to reproduce rheumatic lesions in animal models were attempted for more than 60 years, however, at this time the immune reactions involved with the development of rheumatic lesions were not known. In the 1960's W.J. Cromartie and J.H Schwab developed a murine model injecting bacterial cell wall into mice and they observed that portions of the injected material were taken up by macrophages but not digested. The non degraded substance remained in the phagocytic cells and was able to produce cardiac inflammatory responses [76]. It was demonstrated later that macrophage cells of mice infected with extracts of GAS were able to induce the appearance of heart lesions when transferred to syngeneic receptors and, in vitro, a specific response to syngeneic heart extracts. This model suggested, at the time, that macrophage displayed a key role by selecting determinants for antigen presentation to the TCR [77]. By using M protein synthetic pep-
tides it was shown that some peptides were capable to induce inflammatory heart disease in mice [78] whereas other peptides after imunization induced strong proliferative response, but not heart lesions [49]. An epitope from M protein that contains a repeated residue sequences, named NT4, has been described.This epitope presents shared sequences with cardiac myosin protein region and was able to induce myocarditis in mice [79]. Recently, it was demonstrated that the injection of M6 recombinant protein in Lewis rat induced a myocarditis and inflammatory valvular heart lesions similar to those seen in rheumatic heart disease. A lymph node CD4 § T cell line obtained from immunized rat recognized M6 recombinant protein and cardiac myosin [80]. This cross reactive T cell line produces IL-2 and IFN), when stimulated with M5 peptides from B region of the M protein, which is implicated with heart-cross reactive reactions [79]. Following the experiments in animal models, the same group differentiated segments of cardiac myosin able to induce myocarditis from those inducing valvulitis [81]. The construction of these experimental models of myocarditis and valvulitis induced by streptococcal antigens and cardiac myosin certainly will contribute to better understand the pathogenesis of rheumatic heart disease and could be useful for vaccine designs.
6. CONCLUSIONS Altogether, all the results presented here delineate RF/RHD as a complex autoimmune disease mediated by both humoral and cellular immune response and point out the major role of CD4 § T cells in the development of rheumatic heart lesions. The autoimmune reaction in the heart probably is mediated by a network of immune reactions, involving the recognition of several auto-antigens triggered on the periphery by an immunodominant streptococcal antigen that expands several T cell clones by epitope spreading. These T cell clones migrate to the heart, the local production of inflammatory T-1 cytokines trigger the activation of autoreactive infiltrating T cells, that were able to recognize several auto-antigens with conformational or sequence homologies. New and important data shown here is the degeneracy of antigen recognition suggesting a new type
327
H L A class II molecules DR7 + DR53 +
9 Immunodominant peptides M 5 ( 8 1 - 9 6 ) and M5(83-103) / several heart-tissue derived proteins 9 -
-
-
TCR several oligoclonal expansions in the site of the heart lesions degeneracy of antigen recognition intramolecular epitope spreading
9 Cytokine profile Predominantly T 1-type in the site o f the lesions Figure 3. Model of T cell recognition for RHD patients. After group A streptococcal throat infection, untreated susceptible individuals (5-18 years old) developed RF/RHD. Humoral and cellular immune response against S. pyogenes, mediated mainly by proinflammatory cytokines (Th-1 type) lead to an autoimmune attack to human tissues. Here we propose a model of T cell recognition based on our results. The autoimmune reaction is initiated in the periphery where T cells recognize immunodominant M5 peptides as M5(81-96) and M5(83-103) presented by APC (antigen presenting cells) (macrophage/monocytes) in the context of HLA class II DR7 DR53 molecules. After, the activated T cell clones expanded migrate to the heart (myocardium and valvular tissue), several heart tissue proteins are recognized by molecular mimicry. T cell clones display degenerate TCR (T cell receptor) capable to recognize different antigens. These cells amplify the cross reactivity by intramolecular degenerate reactivity. In the heart tissue the cytokines produced are also predominantly Th-1 type.
of intramolecular epitope spreading that probably amplify the autoimmune reaction. Fig. 3 is a model of RHD development based on our results. One remaining question is the role of these degenerated T cell clones in establishing disease as pathological autoreactive T cells or regulating disease progression as physiological autoreactive T cells.
2. 3.
4. 5.
6. R
E
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N
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59.
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62.
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65.
66.
67.
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Exp Immunol 1993;91:429-436. 70. Narin N, Ktittikqtiler N, Ozytirek R, Bakiler AR, Parlar A, Arcasoy M. Lymphocyte subsets and plasma IL-1 ix, IL-2, and TNF-oc concentrations in acute rheumatic fever and chronic rheumatic heart disease. Clin Immunol Immunopathol 1995;77:172-176. 71. Samsonov MY, Tilz GP, Pisklakov VP, Reibnegger G, Nassonov EL, Nassonova VA, Wachter H, Fuchs D. Serum-soluble receptors for tumor necrosis factor-o~ and interleukin-2 and neopterin in acute rheumatic fever. Clin Immunol Immunopathol 1995;74:31-34. 72. Yegin O, Coskun M, Ertug H. Cytokines in acute rheumatic fever. Eur J Pediatr 1997;156:25-29. 73. Kemeny E, Grieve T, Marcus R, Sareli P, Zabriskie JB. Identification of mononuclear cells and T cell subsets in rheumatic valvulitis. Clin Immunol Immunopathol 1989;52:225-237. 74. Chopra P, Narula J, Kumar SA, Sachdeva S, Bathia ML. Immunohistochemical characterization of Aschoff nodules and endomyocardial inflammatory infiltrates in left atrial appendages from patients with chronic rheumatic heart disease. Int J Cardiol 1988;20:99-105. 75. Fraser WJ, Haffejee Z, Jankelow D, Wadee A, Cooper K. Rheumatic Aschoff nodules revisited. II. Cytokine expression corroborates recently proposed sequential stages. Histopathology 1997;31:460-464. 76. Williams RC Jr. Host factors in rheumatic fever and heart disease. Hosp Practice 1982; 125-138. 77. Dos Reis GA, Gaspar MIC, Barcinski MA. Immune recognition in the streptococcal carditis of mice: the role of macrophages in the generation of heart-reactive lymphocytes. J Immunol 1982; 128:1514-1521. 78. Huber AS, Cunningham MW. Streptococcal M protein peptide with similarity to myosin induces CD4+ T celldependent myocarditis in MRL/++ mice and induces partial tolerance against coxsackieviral myocarditis. J Immunol 1996; 156:3528-3534. 79. Cunningham MW. Molecular mimicry between group A streptococci and myosin in the pathogenesis of acute rheumatic fever. In: Jagat Narula, Renu Virmani, K Srinath Reddy and Rajendra Tandon, eds. Rheumatic Fever. Eds American Registry of Pathology, 1999;135165. 80. Quinn A, Kosanke SD, Fischetti VA, Factor SM, Cunningham M. Induction of autoimmune valvular heart disease by recombinant streptococcal M protein. Infect Immun 2001, 69:4072-4078. 81. Galvin JE, Hemric ME, Kosanke SD, Factor SM, Quinn A and Cunningham M. Induction of myocarditis and valvulitis in Lewis rats by different epitopes of cardiac myosin and its implications in rheumatic carditis. Am J Patho12002; 160 (1):297-306.
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Published by Elsevier B. V 2004. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) Susan E. Swedo, Lisa A. Snider and Marjorie A. Garvey
Pediatrics & Developmental Neuropsychiatry Branch, National Institute of Mental Health, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA
1. INTRODUCTION The relationship between obsessive-compulsive symptoms and Sydenham's chorea (SC) was first noted by Sir William Osier when he described "bizarre" and "perseverative behaviors" of children with "chorea minor" [ 1]. Three subsequent clinical reports noted an association between SC and OCD among children with rheumatic chorea, and adult psychiatric patients with a history of the disorder [24]. A series of studies conducted at the National Institute of Mental Health (NIMH) demonstrated that obsessive-compulsive symptoms were more problematic for children with SC, than for those with rheumatic carditis [5]; and that obsessions and compulsions affected more than 70% of the children in the weeks surrounding the onset of their chorea [6, 7]. The NIMH findings were subsequently replicated and extended by Asbahr and colleagues in Sao Paulo, Brazil, who demonstrated that not only does OCD affect approximately 2/3's of children presenting with an initial episode of SC, but also that the frequency of OCD increases with repeated bouts of chorea, affecting 100% of children experiencing three or more recrudescences [8]. The obsessive-compulsive symptoms in SC are indistinguishable from those of children with primary OCD, and include contamination fears, fear of harm coming to self or others, doubting, symmetry concerns, and other obsessional worries, as well as compulsive washing, checking, ordering, arranging, and hoarding rituals. These clinical similarities, and observations that the OCD in SC begins several weeks prior to the
manifestation of the adventitious movements, led to speculation that post-streptococcal obsessive-compulsive symptoms might occur in the absence of frank chorea [5, 6]. Longitudinal observations of a large cohort of pediatric patients with OCD [9, 10] provided support for this postulate, as a subgroup of patients had an acute symptom onset, an episodic course characterized by periods of complete symptom remission interrupted by abrupt, dramatic symptom exacerbations, and a close temporal relationship between these relapses and preceding Group A beta-hemolytic streptococcal (GAS) infections, (scarlet fever or streptococcal pharyngitis) [11, 12]. The subgroup was labeled with the acronym, "PANDAS", to indicate the shared clinical and presumed etiopathogenic features: Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections [12]. In this chapter, we will review the clinical features of the PANDAS subgroup, and explore the factors postulated to participate in etiopathogenesis.
2. CLINICAL FEATURES OF THE PANDAS SUBGROUP The clinical features of the first 50 children meeting criteria for the PANDAS subgroup were published in 1998 [12]. Those criteria are: 1) The presence of a tic disorder and/or obsessive-compulsive disorder. 2) Prepubertal age at onset, usually between 3 and 12 years of age.
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3) Abrupt symptom onset and/or episodic course of symptom severity. 4) Temporal association between symptom exacerbations and streptococcal infections. 5) Presence of neurological abnormalities during periods of symptom exacerbation. Several unique characteristics of the PANDAS subgroup become apparent when the children are compared to unselected patients with childhoodonset OCD and tic disorders [12-16]. The average age at symptom onset in the PANDAS subgroup is nearly three years younger than that previously reported for childhood-onset OCD [9, 17] and up to two years younger than the average age of onset for tic disorders [ 18]. Further, comparisons of the age and sex-distribution of the PANDAS subgroup with that of other OCD patient groups suggests a bimodal distribution [9, 12], (consistent with the postulate that the PANDAS subgroup is truly distinct from other patient groups); however, this cannot be confirmed without large-scale commumunity-based epidemiologic investigations, or the demonstration of a unique etiopathogenesis for the PANDAS subgroup. The clinical course of the PANDAS subgroup differs markedly from that of other OCD patients [9, 12]. Symptom exacerbations in the PANDAS subgroup are sudden and severe, with parents describing the onset of symptoms as occurring "overnight" or "out of the blue." The symptoms remain at peak severity for a period of several weeks or longer, and then gradually subside in severity, often remitting completely, with patients remaining asymptomatic until they're infected again with GAS. This relapsing-remitting course is in striking contrast to the gradual onset and persistent symptoms typically seen in childhood-onset OCD [9, 10] and also differs substantially from the waxing and waning course of tic disorders [18]. Emotional lability, attentional difficulties, separation anxiety, and motoric hyperactivity frequently accompany the OCD/tics exacerbations in the PANDAS subgroup [12]; these clinical features are shared with Sydenham's chorea. Enuresis and daytime urinary frequency are also common [ 12, 16]. Deteriorations in handwriting also have been noted during the symptom exacerbations in the PANDAS subgroup, and may prove useful as an objective means of tracking
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symptom severity [ 19]. Although the etiology of the handwriting changes isn't known, they appear to parallel the appearance of choreiform movements of the hands and fingers. The presence of choreiform movements during neuropsychiatric symptom exacerbations may prove to be one of the most reliable means of identifying children in the PANDAS subgroup [20]. These mild adventitious movements can be elicited during structured neurological examinations, such as the PANESS [21], and were found to be present in 25 of 26 children in the original cohort who were examined during an exacerbation [12]. These findings were replicated recently in a sample of 17 children in the PANDAS subgroup, all of whom demonstrated choreiform movements on the PANESS examination performed during a symptom exacerbation. The choreiform movements are thought to arise from dysfunction of the basal ganglia of the brain, particularly within the caudate nucleus and putamen. These structures are also implicated in OCD, where symptoms are postulated to result from dysfunction of the corticostriato-thalamocortical circuitry [22]. In Sydenham's chorea, functional imaging studies provide evidence of basal ganglia dysfunction during acute chorea [23, 24], and volumetric abnormalities of the caudate, putamen, and globus pallidus were demonstrated in a cohort of 24 children with Sydenham chorea through the use of structural MRI scans [25]. A volumetric MRI study of 34 children in the PANDAS subgroup also revealed enlargements of the caudate, putamen, and globus pallidus [26]. In some patients, the size of the basal ganglia structures has been found to normalize following successful immunomodulatory therapy with IVIG or plasma exchange (example shown in Fig. 1) [27].
3. MODEL OF ETIOPATHOGENESIS
The etiology of the neuropsychiatric symptoms in the PANDAS subgroup is postulated to be similar to that of Sydenham's chorea, the neurologic manifestation of rheumatic fever. Thus, the etiopathogenesis is hypothesized to occur when "rheumatogenic" GAS bacteria infect a susceptible host and induce an abnormal immune response. As shown in Fig. 2, the
Figure 2. MRI scans of a 14-year old male in the PANDAS subgroup pre-/post-treatment.
proposed model not only provides a framework for understanding the etiology of OCD and tic disor-
ders, but also for the development of novel intervention and prevention strategies.
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4. THE ROLE OF S T R E P T O C O C C A L INFECTIONS IN PANDAS For rheumatic fever, the etiologic role of GAS infections was demonstrated indirectly, through three lines of research: 1) epidemiologic investigations which demonstrated a close temporal relationship between scarlet fever epidemics and subsequent outbreaks of rheumatic fever; 2) the prevention of rheumatic fever recrudescences by penicillin prophylaxis, and 3) demonstration of declining rates of rheumatic fever following the widespread application of antibiotic treatment for GAS pharyngitis [28]. Coburn [29] and Collis [30] are credited with establishing the relationship between GAS infections and rheumatic fever by demonstrating a temporal relationship between epidemics of streptococcal infections (scarlet fever and strep, pharyngitis), and subsequent outbreaks of rheumatic fever. Coburn and Young [31] extended these findings by demonstrating that each time the incidence of scarlet fever increased, it was followed three weeks later by a rise in the rate of rheumatic fever cases. Although epidemiological studies are not usually sufficient to establish causality, the clarity of the relationship in these investigations has been accepted as evidence that GAS infections are the etiologic trigger in rheumatic fever [32]. The associative strategy employed for rheumatic fever can be applied to the question of the relationship between GAS infections and OCD/tic symptoms in the PANDAS subgroup. By demonstrating that each neuropsychiatric symptom exacerbations occurs concurrently with, or is preceded by a GAS infection, it will be possible to demonstrate a temporal association, and possibly, evidence of causality. The PANDAS subgroup is defined by a "temporal association between neuropsychiatric symptom exacerbations and GAS infections" [12]. The "gold standard" for demonstrating such a temporal association is prospective, longitudinal assessments. As shown in Fig. 3, these prospective observations can be quite effective in demonstrating differences between a prototypical child in the PANDAS subgroup (A) and one whose symptoms show no temporal relationship to GAS infections (B). Systematic longitudinal studies are required to establish this relationship, and several investigative groups have such prospective studies underway.
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One caveat in evaluating the relationship between streptococcal infections and neuropsychiattic symptoms is that the disorders are so common that co-occurrence can be a random coincidence, rather than a clinically significant finding. Obsessive-compulsive disorder occurs in 1-2% of schoolage children, and transient motor tics in as many as 10-25% of early elementary students [33, 34]. Further, during regional streptococcal epidemics, the majority of children will be infected at least once during the outbreak [35]. Thus, a single positive throat culture or elevated antistreptococcal antibody titer is not sufficient to determine that a child's neuropsychiatric symptoms are associated with streptococcal infections [12, 34, 36]. The determination that a child fits the PANDAS profile is made through prospective evaluation and documentation of the presence of streptococcal infections in conjunction with at least two episodes of neuropsychiatric symptoms, as well as demonstrating negative throat culture or stable titers during times of neuropsychiatric symptom remission [15]. A child who has multiple symptom exacerbations without evidence of streptococcal infection would not be considered as part of the PANDAS subgroup, nor would a child who has numerous streptococcal infections without subsequent symptom exacerbations. The reduction of rheumatic fever (RF) recurrences by antibiotic prophylaxis against GAS infections was a key factor in determining that GAS played an etiologic role in RF. This was particularly true for Sydenham's chorea, in which evidence of an inciting GAS infection was often unobtainable [28]. Antibiotic prophylaxis not only prevented recrudescences, but also improved the long-term prognosis of RF sufferers, by preventing additional scarring of the cardiac valves [37]. The same goal may apply to OCD. A recent report from Sao Paulo, Brazil demonstrated that the frequency and severity of obsessive--compulsive symptoms increased with repeated bouts of Sydenham's chorea [8]. During the initial choreic episode, approximately 65% of the patients had obsessive--compulsive symptoms, which were reported to be "mild" and non-impairing. If the child had two or more recrudescences, the risk of OCD increased to 100%, and all children reported clinically significant symptomatology [8]. If this pattern applies to the PANDAS subgroup, then secondary prophylaxis against GAS infections
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would be important in preventing their episodic symptom course from becoming chronic and treatment-refractory. In order to determine whether or not antibiotic prophylaxis against GAS infections would be effective in reducing the number and severity of neuropsychiatric symptom exacerbations, we conducted a double-blind crossover comparison of penicillin and placebo for children in the PANDAS subgroup [38]. The hypothesis of the study was that penicillin prophylaxis would prevent GAS infections and therefore, post-streptococcal neuropsychiattic symptom exacerbations, resulting in an overall decrease in OCD/tics symptom severity during the penicillin phase (4 months), as compared with the placebo phase (4 months). However, oral penicillin administration failed to provide adequate prophylaxis against GAS, as evidenced by the fact that 14 of the 35 GAS infections occurred during the pen9
cillin phase. Without significant between-phase differences in infection rates, it was not surprising that ratings of OCD and tics symptom severity were not significantly improved during the penicillin phase. Of note, however, among those children for whom penicillin was an effective prophylactic agent, overall behavior was improved, with the penicillin phase ranked as superior to placebo by 75% of the parents who could discern a between-phase difference [38]. The results of the pilot investigation were sufficiently promising to justify a trial of a potentially more effective prophylactic agent, azithromycin (its once a week dosing schedule is associated with improved compliance). Twenty,two children have participated in a 12-month, parallel-design doubleblind trial of azithromycin and penicillin (Swedo et al, unpublished data, 2003). Although we had expected penicillin to function as an active placebo, it proved effective in preventing GAS infections,
337
perhaps because additional compliance measures had been instituted. The rate of GAS infections among the study participants was significantly less during the year of antibiotics administration than during the year prior to the study, whether children were randomized to penicillin (n = 11; mean number of GAS infections in year prior to study = 2.4 + 0.6, zero during study; p<0.01) or azithromycin (n = 11; year prior to study = 2.0 + 1.0; zero during study; p<0.01). The rate of neuropsychiatric symptom exacerbations was also reduced significantly in both groups (from 2.5 per year to 0.7 per year, on average; p<0.01). Thus, the preliminary data indicate that both penicillin and azithromycin may be effective in preventing post-streptococcal neuropsychiatric exacerbations. Further placebo-controlled investigations of the efficacy and effectiveness of antibiotics prophylaxis are required. Such studies are underway at the NIMH and elsewhere.
5. HOST SUSCEPTIBLITY Host susceptibility for the PANDAS subgroup is likely to be the result of a combination of genetic, developmental and immunologic factors. Developmental vulnerabilities are suggested by the increased rates of disease among grade-school age children. Rheumatic fever is rare among children less than 3 years of age, peaks in incidence during the grade-school years, and declines in frequency at adolescence to become rare again during young adulthood [39]. This pattem might reflect developmental differences in immune responsivity, or may be merely the result of changing risks of exposure (GAS infections are most common during the elementary school years). For the PANDAS subgroup, the peak age at onset of symptoms is 6-7 years, with prepubertal symptom onset serving as a defining characteristic of the subgroup [ 15]. Genetic studies have not been reported for Sydenham's chorea, but family history studies suggested an autosomal (dominant or recessive, depending upon sample studied) pattern of inheritance in the familial clusters [40]. A family history study of 21 children with Sydenham's chorea and 15 children in the PANDAS subgroup revealed significantly increased rates of rheumatic fever among the children's parents and grandparents, in comparison with
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the parents and grandparents of 35 healthy controls [5/126 (4.0%), 6/90 (6.7%), and 3/210 (1.4%), respectively [41]. The between-groups differences were small in this pilot data set, but suggested that children in the PANDAS subgroup may inherit a susceptibility to post-streptococcal sequelae similar to that reported for children with SC. Children in the PANDAS subgroup also appear to have increased rates of OCD and tics among their family members. In a recently completed study of 54 probands in the PANDAS subgroup (24 with a primary diagnosis of OCD and 30 with a primary diagnosis of a tic disorder), 21 (39%) had at least one first-degree relative with a history of a motor or vocal tic, and 14 (26%) had at least one first-degree relative with OCD [42]. Six mothers (11%), nine fathers (19%), and eight siblings (16%) had a motor or vocal tic, while l0 mothers (19%), five fathers (11%) and two siblings (5%) had OCD. These rates are substantially higher than those reported for the general population, and similar to rates previously reported for childhood-onset OCD and tic disorders [42]. The combination of increased familial rates of OCD/tic disorders and increased rates of rheumatic fever suggests that children in the PANDAS subgroup may have a dual genetic vulnerability with inherited susceptibilities to both OCD/tic disorders and post-streptococcal sequelae. Proof of this hypothesis must come from genetic determinations, rather than family history studies, and awaits future testing. In the late 1980's, scientists at the Rocekefeller University identified a monoclonal antibody, labeled D8/17, which was positive (>12% of DRpositive cells reactive) in over 90% of rheumatic fever patients, but only 5-10% of healthy controls [43, 44]. Given the large separation between patients and controls, and the knowledge that rheumatic fever affects only 5-7% of the world's population, it was postulated that the D8/17 marker could serve as a trait marker of rheumatic fever susceptibility. Pilot data from the NIMH cohort of patients with SC was confirmatory, with over 90% of patients, and only 10% of age-/sex-matched controls identified as D8/17 positive [6]. Similar results were obtained in the PANDAS subgroup (85% positive among 27 patients vs. 17% in 24 controls; OR 28.8, p<0.001) [45]. However, almost immediately, the specificity of the D8/17 marker was called into question by a -
study reporting D8/17 positivity in 100% of patients with childhood-onset OCD and/or tic disorders - regardless of their symptom course or association with GAS infections [46]. These results suggested that either the PANDAS subgroup is much more common than previously suspected, or that the D8/ 17 marker is not going to be useful for identifying patients at-risk for post-streptococcal neuropsychiatric sequelae. Of further concern, recent assays show decreased sensitivity of the D8/17 marker, with fewer than 50% of rheumatic fever patients identified as positives [47]. These questions, and others regarding the nature and utility of the D8/17 marker remain under investigation.
6. POST-INFECTIOUS AUTOIMMUNITY - DOES IT PLAY A ROLE?
At present, the "autoimmune" in "Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal infections" is still a postulate, as it has not yet been proven that the neuropsychiatric symptoms arise from post-streptoccal autoimmunity. The etiopathogenesis of OCD and tic disorders in the PANDAS subgroup is postulated to be similar to that of Sydenham's chorea [6, 7, 12, 20]. In SC, certain strains of GAS are thought to initiate the production of cross-reactive antibodies, which react against the host's antigens and set off an "autoimmune" response in the basal ganglia [48]. The basis for this hypothesis is demonstration of antibodies recognizing both GAS antigens and epitopes on basal ganglia tissues (caudate and subthalamic nucleus) in the sera of patients with SC [49], as well as neuroimaging studies demonstrating inflammatory changes in the basal ganglia [23, 24, 50, 51]. Initial investigations found the cross-reactive antibodies only in the sera of children with SC [7, 52], but subsequent reports have suggested that the "antineuronal" antibodies can be found in the sera of healthy children, as well [53-57]. The latter results raise questions about the pathologic nature of the antibodies, as it is not clear whether the antibodies are involved in the pathophysiology of SC and RF, or whether they are merely "red herrings" in the process. The role of the immune system in the neuropsychiatric symptoms of the PANDAS subgroup is
similarly unclear, but clinical observations suggest that symptoms may result from a combination of local, regional and systemic abnormalities [58]. The striking effectiveness of immunomodulatory therapies, such as therapeutic plasma exchange and intravenous immunoglobulin (IVIG) suggests that there is systemic involvement, at least in severely affected individuals [59]. MRI scans reveal enlargements of the basal ganglia, which point to regional inflammatory changes, while local autoimmune reactions are suggested by the presence of serum antibodies cross-reacting with neurons of the caudate, putamen and globus pallidus [7, 25-27, 52-57]. A randomized, placebo-controlled trial conducted at the NIMH revealed that both intravenous immunoglobulin (IVIG) and plasma exchange produced significant improvements in neuropsychiatric symptom s e v e r i t y - obsessive-compulsive symptoms were reduced by 45-58% one month post-treatment with IVIG or plasma exchange (respectively), while placebo (sham IVIG) administration had no effect on OCD symptom severity [59]. Further, one-year follow-up revealed that 14 of 17 children (82%) receiving plasma exchange or IVIG were "much" or "very much" improved from baseline [59]. The effectiveness of these immunomodulatory therapies suggests that circulating immune factors play a role in the pathophysiology of the symptoms. However, both treatments have a broad spectrum of potential mechanisms of action, from clearance of circulating antibodies and cytokines, to activation of subpopulations of T-cells and B-cells, so the specific mecha, nism of therapeutic effect is unknown. If it could be determined, then it might be possible to elucidate the nature of the post-streptococcal autoimmune response in the PANDAS subgroup, as well as to develop targeted therapeutic interventions suitable for use in less severely ill patients. Functional and structural neuroimaging studies suggest a role for regional inflammatory changes in post-streptococcal neuropsychiatric disorders. In SC, functional imaging studies obtained during the acute symptomatic period have demonstrated increased basal ganglia blood flow, as well as disruptions of the blood-brain barrier in the caudate nuclei [23, 24, 50, 51 ]. These abnormalities resolved as the chorea remitted, suggesting that they were etiologically related to the neuropsychiatric symptomatology. Volumetric MRI scans have revealed bilateral
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enlargements of the caudate, putamen, and globus pallidus in a group of patients with SC, and similar abnormalities have been demonstrated recently in patients in the PANDAS subgroup [26]. Further, in a small number of PANDAS patients treated with plasma exchange, baseline caudate enlargements normalized following successful treatment, suggesting that the enlargement might be a reflection of basal ganglia inflammation [27]. Studies which evaluate interstitial edema and disruptions of the blood-brain barrier may prove informative in such cases. 6.1. Cross-Reactive Antibodies
The first report of cross-reactive antibodies in SC came from Rockefeller University, where Husby and colleagues [49] examined sera from patients with SC and compared it with sera from healthy controls and patients with SLE. The SC group had elevated titers of "antineuronal antibodies" which not only recognized neuronal tissue from the caudate nucleus and subthalamic nucleus, but also were absorbed by GAS cellular components. These IgG antibodies were not present in the healthy controls. Some patients with SLE demonstrated antibodies with similar antineuronal reactivity; however, those antibodies did not react with the streptococcal components. Husby thus concluded that the SC patient group had a unique autoantibody present, which was postulated to have occurred as a result of molecular mimicry [49]. A decade later, Bronze and Dale [60] confirmed Husby's findings in a group of patients with acute SC who had serum antibodies that recognized both GAS cellular components and basal ganglia tissue (mainly from caudate nucleus). Crossreactive antibodies directed against human caudate tissue were found in the sera of 20 of 21 patients with acute SC studied at the NIMH [7]. Of note, as choreatic symptoms subsided, the titers of the antibodies fell, and then rose again in the few patients who suffered a recrudescence [7]. Thus, it would appear that antibodies recognizing human neuronal tissue are produced in association with SC. A recent investigation by Kirvan and colleagues [61] suggested that these cross-reactive antibodies may have biological activity. The investigators identified antistreptococcal monoclonal antibodies (mAbs 24.3.1., 31.1.1, and 37.2.1) derived from a patient with SC,
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and selected by crossreactivity with group A streptococcal carbohydrate and brain extracts separated by SDS-PAGE. The mAbs recognized a 55kD brain protein, which sequence analysis revealed had an Nterminal sequence of MREIVHLQ corresponding to tubulin. ELISA analysis demonstrated specificity for mammalian gangliosides, and the mAbs reacted with human caudate and putamen tissue, labeling both intra- and extracellular determinants of a neuronal cell line. Of interest, mAb 24.3.1 induced a 50% increase in Ca+§ dependent protein kinase II activity in a human neuroblastoma cell line, suggesting that the monoclonal antibody can affect cell signaling. Further, increased cell signaling activity was found in the patients' acute, but not convalescent sera [61 ]. Cross-reactive antibodies have also been demonstrated in the PANDAS subgroup. In the study discussed above by Kiessling et al, sera from children with OCD and/or tic disorders were found to have significantly greater frequency of antibodies directed against human caudate tissue than were present in healthy controls' sera [52]. A replicative study utilizing immunofluorescent microscopy revealed that the antibodies recognized both caudate and putamen, and were not present in children with ADHD without tics [53]. Subsequent reports have consistently documented the presence of antineuronal antibodies in the sera of affected children at a higher rate than among controls [54-57], although a substantial proportion of healthy volunteers (40% in one study) [56] also have antibodies present. Recently, Kirvan and colleagues (unpublished data, 2003) have demonstrated the presence of cross-reactive antibodies (similar to those described above for SC) in the sera and CSF of patients in the PANDAS subgroup. The biological activity of these antibodies is less potent than that seen in SC, perhaps explaining the differences in clinical presentation for the two disorders. Further studies are required to determine the target(s) of these antibodies, as well as to determine the mechanism by which they are raised in association with GAS infections. The presence of autoantibodies does not indicate causality, however. One of the key steps in establishing a causal link is to develop an animal model in which the autoantibodies reproduce symptoms of the disorders. Hallett and colleagues [57] infused sera from children with Tourette syndrome into
the dorsolateral region of the striatum of rats and observed an increase in oral stereotypies. Hoffman and Lipkin [62] immunized mice during early development with streptococcal or basal ganglia homogenates, and noted the presence of autoantibodies binding to the basal ganglia and other specific brain regions; additional research has demonstrated strain-specific differences in autoantibody production, as well as an increase in stereotypic behaviors following immunologic challenge. Taylor and colleagues [63] infused sera from 12 TS patients with high levels of autoantibodies bilaterally into the ventrolateral striatum of rats. The rates of oral stereotypies increased significantly, in comparison with a group of rats infused with sera from 12 TS patients with low levels of autoantibodies. In summary, there is a growing body of evidence supporting an etiologic role for molecular mimicry in post-streptococcal neuropsychiatric symptoms (SC and PANDAS). Cross-reactive antibodies recognizing both GAS cellular components and basal ganglia tissue have been observed. The cells recognized by the antibodies are localized to brain regions consistent with the clinical presentation of the disorders, and also, with earlier findings from pathological examinations in SC and neuroimaging investigations of SC, OCD and tic disorders. Recent reports of biological activity for these antibodies, and results of laboratory animal studies, provide further support for the postulate that these antibodies play a role in the etiopathogenesis of both SC and the symptoms observed in the PANDAS subgroup. The mechanism by which these cross-reactive antibodies produce neuropsychiatric symptoms remains to be elucidated.
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18. Leckman JF, Peterson BS, Pauls DL et al. Tic Disorders. Psychiatr Clin N Amer 1997;20(4):839-861. 19. Perlmutter SJ, Garvey MA, Castellanos X et al. A case of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections. Am J Psychiatry 1998;37(2):218-220. 20. Garvey MA, Giedd J, Swedo SE. PANDAS: The search for environmental triggers of pediatric neuropsychiatric disorders. Lessons from rheumatic fever. J Child Neurol 1998; 13(9):413--423. 21. Denckla MB. Revised neurological examination for subtle signs. Psychopharrn Bull 1985;21:773-793. 22. Snider L, Swedo S. Pediatric obsessive-compulsive disorder. JAMA 2000;284(24):3104-6. 23. Goldman S, Amrom D, Szliwowski HB. Reversible striatal hypermetabolism in a case of Sydenham's chorea. Movement Disorders 1993;8:355-358. 24. Weindl A, Kuwert T, Leenders KL et al. Increased striatal glucose consumption in Sydenham's chorea. Movement Disorders 1993;8:437-444. 25. Giedd JN, Rapoport JL, Kruesi MJP et al. Sydenham's chorea: Magnetic resonance imaging of the basal ganglia. Neurology 1995;45:2199-2202. 26. Giedd JN, Rapoport JL, Garvey MA et al. MR/assessment of children with obsessive--compulsive disorder or tics associated with streptococcal infection. Am J Psychiatry 2000; 157(2):281-283. 27. Giedd JN, Rapoport JL, Leonard HL et al. Case study: Acute basal ganglia enlargement and obsessive-compulsive symptoms in an adolescent boy. J Am Acad Child Adolesc Psychiatry 1996;35(7):913-5. 28. Stollerman GH. Rheumatic Fever and Streptococcal Infection. New York: Grune & Stratton, 1975. 29. Coburn AF. The Factor of Infection in the Rheumatic State. Baltimore: Williams & Wilkins, 1931. 30. Collis WRF. Acute rheumatism and hemolytic streptococci. Lancet 1931; 1:1341-5. 31. Paul J. Epidemiology of Rheumatic Fever. New York: American Heart Assoc 1957. 32. Bradford-Hill AB. Proc R Soc Med 1965;58:295-300. 34. Snider LA, Seligman LD, Ketchen BR et al. Tic and problem behaviors in school children: Prevalence, characterization and associations. Pediatrics 2002:110(2 Pt 1): 331-6. 35. Shulman ST. Streptococcal pharyngitis: clinical and epidemiologic factors. Pediatr Infect Dis J 1989;8(11): 816-819. 36. Perlmutter SJ, Garvey MA, Castellanos X et al. A Case of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections. Am J Psychiatry 1998;155(11):1592-1598. 37. Veasy, LG. Rheumatic fever: T. Duckett Jones and the rest of the story. Cardiol Young 1995;5:293-301.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Helicobacter Pylori Infection and Gastric Autoimmunity: Coincidence or Cause-Effect Relationship? Gianfranco Del Prete ~, Mathijs E Bergman 2, Amedeo Amedei 1, Mario M. D'Elios 1, Christina M. Vandenbroucke-Grauls 2 and Ben J. Appelmelk 2
1Department of Internal Medicine, University of Florence, Florence, Italy; 2Department of Medical Microbiology, Vrije Universiteit, Medical School Amsterdam, The Netherlands
1. INTRODUCTION All individuals harbour autoreactive T cells that need activation and critical expansion in order to start active autoimmune disease [ 1]. In a number of human diseases and in their corresponding experimental animal models, it has been suggested that pathogens can induce disease through autoimmune mechanisms [2-6]. Several mechanisms have been proposed for how pathogens might induce activation and critical expansion of autoreactive T cells and start autoimmune disease. Viral and bacterial superantigens, that bind a variety of MHC class II molecules and activate large proportions of T cells, irrespective of their specificity, can also lead to activation of resting autoreactive T cells [7]. Tissue inflammation induced by pathogens may result in local activation of antigen-presenting cells (APC) and in enhanced processing/presentation of self antigens, that causes T-cell priming. T-cell activation would then be followed by expansion ofT cells with additional specificities, a phenomenon referred to as epitope spreading [8, 9]. Another mechanism by which pathogens can start autoimmune disease would imply that the inflammatory setting and the paracrine secretion of T-cell growth factors induce the expansion of activated autoreactive T cells, whose small number was previously insufficient to set up the disease. Such a mechanism is referred to as bystander activation [ 10]. Moreover, a microbial antigen can include an epitope that is structurally similar to an autoantigen epitope, providing the basic element of the mecha-
nism referred to as molecular mimicry [5, 6, 11-14]. A clear example of epitope mimicry in humans is Lyme arthritis, in which Borrelia burgdorferi disseminates to multiple tissues, including joints. In the synovia of patients with specific MHC class II haplotypes, activation of T helper type 1 (Th 1) cells reactive to the 165-173 peptide of the outer surface protein A (OspA) of B. burgdorferi occurs [ 15, 16]. Such an OspA epitope is similar to the L332-340 peptide of the human leukocyte function-associated antigen 1ct (LFA- 1~), whose expression is up-regulated on synoviocytes by the Thl-derived IFN-7 [17, 18]. Since Thl cells dominate the immune response in the synovial fluid of patients with Lyme arthritis, and the Thl cells specific for outer surface protein A of Borrelia burgdorferi persist in patients with prolonged antibiotic treatment-resistant Lyme arthritis, it has been suggested the possibility that such T cells are indeed effectors of an autoimmune process [ 13-18]. Helicobacter pylori is a Gram-negative pathogen that causes persistent infection in half the world population. H. pylori infection results in a series of various clinical outcomes, including transient and almost asymptomatic gastric inflammation, chronic gastritis, peptic ulcer disease, mucosal atrophy, gastric carcinoma, or gastric B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) (reviewed in [19]). Loss of gastric glands in the antrum and corpus, which is referred to as atrophy, is considered a precursor of gastric adenocarcinoma, the second most frequent cause of cancer-related death, and there is strong evi-
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dence that H. pylori infection increases the risk of gastric cancer [ 19, 20]. The trigger of atrophy and the mechanisms involved in its development are still unknown. Although H. pylori is not invasive and usually resides in the antrum, glands located deep in the mucosa of antrum and corpus disappear. Accumulating data in humans suggest that gastric corpus atrophy is caused by an autoimmune process associated with H. pylori infection. In this review, we discuss on the possible linkage between H. pylori infection and gastric autoimmunity in humans. In the first part, current knowledge of the H. pylori-specific immune response occurring at gastric level in H. pylori-infected patients with different clinical outcomes will be summarized. In the second part, the pathogenesis of H. pylori-associated atrophic gastritis associated with the presence of gastric autoantibodies in the serum will be reviewed. In the third part, data on classical autoimmune gastritis (AIG), including recent data on the fine antigen specificity and the functional features of the gastric T-cell response to H§ the major target autoantigen at the gastric level, will be reviewed and compared with data obtained in the experimental autoimmune gastritis (EAIG) in animals. In the last part of this review, the striking similarities between AIG and H. pylori-gastritis accompanied by corpus atrophy and autoantibodies will be discussed, together with recent data that allow to reasonably suggest that in some individuals genetically predisposed to organ-specific autoimmunity due to their MHC class II haplotype, H. pylori infection plays a role in induction and/or exacerbation of gastric autoimmunity through a mechanism of molecular mimicry.
2. 1t. PYLORI INFECTION AND HOST IMMUNE RESPONSE AT GASTRIC LEVEL
Despite a vigorous humoral response against H. pylori antigens, most of infected subjects fail to eliminate the pathogen spontaneously. As in other infectious diseases, antigen-specific T cell responses are essential for the clearance, or at least the control, of the pathogen. Since most of H. pylori-infected patients are unable to clear the pathogen, it has been suspected that H. pylori may somehow hamper
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the host immune response. H. pylori can interfere with protective immunity through the release of factors, such as the vacuolating cytotoxin (VacA), which impairs not only antigen processing and the subsequent priming of efficient immune response [21], but also inhibits T-cell activation by interfering with the T-cell receptor (TCR)/IL-2 signalling pathway [22]. Colonization of the stomach by H. pylori is followed by inflammation of the gastric mucosa, which varies according to the host immune reaction against the pathogen [23]. However, the failure of cleating H. pylori from the gastric environment almost invariably leads to chronic antral gastritis. Activation of macrophages results in the release of cytokines, including IL-12, IL-1, IL-6, TNF-tx, IFN-ct, and chemokines, such as IL-8. Also recruited neutrophils can produce IL- 12 in response to bacterial products [24]. This is an important step of the natural history of H. pylori infection, because the local cytokine "milieu", particularly the IL-12 produced by cells of the natural immunity, is crucial in driving the subsequent specific T-cell response into a more or less polarized Thl pattern. The pattern of cytokines produced by the in vivo activated mononuclear cells recruited in the antral mucosa of H. pylori-infected patients with peptic ulcer was analysed. Antral biopsies from peptic ulcer patients showed IL-12, IFN-y, and TNF-tx, but not IL-4, mRNA expression, whereas no cytokine mRNA signal was found in the mucosa of H. pylori-negative individuals [25]. In the same biopsies, immunohistochemistry showed remarkable in vivo activation of IFNy-producing T cells, without expression of IL-4. Using serial histological sections, a significant correlation was demonstrated between disease severity and the in situ secretion of IFN- 7 and TNF-tx [26]. Increased levels of other Thl-inducers, such as ILl 8 and IL-17, have been found in the gastric mucosa of H. pylori-infected patients [27, 28]. Likewise, a preferential activation of Thl responses has been reported in different animal models, such as mice, beagle dogs, and monkeys experimentally infected with H. pylori or H. felis [29-31]. In the last few years, the antigen specificity and the effector functions of the clonal progenies of in vivo activated H. pylori-specific T cells obtained from the antral mucosa of series of H. pyloriinfected patients with uncomplicated chronic gas-
tritis, or associated with peptic ulcer, or with low grade B-cell lymphoma have been investigated [25, 32, 33]. In all patients studied, most of gastric T-cell clones were CD4 § whereas CD8 § T cells represented a minority of the infiltrating lymphoid cells [34]. While none of the CD8 § clones proliferated to soluble H. pylori antigens, a variable proportion ranging from 2 to 36% of the CD4 § T helper (Th) clones derived from the gastric biopsies of patients with different clinical conditions associated with H. pylori infection showed significant proliferation to H. pylori lysate, and a number of them also to purified urease, CagA, VacA or heat shock protein (HSP) under MHC-restricted conditions. Among the H. pylori-reacfive Th clones from peptic ulcer patients, a half were specific for CagA, whereas in the series of the H. pylori-reacfive clones derived from chronic gastritis patients, about one fourth of clones recognized urease. In the series of H. pylorireactive T-cell clones derived from low grade MALT lymphomas, one fourth were specific for urease, whereas most proliferated only to H. pylori crude extract, suggesting that some still undefined antigens of H. pylori probably drive T-cell responses in low grade gastric MALT lymphoma [33]. The great majority of H. pylori-specific clones derived from gastric biopsies of peptic ulcer patients, and particularly those specific for CagA, showed a polarized Thl profile, with high production of IFN- 7 but not of IL-4, upon stimulation with the specific H. pylori antigen. In contrast, more than a half of H. pylori-specific T-cell clones derived from uncomplicated gastritis showed a Th0 profile, with production of IL-4 and/or IL-5 together with IFN-% whereas polarized Thl profile was expressed by about 40% of H. pylori-specific gastric T-cell clones [32]. Stimulation of T-cell clones derived from the gastric antrum of infected patients with the appropriate H. pylori antigen results in helper function for B-cell proliferation and immunoglobulin (Ig) production [25]. This can explain the intense B-cell activation in the lymphoid tissue associated with the antral mucosa during chronic H pylori infection and the presence of specific antibodies in the serum. In chronic gastritis patients either with or without peptic ulcer, the helper function exerted by H. pylori antigen-stimulated gastric T-cell clones on B cells was negatively regulated by the concomitant perfo-
fin-mediated cytolytic killing of B cells. Moreover, the great majority (88%) Th gastric clones from chronic gastritis induced Fas-Fas ligand-mediated apoptosis in target cells [25, 33]. In mice, T-cell dependent immune responses are needed for protection against H. pylori whereas antibody response is not strictly required for protective immunity [35]. However, if the T-cell response induced against H. pylori is not fully appropriate and balanced, it results in damage to the host, as demonstrated in different animal models [29, 31, 36]. In H. felis-infected mice, neutralization of IFN-~, significantly reduced the severity of gastritis, strongly supporting the concept that preferential long-lasting activation of a Thl-type response contributes to development and maintenance of gastric pathology. The results of these studies provide further evidence that unlimited Thl response is associated with gastric inflammation and disease, whereas a mixed Thl/Th0 response is able to reduce the unbalanced pro-inflammatory Thl response [29].
3. H. PYLORI-INDUCED ATROPHIC GASTRITIS ASSOCIATED WITH GASTRIC AUTOANTIBODIES
The trigger of H. pylori-associated atrophic gastritis and mechanisms involved in its development are still poorly known. While the colonization takes place mainly in the antrum, both infected humans and experimentally infected mice develop atrophy in the corpus [37]. In humans, a series of observations suggests that gastric corpus atrophy can be related to an autoimmune process driven by H. pylori. Approximately a half of H. pylori infected individuals (range 49 to 64%) have in the serum antibodies reactive to autoantigens of the gastric mucosa [38-41]. Irmaaunohistochemistr.r on sections of human gastric tissue from healthy H. pylori non-infected individuals incubated with serum from H. pylori-infected patients allowed to identify two different binding sites for autoantibodies: one on the luminal membranes of the foveolar epithelial cells in the antrum and corpus mucosa, and the other one to the canalicular membranes of parietal cells in the gastric corpus mucosa [40]. Parietal cells in the corpus secrete gastric acid, and their apical secretory canaliculi are rich in H+,K+-ATPase, i.e., the
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gastric proton pump. The gastric H +, K+-ATPase has been identified as the most important target autoantigen in chronic H. pylori gastritis with corpus atrophy [421. Lipopolysaccharide (LPS) of H. pylori expresses Lewis blood group (Le) antigens similar to those expressed on human cells, including gastric epithelial cells [43, 44]. In mice, H. pylori infection can induce autoantibodies through mimicry of Lewis antigens on the gastric proton pump (reviewed in [45]). Mice, experimentally infected with H. pylori, develop antibodies to H. pylori LPS, to Lewis x (Le x) and LeY and to H§ Le x and LeY are expressed on gastric mucin, whereas LeY is expressed on the [3-subunit of gastric H+,K§ Sera of H. pylori-infected mice cross-react with the human gastric mucosa, and these cross-reactive antibodies can be removed by pre-incubation with H. pylori cells [43, 44]. In humans, however, positive sera also react with recombinant H+,K§ ATPase lacking Lewis antigens [42], and reactivity to gastric parietal cells cannot be removed by pre-incubation of serum with H. pylori cells [46]. Therefore, human anti-H+,K+-ATPase autoantibodies associated with H. pylori infection are directed against peptide epitopes and their synthesis does not involve Lewis mimicry. Likewise, infection of ferrets with H. mustelae induces autoantibodies that react with ferret parietal cells and such antibodies cannot be removed with H. mustelae cells or red blood cells expressing Lewis antigens [47]. Both antiluminal and anticanalicular antibodies are significantly associated with H. pylori infection. However, in H. pylori-induced gastritis, only anticanalicular antibodies correlate with a number of morphological and functional alterations of the gastric mucosa as well as with some clinical parameters [40]. In particular, anticanalicular autoantibodies positively correlate with severity of H. pylori gastritis in the corpus, as well as with atrophy and increased apoptosis in the gastric corpus mucosa (Table 1) [39--41 ]. H § K+-ATPase is the target of anticanalicular autoantibodies and patients with autoreactivity against the t~ and or 13 subunit of H § K+-ATPase show the highest prevalence of body mucosa atrophy [42]. In addition, H. pylori-infected patients with anticanalicular autoantibodies have in the serum significantly higher fasting gastrin levels and
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Table 1. Similarities between classical AIG and H. pyloriinduced atrophic gastritis associated with parietal cell autoantibodies
Immunopathological aspects 9 Increased apoptosis of corpus mucosa and its progressive atrophy [40-42] 9 Gastric H§247 is the autoantigen recognized by circulating PCA [42, 61 ] 9 Infiltrates of T cells, B cells and macrophages around glands and into the epithelium [42, 61 ] 9 Expression of MHC class II and B7.1 and B7.2 molecules on gastric epithelial cells, and their putative APC function [81, 84] 9 Increased Fas expression on gastric cells [94]
Clinical aspects 9 Reduced gastric acid secretion [48] 9 Increased serum gastrin levels [40] 9 Reduced pepsinogen I:II ratio [40] 9 Decreased incidence of duodenal ulcer [40]
lower pepsinogen I:II ratio, which is considered a sensitive marker for corpus atrophy, as compared to patients without anticanalicular autoantibodies [40, 48]. A correlation also exists between anticanalicular autoantibodies and decreased gastric acid output in H. pylori-infected patients with non-ulcer dyspepsia [48]. Based on the similar histopathological and clinical aspects between H. pylori-associated atrophic gastritis with anticanalicular antibodies and classical autoimmune gastritis/pernicious anemia (AIG/PA), it has been suggested that H. pylori can represent a causative agent of autoimmune gastritis [45]. This concept is supported by the observations that, in some (but not all) infected patients, gastric autoantibodies decrease after cure of infection [49], and that histologically defined subclinical stages of autoimmune gastritis can be successfully treated by H. pylori eradication [50, 51 ]. However, data regarding the beneficial effects of eradication therapy in atrophic gastritis are not fully consistent, because in individuals with atrophic body gastritis, H. pylori eradication had no effect on either body atrophy or intestinal metaplasia, though increased gastric acid secretion and reduction of hypergastrinemia were achieved [52, 53]. Though a negative association between H. pylori
and pernicious anaemia (PA) had been reported [54], an association between H. pylori infection and gastric autoimmunity is supported by a number of subsequent studies indicating that a substantial portion of patients with autoimmune gastritis have or have had H. pylori-infecfion [55-57]. The conflict about the prevalence of H. pylori infection in AIG/PA, and in the less strictly defined atrophic body gastritis, is largely due to the methods used for the assessment of infection. Although actual colonization could be detected in only 10 to 14% of PA patients and 33% of individuals with atrophic body gastritis [55, 58, 59], serum antibodies to H. pylori were measured in 51 to 83% of patients with PA and in 53 to 86% of patients with atrophic corpus gastritis [55, 56, 58], providing indirect support to the hypothesis that a remarkable proportion of patients with atrophic corpus gastritis had H. pylori-gastritis before the pathogen was cleared by the development of atrophy and hypochloridria.
4. CLASSICAL AUTOIMMUNE GASTRITIS (AIG) AIG is an organ-specific inflammatory disease of the gastric corpus and fundus, that usually does not result in overt symptoms until development of mucosal atrophy and eventually of PA with cobalamin and iron malabsorption [60]. Clinical characteristics of AIG patients are hyperplasia of gastrin-producing cells resulting in high serum gastrin concentration, decreased acid secretion and decreased pepsinogen I:II ratio [61 ]. AIG/PA can associate with thyroid autoimmune disorders, such as Hashimoto's thyroiditis and Graves' disease, particularly in women over the age of 40 [62, 63]. The pathology of AIG is characterized by lymphocytic infiltrates in the gastric mucosa and by ongoing destruction of parietal and zymogenic cells [64]. In the majority of AIG patients, either with or without PA, parietal cell autoantibodies (PCA) are detectable in the serum. The target autoantigen of PCA is the gastric H+,K+ATPase localized in the parietal cell canaliculi [65, 66]. Gastric H+,K+-ATPase is also the major autoantigen in experimental murine EAIG, an organ-specific autoimmune disease, which can be elicited in non-
thymectomized mice by immunization with either gastric mucosal extracts or gastric H+,K§ [67-70], or by neonatal thymectomy [71, 72]. These models of autoimmune gastritis are characterized by an inflammatory infiltrate in the gastric mucosa, subsequent loss of acid-secreting parietal cells and zymogenic cells, and late appearance of circulating autoantibodies directed against the tx and [3 subunits of parietal cell H+,K§ [68-70]. The gastric mononuclear cell infiltrate contains both CD4 + and CD8 + T cells, macrophages and B cells, and histopathology is remarkably similar to that observed in patients with chronic AIG/PA [73, 74]. Like in the human disease, H+,K+-ATPase is the autoantigen recognized by T cells, and H§ CD4 § but not CD8 § T cells mediate gastric pathology and disease in EAIG [75, 76].
4.1. T Cells Specific for H+K+-ATPase Epitopes in the Gastric Mucosa of AIG Patients In the last few years, characterization at molecular level of H+,K+-ATPase T-cell specificity and the effector functions of CD4 + Th cells derived from the gastric mucosa of patients with AIG has been achieved. An efficient method has been developed, that has proved to be useful and accurate for in vitro studies of tissue-infiltrating T cells [77]. Using that approach, the in vivo activated T cells present in the lymphocytic infiltrates of the gastric mucosa of patients with AIG were recovered, efficiently cloned, and studied for their antigen reactivity and for their functional profile [78, 79]. In all AIG patients studied, most of gastric T-cell clones were CD4 +, whereas CD8 § T cells represented a minority of the infiltrating lymphoid cells. None of the CD8 + clones proliferated to H+,K§ whereas a variable proportion (range 12-33%) of the gastric CD4 § Th clones showed significant proliferation to that autoantigen [78, 79]. Upon stimulation with the autoantigen, most of the H+,K+-ATPase-specific Th clones from the gastric corpus of AIG patients produced IFN-y but neither IL-4 nor IL-5 (Thl cytokine profile), whereas only a few clones secreted both Thl- and Th2-type cytokines (Th0 profile) [78]. All the autoreactive H+,K+-ATPase-specific clones also produced high concentrations of TNF-tx. Local WN-y and TNF-ct production by Thl cells can increase the expression
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of MHC class II molecules by gastric cells [80], which in turn would enable these cells to present peptides of gastric autoantigen(s) to T cells that differentiate into Thl effector cells. Since gastric epithelial cells can express either B7-1 (CD80) or B7-2 (CD86) costimulatory molecules, which are up regulated by exposure to IFN-y [81], as well as cathepsins involved in antigen processing, gastric epithelial cells may act in vivo as antigen-presenting cells (APC), as suggested [82]. Virtually all gastric H+,K§ Th clones were able to induce cell death via both Fas-Fas ligand-mediated apoptosis and perforinmediated cytotoxicity against target cells [78]. Considering that parietal cells can express Fas (CD95) molecule [83], and that HLA-DR is ectopically expressed on glandular epithelium in proximity of T-cell infiltrates in human AIG [84], Fas-Fas ligand induced apoptosis and perforin-mediated killing may represent two mechanisms by which H+,K+ATPase-specific autoreactive T cells mediate gastric cell loss. Definite requirement for Fas in development of murine EAIG has recently been demonstrated by the failure of lpr/Tpr mice, deficient in Fas expression, to develop destructive gastritis and autoantibodies upon neonatal thymectomy [85]. In the same study, Fas expression was co-localized with gastric parietal cells in EAIG mice, whereas non-thymectomized, non-gastritic mice did not express Fas in their gastric mucosa. This provides evidence for up-regulated Fas expression on parietal cells in EAIG, and may explain, at least in part, the selective destruction of parietal cells observed in both experimental murine and human AIG. H+,K+-ATPase-specific gastric Th clones from AIG patients were able to provide B-cell help for Ig production in vitro [78], suggesting that chronic autoantigen-induced T cell-dependent B-cell activation at gastric level plays an important role in the synthesis of H+,K+-ATPase autoantibodies that are usually found in the serum of AIG patients. The relevance of H+,K§ Thl effector cells in the genesis of AIG is consistent with data showing predominance of Thl responses in other human organ-specific autoimmune diseases [77], such as thyroid autoimmune disorders [86, 87], and multiple sclerosis [88]. Moreover, a large body of evidence obtained in animal models suggests that in experimental organ-specific autoimmune
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diseases, such as encephalomyelitis, thyroiditis, gastritis, insulin-dependent diabetes mellitus, and myasthenia gravis, a pivotal pathogenic role has to be ascribed to IFN-y-secreting Thl cells that infiltrate the target organ [77, 89]. Epitope mapping of H+,K§ gastric T cells in human AIG showed that more than a half of autoreactive Th clones were specific for peptide epitopes of the [3 chain of H§247 whereas the other gastric autoreative clones (43%) recognized their epitope in the ~ chain. At least 5 different T-cell epitopes were identified in the 13 chain and 6 different T-cell epitopes were recognized by specific autoreactive T cells, all showing 100% overlap with the corresponding epitopes of the porcine H+,K+-ATPase. Since no restricted TCR V[3 usage was found by ~ chain- or [~ chain-specific autoreactive Th clones, it has been speculated that gastric autoimmunity is the result of a polyclonal inflammatory response involving a quite wide variety of H+,K§ effector Th cells in the gastric mucosa [79]. Recognition of the appropriate H+,K§ epitope induced both proliferation and effector functions, such as cytokine production, by the autoreactive gastric T cells. Therefore it is reasonable to suggest that AIG is induced by an autoimmune attack mediated by CD4 § Thl cells specific for either ~ or 13chain epitopes of H+,K§ [79].
4.2. Human AIG and EAIG in Animals Share H§247 Epitopes Precise definition of the relevant H+,K§ epitopes is available also for EAIG. In murine EAIG following neonatal thymectomy, two H§ § ATPase-specific T-cell lines reactive to distinct peptides of the H§247 o~ chain were derived from the gastric lymph nodes [72]. Both T-cell lines were CD4 § TCR~/[3 § I-A d restricted, and equally potent in inducing gastritis in nu/nu recipient animals, but one T-cell line secreted Thl-type, whereas the other one secreted Th2-type cytokines. On the basis of different studies, a major role has been attributed to IFN-y-producing gastric Thl cells in the onset of EAIG and in its pathology [90, 91]. The strong similarity between the data on cytokine profiles obtained in human AIG and murine EAIG strongly supports the concept that H+,K§ -
Table 2. Submolecularpositions and detailed alignment of common T-cell epitopes of the o~chain and the 13chain of H§ +ATPase involved in human AIG and murine EAIG models Source of Sourceof T cells H+,K§
Positionand amino acid sequence of the epitope
Ref.
Human Mouse Mouse Mouse Mouse
Pig Pig Mouse Mouse Human
o~881-TAMAQEGWFP.LLCV G L R P Q W E N H H L QDL-908 (~881-TAM A QE G W F P L L C V G L R P Q W E N H H L Q D L - 9 0 8 0~880-TAMAQEGWFPLLC V G L R P Q W E N H H L QDL-907 o~880-TAM A Q E G W F P L L C V G L R P Q W E N H H L QDL-907 o~882-T AMA Q E G W F P L L C V G L R A Q W E N H H L Q D L-909
[79] [72] [72] [93] [93]
Human Human Mouse
Pig Pig Mouse Human
1376-QLKS P G V T L R P D V Y G E K G L D I S YNV S D S TTWAGL-109 [79] 1376-QLKS P G V T L R P D V Y G E K G L D I S YNVS DS TTWAGL-109 [79] 1376-Q L K S P G V T L R P D V Y G E R G L K I S Y N V S E N S S WAGL-109 [70] 1376-QLKS P G V T L R P D V Y G E K G L E I V Y N V S DNRTWAD L-109
Human Mouse
Pig Mouse Human
13166-P D P T F G F A E G KPCF..!IKMNRIV K F L P G N S-194 1 3 1 6 6 - A D P S F G F E E G K P C F I I K M N R I V K F L P S NN-194 13166-A D P N F G F E E G K P C F I I K M N R I V K F L P S NG-194
specific T cells should have gastritogenic potential also in humans. In mice, multiple immunization with the target autoantigen and subsequent epitope mapping showed that the [3261-274 peptide is a dominant gastritogenic T-cell epitope of the H+,K+-ATPase I~ chain, but there are other immunogenic peptides of the 13 chain [70, 92]. T-cell epitopes in the H+,K+-ATPase o~ chain have also been identified in EAIG. The disease can be transferred by T-cell lines reactive to the 633-641 or 889-899 peptides of the (~ chain [72, 93]. Importantly, ~ chain peptides 891-905 and 892-906 of porcine and human H+,K+ATPase, respectively, but not their homologous counterpart in the ubiquitously expressed human Na+,K+-ATPase, were recognized by one murine T-cell clone [93]. Interestingly, 44% of autoreactive Th clones form AIG patients proliferated in response to peptides that comprise epitopes recognized by gastritogenic T cells in EAIG (Table 2) [72, 79, 93]. The epitope c~881-900, that was recognized by different human Th clones, perfectly overlaps the (~889-899 amino acid sequence that represents the critical epitope for the TXA51 gastritogenic T-cell line in BALB/c mice [72]. In addition, the (x881-900 human T-cell
[79] [7O]
epitope overlaps the epitope of the gastritogenic T-cell clone 11-6 of BALB/cCrSlc mice, that recognizes the (~891-905 and a892-906 epitopes of porcine and human H+,K+-ATPase o~ chain, respectively [79, 93] (Table 2). Of the five different human T-cell epitopes of the 13chain identified so far, three overlapping peptides have a counterpart in EAIG. Peptides 1376-90 and ]381-95 recognized by human T-cell clones are partially overlapping the murine epitope 1385-109, whereas peptide [3166-185 almost exactly overlaps the murine epitope ~169193 (Table 2) [70, 79]. As mentioned above, H+,K+-ATPase-specific T cells infiltrating the gastric mucosa of AIG patients stimulated with the appropriate peptide-epitope expressed a predominant Thl cytokine profile and Fas-Fas ligand-mediated cytotoxic capacity [78, 79]. In EAIG, T-ceU clone 11-6 express Fas ligand upon activation, and caused DNA fragmentation of B cells pulsed with the H+,K+-ATPase (~891-905 peptide [94], which is the epitope overlapping the peptides recognized by different human T-cell clones from AIG patients. In mice, the 13chain of H+,K+-ATPase is expressed in the thymus and its ectopic expression abrogated the induction of gastritis. For this reason the 13chain
351
of H§ has been proposed as the initiating autoantigen of murine EAIG. However, murine T cells that recognize ct chain epitopes were found, possibly due to the phenomenon known as epitope spreading [8, 9]. It is unclear what is the initial factor determining the loss of tolerance in gastric autoimmunity. In mice, it has been demonstrated that depletion of CD4+CD25 + regulatory T cells is necessary but not sufficient for the induction of gastric organ-specific autoimmune disease [95], and it can be presumed that altered function of regulatory T cells may underlie human AIG as well. The results obtained in human AIG indicate that both the ~l and the t~ chain epitopes of gastric parietal cell H+,K§ are targets for specific autoreactive T cells and it is still unclear which portion of H+,K§ is involved in induction and/or maintenance of the disease. The experimental data obtained so far in AIG and in EAIG models indicate that common epitopes of H+,K+-ATPase and effector functions are shared features of autoreactive gastric T cells in both humans or mice. This further supports the hypothesis that in murine EAIG and human AIG similar mechanisms are responsible for target cell destruction.
5. H. PYLORI INFECTION AND GASTRIC AUTOIMMUNITY: COINCIDENCE OR CAUSE-EFFECT RELATIONSHIP? A number of strikingly similar alterations of the gastric corpus mucosa in AIG/PA and H. pylori-associated atrophic gastritis, make it reasonable to suggest an initiating role for H. pylori in both these gastric inflammatory disorders. As mentioned above, however, studies on the association of AIG/PA with H. pylori infection indicate that PA patients are infected with H. pylori less often than age-matched controls [96], though a majority of patients with PA show serological evidence of previous H. pylori infection [56, 58]. An explanation for this discrepancy may be that H. pylori is present in the stomach before the development of AIG/PA and got lost in the course of the process resulting in gastric atrophy. This sequel is supported by a long-term follow-up study, in which a subgroup of H. pylori-infected patients initially had gastritis in the antrum, that later progressed into severe atrophy in the corpus. This proc-
352
ess was accompanied by disappearance of H. pylori and onset of parietal cell antibodies [97]. Also these data would support a causative role of H. pylori in at least a subpopulation of patients suffering from gastric autoimmunity. Infection with H. pylori results in an inflammatory influx of mononuclear cells, including T and B-lymphocytes and macrophages, into the gastric mucosa. Then, under the influence of the predominant Thl cytokine milieu raised in the host by H. pylori-infection, gastric epithelial cells would acquire properties essential for antigen-presentation [31, 98]. Presentation of bacterial antigens by gastric epithelial/parietal cells and local professional APC may result in activation of H. pylori-specific FasL + gastric T cells, that kill the antigen-presenting epithelial cells by H. pylori-antigen-dependent mechanisms (e.g. perforin-mediated lysis) or by inducing apoptosis. Then, nearby Fas + parietal cells may be killed by antigen-independent T-cell mediated bystander lysis. In addition, presentation by professional APC and epithelial cells of gastric H§ to specific autoreactive T cells that escaped negative selection in the thymus, may result in T-cell activation resulting in expansion of H§ +ATPase-specific Thl cells able to provide help for B-cell autoantibody production, and to kill Fas + parietal cells by either antigen-dependent Fas-FasL interaction or by Fas-mediated bystander lysis. The end point of this process is destruction of gastric glands and atrophy. In the course of such cell-mediated inflammatory response, epitope-spreading may occur to other autoantigens such as intrinsic factor and pepsinogen, which may become targets of specific autoantibodies in full blown AIG/PA [61, 99]. Loss of H. pylori and healing of the antrum may parallel increasing corpus atrophy. In the final stage, the autoimmune T-cell response, that was initially triggered by H. pylori infection and associated inflammation, does not require the pathogen anymore, but it is self-maintained by autoantigen and Thl cytokines. This model is supported by the observation in transgenic mice that local expression of granulocyte macrophage-colony stimulating factor (GM-CSF) is sufficient to break tolerance and initiate gastric autoimmunity mediated by H+,K§ ATPase-specific CD4 + T cells [ 100]. A model by which H. pylori infection may mediate autoimmune destruction of the gastric corpus
mucosa, and finally result in AIG/PA, involves a mechanism of molecular mimicry between H§ § ATPase and H. pylori antigens at the level of T-cell epitopes.
5.1. Molecular Mimicry Between H. Pylori Antigens and H+,K+-ATPase in Human Gastric Autoimmunity Recently, Amedei et al have investigated the possible interactions between the T-cell mediated immune response at gastric level to H. pylori antigens and the autoimmune T-cell response to H+,K*-ATPase in a series of patients with AIG, thyroid autoimmunity and current H. pylori infection [101 ]. In vivo-acfivated gastric T cells were recovered from biopsy specimens of gastric mucosa, expanded in vitro with IL-2, and single T-cell blasts were cloned by limiting dilution. Gastric T-cell clones obtained were then screened for their ability to proliferate in response to H+,K+ATPase and/or to a H. pylori lysate. No proliferation was detected in any of the CD8 + clones obtained. In contrast, in the series of 154 CD4 § gastric T-cell clones, 18 were found to proliferate in response to H. pylori lysate, but not to H+,K§ and another 15 CD4 § clones consistently proliferated to H§ but not to H. pylori lysate. More importantly, in all patients, a third group of CD4 + clones (totally 13) was found that proliferated almost equally well to both H§ and H. pylori lysate, but not to a control antigen (Table 3). Each of the 28 clones able to recognize H+,K§ were then screened for proliferation in response to 205 overlapping peptides for the o~ chain and 56 peptides for the ~ chain of H+,K§ In the series of 15 H+,K+-ATPase specific clones that failed to proliferate to H. pylori lysate, 6 clones recognized an epitope in the t~ chain and 9 clones found their epitope in the smaller 13 chain of the proton pump, confirming previous observation in AIG patients without evidence of current H. pylori infection [79], and reinforcing the conclusion that autoreactive gastric T-cell response in AIG patients targets epitopes of either the ot or the 13 chain of H+,K§ In the series of 13 clones that proliferated to both H+,K+-ATPase and H. pylori lysate, 11 recognized their epitope in the tx chain and 2 clones in the 1~ chain. Interestingly, two clones from different patients (bearing different
Table 3. Proliferative response to both H*,K§
and
H. pylori lysate of gastric T-cell clones derived from H. pylori infected AIG patients. T-cell clone
Proliferative response (mean mitogenic index + SD) to: Porcine albumin
H+,K+ATPase
H. pylori lysate
GR.A04
<2
43 + 5
38 + 5
GR.A12
<2
71 + 9
57 + 10
GR.C27
<2
23 + 3
25 + 3
GR.C31
<2
55 + 6
52 + 8
PA.P24
<2
51 + 7
46 + 7
PA.R37
<2
43 + 4
81 + 22
MI.A30
<2
102 + 28
119 + 22
FF.A05
<2
91 + 11
99 + 13
FF.A15
<2
52 + 6
44 + 5
FF.C13
<2
98 + 17
52 + 3
FF.C26
<2
123 + 19
102 + 14
FF.C27
<2
88 + 16
71 + 22
FF.C32
<2
72 + 21
44 + 5
T-cell blasts from 13 CD4§ clones derived from the gastric mucosa of H. pylori infected patients with chronic autoimmune gastritis (4 from patient GR, 2 from patient PA, 1 from patient MI and 6 from patient FF) were cocultured with autologous irradiated APC in the presence of optimal concentrations of H+,K§ H. pylori lysate, or porcine albumin as control antigen. Results represent mean values (+ SD) of mitogenic indexes measured in five consecutive experiments [ 101 ].
TCR-V~) recognized the same ct836-850 epitope and two other clones from different patients in that series recognized the same a 6 2 1 - 6 3 5 epitope. It is also of note that no overlap was found between the H+,K+-ATPase epitopes recognized by clones reactive only to H+,K§ and the H+,K§ epitopes recognized by clones able to proliferate to both H§ and H. pylori lysate (Table 4). Therefore, it has been concluded that a number of H+,K+ATPase epitopes are "private", whereas other epitopes, mainly in the t~ chain, are similar to, or cross-reactive with, epitopes of H. pylori antigens. For 10 H+,K§ pylori cross-reactive gastric clones, bioinformatics offered a wide panel of candidate cross-reactive H. pylori peptides to
353
Table 4. Different epitope specificity of gastric T-cell clones from H. pylori infected AIG patients T-cell clones reactive to H§247
IT, IC-ATPase crchain epitopes recognized (mean MI) PA.Q08 (V~4) oc 1-15 (22) GR.C11 (V135.3) oc 31--45 (65) PA.P34 (V[~19) oc 151-165 (134) FF.C39 (VII9) t~ 351-365 (53) PA.P02 (V[38) oc 881-895 (23) PA.P14 (71319) oc 881-895 (21)
IT, IC-ATPase fl chain epitopes recognized (mean MI) FF.A09 (V1323) 1376-90 (97) FF.A33 (V136.7) 1376-90 (108) FF.C15 (V1322) 1376-90 (72) GR.A01 (V[36.7) [3 81-95 (21) FF.C03 (V~16) [3 81-95 (113) PA.R17 (V134) 13 111-125 (29) GR.C26 (V~13) ~ 166-180 (22) MI. A42 (V~8) [3 231-245 (122) MI.B46 (V~17) 13231-245 (68)
and H. pylori
T-cell clones reactive to both H§247 lysate
PA.P24 (V~4) FF.A15 (V~18) FF.C32 (VI316)
t~ 46-60 ct 181-195 t~ 241-255
(23) (39) (87)
FF.C27 (V115.2)
ct 256-270
(137)
FF.C26 (V~21.3)
oc 516-530
(104)
GR.A12 (VI319)
a 576-590
(64)
GR.C31 (V1313) FF.A05 (V[~16)
t~ 621-635 a 621-635
(79) (99)
GR.A04 (VI]13)
ct 781-795
(194)
PA.R37 (VI315)
a 836-850
(50)
MI.A30 (VI313)
t~ 836-850
(49)
FF.C13 (VI314)
~ 11-25
(91)
GR.C27 (VI]13)
~ 216-230
(57)
Proliferative response to H§247 epitopes by gastric T-cell clones derived from AIG patients with current H. pylori infection. T-cell blasts from 15 CD4§ clones reactive only to H*,K§ and from 13 CD4 § clones reactive to both IT,K§ ATPase and H. pylori lysate were co-cultured for 3 days with autologous irradiated APC in the presence of 15-mer synthetic overlapping peptides covering the entire amino acid sequence of the oc and 13chain of the gastric H+,K§ Results represent mean values of mitogenic indexes (MI) measured in five consecutive experiments [101]. For each of the gastric T-cell clone, TCR VI3 chain expression is reported. The individual donors of gastric clones are indicated by acronimous (GR, PA, MI and FF).
be tested for their ability to induce T-cell clone proliferation. For each of these 10 gastric clones, a cross-reactive H. pylori peptide could be identified (Table 5). Interestingly, two clones from different patients, that shared recognition of the t~836-850 H+,K+ATPase epitope, also shared cross-reactivity to the 11-25 peptide of a lipopolysaccharide biosynthesis protein of H. pylori. In contrast, for another two clones from different patients, both reactive to t~621-635 H§ the bioinfor-
354
matic method had predicted two different series of 11 and 8 cross-reactive candidates, respectively. Indeed, one of such clones reacted well only to the 264-278 peptide of Histidine kinase, whereas the other clone showed cross-recognition of the 35-49 epitope of Porphobilinogen deaminase of H. pylori. In summary, that study led to the identification of nine H. pylori proteins, each harbouring a T-cell epitope suitable for cross-reaction with T-cell epitopes of gastric H+,K+ATPase tx chain [101]. At
Table 5. Cross-reactive IT,K§
T-cell clones
and H. pylori peptides recognized by gastric T-cell clones
Amino acid sequences recognized H§247
epitope
H. pylori protein including the cross-reactive peptide (position)
GR.C31 GR.A04
(tx621-635) (o~781-795)
IRVIMVTGDH.PITAK
VRVDVRRLDHLMNLI ISNL__PYYIA__TRL_VLN
PA.P24
(o~46-60)
KKEMEINDHQLSVAE
PA.R37 MI.A30 FF.A15 FF.C32
(ct836-850) (t~836-850) (o~181-195) (ct241-255)
KAESD_IMItLRPRNPK KAESDIMHLRPRNPK VIRDGDKFQINADQL CTHES_PLET_RNIAFF
FF.C27 FF.C26
(0~256-270) STMCLEGTAQGLWN (o~516-530) VMKGAPERVLERCSS
FF.A05
(tx621-635) IRVIMVTGDHPITAK
NLKKSIAYTL__TKNIP
Histidine kinase (264-278) Dimethyl adenosine transferase (99-113) LNNYQKENSLYNttNL Penicillin-binding protein 2 (104118) LPS biosynthesis protein (11-25) NMRVFIIHLSPKTCK LPS biosynthesis protein (11-25) NMRVF_IIHLSPKTCK VVQGGDKFHAPVLVD Acetate kinase (93-107) Phosphoglucosamine mutase (70VIQIG__PMPTPAIAFL 84) VirB4 homolog (78-92) ALDSL.__EEKVVPdT,L W K Glucose-inhibited division protein VFKGIPGLSL_._E_EAVEK A (571-585) Porphobilinogen deaminase (35L!TdVKTTGDKILDAP 49)
For each gastric T-cell clone reactive to both H*,K*-ATPaseand H. pylori a single H. pylori cross-reactive peptide was identified. Identical amino acid residues in the recognized IT,K§ peptide and the cross-reactive H. pylori peptide are highlighted.
nM concentrations, self and cross-reactive peptides were almost equally potent in inducing T-cell clone proliferation. However, at lower doses, proliferation induced by the appropriate self (H§ peptides was consistently higher than that stimulated by the corresponding microbial peptides, though at concentrations as low as 1 pM, both self and cross-reactive microbial peptides were still stimulatory (Fig. 1). It is of note that none of the bacterial epitopes recognized by the cross-reactive T-cell clones belong to H. pylori immunodominant proteins, i.e., CagA, VacA and urease, which have been identified as targets of the majority of gastric T cells in H. pylori infected patients with chronic antral gastritis [32] and peptic ulcer [25]. Though of potential importance for the induction of gastric autoimmunity, it remains unknown to what extent the H. pylori epitopes recognized by the cross-reactive T cells are relevant to bacterial infection. Based on the reported degeneracy of both TCR and MHC binding [102, 103], there is a theoretical possibility that H+,K+ATPase-specific autoreactive
T cells, which cross-react to H pylori antigens, do it simply by chance. However, such coincidental cross-reactivity and promiscuity of T cell responses to H. pylori lysate could not be found in a large series of CD4 § human T-cell clones isolated from atherosclerotic plaques, which included several clones that recognized Chlamidia pneumoniae antigens [104], although one might expect more similarity between the proteomes of the two bacteria than between the human proteome and that of H. pylori. Since peptide recognition by T cells depends on anchor residues involved in binding to the MHC molecule as well as on TCR contact residues, one would expect that the self peptide and the bacterial peptide recognized by a given T cell, should have considerable homology in their amino acid sequence. However, degeneracy in both TCR [ 105] and MHC binding-motives [ 106] reduces sequencespecific requirement to only a few crucial residues. A clear example is provided by the animal model of myocarditis, where cross-recognition of the autoantigen (myosin) and the microbial peptide (Chlamydia CRP) depends on only 4 identical residues in
355
200
GR.A04
FF.C27
FF.C26
150 xO) 100 "-=
80
c
=', o
60 4O
20
i! ,l !l !! ll m
el el ll
10-310 -210 -] 100 101 10-310-210 -1 100 101 10-310-2 10-1 100 101 200
GR.C31
FF.A05
i FF.C32
150 100 "-=
.u_
80
c
o~ 60 0 4O
20 10.310 .210 -1 10o 101 10.310 .210 -1 10o 101 10. 310.210-1 10o 101 [ peptide] nM Figure 1. Dose-responseeffect of graded concentrations of the self (closed columns) and the corresponding bacterial peptides (open columns) on the proliferative response of gastric T-cell clones reactive to both H+,K+-ATPaseand H. pylori lysate. Results represent mean values + SD of six representative out of ten T-cell clones tested.
the 16 amino acid epitope sequence [11]. In addition, another mechanism of TCR cross-recognition may be at work. MHC-based molecular mimicry has been reported to underlie TCR cross-reactivity in multiple sclerosis [107]. A T-cell clone from a patient recognizes both myelin basic protein (MBP) amino acids 85-99 and Eppstein-Barr virus (EBV) DNA polymerase peptide EBV627-641, but recognition of these peptides is restricted by two different
356
DR2 molecules, i.e., DRBl*1501 and DRB5*0101, respectively. Crystal structure determination revealed structural similarities of both DR-peptide complexes at the surface presented for TCR recognition, thus explaining the mechanism that underlies the mimicry between EBV and MBP. In their study, Amedei et al showed that T-cell recognition of either self or cross-reactive H. pylori epitopes resulted in both proliferation and expres-
sion of functional properties by cross-reactive Tcell clones [101]. In all clones, the Thl cytokine profile expressed upon stimulation with cross-reactive H. pylori peptides overlapped that induced by stimulation with either bacterial lysate or entire H+,K§ or the appropriate H§ epitopes. These data suggest that cross-reactive H. pylori peptides represent signals powerful enough to activate the expression of functional program of cross-reactive Thl cells in the gastric mucosa. Upon stimulation, all H§ pylori cross-reactive gastric clones expressed both perforin-mediated cytolytic activity and the ability to induce Fas-Fas ligand-mediated apoptosis in target ceils to which they were in close contact. Based on these observations, it is tempting to suggest that in the inflammatory setting in which cross-reactive T-cell clones are activated, parietal cells may express APC functions, becoming target, at the same time, of the cytotoxic and pro-apoptotic activity of cross-reactive gastric Thl cells. The end point of this process would be gastric corpus atrophy and hypochloridria. With regard to the question of whether it was H. pylori infection or H. pylori-independent gastric autoimmunity that initiated disease, three hypotheses can be suggested. First, having inherited MHC haplotypes that predispose to organ-specific autoimmunity [108], AIG patients may already have undiagnosed or subclinical disease and H. pylori infection, by providing a number of epitopes cross-reactive to H§ causes a Thl-mediated inflammation leading to the expansion of both cross-reactive and single (H+,K+ATPase)-reactive gastric T cells. The outcome is increased parietal cell destruction and gastric atrophy. A second hypothesis is that H. pylori is indeed the initiating factor, and primary activation of gastric Thl cells reactive to H. pylori peptides that cross-react with H§ results in an inflammatory process in which T cell-derived IFN-y allows H+,K+ATPase bearing parietal cell to act as APC and to become targets of cross-reactive epitope recognition and killing and/or apoptotic suicide. Apoptotic parietal cells would then allow cross-priming of T cells specific for "private" H+,K§ epitopes, ultimately leading to full blown AIG by epitope spreading. Third, H. pylori infection is simply a contingent phenomenon, playing no role in the natural history of gastric autoimmune disease.
6. CONCLUDING REMARKS: THE CAUSATIVE ROLE OF H. PYLORI IN HUMAN GASTRIC AUTOIMMUNITY
Recent data presented by Amedei et al [ 101] seem to fulfil most of the criteria proposed for assessing a case of molecular mimicry [1, 5, 12]. In their patients there was a temporal association between clinical and serological evidence of AIG and H. pylori infection, at least at the time of culture of their in vivo activated gastric T cells. Since exposure to H. pylori is commonly acquired in young life [19], the infection precedes symptoms of AIG that usually arise later in life. Taking into account the wide diffusion of H. pylori [ 19], one can suspect that some AIG patients, who are found H. pylorinegative at the time of their diagnosis of AIG, might have harboured the bacterium previously, and H. pylori was then lost while mucosal atrophy was ongoing. Interestingly, this seems to be the case of all AIG patients studied by Amedei et al [101], who lost H. pylori along the follow up study, possibly due to hypochloridria that made their gastric environment no more appreciated by the bacterium. On the other hand, the possibility that H. pylori is lost due to increasing gastric atrophy and hypochloridria has already been reported [56]. A quite broad repertoire of culprit epitopes have been identified in both the pathogen and in the self gastric protein associated with AIG. The microbial cross-reactive epitopes were able to elicit vigorous responses in the same gastric T cells that responded at comparable levels to both self H+,K+ATPase epitopes and the entire self protein. Finally, crossreactive T-cell clones quantitatively represented a significant component of the T-cell response at gastric level during the autoimmune disease and the concomitant H. pylori infection [101]. This would argue against the possibility that the detection at gastric level of autoreactive, cytotoxic, and pro-apoptotic Thl cells that cross-react to H. pylori epitopes is simply an epiphenomenon. Together, the most recent studies support the idea that, in genetically susceptible individuals, H. pylori infection triggers the development of gastric autoimmunity via molecular mimicry.
357
ACKNOWLEDGEMENTS This study has been supported by grants from the Italian Ministery of University and Research (MIUR), the Italian Ministery of Health, the Italian Association for Cancer Reasearch (AIRC), the Istituto Superiore di Sanit~ and the University of Florence.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Yersinia Enterocolitica Infections and Autoimmune Thyroid Diseases Mark E Prummel and Wilmar M. Wiersinga
Department of Endocrinology & Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
1. INTRODUCTION
Thyroid dysfunction is a common disorder, especially in females. The prevalence of hyperthyroidism in the adult female population ranges between 0.9 and 4.4%, and that of hypothyroidism is 3.0-7.5% [1]. The yearly incidence of hypothyroidism is 3.5/ 1,000 females, whereas per year 0.8/1,000 females will develop hyperthyroidism [2]. Most of the cases of thyroid dysfunction are caused by autoimmune thyroid diseases (AITD). Apart from iatrogenic destruction of the gland, Hashimoto's thyroiditis (HT) is the most frequent cause of hypothyroidism. It is caused by an autoimmune mediated destruction of the thyroid gland, and autoantibodies against the enzyme thyroid peroxidase (TPO) are its hallmark. On the other end of the spectrum, Graves' disease (GD) accounts for approximately 60% of the cases of hyperthyroidism [3]. It is caused by autoantibodies against the Thyroid Stimulating Hormone (TSH) Receptor, which have a stimulating effect. Despite the very different phenotypes, GD and HT share a partly comparable genetic background and both diseases can run in the same family [4]. Hence the term AITD. AITD like most other autoimmune endocrinopathies is considered as a multifactorial condition in which the autoimmune attack occurs in genetically predisposed individuals, possibly provoked by environmental factors. 1.1. Genetics of AITD
The main evidence for a genetic basis of AITD is formed by twin studies in which the concordance
rate in monozygotic twins was found to be higher than that in dizygotic twins. For GD the concordance rates in mono- and dizygotic twins were 35 and 3%, for HT 55 and 0% [5, 6]. These rates also show that AITD does not follow a Mendelian pattern of inheritance, and the risk for AITD declines sharply from 1st to 2nd and 3rd degree family members. In fact affected sib-pairs are not very common. In a Hungarian study, only 5.3% of GD patients had a sibling who was also affected by this disease [7]. The sibling risk (~,s) for GD is 8 [8]. This pattern of inheritance indicates a polygenic, multi-factorial inheritance of several low-penetrance, low-risk alleles of multiple loci [9]. Despite much effort, only a limited number of genes are currently known. The HLA Class II genes located on chromosome 6p21 are consistently linked to autoimmune endocrinopathies, including AITD. The HLA-DR3 haplotype is associated with GD with a relative risk between 1.9 and 3.5 [ 10], and is also associated with HT. Vaidy et al [8] have estimated that the HLA region contributes 17-20% to the familial clustering. The HLA molecules on the surface of antigen presenting cells (APCs) present the digested antigen to the T cell receptors on T lymphocytes. Antigen driven T cell activation will then take place if this presentation is done in conjunction with the B7 molecule on the APC which interacts with its receptor CD28 on the lymphocyte. This co-stimulatory pathway is regulated by the cytotoxic T lymphocyte associated 4 (CTLA-4) molecule that binds to B7 [9]. The genes coding for CTLA-4 and CD28 lie on chromosome 2q33 and polymorphisms in this locus are also associated with AITD [11 ].
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Table 1. Environmental factors possibly involved in the induction of Autoimmune Thyroid disease in genetically predisposed individuals Environmental factor
Mechanism
Low birth weight
Insufficient thymic maturation?
Excessive iodine intake
Thyroidal damage
Stress
Neuroendocrine effects on immune system
Smoking
Unknown
Neck irradiation e.g.: 9 External radiotherapy 9 Radioactive iodine 9 Nuclear fall-out
Thyroid damage
Drugs e.g. amiodarone, iodine
Thyroid damage
Other drugs: 9 Campath-lH 9 HAART 9 Interferon a
Influence on Thl/Th2 balance Influence on T cell availability Dysregulation of immune state
Putative: viruses
Molecular mimicry
Yersinia enterocolitica
Molecular mimicry?
Larger datasets are clearly needed to find further susceptibility genes and indeed several centers are currently involved in this research.
in monozygotic twins the smaller twin had higher levels of TPO antibodies, thus confirming the effect of birth weight on thyroid autoimmunity [17].
1.2. Environmental Factors and AITD
1.2.2. Iodine
Based on twin studies, it has been estimated that 79% of the liability to develop AITD is attributable to genetics, and thus o t h e r - environmental- factors must play a role [ 12, 13]. A number of different environmental factors have been found to be implicated in AITD (Table 1).
Although necessary for thyroid hormone synthesis, excessive iodine intake may damage the gland leading to enhanced presentation of thyroidal antigens to the immune system. High iodine intake is indeed associated with the induction of GD, and a higher prevalence of TPO antibodies and hypothyroidism is observed in areas with a high iodine intake than in regions with a low dietary iodine content [ 18].
1.2.1. Fetal gkowth As early as intrauterine life, the environment can apparently already have deleterious effects on later life. Intrauterine malnutrition (as in the so-called Dutch famine during World War II) is associated with hypertension, hypercholesterolemia, and type II diabetes mellitus in adulthood [14]. Fetal growth however also influences the immune system in later life, because a lower birth weight (but not body weight at one year of age) was found to be associated with the presence of TPO autoantibodies [15, 16]. In a later twin study, the same group found that
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1.2.3. Stress Stress appears to be a riskfactor for GD. In a casecontrol study it was found that GD patients had experienced more negative life events in the year preceding the diagnosis than controls [19]. Similar findings were subsequently reported by others [2022], but prospective studies are necessary to establish a cause and effect relationship. There seems to be no association with HT, and we recently were also unable to find an association between stress
and the presence of TPO antibodies [4].
1.2.4. Smoking Cigarette smoking is weakly associated with Graves' hyperthyroidism (the relative risk being 1.9), but strongly with Graves' ophthalmopathy (RR 7.7) another- less c o m m o n - manifestation of GD [23, 24]. The reason for this association remains unclear. Although thiocyanite is mildly toxic for the thyroid gland, smoking is not associated with hypothyroidism [24].
estrogen use seems to protect against AITD [33, 4]. Lastly, Interferon tx (IFNtx) used in the treatment of hepatitis C is a strong inducer of thyroid autoimmunity, especially in females [34]. It is associated with the induction of TPO antibodies (Relative Risk 4.4; 95% confidence interval 3.2-5.9), and this frequently progresses to overt thyroid disease (both HT and GD). This phenomenon suggests that the same may occur when there are high endogenous levels of IFNtx, such as seen during certain viral infections.
2. VIRAL INFECTIONS AND AITD
1.2.5. Radiation 2.1. Viruses and Autoimmunity Tissue damage in general can elicit an autoimmune attack, because after cell necrosis antigens will be complexed with immunostimulatory molecules such as Heat Shock Proteins 96 and 70 and can activate dendritic cells [25]. For instance, external irradiation to the neck for Hodgkin's disease not only carries a high risk for hypothyroidism (and thyroid cancer), but is also associated with GD [26, 27]. Environmental radiation such as occurred in Eastern Europe after the Chernobyl accident, is also associated with an increase in the prevalence of TPO antibodies [28]. Similarly, treatment of a non-toxic, nodular goiter with radioactive iodine can induce Graves' hyperthyroidism with TSH-R autoanfibodies [29].
1.2.6. Drugs Some forms of amiodarone induced hyperthyroidism may occur due to thyroidal damage [30], but there are several other drugs that are associated with AITD. Campath- 1H (a monoclonal antibody against CD52 bearing T lymphocytes) is used in multiple sclerosis and was found to be associated with the induction of GD [31 ]. This antibody suppresses Thl lymphocytes and thus shifts the Thl/Th2 balance towards antibody production, and hence apparently also against the TSH-R. Highly Active Antiretroviral Therapy (HAART) also affects the T cell availability and three cases of GD have been reported, occurring after 16-21 months of HAART [32]. In view of the definite preponderance of women in AITD, it is surprising that the use of estrogens is not associated with a higher risk of AITD. In contrast,
Indeed, certain viral infections are associated with the development of insulin dependent diabetes mellitus (IDDM), another organ-specific autoimmune disease [35]. Especially the enterovirus CoxsackJe B is associated with the induction of IDDM. In a prospective cohort study, a recent Coxsackie B virus infection (but not an adenovirus infection) was found more frequently in children who developed IDDM than in controls [36]. Also, children with IDDM have strong T cell responses against Coxsackie B viral proteins [37]. Coxsackie B is one of the viruses that cause high endogenous IFNct levels. A recent study found high IFNo~ levels 39/56 (70%) patients with recent onset IDDM, and in half of them Coxsackie b virus could be detected (serotypes CVB 2, 3, and 4), whereas the virus was absent in IFNtx negative patients [38]. In rat strains charecterized by certain MHC Class II genes, which do not spontaneously develop IDDM, diabetes could be elicited by injection with the IFNo~ inducer polyinosinic-polycytidylic acid (Poly IC) [39]. This suggests that non-specific environmental stimuli can induce autoimmunity in susceptible subjects. Coxsackie B virus may, however, also be less specific because an enzyme of the virus (P2-C) shares homology with the autoantigen glutamate decarboxylase (GAD65) [40]. 2.2. Viruses and AITD Much less is known about viral infections and the induction of AITD, and studies similar to those performed in diabetes have not been done in this field.
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Table 2. Some examples of Autoimmune disease linked to bacterial infections via molecular mimicry (Reviewed in Ref. [47]) Disease
Autoantigen
Pathogen
Rheumatic fever Ankylosing spondylitis Rheumatoid arthritis Rheumatoid arthritis Graves' disease
Cardiac myosin HLA-B27 Type XI collagen HSP 60 TSH-Receptor
Stretococcus pyogenes Klebsiella pneumoniae Proteus mirabilis Mycobacterium tuberculosis Yersinia enterocolitica
Viral infections have only been linked to another thyroid disease, De Quervain's subacute, painful thyroiditis [41]. Viruses linked to this disorder include the mumps, measles, influenza, EpsteinBarr, Coxsackie B, and adenovirus. However, subacute thyroiditis is not a true autoimmune disease, but rather an inflammatory disorder with high levels of C-reactive protein [42]. The rare syndrome of congenital rubella infection is the only known viral disease linked to AITD, because children affected by this virus have a higher frequency of TPO antibodies than controls [41 ].
3. BACTERIAL INFECTIONS 3.1. Bacterial Infections and Autoimmunity Bacterial infections have been linked to several autoimmune diseases (Table 2). Infections can lead to an inflammation-induced presentation of autoantigens, for instance in conjunction with heat shock proteins. Bacterial pathogens can also have an antigen that shares homology with a self antigen, e.g. molecular mimicry. An immune reaction against the pathogen can then lead to a breakdown of the tolerance against self, leading to an autoimmune reaction [40]. Molecular mimicry is not limited to simple amino acid sequence homology. Recently, it was shown that an autoreactive T cell line derived from a patient with multiple sclerosis recognized its antigen (myelin basic protein) when presented in the groove of a certain HLA-DR2b molecule. The same cell line also recognized an Ebstein Barr virus peptide (with no amino acid homology to MBP) when presented by a different HLA-DR2a molecule [43]. Thus, it appears that the mimicry also occurs
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at the level of the complex between an HLA molecule together with the antigen: not the antigen has homology, but only the complex [44]. In a number of autoimmune diseases, bacterial infections are thought to play a role. Gram negative bacteria such as Salmonella typhimurium are linked to reactive arthritis and infected mice produce T cells that cross react with the mouse heat shock protein 60. This HSP is also expressed on uninfected but otherwise stressed cells and these cells are indeed attacked by these T cells. The crossreacting bacterial antigen (GroEL) is also present on several other gram negative pathogens [45]. Other diseases are linked to bacterial infections because of cross-reactivity between a bacterial antigen and an HLA molecule, such as HLA-B27 in spondylarthropathies, and HLA-DR4 in rheumatoid arthritis [46, 47]. 3.2. Bacterial Infections and AITD Again, little research on the importance of bacterial infections in AITD has been done [41, 48], with the exception of Yersinia enterocolitica (YE) infections (see below). The only suggestion for a role of bacterial pathogens may be the observation that patients with GD have elevated levels of anti HSP72 antibodies [49, 50], because autoimmunity against HSPs is found in a number of autoimmune diseases and thought to be the result of cross-reactivity between endogenous and pathogen-derived HSP [51 ]. However, Yersinia infections are an attractive etiologic agent because this pathogen bears TSH-R like binding sites for TSH.
4. YERSINIA ENTEROCOLITICA (YE)
5. YERSINIA ENTEROCOLITICA AND AITD
YE is gram negative pathogen belonging to the
5.1. YE and Graves' Disease
Enterobacteriaceae and the Yersinia family, to which also belongs the notorious pathogen Yersinia
pestis [52, 53]. Y. pestis is the cause of the plague and was discovered by Alexandre Yersin [18631943] in 1894. YE is an intestinal pathogen and causes enterocolitis after an incubation time of 4--7 days. It can cause abdominal pain due to mesenteric enteritis, and is a self-limiting disease. It is caused by the ingestion of incompletely cooked pork, and of milk or other food contaminated by pork. Because the organism can grow at 4 ~ C, refrigerated food is often the source of infection. Most cases occur in autumn and winter. The infection often affects children. In 10-30% of adults, it is followed by a reactive polyarthritis which is transient, but persistent lowback pain can occur, especially in HLA B27 positive subjects. Further complications are erythema nodosum and Reiter's syndrome [54]. YE can be cultured from blood and feces, but serologic agglutination tests are also available and become positive soon during infection. Serologic tests typically become negative after 2-6 months. The organism is susceptible to aminoglycosides, trimethoprim-sulfametoxazole, and third generation cephalosporines among others. As stated above, YE infections are usually selflimiting, but in approximately one-third of patients a chronic, low-grade persisting infection seems to occur. In these subjects stool cultures, and conventional agglutination tests are negative, but they are positive for IgG or even IgA (suggesting recent or persisting antigen presentation) against virulence associated proteins. In patients with chronic spondylarthropathy, 25% had IgG or IgA antibodies against virulence associated released proteins and/or Yersinia outermembrane proteins (YOPs). In 83% of the patients with IgA antibodies, YE bacilli could be found in intestinal lymphatic tissue upon biopsy [55]. Chronic YE has even been suspected to cause the chronic fatigue syndrome, but this association was proven to be false. IgG antibodies against YOP were found in 27% of these patients, but also in 25% of controls [56]. Nevertheless, it thus seems that serologic evidence for a past or persistent infection with YE is very common.
In the 1970s two groups reported a higher incidence of YE antibodies in patients with GD then in controis. In Europe, agglutinating antibodies against YE were found in 50% of patients with GD, compared to 28% in controls [57]. In the USA, agglutinating antibodies were found in 25/36 (66%) of GD, and in 10/12 (83%) of liT patients, in comparison to only 9/110 (8%) controls. However, they also found a high number of patients (53%) with non-autoimmune thyroid disease with positive antibodies [58]. Nevertheless, these findings prompted an investigation into the possibility of shared antigens between YE and thyroid membranes [59]. These authors found that YE had specific binding sites for TSH with an affinity in the 10-8 M range, similar to the binding sites on thyroid membranes. It was subsequently shown, that TSH-Receptor autantibodies bind to the same site on YE membranes [60]. TSH binding sites were also found on other gut residing bacteria, and some (but not all) Graves' IgGs could displace TSH binding to YE, but not to several E. coli strains [61 ]. These studies suggested that immunity against YE could result in autoimmunity against the TSH-R and thus to GD on the humoral level. It also appears to happen on the cellular level, because the migration of leucocytes from GD patients was inhibited upon incubation with both YE and thyroid membrane preparations [62]. It was later shown that the epitope cross-reactive with the extracellular domain of the TSH-R is located on the 5.5 kDa lipoprotein of YE. This TSH-R like epitope is unique to the YE lipoprotein, since it is not present in lipoproteins from other Enterobacteriaceae, despite the fact that bacterial lipoproteins are highly conserved. In mice, this cross-reactive envelope protein was further shown to be highly mitogenic to B lymphocytes, which then produce TSH-R antibodies [63]. Recently, it was shown that YE antigen can act as T cell superantigen [64]. These laboratory data strongly suggest an etiological role for YE infections in GD and maybe in AITD at large, and the question arises if this hypothesis is supported by epidemiological data.
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5.2. Epidemiology of YE Infection and AITD 5.2.1. Acute YE infections
The epidemiology of acute, symptomatic YE infections is rather different from the epidemiology of AITD. First, YE infections are common in children, whereas AITD is much more common in adults. Secondly, YE infections occur predominantly in autumn and winter and there is only some evidence for seasonal clustering of GD diagnoses during the warmer part of spring and the summer, probably because of the heat intolerance occurring in GD [65]. YE infections occur predominantly in Northern Europe, the USA, and in Japan. In Europe, the most commonly encountered pathogens belong to the 0:3 and 0:9 serotype, while in the USA other serotypes are seen (0:8; 4,32; 13a; 13,b; 18; 20; 21) It is a rather common cause of gastro-enteritis. In a recent Danish study among 48,857 patients with bacterial enteritis, YE accounted for 4,045 (8.3%) infections, compared to 16,180 (33.1%) caused by Campylobacter, 26,974 (55.2%) by Salmonella, and only 1,658 (3.4%) by Shigella infections [66]. In Ontario, Canada, 44,451 sporadic cases of bacterial enteritis were registered from 1997 to 2001, (a decrease by 22% compared to the period of 1992-1996, and the annual average incidence rate of YE infections was found to be 3 cases per 100,000 inhabitants [67]. A similar annual incidence was found in the Netherlands: 1.2 cases per 100,000 inhabitants [68]. These figures fall well below the annual incidence of GD, which is roughly 100 per 100,000 inhabitants. 5.2.2. Past or Chronic YE infections
As stated above, one-third of YE infections may become chronic, and asymptomatic infections may also occur, because serologic evidence for past or present encounters with YE is present in many more subjects. There are several methods to detect YE antibodies. The oldest is the agglutination assay in which a titer of 1:8 or higher is considered positive, but this assay is not very specific (cross-reactions with other Enterobacteriaceae and Brucella spp.) and may miss infection if the wrong serotype is used [53]. A better assay uses Yersinia outer membrane (YOP) membrane proteins as
368
antigen in immunoblotting or other immunoassays. YOPs are released by the bacteria and are important determinants of their virulence. Using Western blot analysis, IgG antibodies against several YOPs (67-, 46-, 36-, 25-kDa) were found in the sera from 24/25 (96%) GD and from 10/18 (56%) HT patients, but also in sera from 17/24 (71%) of healthy controls [69]. There was no difference in the pattern of the Western blot bands, nor in the density of the several bands between patients and controls. These findings are in contrast to other studies, which did show a difference between AITD patients and controls. In a German study, a higher incidence of anti YOP antibodies was found in GD patients (IgG class: 72%; IgA: 33%) and in HT patients (IgG: 66%; IgA: 37%) than in controls (IgG: 35%; IgA: 11%) [70]. Intriguingly, the presence of these antibodies differed depending on the duration of the GD: no antibodies were present in the first 4 weeks after diagnosis, whereas at 6 months of GD duration antibodies against the 46-, 36-, and 25 kDa proteins were found. In patients with longstanding disease, only anti 36-kDa protein could be detected. YOP antibodies are not only found in GD, but also in HT. In Greece, 25% of HT patients had IgG antibodies (only 2% of controls), and 2.8% had IgA antibodies (0% in controls) [71 ]. Using the agglutination assay, a high prevalence of antibodies against YE 0:5 was found in GD (81%) and HT (91%), in contrast to 59% in controls in Japan [72]. Using the same method, in Turkey more patients with GD and with HT then controls were found to have antibodies against all 4 YE strains tested (0:3, 5, 8, and 9) [73]. In contrast, in a study from Canada, no differences could be found between AITD patients and controls and here in all groups >90% had antibodies against YE [74]. A higher prevalence of YE antibodies is not restricted to patients with established AITD. In a large cohort of 803 female relatives of AITD patients (who are at a higher risk to develop AITD themselves), we found 322 (40.1%) with IgG antibodies and 176 (22%) with IgA antibodies against YOP, in comparison with 24 and 13% respectively in controls. Although 216 (27%) of the female relatives also had TPO antibodies as evidence for thyroid autoimmunity, the presence of these antibodies was not related to YOP antibodies. Of IgG YOP positive females, 23% had TPO antibodies, whereas
25% (not significantly different) of YOP antibody negative subjects had TPO antibodies [75].
3.
6. C O N C L U S I O N
4.
Although there are plausible and attractive laboratory data supporting an etiologic role for YE infections in the initiation of AITD, the epidemiological data are far less convincing. The original hypothesis of cross-reactivity between the TSH-R and YE would suggest a link with GD only. But, most studies find a similar prevalence of YE antibodies in GD and HT patients. Also, if YE infections are one of the environmental risk factors for AITD, the prevalence figures for YE are rather puzzling. First, acute gastro-enteritis caused by YE is rare in comparison with the incidence of AITD (3/100,000 v e r s u s 400/ 100,000 inhabitants per year resp.). And, secondly, if not acute but chronic persisting YE infections are the culprit, there prevalence in controls is surprisingly high: ranging from 17.5 till >90%. In the Amsterdam cohort of healthy female relatives of AITD patients, positive YE serology was not linked to thyroid autoimmunity, but the prevalence of YE antibodies was yet higher than in controls. One hypothesis to explain this, is that these relatives shared a genetic background with an increased susceptibility for chronic, persistent YE infection. This is supported by the observation, that not all rat strains infected with YE develop a chronic persistent infection. In some strains the anti YOP antibodies disappeared when the infection was cleared, while in others antibodies and infection persisted [76]. It thus may be, that a continuous stimulation of the immune system from YE infected plaques of Peyer in susceptible humans stimulates thyroid autoimmunity.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Reactive Arthritis (Reiter's Syndrome): Roles of Infection, HLA-B27 and the Immune Response Maurizio Cutolo, Bruno Seriolo and Alberto Sulli
Research Laboratory and Division of Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy
1. I N T R O D U C T I O N Reactive arthritis is one of the spondyloarthritis family of clinical syndromes. With a calculated prevalence of 1.9%, spondylarthropathies are among the most frequent rheumatic diseases in the white population [1]. The clinical features of reactive arthritis are those shared by other members of the spondyloarthritis family, though it is distinguished by a clear relationship with a precipitating infection (mostly Chlamydia trachomatis-CT) [2]. Susceptibility to reactive arthritis is closely linked with the class I HLA allele B27; it is likely that all sub-types predispose to this condition [3]. The link between HLA-B27 and infection is mirrored by the development of arthritis in HLA-B27-transgenic rats. Nevertheless, the term reactive arthritis it appropriately describes arthritis that is associated with demonstrable infection at a distant site without traditional evidence of sepsis at the affected joint(s) [4]. Although several forms of disease could be described as "reactive", particularly acute rheumatic fever, post-meningococcal septicaemia arthritis and Lyme disease, in clinical practice the term is restricted to an acute spondyloarthritis, usually, but not exclusively, linked to acute genitourinary or gastrointestinal infection [2]. A proportion of patients fulfil criteria for Reiter's syndrome [5]. In addition, reactive arthritis is an infectioninduced systemic illness characterized by an inflammatory synovitis, from which no viable microorganisms can be cultured [5]. Therefore, the paradigm of reactive arthritis is
an infectious trigger that occurs in a genetically susceptible individual in whom the immune system continues to be stimulated or "react" to an ongoing or cleared infection, resulting in intermittent exacerbations of a variety of immune-mediated signs and symptoms. Reactive arthritis is often termed Reiter's syndrome, after Hans Reiter, who was incarcerated at Nuremberg for war crimes [6, 7]. Although the terms Reiter's syndrome and reactive arthritis are sometimes used interchangeably, Reiter's syndrome is actually the complete clinical triad of urethritis, conjunctivitis, and arthritis. Some patients will never fulfil all three components of Reiter's triad, however, and although the term incomplete, Reiter's syndrome is often used in such cases, some authors believe that the term reactive arthritis should be used, as it is less confusing and is inclusive of any extra-articular manifestations. The first description of the syndrome seems done in 1578 in Mexican medical texts [8], Further reports were a clear description of arthritis associated with venereal disease as given by Swediaur (1798/1809) in French. It became relatively common in London from 1820 onwards with 13 cases described in the literature between 1818 and 1836. It accounts for 3% of admissions at University College and St Bartholomew's Hospitals between 1835 and 1839 and also 3% of admissions at the London Hospital from 1895 to 1900 [9]. In view of the above, and because Reiter was not the first to describe reactive arthritis, "Reiter's syndrome" has been suggested only be used to cite an older reference that uses the term or in a historical context [6].
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2. T H E R O L E OF THE I N F E C T I O N The number of pathogens causing reactive arthritis in very limited. In fact, for a particular pathogen to became successful to cause reactive arthritis, it has to be an obligate/facultative intracellular organism and then it must to evolve so that may exert specific activities. The activities of a pathogen include: capacity to travel from the mucosal surface to the joints, ability to adjust its molecular activities to adapt to the joint environment and finally of importance, the pathogen must evade the host defences [ 10]. Characteristically, and in contrast to classical infectious arthritis, the agents of reactive arthritis cannot be cultured and, therefore, may not be "viable" once having reached the joint. However, with regard to the different activities required by a pathogen in reactive arthritis, CT is the best studied, and can be found in about 50% of patients with preceding symptomatic infection of the urogenital tract who developed reactive arthritis [ 11 ]. In addition, positivity for CT can be observed by polymerase chain reaction (PCR) in 65-70% of patients with reactive arthritis. The primary site of infection of CT is the mucosa of the urogenital tract, where the monocytes and or/ dendritic cells are among the host cells that CT enter and survive. After these cells enter the bloodstream, they become carriers to disseminate the bacteria, one of such site being the joints. All the pathogens for reactive arthritis seem to follow this form of dissemination [ 12]. The unique life cycling potential of CT allows this pathogen to have succes as causative of reactive arthritis, in fact, CT infect the cells as infectious elementary bodies (EB). These EB after the binding to the host cell surface, enter inside by endocytosis and after few hours are transformed into larger metabolically active reticulate bodies (RB). The RB form the "inclusion bodies" and reorganize themselves again into EB to be released by both cell lysis or exocytosis. When released outside the cells CT-EB are powerful pathogens that infect other host cells [12]. Once arrived inside the reactive arthritis joint, CT are hosted by monocytes that, as long living cells, allows the survival of the bacteria. The CT modify their normal biphasic life cycle and are metabolically active as demonstrated by the expression
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Table 1. Possible pathogenic organisms in reactive arthritis (frequence of detection in % in reactive arthritis patients) More frequent association
Chlamydia trachomatis Ureaplasma urealyticum Shigella flexneri Salmonella enteritidis Salmonella typhimurium Shigella dysenteriae nk Yersinia enterocolitica Campylobacter jejuni Streptococcus sp nk
65% 20% 15% 6% 3% 2% 1-2%
Less frequent associations
Chlamydia pneumoniae Neisseria meningitidis Bacillus cereus Pseudomona Clostridium difficile Borrelia burgdorferi Escherichia coil Helic obacter pilory Lactobacillus Bacille Calmette-Gu~rin
of very short-lived chlamydial rRNA transcripts and mRNA, in addition to normal RNA and DNA [13, 14]. The expression of several CT antigens is altered, and whereas the synthesis of the major outer membrane protein (MOMP) is reduced, on the contrary, the 57-kDa heat-shock protein (HSP60) and lyposaccaride are highly increased. Changes on structure of CT are demonstrated by electron microscopy that shows aberrant morphology of persisting CT [151. At the same time, genes involved in energy production are altered during different phases of the infection. In early infection the energy is derived from glycolitic and pentose phosphate pathways, whereas in persistent infection the primary source of ATP is obtained from that of the host cell. When CT persist in the joint, one intriguing possibility is that the presence of any bacterial DNA may act as immunostimulant and contribute to the pathogenesis of the disease. As a matter of fact,
Figure 1. The major physiological activity of HLA class I molecules, such as HLA-B27, is to present antigenic peptides derived from bacteria, to the T cell receptor (TCR) of CD8+ lymphocytes in reactive arthritis.
intra-articular immunoglobulin IgG production and a possible role for some CT antigens such as OMP2 in the pathogenesis of reactive arthritis have been reported [ 16]. In order to evade the host defences CT utilize different strategies. Since CT requires that its host cell is alive for their own survival, CT was found to inhibit the apoptosis of the host cell by inhibiting the release of mitochondrial cytochrome c and also by directly engaging the death domains of the TNF receptor family [ 17]. In addition, the monocytes/macrophages infected by CT induce apoptosis of T lymphocytes through the release of TNF-alpha [ 18, 19]. Other infectious agents that have been implicated in the pathogenesis of reactive arthritis, including their frequency are reported in Table 1.
3. THE ROLE OF THE HLA-B27 An ineffective immune response of the infected individuals seems to contribute to the manifestations and course of reactive arthritis [20]. HLA-B27 plays an important role in the pathogenesis of reactive arthritis, although arthritis can also occur in its absence [21 ]. HLA-B27 is an allele of the HLA class I molecules, a major function of which is to present peptides to CD8+ T lymphocytes, and the motif of the peptides carded by each HLA allele is allele specific (see Fig. 1). Of course the identification of HLA-B27-restricted bacterial peptides has been an obvious concern for the major investigations. Several peptides have been identified [22, 23]. Recently, 199 peptides derived from the entire genome sequence of CT have been synthesized and tested [24]. Eleven reactive peptides were identified when tested against the synovial fluid CD8+ lymphocytes of three patients with a CT-induced arthritis in
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Figure 2. HLA-B27 presents some antigenic peptides arising from the arthritis-causing bacteria and carried into the joints by monocytes. These peptides are cross-reactive with autologous peptides derived from proteins present in the joint tissues.
active phase. Since the chance for a particular peptide to be important to reactive arthritis would be high if is also immunodominant and therefore reactive with most patients, one of the 11 peptides from CT genoma was effectively found reactive with the lymphocytes of all three patients tested in the study [24]. Therefore, this peptide (NRAKQVIKL) represent at the present time the most promising identified, however derived from a screening that is far from to be comprehensive of all other possible common peptides [12]. As a matter of fact, this peptide was found able to detect T lymphocytes producing IFN-gamma in response to their contact, but other HLA-B27 CT peptides also react with T lymphocytes without inducing any IFN-gamma production. In conclusion, this study shows that there is an active CD8+ antipathogen activity that can be detected in the joints and this activity may well explain why there is and acute arthritis after urogenital CT infection [24]. The interest in reactive arthritis, is how the acute
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arthritis may shift to chronic arthritis in HLA-B27 positive patients, and the reasonable suggestion is that the early acute antibacterial reaction is followed by a self-maintaining autoimmune response against an autologous petide in the chronic stage. The search was for an autologous peptide that was homologous to those of an Epstein-Barr virus (EBV) protein, and an Italian group recently focused on one that was found previously to provide an immunogenetic peptide for HLA-B27 patients [25]. This identified autologous peptide seems derived from 400 to 408 residues of the vasoactive intestinal peptide receptor 1 (VIP-1R) and represent the important beginning of other screening to identify other autologous peptides that might (see Fig. 2) react with CD8+ lymphocytes from different HLAB27 patients affected by spondyloarthropaties. In addition, CT has been found to downregulate the expression of antigen presentation molecules in host cells, enabling CT evasion from an effective immune response.
Of course, it is not absolutely clear the question whether HLA-B27-related reactive arthritis is always mediated by conventional peptide presentation by class I molecules, since different cell type may act as mediator in the arthritis [ 12]. More recent studies support the concept that HLA-B27 might cause arthritis because it is folded more slowly causing intracellular proinflammatory signals [26]. During the screening of synovial fluid mononuclear cells by microarray, it was very recently discovered the expression of a gene (encoding the proteasome C2) that is linked to an endoplasmic reticulum unfolded protein response, and this high expression is restricted to the macrophage fraction of the synovial fluid cells [27]. Therefore, it is possible that the unfolded protein response is really active at the level of the joints of patients affected by spondyloarthropaties, and the macrophages are the cells playing a central role in the process, as already suggested in the inflammatory synovitis [28].
4. THE ROLE OF THE IMMUNE RESPONSE AND CYTOKINES
The immune system activation undoubtedly plays a crucial role in the pathogenesis of reactive arthritis and the derived autoimmunity (HLA-B27-mediated) seems the most natural mechanism for chronicity. Thl cytokines such as IL-12, IFN-gamma and TNF-alpha are crucial for the elimination of bacteria but a lack of these and/or elevated production of Thl/Th2 cytokines (particularly IL-10) inhibit the effective clearance of the pathogens [29]. A low production of TNF-alpha by peripheral T lymphocytes has been shown at onset of reactive arthritis and the lower levels of TNF-alpha secretion and HLA-B27 status, seems to represent the more likely condition to develop a chronic course, possibly with an additive effect [30] (see Fig. 3). In addition, in reactive arthritis it has been shown that TNF-alpha levels are low in the synovial fluid [31]. Therefore, the diminished TNF-alpha production might reflect a state of relative immunodeficiency contributing to bacterial persistence in reactive arthritis [30]. HLA-B27 might be one reason for low TNFalpha defect, as it can also be observed with healthy
HLA-B27-positive individuals [12]. Interestingly, the TNF locus is located within the MHC only 250 kb centromeric from the class I locus [32]. This has prompted several investigators of rheumatic diseases to undertake a search for other susceptibility genes in this region. Such genes may either function independently of HLA Class I or II genes, or may function as genetic cofactors with the HLA genes. Another reason for low TNF-alpha in reactive arthritis, might be because the disease is associated with low-producing TNF-alpha allele as recently reported in a Finnish population of patients where the increase in the c l allele, independently from HLA-B27, suggests that it might be a new susceptibility marker for the disease [33]. The association of reactive arthritis with other alleles was due to a linkage disequilibrium with HLA-B27. Multiple studies have also reported on TNF-alpha polymorphism in spondyloarthropathies, but further studies need to understand possible roles [32]. In any case, there is a low production of Thl cytokines in reactive arthritis, which might partly explain bacterial persistence. IFN-gamma is a further Thl cytokine that is involved in the antibacterial defenses. Interestingly, contrary to the expectations, the Thl and Th2 cytokine mRNA expression in the synovial tissue (in particular the amount of IFN-gamma), was found lower in the joints of patients affected by spondyloarthropaties compared to rheumatoid arthritis, and reflecting again a lower ratio Thl/Th2 [34] (see Fig. 3). However, the altered ratio Thl/Th2 seems an indicator of disease activity, as the ratio becomes reversed when patients are treated with anti-TNFalpha antibody. Moreover, at the present time, it is impossible to predict whether a cytokine treatment stimulating a Thl response (administration of IL- 12 or IFN-gamma to eliminate the bacteria) or rather the opposite (administration of TNF-alpha blockers) might represent the best new way of approach. In fact, it remains to be determined whether the latter treatment may induce an exacerbation of the infection or just a suppression of the inflammatory reaction without stimulating bacterial growth [ 16]. Finally, the role of IL-10 is quite unlike that of TNF-alpha or IF?q-gamma, since IL-10 inhibits the antibacterial activities of macrophages. Different
377
Figure 3. An ineffective immune response of the infected individuals seems to contribute to the manifestations and course of reactive arthritis low levels of Thl cytokines (mainly TNF-alpha and IFN-gamma) and high concentrations of IL-10 (Th2 cytokine) characterize the production of T cells and monocyte/macrophage in early reactive arthritis joints.
microsatellites of the IL-10 have been investigated with contrasting results, some associated with chronic development, others associated with protection in Finnish reactive arthritis patients [35]. In particular, IL- 10.G 12 and G 10 microsatellite alleles show a strong protective effect against the development of reactive arthritis in Finnish subjects. Since these polymorphic markers themselves do not have direct functional implications, they most likely mark promoter haplotypes with distinct functional properties, suggesting that differential production of IL-10 is a more important susceptibility factor for the development of reactive arthritis [35]. Recently, high concentrations of IL-10 (together with low IFN-gamma levels) have been detected in the joints of patients with early CT-induced arthritis compared with patients with undifferentiated oligoarthritis [4] (see Fig. 3).
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5. POSSIBLE ROLE OF SEX HORMONES
Striking differences in age- and sex-specific rates are seen in many rheumatic diseases. Epidemiological evidence indicates that during the fertile age women are more often affected by rheumatic diseases than men, particularly autoimmune diseases [36]. The pre-or post-menopausal status is a further factor influencing the rate of rheumatic diseases. It is therefore important, whenever possible, to see epidemiologic data broken down into age (for example 10-year age band) and sex-specific group before making inferences [37]. Obviously sex hormones seem to play an important role as modulators of the disease onset/perpetuation [38]. Sex hormones are implicated in the immune response, with estrogens as enhancers at least of the humoral immunity and androgens and progesterone as natural immunesuppressors [39]. Low gonadal and adrenal androgens [testosterone (T) / dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and its sulphate
Figure 4. Since infectious agents are implicated in the etiology of reactive arthritis and androgens exert generally immunosuppressive activities on immune cells (i.e. macrophages), a possible favouring role of androgens on chronic infections (i.e. CT) can not be excluded in reactive arthritis. Therefore, the more frequent appearance of spondyloarthropaty in man might be explained. (DHEAS), respectively] levels, as well as reduced androgens/estrogens ratio, have been detected in body fluids (i.e. blood, synovial fluid, smears, salivary) of male and female rheumatoid arthritis (RA) patients, supporting the possible pathogenic role for the decreased levels of the immune-suppressive androgens [40]. Several physiological, pathological and therapeutic conditions may change the sex hormone milieu and/or peripheral conversion rate, including the menstrual cycle, pregnancy, postpartum period, menopause, chronic stress, inflammatory cytokines, use of corticosteroids, oral contraceptives and steroid hormonal replacements, inducing altered androgen/estrogen ratios and related effects. Sex hormones can exert local actions (paracrine) in the tissues in which they are formed or enter the circulation [41 ]. Both T and 17-[3 estradiol (E 2) seem to exert dose and time-dependent effects on cell growth and apoptosis. In particular, T by inducing cell apoptosis
seems to reduce the immune response. Therefore, lower frequency of autoimmune diseases such as RA or systemic lupus (SLE) in male patients, might be related to androgen inhibition of immune cell proliferation and sequential immune response [42]. Important effects of sex steroids on gene promoter of Thl/Th2 cytokines are also well established and frequently dose-related [43]. Interestingly, if the autoimmune rheumatic diseases prevail in female patients, some other diseases, such as spondyloarthropaties are more frequent in man. Of course, a role for sex steroids in the pathogenesis of these diseases is suggested by the male predominance and by the peak age of onset in young adults. Current data on sex steroid hormones provide no straightforward explanation for the male predominance in spondyloarthritis [44--46]. However, patients with spondyloarthropaties do not show low serum levels of testosterone as it is regularly observed in men affected by rheumatoid
379
arthritis [47]. In addition, normal levels of testosterone and adrenal androgens (DHEA, DHEAS) have been found in synovial fluids of reactive arthritis patients when compared with RA synovial fluids (very low concentrations) [48]. Therefore, is fair to say that present data in patients with long-standing disease are too limited to suggest a role for androgens in the perpetuation of the disease, but a role in the initiation and the early stages of the spondylitis cannot be excluded [49]. In particular, since infectious agents are implicated in the etiology of reactive arthritis and androgens are immunosuppressive, a possible favouring role of androgens on chronic infections can not be excluded and the appearance of spondyloarthropaty to be explained why more frequent in man [50, 51] (see Fig. 4). Male subjects in fact, are characterized by a less efficient immune response also in normal conditions.
6. C O N C L U S I O N S Reactive arthritis, still the most common type of inflammatory polyarthritis in young men. An HLAB27 genotype is a predisposing factor in over two thirds of patients with reactive arthritis and autoimmune mechanisms versus autologous peptides have been shown. Therefore, based on the strength relationship with HLA-B27, reactive arthritis is considered a spondyloarthropathy. The syndrome most frequently follows genitourinary infection with Chlamydia trachomatis, but other organisms have also been implicated. A defective immune response against bacteria seems to characterize reactive arthritis patients, and reduced levels of T h l cytokines are observed in these patients. Since reactive arthritis is more frequent in male subjects, by considering that androgens are immunosuppressive, a possible favouring role of androgens on chronic infections can not be excluded.
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ankylosing spondylitis and healthy controls. Clin Rheumatol 1989;8:37-41. Hedman M, Nilsson E, de la Torre B. Low blood and synovial fluid levels of sulpho-conjugated steroids in rheumatoid arthritis. Clin Exp Rheumatol 1992;10: 25-30. Giltay EJ, van Schaardenburg D, Gooren LJ, PoppSnijders C, Dijkmans BA. Androgens and ankylosing spondylitis: a role in the pathogenesis? Ann NY Acad Sci 1999;876:340-64. Cutolo M. Gender and the rheumatic diseases: epidemiological evidence and possible biologic mechanisms. Ann Rheum Dis 2003;62(sp0005)3. Inman RD, Scofield RH. Etiopathogenesis of ankylosing spondylitis and reactive arthritis. Curr Opin Rheumatol 1994;6:360-70.
9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Bovine Spongiform Encephalopathy as an Autoimmune Disease Evoked by AcinetobacCer: Implications for Multiple Sclerosis and Creutzfeldt-Jakob Disease Alan Ebringer, Taha Rashid and Clyde Wilson
Division of Life Sciences, King's College, University of London, London, UK
1. THE ORIGIN OF THE BSE PROBLEM
"Bovine spongiform encephalopathy" (BSE) is a chronic neurological disease that appeared in British cattle in the 1980's following the introduction of a new form of "winter feed" also called "meat and bonemear' (MBM) produced from abattoir materials containing brain, spinal cord, pancreas, thymus and intestines with their contents [36]. This abattoir material was called "green offal" due to its content of chlorophyll containing grass found in the intestinal contents and following homogenisation with heat treatment, the MBM was given to cattle in a powder form as a food additive in an attempt to increase the protein content of cattle feed. By 1988, some 60,000 cows had been identified as suffering from BSE and the "Ministry of Agriculture, Fisheries and Food" (MAFF) scientists suggested that "brains" in the "green offal" could have been contaminated by "prions" from sheep having had "scrapie" and thereby probably caused the disease in British cattle. A statutory ban on the use of this MBM was introduced in 1988, the incidence of BSE reached a peak in the year 1992 but from then on the disease progressively decreased although not to zero levels (Fig. 1). The drop in new cases of BSE was progressive till in 2001 when there were more cases in the rest of the "European Union" than in the UK. However in 1996, a new disease was described occurring in Great Britain which resembled sporadic-CJD but instead of affecting elderly patients it seemed to be predominantly found among young
people below the age of forty years [38]. The possibility was raised that it could have been caused by the consumption of B~E-affected beef, since it appeared from epidemiological studies that at least one million BSE-affected cattle had been slaughtered in abattoirs and entered the human food chain. This started the world hysteria about meat consumption. The USA then banned Americans from donating blood if they had been on holidays in Europe. The BSE problem continues to dominate public concern about food safety throughout the world.
2. SCRAPIE AS A M O D E L OF A TSE DISEASE The origin of BSE is unknown but it has been suggested that it could have been caused by viruses [10], the use of organo-phosphate pesticides [26] or the consumption of offal from scrapie affected sheep which could have been inadvertently included during the preparation of MBM feedstuffs. Such offal material may have contained denatured "scrapie prions", also known as PrP so, which could have been responsible for the disease in sheep and goats [25]. "Scrapie" is a chronic neurological disease of sheep and goats characterised by generalised ataxia which is endemic in parts of Europe and the United States. The animals appear to be unsteady, tremble especially when excited and often rub themselves against posts and trees hence the name of "scrapie" [23]. It was first described in England in 1732
383
Figure 1. Chronologyof epidemic of bovine spongiform encephalopathy in United Kingdom. (Adapted from Brown P, BMJ 2001 ;322:841-44 with permission.)
whilst a similar disease known as "la Tremblante" was known in France way back in 1690,s during the reign of Louis XIV. The most characteristic findings at p0st-mortem is the presence of intraneuronal vacuoles. The spongy appearance of the neuropil is due to vacuolation of the neuronal processes. Studies in the 1960's had shown that "experimental scrapie" could be produced in goats injected with brain tissues from apparently normal goats [22]. The authors went to point out that similarities existed between the agents of scrapie and those of EAE. Since EAE is considered as an animal model of MS, a disease recognised to be an autoimmune disease, such an observation in this ovine condition raises the question whether scrapie may also be an autoimmune disease. In the 1980's Gajdusek's group from Washington had demonstrated that scrapie animals [2] and patients with kuru and CJD possessed autoantibodies to gray matter neurofilaments [30]. The origin of these brain autoantibodies was unclear since despite an intensive search for pathogenic viruses, no environmental agent causing scrapie has been isolated. If an autoimmune hypothesis is to be entertained either in BSE or scrapie then it is relevant to review some autoimmune diseases in which an external infectious agent has been associated with these conditions.
384
3. RHEUMATIC FEVER, S Y D E N H A M ' S CHOREA, R H E U M A T O I D ARTHRITIS AND A N K Y L O S I N G SPONDYLITIS AS E X A M P L E S OF A U T O I M M U N E DISEASES EVOKED BY I N F E C T I O N Autoimmune diseases are characterised by the presence of antibodies which bind to self-antigens and therefore are known as autoantibodies. In some diseases such autoantibodies can cause tissue damage. Many human diseases, such as juvenile diabetes or rheumatoid arthritis are considered as examples of autoimmune diseases. Two main theories have been proposed for the origin of such autoantibodies: either the immune system spontaneously starts producing tissue damaging immune cells or infection occurs by a microbiological agent, which possesses antigens exhibiting molecular similarity or "molecular mimicry" with some tissues of the host. There is scant evidence for the "lymphocyte mutation" hypothesis. The proposal that a lymphocyte mutates and starts attacking, say synovial tissues to produce rheumatoid arthritis, is to some extent illogical as it involves different pathological processes. A disease involving such a mechanism with an unrelenting clonal proliferation of cytotoxic lymphocytes would then be considered as a neoplastic process and should be called by its proper name of "leukaemia". However leukaemias are not autoimmune diseases
but involve an abnormality in the controls of cell division. The "infection hypothesis" would appear to have greater merit in providing an explanation for the origin of autoimmune diseases. Following infection, an immune response will occur with antibodies being produced against the invading microbe. A portion of these antibodies will bind to self tissues of the host and therefore will act as autoantibodies. When present in high concentrations, such autoantibodies can cause tissue damage and eventually lead to a disease involving the organ possessing structures resembling the invading pathogen. The cytotoxic effect of such anti-bacterial antibodies can be demonstrated using a sheep red cell assay. Such cytotoxic autoantibodies have been demonstrated in rheumatoid arthritis and ankylosing spondylitis
[40]. The classical model of an autoimmune disease evoked by an infection is "rheumatic fever". The microbe Streptococcus possesses molecular sequences, which resemble the human heart [34]. Following an upper respiratory infection or tonsillitis, anti-streptococcal antibodies bind to endocardial antigens and cause tissue damage. The patient then develops a cardiac murmur, fever and joint pains and is then said to suffer from "rheumatic fever". Since autoantibodies can cause tissue damage, this is an example of an autoimmune disease produced by an infection. Rheumatic fever has more or less disappeared in the Westem World over the last fifty years. It has been suggested that the widespread use of penicillin and similar antibiotics since the 1950's has led to the virtual disappearance of rheumatic fever, although in parts of the Third World, such as Egypt or Brazil it is still a relatively common disease. The question arises whether autoimmune diseases could be prevented by the early removal of the offending microbe. Sydenham's chorea occurs in rheumatic fever patients who have a high titre of anti-streptococcal antibodies. Some of these antibodies will bind to the basal ganglia of the brain and produce ataxia and the choreiform movements characteristic of the disease [20]. Involuntary movements are the most prominent symptoms of chorea, usually they are quasi-purposive and are intensified by voluntary effort. Sydenham's chorea usually wanes follow-
ing treatment with high doses of antibiotics, such as penicillin and is an example of a neurological autoimmune disease evoked by an infection. Rheumatoid arthritis is another example of an autoimmune disease evoked by an infection. Over 95% of rheumatoid arthritis patients possess HLADR1/4 antigens while the frequency of these antigens in the general British population is about 35%. A particular amino acid sequence found in these HLA antigens is EQ(K/R)RAA and this shows molecular mimicry with the ESRRAL sequence found in Proteus haemolysin [39]. Elevated levels of antibodies to the urinary pathogen Proteus mirabilis were found in patients with rheumatoid arthritis from more than 14 different countries throughout the world [ 15]. Ankylosing spondylitis, a chronic disease of the spine is also another example of an autoimmune disease evoked by an infection. The HLA-B27 antigen is present in 96% of patients with ankylosing spondylitis but in only in 8% of the general population. Again "molecular mimicry" is found to operate in this condition. A particular peptide sequence present in the HLA-B27 molecule is QTDRED which shows molecular mimicry with a similar sequence located in the nitrogenase enzyme found in the commensal bowel microbe Klebsiella [29]. Antibodies to Klebsiella microbes have been found in to be present in ankylosing spondylitis patients from many different countries, such as Spain [8], the Netherlands [3], Japan [31] and the UK [33]. Furthermore the cytotoxic activity of such anti-Klebsiella antibodies has been demonstrated in ankylosing spondylitis and a similar complement dependent cytotoxicity of anti-Proteus antibodies in rheumatoid arthritis [40]. Clearly the demonstration of four different autoimmune diseases evoked by an infection through the mechanism of "molecular mimicry" raises the question whether a similar approach might be relevant in studying the BSE problem. The presence of clinical similarities between BSE and EAE, especially in relation to hind-quarters paralysis and lower limbs ataxia in MS leads to the question whether some environmental agents might possess antigens resembling or cross-reacting with brain tissues [ 12].
385
4. " E X P E R I M E N T A L A L L E R G I C E N C E P H A L O M Y E L I T I S " AS AN ANIMAL MODEL OF MS "Experimental allergic encephalomyelitis" (EAE) is considered as an animal model of multiple sclerosis. This experimental model was discovered almost by accident in 1880, by Pasteur and his colleagues in Paris. Pasteur was trying to immunise patients who had been bitten by rabid dogs and wolves. To produce anti-rabies immunity, he had available the brains of only two rabid animals, one rabid dog and the other one from a rabid wolf. In an endeavour to increase the quantity of rabies material, he injected the brains of the two rabid animals, into some sixty rabbits. He then used the rabbit brain homogenates to immunise patients who had been bitten by rabid dogs or wolves, and injected them with rabbit brain homogenates. Some patients developed, as expected anti-rabies immunity but a small number of injected subjects, developed a neurological disease which was characterized by ataxia and in some cases led to a fatal outcome. An extensive literature is present in European medical journals describing these serious complications and by the 1940's, the "World Health Organisation" (WHO) in Geneva had between 200 and 300 cases of patients who had died from a disease known as "post-rabies vaccination allergic encephalomyelitis". The cause for this unexpected and lethal response was not explained till the 1930's when it was shown that injection of foreign brain homogenates will evoke an immune response in the immunised individual or animal by the production of anti-brain autoantibodies which will damage the brain tissues of the host [37]. In the 1950's it became apparent that this was a general observation in immunology: immunisation with any organ homogenate would produce an autoimmune disease in the target organ. The classical work of Rose and Witebsky demonstrated that peripheral injection of homogenates of thyroid tissue produced an experimental disease in animals which was similar to the human autoimmune disease Hashimoto's thyroiditis [28]. It was only after the work of Medawar on allogeneic skin transplants when it was recognised that this phenomenon was an example of the homograft response by which the recipient recognises foreign
386
"transplantation antigens" and mounts a powerful immune response against them [4]. Injection of brain homogenates from animals with BSE or scrapie led to a neurological disease and this was described as transmission of the disease. However the question arises whether this is not an example of "allergic encephalomyelitis" the disease Pasteur had observed one hundred years ago.
5. S P O N G I F O R M CHANGES IN EAE, BOVINE M Y E L I N AND ACINETOBACTER In "acute EAE" observed one to three weeks, following immunisation with brain homogenates, there is perivascular infiltration with inflammatory cells leading eventually to the formation of fibrotic plaques resembling those observed in MS patients. This is one of the main reasons why EAE is considered to be an animal model of MS. However in "chronic EAE", observed three to six months following immunisation, characteristic "spongiform changes" have been described, at least in rabbits in 1969 [24] and in guinea pigs in 1974 [27], by Raine's group from New York. It would appear that "spongiform changes" occur not only in TSEs but also in EAE. One of the main components in the central nervous system responsible for the production of EAE is a basic protein present in the white matter of the brain. In 1970, Eylar's group from San Diego, identified a highly active peptide from bovine myelin which when injected in microgram quantities into guinea pigs, would produce hind legs paralysis, tremors, weight loss and eventually death [17]. These features of hind quarters paralysis, tremors, weight loss and death, are also features described in cattle affected by BSE. Furthermore, the biological activity of this peptide was retained when it was heated to 100 ~ C for one hour or treated with 8M urea and these are properties also described for prions. We proposed the hypothesis that there may be in the environment a microbe which could possess proteins resembling brain tissues, similar to the situation of Streptococcus in rheumatic fever and Sydenham's chorea [13]. Computer analysis of proteins in SwissProt data base, using the Eylar
Figure 2. Molecular similarities between Acinetobacter and myelin basic protein molecules. (Adapted from Ebringer et al, Environ Health Perspect 1997;105:1172--4.)
sequence as a probe, revealed that the microbe Acinetobacter which is present in soil, on skin, in contaminated waters and fecal materials had such a sequence (Fig. 2). The sequence is present in the molecule 4-carboxy-mucono-lactone decarboxylase of Acinetobacter [12] and subsequently a similar sequence was found in ~,-carboxy-mucono-lactone decarboxylase of Pseudomonas [191. Both groups of microbes Acinetobacter and Pseudomonas belong to the same family of Gram-negative bacteria and share many antigens. The discovery that a common environmental microbe Acinetobacter had a sequence showing molecular mimicry with bovine brain antigens, suggested a possible mechanism as to how cattle could have developed BSE. Offal material from abattoirs was used in the preparation of the MBM feedstuffs given to cattle and it could have become inadvertently contaminated by Acinetobacter. Although heat treatment was still applied, albeit at a lower temperature, to the preparation of the
MBM, the demonstration by Eylar and coworkers that the myelin peptides were highly resistant to heat denaturation meant that these bacterial fragments retained their biological activity in being able to induce EAE in experimental animals. The presence of such biologically active fragments in MBM, meant that the cows would not only make antibodies against them but because of molecular mimicry or similarity between brain tissues and Acinetobacter, any antibodies produced, especially of the IgG isotype, which can cross the "blood-brain barrier", would also attack the brain and cause a neurological disease.
6. ANTIBODIES TO ACINETOBACTER AND AUTOANTIBODIES TO BRAIN ANTIGENS IN BSE The demonstration that Acinetobacter a common environmental microbe had antigens crossreacting
387
with mammalian brain tissues, raised the question whether BSE animals had been exposed to it or to fragments of the bacterium. An approach was made to the "'Ministry of Agriculture, Fisheries and Food" (MAFF) in the UK with the suggestion that an alternative explanation was possible for the onset of BSE in British cows.
6.1. First Coded Study Approval was given for a small pilot study and access given to BSE and control sera. The Ministry provided sera from 29 animals which had been found at post-mortem to satisfy the criteria of BSE and 18 which did not have the disorder. The 18 animals which did not have histological evidence of BSE had been re/erred to the veterinary services because the animals showed abnormal behaviour involving ataxia which suggested the possibility of a neurological disease. Post-mortem examinations were carried out to exclude BSE. The majority of the BSE-positive animals came from dairy Friesian herds. In addition, sera were obtained from an additional 58 healthy animals to act as extra controls: 30 serum samples were from animals aged less than 30 months consisting mainly of crossbreeds being raised for meat production and 28 serum samples from animals aged more than 30 months, all of them being dairy Friesians. The animals were raised on a farm where no cases of BSE had been reported and were kept under organic farming conditions, with winter feeds consisting of hay and grains but n o ' MBM supplements. Serum samples were obtained during annual herd testing for brucellosis. Antibodies to Acinetobacter caicoaceticus were significantly elevated in the 29 BSE-positive animals, when compared to the 18 BSE-negative animals (p<0.001 ), 30 organically raised cows aged less than 30 months (p<0.001) and to 28 organically raised cows aged more than 30 months (p<0.001) (Fig. 3), but no such elevations were found against two control bacteria E. coli and Agrobacterium [321. High levels of autoantibodies were also tbund against bovine nerofilaments which are components of the gray matter and against bovine myelin which are components of the white matter of the brain (Fig. 4). This is the first report of autoantibodies to brain components being present in animals affected by BSE.
388
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The highest relatively increased levels of antibodies, in comparison to controls were found in the IgA isotype. This suggested that they had been produced by the gut immune system, following transit of Acinetobacter antigens present in the MBM feeds across the gut mucosa and not as a result of brain damage by prions.
6.2. Second Coded Study In a second coded study, antibody responses were measured to 7 different bacteria Klebsiella, Proteus. Serratia, E. coli, Bacillus, Pseudomonas and Acineto&wter in 128 BSE positive animals,
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63 BSE negative animals and 64 healthy controls. Significantly elevated levels of antibodies to Acinetobacter were found in the BSE affected animals when compared to BSE-negative animals (p<0.001) and healthy controls (p<0.001). Antibodies to Pseudomonas were also elevated, although these titres were not as high as those found against Aci-
netobacter bacteria (Fig. 5). This is not surprising as Pseudomonas species belong to the same family as Acinetobacter bacteria. The important specificity observation is that BSE affected animals did not have antibody elevations against six other bacteria, five from this study: Klebsiella, Proteus, Serratia, E. coli, Bacillus and one Agrobacterium from a previous study [41]. It is clear from these studies that specific antibodies to Acinetobacter and maybe Pseudomonas bacteria, as well as autoantibodies to brain antigens are present in BSE affected animals. The conclusions that arise from these investigations is that BSE affected cattle have been exposed to Acinetobacter antigens, probably present in the MBM supplements fed to the cows. The use of MBM supplements was legally banned in 1988 and no residual MBM material has been available for further analysis. The demonstration of specific antibodies to a microbe bearing antigens crossreacting with brain tissues, as well as the presence of autoantibodies to brain components, in a disease showing clinical features resembling those found in EAE and MS, would tend to suggest that BSE could at least be considered as an autoimmune disease that has been evoked by Acinetobacter bacteria similar to the situation of anti-streptococcal antibodies causing a neurological autoimmune disease like Sydenham's chorea. Clearly the presence o f specific antibodies in an animal disease to a bacterium possessing antigens crossreacting with brain tissues could have relevance to human neurological diseases.
7. IMPLICATIONS OF
ANTI-ACINETOBACTER ANTIBODIES IN BSE TO HUMAN DISEASES The discovery that brain antigens have sequences which resemble molecules found in Acinetobacter and Pseudomonas bacteria has opened a new way of looking at neurological diseases in man and animals. The extensive results that cattle affected by BSE have antibodies to Acinetobacter bacteria, raises the interesting question whether similar observations can be made in human diseases. Many clinical studies attest to the observation that lower limb ataxia or paralysis is a feature of MS in man and hindquarters paralysis occurs in both
389
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EAE and BSE. A pilot study was carried out on sera obtained from 53 MS patients, 2 patients with sporadic-CJD, 10 patients with viral encephalitis, 20 patients with cerebro-vascular accidents and 25 healthy blood donors. Elevated levels of antibodies to Acinetobacter were found in the MS and sporadic-CJD patients but not in those with encephalitis or strokes, when compared to blood donors [ 16]. In a more detailed study antibody responses to 5 different strains of Acinetobacter, Pseudomonas, E. coli, myelin basic protein and neurofilaments were measured in 26 MS patients, 20 patients with cerebro-vascular accidents, 10 patients with viral encephalitis and 25 healthy controls. In MS patients elevated levels of antibodies against all 5 strains of Acinetobacter, as well as antibodies to Pseudomonas but no elevations against E. coli were observed when compared to blood donors (Fig. 6). Furthermore specific elevations of autoantibodies to myelin basic protein and neurofilament neuronal antigens were found in the MS patients but not in those with strokes or encephalitis [ 19]. It would appear therefore that in patients with MS and in cattle affected by BSE elevated levels of antibodies to Acinetobacter and autoantibodies to brain components can be demonstrated. These results taken together with the clinical similarities
390
has led to the working hypothesis that BSE and MS are related diseases, associated with exposure to bacteria possessing antigens crossreacting with brain tissues [ 11 ]. In humans, Acinetobacter species usually produce respiratory chest infections, especially in patients lying in intensive care units. Over 50% of MS patients in England suffer from sinusitis [18] and similar results have been published from Scotland [6]. Furthermore investigations of American patients with sinusitis by antral tap and endoscopically directed nasal cultures grow predominantly Acinetobacter and Pseudomonas bacteria [7]. If these results can be confirmed and MS patients are shown to suffer from sinusitis with Acinetobacter and Pseudomonas bacteria, then this opens up entirely new therapeutic possibilities. The use of anfi-Acinetobacter therapy such as antibiotics, drainage of sinuses, immunosuppressive drugs and other measures could be evaluated in the earliest stages of MS, before irreversible neurological changes had occurred in such patients. After all, rheumatic fever and Sydenham's chorea have disappeared in the Western World by the early use of antibiotics.
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Figure 6. Levels of IgA, IgM, and IgG antibodies (mean + standard error [error bars]) to Acinetobacter sp. strain 11171 (a), Acinetobacter sp. strain 19004 (b), A. Junii 17908 (c), A. lwoffii 5866 (d), A. radioresistens (e), P. aeruginosa (f), and E. coli (g) in sera from 26 MS patients, 20 CVA patients, and 25 healthy blood donors. Symbols: open squares = controls, dashed squares = CVA, closed squares = MS. (Adapted from Hughes et al, Clin Diag Lab Immuno12001 ;8:1181-88, with permission.)
8. THE VARIANT-CJD PROBLEM A new disease, variant-CJD (v-CJD) has been described and over one hundred patients have so far died from this condition [38]. It has been suggested
that consumption of meat from BSE affected animals may have caused the disease [5]. However nutritional studies carried out by the "National CJD Surveillance Unit" in Edinburgh and published in their yearly reports have failed to
391
show a higher meat consumption in v-CJD patients compared to controls [42]. Furthermore some vCJD patients had been vegetarians for a number of years. In a review of the v-CJD problem, Dr.G.Venters, an epidemiologist who had investigated the E. coli 0157 epidemic in Lanarkshire, has cast some doubt that there was a link between BSE and v-CJD [35]. The scientific issue relating to the v-CJD problem is the question: To what neurological group did the one hundred v-CJD patients who died from the disease, belong? Is v-CJD a separate, autonomous disease, "sui generis" so to speak or does it belong to a larger group? Since two patients with sporadicCJD had antibodies to Acinetobacter and since MS patients also have antibodies to Acinetobacter, the working hypothesis is that v-CJD patients may also have antibodies to Acinetobacter bacteria. If they do have such antibodies, then the logical conclusion follows that v-CJD, as well as sporadic-CJD and MS are somehow associated with exposure to Acinetobacter bacteria. Approximately 700 persons per year die in England and Wales from MS, which are about two persons per day (Department of Health Statistics, UK: 1995: 685; 1996: 712; 1997: 703; 1998: 801; 1999: 758; 2000: 696). Some 7% of MS patients die before the age of 40 years, which is approximately one person per week. Since the majority of v-CJD patients were below the age of 40 years, could they have belonged to the group of MS patients who died before the age of 40 years? Professor Scholz from Munich has pointed out that the distribution of v-CJD in the UK shows the highest incidence in Scotland [9], which does not tally with the distribution of BSE-affected cattle which occurred predominantly in the south of England [ 1]. However the distribution of v-CJD does fit quite well the distribution of MS in the UK. It is well known that there is more MS in Scotland than in England. The North-South distribution appears to hold for the Northem hemisphere and the reverse is found in the Southem hemisphere. MS is seven times more common in Tasmania and Southern New Zealand [21] than in tropical Queensland, in populations coming predominantly from AngloCeltic stock. Whether this latitudinal effect could be linked to greater prevalence of upper respiratory infections and chronic sinusitis during winter months by Acinetobacter/Pseudomonas bacteria is
392
a question that awaits further studies.
9. CONCLUSIONS The demonstration of a link between Acinetobacter bacteria and BSE has opened a novel approach to the study of MS as an autoimmune disease that is possibly evoked by an infection. Molecular mimicry between Acinetobacter and Pseudomonas bacteria and brain antigens suggests that infections by these microbes involving probably chronic sinusitis could have produced immune responses that led to the neurological and pathological changes of MS. A similar approach should be considered in the study of sporadic and variant-CJD.
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2001;111" 1333-7. Collado A, Gratacos J, Ebringer A, Rashid T, Marti A, Sanmarti R, Munoz-Gomez J. Serum IgA anti-Klebsiella antibodies in ankylosing spondylitis patients from Catalonia. Scand J Rheumatol 1994;23:119-23. Cousens SN, Linsell L, Smith PG, Chandrakumar M, Wilesmith JW, Knight RS, Zeidler M, Stewart G, Will RG. Geographical distribution of variant CJD in the UK (excluding Northern Ireland). Lancet 1999;353:18-21. Dickinson AG, Meikle VM. Host-genotype and agent effects in scrapie incubation: change in allelic interaction with different strains of agent. Mol Gen Genet 1971;112:73-9. Ebringer A. BSE could be an autoimmune disease of cattle caused by Acinetobacters which are microbes found in soil and muddy waters. Cattle Practice 2002;10:235-7. Ebringer A, Thorpe C, Pirt J, Wilson C, Cunningham P, Ettelaie C. Bovine spongiform encephalopathy: is it an autoimmune disease due to bacteria showing molecular mimicry with brain antigens? Environ Health Perspect 1997;105:1172-4. Ebringer A, Pirt J, Wilson C, Thorpe C, Tiwana H, Cunningham P, Ettelaie C. Bovine spongiform encephalopathy: Comparison between the "prion" hypothesis and the autoimmune theory'. J Nut Env Med 1998;8: 265-76. Ebringer A, Ptaszynska, Corbett M, Wilson C, Macafee Y, Avakiasn H, Baron P, James DC. Antibodies to Proteus in rheumatoid arthritis. Lancet 1985:ii:305-7. Ebringer A, Rashid T, Wilson C. Rheumatoid arthritis: proposal for the use of anti-microbial therapy in early cases. Scand J Rheumato12003;32:2-11. Ebringer A, Rashid T, Wilson C, Tiwana H, Boden R, Green A, Thompson E, Chamoun V, Croker J, Binder A. Could a microbially-triggered immune-mediated reaction provide a link between multiple sclerosis and Creutzfeldt-Jakob disease. JAMA (submitted). Eylar EH, Caccam J, Jackson JJ, Westall FC, Robinson AB. Experimental allergic encephalomyelitis: synthesis of disease-inducing site of the basic protein. Science 1970;168:1220-3. Gay D, Dick G, Upton G. Multiple sclerosis associated with sinusitis: case-controlled study in general practice. Lancet 1986;i:815-9. Hughes L, Bonell, Natt RS, Wilson C, Tiwana H, Ebringer A, Cunningham P, Chamoun V, Thompson EJ, Croker J, Voweles J. Clin Diag Lab Immunol 2001;8: 1181-88.
20. Husby G, Van de Rijn I, Zabriskie JB, Abdin ZH, Williams RC Jr. Antibodies reacting with cytoplasm of subthalamic and caudate nuclei neurons in chorea and acute rheumatic fever. J Exp Med 1976;144:1094-110.
21. Miller DH, Hammond SR, McLeod JG, Purdie G, Skegg DC. Multiple sclerosis in Australia and New Zealand: are the determinants genetic or environmental? J Neurol Neurosurg Psychiatry 1990;53:903-5. 22. Millson GC. Distribution of the scrapie agent in the tissue of experimentally inoculated goat. J Comp Path 1962;72:233-44. 23. Pattison IH. Fifty years with scrapie: a personal reminiscence. Vet Rec 1988;123:661-6. 24. Prineas J, Raine CS, Wisniewski H. An ultrastructural study of experimental demyelination. III. Chronic experimental allergic encephalomyelitis in the nervous system. Lab Investig 1969;21:472-83. 25. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science 1982;216:136-43. 26. Purdey M. Are organo phosphates involved in the causation of bovine spongiform encephalopathy (BSE)? Hypothesis based upon literature review and limited trials on BSE cattle. J Nutr Med 1994;4:43-82. 27. Raine CS, Synder DH, Valsamis MP, Stone SH. Chronic experimental allergic encephalomyelitis in inbred guinea pigs. An ultrastructural study. Lab Investig 1974;31:369-80. 28. Rose NR, Witebsky E. Studies on organ specificity. V. Changes in the thyroid glands of rabbits following active immunization with rabbit thyroid extracts. J Immunol 1956;76:417-27. 29. Schwimmbeck PL, Yu DTY, Oldstone MBA. Autoantibodies to HLA-B27 in the sera of HLA-B27 positive patients with ankylosing spondylitis and Reiter's syndrome. Molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease. J Exp Med 1987;166:173-81. 30. Sotelo J, Gibbs CJ Jr, Gajdusek DC. Autoantibodies against axonal neurofilaments in patients with kuru and Creutzfeldt-Jakob disease. Science 1980;210:190-3. 31. Tani Y, Tiwana H, Hukuda S, Nishioka J, Fielder M, Wilson C, Bansal S, Ebringer A. Antibodies to Klebsiella, Proteus and HLA-B27 peptides in Japanese patients with ankylosing spondylitis and rheumatoid arthritis. J Rheumatol 1997;24:109-14. 32. Tiwana H, Wilson C, Pirt J, Cartmell W, Ebringer A. Autoantibodies to brain components and antibodies to Acinetobacter calcoaceticus are present in bovine spongiform encephalopathy. Infect Immun 1999;67: 6591-5. 33. Trull A, Ebringer R, Panayi GS, Colthorpe J, James DC, Ebringer A. IgA antibodies to Klebsiella pneumoniae in ankylosing spondylitis. Scand J Rheumatol 1983;12:249-53. 34. Van de Rijn I, Zabriskie JB, McCarty M. Group A streptococcal antigens crossreactive with myocardium. Purification of heart-reactive antibody and isolation
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39. Wilson C, Ebringer A, Ahmadi K, Wrigglesworth J, Tiwana H, Fielder M, Binder A, Ettelaie C, Cunnigham P, Joannou C, Bansal S. Shared amino acid sequences between major histocompatibility complex class II glycoproteins, type XI collagen and Proteus mirabilis in rheumatoid arthritis. Ann Rheum Dis 1995;54:216-20. 40. Wilson C, Rashid T, Tiwana H, Beyan H, Hughes L, Bansal S, Ebringer A, Binder A. Cytotoxicity responses to peptide antigens in rheumatoid arthritis and ankylosing spondylitis. J Rheumato12003;30:972-8. 41. Wilson C, Hughes LE, Rashid T, Ebringer A, Bansal S. Antibodies to Acinetobacter bacteria and bovine brain peptide measured in bovine spongiform encephalopathy (BSE) in an attempt to develop an ante-mortem test. J Clin Lab Immunol (in press). 42. UK Creutzfeldt-Jakob Disease Surveillance Unit, Edinburgh. Eighth Annual Report 1999. www.cjd.ed.ac.uk.
9 2004 Elsevier B. V All rights reserved.
Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Autoimmunity in Patients with Antibiotic TreatmentResistant Lyme Arthritis Allen C. Steere
Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital Harvard Medical School Boston, MA, USA
1. INTRODUCTION In about 10% of patients with Lyme arthritis in the United States, joint inflammation persists for months or even several years after the apparent eradication of the spirochete, Borrelia burgdorferi, from the joint with antibiotic treatment. We propose a model of infection-induced autoimmunity to explain this treatment-resistant course. The majority of patients with treatment-resistant Lyme arthritis have HLADRB 1"0401, 0101, 0404 or related alleles. These alleles, which have a shared sequence in the third hypervariable region of the DRB 1 chain, are also associated with the severity of rheumatoid arthritis. Clinical correlations have linked treatment-resistant Lyme arthritis with cellular and humoral immune responses to outer-surface protein A (OspA) of the spirochete. The immunodominant epitope of OspA presented by the implicated MHC molecules is located at aa 165-173 (OspA165_173). In an epitope mapping study, 15 of 16 patients with treatmentresistant Lyme arthritis had T cell reactivity with this OspA epitope compared with 1 of 5 patients with treatment-responsive arthritis (P=0.004). Molecular mimicry between this dominant T cell epitope of OspA and a host protein may be an important factor in the persistence of joint inflammation in genetically susceptible patients with treatment-resistant Lyme arthritis. Lyme arthritis was recognized as a separate entity in 1976 because of geographic clustering of children in Lyme, Connecticut, who were thought to have juvenile rheumatoid arthritis [1]. Lyme disease or Lyme borreliosis is now know to be a complex
multi-system infection caused by the tick-borne spirochete, Borrelia burgdorferi sensu lato [2]. According to current knowledge, B. burgdorferi sensu lato (B. burgdorferi in the general sense) consists of three pathogenic species, B. burgdorferi sensu stricto (B. burgdorferi in the strict sense), B. afzelii and B. garinii [3, 4]. Only B. burgdorferi sensu stricto strains have been found in the United States. In contrast, most of the illness in Europe is due to B. afzelii and B. garinii, and only these two species have been found in Asia. Of the 3 species, B. burgdorferi appears to be the most arthritogenic.
2. CLINICAL PICTURE OF LYME ARTHRITIS IN THE UNITED STATES After an incubation period of 3 to 32 days, the illness usually begins in summer with a characteristic expanding skin lesion, erythema migrans, which occurs at the site of the tick bite [5, 6]. Within several days to weeks, the spirochete may spread to many other sites, including to other skin sites, the nervous system, heart, or joints. Months after the onset of illness, about 60% of untreated patients begin to experience intermittent attacks of joint swelling and pain, primarily in large joints, especially the knee (Fig. 1) [7]. In untreated patients, attacks of arthritis generally last from a few weeks to months separated by periods of complete remission. However, in about 10% of patients, chronic arthritis develops, defined as one year or more of continuous joint inflammation (Fig. 1). The synovial histology in these patients, which shows synovial hypertrophy,
395
EM & chronic arthritis (n=6)
EM alone (n-11 )
:,, ,' ',
EM & arthralgia
alone (n-lO) EM & intermittant episode of arthritis (n=28) Figure l. The frequency of types of joint involvement in 55 untreated patients with erythema migrans (EM) followed prospectively throughout the illness. The joint involvement ranged from brief episodes of joint pain, to intermittent attacks of arthritis, to chronic arthritis, affecting primarily knees. vascular proliferation, fibrin deposition, infiltration of mononuclear cells, and marked expression of adhesion molecules, is typical of that found in all of the various forms of chronic inflammatory arthritis, including rheumatoid arthritis [8-10]. Most patients with Lyme arthritis can be treated successfully with appropriate antibiotic therapy [ 11, 12]. However, about 10% of patients in the United States have persistent arthritis, typically affecting one or both knees, for months to even several years after two months or more of oral doxycycline or one month or more of intravenous ceftriaxone therapy [ 12]. Thus, persistent synovitis develops in a similar percentage of both untreated and antibiotic-treated patients with Lyme arthritis. Although B. burgdorferi DNA can usually be detected in joint fluid by polymerase chain reaction (PCR) prior to antibiotic therapy [ 13], in our experience, the PCR results are almost always negative in synovium and synovial fluid after antibiotic treatment of this duration [ 13, 14]. This suggests that Lyme arthritis may persist in certain individuals after the apparent eradication of the spirochete from the joint with antibiotic therapy. Thus, the question arises: why does arthritis persist in these patients after the causative agent has been eradicated?
396
3. HLA ASSOCIATIONS The first indication that antibiotic treatment-resistant Lyme arthritis might have an autoimmune pathogenesis was a study of human lymphocyte antigen (HLA) alleles among patients with Lyme arthritis of brief, moderate or chronic duration. An increased frequency of the HLA-DR4 specificity, determined by serologic typing methods, was found in the patients with chronic arthritis [15]. In addition, the HLA-DR4 specificity was associated with lack of response to antibiotic therapy. More recently, using contemporary molecular techniques, the majority of patients with chronic, treatmentresistant Lyme arthritis had HLA-DRBI*0401, 0101, 0404 or several other related alleles [16]. These alleles, which have a similar shared sequence in the third hypervariable region of the HLA-DRB 1 chain [17, 18], are associated with the severity of rheumatoid arthritis [ 19, 20]. In rheumatoid arthritis, the cause of the disease and the critical antigen peptides are not known. However, in Lyme arthritis, it is possible to ask the question: which spirochetal peptide presented by these MHC molecules may trigger a pathogenic T cell response in treatmentresistant Lyme arthritis?
nTreatment- responsive (N=16) I I T r e a t m e n t - resistant (N=14)
xJ 10 r .o
r
8
tin
i
r~
r" Q. -r"
~.
4
0
Antigen = P value =
P1
P2
P3 .004
P4
P5
P6
P7
P8
P9 .01
P10
Pll
P12
P13
P14
P15 .009
P16
P17
P18
P19
P20
P21 .006
P22
P23
P24 .0004
P25
Figure 2. T cell responses to overlapping synthetic 20-mers of OspA in peripheral blood from patients with treatment-responsive or treatment-resistant Lyme arthritis, determined by proliferation assay. 2 • 105 cells were stimulated with 2.5 ~g/ml of each OspA peptide. Bars show the median values (half of the samples); I-bars represent the third quartile values (three-quarters of the samples). Compared with patients with treatment-responsive arthritis, the median responses of PBL in those with treatment-resistant arthritis were greater with 5 OspA epitopes. Comparisons were analyzed by Kruskal-Wallis test, a non-parametric statistical method. (From Chen Jet al, Arthritis Rheum 1999;42:1813-1822, used with permission.)
4. I M M U N I T Y TO OSPA Patients with untreated Lyme disease have a complex, immune response to an increasing array of spirochetal proteins, some of which are differentially expressed, over a period of months to years. Common early responses are to outer-surface protein C (OspC), the P41 flagellar antigen, and a 45-kD fibronectin-binding protein, followed within weeks by reactivity with P39, P93, OspE, and to a lesser extent, OspF [21]. Months to years later, approximately 70% of patients with Lyme arthritis develop an IgG antibody response to OspA and OspB, usually after several brief attacks of arthritis, near the beginning of prolonged episodes of arthritis [21, 22]. The association of class II MHC molecules with treatment-resistant Lyme arthritis suggests that T cell reactivity is likely to be important in the persistence of synovial inflammation. As with antibody responses, T cell reactivity in Lyme disease is
directed against multiple spirochetal antigens [2325]. Antigen-specific peripheral blood lymphocytes (PBL) and synovial fluid lymphocytes (SFL) secrete primarily the pro-inflammatory T helper (Th) 1 cytokine, IFN- 7 [25, 26], and a Thl response is dominate in the local immune response in synovial tissue [27]. Thl responses to OspA, and to a lesser degree to OspB, correlate directly with the severity and duration of Lyme arthritis [25]. In an epitope mapping study (Fig. 2), 14 of 16 patients with treatment-resistant arthritis had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes) (P = 0.003) [25]. According to a computer algorithm [28], the immunodominant epitope of OspA presented by the DRB 1"0401 molecule was predicted to be amino acids (aa) 165-173 of the protein (OspA165_173) [29]. This prediction was confirmed using DRB 1"0401 transgenic mice that lacked their own murine class II
397
T a b l e 1. Sequences of the implicated epitope of OspA of B. burgdorferi (strain N40) compared with the equivalent epitope in representative strains of related Eurasian borrelial species and in a candidate autoantigen, LFA-1
Peptide
Sequence P1
P4
P6
P9 a
OspAI65_173
B. burgdorferi (N40) B. garinii (PKO) B. afzelii (Phei)
hLFA- 10~332_340
Y
V
F
L
E
G
T
pp
pp
pp
F
T
H
H
tP
tp
pp
I
"
"
T
L
T
A
H
A
K
V
A
N
"
S
K
Q
The peptide is constrained such that the amino acid side chains at position 1 (P1), P4, P6, and P9 interact with the MHC binding pocket. a
molecules. Moreover, in the epitope mapping study [25], the major difference between treatment-resistant and treatment-responsive patients was that 15 of the 16 patients with treatment-resistant arthritis had reactivity with this OspA epitope compared with 1 of 5 patients with treatment-responsive arthritis (P = 0.004). Thus, T cell recogition of the OspA165_173 epitope would appear to be a critical step in the pathogenesis of treatment-resistant Lyme arthritis.
5. OSPA SEQUENCES A M O N G T H E 3 PATHOGENIC B O R R E L I A L SPECIES When the sequences of this immunodominant epitope of OspA were compared among representative strains of the 3 pathogenic species of B. burgdorferi sensu lato, a number of residues were disparate. Compared with the B. burgdorferi peptide, the corresponding B. garinii peptide differed in three of the nine core residues, and the B. afzelii peptide differed in six of the nine core residues (Table 1). Thus, the Eurasian causative agents, with the possible exception of a few strains of B. garinii, lack the putative pathogenic T cell epitope of OspA, which would explain the rarity of treatment-resistant Lyme arthritis in Europe.
398
6. LFA-1 AS A CANDIDATE AUTOANTIGEN With the implication of the Ospml65_t73 epitope in treatment-resistant Lyme arthritis, we looked at whether this peptide sequence might serve as a molecular mimic of a host protein. A search of human gene sequences revealed a homologous sequence on the light chain of human leukocyte adhesion molecule- 1 (hLFA- 1(XL332_340) that was also predicted to bind the 0401 molecule (Table 1) [29]. This peptide is located extracellularly in the region of LFA- 1(~ called the "interactive" or "I-domain", which mediates the binding of LFA-1 to its ligand, intracellular adhesion molecule- 1 (ICAM- 1). Using an ElisaSpot assay, synovial fluid mononuclear cells from 10 of 11 patients produced IFN- 7 when stimulated with OspA, LFA-1, or both, whereas patients with other chronic inflammatory arthritides, including 5 patients with rheumatoid arthritis, did not [291. As the next step, antigen-specific CD4+ T cells from peripheral blood and, in some cases, synovial fluid of 6, DRB l*0401-positive patients with treatment-resistant Lyme arthritis were enumerated using an 0401 tetramer reagent that was covalently loaded with the OspA165_173immunodominant epitope [30]. Direct analysis of OspA165_t73 tetramer binding cells revealed frequencies of between <0.005 and 0.1% in peripheral blood, and between <0.005 and 3.1% in synovial fluid. When the OspAl65_173-reactive tetramer bound cells were cloned at one cell per well, 168 T cell clones were generated from synovial fluid [31 ]. When stimulated with the OspA peptide, 95% of the clones responded, as measured by 3H-thymidine incorporation, and they secreted large amounts of IFN- 7 and lower amounts of IL4 and IL-13. When these OspA-reactive clones were stimulated with the human LFA-1 peptide (hLFA-1 (zL332_340), approximately 50% of the clones secreted Th2 cytokines, particularly IL-13, and about 10% proliferated and secreted small amounts of IFN- 7 [31]. Compared with the OspA peptide, these clones required a 100,000-fold greater concentration of the LFA-1 peptide to reach half-maximal response. Thus, hLFA-10~L332_340may behave as a weak, partial agonist for OspA165_173-reactive T cells in DRB 1"0401-positive patients.
Table 2. Frequency of various HLA-DRB 1 alleles in patients with treatment-responsive or treatment-resistant Lyme arthritis of moderate, or prolonged duration correlated with the binding of these DRB 1 molecules to OspA and hLFA-1 peptides Percentage of Patients Positive Duration of Arthritis after Initiation of Treatment
HLA-DRB 1
TreatmentResponsive
Treatment-Resistant
< 3 mos.
4-11 mos.
12-48 mos.
N = 47
N = 32
N = 26
P Valuea
OspA163_175
HLFA-1~330-342
In vitro Binding of DRB 1 Molecule to
0401
6
6
23
.05
Strong
Strong
0101
15
9
35
.03
Strong
Weak
0404
6
31
8
.004
Moderate
Moderate
0801
13
0
0
.02
Weak
Weak
1101
17
3
8
.1
Weak
Weak
"3 x 2 tables were analyzed by Chi-square analysis. The percentages shown in boldface were increased in frequency compared with the other 2 percentages shown, for each allele.
7. OSPA AND LFA-1 P E P T I D E B I N D I N G T O IMPLICATED MHC MOLECULES We next used an in vitro binding assay to assess the binding of the OspA and hLFA-1 peptides to 5 MHC molecules that are increased or decreased in frequency in patients with treatment-resistant Lyme arthritis [ 16]. The frequencies of DRB 1 alleles among 105 patients with treatment-responsive arthritis of brief duration (<3 months) or treatment-resistant arthritis of moderate (4-11 months) or prolonged (12-48 months) duration are shown in Table 2. The DRB 1"0401 and *0101 molecules, which bound the OspA165_173peptide strongly, were significantly increased in frequency among patients with treatment-resistant arthritis of prolonged duration. The DRBI*0404 molecule, which bound the ()spA peptide moderately well, was increased in frequency in patients with treatment-resistant arthritis of moderate duration. The DRB 1"0801 and 1101 molecules, which bound this peptide weakly, if at all, were found more often in patients with treatment-responsive arthritis. Thus, the strength of OspA165_173binding correlated well with the frequencies of the various DRB1 alleles among patients with treatment-resistant Lyme arthritis, which further supports the hypothesis that recogni-
tion of this T cell epitope is of critical importance in this treatment-resistant course. As with the OspA peptide, the DRB 1"0401 molecule bound the hLFA-lff, L332_340peptide strongly and the 0404 bound it moderately well. However, in contrast with the OspA peptide, the DRB 1"0101 molecule bound the LFA-1 peptide weakly, if at all. Because the LFA-1 peptide does not bind the 0101 molecule and because this peptide serves as only a weak partial agonist even in 0401-positive individuals, it seems unlikely that hLFA-1 could be a pathogenic autoantigen in treatment-resistant Lyme arthritis.
8. MOUSE MODELS OF LYME ARTHRITIS Most inbred strains of mice can be infected with B. burgdorferi, and some strains, such as C3H/He and BALB/c mice, develop arthritis [32, 33]. The clinical picture in these mice is reminiscent of the acute, infectious phase of human Lyme arthritis. Studies in inbred strains have allowed an understanding of individual components of the immune response in susceptibility to arthritis and control of the spirochete. However, a mouse strain has not yet been identified that develops the equivalent of human
399
treatment-resistant Lyme arthritis.
ACKNOWLEDGEMENTS
9. C O N C L U S I O N S
Supported by NIH grants AR-20358, and by the Mathers Foundation, the Lyme/Arthritis Research Fund, and the Eshe Fund.
Among patients with Lyme arthritis who develop persistent synovial inflammation, infection and autoimmunity presumably occur together in the joint. The putative autoimmune phase of the disease becomes apparent only after antibiotic treatment and spirochetal killing. These patients have an increased frequency of DRB 1"0401 or related alleles, T cell reactivity with a particular epitope of OspA or B. burgdorferi, and lack of response to antibiotic therapy. To explain these findings, we have proposed that autoimmunity may develop within the pro-inflammatory milieu of the infected joint because of molecular mimicry between an immunodominant T cell epitope of OspA and a host protein. However, a pathogenic autoantigen remains to be identified. Several caveats should be stressed. First, some patients, even those with the appropriate genetic susceptibility, may not develop treatment-resistant arthritis because the spirochete may not up-regulate OspA expression in the joint or because not enough time elapses prior to treatment for the development of this pathogenic response. Second, one might anticipate that joint-specific autoimmunity would be caused by a joint-specific autoantigen, but the model of autoimmune arthritis in transgenic NOD mice shows that antibody to a systemic autoantigen (glucose-6-phosphatase isomerase) may lead to arthritis [34]. Third, although an autoantigen would be necessary for the induction of autoimmunity, other factors surely play a role in treatment-resistant Lyme arthritis. For example, genetically determined differences in cytokine responses, which may influence the breaking of tolerance, could also be an important factor. In conclusion, treatment-resistant Lyme arthritis seems to be an important human model of infectioninduced autoimmunity causing a chronic joint disease. Studies of this entity may serve as a model for helping to understand not only Lyme arthritis, but also other types of chronic inflammatory arthritis, including rheumatoid arthritis.
400
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Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross MR et al. Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three Connecticut communities. Arthritis Rheum 1977;20:7-17. 2. Steere AC. Lyme disease. N Engl J Med 2001;345: 115-125. 3. Baranton G, Postic D, Saint-Girons I, Boerlin P, Piffaretti JC, Assous Met al. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Inter J Syst Bacteriol 1992;42:378-383. 4. Canica MM, Nato F, Du Merle L, Mazie JC, Baranton G, Postic D. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis. Scand J Infect Dis 1993;25:441-448. 5. SteereAC, Bartenhagen NH, Craft JE, Hutchinson GJ, Newman JH, Rahn DW et al. The early clinical manifestations of Lyme disease. Ann Intern Med 1983;99: 76-82. 6. Smith RP, Schoen RT, Rahn DW, Sikand VK, Nowakowski J, Parenti DL et al. Clinical characteristics and treatment outcome of early lyme disease in patients with microbiologically confirmed erythema migrans. Ann Intern Med 2002; 136:421-428. 7. SteereAC, Schoen RT, Taylor E. The clinical evolution of Lyme arthritis. Ann Intern Med 1987;107:725-731. 8. Johnston YE, Duray PH, Steere AC, Kashgarian M, Buza J, Malawista SE et al. Lyme arthritis: spirochetes found in synovial microangiopathic lesions. Am J Path 1985;118:26-34. 9. SteereAC, Duray PH, Butcher EC. Spirochetal antigens and lymphoid cell surface markers in Lyrne synovitis: comparison with rheumatoid synovium and tonsillar lymphoid tissue. Arthritis Rheum 1988;31:487-495. 10. Akin E, Aversa J, Steere AC. Expression of adhesion molecules in synovia of patients with treatment-resistant lyme arthritis. Infect Immun 2001;69:1774-1780. 11. Steere AC, Green J, Schoen RT, Taylor E, Hutchinson GJ, Rahn DW et al. Successful parenteral penicillin therapy of established Lyme arthritis. N Engl J Med 1985;312:869-874.
12. Steere AC, Levin RE, Molloy PJ, Kalish RA, Abraham JHIII, Liu NY et al. Treatment of Lyme arthritis. Arthritis Rheum 1994;37:878-888. 13. Nocton JJ, Dressier F, Rutledge BJ, Rys PN, Persing DH, Steere AC. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid in Lyme arthritis. N Engl J Med 1994;330:229-234. 14. Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC. Lack of Borrelia burgdorferi DNA in synovial samples in patients with antibiotic treatmentresistant Lyme arthritis. Arthritis Rheum 1999;42: 2705-2709. 15. Steere AC, Dwyer E, Winchester R. Association of chronic Lyme arthritis with HLA-DR4 and HLA-DR2 alleles. N Engl J Med 1990;323:219-223. 16. Steere AC, Falk B, Drouin EE, Baxter-Lowe LA, Hammer J, Nepom GT. Binding of outer surface protein A and human lymphocyte function-associated antigen 1 peptides to HLA-DR molecules associated with antibiotic treatment-resistant Lyme arthritis. Arthritis Rheum 2003;48:534-540. 17. Gregersen PK, Silber J, Winchester RJ. The shared epitope hypothesis: an approach to understanding the molecular genetics of rheumatoid arthritis. Arthritis Rheum 1987;30:1205-1213. 18. Winchester R, Dwyer E, Rose S. The genetic basis of rheumatoid arthritis. Rheum Dis Clin N Am 1992;18: 761-783. 19. Weyand CM, Hicok KC, Conn DL, Goronzy JJ. The influence of HLA-DRB1 genes on disease severity in rheumatoid arthritis. Ann Intern Med 1992;117: 801-806. 20. Nepom BS, Nepom GT. Polyglot and polymorphism: an HLA update. Arthritis Rheum 1995;38:1715-1721. 21. Akin E, McHugh GL, Flavell RA, Fikrig E, Steere AC. The immunoglobin (IgG) antibody response to OspA and OspB correlates with severe and prolonged Lyme arthritis and the IgG response to P35 correlates with mild and brief arthritis. Infect Immun 1999;67: 173-181. 22. Kalish RA, Leong JM, Steere AC. Association of treatment resistant chronic Lyme arthritis with HLA-DR4 and antibody reactivity to OspA and OspB of Borrelia burgdorferi. Infect Immun 1993;61:2774-2779. 23. Yoshinari NH, Reinhardt BN, Steere AC. T cell responses to polypeptide fractions of Borrelia burgdorferi in patients with Lyme arthritis. Arthritis Rheum 1991;34:707-713.
24. Lengl-Janssen B, Strauss AF, Steere AC, Kamradt T. The T helper cell response in Lyme arthritis: differential recognition of Borrelia burgdorferi outer surface protein A (OspA) in patients with treatment-resistant or treatment-responsive Lyme arthritis. J Exp Med 1994; 180:2069-2078. 25. Chen J, Field JA, Glickstein L, Molloy PJ, Huber BT, Steere AC. Association of antibiotic treatment-resistant Lyme arthritis with T cell responses to dominant epitopes of outer-surface protein A (OspA) of Borrelia burgdorferi. Arthritis Rheum 1999;42:1813-1822. 26. Gross DM, Steere AC, Huber BT. T helper 1 response is dominant and localized to the synovial fluid in patients with Lyme arthritis. J Immunol 1998;160:1022-1028. 27. Harjacek M, Diaz-Cano S, Alman BA, Coburn J, Ruthazer R, Wolfe H et al. Prominent expression of mRNA for proinflammatory cytokines in synovium in patients with juvenile rheumatoid arthritis or chronic Lyme arthritis. J Rheumatol 2000;27:497-503. 28. Hammer J, Bono E, Gallazzi F, Belunis C, Nagy Z, Sinigaglia E Precise prediction of major histocompatibility complex class-II peptide interaction based on peptide side chain scanning. J Exp Med 1994;180:2353-2358. 29. Gross DM, Forsthuber T, Tary-Lehman M, Etling C, Ito K, Nagy ZA et al. Identification of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 1998;281:703-706. 30. Meyer AL, Trollmo C, Crawford F, Marrack P, Steere AC, Huber BT et al. Direct enumeration of Borreliareactive CD4+ T cells ex vivo by using MHC class II tetramers. Proc Natl Acad Sci USA 2000;97:1143311438. 31. Trollmo C, Meyer AL, Steere AC, Hailer DA, Huber BT. Molecular mimicry in Lyme arthritis demonstrated at the single cell level: LFA-ltx(L) is a partial agonist for outer surface protein A-reactive T cells. J Immunol 2001; 166:5286-5291. 32. Barthold SW, Beck DS, Hansen GM, Terwilliger GA, Moody KD. Lyme borreliosis in selected laboratory strains and ages of laboratory mice. J Infect Dis 1990;162:133-138. 33. Barthold SW, DeSouza MS, Janotka JL, Smith AL, Persing DH. Chronic Lyme borreliosis in the laboratory mouse. Am J Path 1993;143:959-971. 34. Matsumoto I, Staub A, Benoist C, Mathis D. Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 1999;286:1732-1735.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Sh(~nfeld and N.R. Rose, editors
Post-Infectious Arthritis: Reactive Arthritis or Slow Infectious Arthritis? Jean Sibilia x,2and Dominique Wachsmann 2
ZRheumatology department, CHU Hautepierre, HOpitaux Universitaire de Strasbourg, Universitd Louis Pasteur, Strasbourg, France; 2Unit~ INSERM U392, "ImmunitY-Infection", Universit~ Louis Pasteur, lllkirch-Graffenstaden, France
One of the major conceptual revolutions has been the discovery that a large number of inflammatory disorders are the consequence of an interaction between microbial and human immunogenetic factors. Schematically, the result of these sometimes complex interactions is the induction of an inflammatory response which may or may not be controlled by the physiological mechanisms of regulation. Two of the most spectacular examples are the peptic ulcer related to Helicobacter pylori and microbial effects in atheromatosis. This concept also applies with a great deal of pertinence to arthritis, where many forms which are useful to explain inflammatory phenomena are induced by microbial compounds (adjuvant arthritis, streptococcal wall arthritis). In man, the best examples are the various types of post-infectious arthritis, which include not only reactive and post-streptococcal arthritis, but also post-infectious forms occurring as the result of viral or parasitic infections. The mechanisms underlying these postinfectious disorders are most certainly of interest for our understanding of the common forms of inflammatory rheumatism such as rheumatoid arthritis. In this chapter, different mechanisms will be discussed which allow one to explain the occurrence of post-infectious arthritis of bacterial origin. More precisely, an attempt will be made to answer the following questions: 1. Is the articular cavity really sterile? 2. How can a bacterium find its way into the articular cavity and remain there without being eliminated by the immune system of the host?
3. How can a bacterium trigger arthritis? What are the bacterial virulence factors and the immunogenetic factors of the host which may predispose to arthritis? 4. What treatment may be envisaged for these postinfectious forms of arthritis? In order to reply to these questions, use will be made of the abundant literature relating to post-infectious arthritis, in particular reactive arthritis which will serve as a guideline for this chapter.
1. IS THE ARTICULAR CAVITY A STERILE MEDIUM? The articular cavity may be contaminated by direct injection or during bacteremia, but this is septic arthritis which is a very particular case. Conversely, in all other forms of post-infectious arthritis and in inflammatory rheumatism (rheumatoid arthritis, spondylarthropathies), the articular cavity is considered to be sterile despite the existence of arguments in favor of an arthritogenic role of certain bacteria. In fact, contrary to what is commonly believed, there are various elements to suggest that the joint is probably not a sterile medium, but rather an exchange zone serving as an interface with the environment. The synovial membrane is very rich in cells, including intrinsic immune cells (macrophages, mastocytes, granulocytes), resident endothelial cells and fibroblast-like synoviocytes capable of acting as intrinsic immune cells (syn-
403
thesis of IL-6, IL-8 and proteases). This membrane also contains dendritic cells able to take up, store, process and present different antigens. The maturation of these dendritic cells seems to be induced by various cytokines, like the TNF0~ produced by the cells of the intrinsic immune system. These phenomena permit the establishment in the synovium of an adaptative T and B lymphocyte immunity. In certain forms, this immune reaction leads to the appearance of veritable intrasynovial lymphoid follicles, reflecting the importance and intensity of the intrasynovial antigenic stimulation. All these phenomena are observed in different types of inflammatory rheumatism, notably in rheumatoid polyarthritis, but also in reactive arthritis [1--4]. It remains to be clarified whether under physiological conditions in healthy subjects there also exists an intrasynovial passage of microbial compounds and what is the immune response of the synovium. Many studies have been devoted to the search for intra-articular microbes in presumably sterile forms of arthritis. The results and conclusions of such work are dependent on a number of factors: - the nature of the microbe, which is sometimes difficult to grow in culture and/or present in small quantity, - the method of detection, which may be indirect (search for antibodies), or direct but of low sensitivity (immunohistochemistry, immunomicroscopy), or in recent years much more sensitive (gene amplification of the microbial nucleic acid), - the affection in which the study was carried out.
1.1. Microbiological Exploration of the Synovium in Reactive Arthritis (ReA) A study of the microbiological history of ReA is instructive as it illustrates well the influence of technological progress and in particular the impact of molecular biology on the evolution of such concepts
[5, 6]. 9 As early as the 1970s, the first studies revealed microscopic intracellular inclusions in synovial tissues which could correspond to Chlamydia trachomatis, the principal arthritogenic microbe involved in ReA [7]. Confirmation of this work was however hindered for many years by the fact that the majority of these bacteria are very diffi-
404
cult or almost impossible to cultivate from synovial samples [8-13]. Subsequent studies likewise demonstrated the existence of antigens (Yersinia, Salmonella, Shigella...) [ 14, 15]. 9 Since the beginning of the 1990s, the explosive development of new molecular biology techniques has led to the detection (by polymerase chain reaction methods) of small quantities of C. trachomatis DNA in the articular cavity [16-22]. * The phenomenon has grown since 1995 with the discovery of DNA of most other classical arthritogenic agents in synovial samples from ReA patients [23-26]. One must nevertheless take a fairly critical standpoint, since if the results are convincing for C. trachomatis, they are much less so for enterobacteria. It is true that DNA of Yersinia, Shigella or Campylobacter has been identified in some studies, but these are few and include very few patients [23, 24, 26-34]. Thus, Ekman et al identified DNA of Salmonella in synovial samples but were not able to repeat their results [35]. This could have been due to technical artefacts which led to false positives or to a very small amount of bacterial DNA in the synovium. Although DNA of Ureaplasma urealyticum and other Mycoplasma (M. fermentans, M. genitallum) is detectable in synovial fluid, it has been reported in only a small number of cases of ReA [36]. Despite these reservations, the list of arthritogenic agents continues to grow from year to year, even if for many of them there is no confirmation of their intrasynovial persistence (Table 1) [37-48]. 9 These results immediately raised a large number of questions. Did they indicate the presence of viable bacteria in the joint, or were these simply genomic vestiges of bacteria passively transported into the joint by monocytes? The reply was provided by the discovery of messenger and ribosomal RNA of C. trachomatis using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique [49]. The presence of these nucleic acids, which have a very short half-life in tissues (some minutes), implies the occurrence of transcription and hence active multiplication of the bacteria. In an other work, COX et al studied the synovial fluid of 12 patients suffering from different forms of post-infectious arthritis
Table 1. List of the "classical" and "new" arthritogenic agents implicated in reactive arthritis The "classical" candidates
The "new" candidates
9 Chlamydia trachomatis
9 Streptococcus sp
9 Ureaplasma urealyticum
9 Chlamydia pneumoniae
9 Yersinia enterocolitica and pseudotuberculosis
9 Mycoplasma hominis and fermentans
9 Shigellaflexneri, dysenteriaeandsonnei
9 Neisseria gonorrhoeae and meningitidis (serogroup B)
9 Salmonella typhimurium and enteritidis
9 Borrelia burgdorferi
9 Campylobacterjejuni
9 Clostridium difficile 9 fl-haemolytic streptococci 9 Propionibacterium acnes 9 Escherichia coli 9 Helicobacterpylori 9 Brucella abortus 9 Pseudomonas aeruginosa, fluorescens, migulae, putida
9 Calmette-Guerin Bacillus 9 Leptospira 9 Bartonella 9 Tropheryma whipplei 9 Lactobacillus 9 Bacillus cereus 9 Hafnia alvei 9 Gardnerella vaginalis 9 Giardia lamblia
The majority of "classical" candidats are dependent on HLA B27.
including 10 cases of ReA using various RT-PCR methods, one of which employs universal primers capable of amplifying bacterial 16s-RNA sequences. RNA of classical arthritogenic bacteria (Chlamydia, Yersinia, Campylobacter) was detected, but only by specific RT-PCR techniques and not by universal RT-PCR. On the other hand, in the majority of samples universal RT-PCR revealed the presence of RNA originating from various digestive and cutaneous commensal bacteria, despite drastic precautions to avoid contamination. These findings, since confirmed by others, suggest that microbes can survive in small numbers in the articular cavity in certain forms of ReA (Table 2).
1.2. Microbiological Exploration of the Synovium in Other Types of Inflammatory Rheumatism One of the most disturbing reports has been the detection of bacterial DNA and sometimes RNA in synovial samples from other forms of inflammatory rheumatism, in particular RA and undifferentiated oligoarthritis [16, 50-53]. C h l a m y d i a p n e u m o n i a e and B o r r e l i a b u r g d o r f e r i , for instance, have been associated with cases of monoarthritis or oligoarthritis resembling ReA [54-57]. In this context, it is interesting to return to the case described by Reiter in 1916 and his publication entitled "Ober eine bisher unerkannte Spiroch~iteninfektion ( S p i r o c h a e t o s i s a r t h r i t i c a ) " [58]. One might wonder, in view of the high prevalence of B. b u r g d o r f e r i infections in Central Europe, whether this spirochete rheumatism was not one of the first reports of Lyme arthritis?
405
Table 2. The principal detectable microbes in reactive arthritis and undifferentiated arthritis: analysis of the different methods of identification Antigens
DNA
RNAc
Culture
C. trachomatis
+
+
+
+/-
Y. enterocolitica
+
+a
ND
-
Y. pseudotuberculosis
+
+a
+b
_
S. flexneri and sonnei
+
+a
ND
-
S. typhimurium and enteritidis
+
+~
ND
-
C. jejuni
_
+a
ND
-
U. urealyticum
+
+
-
+
C. pneumoniae
+
+
+
-
B. burgdorferi
+
+
ND
+
T. whipplei
+
+
ND
+
ND: not done. aThe detection of nucleic acid of enterobacteria in the synovium is subject to caution for reasons related to the different techniques employed and the very small numbers of patients studied. bRNA has only been detected in one case. tin two recent studies [49, 50] it was possible to identify, in undifferentiated arthritis, DNA and rRNA of various commensal bacteria of which the pathogenic role is uncertain.
Different
studies
concerned
RA
patients
and
u n c l a s s i f i e d arthritis, C. t r a c h o m a t i s and p n e u m o n i a e are present in the synovial fluid of RA patients
in respectively 30 and 15% of cases and comparable observations have been made for M y c o p l a s m a p n e u m o n i a e and f e r m e n t a n s [36, 51, 53]. Gerard et al have studied samples of synovial fluid and tissue from 237 patients including RA, R e A undifferentiated oligoarthritis a n d osteoarthritis (OA) [59]. A universal PCR method with pan-bacterial primers was used to analyse synovial samples containing no DNA of Chlamydia, Mycoplasma or Borrelia detectable by specific PCR. DNA of other bacteria was detected in 23 of the 237 patients (10%), in 22 cases in the synovial tissue, but in all the pathologies studied without distinction: RA (4/39, 10.3%), OA (3/61, 4.9%), ReA (3/36, 8.3%) and unclassified oligoarthritis (1/85, 1.2%). In 8 of these 23 patients, DNA of several bacteria was detected simultaneously: Klebsiella, Pseudomonas, Salmonella, Shigella, Neisseria and Acinetobacter. Kempsell et al, employing RT-PCR and sequencing, identified respectively 92 and 50 different bacterial species in the synovial fluid of RA and osteoarthritis patients [50]. Only 6 species ( C o r y n e b a c t e r i u m , E s c h e r i c h i a coli, S t r e p t o c o c c u s ,
406
Pseu-
d o m o n a s , L e p t o s p i r a and M e t h y l o b a c t e r i u m )
were found exclusively in RA, although without proof of this being an argument in favour of their pathogenic role. The simultaneous presence of DNA and/or RNA of numerous bacterial species in synovial samples from patients with RA a n d unexplained arthritis was also observed in other studies [60]. Thus, the "genetic" technique of universal bacterial gene amplification has permitted the detection of a certain number of unexpected intrasynovial microbes. The pathogenic significance of these findings nevertheless remains to be confirmed, since this type of method carries the risk of amplifying a commensal bacterium or contaminant gene of no particular importance [61 ]. For example, one other report suggests that the presence of intrasynovial bacterial DNA might in fact be an exceptional phenomenon. In this work, a search by PCR analysis for the bacterial 16S and 23S ribosomal RNA genes proved negative in the synovium of 81 patients (42 cases of RA, 31 of OA, 8 other cases of inflammatory rheumatism) when using drastic conditions of sterility [62].
1.3. Original Microbiological Observations in Other Forms of Inflammatory Arthropathy Propionibacterium acnes, a microbe implicated in inflammatory outbreaks of acne, was recently identified in articular samples from SAPHO patients, suggesting an infectious origin of this syndrome which is frequently regarded as a form of spondylarthropathy [63, 64]. Mycobacterium bovis, used in BCG (Bacille Calmette-Gurrin) therapy, is known to cause presumably aseptic oligo and polyarthritis [65-67], but bacterial DNA has also been detected in synovial fluid from cases of arthropathy triggered by intravesical injection of BCG. In other situations, the discovery of inflammatory articular manifestations and sometimes of unexplained recurrent arthritis following documented infection, would suggest the possibility of reactive arthritis "in the wide sense of the term" [40, 41, 68-73]. However, in the majority of these cases, the proof is not tangible although sufficient that one may keep the hypothesis in mind.
1.4. Microbiological Exploration of the Synovium in Osteoarthritis (OA) and Healthy Subjects One of the most astonishing discoveries was the identification by PCR of DNA of C. trachomatis in synovial samples from healthy volunteers and OA patients (in respectively 9% and 20% of cases) [74]. In this study, only PCR amplifications using primers hybridising with 16S RNA or plasmid genes were positive, while attempts to amplify the MOMP gene (Major Outer Membrane Protein) were always negative. These results have been confirmed in other studies in OA patients [50, 60, 75, 76], but not by all authors especially for healthy subjects [60, 76]. This would suggest, assuming the results to be pertinent, that the bacteria persist in a "particular" form. It should nevertheless be noted that the variability of the PCR results can equally be explained by differences in sensitivity related to the amplification target.
1.5. What Can We Conclude? It is possible to detect bacterial nucleic acid (DNA or RNA) not only in certain forms of ReA, but also
in other types of inflammatory rheumatism and even in OA or presumably healthy subjects [77, 78]. Since these results are fairly reproducible, they are probably not due to artefacts linked to technical errors. Altogether, two observations may be made: 9 DNA (or RNA) of classical arthritogenic bacteria like Chlamydia or Mycoplasma is detectable in certain forms of arthritis, most often post-infectious. This is probably a significant arthritogenic event, but not specific to a particular affection. 9 DNA (or RNA) of numerous commensal bacteria, most frequently digestive or cutaneous (sometimes associated), can be found in the synovium in cases of arthritis (or more rarely in OA). The passage of these bacteria into the synovium may be considered to be a relatively common occurrence and not necessarily of pathological significance. Nevertheless, a sometimes polymicrobial colonisation of the synovium, even transitory, can trigger or aggravate synovitis, or inversely inhibitory phenomena [78, 80]. In practice, the identification of a microbe in the synovial fluid or tissue by sensitive molecular methods is not sufficient to conclude that the microbe is responsible for arthritis. These models of arthritis linked to host-environment interactions do not in fact satisfy the postulates of KOCH [81]. Hence KEAT and TAYLORROBINSON have proposed a certain number of causal criteria which take into account the particularities of the microbes in question and their new methods of detection by PCR [82, 83]. Moreover, an analysis of the currently available microbiological data relating to ReA allows one to distinguish two different forms. 9 Reactive arthritis of the type chronic infectious arthritis Certain forms of ReA could represent authentic chronic infectious arthritis caused by slow growing organisms which are very difficult to culture and hence impossible to identify by the usual microbiological methods. In the light of current knowledge, this hypothesis would appear to hold for Chlamydia, Mycoplasma and Borrelia although not for enterobacteria. These microbes have an attenuated virulence,
407
unlike those responsible for septic arthritis. Such forms of ReA are thus related to a "slow" intrasynovial infection, a condition also called "slow infectious arthritis" or "infection reactive arthritis" [84, 85] Similarly, it is by invoking the same mechanisms that one explains today the arthritis of Whipple's disease, whereas for many years it was not possible to identify or culture this slow growing organism [42, 43].
9 Reactive arthritis of the type infection triggered aseptic arthritis Some forms of ReA are probably aseptic and if so it is the persistence of bacterial antigens (heat shock proteins, lipopolysaccharides and other PAMPs) which could explain the appearance of an inflammatory reaction in the synovium. This hypothesis applies above all to enterobacteria (Yersinia, Salmonella...), microbes not found in the joint except possibly in authentic (but rare) incidents of acute septic arthritis [14, 15, 28, 86, 87]. In chronic forms, it is unlikely that viable and active bacteria persist in the synovium, although it has occasionally been possible to detect bacterial DNA [23, 24, 26, 27, 35] and even recently intrasynovial RNA, in a case of ReA caused by Yersinia pseudotuberculosis [27] and in some other rare cases [60]. This type of arthritis, triggered by bacterial antigens originating from an extra-articular site in the absence of any viable intra-articular microbe, may be called "infection triggered reactive arthritis".
2. HOW CAN A BACTERIUM FIND ITS WAY INTO AND PERSIST IN THE ARTICULAR CAVITY? The most original microbiological observations described in the foregoing section raise a number of questions, the answers to which help to understand the pathogenesis of reactive arthritis.
2.1. How Do These Bacteria Spread into the Joint? The majority of arthritogenic bacteria are obligatory or facultative intracellular microbes originating from mucous sites. In most cases, these bacteria are transported into the synovium by monocytes and
408
dendritic cells which they have infected [88-90]. 9 Certain microbes like Chlamydia can survive in small numbers in a "vegetative" state, probably with intermittent periods of replication triggered by still unknown phenomena [10, 91-93]. This has been clearly demonstrated for C. trachomatis, which persists in the form of "atypical" reticulated bodies [94, 95]. The intracellular persistence of this bacterium is made possible by its capacity to inhibit fusion between Chlamydia vacuoles and lysosomes [96]. It has also been demonstrated recently that the metabolic activity of persistent forms of Chlamydia trachomatis is slower that of replicative forms [97]. Other bacteria originating from the aerodigestive pathways or the skin (Streptococci ...) probably employ identical but less well known mechanisms. 9 Conversely, it is likely that enterobacteria survive at an extra-articular site, in particular in the mucosal membranes of the digestive system and/ or the lymphatic ganglions, and are carded to the joint by monocytes, probably in recurrent fashion [98-100]. In support of this theory, there is some evidence that a preferential connection exists between the digestive system and the joints. It has been observed that mucosal leukocytes collected from patients with inflammatory bowel disease bind well to synovial vessels [101]. This homing involves many receptors and their ligands, which differ according to the leukocyte subset, while mononuclear cells from peripheral blood do not share the binding characteristics of cells of digestive origin. Although these results concerned only inflammatory bowel disease, the concept can probably be extended to enteric ReA [79, 102-104]. After homing, the bacterial antigens can subsequently persist sometimes for a long time in the synovium, in certain cases in the form of bacterial "ghosts" without nucleic acid [28, 86].
2.2. How Do These Bacteria Persist in the Articular Cavity and Escape from the Immune System of the Host? (Fig. 1)
2.2.1. Role of antigenic modulation Several recent studies have shown that C. trachomatis can survive in a particular form whereby
1. Mechanisms of Chlamydia trachomatis (CT) persistence. Antigenic and metabolic modifications of CT (persistence in a vegetative state ~ atypical reticulated bodies). m - CT can inhibit monocyte apoptosis: inhibition of mitochondrial cytochrome release. 9 CT can provoke TNFtx release: induction of T cell (antichlamydial response) apoptosis. 9 CT can induce IL-10 production: IL-10 inhibits the antibacterial activity of macrophages. - C~ down-regulates IFNT induced MHC class I and class II expression. - CT can modulate splicing of HLA B27 and induce the liberation of soluble HLA B27 form which inhibits the antibacterial T-cell response. ,
-
it down-regulates the expression of membrane antigens (MOMP), while continuing to synthesise immunomodulatory proteins like heat shock proteins and LPS [91]. It has further been shown that these modifications can be induced in vitro by prolonged antibiotic treatment (Ciprofloxacine), which could have important practical implications for future therapeutic strategies. B. burgdorferi can likewise modulate its expression of surface antigens. On entering the host, this bacterium down-regulates expression of the principal membrane protein OspA (Outer surface protein A) and expresses larger quantifies of another membrane protein OspC [105]. Such antigenic modifications could permit these bacteria to escape from the immune system of the host.
2.2.2. Role of the intraceUular localisation of the bacterium As pointed out by R. Z'mckernagel, there exist numerous circumstances in which micro-organisms (especially viruses) escape from the immune system by persisting in non lymphoid cells (papillomavirus in keratinocytes, Epstein-Barr virus in epithelial cells) [106]. Similarly, certain arthritogenic bacteria can infect and persist in long-living syno-docytes and monocytes [10, 107] (and other cells such as endothelial cells), sometimes in spite of antibiotics, as has been demonstrated for C. trachomatis and B. burgdorferi [90, 92, 93, 95, 108-113]. The situation is less clear in the case of enterobacteria and differs according to the microbe. Yersinia and Salmonella can persistently infect the mucosa of the intestine and the digestive ganglions but not the synovium. These microbes are also present in monocytes, which serve perhaps as a reservoir
409
[98-100]. Shigella in contrast only infects digestive epithelial cells and cannot survive in monocytes and its mechanisms of persistence and transport are less well known. The bacteria can moreover facilitate the survival of the infected cells which should be eliminated by the host. In particular, it has been shown that C. trachomatis can block the apoptosis of such cells by inhibiting the release of mitochondrial cytochrome C and by directly engaging the death domain of the TNF receptor family [ 114]. Intracellular persistence is linked to different mechanisms, some of which are related to HLA B27 (see sections 3.2.2 and 3.2.3). A recent study investigated the factors likely to influence the synoviocyte clearance of arthritogenic bacteria (S. typhimurium and Y. enterocolitica) in SE Although a direct effect of HLA B27 was found to be unlikely, alteration of the cytokine response profiles could play a contributory role. Thus, an increase in intracellular bacteria was correlated with a rise in IFNy, which suppressed cellular NO production in HLA B27 positive but not in HLA B27 negative synoviocytes [ 115].
the homology between this bacterial constituent and an antigen of the articular cavity can induce "autoimmune" synovitis, as will be discussed later. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease- induced self-reactivity. The immunodominant epitope GROEL from S. typhii murium presented by the mouse H2-T23-encoded class Ib molecule was recognized by CD8+ CTL after natural infection. These CTL recognize the GROEL epitope cross-reacted with a peptide derived from mouse hsp 60 and recognized stressed macrophages [ 116]. Molecular mimicry could likewise have implications in various other situations. A recent discovery has been the identification of a 7 amino acid peptide (DH5YQEE) specific to the myelin oligodendrocyte glycoprotein and homologous to a C. trachomatis protein of unknown function. This unexpected homology could be involved in the pathogenesis of multiple sclerosis or in other diesase associated with arthritogenic bacteria such as Chlamydia [ 118, 119].
92.2.3. Role ofmolecularmimicry
2.2.4. Role of bacterial interactions with the immune response of the host
Models proposed to account for the link between infection and autoimmunity induced inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Certain bacteria have constituents which display strong homology with proteins of the host (YopH of Y. pseudotuberculosis and CD45, M. fermentans antigens and CD4). This molecular mimicry can give rise to a tolerance with respect to some microbes, which thereby escape from the immune system of the host [116]. One recently described example is the case of Lyme borreliosis. It has been shown that a dominant epitope of OspA of B. burgdorferi (usually presented by HLA DRB 1"04 01) has close sequence homology with LFA-1 (Leukocyte Function-associated Antigen-l), which is a beta2integrin expressed at the surface of lymphocytes, polynuclear granulocytes and monocytes [ 117]. As a result, OspA can bind to ICAM-1 (Intracellular Adhesin Molecule-I), a ligand of LFA-1 expressed by synoviocytes, enabling the bacterium to persist in the synovium. On the other hand, the mimicry may also be differently interpreted by the host, since
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Cytokines like the IFNy and TNF~ produced by T cells play a major part in the elimination of intracellular bacteria [120]. It has been found that in ReA the anti-bacterial Thl cytokine response (IFNy, TNFa, IL-2 and IL-12) is impaired in favour of a Th2 response (IL-4 and IL-10) [121-127]. Hence in the absence of a "good" Thl anti-bacterial reaction, the microbes are able to survive. 9 The role of the dendritic cells The role of dendritic cells (DC), the most potent antigen-presenting cell (APC) in the course of arthritogenic infections could be important. Thus Yersinia enterocolitca is able to invade DC, does not induce necrosis or apoptosis, but affects maturation. The diminished immunostimulating capacity of DC following infection with Y. enterocolitica in vitro might impair or delay elimination of bacteria thereby contributing to pathogenesis of extra intestinal manifestation [90, 128, 129].
9 The genetic polymorphism ofcytokines Although little is known about the pathogenesis of this Thl/Th2 imbalance, it is probable that genetic factors of the host are causally involved, including in particular the polymorphism of cytokdne genes. Thus, in Finnish patients, the microsatellites ILIO.GIO and IL10.G12 from the promoter region of the IL-10 gene seem to protect against the development of ReA. IL-10 on the other hand inhibits the antibacterial activity of macrophages [ 130]. In a German study, it was shown that the level of TNFt~ secretion from peripheral blood T cells at the onset of ReA was inversely proportional to the duration and severity of the disease [121]. TNFt~ does in fact appear to participate in anti-infectious defence. TNF R p55 -/- knockout mice are more susceptible to infection by Yersinia and develop more severe arthritis [ 131 ]. In humans, ReA seems to be associated with low-producing TNFt~ alleles and an HLA B27-independent increase in the low-producing TNF C1 allele has been observed in Finnish ReA patients [123]. The situation is however complex, as it has also been shown that the infection of monocytes by C. trachomatis triggers release of TNFct, which will induce the apoptosis of autologous T lymphocytes normally responsible for eliminating microbes [132]. The polymorphism of other genes certainly also enters into the capacity of the host to eliminate bacteria and notably IL-1 Ra et ILR-4 and even cytochrome P450 are of importance [ 133]. 9 Effects of the bacteria on cytokine production Some bacteria are capable of influencing the immune response of the host by inducing the synthesis of cytokines by cells of the peripheral or synovial immune system [90, 112]. Campylobacter jejuni, for example, on infecting a lineage of human intestinal epithelial cells (IMT 407), triggers the synthesis of both pro-inflammatory Thl (IFN% TNFct) and anti-inflammatory cytokines (IL-10, IL4). This synthesis is only observed with viable bacteria while sonicated microbial debris induces no pro-inflammatory response [134]. Another original example is the immunosuppression induced by B. burgdorferi, as this bacterium has numerous ways of escaping from the immune system, not only by interacting with the complement pathway [135] or blocking the synthesis of TNFct and IFN~/[ 136], but also by triggering the production of IL-10 which is
able to inhibit the pro-inflammatory response of the host [137, 138]. B. burgdorferi can further induce tolerance by desensitising human blood monocytes through the Toll-like receptor 2 (TLR2). There is moreover considerable evidence to support the importance of the intrinsic immunity dependent on TLR2 and ILR4 in the bacterial induction of tolerance [133, 139, 140]. 9 Role of bacterial interactions with the genetic characteristics of the host However, ReA cannot be attributed merely to cytokine production or polymorphism and other susceptibility factors are undoubtedly involved. One of these is the fact that arthritogenic bacteria like Chlamydia, Yersinia or Salmonella are capable of modulating HLA B27 (modification of the messenger RNA splicing) and can induce modification in the B27-bound peptide repertor, which may includes peptides of host origin potentially through modulation of proteasome LMP2 subunit expression [141,142]. C. trachomatis also downregulates IFNq,-induced major histocompatibility complex (MHC) class I and II expression. Secretion of proteosome-like activity factor (CPAF) into the host cell cytosol causes degradation of the host transcription factor RFX5 which is crucial for MHC class I and II expression [143]. Persistence of C. trachomatis in the synoviocytes can induced up regulation of HLA class I heavy chain. This up regulation is caused by the induction of IFN-~, which in turn stimulates the synthesis of interferon-stimulated gene factor 3 gamma (IS GF3 gamma), a transcription factor participating in the regulation of HLA genes [127]. These different interferences facilitate the persistence of the bacteria within cells or tissues. An original approach to attempt to understand the influence of a bacterial infection is to study the genomic modifications induced by the bacterium in the cells of the host. Use of microchips allows one to examine the expression of many different genes in very small quantifies of tissue. Several recent studies have been devoted to this approach. 9 Monocytes from healthy subjects were infected with C. trachomatis. After 1 to 7 days of infection, it was possible to observe a significant increase in the expression of 18 genes, notably those of TNFt~, IL-8 and MIPI-[3 [ 144].
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9 A detailed analysis of the genetic expression of cultured Hela cells infected with C. trachomatis was carried out using affymetryx microchips. Hyperexpression of 14 genes was observed, including IL-11, LIF, MIP2-~, different transcription factors (EGR1, ETR101, FRA1, C-jun), anti-apoptotic genes (IEX-1L, MCL1), adhesion molecules (ICAM-1) and other proteins having diverse functions. The results were confirmed by a semi-quantitative RT-PCR study and enzymatic assays [ 145]. This work revealed a certain number of elements: - the transcription responses induced by bacteria of the Chlamydia family (pneumoniae and trachomatis) are fairly comparable, but differ from those induced by S. typhimurium; - t h e r e is no significant hyperexpression of pro-inflammatory genes like IL-6 or TNF~, but rather of genes implicated in cellular differentiation and apoptosis and of transcription factors like EGR1 (Early Growth Response protein 1) capable of promoting the synthesis of metalloproteinases. This elegant and modem approach demonstrates that a cellular infection can modify the expression of genes of the host cell, with differences according to the cell type and the duration of infection. Results indicate that close to 1.5% of the genes analysed are over-expressed and these genes are in most cases regulatory or involved in important cellular functions. This technique has the main disadvantage of not allowing one to assess post-transcriptional or post-translational phenomena. Nevertheless, it is destined to transform our understanding of postinfectious arthritis by permitting analysis of synovial samples at different time points in the evolution of the disease. Such results will perhaps enable us to explain the passage to chronic or recurrent arthritis.
3. HOW CAN BACTERIA OR THEIR ANTIGENIC DEBRIS TRIGGER ARTHRITIS? (FIG. 2)
Although the above description of the two forms of reactive arthritis gives a fairly simple picture, it is possible that the future will reveal a more "heterogeneous" reality. In any case, the appearance of
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arthritis is a consequence of the encounter between an arthritogenic bacterium and a predisposed host. Hence the key to the mystery of ReA will no doubt lie in a detailed study of these host-bacterium interactions, of which we already know some of the subtleties. 3.1. Role of Bacterial Virulence Factors
All bacteria do not have the same arthritogenic potential. As an example, certain strains of Shigella flexneri contain a plasmid with a gene coding for a peptide sequence homologous to the o~1 domain of the HLA B27 molecule, which could confer particular arthritogenic properties [ 146]. The arthritogenic strains of Yersinia also possess plasmid and chromosome virulence factors capable of modulating cellular adhesion and invasion, in particular a plasmid coding for the protein Yad A which is an adhesin and could use HLA B27 as a ligand [147, 148]. Other examples may exist like for instance Lyme arthritis in Europe, which would seem to be preferentially related to B. burgdorferi sensu stricto [ 149], although the virulence factors of this species are not yet known. 3.2. Role of the Immunogenetic Characteristics of the Host
There are various cases illustrating the involvement of the immunogenetic factors of the host, but the best example is undoubtedly HLA B27. In the following sections, we will attempt to describe the role of this HLA molecule in the host-bacteria interactions regulating the pathogenesis of SP, especially in ReA. HLA B27 plays an important part in SP, although not necessarily as an antigen presenting molecule since evidence for the participation of this mechanism remains inconclusive. The existence of a significant interaction between bacteria and HLA B27 is supported by the finding that 30 to 60% of HLA B27+ ReA patients progress to ankylosing spondylitis (AS) after 10 to 20 years. The role of HLA-bound peptides has also been studied in transgenic HLA B27+ rats. These animals develop arthritis only in a non sterile environment, which again points to the importance of microbial peptides presented by HLA B27. On the other hand, some
Figure 2. Mechanisms of reactive arthritis.
types of ReA are not linked to HLA B27 but probably to other immunogenetic factors [146, 150] and one distinguishes at present the forms dependent on and independent of HLA B27. 3.2.1. Role of HLA B27 as an arthritogenic antigen presenting, molecule
Since the main function of HLA class I molecules is to present peptides to cytotoxic T lymphocytes (CTL), it has been proposed that the antigen presenting properties of HLA B27 could be crucial to the pathogenesis of AS. This hypothesis nevertheless encounters certain contradictions. Thus, it is very difficult to demonstrate the presence in man of synovial CTL clones specific for HLA B27 restricted bacterial epitopes. Moreover, HLA B27+ transgenic mice develop arthritis even when they are deficient in ~12mor TCD8 [ 151 ]. If HLA B27 intervenes as an antigen presenting molecule, the different sub-types do not have the same capacities. HLA B27 sub-types associated with AS (*B 27 02, 05, 04 and 07) are capable of
presenting arthritogenic bacterial peptides, whereas those not associated with AS (*B 27 06 and 09) are not. Various arthritogenic microbial peptides presented by HLA B27 have been described in SA and ReA [152]. Certain peptides display homology with self-peptides and hence are able to stimulate auto-reactive (CD8+) CTL [153-156]. In HLA B27+ ReA, these peptides are capable of inducing an oligoclonal proliferation of synovial CTL, which suggests that the number of arthritogenic peptides is fairly limited [102, 157-159]. It remains to determine the nature of these microbial peptides and their eventual homology with self-peptides, since the natural hypothesis is that in chronic ReA the acute antibacterial response is followed by a selfperpetuating auto-immune response against autologous peptides [ 160]. The detection of HLA B27 restricted bacterial antigens capable of stimulating synovial CTL is technically not easy. Chlamydia specific CDS+ T cell lines cultured from infected mice have been used to screen expressing librairies of C. trachomatis genes. An antigen encoded within an open
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reading frame of the C. trachomatis genome and designated Cap l (Class I accessible protein 1) was identified, but no homology to any known functional protein could be found [ 161]. In another study, Fiorillo et al characterised an EBV protein previously found to be an immunogenic peptide for HLA-B27+ subjects. This protein is mimetic with an autologous peptide derived from the vasoactive intestinal peptide receptor 1 (VIP-1R) and reacts specifically with CD8+ CTL from AS cases associated with the predisposing B'27 05 sub-type, but not with CTL from cases having the non predisposing B ' 2 7 09 sub-type [ 154]. The recent characterisation of the genome of C. trachomatis, which codes for 904 proteins susceptible to be presented by HLA B27, has permitted a different approach. This uses a combination of two computer based algorithms. The first is designed to predict the probability that nonamer peptides with an arginine at position 2 will bind to HLA *B27 05, while the second exploits the fact that peptides presented by MHC class I molecules to CD8+ T cells are processed in the cytoplasm in proteasomes [162]. Combining these two computer algorithms, 199 peptides were selected and tested for immunogenicity. Peripheral blood mononuclear cells and cells derived from the synovial fluid of patients with C. trachomatis induced ReA were stimulated with the selected peptides. Eleven HLA B27 restricted nonamer peptides could be identified using CD8+ T lymphocytes from three HLA *B27 05+ ReA patients. Only one of these 11 peptides from the Chlamydia genome reacted with CTL from all three patients. However, this screening was based solely on IFNy production in response to the peptides and it is very likely that several other HLA B27 restricted Chlamydia peptides also react with CD8+ T cells but do not induce IFN 7. An HLA B27 tetramer formed with bacterial-selected antigens could be very useful for analysis of the synovial or peripheral blood CD8+ CTL response. The frequency of CD8+ T cells in peripheral blood is nevertheless too low to allow their detection by this technique without prior T cell expansion. In recent work, using dendritic cells infected with C. trachomatis for T cell expansion, it was possible to identify Chlamydia specific CD8+ CTL in peripheral blood [90, 163]. Such an HLA B27 tetramer further has the potential advantage
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of enabling a search for the presence in tissues of CD8+ T cells specific for these chlamydial peptides [ 164, 165]. This approach is very important as it has demonstrated that proteasomes are able to produce CD8+ T cell epitopes which are specific to certain tissues on account of their organ-specific subunit composition [166, 167]. It was shown for example that T cell epitopes of mycobacterium (Hsp 60 kD) are only present in tissues after their infection with this bacterium. Thus, there could exist a tissuespecific hypersynthesis of self-peptides capable of inducing an organ-specific immune reaction. This study has to date not yet yielded results permitting the identification of these self-peptides homologous to microbial peptides. However, work has been devoted to the study of other target autoantigens including notably some macromolecules of cartilage. As an illustration, among the 19 B27-binding peptides derived from different human procollagens (I, II, XII), one was found to stimulate HLA B27 restricted CTL. The identification of HLA B'27 05 restricted peptides from collagens type II and VI which are capable of activating CTL in AS patients also suggests that a CD8+ T cell response against cartilage collagens could play a critical role in the local immunopathology of AS [168, 169]. Another potential target is the G 1-domain protein of cartilage proteoglycan (aggrecan), which has been shown to induce arthritis and spondylitis in Balb/C mice and to stimulate T cells in SA patients [ 170, 171 ]. The immunogenicity of 28 (among 76) HLA B27 restricted nonamer peptides from human aggrecan was recently studied in a Balb/C B'27 05+ transgenic mouse model and four peptides were found to stimulate CD8+ T cells [162]. Other observations indicate that humoral immunity to the G 1-domain of human cartilage proteoglycan is also involved in the induction of experimental spondylitis and sacroiliitis in Balb/C mice, although it is not associated with polyarthritis probably due to the lack of cross-reactive T cell immunity [172]. These results would suggest that SP is primarily an inflammatory disorder of the subchondral bone or an osteitis largely resulting from cartilage autoimmunity. However, the convincing data for fibrocartilage autoimmunity in experimental models of SA need to be confirmed in man [173].
3.2.2. Role of HLA B27 as an adhesin molecule for bacterial invasion
It has been reported that the adhesion molecules of certain bacteria (Yersinia, Salmonella) use HLA B27 as a ligand to attach to cells of the synovial environment [174, 175]. HLA B27 facilitated the entry of Salmonella into intestinal epithelial cells (a human intestinal cell line (Henle-407) transfected with HLA B27) and hence could increase the Salmonella load in intestinal tissue. The same phenomenon was not observed for Yersinia infection [ 176]. 3.2.3. Role of HLA B27 in bacterial persistence
In some genetically predisposed individuals, HLA B27 (unlike HLA A2) would appear to lack the capacity to mediate the elimination of infected macrophages normally, thus facilitating the intraarticular persistence of microbes like Salmonella, Yersinia or Chlamydia [177-180]. Although several groups have addressed this question, there is at present no consensus of results from the various studies [181 ]. Penttinen et al have suggested that the impaired resistance of HLA B27-expressing U397 cells to the intracellular replication of Salmonella enteritidis is dependent on the glutamic acid moiety at position 45 in the B pocket of HLA B27 [182]. Mutation of this amino acid has been reported to prevent misfolding of the HLA B27 molecule. Thus, it may be speculated that misfolding of HLA B27 influences its ability to modulate host-bacteria interactions. On the other hand, in a study of 198 patients with Salmonella infection, HLA B27 was not found to modify the susceptibility to infection, the duration of symptoms or the time to excretion of the bacteria [183]. An in vitro infection model has also demonstrated that HLA B27 suppresses Chlamydia replication. A variety of mechanisms could be invoked here, but in particular the fact that the soluble HLA B27 induced by ReA-triggering bacteria is able to inhibit HLA B27 restricted T cells [ 184, 185]. 3.2.4. The HLA B27 misfolding hypothesis
A new attractive hypothesis concerning the role of HLA B27 in ReA was recently proposed by Colbert's group [186]. The HLA B27 misfolding
hypothesis holds that HLA B27 itself is directly implicated in the pathology of AS through a misfolding involving the B pocket, caused by a particular form of HLA B27. During its processing and assembly in the endoplasmic reticulum, HLA B27 has in fact a tendency to misfold even without any ~2-microglobulin (~i2m) or peptide deficiency. This results from the fact that the two heavy chains can unite to form a homodimer before either of them binds to ~12m [187]. HLA*B27 05 seems to fold and associate with 1~2m more slowly as compared to other MHC class I molecules. Deglycosylated heavy chains are thought to be dislocated from the endoplasmatic reticulum as a consequence of misfolding. An accumulation of free HLA B27 heavy chains could thus induce the formation of abnormal homodimers and multimers at the cell surface and in this way activate the immune systeme [186, 188, 189]. The pathogenic role of free HLA B27 heavy chains is further supported by the amelioration of murine disease by treatment with the human heavy chain-specific monoclonal antibody HC10 [189]. Such HLA B27 homodimers form class-H-like structures, present peptides to CD4+ T cells [151, 190] and are recognised by the TCR and also by receptors belonging to the KIR (killer cell immunoglobulin-like receptor) and ILT (immunoglobulin-like transcriptor or leukocyte Ig-like receptor) families [191]. These receptors are expressed on certain groups of NK, T and B monocytic macrophages and dendritic cells [ 192]. It remains to determine the manner in which this phenomenon is involved in the pathogenesis of HLA B27 mediated ReA and AS. In particular, studies are under way to attempt to identify the peptides presented by 132m-free HLA B27 heavy chain homodimers [ 193]. The misfolding of HLA B27 can further lead to a stress response which could increase the production of proinflammatory cytokines through activation of NF-rd3 [194]. It has been possible to test this hypothesis by using a microarray to perform a comparative study of gene expression in SP and RA. A small cluster of genes appears to be preferentially expressed in cells from the synovial fluid of SP patients. One of these genes encodes the proteasome subunit C2 the expression of which is linked to an endoplasmic reticulum unfolded protein response and this over-expression seems to be restricted to
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the synovial macrophages [195]. Another recent microarray analysis has revealed striking differences in gene expression between bone marrowderived macrophages from HLA B27+ transgenic and wild type rats. The genes over-expressed in HLA B27+ rats comprised several components of the unfolded protein response including notably B ip (an endoplasmic reticulum chaperone) [ 196]. Recently, TSAI et al have demonstrated that free heavy chain expressing monocytes infiltrated the synovium of an involved hip joint of a patient with AS. This is one of the first patient-related evidence that surface free heavy chains of HLA B27 have to be implicated in human AS [ 188]. 3.2.5. Role of HLA B27 as a self-peptide A novel HLA B27 self-derived dodecamer peptide originating from the intracytoplasmic tail of the molecule was recently reported [197]. This peptide displayed striking homology with a sequence derived from the DNA primase of C. trachomatis, which was a natural ligand of the disease-associated subtypes HLA B 27 02, 27 04 and 27 05, but not of the non associated subtypes HLA B27 06 and 27 09. Intriguingly, two peptides from the second extracellular domain of HLA*B27 05 also share sequence homologies with several enterobacterial antigens. Thus, the HLA B27 168-176 peptide was recognised by peripheral blood lymphocytes from AS patients but not from B27+ healthy controls [198]. Autoantibodies to HLA B27 further crossreact with Shigella proteins to form circulating antigen-antibody complexes although the pathogenic significance of this finding is not known. Such observations demonstrate that an HLA B27 self-derived peptide can mimic a sequence from an arthritogenic bacterium and hence could play a role in AS. However, this mechanism of rupture of tolerance through molecular mimicry is probably not exclusive, since it has been observed that autoreactive T cells activated by C. trachomatis recognise a B27 self-derived peptide having no homology with C. trachomatis sequences [ 199, 200]. 3.2.6. Role of genetic factors other than HLA B27 The polymorphism of genes coding for other structures involved in antigen presentation like the Tap
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(transporter associated with antigen processing) and LMP (low molecular weight proteasome) molecules and in the cytokine cascade (TNFo~ promoteur, IL-6, IL-10, IL-1 Ra) also contributes to a genetic predisposition to SP [201]. As discussed above, a certain number of these factors (TNFc~, IL-10) play a part especially in the elimination of microbes. This is likewise the case for cytochrome P450. One P450 variant associated with an increased risk of AS corresponds to a "weakly metabolising" cytochrome, which could be responsible for an impaired ability to eliminate certain bacterial compounds [202]. In a recent study, the expression of macrophage scavenger receptors (SRs) that are responsible for innate immunity against gram-negative bacteries were analyzed. Expression of host defense gene, such as the macrophage receptor with collagenous structure (MARCO) was susceptible to modulation not only during infections, but also in the RA and SA. SRs could be considered as candidate factors that might contribute to ReA [203]. The role of NOD2 gene in SP is also discussed but NOD2 variant do not significantly affect the risk of developing primary AS but may influence susceptibility to colitic spondylarthritis [204].
3.3. The Immunopathological Mechanisms of Post-Infectious Arthritis The role of bacteria has been studied not only in SP and various other forms of post-infectious arthritis but also in RA [205]. It is interesting to note that the mechanisms inducing an inflammatory reaction of the synovium are basically the same in these different forms of inflammatory arthritis. In general, the immune response whether HLA B27 restricted or not develops into arthritis for two principal reasons. 1) The intrinsic and adaptative immune system has not been able to eliminate the micro-organism rapidly enough and it continues to have a proinflammatory effect. 2) The immune system exceeds its main objective, namely the elimination of exogenous agents. This leads to chronic inflammatory phenomena characterised by oligoclonal expansions of synovial T cells [102, 154, 157, 193,206-208]. It remains to
determine the mechanisms underlying this proinflammatory disequilibrium which seems to be related to a number of different processes. 9 As described above, there exists a deregulation of the cytokine response, notably in the case of the intrinsic immune system. This deregulation is linked to the immunogenetic characteristics of the host and also to the influence of the microbes on the immune response, in particular through the action of PAMPS (section 3.3.1.). 9 The TCD8 CTL response (HLA B27 restricted or not) is directed against different bacterial antigens. It is thought that this acute reaction is followed by a self perpetuating autoimmune response against autologous intracellular peptides, which would explain the chronic nature of the reaction. A study of this oligoclonal reaction could permit the identification of new target auto-antigens [ 159, 206]. 9 The TCD4 response, which could be HLAB27 restricted, is likewise relevant as it undoubtedly contributes to maintaining the inflammatory reaction in the synovium [ 190]. It is noteworthy that in B27+ transgenic mice, knockout of the CD4 gene but not of MHC class II genes will prevent the development of arthritis. A certain number of target bacterial antigens have been identified, especially for Chlamydia bacteria. Thus, in arthritis induced by Chlamydia, the chlamydial hsp60, HC1 protein (histone), Omp2, pmpD, enolase and CT579 epitopes are recognised by CD4+ T cells [209], although other microbial antigenic determinants probably remain to be discovered [210]. In arthritis induced by Yersinia, the strongest synovial CD4 responses are those against the ~-subunit of urease (19 KD) and hsp60 [208, 211,212]. CD4+ and CD8+ T cell responses against bacterial hsp60 both s e e m to be relevant in ReA. However, since there is little or no cross-reactivity among different bacteria or between bacterial and self hsp60, there does not appear to exist a single relevant hsp60 epitope on which to base a unifying pathogenetic hypothesis. 9 The B lymphocytes response, which could be modulated by the innate immunity, is probably very important. However, in contrast with
RA, the importance of this B response is still misunderstood [ 1, 2, 213-216]. 9 One important point, little studied as yet, is the role of innate immunity and the question of the articular consequences of these immune phenomena. There exists a cascade of inflammatory events which explains the occurrence of arthritis and its complications. The role of the complement network has been recently demonstrated in K/BxN T cell receptor transgenic mice which is a model of inflammatory arthritis similar to RA. In SP, its implication is still probably underestimated [217, 218]. Recently, it was shown that arthritogenic bacteria (S. typhymurium) can alter bone structure by infecting synoviocytes. The infected cells upregulate RANK-L expression and thereby enhance the maturation of osteoclast precursors into multi-nucleated TRAP-positive osteoclast-like cells [219].
3.3.1. The role of innate immunity: bacterial PAMPS (Fig. 3) It is now well established that pathogen-associated molecular patterns (PAMPS), which are found in microbial membranes, cell walls and DNA, may be regarded as a molecular signature indicating the presence of bacteria at a specific time or the possible transport of bacterial components to a site during local inflammation. If no bacterial species has been reproducibly identified as a causative agent in different forms of arthritis, some data suggest that bacterial products persisting chronically in the joint could through various mechanisms contribute to articular inflammation.
9 What PAMPS might be involved in joint inflammation in arthritis ? PAMPS are in general chemically diverse. Although endotoxin is often used as a model stimulus, since the most prevalent and severe infections are caused by Gram-positive bacteria, one key microbial element which could trigger an inflammatory response is the cell wall [220, 221 ]. This consists of teichoic acid and peptidoglycan associated with proteins and lipids [222-225]. A complete pro-inflammatory response including TNFct, IL- 1, IL-6, IL- 10, IL- 12 and NO production and expression of endothelial
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Figure 3. The innate immunity response. adhesion molecules is induced by the bacterial cell wall through activation of MAPKinases and translocation of NF-w.B to the nucleus. The crucial inflammatory element is peptidoglycan or more precisely the glycan backbone, composed of Nacetyl-glucosamine and N-acetyl muramic acid repeats and crosslinked to other backbones through various stem peptides [223, 226]. Peptidoglycan is covalently linked to teichoic acid, which although very similar to Gram-negative bacterial endotoxin, does not seem to play a major role in the inflammatory process [227, 228]. In previous studies, using immunohistochemical analyses, peptidoglycan was detected in synovial tissues notably in macrophages, but it is not known whether this material originated from bacteria already present in the tissues or had been transported in mononuclear cells [229, 230]. In more recent work using gas chromatography-mass spectrometry, a method which has proved considerably more sensitive for the detection of traces of bacterial components other than DNA, Chen et al were able to demonstrate the presence of muramic acid in synovial tissue from some RA and OA patients, despite an absence of bacterial DNA [76].
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It is likely that in the future, use of such techniques will allow the detection of, bacterial components in joint tissue more frequently than before. 9 What mechanisms might enable these bacterial components to participate in the inflammatory reaction ? The detection of microbial antigens by the host involves numerous receptors located on cells involved in the etiopathology of RA and other forms of arthritis: leucocytes, dendritic and epithelial cells, fibroblasts and mastocytes. A crucial role may be attributed to Toll receptors. The known TLR family consists of 10 receptors, each of which appears to interact with specific components of pathogens and could thereby contribute to the synovial inflammatory reaction [141, 231].
- TLR4 is the LPS receptor. Several structures are involved in the recognition of LPS including CD 14, LBP and MD-2, as has been demonstrated using mice deficient in these molecules. Additional receptors have been described on B cells, notably RP105 which associates with TLR4 and
MD-1, an analogue of MD-2 [232]. Other endogenous TLR4 ligands implicated in the inflammatory response have been reported, including Hsp60, Hsp70 and ECM components (extracellular domain A of fibronectin, hyaluronic acid, heparan sulphate) [233]. However, the results obtained for such molecules are questionable since recombinant molecules were used in the studies and contamination with LPS cannot be excluded. In addition, interactions with the ECM are more likely to be involved in tissue repair. - TLR2 recognises different components from various pathogens, among which the most important are lipoproteins, lipoarabinomannan from mycobacteria, lipoteichoic acids from Gram-positive bacteria, some atypical LPS and peptidoglycan [225, 234, 235]. This recognition of such a variety of components is related to the dimerisation of TLR2 with other TLRs such as TLR1 and TLR6. Blockade of TLR6 on macrophages leads to inhibition of the cytokine response to peptidoglycan, while the response to lipopeptides is not modified. - TLR1 and TLR6 are involved in lipopeptide recognition in association with TLR2. As demonstrated using TLR6- and TLRl-deficient mice, these receptors are able to detect subtle structural differences between lipopeptides insofar as they discriminate between diacyl (TLR1/TLR2) and triacyl peptides (TLR6ffLR2) [232]. - TLR5 is activated by bacterial flagellin which is present in some arthritogenic bacteria, especially Borrelia [235]. A great deal of attention has been paid to TLR9 as it is implicated in the recognition of bacterial CpG oligonucleotides [236, 237]. Bacterial DNA contains unmethylated CpG motifs which are not found in vertebrates and therefore have immunostimulatory or suppressive effects [214, 216, 238-243]. TLR9 is not expressed at the surface of cells but is localised in the endosomal compartment, where it interacts with non specifically internalised CpG.
-
Classically, signalling pathways triggered by TLR activation and leading to the production of unique cytokine combinations are thought to involve the adaptor molecule MyD88, IL-1Rl-associated kinases (IRAKs), the TGF~ associated kinase
TAK1, TAK1 binding proteins (TAB 1 and TAB2) and tumor necrosis factor receptor associated factor 6 (TRAF6). Other adaptor molecules have been identified more recently: TIRAP which is implicated in MyD88-dependent signalling downstream of TLR4, TLR1 and TLR2/TLR6 and TRIF, a third TIR-containing adaptor which plays an essential role in the MyD88-independent pathway downstream of TLR4 and TLR3 leading to IFNy release [80, 231]. After TLR activation, MyD88 recruted through its death domains IRAK-1 and -4 and interacts with the receptor through its TIR domain. The formation of this complex activates IRAK-4 which in turn phosphorylates and activates IRAK-1. IRAK1, which contains three TRAF6 binding motifs, then interacts with TRAF6. The resulting complex (IRAK-4/IRAK-1/TRAF6) dissociates from the receptor and associates with another complex consisting of TAK1 (MAPKKK) and two adaptor proteins (TAB 1 which directly activates TAK1 and TAB2 which links TAK1 to TRAF6, thus facilitating its activation by TRAF6). Activation of TAK1 leads to activation of IKKs and NF-r,B and of MAPKs and JNKs [140]. Other molecules have been shown to negatively regulate this pathway, in particular IRAK-M which is a negative regulator of TLR signalling and Tollip which is believed to terminate TLR signalling [244]. 9 What role might TLR-bacterial component interactions play in RA ?
Several studies provide evidence for a link between TLRs and autoimmunity [80, 225]. Activation of TLRs on dendritic cells by PAMPS, induces IL-6 synthesis, which is known to stimulate the effector T cells usually inhibited by CD4CD25+ repressor T cells [240, 245]. IL6-/- mice are in fact more resistant to RA [246]. Furthermore, Leadbetter et al have shown that the proliferation of autoreactive B cells recognising chromatin-IgG2a complexes requires the synergistic engagement of the BCR specific for rheumatoid factor and a member of the TLR family, probably TLR9 (RUI 2003) [216, 247]. In RA, it is now well established that synovial fibroblasts play a key role in the change from the acute phase to the chronic phase by modifying the microenvironment, or more precisely the cytokine, chemokine and other factors acting on infiltrating
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leucocytes. Since these cells have a stable phenotype, their interaction with bacterial components will certainly be important. On the other hand, whereas the expression of TLRs on antigen presenting cells such as dendritic cells, macrophages and B lymphocytes is well documented, their presence on synovial fibroblasts is less clear. It was previously hypothesised that these fibroblasts were indirectly stimulated through monocytes/macrophages. However, recent data from Kyburz et al indicate that the basal expression of TLR2 mRNA, which is very low on synovial fibroblasts, is greatly enhanced under conditions prevailing in the synovial cavity, namely in the presence of cytokines like TNFcz and IL-1 [248]. This may explain why these cells respond strongly to peptidoglycan. These results have been confirmed by Seibl et al, who demonstrated that stimulation with IL-1, TNFcz, LPS or sBLP enhanced TLR2 expression in synovial tissues and that the elevated expression of TLR2 observed in RA synovial fibroblasts was due to the presence of inflammatory mediators or microbial components in the synovial cavity [249]. Thus synovial fibroblasts could through TLR2 expression directly contribute to the inflammatory reaction in response to certain bacterial components. In contrast, these cells express very little TLR9 and therefore do not respond to CpG oligonucleotides [248]. Among the other receptors likely to be involved, integrins could play an important role. Neff et al have reported that protein UII, a PAMP from oral streptococci for which mRNA has been identified in the joint cavity of RA patients induces synthesis and release of IL-6 and IL-8 in synovial fibroblasts [50, 250]. Recognition of integrin cz5 131 was found to be responsible for the production of these proinflammatory cytokines. Furthermore, these authors showed that ERK1/2 and JNKs, together with AP-l-binding activity and nuclear translocation of NF-v,B, were essential for this protein I/II-mediated synthesis of IL-6 and IL-8, which was independent of FAK-mediated kinase activity. It is not known whether these PRRs-PAMPs interactions are sufficient to initiate and/or perpetuate the disease, although they are probably not. Bacterial cell wall components like peptidoglycan have also been found in joint tissues from OA patients and hence additional factors such as genetic poly-
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morphisms must be present to explain the development of ReA [76]. In support of this hypothesis, Choe et al showed that administration of LPS and arthritogenic sera from K/BxN mice resulted in joint swelling and destruction in IL-1R-deficient but not in MyD88-deficient mice. TLR4 signalling could in fact replace IL-1 activation in chronic joint inflammation [251 ]. This suggests for the first time that innate receptors like TLRs might additionally participate in and perpetuate joint inflammation. 9 Do P A M P - T L R interactions play a role in SP?
Recent insights into innate immunity have suggested that PAMPs-PRRs interactions may also be involved in the development of SP. Bacterial components such as Yersinia or Chlamydia outer membrane proteins and components of the cell wall (peptidoglycan) from Gram-positive bacteria resident in gut flora were found in the joints of patients with different arthropathies and shown to contribute to arthritogenicity in experimental models. Of interest are also the findings that bacterial CpG DNA is present in SA patients with early or long-standing disease and aggravates the course of experimental disease [252]. In this context, the persistence of these bacterial components might constitute a permanent stimulus and their interaction with TLRs having functional polymorphisms could explain their arthritogenicity. There nevertheless exist few data to corroborate this hypothesis. TLR2 which responds to Chlamydia spp might play a role in Chlamydia-induced SA [253]. TLR4 is over-expressed in the gut epithelia in IBD [254], but there is tittle evidence for its eventual involvement in SA, apart from the fact that like TLR2 it can be activated by endogenous components such as fibronectin, fibrinogen or HSPs [252, 255,256]. In a recent work, Bulut et al demonstrated that chlamydial hsp60 activates macrophages and endothelial cells through TLR4 [257]. The role of HLA B27 in the innate immunity response is discussed. HLA B27 has effects on host response to LPS that are unrelated to antigen presentation. The activation of NF-v,B and the secretion of TNFcz were found to be enhanced in HLA B27 expressing cells (U397) upon LPS stimulation. The enhanced inflammatory response of these HLA B27 positive cells offers an attractive explanation for the role of HLA B27 in the development of ReA [ 194].
3.3.2. Bacterial antigens: the role of adaptative immunity Bacterial antigens, whether produced by a viable intrasynovial bacterium or brought into the articular cavity by monocytes, have an immunostimulatory action. These antigens can most likely persist in the joint either "stuck" to the extracellular matrix or within antigen presenting cells [221, 258, 259] and as described above, are able to specifically stimulate CD8+ and CD4+ lymphocyte populations [207]. This lymphocyte activation has numerous consequences which could explain the appearance of synovitis, but a description of these mechanisms is not the object of this review. It remains to explain why this anti-bacterial response can in certain individuals exceed its physiological protective role and induce synovitis. 9 The bacterial antigens can be simple polyclonal lymphocyte activators capable of stimulating B or T clones in a non selective manner, as has been demonstrated for Mycoplasma. Various bacterial components of Y enterocolitica can likewise induce polyclonal activation of B lymphocytes
[215]. 9 Some antigens can behave as superantigens able to stimulate whole families of T lymphocytes expressing a particular T receptor, as in the case of Yersinia and Mycoplasma. 9 Certain bacterial antigens display close homology with a "self' antigen (molecular mimicry), which as already mentioned can induce a form of tolerance enabling the bacterium to avoid elimination (section 2.2.3). However, this mimicry can likewise trigger veritable "auto-immune" intraarticular inflammatory reactions in predisposed individuals. There are many known examples of microbial homology: some epitopes of Shigella and Ureaplasma display strong homology with HLA B27, M. fermentans has an epitope sharing sequence homology with CD4, while one of the best examples is probably the recently discovered sequence homology between OspA and LFA-1 described in section 2.2.3. This induces in certain subjects a strong intra-articular anti-OspMLFA-1 lymphocyte response, which could be involved in the pathogenesis of Lyme arthritis [ 117]. Nevertheless, there are several reasons (modification of the expression and species diversity of OspA)
to consider that this mechanism is of no major importance, even if it may exist in some chronic forms of the disease.
3.3.3. The role of antimicrobial peptides in innate and adaptative immunity A wide variety of host proteins has been shown to have antimicrobial activity. These antimicrobial peptides are produced not only by leucocytes, phagocytes cells and lymphocytes, but also by the epithelial cell lining of the gastrointestinal, genito-urinary and tracheobronchial tracts. They include defensins, cathelicidin, histatins, cathepsin G, azurocidin, lactoferin and many others. Some are produced constitutively, whereas others are induced by proinflammatory cytokines and exogenous microbial products. These proteins, such as defensins, produced in the course of innate host defense severe as signals which initiale and amplify adaptative Thl and Th2 immune host defenses. The role of antimicrobial peptides in the pathogenesis of ReA and other forms could be an important point, but there is very few data in this topic for the moment [260].
4. WHAT IS THE EFFICACY OF ANTIBIOTIC THERAPY IN REACTIVE ARTHRITIS? Reactive arthritis is one of the best disorders to attempt to evaluate the interest of anti-infectious treatment in post-infectious arthritis. The work of Lauhio and Bardin suggested a decade ago that in post-venereal ReA, antibiotic therapy of at least three months could be beneficial [261, 262]. A number of recent studies are however less optimistic.
4.1. Evaluation of Ciprofloxacine The efficacy of a three month course of ciprofloxacine (2 • 500 mg/day) as compared to placebo was evaluated in four studies [263-267]. In these reports, although the therapeutic schemas were fairly comparable, the study populations were different and included not only authentic ReA but also undifferentiated oligoarthritis. The evolution of the
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disease before treatment was quite variable with durations of 39 to 52 days and 84 to 210 days in the Finnish and German studies, respectively. The criteria of evaluation were very different, but globally no study was able to demonstrate a clinical benefit with regard to variables like pain, arthritic symptoms, functional scores or the onset of remission. Nevertheless, a detailed analysis reveals certain elements. 9 In the work of Sieper et al, a benefit was observed concerning the percentage of patients achieving remission in the Chlamydia induced ReA group, although this difference was not significant probably due to the small number of individuals with documented infection (13 among the 126 patients of the study) [264]. 9 Hoogkamp-Korstanje et al found that ciprofloxacine could significantly reduce levels of serum IgA directed against a major antigen of Y enterolitica known as Yop (Yersinia outermembrane protein) [266]. These antibodies are considered to be markers of bacterial persistence. In this work, the authors also observed the disappearance of microbial antigens from lymphoid tissues of the digestive tube in 6 of the 7 patients receiving ciprofloxacine, whereas after 3 months of treatment these antigens were still detectable in the digestive tube of the 9 subjects receiving placebo. 9 Yli-Kerttula et al recently reported on the 4 to 7 year follow-up of 53 of the 71 patients included in a placebo-controlled study published in 2000 [263]. In the placebo group, 11 of the 27 patients (40%) developed manifestations of chronic inflammatory rheumatism (oligoarthritis, enthesitis and two typical cases of ankylosing spondylitis). These patients were mainly HLA B27+ individuals (10/11). Conversely, inflammatory symptoms were observed in only 2 of the 26 patients (8%) initially treated with ciprofloxacine. 9 Hannu et al (ARD 2002) followed 78 individuals who had suffered from gastro-enteritis induced by S. typhimurium (with a positive coproculture) during an epidemic in Finland in 1999 [268]. Among these 78 patients, 5 (8%) developed ReA. This study revealed that those patients who had benefited from antibiotic therapy had fewer musculoskeletal complications. Thus, none of the 5
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subjects who developed ReA had received early antibiotics for gastro-enteritis. 4.2. Evaluation of Cyclines
A single recent study has reassessed the value of doxicycline (200 mg/day). This three month placebo-controlled trial included 60 patients who had been suffering from seronegative chronic arthritis for an average of 24 months (4 to 432 months). The criteria evaluated (pain, functional status on the AIMS 2 (Arthritis Impact Measurement Scale, version 2) and different articular indices) were comparable in the doxicycline and placebo groups. However, although well conducted on the etiological level, this work concerned mainly seronegative chronic arthritis evolving for several months and few cases of recent ReA [269]. Laasila et al reported on the follow-up of patients included in the placebo-controlled study of Lauhio published in 1991 [270]. Among the 40 patients treated with lymecycline or placebo in 1987-1988, 17 agreed to undergo a clinical control and complementary examinations. No significant difference was observed between the placebo and cycline groups with respect to the appearance of chronic arthritis, sacro-iliitis or authentic ankylosing spondylitis. Hence treatment with lymecycline did not appear to have changed the natural history of the articular disease. In summary, a short conventional course of antibiotics can eradicate the triggering infection in the majority of patients and it appears to be effective in preventing the development of ReA if given early enough. On the contrary, no definite effect is observed in established arthritis. There nevertheless exists to date no formal demonstration of the interest of antibiotic therapy (monotherapy), in particular for post-dysenteric arthritis, even if in the case of post-venereal arthritis clinical experience and some studies indicate a possible benefit. Controlled studies are in fact difficult to realise, since authentic cases of ReA are rare and often poorly documented at the bacteriological level, which means that one has to maintain a critical attitude towards the literature. The available reports have always evaluated the interest of a monotherapeutic anti-infectious treatment. However, the characteristics of these microbes and the chronic
Figure 4. The spectrum of arthritis induced by a bacterial infection. The host-bacterium interactions and in particular the bacterial virulence factors lead to different forms of arthritis. In some cases, there may be no more than a simple "bystander" bacterial presence in the synovium. evolution of these forms of arthritis suggest that as in infections like tuberculosis and brucellosis, other strategies could be envisaged (association of antibiotics, prolonged treatment, etc.) On the other hand, one of the principal restrictions is the fact that prolonged use of an antibiotic (ciprofloxacine or cycline) could select resistant strains notably of C. trachomatis [271,272]. Everyday experience has shown that administration of NSAIDS and/or intra-articular injections of corticoids is justified in the control of ReA. Recently, first results were reported concerning the use of anti-TNF agents [273, 274]. The effects of these molecules will nevertheless require precise analysis, since if anti-TNFt~ compounds would seem to be highly efficacious in SP, it will be necessary to confirm that their prolonged use does not have a detrimental effect by inhibiting the elimination of the triggering microbial agents. One could therefore imagine combined use of anti-inflammatory and anti-infectious treatment as
in other situations like the gastro-duodenal ulcer induced by H. pylori. In this case, only the association of proton pump inhibitors and double antibiotic therapy is effective.
5. CONCLUSION: T H E I M P O R T A N C E OF H O S T - B A C T E R I A INTERACTIONS (FIGS. 4, 5) All these points illustrate well the fundamental importance of host-bacterium interactions in the pathogenesis of inflammatory arthropathy, which largely exceeds the context of ReA. The important facts may be summarised as follows: 9 The synovial cavity is not as previously believed a sterile medium, but rather a site accessible to microbes, either directly during recurrent episodes of bacteremia or by transport within lymphoid cells or monocytes. The frequency and banality of these infections explains the presence
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Figure 5. Natural history of arthritogenic infections in reactive arthritis. 9 Arthritogenic microbes gain access to different extra-articular sites, especially mucous membranes ("exchange zone"). 9 The microbes can persist within these sites and/or disseminate to the articular cavity through recurrent bacteremia (Streptococcus, Borrelia...) or transported by monocytes (Chlamydia, Yersinia, Salmonella...). 9 Viable and active microbes (Chlamydia, Mycoplasma, Borrelia...) or bacterial antigenic debris (Yersinia, Salmonella, Shigella...) reach the synovial membrane. 9 This "contact" with the synovium can lead to 3 sequences of events: elimination of the microbes or their debris by the immune system of an immuno-competent host; persistence of viable and active microbes or antigenic debris in a "tolerant" host, particularly as a result of molecular mimicry; excessive stimulation of the immune system of a predisposed host which triggers synovial inflammation (arthritis). -
-
-
of "bystander" bacterial constituents in synovial samples from patients with osteoarthritis and healthy volunteers. Such intra-articular microbes may then be eliminated, trigger a sterile inflammatory reaction, or provoke a slow synovial infection, depending on the characteristics of the host and different factors controlling the synovial micro-environment. 9 Certain presumed forms of ReA thus sometimes correspond to authentic slow infectious arthritis
(C. trachomatis, Mycoplasma, B. burgdorferi ...), while "reactive" arthritis of the type infection triggered aseptic arthritis certainly exists. Moreover, there is nothing to indicate that the two mechanisms are exclusive, notably for Borrelia and perhaps for some enterobacteria. At
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the present stage of our knowledge, one may continue to use the term ReA provided one distinguishes clearly between the chronic infectious forms related to the intrasynovial persistence of viable microbes and the infection triggered forms linked to an extra-articular site of infection (Figs. 4 and 5). The concept of inflammatory arthritis is probably going to be transformed by study of these slow growing bacteria which have the particularity of being able to enter the host easily (by binding to mucosal adhesion molecules) and persist there by "hiding" in certain cells and/or by inducing a specific immune tolerance through molecular mimicry. It is perhaps these "parasite" bacteria, perfectly
adapted to their host, which have been selected in the course of the parallel evolution of the bacterial w o d d and the human species. This cohabitation results from a subtle equilibrium between the i m m u n e response of the host and the virulence of the bacterium. Any modification of one of these factors can lead to the appearance of inflammatory arthropathy of the type ReA and probably also other forms of peripheral inflammatory rheumatism. However, numerous questions still remain to be answered: 9 What are the most important factors which regulate the bacterial virulence or modify the response of the host in these types of arthritis? 9 How does one explain the stereotyped clinical and radiological manifestations of some forms of inflammatory rheumatism?
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ACKNOWLEDGEMENT The authors would like to thank Beno~t Jaulhac and Yves Pirmont of the Institut de Bactrriologie de la Facult6 de M r d e c i n e de Strasbourg.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Parasitic Infection and Autoimmunity Mahmoud Abu-Shakra 1 and Yehuda Shoenfeld 2
tAutoimmune Rheumatic Diseases Unit, Soroka Medical Center and Ben-Gurion University, Beer-Sheva, Israel; 2Research Center for Autoimmune Diseases, Department of Medicine 'B', Sheba Medical Center, Tel-Hashomer and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
During the last 4 decades, it has been suggested that microbial antigens can play a significant role in the pathogenesis of the immune disregulation leading to autoimmune disorders. Viral, bacterial or parasitic infection of subjects with specific genetic background, immune abnormalities, or hormonal constellation may trigger autoimmunity that leads to the development of an overt autoimmune disease [1-3]. Activation of autoimmune mechanisms has been associated with infection with various parasites. Observations that link autoimmunity and parasitic infections include: the presence of pathogenic autoantibodies and autoreactive cytotoxic T cells to heart and nerve cells in mice and in patients with Chagas' disease [4, 5], the detection of antibodies directed against self antigens of the inner retina in the sera of patients with onchocerciasis [6] and the development of complement-mediated hemolytic anemia associated with autoantibodies reacting with triosephosphate isomerase in patients with long standing malaria [7]. Autoantibodies associated with parasitic infections bind various self and foreign antigens (polyreactive antibodies) [8]. Autoantibodies to nuclear antigens detected in the sera of schistosoma infected mice bind DNA, polynucleotides and malaria and cercarial antigens [9]. This pattern of autoantibody binding similar to that of human monoclonal antiDNA antibodies derived from patients with SLE [10]. The clinical significance of autoimmune activity in patients with chronic infections is not clear. Autoimmunity related clinical manifestations
occur in chronic parasitic infections [ 11, 12]. However, other reports have suggested that a principal autoimmune etiology is unlikely for cardiac disease in patients with Chagas' disease [13]. In addition, a protective role for autoimmune mechanisms has been proposed. Autoantibodies in malaria may protect against infection by destroying infected cells, or blocking receptors for Plasmodium on the surface of RBC [14]. Parasitic antigens may induce autoimmune activity and immune-mediated damage to self antigens by several mechanisms including: molecular mimicry between host and parasites, induction of pathogenic autoantibodies, polyclonal activation of B cells, and manipulation of the idiotypic network [ 1-3]. In this chapter we summarize the link between parasitic infections, autoimmunity and autoimmune diseases. The association between autoimmunity and malaria, leishmania, schistosomiasis, and onchocerciasis will be detailed. Autoimmunity and Chagas' disease is discussed in the other chapters.
1. MALARIA AND AUTOIMMUNITY Malaria is a protozoan disease transmitted to humans by the bite of Anopheles mosquitoes. It remains among the main causes of human sickness and death in the world. Over 1 billion people reside in malarious endemic regions and about 200 million of these are infected at any given time. Malaria is characterized by fever, rigors, splenomegaly and hyperglobulinemia. Various autoantibodies were detected in the sera
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of patients with malaria. Shaper et al [ 15] were the first to detect anti-nuclear antibodies (ANA) and antibodies reacting with heart tissue in the sera of patients with malaria. Subsequent studies have found the sera of patients with malaria to bind: ssDNA, dsDNA [16], smooth muscle, parietal cells [17], cardiolipin [18], red blood cells (RBC) [19], lymphocytes [20], IgG (Rheumatoid factor) [21], neutrophil cytoplasmatic enzymes (ANCA) [22], and ribonecleoproteins (RNP) [ 16]. Autoantibodies are detected in patients with acute malaria, chronic malaria, and in the sera of healthy subjects living in endemic malarious area [23]. A correlation was found between the presence of autoantibodies and high titers of anti-malarial antibodies, suggesting that acute malaria trigger the generation of ANA and possibly other autoantibodies. However, the persistence of these autoantibodies did not correlate with the level of parasitemia once the acute infection was treated, indicating that autoantibodies are associated with chronic infection [24]. IgG anti-cardiopilpin (ACL) were detected in high titers in asymptomatic plasmodium falciparum carders and IgM ACL titers correlated with cerebral malaria [ 18]. Autoantibodies are seen in all ethnic groups. The frequency of ANA and anti-smooth muscle was similar in the sera of Caucasians, Asians and Africans with acute malaria, chronic malaria and those chronically exposed to malaria [25]. The clinical significance of autoantibodies in the sera of patients with malaria is not clear. Autoantibodies derived from splenocytes of a plasmodium chabaudi infected BALB/c mice exhibited characteristics similar to those of natural autoantibodies, suggesting that the generation of autoantibodies in the sera of patients with malaria has no clinical significance [8]. In addition, the prevalence of classic autoimmune diseases like systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are not higher in endemic malarious areas compared with the Caucasian population [26]. However, the association between malaria, hemolytic anemia, thrombocytopenia and nephritis indicates an autoimmune mechanism and a pathogenic role for malaria associated autoantibodies. The appearance or the persistence of hemolysis or nephritis in the chronic phase of the malaria, which is associated with low parasitic antigens load, indi-
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cates that pathogenic autoantibodies may have a role in nephritis and hemolysis in patients with malaria. Anti-RBC antibodies that bind red cells are present in the sera of patients with malaria [19]. The origin of those antibodies is not clear. Two main hypotheses have been suggested. Sayles and Wasson [27] have reported that anti-RBC is the result of anti-idiotypic antibodies of anti-plasmodial antibodies. A molecular mimicry between the anti-idiopypic antibodies and plasmodial peptides leads to binding of the anti-idiopypic antibodies to RBC. Others have suggested that parasite induced mechanical hemolysis of RBC leads to the development of autoantibodies against the cytoplasmatic enzyme triosephosphate isomerase (TPI). Anti-TPI is found in the sera of patients with malaria and is associated with persistent hemolysis [7]. Renal disease, manifested mainly by nephritic syndrome, contributes significantly to the morbidity and mortality of malaria. Deposition of immune complexes, composed of plasmodium antigens and their antibodies, is the main mechanism of this sequel of malaria. However, anti-DNA antibodies and possibly their idiotypes may have a role in the development of nephritic syndrome. Wozencraft et al [28] have reported that anti-DNA antibodies can contribute to the renal damage of mice infected with plasmodium berghei. Monoclonal anti-DNA antibodies were detected in the glomeruli of mice with malraia and renal damage. In a challenging review Daniel-Ribeiro and Zanini [ 14] have proposed that malaria may protect against autoimmune disease, by preventing the development of autoimmune diseases or by diminishing its severity. They hypothesized that autoantibodies in malaria participate in the immune protection against malaria. The generation of autoantibodies reacting with cryptic or neo-antigens [29] or anti-idiotypic antibodies against plasmodial antigens may have a role in destroying infected cells or by blocking R33C invasion. In addition the autoantibodies may bind parasitic antigens and inhibit their activity. All of these plausible mechanisms need further research.
2. LEISHMANIA AND AUTOIMMUNITY Leishmania are intracellular protozoa that reside within mononuclear phagocytes of the host.
Depending on the Leishmania species, the infected host develops localized or disseminated disease. The localized form of leishmaniasis is characterized by cutaneous or mucocutaneous lesions which are caused by L. tropica, L. major, L. aethiopica, L. mexicana, and L. braziliensis. Visceral leishmaniasis or kala-azar is caused primarily by L. donovani. Leishmaniasis is a public health problem in tropical areas and most countries bordering the Mediterranean basin. As with malaria, sera of animals and humans with cutaneous and visceral leishmaniasis, recognize various autoantibodies including: nuclear antigens (ANA), native DNA, immunoglobulin (rheumatoid factor), Sm, RNP, SS/A, SS/B, cardiolipin, beta 2-glycoprotein I, actin, tubulin, smooth muscle and others [30-34]. The autoantibody activity was seen in the sera of patients with localized and systemic leishmaniasis. However, the frequency and titers of autoantibodies were higher in the sera of patients with kala-azar compared with cutaneous leishmaniasis. Anti-Sm, RNP, SS/A, SS/B were detected in high titers in 83, 86, 36, and 73% respectively of the sera of patients with visceral leishmaniasis compared with, 7, 14, 25 and 25% respectively in the sera of patients with cutaneous leishmaniasis. The binding of visceral leishmaniasis sera to ribonucleoproteins was inhibited by prior incubation of the sera with either leishmanial membrane antigens from four different species of Leishmania, or with intact cells of Leishmania donovani, suggesting a similarity between host and leishmania antigens [30]. In patients with Kala-azar a high prevalence of anti-beta2GPI was seen. The prevalence of IgG aCL, IgM aCL and anti-beta2GPI was 6% (2/30), 3% (1/30) and 53% (16/30), respectively [31]. No statistical correlation between aCL and antibeta2GPI antibodies was found. A pathogenic role for autoimmunity in leishmaniasis was reported. Three cases of pancytopenia in patients with kala-azar were described. Antiplatelet, antineutrophil and antierythrocytic IgG antibodies were documented on the cell surface of platelets, RBC and neutrophilis in all three cases. Bone marrow suppression was not found, suggested that pancytopenia resulted from rapid destruction of antibody-coated blood cells [33]. Recently, a case of patient who developed SLE
concurrently with leishmaniasis was reported [35]. The SLE was characterized by thrombocytopenia, nephritis, skin lesion, and positive ANA and anticardiolipin. Renal biopsy showed lupus nephritis without evidence of infection with leishmania. A complete and prolonged remission of SLE and disappearance of ANA was observed after the treatment of the leishmanial infection. Renal involvement in leishmaniasis; manifested by acute glomerulu-nephritis, nephritic syndrome, and interstitial nephritis; has been reported. Renal biopsies have shown leismanial antigens in some cases and deposition of immunoglobulins, complement and fibronigen without direct infection of the kidneys in others [36, 37]. Although IgG-anti-IgG immune complexes were detected in the sera of patients with leishmaniasis, no direct pathogenic role for those complexes was documented [38].
3. SCHISTOSOMIASIS AND AUTOIMMUNITY Three main schistosome species infect humans: Schistosoma mansoni and Schistosoma japanicum mostly infect the liver and Schistosoma haematobotium infect the venules of the urinary tract, mainly the ureters and bladder. Schistosomiasis is a granulomatous disease characterized by the development of granulomas surrounding the helminth's eggs. Autoantibodies were detected in the sera of humans and animals infected with various schistosoma species including: Anti-DNA, ANA, RF, anti-Sm, anti-sperm, and anti-lymphocytic, antithyroid, anti-tubulin, anti-parietal cells [9, 39--44]. The sera of 234 patients with chronic schistosoma mansoni infection were screened for a wide range of autoantigens [44]. Fifteen percent were positive for ANA, smooth muscle or gastric parietal cell antibodies and 27% had antibody against the hepatic asialoglycoprotein receptor (ASGP-R). Anti-ASGP-R occurred more commonly in patient with hepatosplenic involvement than patients with hepatointestinal disease or patients who underwent splenectomy (33%, 4.5%, 5.8% respectively). The data suggest that anti-ASGP-R may have role in the pathogenesis of hepatosplenic schistosomiasis. Similarly, Mice infected with Schistosoma mansoni generated autoantibodies and T-cells responsive
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to collagen [45]. Anti-collagen antibodies have been identified in a variety of autoimmune rheumatic diseases; including scleroderma, rheumatoid arthritis, and relapsing polychondritis. A role of anti-collagen activity in the hepatic and other visceral fibrosis of patients with schistosomiasis has been reported [45]. It has been postulated that the anti-collagen activity may result in the secretion of proinflammatory cytokines that stimulates fibroblasts to migrate and synthesize collagen. In a subsequent study [9], various autoantibodies to nuclear antigens, including anti-dsDNA, antipoly (I), anti-Sm and anti-SS/B were detected in the sera of 15 mice, 9 weeks after infection with schistosoma. The binding of the sera of the mice with schistosomiasis to cercarial extract was inhibited by prior incubation of the sera with DNA, polynucleotides and cercarial extract. A possible link between SLE and schistosomiasis has been suggested. High levels of a pathogenic anti-DNA idiotype (16/6 Id) and anticardiolipin antibodies were reported in the sera of patients with schistosomiasis [46]. In addition, sera of mice with experimental SLE and patients with SLE reacted with the schistosomal extract. Immunoblot analyses showed that the sera of mice and patients with SLE reacted with 60, 85 and 94 kDa cercarial proteins while the sera of patients with SLE also reacted with 10-18, 29 and 52 kDa cercarial proteins [9]. The significance of this binding of SLE sera to schistosoma extracts is not clear. It may indicate that antigens with homology to schistosoma may trigger SLE.
4. ONCHOCERCIASIS AND AUTOIMMUNITY
Onchocerciasis, or river blindness, is a tropical infection caused by the filarial nematode Onchocerca volvulus. In endemic areas, an estimate of 50% of adults over 40 years may be blind as a result of corneal or retinal lesions induced by O. volvulus. Two forms of corneal disease occur in onchocerciasis. Punctate keratitis occurs within the first 10 years of infection and is characterized by acute inflammation and edema around dead microfilaria. A complete resolution of the infection without residual damage may occur. Sclerosing keratitis is associated with
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prolonged infection and consists of a fibrovascular pannus which may affect the entire cornea. Autoimmune mechanisms have been associated onchocerciasis. Autoantibody activity has been observed in the sera of patients with onchocerciasis and includes: Anti-tubulin, rheumatoid factor, antilaminin antibodies [43, 47, 48] and antibody activity directed against corneal and retinal antigens [6]. Anti-tubulin was found in the 89% of the patients [43]. It is also seen in various parasitic infections including: visceral leishmaniasis (67%), cutaneous leishmaniasis (60%), and all patients with schistosomiasis. Autoantibodies that bind retinal extract were generated in the sera of guinea-pigs after subconjunctival or subcutaneous injections of microfilaria over 4-14 weeks [49]. Severe corneal inflammation was induced by autoimmunization with the comeal extract followed intra ocular injections of cytokines, suggesting that exposure to microfilaria may induce pathogenic anti-corneal autoantibodies [50]. In another study 10 of 12 sera and ocular fluid from patients with onchocerciasis reacted with autoantigens of the inner retinal; including nerve fiber, ganglion cells and Muller cell. Antibodies against the outer segment of the photoreceptor were noted in 7 of the 12 patients [51 ]. Another study have identified the presence of autoantibodies, directed against human S-antigen and interphotoreceptor retinoid binding protein (IRBP) in the sera of patients with onchocerciasis [52]. The frequency of all autoantibodies was significantly higher in patients with onchocerciasis compared with controls living in the same area, however no correlation was found between their presence and the clinical features of the disease. Subsequent studies identified autoantibody reactivity against five major cytoplasmic non-RNAassociated autoantigens with molecular weight of 35, 51, 64, 83, and 110 kDa [53]. In addition, anticaketiculin reactivity [54], the human homologue of the onchocercal Ag RAL-1, anti-retinal pigment epithelial and neural retinal cells [6], and antibody reactivity against the 65-kDa arthritis-associated mycobacterial heat shock were also seen in onchocerciasis [53]. Recently, anti-calreticulin is considered to have a significant role in autoimmunity. Calreticulin is implicated in a number of autoimmune processes,
including molecular mimicry, epitope spreading, complement inactivation and stimulation of inflammatory mediators, such as nitric oxide production. It binds to the Ro/SS-A antigen complex, and forms a common target for autoimmune responses. Anticalreticulin are present in sera from patients with systemic lupus erythematosus, Sjtigren's syndrome, and other rheumatic disease [54, 55].
5. MECHANISMS FOR AUTOIMMUNITY IN PATIENTS W I T H PARASITIC INFECTIONS Parasites may induce autoimmune activity by several mechanisms (Table 1) including: molecular mimicry between microbial polypeptides and host antigens, alteration of host antigens, polyclonal activation of B cells and manipulation of the idiotypic network [ 1-3]. All of these mechanisms are detailed in chapters 1-4. Following parasitic infections the host immune system responds by recruitment and activation of inflammatory cells and by the secretion of various inflammatory mediators. This process may be associated with damage and alteration of the structure of host antigens, trigger the release of sequestered antigens, or exposure of cryptic antigen. Those modified host antigens may no longer be recognized as self and may induce the secretion of autoantibodies and the generation of auto-reactive T cells [ 1-3, 14]. Infection of RBC with Plasmodium falciparum results in the exposure of band 3 related neoantigens to the immune system and subsequently the development of autoantibodies reacting with those antigens [29]. Parasites can trigger autoimmunity by polyclonal activation and expansion of B cell clones that produce a large amount of natural autoantibodies. Plasmodium strains were shown to release mitogens that activate B cells [56]; incubation of Trypanosoma cruzi with human peripheral mononuclear cells leads to blast transformation and proliferation of lymphocytes [57]. Recently, a 33 kDa alkaline fraction of T. cruzi was identified. This fraction stimulates proliferation and promotes differentiation into antibody-secreting cells of normal B cells in a T-cell independent mechanism [58]. The released antibodies are mainly IgM and IgG3 which
Table 1. Possible mechanisms for parasite-induced autoimmunity Polyclonal activation of B cells Modified self antigens Release of sequestered antigens Alteration of host cell proteins Neoantigens Idiotypic network Molecular mimicry Linear sequence homology Three dimensional structural similarity Over-representation of specific motifs
do not specifically recognize the mitogenic antigen. In addition they exhibit characteristics similar to those of natural autoantibodies namely polyreactivity and low binding affinity to antigens [8]. Manipulation of the idiotypic network [59] may occur in patients with parasitic infection. The idiotypic network indicates that idiotypes and their antiidiotypes antibodies have a major role in regulating the immune response to self and foreign antigens. Auto-anti-idiotypes are a normal component of the immune system and their role is to down regulate autoactive clones [59]. In people susceptible to autoimmune diseases (those with the appropriate genetic, hormonal, immunological and environmental background), the parasites induced activation of the idiotypic cascade may proceed from anti-idiotypes (anti-anti-parasite) to the generation of antianti-idiotypes (Ab3) that may be pathogenic. This process may be the result of exposure of cryptic antigens by Ab2, or as a result of epitope spreading based on molecular mimicry existing between the idiotype structure and intracellular or intranuclear antigens [59]. In malaria, anti-merozite ligand for RBC receptors triggers the production of anti-idiotypic antibodies mirroring the merozite ligand. Those autoantibodies bind RBC receptor and induce hemolysis [14,27].
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6. M O L E C U L A R MIMICRY BETWEEN HOST AND PARASITES The Molecular mimicry theory is based on antigenic similarity between host tissue and infectious agents. Antigenic cross-reactivity occurs between host and bacterial, viral or parasitic antigens. The amino acid sequences of peptide fragments derived from the extracellular domain of the human thyrotropin receptor were found to be identical to those of envelope lipoproteins produced by the Yersinia species. Following stimulation with Yersinia lipoproteins, mice spleen cells produced autoantibodies reacting with the thyrotropin receptor [60]. The similarity between host and microbial antigens may suppress the immune system and protect invading parasites and other infectious agents from being eliminated by the immune system. AntiGP50/55 is a monoclonal antibody derived from a patient with Chagas' disease that binds a GP-linked 50/55 kDa antigen (GP50/55) presented on the Trypanosoma cruzi membrane. This antibody reacts with a 28 kDa antigen expressed on the membrane of activated human T and B cells and suppresses proliferation of B and T cells expressing the 28 kDa antigen following mitogenic stimulation. This indicates that molecular mimicry between T. cruzi and human antigens may have a possible role in the immunosuppression seen in patients with Chagas' disease [61 ]. The presence of common epitopes shared by microbial and host antigens may lead to the expansion of self reacting lymphocytes and to the induction of autoimmune phenomena [62-63]. This is supported by the following observations: binding of autoantibodies to parasitic antigens, inhibition of binding of autoantibodies to their respective autoantigens by parasitic determinants, sequence homology between various parasitic antigens and self antigens and genetic studies showing that parasite genomes contain genomic sequences that encode proteins close to self antigens. Monoclonal antibodies derived from patients with parasitic infections bind self antigens. CAK20.12 is a monoclonal antibody derived from patients with Chagas' disease that bind specifically to an epitope of a 150-kDa antigen expressed on the surface of several strains of Trypanosoma cruzi. It also identifies a common epitope presented on: human striated
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muscle tissue, the smooth muscle layer of cardiac arteries, the lamina muscularis mucosa and the striated muscle layer of the esophagus [64].
7. H O M O L O G Y BETWEEN SELF AND
PARASITIC ANTIGENS Observations that suggest molecular mimicry between parasitic and human antigens include: 1. The binding of the monoclonal anti-circulating cathodic antigen (CCA) antibodies from patients with schistosomiasis to the repeating L (ex) units on the surface of human granulocytes. The CCA, a glycoprotein which contains polysacharide side chains with the trisacharide Lewis-x L(ex) being a repeated unit [65]. This autoactivity was associated with granulocyte lysis in a complement-dependent cytotoxic assay, suggesting that autoimmune mechanisms may be associated with the development of moderate neutropenia seen in patients with schistosomiasis [65]. 2. Amino acids sequence homology between a 2.5 kDa Onchocerca volvulus antigen and human defensins 1-3 of neutrophils. The titer of anti-2.5 kDa antibody is directly related to the disease activity and it has an anti-defensins activity [66]. 3. A sequence homology was identified between a Plasmodium falciparum merozite surface antigen and host intermediate filament proteins. As well, homology between the conserved part of variable region II of the circumsporozite of Plasmodium and region E of thrombospondin, a platelet derived adhesion molecule that mediates cytoadherance between infected erythrocytes and vascular endothelium [67, 68].
8. GENETIC SIMILARITY BETWEEN PARASITES AND HUMANS The following studies have pointed to a sequence homology between genes encoding parasitic and human antigens. 1. FL-160-1 is a gene encoding the COOH terminus of a protein associated with the flagellum of Trypanosoma cruzi trypomastigotes. The COOH terminus of FL-160 has an epitope of 12 amino acids which molecularly mimics a
nervous tissue antigen of 48 kDa present in the myenteric plexus, sciatic nerve and a subset of cell in the central nervous system. The FL-160 genes belong to a family of highly related genes. More than 750 copies of FL-160 are present in the DNA of the parasite. Sequence analysis of genes revealed that all members of the FL-160 gene family retain the 12 amino acid molecular mimicry epitope. Homology was also found between an epitope located on the NH2 terminus of FL-160 and nervous tissues. Antibodies directed against this epitope were also found to bind the epineurium [69]. 2. A high sequence homology between the Onchocerca volvulus RAL-1 antigen; one of the subunits of human and mouse Ro antigen and calreticulin, a high affinity calcium binding protein [70]. These findings may indicate that Ro antigen has a very basic cellular function and its presence in parasites may explain the development of autoantibodies to nuclear antigens seen in patients with parasitic infections. 3. In patients with malaria, cloning of a Plasmodium falciparum gene related to the human 60kDa heat shock protein revealed more than 50% homology to the hsp60 from human and other eukaryotes [71 ].
9. SIGNIFICANCE OF MOLECULAR MIMICRY The biologically significant homology between microbial and self antigens may be the result of linear sequence homology of amino acids motif, over-representation of a specific motif, and /or three dimensional structural similarity between the pathogenic peptide and MHC antigens. This homology results in interaction of the T cell receptor with specific MHC-peptide ligands of parasites that leads to proliferation and clonal expansion of autoreactive cells [62, 63]. A similarity between peptides located on the beta chain of human MHC and viral and bacterial peptides have been identified. In patients with rheumatoid arthritis (RA), the QKRAA/QRRAA motif of the beta chain of HLA-DR4 is located on peptides from HIV, EBV g p l l 0 and E. coli dnaJ [72, 73]. This motif is highly associated with RA, suggest-
ing that linear molecular mimicry (shared epitopes) between MHC antigens and self and microbial antigens is associated with activation of T cells and induction of autoimmunity in patients with RA. Others have indicated that the activation of T cells and the induction of cross reactive immunity require homology between the three-dimensional structure of epitopes of MHC and pathogenic antigens and not necessarily linear sequence homology. In a model of experimental autoimmune uveitis, a strong three dimensional similarity was found between peptides of three antigens; retinal S-antigen, HLA-B27 and B7, although the linear structure of the S and HLA peptides revealed homology only in 5 out of 14 amino acids [74]. Roudier et al [75] have performed statistical and mathematical analyses on the amino acids database of proteins using the SWISS-PORT database and suggested that in patients with autoimmunity overrepresentation of certain amino acids motifs may result in an overt clinical disease. The QKRAA motif, associated with RA, was found to be 37 times more common than the expected number of matches for QKRAA on theoretical calculations in the database. Their analyses revealed that because of the large size of the protein databases it is easy to identify a 5-7 amino acids match between unrelated proteins and molecular mimicry between proteins is a common finding and not necessarily associated with pathogenic significance. While similarity between self and parasitic antigens has been suggested, there has been no report showing the biologic significant of specific parasitic epitopes in generating autoimmunity at the level of T-cell recognition.
I0. SUMMARY The studies summarized in this paper suggest that parasites may trigger activation of autoimmune mechanisms. The association between parasites and autoimmunity could by manifested by the development of pathogenic anti-parasitic antibodies and cytotoxic T cells that attack and damage self tissues as a result of molecular mimicry between host and parasites. On the other hand, the homology between self and parasitic antigens may enable parasites to protect themselves from the immune system and to
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induce a state o f immunosuppression.
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9 2004 Elsevier B.V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Autoimmunity in Chagas' Disease Edecio Cunha-Neto, Leo Kei Iwai, Angelina Morand B. Bilate, Simone Gonqalves Fonseca and Jorge Kalil
Laboratory of Immunology, Heart Institute (Incor) and Division of Clinical Immunology and Allergy, University of Sao Paulo School of Medicine, Sao Paulo, Brazil; Institute for Investigation in Immunology, Millenium Institutes, Brazil
1. INTRODUCTION Chronic Chagas' Disease Cardiomyopathy (CCC) is one of the few well-defined examples of human post-infectious autoimmunity, as documented by several groups in over 50 publications. This lifethreatening heart disorder affects ca. 6 of the 20 million patients infected by Trypanosoma cruzi in Latin America, being a major public health problem in this region. The time scale dissociation between primary infection with high tissue and blood parasitism and tissue pathology, allied to the scarcity of T. cruzi in CCC heart lesions prompted investigators as early as 70 years ago [1] to suggest that the mononuclear cell infiltrate should directly damage the heart in an autoimmune fashion. Here we will review evidence for autoimmunity as a major factor for heart tissue damage in CCC. Unraveling the mechanism by which an infectious agent can trigger organ-specific autoimmunity may lead to reverse strategies for identifying putative triggering infectious agents in autoimmune diseases of suspected infectious etiology. In addition, testing current concepts on molecular pathogenesis of human autoimmune disorders on proven post-infectious autoimmune disease, like CCC and rheumatic fever [2] may allow the early identification of susceptible individuals and therapy of affected patients.
2. THE SPECTRUM OF CHAGAS' DISEASE
In 1908, the Brazilian physician scientist Carlos Chagas described American Trypanosomiasis (later
dubbed Chagas' disease) identifying its etiologic agent, the flagelate protozoan Trypanosoma cruzi, its full life cycle, and the clinical spectrum of the disease [3]. Human T. cruzi infection occurs after a blood meal by infected hematophagous hemipteran bugs (family Triatominae), where metacyclic trypomastigotes, the infective form of T. cruzi, from the insect's fecal material invade the cytoplasm of many different types of host cells. Once inside the cytoplasm, trypomastigote forms change into the replicative amastigote forms, which multiply by binary fission, changing back to trypomastigote forms just prior to host cell lysis, releasing hundreds of infective forms of T. cruzi ready to invade new target cells. After several cycles of invasion-replication-release, parasitism is widespread in several tissues and blood; acute asymptomatic myocarditis is thought to occur nearly always [4]. The acute phase of infection, which lasts about two months, is usually asymptomatic, but flu-like symptoms and, more rarely, fulminant myocarditis may occur in 10-30% of infected individuals. The high parasite load ensues a strong cellular and humoral immune response against T. cruzi, leading to the control - but not the complete elimination - of tissue and blood parasitism. Thus, a low-grade persistent infection is established regardless of the clinical progression of the disease, as indicated by the invariable reactivation of parasitemia in chronically infected individuals after immunodeficiency or pharmacological immunosuppression [5-9]. For yet unknown reasons, probably due to multiple immune escape mechanisms, there is no "sterile immunity" [10].
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Chagas' disease is endemic in many countries of Latin America, where 20 million people may be infected. Since there are no anti-T, cruzi vaccines or highly effective chemoterapeutic agents, disease control is based on vector control in endemic areas and serological screening of donor blood. Up to 25 %, or 5 out of the 20 million Chagas' disease patients, develop Chronic Chagas disease cardiomyopathy (CCC) 5 to 30 years after primary T. cruzi infection. CCC is an inflammatory cardiomyopathy with characteristic heart conduction defects; one-third of CCC patients develop severe forms including arrythmia, ventricular dilation and refractory heart failure [11, 12]. CCC is a particularly lethal form of dilated cardiomyopathy, as survival after presentation is 2--4 fold shorter than that of idiopathic dilated cardiomyopathy [13, 14]. The remaining 60-70% chronically T. cruzi-infected individuals either remain asymptomatic (ASY, also known as indeterminate patients) or develop denervation of parietal smooth muscle in the digestive system, generally the oesophagus or colon (5-10%) [ 15]. A functional damage to the autonomic nervous system is also observed, affecting a subgroup of symptomatic and "indeterminate" asymptomatic patients [16, 17]. In CCC, dilated cardiomyopathy, arrythmia, and arterial thromboembolism may occur as single forms or in combination, and patients usually die of refractory heart failure or sudden arrythmic death. Cardiac or digestive "syndromes" of chronic Chagas' disease may also present as isolated or overlapping forms [ 18].
3. PATHOGENESIS OF HEART-SPECIFIC INFLAMMATORY LESIONS IN CCC: ROLE OF LOCAL PARASITISM AND AUTOIMMUNITY
The major histopathological feature attending dilated cardiomyopathy in CCC is the presence of a diffuse myocarditis with a significant lymphomononuclear infiltrate, intense heart fiber damage and fibrosis, in the presence of very scarce T. cruzi forms [19, 20]. Histiocytes and endothelial cells in CCC heart tissue display increased expression of human leukocyte antigen (HLA) class I and class II molecules; CCC cardiomyocytes only display increased levels of HLA class I [21]. The inflammatory infiltrate is
450
composed by macrophages (50%), T cells (40%) and B cells (10%) [22]; among T cells, a 2:1 predominance of the CD8 + over the CD4+ subset was observed [20, 23]. The demonstration of restricted heterogeneity of T-cell receptor Vt~ transcripts in heart biopsies from CCC patients [24] is in line with similar findings in established autoimmune diseases [25]. Heart-infiltrating T cell lines obtained from CCC patients produce IFN-~, and TNF-ot in the absence of IL-4 upon PHA stimulus [26], in line with the predominant detection of IFN- 7 and TNF-ct in immunohistochemical studies of CCC heart tissue [27, 28]. Chronic infection with T. cruzi induces a systemic shift in the PBMC cytokine profile towards T~ type cytokines with suppression of T 2 type cytokines [26, 29, 30]. The increased production of IFN- 7 in CCC patients [30, 31] as compared to ASY patients has been linked to decreased production of IL-10 [30]. A recent report from our group showed that all chronically T. cruzi-infected patients- even from the asymptomatic ASY form - d i s p l a y increased plasma levels of TNF-t~. Furthermore, patients displaying severe CCC present significantly higher plasma levels of TNF-ct [32]. The proinflammatory and Tl-type cytokine profile described above among chronically T. cruzi infected patients may be related to the ability of mucin-like glycoconjugates from persisting T. cruzi infection to induce the production of IL-12 [33]. Since it is known that T. cruzi establishes a lifelong, low-grade infection, the possibility that heart tissue damage in CCC is a mere result of recognition of parasite antigen on target tissue, with attending inflammation, must be entertained [12, 34]. Even though heart-infiltrating T cells have been implicated as the ultimate effectors of heart tissue damage, a direct role for heart parasitism has been proposed after the identification of T. cruzi antigen and DNA in CCC hearts by immunohistochemical and PCR techniques, respectively [20, 35]. However, evidence indicates that low-grade parasite persistence is universal in CCC and ASY patients (3638) and not linked to the development of CCC in follow-up studies [39]. Furthermore, both CCC and ASY patients display increased levels of TNF-ct, possibly in response to the proinflammatory stimulus of chronic low-grade T. cruzi infection [32]. Recent studies in heart tissue samples from CCC patients using immunohistochemistry to detect
T. cruzi failed to disclose an association between parasite presence and inflammatory lesions in hearts from Chagas' disease patients [40]. A recent report using in situ hybridization with T. cruzi DNA probes failed to detect intact T. cruzi even at sites of strong inflammation [41]. T. cruzi DNA has been detected in hearts of both CCC and ASY individuals [42, 43], and CD4+ T cell clones obtained from the heart tissue of a CCC patient failed to recognize recombinant and crude T. cruzi antigens [44]. The bulk of the evidence indicates that chronic Chagas' disease patients undergo low-grade tissue parasitism, but only in the hearts of CCC, rather than ASY patients, is there diffuse myocarditis. The overwhelming majority of microscope fields in CCC hearts show inflammation but is devoid of T. cruzi even using immunohistochemical detection of T. cruzi antigen [40] or T. cruzi DNA [41 ]. This may indicate that while direct T. cruzi tissue parasitism might induce focal inflammatory foci, this is apparently unable to evoke sufficient heart damage to lead to dilated cardiomyopathy. Thus, some other factor must be operating along with parasite persistence, to lead a subgroup of T. cruzi-infected individuals towards heart damage. The recent identification of persistent virus infection in patients with bona fide human autoimmune diseases like multiple sclerosis [45] and insulin-dependent diabetes melitus [46] may indicate that this might be a common theme among such diseases.
or murine models and could generate experienced, effector autoreative T or B cells capable of inducing tissue damage. (i) Antigen exposure. T. cruzi infection promotes tissue damage and a consequent exposure of intracellular proteins, such as myosin, along with myocardial inflammatory response during the acute and chronic phases of infection, with upregulation of MHC class I and class II proteins [49]. Self-epitopes may be presented in the context of MHC and upregulated costimulatory molecules [50]. T cell sensitization to cardiac myosin has been shown to occur during acute T. cruzi infection [51 ]; (ii) Molecular mimicry. T and B cells recognize parasite antigens that present molecular mimicry with structurally similar epitopes in host antigens, generating crossreactive autoimmune responses (see Table 1). (iii) Polyclonal activation. Acute murine T. cruzi infection induces antibody production that lack a T. cruzi specificity and include self antigens, suggesting polyclonal B cell activation [52]. The T. cruzi secreted protein TcPA45 has been described as a T cell independent B cell mitogen in mice, and the inhibition of its proline racemase activity blocked its mitogenic effects [53]. Here we critically review the reports on autoimmunity in Chagas' disease patients and experimental models, and discuss their possible pathogenic role under the light of current criteria to establish an autoimmune etiology. 4.1. Animal Models
4. IMMUNOPATHOGENESIS OF CHAGAS' DISEASE The time scale dissociation between high parasitemia and tissue pathology, allied to the scarcity of T. cruzi in CCC heart lesions as discussed in the previous section [40, 41, 47, 48] prompted early investigators [1] to suggest that the mononuclear cell infiltrate lymphocytes in the heart should recognize and mount delayed-type hypersensitivity responses towards a tissue-specific heart component as a result of chronic T. cruzi infection, the so-called autoimmune hypothesis of pathogenesis. Several mechanisms have been suggested to play a role in the triggering of autoimmunity after infection. The three mechanisms described below have been demonstrated in Chagas'disease patients
One of the critical points in validating autoimmunity and molecular mimicry in Chagas' disease is the use of animal models that reproduce all the components of human CCC. Chronically infected mice and rats may develop an inflammatory infiltrate and fibrosis in the heart [54-56] without dilated cardiomyopathy. In spite of the lack of progression to the life-threatening stages of Chagas heart disease, the availability of molecular tools has made the T. cruzi infected mouse a preferential model for studying the pathogenesis of Chagas' disease.
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Table 1. Autoreactivity after T. cruzi infection
.Host component
Host
Molecular definition
Reference
Cardiac myosin Cardiac myosin, p 150 Actin Desmin Tubulin Heart homogenate 43 kDa muscle glycoprotein Nervous tissue, Heart and skeletal muscle 2nd extracellular loop, M2 cholinergic receptor 2nd extracellular loop, 131adrenergic receptor M2 cholinergic receptor M2 cholinergic receptor 2nd extracellular loop, M2 cholinergic receptor Neurons Sciatic nerve homogenate Small nuclear ribonucleoprotein Heart homogenate Cardiomyocytes Cardiomyocytes
M M M M M M M M M M H
CD4+ T cells Serum IgG Serum IgG Serum IgG Serum IgG T cells Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG Serum IgG T cells T cells T cells
[67] [51, 62] [51] [62] [61] [65, 66, 68] [57] [58] [60] [164] [124] [119, 120, 165] [ 121] [110] [111] [129] [133, 134] [137] [107]
H H
H H H H H
Rb
Abbreviations: M, mouse; H, human; Rb, rabbit. 4.1.1. Murine models 4.1.1.1. Autoimmunity Autoantibodies. During T. cruzi infection, mice can display antibodies specific for various autoantigens contained in target tissues such as cardiac [57] or nervous tissue [58, 59]. Autoantibodies against several defined autoantigens have been described in the last decades in sera from T. cruzi-infected mice. Acutely and chronically T. cruzi infected mice develop antibodies that recognize epitopes from the second extracellular loop of the 131-adrenergic and M2-muscarinic receptors and these antibodies are able to activate and regulate calcium channels in isolated cardiomyocytes [60]. T. cruzi infected C57BL/6 X BALB/c F1 mice display high affinity IgG against tubulin that are detected during chronic infection [61]. Sera from acutely infected A]J mice or chronically infected C57BL/6 mice strongly recognized contractile/cytoskeletal proteins such as cardiac myosin, desmin [51, 62] and actin [51]. The
452
novel autoantigen Cha was strongly recognized by sera from infected mice during different stages of infection [63]. This autoantigen was identified by screening of cDNA libraries with sera from Chagas' disease patients and its mRNA is expressed in several tissues including heart and skeletal muscle [63]. Autoreactive T cells. Early evidence for T cell autoreactivity against heart antigens has come from experiments reporting that splenic lymphocytes from chronically T. cruzi infected mice displayed cytotoxicity against normal syngeneic neonatal cardiac myofibers in vitro [64]. Peripheral T cells from infected mice showed in vitro proliferative responses to heart homogenates highlighting the participation of autoreactive T cells in experimental T. cruzi infection [65, 66]. The first evidence for the T cell recognition of a defined heart-specific autoantigen was provided by Rizzo and co-workers [67], who showed that lymph node cells from chronically T. cruzi-infected BALB/c and CBA
mice proliferated in vitro in the presence of syngeneic cardiac myosin. Acutely T. cruzi infected AJJ mice developed delayed type hypersensitivity (DTH) response against cardiac myosin and displayed intense myocarditis [51 ]. Induction of tolerance with a myosin-enriched cardiac homogenate plus anti-CD4 antibody prior to T. cruzi infection resulted in less intense myocarditis and fibrosis when compared to non-tolerized infected mice [68]. Since cardiac myosin is still the single heart-specific defined autoantigen recognized by T cells from T. cruzi-infected mice, these results reinforce the participation of cardiac myosin autoimmunity in the pathogenesis of chronic murine T. cruzi infection. Myosin is the most abundant heart protein, making up to 50% of muscle protein by weight [69]. It is a major antigen in several instances of heart-specific autoimmunity [70-73]; moreover, immunization with cardiac myosin in complete Freund's adjuvant induces severe T-cell dependent myocarditis in genetically susceptible mice [74-77]. The passive transfer of lymphoid cells can validate the pathogenic role of autoimmune T cells. The transfer of T cell populations from chronically infected mice to naive recipients induced nerve inflammatory lesions [78]. Moreover, injection of CD4+ T cells from BALB/c mice chronically infected with T. cruzi (Colombiana strain) adjacent to newborn syngeneic hearts that had been grafted into naive BALB/c recipients resulted in complete rejection of the transplanted heart [66] in the absence of T. cruzi [79], suggesting a pathogenic role for autoreactive T cells. The autoreactive nature of such T cells was disputed using a passive transfer model similar to that used by Ribeiro-dosSantos [66] but using different T. cruzi-mouse strain combinations [80]. A heart-specific CD4+ T cell line (with a Thl cytokine profile) from a chronically T. cruzi-infected BALB/c mouse was able to cease the beating of intact cardiac cells cultivated in vitro leading to their destruction [81]. Transfer of this line to BALB/c nude mice simultaneously immunized with syngeneic heart homogenates resulted in intense myocarditis [81]. Girones and co-workers showed that adoptive transfer of splenic T cells from chronically infected mice to naive recipients induced myocarditis in the latter and triggered antibody response against the Cha autoantigen [63]. However, the presence of T. cruzi in the T
cell suspension was not tested and the possibility of myocarditis secondary to T. cruzi infection cannot be ruled out. 4.1.1.2. Molecular mimicry Crossreactive antibodies. Monoclonal antibodies recognizing T. cruzi crossreactively recognized mouse cerebellar neurons and astrocytes [82]. Later it was shown that sulphated glycolipids were the target antigens; administration of such monoclonal antibodies induced paralysis and death by respiratory failure [83, 84]. Van Voorhis & Eisen (Table 3) described a crossreactive epitope (TPQRKTTEDRPQ) between FL- 160, a 160 kDa T. cruzi flagellar protein, and a neuronal 47 kDa protein; the peptide was capable of inhibiting the binding of anti-FL-160 antibodies to the neuronal antigen [85-87]. Crossreactive antibodies between T. cruzi and myelin basic protein was observed in experimentally infected mice [88] (Table 2). Sera from T. cruzi infected mice contained crossreactive antibodies recognizing microtubule-associated proteins (MAP) from T. cruzi, rat fibroblasts and bovine brain; such antibodies were also present in patient's sera, but without association with clinical symptoms [89]. A functionally active monoclonal antibody against M2 muscarinic receptors obtained from T. cruzi infected mice was shown to be crossreactive with a 150 kDa polypeptide from T. cruzi [90]. Peptides from ribosomal proteins P0 and P213 could block serum antibodies with muscarinic activity [91], probably by an interaction with the second extracellular loop of the receptor. The immunization of BALB/c mice with R13 synthetic peptide from C-terminal of T. cruzi ribosomal proteins P0 [92], P1, P2 [93, 94] and P2~ [92, 95] induced increased heart frequency and heart functional alterations without inflammatory lesions, as well as anti-ribosomal protein and anti-heart autoantibodies. A murine monoclonal antibody that recognizes R13 peptide (a segment of the P21~) had a positive chronotropic effect on cultured neonatal rat cardiomyocytes [96]. Further in support of the concept of crossreactivity between T. cruzi and heart antigens, it has been reported that mice with experimental autoimmune myocarditis induced by immunization with heart
453
Table 2. Molecular mimicry after T. cruzi infection Host component
T. cruzi antigen
Host
Molecular definition
Reference
Neurons, liver, kidney, testis Neurons Neurons Heart tissue Heart and skeletal muscle Human cardiac Myosin heavy chain Human cardiac Myosin heavy chain 95KDa myosin tail Skeletal muscle Ca++ Dependent SRA Smooth and striated muscle Glycosphingolipids MAP (brain) Myelin basic protein 28 kDa lymphocyte membrane protein 47 kDa neuron protein 23 kDa ribosomal protein Ribosomal P protein [~1-adrenoreceptor M2 muscarinic receptor
? ? Sulphated glycolipids ? Microsomal fraction B13 protein
M, R R H M H H
[ 166] [167] [82, 84, 168, 169] [ 170] [98, 171] [44, 145, 147]
Cruzipain
M
Mab Mab Mab Serum IgG Mab rDNA, Ab* T cell clones Ab
T. cruzi cytoskeleton SRA
M Rb, H
Mab AS
[ 173] [ 105, 138]
150 kDa protein glycosphingolipids MAP T. cruzi soluble extract 55 kDa membrane protein
H, M H, M H, M M H, M
Serum IgG Serum IgG r D N A ,AS Serum IgG, T cells Mab
[174] [ 175] [89] [88] [ 176]
FL- 160 23 kDa ribosomal protein Ribosomal P protein Ribosomal P0 and P213proteins 150 kDa protein
H H H H
rDNA, AS Ab rDNA, Ab, SP rDNA, Ab, SP
H, M
Mab
[85-87] [ 142] [ 140] [91, 92, 95, 96, 123, 143] [90]
R 13 peptide from ribosomal protein P1, P2 ?
M
IgG 1, IgG2
[ 177]
H
Ab
[128]
SAPA, 36KDa TENU2845
M
Ab, T cells
[63]
131-adrenoreceptor M2 cholinergic receptor 38-kDa heart antigen Cardiac muscarinic Acetylcholine receptors (mAChR) Cha antigen
[ 172]
Abbreviations: M, mouse; H, human; Rb, rabbit; R, rat; AS, antiserum; Ab, patient antibody; Mab, monoclonal antibody; rDNA, recombinant DNA; SP, synthetic peptides.
homogenate developed anti-T, cruzi antibodies [97]. Monoclonal antibodies produced against T. cruzi microsomal fraction crossreacted with myocardial and skeletal muscle [98]. Giordanengo and coworkers (2000) showed that mice immunized with cruzipain (a major cystein protease from T. cruzi) devoid of enzymatic activity developed crossreactive anti-cardiac myosin heavy chain autoantibodies, eletrocardiographic conduction disturbances and myositis [99, 100].
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Crossreactive T cells. Girones and co-workers [63] showed that spleen cells from mice immunized with a peptide derived from the shed acute phase protein from T. cruzi (SAPA 782-800) display in vitro proliferative response against the Cha autoantigen or a homologous peptide epitope Cha [254-272], suggesting the existence of T cell cross-reactivity between T. cruzi and host antigens. However, definite proof of T cell cross-reactivity can only be demonstrated with T cell clones.
4.1.2. Other animal models
Other animals infected by T. cruzi, such as rabbits, dogs and monkeys have been shown to develop the dilated cardiomyopathy typical of end-stage CCC patients [ 101-103] but these larger outbred animals require long-term follow up, appropriate facilities and expensive experiments. T. cruzi infected rabbits can develop cardiac and pathological alterations such as cardiomegaly, focal myocarditis, fibrosis and myocardial as well as ECG dysfunction [101, 104] consistent with CCC. Serum antibodies from rabbits immunized with microsomal membranes of T. cruzi recognize Ca2+-dependent ATPase from the sarcoplasmic reticulum membranes from skeletal and heart muscle (SRA) indicating crossreactive recognition between T. cruzi and host antigens [105]. Repeated injection of T. cruzi subcellular fractions in rabbits resulted in myocardial inflammatory lesions [106] and peripheral T cells from rabbits experimentally infected with T. cruzi also displayed cytotoxicity to uninfected cardiomyocytes [107]. T. cruzi infected monkeys developed cardiac dysfunction including ECG alterations and ventricular dilation [ 108]. Recent reports have shown that chronically T. cruzi infected Syrian hamster develops a cardiomyopathy resembling the human cardiac disease, including multi-focal or diffuse myocarditis, intense fibrosis, cardiac dysfunction, macroscopic ventricle dilation and associated deaths (109; Bi!ate et al, submitted], suggesting that the Syrian hamster may be a promising small rodent model for the study of immunological intervention protocols. However, the small number of hamster-specific immunological/molecular tools limits the studies of pathogenic mechanisms.
4.2. Human Chagas' Disease 4.2.1. Autoimmunity Autoantibodies. In human Chagas' disease, there is a net loss of neurons from the autonomic system along the hollow viscerae and the heart [15], and sera from over 80% of Chagas' disease patients contained anti-neuron autoantibodies [ 110] (Table 1). Antibodies against sciatic nerve homogenate have been found in sera from Chagas disease
patients [111]. Furthermore, the autonomic nervous system dysfuction observed in symptomatic and asymptomatic patients may be related to such a denervation [112, 113]. This clinical observation prompted investigators to study the possible involvement of antibodies against adrenergic or muscarinic cholinergic receptors. Functional antibodies against adrenergic G-protein-coupled receptors were first demonstrated in serum from Chagas' disease patients as early as 1984 [114-117] and agonistic antibodies against muscarinic (M2) cholinergic receptors received more attention along the last decade [118-120]. Recently, it was shown that functional anti-receptor antibodies bind to the second extracellular loop of the M2 muscarinic receptor [121]. It was also demonstrated that sera from chronic Chagas' disease patients interfere with electric and mechanical activities of embryonic myocardial cells in vivo [122, 123]. These alterations in heart cell function seem to result from the previously demonstrated binding of antibodies to ~-adrenergic and M2-cholinergic receptors on the myocardial cell surface [122-124], and sera from CCC patients induce arrythmia in rabbit hearts [124]. Authors have demonstrated that the presence of such functionally active anti-receptor antibodies does not correlate with heart symptomatology but rather with dysfunction of the autonomic nervous system [121]. Similarly, antibodies against the [~-adrenoreceptor are found in patients with severe idiopathic dilated cardiomyopathy and may be functionally active [125, 126]. Chagas' disease patients showing colonic denervation syndrome display antibodies that recognize and activate M2 muscarinic acethylcoline receptors (mAChR) [127]. Recently, Hernandez [128] showed that antibodies from chronic chagasic patients bind to mAChR in a non-competitive manner and are able to activate the receptor in an agonist-like form resulting in L-type C a 2+ current inhibition. Antibodies against ribonucleoproteins [ 129] have been detected during T. cruzi infection. Autoantibodies against galectin- 1(Gal- 1), a protein expressed in human heart tissue, are correlated with the severity of cardiac damage in CCC [130]. Gironrs et al [63] showed that the Cha human autoantigen and its major B cell epitope Cha(120-129) (peptide R3) are recognized by sera from Chagas' disease patients [131].
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Evidence suggests that fuctional autoantibodies may play a role in the pathogenesis of Chagas' disease. Whether or not the remaining autoantibodies play a pathogenic role or are mere epiphenomena resulting from antigen exposure or immune system activation is still a subject of intense debate. A recent report identified complement C5-C9 membrane atack complexes formed cardiomyocyte membranes from CCC heart tissue [132], suggesting that complement activation - perhaps induced by autoantibodies - could play a role in heart tissue damage~ Autoreactive T cells. Early studies have shown that
cardiac tissue homogenate induced lymphokine production [133, 134] but not proliferative responses among CCC peripheral blood T cells [135, 136]. Non-infected cardiomyocytes were targets of cytotoxicity by CCC PBMC [137]. Interestingly, tests related to effector function (lymphokine production, cytotoxicity) yielded positive results in patients, whereas T cell proliferation assays apparently had uniformly negative results.
ergic receptor sharing an acidic sequence stretch, AESDE, and the main epitopes of the C termini of the T. cruzi ribosomal P0, and the P 1 and P2 proteins has also been shown [ 123, 143, 144]. A recent study demonstrated that peptides from ribosomal proteins P0 and P2b bearing acidic epitopes or the second extracellular portion of the muscarinic acetylcholine receptor could block antibodies from CCC sera with muscarinic activity [91 ]. Our group has shown that affinity-selected antihuman ventricular cardiac myosin heavy chain antibodies from Chagas' disease patients' sera specifically recognized a defined T. cruzi antigen [ 145], the recombinant tandemly repetitive protein B13 [ 146]. Cardiac myosin-B 13 crossreactive antibodies (116/140kDa) were present in sera from 100% of CCC patients but only 14% of ASY patients [145]; sera from 100% of both CCC and ASY patients recognized cardiac myosin. In addition, a monoclonal antibody raised against solubilized T. cruzi cytoskeletons crossreactively recognized a cardiac myosin tail epitope (95kDa) [30]. Crossreactive T cells. CD4+ T cell clones expanded
4.2.2. Molecular mimicry
Crossreactive antibodies. Several reports of immunological cross-reactivity/antigenic mimicry between defined T. cruzi and host self-antigens have been described. Given the evolutionary conservation of primary sequences of many key structural proteins or enzymes from protists to humans, it is not surprising that this kind of crossreactive antigens can be detected. CCC patients' sera contained antibodies that crossreactively recognized a conserved Ca2§ ATPase present in T. cruzi microsomal membranes as well as heart and skeletal striated muscle sarcoplasmic reticulum membranes (SRA) [138]. Sera from CCC patients possessed antibodies against a C-terminal epitope of T. cruzi ribosomal P2[~ protein (R13: EEEDDDMGFGLFD), more frequently than ASY patients. This peptide is similar to the corresponding C-terminus of mammalian ribosomal P protein (H13: EESDDDMGFGLFD) [139, 140]. It was claimed that such antibodies were indeed autoreactive, and able to bind to mammalian P protein [ 123, 139] but some authors could not reproduce this finding [ 141, 142]. Evidence of cross-reactivity between a ~ 1-adren-
456
in the absence of exogenous antigen - from heart tissue of a CCC patient crossreactively recognized cardiac (but not skeletal) myosin heavy chain and T. cruzi protein B 13 [44]. However, none of the 17 tested clones responded to the immunodominant recombinant T. cruzi antigens CRA, FRA, JL5 or B 12 or to T. cruzi trypomastigote lysate [44]. Authors hypothesized that autoimmunity/molecular mimicry targets, rather than the direct antigenic stimulus of T. cruzi were the primary stimuli of the heart tissue-damaging T cell infiltrate. For that matter, in vitro sensitization of peripheral lymphocytes from a T. cruzi seronegative individual with B 13 protein elicits cardiac myosin-crossreacfive T cell clones [ 147]. Authors hypothesized that in vivo challenge with B 13 antigen along T. cruzi infection could break immunological tolerance towards cardiac myosin and elicit cardiac myosinresponsive T cells in vivo. Variant B13 epitope S 15.4 was shown to be preferentially recognized by CCC but not ASY patients lAbel et al, submitted] and also induced myosin-crossreactive T cell lines and clones [ 148].
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5. FINAL R E M A R K S AND HYPOTHESIS
Attributing autoimmune disease status to Chagas' disease, a low-grade, systemic chronic infection with documented autoimmune phenomena, is a key issue. Inasmuch as some authors refuse to classify diseases associated to a known infectious agent as autoimmune, [149, 150], the autoimmune component becomes important when tissue damage is secondary to autoimmune mechanisms. When confronting the available data with the criteria of autoimmune disease described by different authors [ 151,152], it can be seen that CCC fulfills several of them. The identification of T cell crossreactive antigens (Table 2), with reproduction of pathobiological changes by passive transfer in murine models in the absence of T. cruzi parasites [66, 81], and the amelioration of inflammation as a consequence of tolerance induction to myocardial antigens [68] together with the induction of autoimmune disease after immunization with cardiac myosin, the major candidate self antigen in Chagas' disease cardiomyopathy [71], have all been shown. The isolation of cardiac myosin-autoreactive T cells in molecular mimicry with T. cruzi B 13 protein from affected tissue [44] is considered important indirect evidence. TCR Vo~ region usage restriction [153] is considered circumstantial evidence according to Rose and Bona's criteria [154]. Together with the demonstration that in vitro immunization with B 13 protein or B 13 epitopes elicits T cell lines and clones crossreactive with cardiac myosin [147] or its epitopes [148], results are in line with amajor role for autoimmunity in CCC. Our findings, together with the available literature, allow us to suggest a hypothesis for the development of autoimmune cardiomyopathy upon T. cruzi infection of a susceptible individual. During the acute phase of T. cruzi infection, CD4+ T cell clones are sensitized in the periphery by macrophages presenting T. cruzi antigens (e.g. B 13 protein or ribosomal proteins) after endocytosis of the parasite, which will induce IL-12 production [155] and expression of the costimulatory molecule B7 [156]. Such T cells become experienced, Tl-type T cells [31,157, 158]. Susceptible individuals possess T cell clones that crossreactively recognize T. cruzi and heart epitopes (e.g. cardiac myosin) [147]. During the acute phase of disease, CD4+ T cells
and CD8+ T cells migrate to the heart driven by chemokines such as RANTES, MIP-lct and MIP113 produced by T. cruzi infected cardiomyocytes and macrophages, where they accumulate and may participate in the immune response against T. cruzi [159]. As heart tissue parasitism decreases, the persistence of inflammation may be maintained by inflammatory cytokines such as IFN-y [31,160] produced by intralesional heart antigen-specific CD4+ T cells (present only in susceptible individuals) [44, 51, 67], upon recognition of T. cruzi-crossreactive heart antigen epitopes (e.g. from cardiac myosin) constitutively presented by HLA class II+ interstitial macrophages [50] (Fig. 1). It has been demonstrated that PBMC from CCC patients produce more IFN-y [31, 161 ] and less IL- 10 [30] than ASY patients. T. cruzi infection causes an increase in plasma TNF-t~ levels, which are further increased among severe CCC patients [32]. Severe CCC patients carrying high TNF production alleles display significantly shorter survival than carriers of other alleles (unpublished observations from our group), in line with the known cardiotoxic effects of TNF-ct [162]. Thus, we postulate that the differential susceptibility to CCC development is secondary to checkpoints that involve at least the regulation of inflammatory cytokine production and the ability to crossreactively recognize the target antigens. The dichotomy between tissue-damaging autoimmunity and systemic parasite persistence may indeed be false in the case of Chagas disease. Whereas effective measures to control the parasite burden should be welcome and probably would reduce the tissue damage, published evidence apparently fail to indicate that currently available anti-T, cruzi drugs are effective. On the other hand, immunological intervention such as cytokine blockade and tolerance induction are immediately available and perhaps should be of more immediate use to the millions of patients already infected by T. cruzi in the Americas. The identification of relevant heartspecific antigenic targets may allow the future use of therapeutic antigen-specific immunomodulation (e.g. tolerance induction) to avoid progression of chronic inflammation and heart tissue damage without interfering with the protective immune response against the parasite, as recently shown in murine models with cardiac antigens [68]. The T 1type cytokine profile observed in CCC heart lesions
457
Figure 1. Demonstrated events in the immunopathogenesis of CCC. Tissue sections come from chronically T. cruzi-infected Syrian hamsters.
458
is consistent with a delayed-type hypersensitivity mechanism of tissue damage. This may open the possibility of using immune deviation therapy, in which cytokines produced in the target organ switch from inflammatory to anti-inflammatory, to control tissue damage in CCC. Since TNF-ct plays an important role in the pathogenesis of CCC, as addressed before, blocking this cytokine could prevent progression of cardiac lesions. Another novel and promising strategy to regenerate myocardium from T. cruzi chronically infected patients is the autologous bone marrow stem cell transplantation. Injection into coronary arteries of bone marrow cells regenerate infarcted myocardium in mice [ 163]. The effectiveness of bone marrow cell transplantation in the ameliorating heart inflammation and fibrosis and its consequent clinical response is currently being assessed in murine models and CCC patients. If successful, this kind of therapy could be an alternative therapy for the severe CCC patients in the near future.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Anti-Saccharomyces Cerevisiae Antibodies and Autoimmune Diseases Ilan Krause ~,2and Gui Milo ~
1Department of Medicine E, Rabin Medical Center, Beilinson Campus, Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer; 2Sackler Faculty of Medicine, Tel-Aviv University, Israel
1. CROHN'S DISEASE
Inflammatory bowel diseases (IBD) are subdivided into ulcerative colitis (UC) and Crohn's disease (CD) [1, 2]. Several lines of evidence suggest that CD and UC are different diseases. However, some patients (10-20%) cannot be easily classified into either and a final diagnosis of intermediate colitis is made. Making an earlier, more accurate diagnosis of IBD is important, as the management of CD and UC is different, especially when surgery is planned. A search for serological tests to differentiate CD from UC has been underway for a long time. An ideal serological marker should have high sensitivity, high specificity, and high predictive values. In 1988 Main et al [3], using a boiled suspension of Saccharomyces cerevisiae as antigenic substrate in an enzyme linked immunosorbent assay (ELISA), were the first to show a high titer of antibodies against Saccharomyces cerevisiae among patients with CD but not in patients with UC. Reactivity of sera from patients with CD has been subsequently described, regardless of the Saccharomyces cerevisiae strain used [4, 6, 7]. It seems that ASCA is more prevalent in patients with small bowel Crohn's disease than those with colonic disease [5, 8, 9], and is also correlated with a younger age of disease onset. Furthermore, although the presence of ASCA does not correlate with disease activity, titers were found to decrease after resection of damaged gut tissue [10]. It was also reported that ASCA titer was significantly lower in patients with CD taking mesalazine than in those not taking it, although administration of corticosteroids did not affect ASCA levels [11]. Despite the lack of
standardization within this area, in some studies elevated titers of IgG and/or IgA ASCA have been found in 50-80% of CD patients but rarely (<10%) at similar titer levels in UC patients or in healthy controls (<5%) [5, 10, 12]. Quinton et al [2] studied 100 CD patients, 101 UC patients and 163 healthy controls in order to determine the diagnostic role of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) and ASCA in inflammatory bowel disease. The combination of a positive ASCA and a negative pANCA tests yielded a sensitivity, specificity, and positive predictive value of 49%, 97%, and 96% respectively for CD. In the other hand, a positive pANCA and a negative ASCA tests yielded a sensitivity, specificity, and positive predictive value of 57%, 97%, and 92.5% respectively for UC. Similar results were obtained in another study, encompassing 582 IBD patients [12]. It seems, therefore, that combined measurement of both pANCA and ASCA may be used advantageously in the subclassification of IBD patients with intermediate colitis. Family studies indicate that ASCA may be used to identify individuals at risk for CD. Lindberg et al [ 13] studied 26 monozygotic twin pairs with inflammatory bowel disease for serum antibodies against whole Saccharomyces cerevisiae and yeast cell wall mannan. The twins were made up of five pairs concordant and nine pairs discordant for Crohn's disease, and two pairs concordant and 10 pairs discordant for ulcerative colitis. Twins who had developed Crohn's disease displayed high antibody titres towards yeast cell wall mannan in particular, but also to whole Saccharomyces cerevisiae yeast of all antibody types (IgA, IgG, and IgM). In families in which at least two members were affected with CD,
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ASCA were detected in 69% patients with CD and in 20% healthy relatives, parents or siblings [9]. Similar rates of ASCA in first-degree relatives of patients with CD (16-25%) were reported in other studies [14-16]. Interestingly, the prevalence of ASCA in relatives did not depend on the ASCA status of affected members [9]. Furthermore, ASCA were significantly increased in a considerable number of unaffected relatives of IBD families, irrespective of the characteristics of their disease (UC, CD, mixed, ASCA positive or ASCA negative) [ 17].
2. BEH(~ET'S DISEASE Behqet's disease (BD) is a multi-system disorder, the clinical expression of which may be dominated by mucocutaneous, articular, neurologic, urogenital, vascular, intestinal or pulmonary manifestations [18]. BD and CD share various clinical similarities, including mucocutaneous manifestations (recurrent oral ulcers, erythema nodosum), gastrointestinal disease favoring the terminal ileum, recurrent arthritis as well as uveitis, thus raising the possibility of certain etiologic and pathogenic factors common to both diseases. In a recent study, Krause et al [19] found a high rate (48.1%) of ASCA in a group of 27 BD patients, compared to 10% in three control groups of patients with recurrent oral ulcers, systemic lupus erythematosus or healthy volunteers No correlation was found between ASCA and any B D-associated clinical manifestation nor the presence of HLA-B5. Furthermore, no difference was found in the rate of major oral ulcers nor in disease severity between positive- and negative-ASCA BD patients. These findings are supported by two other recent studies. Oshitani et al [20] analyzed ASCA IgG subclasses in sera from patients with IBD, healthy controls, and seven patients with intestinal BD. IgG4 ASCA was significantly increased in patients with IBD. In the patients with intestinal BD, IgG1, IgG3, and IgG4 ASCA were increased. Similarly, Kim et al [21] reported on a high prevalence (41.7%) of ASCA in a group of 36 patients with Behqet colitis. Furthermore, four out of eight healthy relatives of the B D patients also tested positive for ASCA [21]. It is possible that BD has similar immunologic pathogenesis to that of CD, and that the presence of ASCA may prove valuable
468
as a preclinical marker of the disease. It is also conceivable that the presence of ASCA do not pose an increased risk for a more severe disease course in B D. Further prospective studies are needed to evaluate whether ASCA titers are correlated with clinical relapses of BD.
3. CELIAC DISEASE Celiac disease is defined as a disease of the proximal small intestine, characterized by damage to the small intestinal mucosa, and is associated with permanent intolerance to gluten [22]. Clinical symptoms and abnormal small bowel histology resolve on removal of gluten from the diet. Whenever suspected, the diagnosis of celiac disease is verified with an intestinal biopsy and an unequivocal clinical response to a gluten-free diet. Screening for elevated levels of several circulating autoantibodies is helpful in selecting suspected patients for the requirement of an intestinal biopsy. These serological tests include the detection of anti-endomysium (EMA) and antigliadin antibodies. The endomysial antigen has been identified as tissue transglutaminase (fiG). EMA, and consequently anti-fiG antibodies are considered highly specific and sensitive for celiac disease [22]. Few studies, on small numbers of patients with celiac disease, have addressed the relation between ASCA and the disease. Giaffer et al [8] reported on a group of 14 patients with celiac disease, in whom high levels of IgG, but not IgA ASCA were found, the antibody responses being indistinguishable from those found in Crohn's disease. In a recent study of 37 patients with biopsy-confirmed celiac disease, the prevalence of ASCA was found to be high for both IgA (29.7%) and IgG (32.4%), the combined prevalence being 43.2% [23]. In contrast, Darroch et al [24] did not observe increased levels of either IgG or IgA ASCA in 17 patients with celiac disease. Large-scale studies are still needed to confirm a possible relation between ASCA and celiac disease.
4. ANKYLOSING SPONDYLITIS Ankylosing spondylitis (AS) is an inflammatory disorder of unknown cause that primarily affects the axial skeleton. AS belongs to the spondyloar-
thropathies (SPA) which bold phathophysiologocal similarities with IBD. Subclinical bowel inflammation is present in up to 68% of patients with SpA, and 7% of these patients develop CD after 24-92 months [25]. On the other hand, rheumatic manifestations are common extraintestinal manifestations of CD, and 35% of patients with CD fulfill the criteria for SpA [26]. Recently, Hoffinan et al [27] studied the levels of ASCA in 26 patients with CD, 108 patients with SpA (including 43 patients with AS), 56 patients with rheumatoid arthritis (RA) and 45 healthy controls. ASCA IgA levels were significantly higher in SpA, and more specifically in AS, than in healthy controls and patients with RA. No correlation between the presence of subclinical bowel inflammation and ASCA IgA levels was noted. However, it remains to be evaluated whether patients with SpA with ASCA have an increased risk of developing CD.
5. A U T O I M M U N E LIVER DISEASES The autoimmune liver diseases (namely, autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC)) are chronic inflammatory disorders of unknown etiology that are characterized by immunological features generally including a variety of circulating and characteristic autoantibodies [28]. At the light of the epidemiological association between IBD and PSC, the prevalence of ASCA in autoimmune liver diseases was recently studied. Reddy et al [29] reported an ASCA rate of 22% in 80 patients with AIH, 19% in 31 patients with PBC and 20% in 15 PSC patients. Muratori et al [30] on the other hand, found higher ASCA rates in 17 anti-mithochondrial antibody-negative PBC patients (44%) and in 25 PSC patients (53%). ASCA reactivity did not correlate with biochemical parameters associated with liver disease. Furthermore, in PSC patients ASCA positivity did not predict the presence of concomitant IBD. The relatively low prevalence of ASCA in autoimmune liver diseases (20-50%) suggests it may have lower diagnostic significance. Yet, it may reflect common background susceptibility with IBD with a higher level of expression in CD. Given that only few studies evaluated ASCA in autoimmune liver diseases, the actual incidence as well its
clinical significance in terms of disease progression and expression still awaits further prospective largescaled studies.
6. C O N C L U D I N G R E M A R K S The introduction of ASCA in the last decade has provided a major diagnostic tool for the differentiation between CD and UC in IBD patients. Furthermore, ASCA may be useful in identifying IBD patients-relatives who are at risk for future development of the disease. Although ASCA were claimed to be quite specific for CD, a growing number of studies have identified high levels of ASCA in several other autoimmune diseases, among them Beh~et's disease, spondyloarthropathies, primary sclerosing cholangitis and celiac disease. We may, therefore, deal not with a CD-specific antibody, but rather a panel of ASCA-associated diseases, of which CD is the prototype one. There is yet no evidence that ASCA are involved in the pathophysiology of CD, e.g. by mediating cytotoxicity against intestinal epithelial cells. Instead, they may be the result of a cross-reactivity between the respective target antigen (or tissue) used for their detection and yet undetermined luminal antigens. The possible pathogenic potential of ASCA still awaits further studies, in human patients as well as experimental animal models.
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Su C, Lichtenstein GR. Recent developments in inflammatory bowel disease. Med Clin North Am 2002;86: 1497-523. BanerjeeS, Peppercorn MA. Inflammatory bowel disease. Medical therapy of specific clinical presentations. Gastroenterol Clin North Am 2002;31:185-202. Main J, McKenzie H, Yeaman GR et al. Antibody to Saccharomyces cerevisiae (bakers' yeast) in Crohn's disease. BMJ 1988;297:1105-6. Sendid B, Colombel JF, Jacquinot PM et al. Specific antibody response to oligomannosidic epitopes in Crohn's disease. Clin Diagn Lab Immunol 1996;3: 219-26. QuintonJF, Sendid B, Reumaux D et al. Anti-Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflamma-
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McKenzie H, Parratt D, Main J et al. Antigenic heterogeneity of strains of Saccharomyces cerevisiae and Candida albicans recognised by serum antibodies from patients with Crohn's disease. FEMS Microbiol Immunol 1992;4:219-24. McKenzie H, Main J, Pennington CR et al. Antibody to selected strains of Saccharomyces cerevisiae (baker's and brewer's yeast) and Candida albicans in Crohn's disease. Gut 1990;31:536-8. Giaffer MH, Clark A, Holdsworth CD. Antibodies to Saccharomyces cerevisiae in patients with Crohn's disease and their possible pathogenic importance. Gut 1992;33:1071-5. Sendid B, Quinton JF, Charrier Get al. Anti-Saccharomyces cerevisiae mannan antibodies in familial Crohn's disease. Am J Gastroenterol 1998;93:1306-10. Ruemmele FM, Targan SR, Levy G et al. Diagnostic accuracy of serological assays in pediatric inflammatory bowel disease. Gastroenterology 1998; 115:822-9. Oshitani N, Hato F, Matsumoto T et al. Decreased antiSaccharomyces cerevisiae antibody titer by mesalazine in patients with Crohn's disease. J Gastroenterol Hepato12000; 15:1400-3. Peeters M, Joossens S, Vermeire S et al. Diagnostic value of anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease. Am J Gastroentero12001 ;96:730-4. Lindberg E, Magnusson KE, Tysk C et al. Antibody (IgG, IgA, and IgM) to baker's yeast (Saccharomyces cerevisiae), yeast mannan, gliadin, ovalbumin and betalactoglobulin in monozygotic twins with inflammatory bowel disease. Gut 1992;33:909-13. Seibold F, Stich O, Hufnagl R et al. Anti-Saccharomyces cerevisiae antibodies in inflammatory bowel disease: a family study. Scand J Gastroentero12001 ;36: 196-201. Vermeire S, Peeters M, Vlietinck R et al. Anti-Saccharomyces cerevisiae antibodies (ASCA), phenotypes of IBD, and intestinal permeability: a study in IBD families. Inflamm Bowel Dis 2001;7:8-15. Glas J, Torok HP, Vilsmaier F et al. Anti-Saccharomyces cerevisiae antibodies in patients with inflammatory bowel disease and their first-degree relatives: potential clinical value. Digestion 2002;66:173-7. Annese V, Andreoli A, Andriulli A et al. Familial
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9 2004 Elsevier BoV All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
The Infectious Origin of the Antiphospholipid Syndrome M. Blank 1, M. Eisenstein 2, R.A. Asherson 3, R. Cervera 4 and Y. Shoenfeld 1,5
1Internal Medicine 'B' and The Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, and Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; 2Department of Chemical Services, The Weizmann Institute of Science, Rehovot Israel; 3Rheumatic Diseases Unit, University of Cape Town School of Medicine, Cape Town, South Africa; 4Department of Autoimmune Diseases, Institut Clinic d'Infeccions i Immunologia, Hospital Clinic, Barcelona, Catalonia, Spain; 5Incumbent of the Laura Schwarz-Kipp Chair for Autoimmunity, Tel-Aviv University, Tel-Aviv, Israel
1. INTRODUCTION There is a general consensus that autoimmune diseases have a multifactorial etiology, depending on both genetic and environmental factors. Microbial agents or viruses can induce autoimmune diseases by a variety of mechanisms [1-5]. For example, proteins of certain infectious agents can act as polyclonal activators on unique lymphocyte subsets. Viruses can preferentially infect/destroy a particular T cell subset, leading to an imbalance in the immune response. In other instances, infectious agents can up-regulate Thl cytokines, thereby increasing selected expression of molecules such as MHC glycoproteins, as well as activation of costimulatory molecules. Several microbial agents have been found to encode superantigens that can selectively activate subset(s) of T cells. Microbes can also direct the release of cytokines and chemokines, which can act as growth, differentiation, or chemotactic factors for different Th populations and regulate expression of MHC class I and class II molecules [1-5]. The healthy immune system is tolerant to the molecules of which the body is composed of. However, one can find that among the major antigens recognized during a wide variety of bacterial, viral and parasitic diseases, many belong to conserved protein families, sharing extensive sequence identity or conformational fits, with host's molecules, namely molecular mimicry. Antigenic similarity of
either molecules' linear amino-acid sequences or their conformational structure between antigens of infectious agents and host tissues might trigger an immune response against the shared determinant. As a result, the tolerance to autoantigens breaks down, and the pathogen-specific immune response that is generated cross-react with host structures to cause tissue damage and disease. A role for molecular mimicry in the pathogenesis of autoimmune diseases has recently been shown in several animal models such as allergic encephalomyelitis, experimental myocarditis and experimental autoimmune uveitis and keratitis [5-10]. Recently, two groups of researchers found that molecular mimicry between common pathogens and [~2-glycoprotein-I ([~2GPI) may be one of the main causes for induction of antiphospholipid syndrome [ 11-14]. ,
,
2. THE ANTIPHOSPHOLPID SYNDROME
The classical "Hughes Syndrome"- antiphospholipid syndrome (APS) is characterized by the presence of direct binding anti-phospholipid antibodies (aPL) or aPL which bind target phospholipid molecules mainly via 132GPI, and/or lupus anticoagulant associated with recurrent fetal loss, thromboembolic phenomena, thrombocytopenia, neurological, heart, skin and other organ disorders [15-20]. Recently defined as systemic APS [21]. The human [32GPI molecule is a heavily glyco-
473
sylated membrane-adhesion glycoprotein (326aa), present in blood plasma at a concentration of --150-300 ~tg/ml [22, 23]. ~2GPI exhibits several properties in vitro which define it as an anticoagulant (e.g inhibition of prothrombinase activity, ADP-induced platelet aggregation and platelet factor IX production) [24, 25]. It has a role in the clearance of apoptotic bodies from the circulation [26]. ~2GPI molecule was found to be immunogenic in vivo. Immunization of BALB/c, PL/J mice, or New Zealand white rabbits with 132GPI resulted in generation of anti-132GPI Abs [27-30]. ~2GPI-immunized mice developed high titers of 132GPI-dependent aCL, associated with an increased percentage of fetal resorptions (the equivalent of fetal loss in human APS), thrombocytopenia, and prolonged activated partial thromboplastin time (aPTT), indicating the presence of lupus anticoagulant, a presentation characterized as experimental APS [29]. Acceleration of APS manifestations was observed in MRL/Ipr mice (a mouse model of APS with a genetic background) immunized with ~I2GPI [31 ]. Furthermore, experimental APS was prevented in ~2GPI orally fed mice in which tolarization was induced [32]. Anti-132GPI Abs exert a direct pathogenic effect by interfering with homeostatic reactions occurring on the surface of monocytes, platelets or vascular endothelial cells [33-35]. Anti-132GPI Abs were found to activate monocyte leading to tissue factor release [34, 35] and activate endothelial cells via induction of adhesion molecule expression including E-selectin, ICAM-I, and VCAM-I, NFkB expression [37-39]. In an ex-vivo model of thrombosis these family of Abs induce thrombus formation [40, 41 ]. Passive transfer of these Abs, or Abs directed to ~2GPI related synthetic peptides with homology to common bacteria, into naive mice, resulted in induction of experimental APS [38, 42, 43]. Exchanging of heavy and light chains between pathogenic and non-pathogenic anti-~12GPI single chain Fv, showed that the pathogenic part of the anti-~2GPI molecule is located on the CDR3 of the heavy chain of the immunoglobulin [44]. The target epitopes for anti-132GPI binding to the ~2GPI molecule, as shown by us [38] and other groups [45-53], are spread at several locations over the five domains of the molecule. Various APS patients present differentially diverse panel of anti-1~2GPI Abs directed
to different epitopes [38, 54].
3. ORIGIN OF ANTI-[32GPI ANTIBODIES IN THE PLASMA The ~2GPI molecule and cardiolipin are ubiquitous molecules. Several pathways were proposed to explain the generation of pathogenic Abs toward them. ~I2GPI cryptic epitope was suggested to be exposed upon binding to oxidized surface [55]. Others proposed that epitopes recognized by many aPL Abs are adducts of oxidized phospholipid and associated proteins, including 132GPI molecule [56]. Oxidized form of 132GPI undergo conformational changes presenting neoepitopes which may induce anti-1~2GPI Abs [56, 57]. Presentation of 132GPI molecules on apoptotic cells via binding to phosphatidylserine, could induce B cells with Ig receptors for apoptotic cells and DNA which are positively selected and could cause generation of aPL in appropriate condition [58-61]. During the last few years common bacteriae and viruses were proven to evoke experimental APS [11-14].
3.1. Infection and Anti-Phospholipid Antibodies Many infections may be accompanied by antiphospholipid antibody (aPL) elevations and, in some, these elevations may be accompanied by clinical manifestations of the antiphospholipid syndrome (APS). Several reviews on this important topic have recently deeply detailed in ref 62-65 and all summarized in Tables 1, 2. Skin infections (18%), Human immunodeficiency virus infection (HIV) (17%), pneumonia (14%), Hepatitis C Virus (HCV) (13%) and urinary tract infections constituted the most common infections found as "triggering" factors in the most recent review by Cervera et al [66]. In 9 cases, more than one agent/organ was identified as the source of infection. Other infections less frequently associated with APS included mycoplasma (3 cases), pulmonary tuberculosis (2 cases), malaria (2 cases), P. carinii and leptospirosis in 1 case each. Although IgM isotypes of the anti-cardiolipin Abs seem mainly to be produced, IgG elevations have also been detected in some sera.These infections include, viral, bacterial, as well as parasitic.
Table 1. APS manifestations associated with viral infections
Table 2. Prevalence of aCL antibodies in bacterial, rickettsial, yeast, parasite and spirochetal infectionsa .
Infectious agent
cardiolipin 132GPI APS manifestations
Hepatitis C IgG
+
EBV Varicella Parvovirus B19 CMV HTLV-1 HIV
IgG, IgM IgG, IgM IgG
+ +
IgG, IgM IgA IgG, IgM, IgA
+
Thrombosis NDb
+
Leg ulcer necrosis, PE, VE~ arterial & vein thrombosis, vasculifis, livedo reticularis Thrombocytopenia
Adenovirus IgG
+
Thrombosis,brain infarction PEa, thrombosis PE, thrombosis Thrombosis
aPE: pulmonary embolus. bND: not detected. eVE: Venous thromboembolism.
3.2. The Catastrophic APS and Infections
This recently characterized, unusual and potentially fatal subset of the APS was first defined in 1992 [67]. Since then, more than 150 patients [68, 69] have been fully analyzed and documented in major publications although now more than 200 individual cases have now been reported [70] and its pathogenesis has been reviewed [71]. "Triggering" factors have become increasingly apparent and were present in 51% of cases in the latest analysis [70]. These have included trauma (including surgical, both major and minor) anticoagulation withdrawal, a variety of carcinomas and, most importantly and commonly, infections, which were identified in 24% of these patients. Infections preceding the appearance of CAPS were reported in eight patients by Rojas-Rodrfguez et al [72] and three episodes of CAPS in one patient ("recurrent" CAPS), all of which were precipitated by an infection were reported from Poland by Undas et al [73]. The latest comprehensive analysis of infections and aPL [65] has shown that forty of these 100 cases (40%) manifested CAPS which now seems to be almost
.
.
.
.
.
.
.
.
Infection/ Organism
Frequency Isotype (%)
Typhus Leprosy TB Bacterial endocarditis Helicobacter pylori Mycoplasma pneumonia S. aureus Streptoccocus Streptoccocus pyogenus Salmonella E. Coil Ornithosis Coxiella butneti Leptospirosis Borrelia burgdorferi Saccharomyces Cervevisiae Malaria Kalaazar
20 33-67 27-53 5-44 NDa 20-53 43 80 0-80 60 67 33 42-84 50 14--41 ND
IgG, IgM, IgA IgG, IgM IgG, IgM IgG, IgM IgG, IgM, IgA IgG, IgM, IgA IgG, IgM, IgA IgG, IgM IgG, IgM, IgA IgG, IgM, IgA IgG, IgM, IgA IgG, IgM IgG IgG, IgM IgG
30 ND
IgG, IgM IgG
aND: not defined.
as commonly seen as the "classic" APS following a triggering infection and this number is clearly disproportionate, considering the small number of CAPS reported to date compared with the several thousands of simple or "classic"APS. These infections comprised respiratory (10%), cutaneous (including infected leg ulcers) (4%), urinary tract (45%), gastrointestinal (2%), general sepsis (1%) and others (3%). In the latter group was one patient who developed CAPS following typhoid fever and this patient has been reported in detail [74]. Another patient who developed two large vessel occlusions following typhoid fever has also recently been documented [75] and, although this patient was represented as suffering from CAPS, small vessel occlusions, essential for the diagnosis of CAPS and included as one of the major guidelines for diagnosis were absent [76]. Two patients who developed
Figure 1. Location of the 132GPIrelated peptides identified by the peptide phage display library.
CAPS following malaria have been recently been reported [77] and it has been described following Dengue fever in a patient from Brazil by Levy [78] as well as after immunization for Japanese B Encephalitis in an Israeli patient [79]. Resolution of CAPS following amputation of a gangrenous limb has also been described in two patients with CAPS [80]. Molecular "mimicry" has been proposed as one of the major mechanisms responsible for the development of CAPS following infections [81 ] but there may be an interplay of other mechanisms.
3.3. The Infectious Origin of Circulating Anti~2GPI Abs 3.3.1. Molecular mimicry between common pathogen and anti-~2GPI peptide epitopes as a possible origin for anti-~2GPI Abs We hypothesize that molecular mimicry mechanism between pathogen and [32GPI molecule may be the dause for APS, based on: a) a correlation between APS clinical manifestations and infectious agents in human; b) strong homology between ~2GPI related
476
peptides (target epitopes for anti-132GPI Abs) and different common pathogens, in the protein data bases, summarized in Table 3. Previously, introducing human anti-~2GPI monoclonal Abs derived from APS patients to hexapeptide phage display library, we have identified several synthetic peptides as target epitopes for anti-~2GPI Abs. These [~2GPI related peptides were found to be located on domain I-II (mimotope), domain-M and domain-IV (both linear sequences) (Fig. 1). All the 3 synthetic peptides inhibited activation of endothelial cell in-vitro and induction of experimental APS in naive mice via neutralizing the pathogenic anti-~12GPI Abs [38]. Sera of one APS patient contain diverse panel of anti-peptides Abs [38]. The prevalence of circulating anti-peptide A-C Abs in sera of 295 APS patients ranged between 18% up to 47.5% [54]. Employing the protein data base we found homologies between our and others peptides with common bacteria viruses, yeast and tetanustoxin (Table 3). In order to prove the involvement of molecular mimicry mechanism between the pathogen and ~2GPI molecule as a cause for experimental APS, we immunized naive mice with microbial
pathogens, which share structural homology with the TLRVYK hexapeptide. Anti-TLRVYK were found to Mouse IgG specific to the TLRVYK peptide were affinity purified from the immunized n-rice on a TLRVYK-column and passively infused i.v. into naive mice at day 0 of pregnancy. APS-clinical parameters were evaluated in the infused mice on day 15 of pregnancy. Following immunization, various levels of mouse anti-132GPI Abs were observed, the highest being detected in those mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae or Tetanus toxoid, and were specific for the molecular weight defined in the protein data base, as shown by Western blot. Mice infused with these anti-[~2GPI Abs had significant thrombocytopenia, prolonged aPTT and elevated percentage of fetal loss, similarly to a control group of mice immunize with a pathogenic anti-~2GPI monoclonal Ab [ 11]. Hence, our study established a mechanism of molecular mimicry in experimental APS, demonstrating that 132GPI-structure homologous bacteria are able to induce the generation of pathogenic anti132GPI Abs along with APS manifestations [ 11]. In parallel Gharavi et al [82, 83] induced circulating anti-~2GPI Abs in nai've mice by immunization with synthetic peptides conjugated to BSA which share some similarity with 72 kd human adenovirus type 2 DNA-binding protein, CMV, HCMVA and Bacillus subtilis. We believe that pathogen particles are digested and presented on macrophages, dendritic cells or on B cells. These pathogen particles are presented to T cells which in concert with appropriate HLA presentation and Thl/Th2 activated cytokine cascade expression, will lead to the generation of plasma cells secreting anti-~2GPI Abs, directed to the pathogen particles, which share structural homology (molecular mimicry) with the [~2GPI molecule (Fig. 3). Whether an individual will develop APS will depend mainly on his genetic predisposition. 3.3.2. Lessons from data base for correlations between various common pathogens and ~2GPI structure and ~2GPI related peptides Table 3 summarize linear homologies between the [~2GPI molecule related peptides and common pathogen, using the Swiss Protein Database. Previously, we have shown molecular mimicry between
common pathogens and ~2GPI related peptides, in which 2 out of 7 ~2GPI/pathogen homologies were found to have functional biological significance and the antibodies to these shared epitopes induced experimental APS [11]. Therefore, a) A limitation in mismatches should be considered, b) One should keep in mind that probably many conformational structures are shared between 132GPI molecule and pathogens, an example will be given bellow for vaccinia which share high tertiary structural similarity with the 132GPI molecule and no significant shared homology in the amino acid sequences (Fig. 2). 9 Helicobacter pylori anti-~2GPI Abs and APS Helicobacter pylori (H. pylori), one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induce chronic gastric inflammation which can progress to a variety of diseases ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and participate in the lifelong chronicity of infection [84]. Recently, a disappearance of antiphospholipid antibodies syndrome after Helicobacter pylori eradication was reported [85]. Accumulating data show that H. pylori infection has diverse clinical effects: a) H.pylori infection in pregnant women may affect fetal intrauterine growth [86], increasing the risk of developing reproductive disorders [87]; b) H.pylori infected mice showed increased platelet embolization followed by damage to arterioles [88], c) H.pylori infection was suggested to be a risk factor for cerebral ischemia [89] and a risk factor for coronary heart disease [90]. In all these cases, the correlation to anti-~12GPI or to anti-1~2GPI related peptides should be analyzed. Screening of 50 patients with H.pylori infection for the presence of anti-l]2GPI revealed prevalence of 33.3% in one study [91]. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as
477
T a b l e 3. Anti-phospholipid Abs detected in diverse infections and 132GPI peptide homologies shared with structures in these pathogens
Infections associated with circulating anti-PL Abs
TLRVYK" [38]
LKTPRV [38]
KDKATF [38]
GDKVSFF [49]
GRTCPKPDDLP [53]
Viral:
CMV
+
EBV
+
HIV
+
Hepatitis C
+
Parvovirus B 19
+
Adenovirus
+
Varicella
+
Vaccinia
+
Mumps
+
Rubella
+
HTLV-1
+
+
+ +2 b
+
+
+ +2
Herpesvirus Bacterial:
Leprosy
+
Tuberculosis
+
M pneumoniae, M penetrans
+
Salmonella
+
Staphylococci
+
+ typhi +
+
Streptococci
+
+ 7pyogenes
+ pyogenes
Trypanosome brucei rhodesiense
-
+
Coxiella burnetii (riccecia, Q fever)
+
+
+
Chlamydia
+
Porphyromonas gingivalis Helicobacter pylori
+
+
Haemophilus influenzae Neisseria gonorrhoeae
+ + +
Neisseria meningitidis Shigela dysenteriae Pseudomonas aeroginosa Yersinia pseudotuberculosis (Continued.)
478
+2
+2 -
+
+3
Table 3. Continued
Infections associ- TLRVYKa ated with circulat- [38] ing anti-PL Abs
LKTPRV [381
KDKATF [38]
GDKVSFF GRTCPKPDDLP [49] [53]
Klebsiella pneumoniae Campylobacter jejuni E.Coli Brucella melitenensis Spirochaeta:
Treponema Palidum (Syphilis) Leptospira Borrelia burgdofferi
+ + +
Parasit:
Kala azar Schistosoma mansoni Toxoplasmosis
+
+
Yeast:
Saccharomyces Cervevisiae (crohn) Candida albicans Streptomyces lividans Mycoplasma
+
+
+ pulmonis genitalium
+ pneumonia capricolum genitalium pulmonis
a Homologies between the 2GPI related peptides and different pathogens (1-2 mismatches) using the Swiss protein database, updated until April 2003. bTwo shared structures.
mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH [92]. Using the Swiss protein database, we were able to find homology (misl-2) between anti-~2GPI peptides target epitopes and H.pylori structures (Table 3). For example: Q9zmk9
- a DNA repair protein radA homolog, which may play a role in the repair of endogenous alkylation damage; Q9zkm3 - c e l l division protein ftsA, which may be involved in anomalous filament growth (by similarity); P 5 5 9 8 0 - Cytotoxicity associated immunodominant antigen, which may be necessary for the transcription, folding, export or function of the cytotoxin; P 9 6 7 8 6 - Flagellar hook-associated protein 2 (HAP2), an essential factor in flagellar structure and motility; Q9zmz4 and P14916
479
Figure 2. Ribbon diagram of alignment of I]2GPI (a) and vaccinia virus homolog (b) and both molecules (c).
- Urease alpha subunit (Urea amidohydrolase). 9 Streptococcus pyogenus, anti-~2GPl Abs and APS Rheumatic fever (RF) and subsequent rheumatic heart disease (RHD) represent a relatively common connective tissue diseases, caused by Streptoccocus pyogenus. Molecular mimicry mainly between the pathogenic M protein and self structures has been thought to be a mechanism for the development of acute rheumatic fever (ARF) after streptococcal pharyngitis. M protein and other not-well-defined streptococcal antigens from the bacterial cell have been related to the cross-reaction with human proteins having coiled-coil structures, such as myosin, tropomyosin and valvular proteins. Besides the appearance of cross-reacting anti-streptococcal antibodies the immunology of rheumatic fever is complicated by heart autoantibodies under the form of immunoglobulins bound to the myocardium and endocardium, as well as by circulating serum autoantibodies against the heart. A variety
480
of autoantibodies reactivities have been reported in rheumatic fever and this response may be consistent with either a hyperactivation of B cells or an underlying specific antigen driven cross-reaction [93-95]. Rheumatic fever (RF) and antiphospholipid syndrome (APS) share partial common clinical picture such as CNS and heart involvements. We assumed that it may be a consequence of a cross-reactive epitope between the M-protein and the I]2GPI. The ~2GPI related peptides TLRVYK and LKTPRV share homology with Streptoccocus pyogenus M-protein (mismatch=2). [~2GPI related peptide TLRVYK inhibited the binding of anti-M protein Abs from RF patients, to M protein by 37%. Anti~2GPI could be inhibited by M protein for binding to ~I2GPI by 23% (personal communication). 9 Borrelia burgdorferi, anti-f52GPI Abs and APS The spirochaete Borrelia burgdorferi is the cause of the neurological Lyme disease. A subset of patients
(50%) with neuroborreliosis (Lyme disease) showed IgG reactivity to cardiolipin in a solid phase ELISA [96, 97]. Since the assay was conducted at 1987 with serum as blocker, probably the reaction is ~2GPI dependent. The 132GPI related peptides TLRVYK and LKTPRV share homologies (mismatch l) with Borrelia burgdorferi: Q51257 - a glycerol kinase, 051376 protein-glutamate methylesterase. 9 Saccharomyces cerevisiae, anti-~2GPI Abs and APS Presence of circulating anti-Saccharomyces cerevisiae IgA/IgG Abs (ASCA) is one of the main markers for Crohn's disease, an idiopathic chronic inflammatory bowel disease [98]. The serologic responses seen in Crohn's disease include antibodies to Saccharomyces cerevisiae, mycobacteria, bacteroides, listeria and E. coli. Many of these organisms may be involved in the pathogenesis of Crohn's disease. Oligomannose, paratuberculosis p35 and p36 antigens are epitopes of the Saccharomyces cerevisiae, defined by now and demonstrated in 60-70% of the patients with Crohn's disease. Patients with inflammatory bowel diseases have elevated titers of circulating anti-cardiolipin / anti-~2GPI Abs [99-101]. Episodes of thrombosis associated with Crohn's disease were described accompanied with elevated titers of anti-I]2GPI Abs is still not clear [99-101]. The association of ASCA was described also in patients with other autoimmune condition - Behqet's disease with no relevance to the clinical picture [102]. Some of the patients (30-32%) with BD have circulating anti-~2GPI Abs [103, 104]. Thrombosis, usually venous, occurs in 10% to 25% of patients with Behqet's disease, but its pathogenesis is poorly understood [104]. The association of antiphospholipid antibody syndrome with Behqet's disease was raised as a cause for the total thrombotic occlusion of the vena cava in the case [105]. Famularo et al [106] described a case of a patient who had a life-threatening relapse of Behqet's disease associated with a catastrophic antiphospholipid syndrome. The patient experienced over a short time a recurrent acute myocardial infarction, multiple venous thromboses, uveitis, and erythema nodosum. Search for thrombophilic factors was positive only for lupus anticoagulant (LAC) and cri-
teria for the diagnosis of the antiphospholipid antibody syndrome were fulfilled [ 106]. The functional cross-reactivity between ASCA and anti-[32GPI or anti-132GPI peptides should be analyzed. Using the Swiss protein database, several homologies between 1~2GPI related peptides and Saccharomyces cerevisiae (mismatch l or 2) (Table 3). TLRVYK: Q07878 - Vacuolar protein sortingassociated protein VPS13, promotes endosomal cycling of TGN membrane proteins by modulating the function of two cytosolic TGN localization signals. P 5 3 0 7 0 - Mitochondrial translation optimization protein. LKTPRV: P41911 - glycerol-3phosphate dehydrogenase [NAD+] 2. P40550 and Q04182 - ATP-dependent permeases PDR11 and PDR15 a multidrug ATP binding cassette transporter of the yeast plasma membrane. 3.4. Anti-[~2GPI Tentative Dual Activity Associated with Infections
A wide range of specific protein-protein interactions at the cell surface and in the serum are mediated by functionally versatile 60-amino acid residue protein domains called complement control proteins- CCP (also known as Short Consensus Repeats) which are characterized by unique consensus sequence. Among the ligands we can find the ~I2GPI molecule which contain four typical CCP modules/one atypical and key complement system proteins such as C3b and C4b anti-coagulant vitamin K-dependent protein S; heparin; viruses such as Epstein-Barr virus (EBV), the measles virus, enterovirus 70, echovirus and bacterial proteins such as M-protein of the group A Stapholococcus pyogenes and the adhesion of E.Coli. CP-modules have been identified so far in 50 distinct mammalian proteins from plasma, the surface of many cell types, spermatozoan acrosomal matrix, retina, brain and more [ 107]. Using Gapped-BLAST, PSI-BLAST of Swiss protein database, and apoH_fasta-file, we found a strong significant alignment (> l e-ll) between the ~2GPI molecule and several infection-relevant sequences or pathogen control molecules. Herein, we present few examples and discuss the functional relevance of anti-[32GPI Abs as natural autoantibodies or pathogenic ones: Vaccinia virus: Vaccinia and/or vaccinia gamma globulin are used for smallpox vaccination. Cases
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Figure 3. Molecular mimicry between [32GPIand common bacteria/viruses. of deaths were attributable to smallpox vaccination mainly by transmission of vaccinia from smallpox vaccination, although the disease is now eradicated in most of the countries [108]. Domains 3 4 of the 132GPI molecule, share high tertiary structural similarity (> le- 19) with vaccinia virus complement control protein (VCP) (Q89859) (Fig. 3). This 243residue protein is a regulators of complement activation, its role is to defend the virus against attack by the host complement system. [~2GPI may compete with this VCP protein residue for its receptor recognition and facilitate complement activation as a response to the virus. Anti-[~2GPI may act on the VCP via virus neutralization thus enables complement activation to occur. P10998 - Complement control protein precursor (VCP) (Secretory protein 35) (Protein C3) (28 kDa protein). Vaccinia virus encodes a secretory polypeptide structurally related to CCP, (>9e-
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20). This polypeptide protect the virus against complement attack by inhibiting both classical and alternative pathways of complement activation. Binds C3b and C4b. Preventing this kind of via'us protection by circulating anti-132GPI Abs will cause response to the virus.
3.5. EBV -[~2GPI/Anti-[~2GPI Interrelations Q9DC83; 046545; Q99254 - complement receptor 2 - CR2. CR2 is the receptor for C3d, the 33kd fragment of the third complement component. CR2 is also the EBV receptor(EBV/C3d, CD21). CR2 bind to its 2 extracellular ligands, C3d and the EBV capsid glycoprotein gp350/220through two distinct binding sites. CR2 allows C3d and EBV to induce proliferation [ 109]. Theoretically, different scenario can be envisioned regarding the EBV-I]2GPI/anti[~2GPI interrelations: Anti-~2GPI may be generated
by a molecular mimicry mechanism as described in Fig. 2. These Abs may neutralize the EBV or the CR2 if they recognize a shared epitope and thus prevent infection mononucleosis. On the other hand, they can enhance the disease if the anti-~2GPI Abs recognize a different epitopes on the EBV and on CR2. Presence of circulating anti-~2GPI Abs was described in patients with infectious mononucleosis with no correlation to disease activity [ 110]. However, a wide range of populations were exposed to EBV, have anti-~2GPI and no infectious mononucleosis. APS patients have very high titers of antiEBV associated with anti-~2GPI with no infectious mononucleosis (unpublished data). Q16744; Q29530 - complement receptor 1 CR1. Controversy was shown regarding the CR1 as inducer or inhibitor of B cell activation. At this stage, the question whether [~2GPI can function as a mimetic of CR1 or CR2 is speculative, as well as the role of anti-~2GPI in B cell activation in response to the pathogen. Previously, anti-I]2GPI have been shown to be protecting antibodies in different systems: a) Opsonizing apoptotic cells and accelerating clearance by macrophage scavenger receptor and dendritic cells, preventing long exposure of intracellular cell components to the immune system as a risk factor for generation of pathogenic autoantibodies [111, 112]. b) Enhanced uptake of oxidized low density lipoprotein via a specific ligand on oxLDL by macrophage scavenger receptor and promote atherosclerosis [113], unless the 132GPI compete on the same receptor and protect from atherogenic process.
3.6. Anti-[~2GPI Involvement in Controlling the Complement Activation As previously discussed, exposure to a pathogen might induce the generation of pathogenic anti132GPI Abs to the shared epitopes between the relevant pathogen and the ~2GPI molecule. Having a high alignment between [~2GPI and some CCPs, anti-132GPI may interfere with complement activation. One of the proposed mechanisms for accelerating APS is the enhanced complement activation [114, 115]. Furthermore, several studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. In-vivo inhibition
of complement cascade prevented fetal loss induced by anti-I]2GPI Abs, using the C3 convertase inhibitor CRl-related gene/protein y (Crry)-Ig [116]. Theoretically, ~2GPI can be a mimetic of CR1, thus anti-132GPI Abs can shadow complement inhibitor and enhance complement activation (Q16744; Q29530 - complement receptor 1 CR1 and the Q9DEGO; Q163135 - complement regulatory membrane proteins). In addition, strong alignment (le-18) is demonstrated between [32GPI and C4b-binding protein alpha chain precursor (Proline-rich protein) (P04003; Q99JA1). C4bp controls the classical pathway of complement activation. It binds as a cofactor to C3b/C4b inactivator, which then hydrolyzes the complement fragment C4b. It also accelerates the degradation of the C4bC2a complex (C3 convertase) by dissociating the complement fragment C2a. Alpha chain binds C4b. it interacts also with anticoagulant proteins and with serum amyloid p component (SAP). Thus, anti-l]2GPI Abs will neutralize this protein enabling increase in complement activation and the pathogenesis of the disease.
3.7. Anti-[32GPI and Membrane Cofactor Protein CD46 A strong alignment between ~2GPI and a membrane cofactor protein (MCP) CD46 (5e-18) tertiary structure was detected in the database. The MCP was previously identified as the host cell receptor for the Edmonston laboratory strain of measles virus. CD46 is a ubiquitous molecule, a member of the regulators of complement activation family proteins and interfere with the formation of the complement attack complex on the membrane of normal cells and prevent deregulated complement cell lysis of the host cell [ 117]. Therefore, CD46 plays a critical role in prote~ting uninfected self-tissue from complementmediated damage. It is a type I membrane glycoprotein with different isoforms of 57-67 molecular masses. Thus, anti-~2GPI Abs will neutralize this protein enabling increase in complement activation and the pathogenesis of the disease. Still, the CD46 has an additional role. It binds by an hydrophobic interaction, the measles H protein. As a possible mimetic of the CD46, 132GPI molecule could bind the measle H protein and protect the normal cell.
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4. A CROSS-POINT WITH INNATE
IMMUNITY Toll-like receptors (TLRs) are pattern recognition receptors that trigger innate immunity, providing both immediate protective responses against pathogens and instructing the adoptive immune response [ 118]. Recently, a link was introduced between the innate and adoptive immune systems, in the development of systemic autoimmune diseases such as SLE [ 119]. Accumulating evidences paved the way to explain the importance of TLRs in promoting autoimmune conditions [ 120, 121 ]. B cells play an essential role in adaptive immune response and in innate immunity. Chromatin-IgG complexes were found to activate B cells by a dual engagement oflgM to the TLR9 [ 122], which is known to detect bacterial CpG DNA [ 123]. Recent studies have revealed that common molecular patterns of microorganisms such as lipopolysaccharide (LPS) are recognized by TLRs. B cells have two known TLRs that mediate LPS signaling, TLR4 and PR105 (CD 180). The role of pathogens mimicking the 132GPI in B cell TLR triggering, may promote the humoral response leading to the development of APS. The importance of TLRs in the procoagulative state of endothelial cells in APS was proposed recently by Meroni PL et al [ 124] showing the role of MyD88 transduction signaling pathway in endothelial cell activation by anti132GPI. The Abs reacted with 132GPI in association with TLR/IL-1 receptor family on the endothelial cells surface [ 124].
5. THERAPEUTIC CONSIDERATIONS As has been summarizied above, there is quite strong evidence for an infectious etiology in APS. This raises the big question of instituting antibiotic therapy as prevention and/or interventional therapy especially in catastrophic APS. More information will be required in the future to solve this enigma. It will depend if the mechanism of the disease is "hit and run" [ 125, 126], or a continuous stimulation of the immune system. The study where the APS was abrogated by eradication of Helicobacter pylori with antibiotic therapy [85], points to the impact of the continuous presence of the bacteria on the induction of the disease. In another study employ-
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ing an experimental APS model, treatment with ciprofloxacin improved the clinical picture by induction of IL-3 and GM-CSF expression [127]. These seminal works support the addition of antibiotics to combination therapy to patients with APS. The employment of IVIG for severe cases entailing also catastrophic APS would be warranted, since IVIG in addition of having an anti-idiotypic mechanism [ 128-130], also has a broad spectrum of anti-bacterial and anti-viral effects [ 131,132].
6. CONCLUSIONS Molecular mimicry is one of the mechanism by which experimental APS can be intiated in association with the presence of pathogens. Like in other autoimmune diseases, the concept of molecular mimicry remains a viable hypothesis for framing questions and approaches to decipher and understand the pathogenic mechanisms involved, and to design strategies for novel therapies. Studies on experimental APS and synthetic peptides which share common epitopes between bacteria and viruses, and the [~2GPI molecule, prove the existence of molecular mimicry between pathogens and autoantigens in APS. We speculate that a mimicking antigen, similar in only one epitope, may initiate a primary cross-reactive response to that epitope that subsequently results in recognition of numerous epitopes on the host ~2GPI. Mimicry may be one of the mechanisms for braking the tolerance and triggering the autoimmune response. Yet, the mere presence of a self determinants on a virus or bacteria, not necessarily will result in disease. The full blown APS will emerge only if the appropriate genetic predisposition exists.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
SLE and Infections Gisele Zandman-Goddard and Yehuda Shoenfeld
Center for Autoimmune Diseases and Department of Medicine 'B', Sheba Medical Center, Tel Hashomer; Sackler Faculty of Medicine, Tel-Aviv University, Israel
1. INTRODUCTION Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by a myriad of autoantibody production. Immunological aberrations may play a role in the susceptibility of SLE patients to infections. Furthermore, immunosuppressive agents utilized in the treatment of moderate and severe lupus give rise to a tendency for infections including opportunistic ones. Infections may mimic exacerbations of SLE, leading to confusion over the diagnosis and appropriate treatment. Despite increased awareness of the disease and multiple antibiotics, infections remain a major source of morbidity and mortality. Although the mechanism of disease is yet unknown, multiple factors including environmental factors, genetic susceptibility, and immunological alterations are suspected in the development and/or trigger exacerbation of disease. One of the postulated mechanisms for the induction of disease is molecular mimicry where a microbial pathogen may be the inciting agent in the development of autoantibodies. We review infections in SLE and possible evidence for molecular mimicry in this chapter.
2. SLE MORTALITY FROM INFECTION
Infection remains a major cause of morbidity and mortality in systemic lupus erythematosus (SLE) [1-3]. The studies on morbidity and mortaliy in SLE until 1996 have been previously reviewed [ 1]. More recent studies are reviewed here. A casecontrol study investigated the nature, outcomes of
infection and associated risk factors in patients with SLE. Ninety-three patients had 148 infection episodes; the majority were bacterial, but viral, fungal and protozoan organisms were also identified (multiple organisms in 7 patients). Forty-eight patients required hospital admission and three patients died. Steroids at time of infection, as well as use ever, duration and dose, immunosuppressives at time of infection and use ever, active renal disease, CNS damage, SLEDAI at the time of infection, adjusted mean SLEDAI and variability measure were significantly associated with infection by univariate analysis. The only factor that remained statistically significant, by multivariate analysis, was the use of steroids ever (P = 0.029) [4]. The frequency, characteristics of the main causes, and the prognostic significance for morbidity and mortality in 1,000 patients with SLE during a 5-year period were analyzed in another study. Two hundred seventy patients (27%) presented infections. Forty-five patients (4.5 %) died; the most frequent causes of death were divided similarly among active SLE (28.9%), infections (28.9%), and thromboses (26.7%) [5]. A monocentric cohort of 87 adults with SLE over a 37-year period was assessed to determine the risk factors for infection by comparing patients who had suffered at least one infectious episode (n=35; 40%) with non-infected patients (n=52; 60%). The prognostic indicators were assessed by comparing the survivors at 10 year interval with the non-survivors. Of the 57 infectious episodes, 47 (82%) were of bacterial origin, 16 (28%) were pneumonia, and 46 (81%) were community acquired. According to univariate analysis, significant risk factors for infection were severe flares, lupus glomerulonephritis,
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Table 1. Rates and characteristics of infections in SLE patientsa Study [Ref.] Gladman [4] Cervera [5] Noel [6] Zonana-Nacach [7] Li [8]
# of infections (%)
Pathogens
Risk factors
Mortality rate
148
B, V, F, P
Steroids, immunosuppression, active renal disease, CNS, SLEDAI
3 died
1000 87
270 (27%) 57
82%-B,
200
65 (32%)
Severe flares, renal, steroids, IV cytoxan, plasmapharesis SLEDAI, renal, steroids, IV cytoxan Cardiac, pulmonary, renal, high dose steroids
#.of patients 93
86
133
Wongchinsri [9]
488
191
Bouza [ 10] Alarcon [11] Suh [50]
662 288
28.9%
Sites: UTI, skin, sepsis, vaginal B, F, nosocomial
27%
Salmonella, E. Coli, TB
27% 11/288 120
High dose steroids, SLEDAI, anemia, renal, high CRP
aPrevious studies in Ref. [1]. B, bacterial; V, viral; F, fungal; P, parasite; SLEDAI, SLE disease activity index. oral or intravenous corticosteroids, pulse cyclophosphamide, and/or plasmapheresis. No predictors were identified at the time of SLE diagnosis. Multivariate analyses retained intravenous corticosteroids (p<0.001) and/or immunosuppressants (p<0.01) as independent risk factors for infection, which was the only factor for death after 10 years of evolution (p<0.001) [6]. The incidence and risk factors of infections in 200 SLE outpatients with active or inactive SLE was assessed. Major infections were those requiring hospitalization and parental antibiotic therapy; minor infections required oral or topical therapy. After a follow-up of 22+/-7 months, 65 (32%) patients had infections; 35% of those were major. The most common sites for infection were urinary (26%), skin (23%), systemic (12%), and vaginal (9%). At infection onset, 50 of 65 patients (77%) had disease activity, with a mean SLEDAI score of 6.1. The variables significantly associated with infection in the univariate analyses were the presence of disease activity, SLEDAI score, renal activity, prednisone dose, and IV cyclophosphamide. The only variable associated with infection in the multivariate analyses was a SLEDAI score of 4 or higher. Most infections in SLE outpatients were
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single, minor, non-life threatening, and associated with disease activity independently of sociodemographic and therapeutic factors [7]. The aim of another study was to investigate the clinical and bacteriologic features of 86 patients SLE complicated by bacterial and/or fungal infections. One hundred and thirty-three episodes of infections occurred in 86 patients with SLE, in which 51.13% were nosocomial infections and 76.69% occurred in the blood system, respiratory tract, lungs and urinary tract. Gram-negative bacilli, gram-positive cocci, fungal and other bacterial infections accounted for 39.85%, 31.58%, 18.80% and 9.77%, respectively. In the bacterial infections, 18.52% were caused by L-form bacteria and more than 60% of the patients had no apparent toxic manifestations. The odds ratio of infection increased significantly in patients with damaged functions of the heart, lungs and kidneys, and in those who received highdosage steroids. Patients with SLE tend to develop nosocomial infections with gram-negative bacilli that are the most common pathogens. The clinical manifestations of the infection are atypical. Careful inspection and monitoring, timely collecting the specimens for L-form bacterial culture can reduce misdiagnosis and missed diagnosis of the infection
[8]. A retrospective review of 488 hospitalized SLE patients during a 5-year period was performed to determine the infectious complications in these patients. One hundred ninety-one patients with SLE were admitted because of infection. Lower respiratory tract infection was the most commonly found in these patients (24.6%) followed by infections of the urinary tract (15.7%), skin (15.7%), septicemia (13.6%) and the musculoskeletal system (11.5%). The most common pathogens were Salmonella spp (12.6%), Escherichiae coli (9.9%) and Mycobacterium tuberculosis (8.4%), respectively [9]. Another retrospective study examined the causes of mortality in 662 SLE patients spanning a 34-year period at the University of Puerto Rico Hospital. Out of 662 patients diagnosed with SLE, 161 (24%) patients died. The mean duration of disease was 11.5 years and the mean age at death was 37 years. Infection was the primary cause of death in 44 (27%) of patients [ 10]. The features associated with mortality in a multiethnic US cohort of patients with SLE within 5 years of study onset were evaluated among survivors and deceased patients. Within 5 years of study onset, 34 of 288 patients died. Fourteen deaths could be directly attributed to SLE and 11 to infections. There were 10 deaths among Hispanics, 18 among African Americans, and 6 among Caucasians (P < 0.05). Disease activity, disease damage, and poverty appeared to be the most important determinants of mortality in this multiethnic US cohort of SLE patients [ 11]. Factors associated with mortality, survival and causes of death in a series of 366 patients with SLE (45 men and 321 women), mean age 29 y (range 11-70 y) and mean disease duration of 6 years, were evaluated from 1990 to 1998 in Argentina. A total of 57 clinical, serological and therapeutic variables were studied. Five- and 10-year survival was 91% and 85% respectively. Forty-four patients died (12%): 54% due to sepsis and 32% due to active SLE [12]. A multi-center cohort of 513 clinic attenders with SLE was retrospectively identified, representing 4185 patient-years of follow-up. Expected numbers of death were calculated by means of age- and sex-specific mortality rates of the general Danish population. The observed number of deaths was
122. The survival rates were 97%, 91%, 76%, 64% and 53% after 1, 5, 10, 15, and 20 years respectively. The overall mortality rate was 2.9% per year (95% CI 2.4-3.5). The cause of death was infection in 25 patients. SLE patients in the present cohort had a 4.6-fold increased mortality compared with the general population and half of the deaths were caused by SLE manifestations or infections, especially in young patients during the early period of the disease [13]. When compared to previous studies [1, 2], the morbidity and mortality rates have not decreased over the past 5 years.
3. BACTERIAL I N F E C T I O N S A wide variety of infectious pathogens are recognized in SLE. Most infections are caused by common pathogens and include Staphylococcus aureus, Streptococcal pneumonia, E. Coli, and Pseudomonas aeruginosa. However, an increased incidence of Salmonella infection and pneumococcal sepsis is also observed [2]. 3.1. Salmonella
Infection with Salmonella species is recognized to be more common in SLE patients than the normal population and may be due to splenic dysfunction [ 1]. Recently, the risk factors of mortality for Salmonella infection in patients with SLE were reviewed in 37 cases of Salmonella infection in 31 patients with SLE, from a total of 1191 hospitalized patients with SLE at a medical center in Taiwan. Contrasting cases of patients who died with those who survived, a comparison of clinical and laboratory characteristics of SLE at the time of Salmonella infection, with special attention to potential risk factors were evaluated. The patients with SLE who were older or had associated infections other than Salmonella had an increased mortality rate when they have concurrent Salmonella infection. Patients with Salmonella infection occurring concurrently with the first presentation of SLE and patients with SLE reinfected with Salmonella species were at a higher risk of mortality [ 14]. In another study, the clinical profiles of lupus patients with non-typhoidal salmonellosis were assessed. A retrospective review of the clinical charts of 50 lupus patients diagnosed with bacterio-
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logically proven non-typhoidal salmonellosis over a 20 year period was undertaken, paying special attention to risk factors, clinical presentation and treatment outcome. Most episodes were bacteraemic without a localizing focus; and some patients were afebrile. They usually occurred in patients prone to opportunistic infections, and at times of increased immunosuppression given for lupus flares (especially nephritis). However, salmonellosis also occurred in some patients presenting with lupus. Mortality occurred in the setting of septic shock from mixed-microbial sepsis and major organ failure from active lupus. There is a high association of non-epidemic, non-typhoidal salmonellosis with SLE, especially in patients with active disease on intensified immunosuppression [ 15].
3.2. Klebsiella Sera from patients with Klebsiella pneumonia were found to contain high titers of the common antiDNA idiotype (16/6Id) [16-18]. In one study, the sera of 52 patients with urinary tract infection or septicaemia from this Gram-negative pathogen was examined for the presence of antibodies to DNA, polynucleotides, cardiolipin and a common antiDNA idiotype 16/6. Up to 27% of these patients had detectable anti-polynucleotide antibodies, and in 37% the 16/6 idiotype was found. Absorption of the sera of two patients, with no DNA binding, against the Klebsiella polysaccharide K-30 induced a significant fall in both their anti-K30 antibody and 16/6 idiotype levels. Among 52 patients with other Gram negative infections a maximum of 17% and 19% respectively, had anti-DNA antibodies and the 16/6 idiotype present in their serum. In 37 normal controls, the rate of antibody and idiotype detection was 5% or less. The presence of autoantibodies in the serum of patients with Klebsiella infections may be the result of non-specific stimulation due to bacterial polyclonal activation. However, there might also be a specific stimulus triggered by idiotypic cross-reaction between autoantibodies and anti-Klebsiella antibodies [ 17].
3.3. Mycobacterium Tuberculosis The impact of M. tuberculosis infection in SLE patients in endemic regions is emerging and the
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prevalence ranges from 5-30% [2]. Recently, a retrospective study from Korea, an endemic area for pulmonary TB, investigated the incidence and clinical characteristics of M. tuberculosis infection in 283 SLE patients. Tuberculosis was documented in 15 SLE patients with an incidence rate of 7.9/1,000 patient-years. The characteristics of tuberculosis in SLE patients were: (1) a higher incidence rate, (2) more frequent extra-pulmonary involvement, (3) more extensive pulmonary involvement, and (4) a higher relapse rate than in rheumatoid arthritis. The contributory role of M. tuberculosis infection in the morbidity and mortality of patients with SLE should be emphasized, especially in areas in which this bacteria is endemic [19]. In another retrospective analysis of 146 SLE patients from India, seen over a 5 year period, 17 patients with tuberculosis were identified yielding a prevalence rate of 11.6%. The median duration of SLE was 12 months and 12/17 patients had disease activity score of more than five. The median duration of steroid treatment was 12 months and the median cummulative dose of steroid was 7.75 gms. Pulmonary TB (miliary5, nonmiliary infiltrates-7 and pleural effusion-2) was the commonest type and there was an average diagnostic delay of approximately 1 month. While the majority of the patients responded adequately to treatment, 1 patient had a relapse and 1 expired due to a combination of active lupus and disseminated TB [20]. In a retrospective case series of 30 patients, from Mexico City, with autoimmune diseases and TB, 20% were SLE patients, and 6/7 SLE patients had military TB [21 ]. Mycobacterial infection may present as a mimicker of vasculitis and patients with SLE may present with skin disease posing a diagnostic challenge. The skin disease can reflect an increase in systemic disease activity suggested by other features of active lupus and, as such, usually responds well to aggressive immunosuppressive therapy. In patients who show a poor response to more aggressive immunosuppressive therapy, consideration must be given to the possibility of opportunistic infection. A high index of suspicion will allow prompt treatment. Atypical mycobacterium infection in SLE patients is rare. Two patients with SLE were described who developed cutaneous atypical mycobacterial infection during immunosuppressive therapy. The diagnosis of cutaneous vasculitis was con-
sidered in both cases, but subsequent skin biopsy revealed the correct diagnosis. This report illustrates the importance of skin biopsy in patients with suspected cutaneous lupus who are not responding to immunosuppressive therapy [22]. Hemophagocytic syndrome (HPS) in systemic lupus erythematosus (SLE) patients has not commonly been reported. In a case study, a lupus patient with Mycobacterium avium complex (MAC)-associated hemophagocytic syndrome was described. The patient had received high dose prednisolone (> 0.5mg/kg/day) for more than 3 years. She presented with a spiking fever, hepatosplenomegaly, pancytopenia, hyperferritinemia and adult respiratory distress syndrome. Bone marrow examination revealed hemophagocytosis as well as non-caseating granulomatosis [23]. Cross reactivity between anti-DNA antibody idiotypes in the sera of patients with active TB has been previously described [24]. In addition, heat shock proteins (hsp) may be involved in the initiation and perpetuation of autoimmune diseases. In order to investigate the possible role of hsp and other intracellular proteins of M. tuberculosis in the autoantibody production in SLE, the immunocrossreactivity of SLE autoantibodies with Mycobacterium tuberculosis sonic extract and hsp-70 kDa was investigated. These proteins showed significant binding with Protein A-Sepharose isolated SLE IgG. Western blotting of hsp-70 with SLE IgG showed strong recognition, suggesting possible involvement of hsp and other intracellular proteins of Mycobacterium tuberculosis in the autoantibody induction in SLE [25].
4. OPPORTUNISTIC INFECTIONS Increasing evidence indicates that opportunistic infections contribute to the infectious mortality in SLE. Opportunistic infections are considerably under-reported due to difficulties in diagnosis pre-mortem and the fact that they can mimic or be superimposed upon active lupus. The presenting features of tuberculosis, listeriosis, nocardiosis, candidiasis, cryptococcal meningitis, Pneumocystis carinii pneumonia and invasive aspergillosis are described in patients with SLE [1-3]. In addition, there have been many reports of toxoplasmosis in SLE patients, presenting as cerebritis and pericar-
ditis mimicking SLE manifestations [26-29]. Seropositivity for Toxoplasma gondii is more common among SLE patients than control individuals [24]. Toxoplasma infection can also occur in patients with low disease activity and low steroid dosage [26]. Normalization of immunity in patients with inactive SLE may contribute to the manifestations of opportunistic infections [26]. Heightened awareness of the potential for opportunistic pathogens to infect SLE patients, together with earlier investigation and appropriate therapy for such infections, are likely to make a significant contribution to decreasing the mortality in patients with SLE.
5. VIRAL INFECTIONS SLE patients show an increased risk for viral diseases. Viruses such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), and parvovirus B 19 seem to play a role as environmental agents that may trigger the development of SLE. 5.1. Parvovirus B19
The clinical significance of the presence of B19 DNA in 72 patients with SLE was studied and compared to patients with other autoimmune diseases. Parvovirus B 19 DNA was detected in 17 of 72 patients with SLE (by serological assays, nested PCR and Southern blotting), but not in patients with other systemic rheumatic diseases. Of the 17 patients with B 19 DNA, only one had IgG anti-B 19 antibody and two had IgM anti-B 19 antibodies, whereas IgG and IgM anti-B 19 antibodies were detected in 27 (49.1%) and 21 (38.2%) of 55 SLE patients without B 19 DNA respectively. B 19 DNA was found more commonly in sera from SLE patients without antiB 19 antibodies than in those with anti-B 19 antibodies (P<0.05). B19 infection in patients with SLE may be due to lack of anti-B 19 antibodies because of either the immunocompromised nature of the host or the use of immunosuppressive drugs [30]. The association of parvovirus and SLE [31 ] or the induction of SLE by parvovirus infection are anecdotal [32], but may provide insight into possible molecular mimicry. The occurrence of HPV-B19 infection has been documented in patients with SLE, in particular in relation to disease onset. Simi-
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larities in clinical and immunological features of viral infections and SLE at presentation may hinder the differential diagnosis between these two conditions. In a study of 4 patients, HPV-B 19 infection may have mimicked the onset of SLE in three cases, but triggered the disease in one [33].
5.2. Cytomegalovirus (CMV) A recent report of 10 patients with spatially related SLE and CMV infection identified 3 aspects of the viral infection. First, CMV infection and SLE exacerbation may be difficult to distinguish because of similar clinical manifestations. Second, the development of SLE may be triggered by a CMV infection. Third, existing SLE may undergo an exacerbation following a CMV infection [34]. Furthermore, CMV infection may be an opportunistic infection, affecting specifically SLE patients on chronic steroid and/or immunosuppressive agents [1]. In the antiphospholipid syndrome, associated with SLE in 40% of cases, new evidence for molecular mimicry is provided in mouse models. Antiphospholipid antibodies (aPL) induced by immunization of mice with a phospholipid-binding CMV peptide are pathogenic in vivo. The results suggest that molecular mimicry may be the mechanism by which pathogenic aPL may be generated in patients with antiphospholipid syndrome [35].
5.3. Epstein-Barr Virus (EBV) The literature remains controversial concerning the connection between Epstein-Barr virus (EBV), a common infection, and the development of SLE. For many years, investigators have suspected that EBV might somehow be involved in the etiology and/or pathogenesis of SLE. Numerous studies have examined this possibility from various angles and have arrived at different conclusions [36]. The possible molecular mimicry of the Epstein-Barr virus (EBV) peptide PPPGRRP by the peptide PPPGMRPP from Sm B'/B of the human spliceosome is consistent with the possibility that EBV infection is related to the origin of SLE in some patients. Association of EBV exposure with SLE was tested for and subsequently found in children and adolescents. The results were confirmed at the level of EBV DNA. Much smaller seroconversion rate dif-
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ferences were found against 4 other herpes viruses. Herein, these studies were extended to adults to test the hypothesis that EBV infection is associated with adult SLE. The selection included 196 antinuclear antibody-positive adult SLE that were tested for evidence of previous infection with EBV, cytomegalovims (CMV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), or varicella-zoster virus (VZV) by standardized enzyme-linked immunosorbent assays. Of the 196 SLE patients tested, all but 1 had been exposed to EBV (p = 0.014). No differences were observed between SLE patients and controls in the seroconversion rate against CMV, HSV-2, or VZV. These new data from adults, along with the many suggestive features of EBV infection, are consistent with a possible contribution of this infection to the etiology of SLE [37].
5.4. HIV SLE has a decreased incidence in the HIV infected population than would be expected in the general population [38, 39]. This inconsistency suggests that the immunosuppressive effect of HIV may inhibit the development of autoimmune diathesis. On the other hand, in a setting of HIV infection that is controlled by protease inhibitors and other antiretroviral agents, the immune system is no longer immunodeficient. There is immune restoration with normalization of the CD4 count and functional T cell reconstitution [40], so that a genetically predisposed host can develop autoimmunity. This has been postulated in the coexistence of HIV with SLE [41]. Systemic lupus erythematosus (SLE) may be influenced by human immunodeficiency vires type1 (HIV) infection. It has been suggested that the immunosuppression resulting from HIV infection can prevent the emergence of SLE. To date, 29 cases of association between the two diseases have been reported, but the diagnosis was simultaneous in just two of these and only 18 fulfilled the ACR criteria for the diagnosis of SLE. Most patients experienced an improvement in their SLE after development of their HIV associated immunosuppression and a reactivation of lupus manifestations has also been noted after immunological recovery secondary to antiretroviral therapy [42]. One report described a female patient with SLE who was infected with the human immunodefi-
ciency virus (HIV). Using stored serum, the precise timing of HIV seroconversion was determined and the early effects of HIV infection on SLE examined. This infection resulted in clinical improvement and the disappearance of autoantibody production [43]. On the other hand, another report described a patient with HIV infection who developed SLE after the initiation of highly active antiretroviral therapy [41]. A number of clinical and laboratory features of HIV infection are found in SLE. The presence of circulating antibodies to small nuclear ribonucleoproteins (snRNP) was analyzed in patients with HIV or SLE [44]. Sera from 44 HIV-infected children, from 22 patients with childhood-onset SLE, and from 50 healthy children were studied. Results included the detection of anti-snRNP antibodies by ELISA in 30 HIV-infected patients (68.1%) and 19 SLE patients (86.3%). These antibodies were directed against U1-RNP (61.3% and 77.2%, respectively), Sm (29.5% and 54.5%, respectively), 60 kDa Ro/ SS-A (47.7% and 50%, respectively), and La/SS-B proteins (18.1% and 9%, respectively). None of the HIV-infected children and 11 SLE patients (50%) showed anti-snRNP antibodies by counter immunoelectrophoresis (CIE). None of the HIV-infected patients showed anti-70 kDa U1-RNP or anti-DSm antibodies by immunoblotting. No differences between the two groups were noted on the presence of nonprecipitating anti-snRNP antibodies. No such reactivities were observed among the normal sera tested. The authors concluded that non-precipitating anti-snRNP antibodies in HIV-infected children are as frequent as in childhood-onset SLE. The significance of these antibodies is not clear at present. Although polyreactive and low-affinity antibodies and a mechanism of molecular mimicry may explain these results, a specific stimulation of B cells by nuclear antigens could not be excluded. Review of the literature revealed only 29 cases of concomitant SLE and HIV. In these cases, rheumatologic signs and symptoms were common in HIV and overlapped significantly with SLE. Autoantibodies also occurred frequently in both diseases [44]. SLE patients produce high titer antibodies to various retroviral proteins, including Gag, Env, and Nef of HIV and HTLV, in the absence of overt retroviral infection. In particular, the role of HTLV1-related endogenous sequence (HRES-1) should
be considered in SLE. Molecular mimicry may be a mechanism between HRES-1 and the small ribonucleoprotein complex that initiate the production of autoantibodies, leading to immune complex formation, complement fixation, and pathological tissue deposition [45]. The role of HIV and endogenous retroviral infections in autoimmunity are further discussed in other chapters.
6. GENETIC FACTORS PREDISPOSING TO INFECTION Suceptibility to infections in SLE patients may be related to genetic factors. Mannose-binding lectin (MBL) variant alleles may play a genetic role in the development of infections in SLE [2]. In a population-based cohort, MBL alleles were determined in 99 SLE patients recruited from a representative Danish region. A meta-analysis of eight previously published studies suggested that the presence of MBL variant alleles confer a 1.6 times overall increased risk for D-SLE (P < 0.00001). MBL variant allele carriers had higher disease activity (SLEDAI-index) in a 2-year follow-up period (P = 0.02) and had an increased risk of acquiring complicating infections in general (P = 0.03) and respiratory infections in particular (P = 0.0006). MBL variant alleles were also associated with increased risk of disease activity and of complicating infections indicating that the MBL gene is an SLE disease modifier locus [46]. Complement deficiency states, disease susceptibility, and infection risk in SLE have been widely reported [1, 47]. Recently, complete deficiency of factor I (fl) was described in two sisters from a consanguineous Brazilian family. The eldest sibling (20-year-old) developed SLE early during childhood while the youngest had been committed on several occasions owing to repeated infections although she was asymptomatic for autoimmune diseases. Lower concentrations of C3 and factor B in both sisters were detected. Biological functions dependent on complement activation such as the production of opsonins and killing of phagocytozed micro-organisms, chemotactic factors and haemolytic activity were all significantly reduced in both probands. Consistent with the absence of fl and low levels of fH, a deregulated production of C3b was
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observed by bidimensional electrophoresis in sera of both the probands [48]. Osteopontin (SPP1) is a soluble ligand with pleomorphic immunologic activities including activation of macrophage chemotaxis, promotion of Thl responses, and activation of B 1 B cells. It has been implicated in the development of murine lupus and is overexpressed in humans with SLE. A polymorphism of osteopontin was examined for an association with lupus in humans in an effort to determine whether there is any evidence that a genetic predisposition to altered osteopontin expression might explain the overexpression seen in human SLE patients. A silent polymorphism (707C>T, rs1126616) of osteopontin was significantly associated with SLE. Additional associations with renal disease and opportunisitic infections were suggested. This is the first phenotypic association with a polymorphic variant of osteopontin [49].
7. SEROLOGICAL MARKERS OF INFECTION IN SLE Serological parameters may be useful to distinguish a lupus flare from infection. CRP is an acute phase reactant that is found to be elevated in infections and many autoimmune states. However, CRP levels are normal in SLE patients and do not reflect disease activity. To evaluate risk factors for infection and the role of CRP in the diagnosis of infection, a retrospective case control study was performed among Korean SLE patients. Of 120 proven infections, 31 episodes (25.8%) occurred in patients taking no corticosteroids (CS). The CRP was higher in the patients with infection than controls and the CRP levels over 50 mg/l were observed only in infection [50]. In contrast, normal CRP levels may be found even in the setting of infection in SLE patients
[51]. Serum levels of soluble Fcgamma receptor III and granulocyte colony-stimulating factor (G-CSF) were determined by ELISA to evaluate the risk of infection in 10 patients with SLE. Two lupus patients had one or more infections within 3 months before and after blood samples were obtained. Absolute neutrophil counts were similar in neutropenic patients who did or did not have infections. However, the median level of soluble Fcgamma
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receptor III was significantly lower among patients who developed infections (p = 0.005), whereas the median level of G-CSF was significantly higher compared with patients without infections (p = 0.04). In patients with chronic neutropenia due to rheumatic diseases, low soluble Fcgamma receptor III levels and elevated G-CSF levels are better indicators of the risk of infection than is the neutrophil count [52]. Soluble markers of activity of the monocyte/ macrophage system (sCD14) and the vascular endothelium (sE-selectin, slCAM-1) in patients with SLE and primary Sj6gren's syndrome (pSS) in comparison to patients with infections or sepsis was investigated utilizing the ELISA method. Elevated levels of sE-selectin and slCAM-1 were detected in patients with SLE as well as sepsis, in contrast to patients with a localized infection (SLE and sepsis, respectively, versus infection, p<0.001). Levels of sCD14 were persistently elevated in sera from patients with SLE, whereas these values decreased rapidly after effective therapy in patients with sepsis or infection. A continuous elevation of all of these three parameters was associated with a fatal outcome in patients with sepsis as well as in patients with SLE. Combined elevation of sCD 14, slCAM- 1 and sE-selectin correlates with the prognosis in patients with active SLE and indicates a remarkable immune activation involving the monocyte/macrophage system and the endothelium comparable to an activation found only in patients with sepsis [53].
8. M O L E C U L A R MIMICRY IN SLE One of the most distinguishing features of SLE is the presence of high concentrations of autoantibodies that recognize a limited number of self-antigens. Even though many lupus autoantigens have been identified, the inciting triggers of these abnormal immune responses are not fully understood. One mechanism that could generate these autoanfibodies is a normal immune response toward a foreign epitope that mimics a common antigenic target of an autoantigen. Antibody generated toward the foreign epitope could also bind the autoantigen. This "cross-reactivity" would result in the presentation of the autoantigen to the immune system. Under autoimmune-prone conditions, tolerance toward
the native protein is broken and an autoimmune response is initiated. Research utilizing murine models, monoclonal antibodies, combinatorial and peptide phage display library technologies has enhanced our understanding of the mechanism. To date, there is no direct evidence rather a casual one that molecular mimicry exists. The following data provides insight to the existing association between infections as a trigger for the development or exacerbation of SLE.
8.1. Murine Models Anti-double-stranded (ds) DNA antibodies play an important role in tissue injury and microbial antigen may be a stimulus for the production of these antibodies. In one study, an IgG2b monoclonal antibody (99D.7E), from a nonautoimmune BALB/c mouse that is cross-reactive with both dsDNA and phosphorylcholine, the dominant hapten on the pneumococcal cell wall was isolated While partially protective against a bacterial challenge, 99D.7E is also pathogenic to the kidney. To identify those molecular motifs that confer on anti-PC antibodies the potential for autoreactivity, a panel of 99D.7E mutants with single amino acid substitutions in the heavy chain was created and the changes in antigen binding and renal deposition was examined. High affinity IgG antibodies were cross-reactive with bacterial and self antigen and displayed pathogenic potential, suggesting that defects in peripheral regulation of B cells, activated by foreign antigen but cross-reactive with self antigen, might lead to autoimmune disorders [54].
8.2. Phage Display Technology Clinical observations indicate that anti-viral and antibacterial responses are often accompanied by self reactivity, and anti-pneumococcal antibodies elicited in non-autoimmune individuals by pneumococcal vaccine express lupus-associated anti-DNA idiotypes. To explore the relationship between protective and pathogenic antibodies in humans, a phage display immunoglobulin expression system was ulilized to generate a combinatorial library from spleen cells of a lupus patient immunized with a polyvalent pneumococcal polysaccharide vaccine prior to splenectomy. From this library,
monovalent antigen-binding fragments expressing the 31 Vkappal-associated idiotype were isolated. This idiotype is expressed on up to 90% of antiDNA antibodies in the serum of lupus patients and on anti-pneumococcal antibodies in the serum of non-autoimmune individuals. Eight 31+ monovalent antigen-binding fragments reacting with pneumococcal polysaccharide, DNA or both were analyzed. Four of these fragments were cross-reactive with both foreign and self antigen, demonstrating that a high percentage of anti-bacterial antibodies produced in a patient with lupus bind double-stranded DNA. These studies provide support at the molecular level for a potential role of molecular mimicry in the generation of anti-DNA antibodies [55].
8.3. Peptide Technology L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases. A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264-286 of the RNA polymerase major sigma factor of the eubacterium Chlamydia trachomatis. Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant sigma factor. Immunofluorescence labeling experiments on C. trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified antipeptide II autoantibodies. Binding of autoantibodies to peptide II was inhibited by the homologous sigma peptide. This study was the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells. Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C. trachomatis infection in patients with SLE and mixed connective tissue disease. Molecular mimicry may be involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases [56]. The aim of another study was to investigate the epitope recognition pattern of La(SS-B) autoantibodies in sera from patients with Sj6gren's syndrome (SS) and SLE using overlapping synthetic
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decapeptides on solid phase. Eighty different decapeptides with five amino acids overlap from the human La(SS-B) autoantigen were synthesized on cellulose paper using F-moc chemistry. Tests were performed with 14 SS and six SLE sera. The results showed that the immune response to the La(SS-B) oligopeptides was restricted and unique for each individual with no particular pattern typical for each of the two diseases, apart from the fact that SLE sera gave positive reaction with fewer peptides. Regions within the N- and C-termini harboured most of the positive sequences. The authors specifically addressed the possibility of a viral aetiology for disease development or autoantibody generation. In this context the most frequently recognized linear epitopes on the La(SS-B) autoantigen showed sequence similarities with proteins from a range of ubiquitous human viruses, in particular from the herpes virus group. The La(SS-B) autoantibodies may thus be generated through molecular mimicry
[57]. Previously, it was suggested that Epstein-Barr virus might use molecular mimicry to initiate an autoimmune response. Cross-reactive epitopes may have a similar amino acid sequence or a similar tertiary structure that is independent of amino acid sequence. A major, and likely initial, target of the lupus anti-SmB' response is a repeated, proline-rich sequence, PPPGMRPP. To identify potential crossreactive targets, affinity-purified autoantibodies specific for PPPGMRPP were utilized to screen a random heptapeptide phage display library. Eightyfive clones were isolated and sequenced with eleven distinct sequence motifs being identified. Two of these motifs were homologous to the SmB' epitope, while the other nine were not. Interestingly, one of the peptide motifs that mimicked the SmB' epitope is identical to a peptide sequence found in the Epstein-Barr virus major DNA binding protein [58]. 8.4. Monoclonal Antibodies
To evaluate further bacterial DNA immunization as a model to study antigen drive in the anti-DNA response, the specificity of induced monoclonal anti-DNA antibodies was characterized. A panel of IgM and IgG monoclonal anti-DNA antibodies was produced from spleen cells of BALB/c mice
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immunized with single-stranded DNA from E. coli complexed to methylated bovine serum albumin in complete Freund's adjuvant. The binding of these antibodies to DNA and non-DNA antigens, was tested by ELISA to assess their range of polyspecificity. The monoclonal antibodies were found to bind to nucleic acid as well as non-nucleic acid antigens, such as beta-galactosidase, cardiolipin, Ro, La and Sin. The study demonstrated that anti-DNA antibodies from normal mice, although induced by bacterial DNA, may display a broad range of antigen recognition and thus resemble lupus anti-DNA antibodies, many of which are polyspecific, in their pattern of cross-reactivity [59]. Classical models of experimental autoimmune diseases, such as adjuvant arthritis entail the use of mycobacteria. Furthermore, BCG immunotherapy may be followed by arthritic symptoms. To test the infection-autoimmunity relationship of mycobacteria, monoclonal antibodies raised against M. tuberculosis and against DNA were utilized. Murine monoclonal anti-TB antibodies were found to react with ssDNA, dsDNA and other polynucleotides. Monoclonal anti-DNA autoantibodies derived from patients and mice with SLE bound to three glycolipids shared among all mycobacteria and derived from mycobacterial cell wall. Prior incubation of the antibodies with ssDNA and other polynucleotides or with glycolipid antigens inhibited binding. These results indicate that infecting mycobacteria share antigens with human tissue, thus accounting in part for the production of autoantibodies in mycobacterial infections [60].
9. CONCLUSION Infections are an important cause of morbidity and mortality in patients with SLE. Patients with SLE have a higher infection rate than the general population. It is estimated that at least 50% of them will suffer a severe infectious episode during the course of the disease [3]. The rate of infection and concomitant morbidity has not decreased. Meticulous exclusion of infection is mandatory in patients with SLE, because infections may masquerade as exacerbation of underlying disease; and the immunosuppression used to treat severe forms of exacerbation of lupus can have catastrophic consequences in patients
with infections. Further research in the identification of genetic factors m a y isolate S L E patients at increased risk for infections. To date, only highly specific serological markers aid to distinguish infection from lupus flare. Indirect evidence for molecular mimicry as a m e c h a n i s m for the development of SLE exists.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Type I Diabetes Mellitus, Infection and Toll-Like Receptors Francisco J. Quintana and Irun R. Cohen
The Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
1. EPIDEMIOLOGY OF TYPE-I DIABETES
2. THE HYGIENE HYPOTHESIS
During the last forty years, the incidence of type I diabetes mellitus (TIDM) has shown a significant increase in developed countries [1]. Gross geographical differences can be seen in the increase of TIDM, and Europe provides us with a good example [ 1]. Northern European countries have higher incidences of TIDM than southem European countries, with Finland showing the highest European (and world) incidence [2]. The genetic background is an important factor in determining the susceptibility to autoimmune disease [3], however several observations indicate that genetic differences cannot account for the uneven distribution of TIDM. First, epidemiological studies carded out in Northern Ireland [4] and England [5] have found a positive correlation between a low incidence of type I diabetes and poorer socioeconomic conditions. Second, populations migrating between countries with different values of TIDM incidence are interesting examples: children born to parents who migrated to Yorkshire from Pakistan show an incidence of TIDM indistinguishable from that seen among non-migrants in England (11.7/100,000), which is significantly higher than Pakistan's incidence of TIDM (1/100,000) [ 1, 6, 7]. Third, in a recent paper Hyttinen and co-workers report that after studying 22,650 twins, the concordance rate for TIDM was only 27.3% in monozygotic twins and 3.8% in dizygotic twins [2]. In summary, these data highlight the importance of environmental factors in the worldwide increase of TIDM.
The hygiene hypothesis associates the increase in the incidence of autoimmune diseases and allergy in developed countries with the effects of the environment on the immune system. It postulates that childhood infections educate the immune system on how to react to antigenic challenge. The immune system is a self-organizing system and like the brain it requires experience to learn how to behave [8]. However, as a consequence of improved hygiene, vaccination campaigns, and the use of antibiotics in industrialized countries, the "education" of the immune system has been significantly diminished, and the immune response runs out of control upon stimulation with otherwise innocuous substances. The data obtained in the non-obese diabetic (NOD) mouse, a laboratory model of TIDM, seems to support the hygiene hypothesis. TIDM can be prevented in NOD mice by infection with bacteria, viruses or parasites [9-12]. It should be noted that TIDM protection does not need these agents to be alive: preparations of dead mycobacteria [13-15]; streptococci [ 16] or parasites [ 17, 18] can also halt the process that leads to overt TIDM. Thus, microbial components supply the immune information needed to shut off autoimmune diabetes.
3. MECHANISMS OF TIDM PREVENTION
Several mutually non-exclusive mechanisms have been invoked to explain the protection from autoimmune diseases afforded by infections. Microbial epitopes can share sequence homology with regulatory self-epitopes; this "molecular mimicry"
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[19] might allow infections (or more specifically, microbial molecules) to activate built-in regulatory networks. Although such a mechanism has been reported to inhibit experimental autoimmune encephalomyelitis [20] and adjuvant arthritis [21], it has not yet been reported for TIDM. In fact, molecular mimicry with microbial antigens seems to accelerate, rather than inhibit TIDM [22]. The contribution of molecular mimicry, and other mechanisms involving the adaptive immune response (antigen competition, bystander suppression or microbial superantigens) to the control of autoimmune diseases by the environment has been recently reviewed elsewhere [ 1].
molecule involved in TLR-triggered signaling, MyD88-independent signaling pathways have also been described [30-32]. Functional studies indicate that TLRs can dimerize, generating both homo and heterodimers [33-35]. Their ability to dimerize and form complexes with other surface molecules like MD2 [36] might explain the structural diversity of TLR ligands. A striking example of ligand diversity is given by TLR4, involved in the recognition of both lipopolysaccharide (LPS) [36] and chlamydial 60 kDa heat shock protein (HSP60) [37]. Although they were initially thought to recognize only pathogen-associated molecules [38], TLRs respond both to self and non-self molecules [39]. Table 1 lists some microbial and host TLR ligands.
4. TOLL-LIKE RECEPTORS
Innate immunity might also play a role in the modulation of TIDM by the environment. Toll-like receptors (TLRs) constitute a family of innate receptors recently identified in mice and humans [23-25]. TLRs were identified based on their homology with the Drosophila melanogaster toll receptor, which is involved in the development and the immune response of the fly [26]. Ten different TLRs have been identified thus far in mice and humans [23, 25]. However, none of the TLR knock-out mice described so far has developmental disorders, suggesting that mammalian TLRs lack a role in development or have some degree of redundancy. Mammalian TLRs are type I transmembrane receptors that share several structural/functional features [23, 25, 27]. They present an extracellular leucine-rich (LRR) repeat whose length varies between different TLRs. LRRs are thought to mediate protein-ligand interactions; they are found in proteins with several functions not restricted to the immune response. The LRR domain of the TLRs is separated from the single transmembrane domain by a characteristic cysteine-rich domain. TLRs also share a cytoplasmic Toll/Inerleukin-1 receptor homology (TIR) domain. TIR domains are protein interaction modules shown to recruit adaptor molecules. Finally, all TLRs have at least one MyD88-dependant signaling pathway. MyD88 is a 35 kDa adaptor protein that, through its own C-terminal TIR, interacts with the TIR of activated TLRs [28, 29]. However, MyD88 is not the only adaptor
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5. TLRS AND THE IMMUNE RESPONSE
Dendritic cells (DC) are antigen-presenting cells that trigger and influence several aspects of the immune response, including the differentiation of na'fve CD4 + T cells into either Thl or Th2 effector/ memory cells [40, 41]. DC express several TLRs whose levels are adjusted according to the state of activation of the cell [42-46], thus by modulating DC activity, TLR ligands could potentially influence the adaptive immune response. TLR activation by self [47-49] or non-self [37, 42, 50-54] TLR ligands promotes DC maturation. Indeed, several experimental reports seem to indicate that the activation of TLRs on DC has strong effects on the immune response. MyD88 is a pivotal molecule in the signal transduction pathway of TLRs [28, 29]. Schnare and colleagues showed that the immune response triggered by immunization with antigen in CFA, marked by Thl-type cytokines, cytotoxic activity and specific IgG2a in wild type mice, was highly impaired in MyD88-deficient mice [55]. The inability to mount a vigorous Thl response might have been associated with a defective maturation of DCs in response to the mycobacterial component of CFA, as reported by the authors [55]. Therefore, the triggering of TLR-dependant signaling pathways are needed for the maturation of DC and the induction of a vigorous Thl response when microbial products are used as adjuvants. Further studies have also included TLRs
Table 1. Self and non-self TLR ligands
TLR
Non-self Ligands
Self Ligands
TLR1
Mycobacterial lypoprotein Triacylated lipopeptides B. burgdoferi OspA
Unknown
TLR2
P. gingivalis LPS Zymosan Peptidoglycan (bacteria) Lipoproteins (bacteria and mycoplasma) T. cruzi GPI anchor B. burgdoferi OspA
HSP60 Surfactant protein A HSP70
TLR3
Poly (I:C) dsRNA
Unknown
TLR4
LPS Respiratory syncytial virus GroEL HSP60 Chlamydia HSP65
HSP60 HSP70 Saturated fatty acids Unsaturated fatty acids Hyaluronic acid Surfactant protein A Fibronectin
TLR5
Flagellin
Unknown
TLR6
Mycoplasma lipoproteins Lipoteichoic acid Peptidoglycan (bacteria)
Unknown
TLR7
Resiquimod Imiquimod
Unknown
TLR8
Resiquimod Imiquimod
Unknown
TLR9
CpG DNA
dsDNA
TLR10
Unknown
Unknown
in the induction of antigen specific Th2 responses [56, 57]. Overall, these results suggest that TLRs are the receptors involved in the adjuvant properties of several microbial preparations [58]. TLR ligands can also be the targets of the adaptive immune response. OspA is an outer-surface lipoprotein from B. burgdoferi that activates macrophages through a TLR1 and TLR2, probably complexed in a heterodimer [59]. Vaccination with OspA is being studied as a tool to fight Lyme disease [59]. Strikingly, TLR1 or TLR2-deficient mice do not mount OspA-specific immune responses upon vaccination [59]. Furthermore, humans with reduced TLR1 surface expression on CD4 + cells did not mount a detectable immune response to
OspA after repeated vaccination [59]. These results highlight the importance of TLRs for the design of vaccines and demonstrate that TLR ligands can simultaneously work on several components of the immune response, including CD4 § T cells. TLR signaling has been classically associated with the promotion of a Thl, pro-inflammatory, immune response. TLR activation, however, could also lead to the release of Th2 cytokines [56, 57]. Moreover, TLR signaling is needed for the induction of Th2 responses [56, 57, 60, 61] and for the maintenance of B cell memory in humans [62-64]. Thus, microbial antigens activate specific TLRs and in this way influence the immune response directed against them (and the microbe). However,
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as we have already mentioned, microbial infection can also lead to the inhibition of autoimmune diabetes; is there a role for TLRs?
suggest that immunostimulation by bacterial DNA motifs can modulate spontaneous HSP60 autoimmunity and inhibit NOD diabetes.
6. TLRS AND AUTOIMMUNITY
8. LPS
A healthy immune system harbors self-reactive T and B cells [65-69]. These self-reactive clones are constantly kept under the active control of regulatory cells [70-72]: the removal of the regulators leads to the onset of autoimmune disease [73]. Remarkably, regulatory T cells express several TLRs and are stimulated by TLR ligands like LPS [74]. In addition, upon activation of their own TLRs, DC can have opposite effects on regulatory cell function. DC can secrete immunomodulatory cytokines that favor induction of regulatory cells, like IL-10 [53, 60] or they can inhibit regulatory T-cells via an IL6-mediated mechanism [75]. Accordingly, several recent papers have shown that TLRs can play a role in the modulation of TIDM by the environment.
LPS from E. Coli stimulates innate immunity via TLR4 [23, 25]. Moreover, administration of LPS has been shown to inhibit spontaneous diabetes [80]. Furthermore, LPS-activated B cells express the Fas ligand, secrete TGF~ and can induce apoptosis of diabetogenic T cells [81]. The transfer of LPS-activated B cells could temporarily impair APC function and thereby prevented the onset of diabetes in NOD mice, and halted the pathogenic process in an adoptive transfer model of type I diabetes mellitus (T1DM) to NOD/scid mice [81]. However, the transfer of LPS-activated B cells did not promote Th2 responses to [I-cell antigens. Thus, B-cell activation through TLR4 by LPS can inhibit spontaneous autoimmune NOD diabetes.
7. I M M U N O S T I M U L A T O R Y BACTERIAL DNA MOTIFS
9. DOUBLE STRANDED RNA
Bacterial DNA, like that present in CFA [76], is rich in DNA motifs that stimulate the innate immune system via TLR9-mediated mechanism [77]. These immunostimulatory DNA sequences consist of a central unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines [76]; such a sequence is referred to as a CpG motif. We have demonstrated that CpG motifs present in bacterial DNA can inhibit spontaneous diabetes of the NOD mouse [78], but not the more aggressive cyclophosphamide-accelerated diabetes [79]. Prevention of diabetes was characterized by a decrease in insulitis, and a down-regulation of the spontaneous proliferative T cell responses to HSP60 and to its 437-460 peptide (p277) which characterize NOD diabetes [78]. Moreover, we detected a concomitant increase in IgG2b antibodies to HSP60 and to p277, and not to other islet antigens (GAD or insulin) or to control antigens. The IgG2b isotype of the specific antibodies, together with the decrease in T cell proliferative responses, indicated a shift of the autoimmune process to a Th2-type in treated mice. These results
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Polyriboinosinic:polyribocytidylic acid (poly I:C) is an analogue of viral double-stranded RNA that has been shown to activate the innate immune system via TLR3 [23, 25]. Serreze and co-workers found that the repeated administration of poly I:C, alone or in combination with IL-2, completely prevented the onset of diabetes [82]. However, the therapeutic effect required continuous administration of the immunostimulants since pancreatic insulin content declined and severity of insulitis increased following cessation of treatment. T cells isolated from Poly I:C-treated mice were capable of suppressing NOD T-cell responses to alloantigens in a mixed lymphocyte culture, indicating that regulatory T cells can be induced in NOD mice by TLR3-mediated signaling pathways.
10. HSP60 AND PEPTIDE P277 Self-HSP60 is an endogenous ligand for TLR2 and TLR4 [83]. The TLR4 molecule does not seem to bind HSP60 directly, but TLR4 is required to
transduce a signal [83-85]. Macrophages exposed to soluble HSP60 secrete pro-inflammatory mediators such as TNF
and IL-2-activated CD45RO+ T cells responded optimally at low concentrations (0.1-1 ng/ml), but non-activated CD45RO+ T cells required higher concentrations (> 1 l.tg/ml) of HSP60. T-cell HSP60 signaling was inhibited specifically by a mAb to TLR2, but not by a mAb to TLR4. The human Tcell response to soluble HSP60 depended on PI-3 kinase and PKC signaling, and involved the phosphorylation of Pyk-2. Soluble HSP60 also inhibited actin polymerization and T-cell chemotaxis through ECM-like gels towards the chemokines SDF-1 cz or ELC. Exposure to HSP60 could also down-regulate the expression of chemokine receptors CXCR4 and CCR7. Most importantly, HSP60 down-regulated the secretion of IFN7 by activated T cells (unpublished observations). These results suggest that soluble HSP60 (and its fragments), through TLR2dependent interactions, can down-regulate T-cell behavior and control inflammation. Thus, HSP60 can have both pro- and anti-inflammatory effects on various cell types. HSP60 works as a ligand both for antigen receptors on T cells and B cells (and autoantibodies) and for innate receptors TLR4 and TLR2 on various cells types. To further examine the contribution of innate immune signaling to autoimmune diabetes, we inserted a TLR4 mutation into NOD mice. As we mentioned above, TLR4 is needed for the activation of macrophages by HSP60 [84, 85]. Mutated TLR4 appears to markedly increase susceptibility to autoimmune type 1 diabetes (Dr. G. Nussbaum, unpublished observations). Apparently TLR4 signaling, whether by endogenous ligands such as HSP60 or foreign ligands such as LPS, can educate the immune system to avoid pathogenic autoimmunity.
11. CONCLUDING REMARKS TLR-activation has usually been associated with the induction of pro-inflammatory immune responses. Thus the inhibition of TIDM (an inflammatory condition) by TLR-ligands is controversial. Several mechanisms might account for this paradoxical observation. First, the direct activation of regulatory cells via TLR [94]. Second, the activation of TLRdependant anti-inflammatory responses on effector T cells [94]. Third, by inducing pro-inflamma-
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tory responses, TLR ligands might trigger built-in anti-inflammatory responses. Indeed, a controlled inflammatory response has been shown to be necessary for the inhibition of NOD diabetes by CFA [95]. These mechanisms are not exclusive, and further experiments should be directed at determining the contribution of each one of them in the control of diabetes via TLR activation. Through their interaction with microbial molecules, TLRs sense the environment. The experimental data showing that TLR-mediated activation of the immune system can inhibit the progression of NOD diabetes is in accordance with the hygiene hypothesis. The hygiene hypothesis could then be partially explained based on the "education" of the immune system by microbial TLR ligands. The role played by endogenous TLR ligands is still not clearly understood. However, the preliminary results obtained using NOD mice bearing a nonfunctional TLR4 might suggest that TLR activation by self ligands is involved in the control of diabetogenic T cells. Maybe the "lesson" thought by exogenous TLR ligands is continuously reinforced via the activation of TLRs by endogenous ligands, like HSP60. Thus, the stimulation of TLRs with defined TLR ligands might allow us to translate the hygiene hypothesis into immunotherapy. New therapeutic approaches for TIDM aiming at the "re-education" of the immune system might be designed using TLR-ligands, but without the risk of infection with life-threatening pathogens. The initial success of p277 in treating TIDM might then be the first lesson to learn, in a whole new program on the treatment of autoimmne disease.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Autoimmune Thyroid Diseases Yaron Tomer and Ronald Villanueva
Division of Endocrinology, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA
1. INTRODUCTION The autoimmune thyroid diseases (AITD) include a number of conditions which have in common cellular and humoral immune responses targeted at the thyroid gland. The AITD include Graves' disease (GD) and Hashimoto's thyroiditis (HT), both of which involve infiltration of the thyroid by T and B cells reactive with thyroid antigens, production of thyroid autoantibodies, with the resultant clinical manifestations (hyperthyroidism in GD and hypothyroidism in HT) (reviewed in [1, 2]). While the etiology of the immune response to the thyroid remains unknown, the current paradigm is that AITD are complex diseases in which susceptibility genes and environmental triggers act in concert to initiate the autoimmune response to the thyroid. In this review we focus on the contribution of one environmental factor, namely infection, to the pathogenesis of AITD. We will examine the pertinent data relating to the role of infectious organisms in the development of autoimmune thyroid diseases (AITD), with an emphasis on the mechanisms by which infection may trigger AITD.
2. GRAVES' DISEASE AND INFECTION Graves' disease is an autoimmune disease characterized by hyperthyroidism and diffuse goiter with or without the associated ophthalmopathy and dermopathy [1]. Graves' disease is an organ-specific autoimmune disease caused by the production of thyrotropin receptor autoantibodies (TSHR-Ab). These autoantibodies stimulate the TSH receptor to increase iodide uptake and cyclic-AMP production,
thereby inducing production and secretion of excess thyroid hormones [3].
2.1. Epidemiological Data Evidence for genetic factors being implicated in the development of autoimmune thyroid disease (AITD) is now widely accepted. However, it is also recognized that the development of AITD may be triggered by environmental factors, including infectious agents. Several studies have suggested that infectious agents marc be involved in the mechanisms triggering the breakdown of tolerance for the TSH receptor in Graves' disease. Cox et al [4] found seasonality in the diagnosis of Graves' disease but the data failed to reach statistical significance. In another study, Phillips et al [5] found that the incidence of thyrotoxic patients who had TSH receptor and anti-microsomal antibodies varied markedly between towns in England, but this study remains to be confirmed. Moreover, significantly increased prevalence of non-secretors (individuals with inability to secrete the water soluble glycoprotein form of the ABO blood group antigens into saliva) was reported in patients with Graves' disease [6-8]. Since non-secretors are known to have increased susceptibility to infection [7], these findings lend further support to the notion that an infective agent may play a part in the pathogenesis of Graves' disease. Indeed, Valtonen and co-workers found serological evidence for a recent bacterial or viral infection in 36% of newly diagnosed Graves' patients and in only 10% of controls [9]. An increased frequency of antibodies to the influenza B virus has also been found in patients with thyrotoxicosis [ 10]. To date, only one report of "clustering" of Graves'
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disease patients exists [ 11 ]. Recently a TSH-binding protein was reported to exist in a number of gram-positive and gram-negative bacteria suggesting the presence of TSH receptor antigen [12]. Moreover, 5 of 12 Graves' IgG preparations displaced radiolabelled bTSH from the bacterial binding proteins [12]. These findings imply that anti-TSH receptor antibodies could be produced by cross-reaction between bacterial proteins and the TSH receptor. 2.2. Yersinia Infection and Graves' Disease
Primarily known to cause outbreaks of food poisoning, Yersinia enterocolitica has also been implicated in the pathogenesis of Reiter's syndrome, and is associated with various autoimmune phenomena [13]. Studies on patients with Yersinia enterocolitica infections have demonstrated that the sera of these patients contained autoantibodies to thyroid epithelium [14, 15] although these may be of the "natural" autoantibody variety [16]. Conversely, a large proportion of patients with Graves' disease and autoimmune thyroiditis were reported to have antibodies to Yersinia [17], and many different Yersinia antigens cross-react with thyroid antigens [18] although with uncertain affinity. Wenzel et al found antibodies to plasmid encoded release proteins of Yersinia enterocolitica in 72% of Graves' disease patients, in 81% of patients with recurrent disease, but also in an unmatched 35% of controls [19]. Arscott et al examined the serological reactivity of sera taken from GD patients to Yersinia release proteins (YOP2_5), as well as the ability of YOP2_5 to stimulate T-lymphocyte proliferation from patients with GD but they could not confirm the findings of Wenzel et al. No unique serological reactivity to any specific Yersinia release protein could be demonstrated in GD patients. However, 2 GD patients demonstrated significant proliferative responses to the release proteins in their PBMC, as well as showing significant proliferation of intrathyroidal lymphocytes to the release proteins, suggesting that the relationship of this pathogen with GD may be due to T-cell cross-recognition and not serological cross-reactivity [20]. A recent study in Turkey also showed a high incidence of Yersinia agglutinating antibodies measured in blood samples from 65 GD patients compared to patients with
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HT, multinodular goiter and controls [21]. A large study from the Netherlands of 803 female relatives of AITD patients and 100 healthy controls showed that IgG and IgA antibodies to the outer membrane of Yersinia were higher in AITD relatives compared to controls. Consistent with the literature, TPOAbs were more prevalent in the AITD relatives; however, among euthyroid AITD relatives, no difference in the prevalence of TPO antibodies was found between those who were Yersinia Ab positive or negative. Nevertheless, these findings suggested a higher rate of Yersinia infection in AITD relatives [22]. In contrast, an earlier study from Canada showed no differences in the mean levels of Yersinia antibodies to serotypes 3 and 9 of Yersinia between patients with with GD, nontoxic goiter, autoimmune rheumatic disease and normal controls [23]. While Weiss et al demonstrated a saturable binding site for TSH on Yersinia enterocolitica [24], and the binding of radiolabeled TSH to Yersinia has been shown to be inhibited by Graves' immunoglobulins [25], none of these obServations have shown that infection with Yersinia leads directly or indirectly to the development of AITD. However, it is possible that the hypothesized cross-reaction between Yersinia antibodies and thyroid antigens is secondary to a recurrent epitope or a reflection of a related but unknown infection [26]. Additionally, a 200 bp fragment of Yersinia cDNA was successfully amplified using TSH receptor oligonucleotide primers and a radiolabled TSHR probe gave several discrete bands of hybridization with digested Yersinia enterocolitica DNA but under low stringency conditions [27]. Further evidence supporting the role of Yersinia infection in thyroid autoimmunity comes from the study of rats which demonstrated induction of lymphocytic thyroiditis after immunization with the Yersinia enterocolitica purified outer membrane protein [28]. In addition, T-cell mediated immunity towards Yersinia enterocolitica has been reported in patients with Graves' disease [29]. However, similar data have been demonstrated in rheumatoid arthritis [30]. The possibility of Yersinia-specific immune responses being merely reflective of the "natural" immune response deserves serious consideration in view of the low affinity interactions and lack of predictability in the published studies. Moreover, most patients with Yersinia infection (including those
who produce anti-TSH receptor antibodies) do not develop Graves' disease [31]. Hence, it is possible that while Yersinia antigens which are homologous to the TSH receptor can induce production of antiTSH receptor antibodies, this immune response is transitory and does not precipitate Graves' disease.
2.3. Enterovirus and Graves' Disease It has also been hypothesized that certain HLA subtypes interacting with an infectious triggers, including enteroviruses, can contribute to the development of GD. Kraemer et al, HLA-typed serum from 40 GD patients and 157 healthy individuals and found that the frequencies of HLA B-15, B21 and -DR3 were increased in GD, and that an association between HLA-DR3 antigen and lymphocytotoxic antibodies was observed (i.e., IgGs from GD patients were cytotoxic to HLA-DR3+ normal B cells). Absorption with Coxsackie B virus completely inhibited the lymphocytotoxic reactions against HLA-DR3+ B cells, suggesting a role for Coxsackie-reactive HLA-DR3 antibodies as a contributing factor to the development of GD [32]. Recently however, nested PCRs performed on blood samples from 21 newly diagnosed GD patients were investigated as to whether they had subclinical enterovirus infection. Nested PCRs were performed on these patients' blood samples seeking to amplify enterovirus genome, but no RNA from Coxsackie or related viruses were detected [33].
2.4. Endogenous and Exogenous Retroviruses and Graves' Disease Retroviruses have been implicated in the induction of Graves' disease but data supporting this view remains unsubstantiated. Bottazzo and colleagues reported the existence of retroviral sequences in the thyroid and peripheral blood mononuclear cells (PBMC) of Graves' patients [34]. Using antibodies against foamy virus (FV) gag protein, Wick and others detected a signal by immunofluorescence in thyroid tissue sections from GD patients [35]. Lagaye at al, detected the presence of FV DNA in 19 of 29 French GD patients using PCR [36]. Subsequent analysis of DNA from four of these patients by Southern blot and PCR confirmed the presence of FV DNA, but the same study did not detect any FV DNA in samples from another cohort
of 41 German GD patients [37]. Another study of 28 GD patients found no FV DNA using immunofluorescence, RIPA and Western blot [38]. The last two negative studies leave the possibility of artifactual contamination of the French GD samples. Analyses by ELISA and Western Blot also found no FV antibodies in 45 African GD patients [39]. More recently, FV gag, env and LTR sequences were detected by nested PCR in 13 of 24 Korean GD patients, and in 7 patients all three regions were amplified. However, 9 of 23 normal controls were also positive for at least one locus under the same conditions [40]. Thus, there is presently no evidence for FV as a causative factor for GD. DNA extracted from thyroid glands of 5 Graves' disease patients was hybridized with a probe containing the gag region of HIV-I. The results showed positive bands in each of the patients thyroid DNA which apparently were absent in control samples. However, these results were not confirmed by Humphrey et al [41 ] and Tominaga et al [42]. More recently, high reverse transcriptase (RT) activity, a marker for retroviral infection, was demonstrated in thyroid tissue extracts from patients with GD, compared to thyroids from patients with thyroid adenomas or carcinomas. Furthermore, the RT activity was also confirmed not to be due to other DNA polymerases [43]. An uncharacterized retroviral-like factor (pl5E) has also been detected in the serum of Graves' patients and absent in controls, suggesting involvement of endogenous retroviruses in the pathogenesis of the disease [44]. Tas et al have suggested that the origin of the p l5E related factors may be an exogenous infection with an as yet unknown retrovirus possessing determinants with structural homology with the p l5E, or an endogenous retrovirus [45]. Supporting these findings are the studies in Obese strain chickens discussed above which also demonstrated expression of a new retrovirus (ev 22) in susceptible chickens' thyroid tissue [46]. Jaspan et al studied 40 patients with GD and compared them with the healthy subjects, patients with multinodular goiter (MNG), and other patients with autoimmune disease (type 1 DM and other endocrine autoimmune disorders). The authors found that 87.5% (35/40) of patients with GD had a positive reaction against a prototypic strain of a human intracisternal A-type retroviral particle type 1 (HIAP-1) compared with only 2% of healthy
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controls, 10% of patients with MNG, and 15% of patients with type 1 DM [47]. A study performed on 35 members of 3 kindreds with a high prevalence of GD reported a possible interaction between HIAP-1 and HLA-susceptibilty haplotypes. When HLA-susceptibility genes and HIAP-1 (indicating retroviral exposure) were co-detected (in a total of 15 members in the 3 kindreds), the incidence of GD was 100%, 67% and 80% respectively. Four additional members (one from one family and three from another) with both viral and genetic susceptibility were found to have serological abnormalities and/or goiter and ocular signs consistent with evolving or preclinical GD. The association between the occurrence of both anti-HIAP-1 antibody positivity and HLA susceptibility and the presence of GD was highly significant (p<0.001) [48]. Another group further investigated these findings in the background of recent experiments showing that HIAP-1 was found in H9 cells co-cultured with homogenates of salivary glands from patients with SjSgren's disease [49]. Fierbracci et al co-cultured the H9 T-cell line with thyroid homogenates and viable thyrocytes prepared from the thyroids of patient's with GD, but after 24 weeks no HIAP-1 particles could be detected by electron microscopy from either thyroid preparation [50]. Other retrovimses have also been studied as possible factors in the development of GD. Yokoi et al, found a high incidence of HTLV-II proviral DNA fragments in the DNA of peripheral blood leukocytes from patients with AITD, demonstrating this in 51.5% of patients with HT, 11.8% of patients with GD, compared to only 1.9% of disease controls, and 1.0% of healthy controls. However, no antibodies to HTLV-II could be detected [51 ]. However, another study did not implicate the involvement of retroviruses in the development of GD. Yanagawa et al detected the gag region sequence of human spumaretrovims from DNA extracted from peripheral blood lymphocytes and thyroid tissue of GD patients and controls and found no significant difference between the two groups [52]. In order to help explain a possible relationship between retroviruses and thyroid autoimmunity, a homology has been suggested between the HIVI Nef protein and the human TSH receptor [53]. There was 66% homology demonstrated within a 166 bp region encoding a unique portion of the hTSH-R with a segment in which 7 of 10 consecu-
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five amino acids were identical. However, when sera from 10 patients with Graves' disease and 10 controls were tested for reactivity against an 18 amino acid peptide containing this segment of homology there was no significant difference in the degree of interaction [54]. Nevertheless, this does not exclude a conformational B-cell epitope or the presence of a T-cell epitope.
3. AUTOIMMUNE (HASHIMOTO'S) THYROIDITIS AND INFECTION Autoimmune (Hashimoto's) thyroiditis is characterized by infiltration of the thyroid by lymphocytes, gradual destruction of the gland associated with cytotoxic T-cells, and production of various secondary polyclonal thyroid autoantibodies, notably anti-TPO and anti-Tg [2]. The etiology of Hashimoto's disease is still unknown; however, genetic and environmental factors have been implicated in the pathogenesis of the disease [2].
3.1. Evidence for Involvement of Infection in the Etiology of Autoimmune Thyroiditis Serological evidence for a recent bacterial or viral infection has been demonstrated in patients with Hashimoto's thyroiditis [9], and lymphoid thyroiditis has been described after immunization with group A streptococcal vaccine [55]. Additionally, it has been reported that autoimmune thyroiditis may be associated with significantly increased frequency of antibodies that react to HIV-1 western blot proteins in patterns not diagnostic for HIV infection [56]. These data suggested that autoimmune thyroiditis patients were infected with non-HIV retrovirus producing antibodies to a viral protein cross-reactive with HIV proteins [57]. Viral-like particles have been detected in thyroids of humans with autoimmune thyroid diseases; however, since these viral-like particles were also demonstrated in normal thyroids and in other tissues, their significance is unclear at present [58]. Yersinia enterocolitica infection has also been implicated in the pathogenesis of Hashimoto's thyroiditis as well as Graves' disease. Wenzel et al reported that antibodies against Yersinia release proteins (RP) raised in rabbits showed specific
bands on Western blots with thyroid epithelial cell homogenates, and one band could be blocked by purified TPO [59]. Further evidence suggesting that infection is involved in the etiology of Hashimoto's thyroiditis comes from studies of T cell function in the disease. It has been reported that the lymphocytes in Hashimoto's thyroids have restricted antigenic response, suggesting their triggering by a specific antigenic stimulus, perhaps an infection [60]. Clearly, isolation of infecting organisms from thyroid tissue is needed to substantiate these indirect data. 3.2. Viral Hepatitis and Hashimoto's Thyroiditis The association of Hepatitis C with Hashimoto's thyroiditis (HT) has been widely studied. There have also been few reports of Hepatitis B as possibly being associated with HT. Because of the increasing prevalence of viral hepatitis, especially Hepatitis C, the use of interferon (IFN) has also become widespread. To assess whether these hepatitis viruses play a role in the development of AITD, it is thus important to study patients with viral hepatitis prior to receiving IFN, as IFN is a known immunomodulator that can trigger MTD. Both Hepatitis B and HT occur frequently in Down's syndrome, and so a cause and effect relationship between the Hepatitis B and Hashimoto's has been investigated. May and Kawanishi found a threefold increase in frequency of HT in patients with Down's syndrome who were carriers of HbsAg compared to patients with Down's syndrome and HbsAg negative. They postulated that this might be explained by cytokine/cellular immune abnormalities due to extra genetic material in chromosome 21 [61]. However, Hepatitis B alone, has not been found to be associated with an increased incidence of thyroid autoimmunity [62]. Data for Hepatitis C as a possible factor in the development of HT have been mixed. There have been numerous studies reporting varying concentrations of abnormal thyroid antibodies in patients with hepatitis C. Several studies have not found any significant correlation between the presence of Hepatitis C, prior to IFN treatment, and the presence of thyroid antibodies [63-67]. Other studies have shown a significant association between hepatitis C and Hashimoto's thyroiditis and/or thyroid anti-
body positivity. Abnormal concentrations of thyroid antibodies in up to 42% of patiems with Hepatitis C have been reported [68-74]. Some studies have shown that these IFN-naive Hepatitis C patients with associated HT or positive thyroid antibodies tended to be older and female [62, 68, 71, 75]. There have also been reports of a high prevalence of antiHCV antibodies in patients with HT suggesting this could be induced by HCV infection [76, 77], but this was not confirmed by other investigators [72, 78]. In summary, epidemiological studies measuring the frequencies of AITD and the presence of thyroid antibodies in patients with Hepatitis C patients have been inconclusive.
3.3. Retroviruses and Hashimoto's Thyroiditis Similar to Graves' disease, retroviruses have also been purported to be involved in the development of Hashimoto's thyroiditis. A high incidence of HTLVII proviral DNA fragments was found in DNA of peripheral blood lymphocytes from patients with AITD. Although no antibodies for HTLV-II could be detected, and thus HTLV-II infection could not be confirmed, amplified HTLV-II proviral DNA fragments were demonstrated in 51.5% of HT patients and 11.8% of GD patients compared to 1.9% disease controls, and 1% healthy controls [51]. The same authors found HTLV-I protein and messenger RNA (mRNA) in thyroid follicular cells in one of two patients with HT, although no virus particles were found by electron microscopy [79]. This same group measured thyroid antibodies (TAbs) in blood donors with or without HTLV-I antibodies, as well as HTLV-II proviral DNA in leukocyte DNA. Donors with both HTLV-I antibodies and (TAbs) tended to be more prevalent compared to donors without HTLV-I antibody. Furthermore, in young male donors with HTLV-I Ab, the frequency of TAbs was significantly higher than those without HTLV-I Ab (P<0.05). Also, detection of HTLV-II proviral DNA was significantly higher (p<0.001) in donors with TAb than in those without, regardless of HTLV-I infection. The authors suggested that HTLV-I infection and the presence of HTLV-II proviral DNA may be independently related to the development of AITD [80]. Akamine et al found that in patients with adult T-cell leukemia (ATL), TAbs were present in 40.4% compared to 30% in
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HTLV-I carriers and 13.7% in healthy controls. The difference in HTLV-I infected patients and controls was statistically significant (P<0.005). Furthermore, for those subjects positive for TAbs, 7.5% of controls, 19% of ATL patients and 40% of HTLV-I carriers had hypothyroidism with a statistically significant difference between HTLV-I infected patients and controls (P<0.005) [81 ]. A more recent study of CTLA-4 gene polymorphisms and HTLV-I infection in Japanese patients with HT showed that there was a significantly higher frequency of exon 1 G allele of CTLA-4 in HT patients with HTLV-I Ab compared to those without HTLV-I Ab. However, HTLV-I Ab was not associated with CTLA-4 polymorphisms in either HT or controls, suggesting that if indeed HTLV-1 is a factor in the development of HT, it is a purely an environmental one [82].
3.4. Yersinia and Hashimoto's Thyroiditis Yersinia enterocolitica has also been suspected of being involved in the development of Hashimoto's thyroiditis. Early studies have reported finding Yersinia antibodies in 24 of 36 GD patients, and in all seven patients with Hashimoto's thyroiditis compared to a low prevalence (<8%) in controls [17]. The most prevalent serotype, with the highest titers was serotype 3. Chatzipanagiotou and co-workers reported a 14-fold prevalence of class-specific antibodies to Yersinia plasmid encoded outer proteins (Yops) in HT patients compared to healthy blood donors and non-postinfectious rheumatic disorders. These antibody measurements (ELISA) were confirmed by Western blots [83]. A Japanese study found that antibodies to serotype 5, but not serotype 3, was significantly higher in GD (81.4%) and HT (91%) (p<0.001 vs. controls), and that a significantly increased incidence of antibodies to serotype 6 and 9 was seen only in HT patients [84]. This was not confirmed by a Canadian study of patients with GD, HT and nontoxic nodular goiter (NTG) and autoimmune rheumatic disease as well as normal controls; no significant difference in the serological reactivity was found to serotypes 3 and 9 of Yersinia enterolitica in AITD versus controls [23].
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3.5. Helicobacter Pylori Another infectious agent purported to be involved in the development of Hashimoto's thyroiditis is Helicobacter pylori. De Luis, and co-workers determined the prevalence of H. pylori infection in patients with HT, non-autoimmune thyroid disease and Addison's disease. H pylori infection was markedly increased in patients with HT (85.7%) compared with multinodular goiter (MNG) controls (40%), and Addison's disease (45.4%). Furthermore, a positive linear regression was found between levels of antiTPO antibodies and those of anti-H, pylori IgG in patients with HT (n=21; r=0.79; p<0.01), and antiH.pylori IgG levels were higher in patients with HT than controls [85]. Figura et al found that the prevalence of infection by CagA-positive H. pylori was significantly higher in patients with AITD (56%) compared to controls (24.2%) (p = 0.006) [86].
4. MECHANISMS OF INDUCTION OF AUTOIMMUNITY BY INFECTIOUS AGENTS The central feature of the immune system is specificity. The specificity of the immune response is maintained by complex mechanisms, which include the unique structures of secreted and non-secreted antigen receptors on lymphocytes and the elaborate interactions which occur between cells involved in the immune response. Together they form an interconnecting regulatory network which controls immune reactions. Infectious agents may induce autoimmunity by influencing any of the stages of the immune response, namely, the encounter with an antigen, the recognition of an antigen as non-self, the activation of the various effector arms of the immune system, and the regulation of the immune response.
4.1. Viral Induced Changes in Self Antigen Expression Viral infection, theoretically, may (a) alter self, (b) lead to the virus becoming a persistent endogenous antigen, or (c) cause the revelation of previously non-exposed, or rarely exposed, antigens. Alterations in normal body components can
develop during infections as a result of the release of bacterial or viral products, the expression of viral antigens on host cells, or the tissue injury caused by the accompanying inflammatory reaction [87]. For example, it has been shown that Coxsackie virus B4 infection produces diabetes in mice by inducing increased expression of a 64,000-M islet autoantigen in infected mice [88]. The 64,000-M antigen is believed to be the primary target protein in the antibeta cell autoimmune reaction which characterizes the insulitis of insulin dependent diabetes mellitus [89]. Further evidence that viral induced modification of self constituents may cause autoimmune disease comes from studies of Whittingham et al on a patient who developed Sjrgren's syndrome after protracted infectious mononucleosis [90]. The authors were able to show that the autoimmune reaction developed as a consequence of the association of viral RNA with the La nucleoprotein resulting in a break in immunologic tolerance and induction of anti-SSB antibodies leading to autoimmune sialoadenitis. The mechanism of altered self has not been reported to operate in thyroid autoimmunity. However, one observation of interest is the finding of Graves' disease and Graves' ophthalmopathy in patients who have received external radiation over the thyroid region. The radiation may have altered thyroid antigens or their expression thereby inducing an autoimmune reaction [91 ]. However, it cannot be ruled out that the development of Graves' disease in these patients was a consequence of immune dysregulation induced by irradiation of the thymic region. Another mechanism by which an infecting organism, mainly viruses, may alter self constituents and induce autoimmunity is via persistent expression of viral antigens on host cells. This phenomenon has been shown to occur in infections with endogenous retroviruses. Adams et al [92] have reported that if endogenous retroviral expression does not develop neonatally and is delayed until adulthood, then an autoimmune reaction ensues. As discussed earlier, endogenous retroviral protein expression has been reported in autoimmune thyroid disease [34, 44, 56] and in SjOgren's syndrome [93-95], and it has been shown that when transgenic mice expressing LCMV antigens in pancreatic beta cells are challenged with LCMV they produce lymphocytic infiltrates in their beta cells and develop signs of IDDM [96, 97].
Additionally, antigens not normally exposed to the immune circuit may become significant antigens for the first time. This may be important in viral induced murine encephalitis and cardiac myositis, and in the formation of sperm antibodies after vasectomy [98].
4.2. Molecular Mimicry Molecular mimicry is defined as structural similarity between antigens coded by different genes. Molecular mimicry has long been implicated as a mechanism by which microbes can induce autoimmunity [99]. The best known example of molecular mimicry and autoimmunity is rheumatic fever, in which antigenic cross-reactivity between cardiac tissue and streptococcal polysaccharides is believed to induce an autoimmune reaction targeted at the heart valves [100]. Antigenic similarity between infectious agents and host cell proteins is common, and in one analysis of 600 monoclonal antibodies raised against a large variety of viruses it was found that 4% of the monoclonal antibodies cross-reacted with host determinants expressed in uninfected tissues [101]. The clinical importance of molecular mimicry between mycobacteria and self-antigen was highlighted by the observation that patients treated with BCG immunotherapy developed arthritis [102]. Furthermore, anti-DNA antibodies were shown to have amino acid sequence homology with anti-Klebsiella pneumonia Waldenstrom monoclonal antibody, and when normal peripheral blood mononuclear cells were stimulated with Klebsiella antigens they secreted a common anti-DNA idiotype (the 16/6 Id) [103]. The 16/6 Id has also been found in the serum of patients with the parasitic infections filariasis and schistosomiasis [ 104]. Mice infected with reovirus type 1 developed an autoimmune polyendocrinopathy and generated a panel of autoantibodies directed against normal pancreas, pituitary, and gastric mucosa, suggesting an antigenic similarity between a reoviral antigen and an endocrine tissue antigen [105, 106]. Serreze and colleagues [107] reported that antibodies directed against the p73 antigen, an endogenous retroviral gene product, are cross reactive with anti-insulin antibodies. The authors suggested that anti-p73 autoantibodies are involved in inducing beta cell destruction in NOD mice. Likewise, Talal
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et al have demonstrated that 22 of 61 SLE patients produced antibodies to the p24 gag protein of HIV- 1 [108]. Moreover, Sm (a ribonucleoprotein involved in the generation of messenger RNA) was shown to partially inhibit the antibody binding of p24 gag, suggesting immunologic cross-reactivity between the retroviral antigen p24 gag and the autoantigen Sm [99]. Homology between microbial and host tissue antigens does not necessarily mean that an autoimmune response will emerge upon infection with that microbe. In order to prove that a sequence homology leads to autoimmunity it is necessary to show that challenging the host with the microbial antigen leads to an autoimmune response unrelated to direct infection of the target tissues. An example of such an experiment was provided by Oldstone and co-workers. They have used myelin basic protein which has been shown to have significant homology with several viral proteins including hepatitis B virus polymerase (HBVP) [109]. When the authors injected a HBVP derived peptide into rabbits, the animals developed lesions in the central nervous system similar to the autoimmune disease induced by injection of myelin basic protein [ 109]. As discussed earlier, molecular mimicry has been reported between Yersinia enterocolitica and the TSH receptor based on the observed cross reaction between sera from Yersinia and Graves patients [ 15, 17]. Moreover, a saturable binding site for TSH was demonstrated on Yersinia enterocolitica [24]. Recently, Wolf and co-workers have demonstrated that IgG of individuals convalescing from Yersinial infections produced concentration dependent inhibition of TSH binding to thyroid membranes and stimulation of adenylate cyclase activity [31]. This study demonstrates that IgG from patients with Yersinial infections can react directly with the thyrotropin receptor, perhaps as a consequence of cross reactivity between antigenic determinants on Yersinia enterocolitica and the TSH receptor. However, since a definite association between Yersinia enterocolitica infections and increased incidence of Graves' disease has not been demonstrated, the pathogenetic importance of Yersinia infection in the development of Graves' disease remains to be unravelled. Molecular mimicry has been suggested between retroviral sequences and the TSH receptor [54],
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however these findings remain to be confirmed. Another finding suggesting a possible role for molecular mimicry in autoimmune thyroid disease was that 42% of sera from lepromatous leprosy patients contained anti-thyroglobulin antibodies compared to 3% in the controls [ 110].
4.3. Alterations in the Idiotypic Network As first envisioned by Jerne [111], auto-anti-idiotypic antibodies are generated in the course of the normal immune response to a foreign invading pathogen, and serve to regulate the normal immune response. Anti-idiotypic antibodies produced during the primary immune response to an exogenous pathogen carry the internal image of the epitopes on the pathogen which bind to its receptor in the host. Consequently, the development of receptor binding anti-idiotypes can be hannfial to the host and initiate autoimmunity [112]. For example, the antibodies to reovirus type 3HA (HA3) have been studied extensively. Williams and co-workers have been able to produce in BALB/c mice a monoclonal antibody (called 9BG5) which could strongly bind to reovirus type 3 and neutralize its infectivity. Moreover, an anti-idiotype monoclonal antibody was produced which was found to bind to 9BG5 and to inhibit its interaction with HA3. The workers then demonstrated that the anti-idiotype bore the internal image of the receptor binding epitope on reovirus type 3; this anti-idiotype could induce changes in cells upon binding to their HA3 receptors similar to those induced by the virus itself [113]. Therefore, the damage caused by certain viruses need not involve their direct effect on target cells but rather an immunologic attack by anti-idiotypic antibodies on viral receptors. There are now reports supporting the notion that anti-idiotypes bearing internal images of viral antigens participate in the induction of several autoimmune diseases (reviewed in [112]). Myasthenia gravis developed in five individuals a few weeks after rabies virus vaccination. The rabies virus has been shown to bind to acetyl-choline receptor [114]; therefore it was possible that the anti-viral response induced anti-idiotypic antibodies which acted also as anti-receptor antibodies and bound to the acetyl-choline receptor, thus inducing an autoimmune disease. Alterations in the idiotypic network have been
implicated in the pathogenesis of Graves' disease based on the findings that immunization of animals with TSH has led to the development of anti-idiotypic antibodies that recognized and were able to activate the TSH receptor [ 115]. Moreover, we were able to induce experimental autoimmune thyroiditis in mice (a model of human autoimmune thyroiditis) by immunizing them with anti-thyroglobulin antibodies which induced anti-idiotypic reaction [ 116].
4.4. Immune Complex Formation Antigens produced by an infectious organism can form immune complexes with antibodies generated against them thus leading to the development of autoimmunity. The best known example of this phenomenon is the immune thrombocytopenia that follows viral infection. The virus triggers production of antibodies which can form immune complexes with the viral antigens. The immune complexes then attach to platelets which are damaged as innocent bystanders [87]. Immune complex formation has also been implicated in the pathogenesis of certain parasitic diseases (for a review see [ 117]). In malaria patients the development of nephrotic syndrome has been associated with deposition of immune complexes in the renal glomeruli [117]. Similarly, immune complex deposition has been implicated in the pathogenesis of glomerulonephritis associated with schistosomiasis, and circulating immune complexes have also been found in patients with leishmaniasis [117]. Immune complexes have been well documented in autoimmune thyroid diseases [118-120], and there are reports of glomerulonephritis and immune complex deposition with thyroglobulin antigen recognized within the complexes [121]. However, it remains to be shown whether these immune complexes are induced by infection.
4.5. Heat Shock Proteins and Thyroid Autoimmunity Ceils in different organisms respond to elevated temperatures by synthesizing new proteins termed heat shock proteins (hsps). Hsp synthesis can be induced not only by heat shock but also by other stressful stimuli, including exposure to oxidative radicals, alcohol or heavy metals, anoxia and infec-
tion [122]. Since hsps are highly conserved among different species and are widespread in bacterial cells, it is believed that they carry vital cellular functions during stress and in resting conditions (reviewed in [123]) and, therefore, hsps are produced in small quantities under normal conditions. By interacting with other intracellular proteins, and altering their folding and unfolding, hsps serve 4 vital cellular functions: (1) hsps assist in the assembly of polypeptides into their final tertiary structures [124], (2) hsps assist in intracellular transport of other proteins by maintaining them in a conformation suitable for transport into cellular organelles (e.g. mitochondria) [125], (3) hsps can bind and temporarily inactivate other proteins such as the steroid receptor [126], and (4) hsps participate in protein degradation [ 123]. Hsps have been shown to be strongly immunogenic and in view of their high degree of conservation between different species and their presence in many infectious agents they may be involved in the induction of autoimmune diseases. During the course of a bacterial infection an antibody and T-cell response to the microbe's hsps is induced. Theses antibodies and T-cells may then cross react with self hsps containing conserved epitopes. Moreover, the stress of the infection itself and the inflammation accompanying it may generate increased synthesis of self hsps, thereby enhancing the autoimmune response [127]. Self hsp production can also be induced by a concomitant viral infection. Increased levels of anti-hsp antibodies have been reported in SLE [128], and in rheumatoid arthritis [129]. However, it is possible that self hsp production is not the primary event triggering autoimmunity, but a secondary response to the tissue damage induced by the autoimmune process itself. Recent studies have suggested a role for hsps in the pathogenesis of insulin dependent diabetes mellitus (IDDM). Cohen and co-workers have demonstrated that a pancreatic beta cell target antigen in NOD mice is a molecule cross reactive with hsp 65 of Mycobacterium tuberculosis. The authors have shown that the onset of beta cell destruction in the mice was associated with spontaneous development of anti-hsp 65 T lymphocytes and antibodies [130]. Only a few studies have examined the possible association of hsps with AITD although this is an active area of investigation. Ratanachaiyavong
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et al reported an association between Graves' disease and a specific RFLP of the hsp 70 gene [ 131 ]. Additionally, hsp 72 was recently demonstrated in Graves' disease and Hashimoto's thyroiditis thyroid specimens and not in controls [132]. Heufelder et al [133] reported increased surface expression of a 72 kDa hsp in retro-ocular and pretibial fibroblasts from Graves patients with severe ophthalmopathy and pretibial myxedema. This expression was not observed in fibroblasts from unaffected sites from the same patients and in fibroblasts from controls. Moreover, the same group has suggested that Graves' disease patients IgG enhanced hsp 72 expression in cultured retroorbital fibroblasts in a dose-dependent manner [134]. These studies imply that AITD is associated with an autoimmune response to certain hsps. Their role in etiology, however, requires clarification. 4.6. Induction of MHC Antigens on NonImmune Cells
Infection can lead to an autoimmune reaction associated with expression of MHC antigens on non-immune cells. As proposed by Bottazzo et al [135], a local viral infection may cause production of gamma interferon or other cytokines in the target organ, which in turn induce HLA class II expression, for the first time, in non-immune cells (eg. epithelial cells), and this can lead to presentation of autoantigens and activation of autoreactive T cells. Indeed, expression of HLA-DR antigens by thyroid epithelial cells has been demonstrated in thyroids from patients with AITD which was absent in normal tissues [ 136]. The mechanisms leading to this unusual MHC class II molecule expression in thyroid cells have been investigated (for a review see [ 137]). Rat and human thyroid cells can be induced to express MHC class II antigens by recombinant gamma interferon, TNF" and TSH itself [ 138]. Neufeld et al demonstrated that cultured rat thyroid cells (derived from a rat thyroid cell line) infected with reovirus types 1 and 3 were induced to express class II MHC antigens in a dose dependent manner [ 139]. Furthermore, a viral thyroiditis, caused by infecting either the thyroid or the immune cells, has been demonstrated in an avian model as discussed earlier [140]. Thyroid follicular epithelial cells bearing MHC class II determinants have been shown to be
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able to present pre-processed viral peptide antigens to cloned human T cells [141] but unfortunately the thyroid cells utilized contained other potential antigen presenting cells. Further evidence, however, came from studies in which thyroid-reactive T cell clones were specifically reactive to cloned autologous thyroid cells in the total absence of antigen presenting cells [142]. Moreover, Kawakami et al have recently shown that in vivo induction of MHC class II molecules on thyrocytes by interferon gamma can induce autoimmune thyroiditis in mice [143]. Viruses can also induce class II MHC molecule expression independent of cytokine secretion. Massa et al have shown that a neurotropic murine hepatitis virus can induce expression of Ia antigen on astrocytes in tissue culture directly and not through release of cytokines [144]. Retroviruses have also been shown to enhance class I MHC expression [145]. However, there are also reports showing reduced expression of MHC molecules by cells infected in vitro with some viruses [146-148]. It has been reported that cytomegalovirus (CMV) infection of primary cultures of thyroid cells resulted in induction of HLA-DR expression on thyroid follicular cells [149]. These findings support the view that infection may induce MHC class II molecule expression on thyroid cells for the first time and that these cells may act as antigen presenting cells and may be involved in the induction of thyroid autoimmune disease.
5. C O N C L U S I O N The literature examined in this review points to the possibility of involvement of an infectious agent in the pathogenesis of autoimmune thyroid diseases (AITD). Several studies point to the involvement of bacterial and viral agents in the pathogenesis of Graves' and Hashimoto's diseases. Various mechanisms have been proposed to explain induction of autoimmunity by infection but it seems that two possibilities may be important to thyroid autoimmunity. Namely, molecular mimicry (perhaps to retroviruses), and MHC class II antigen induction. However, it should be remembered that the association between AITD and infections may be merely coincidental and not etiological. To address this
question more studies utilizing direct approaches (e.g. isolation of the infecting organisms from thyroids of patients with AITD and induction of AITD in experimental animals by viruses) are needed.
12.
13.
ACKNOWLEDGEMENTS This work was supported in part by DK61659 and DK58072 from N I D D K D (to YT) and the Thyroid Research Advisory Council (Abbott) research grant
(to RV).
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15.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Pemphigus and Infection Daniel Mimouni ~,2,3and Michael David 2,3
~Research Center for Autoimmune Diseases, Sheba Medical Center, Tel Hashomer, Tel-Aviv, Israel; 2Department of Dermatology, Rabin Medical Center, Petah-Tiqva, Israel; 3Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel
1. INTRODUCTION Autoimmune diseases are assumed to be initiated or triggered by a complex interaction of environmental factors such as infections and drugs in a genetically susceptible individual. Among the autoimmune diseases, pemphigus is a group of organ-specific autoimmune mucocutaneous blistering disorders with an established immunological basis. The autoimmune mechanism in pemphigus has been elucidated by several steps: initially, Beutner and Jordon in 1964 [ 1] discovered the presence of deposition of IgG in the intercellular space as well as circulating autoantibodies in the sera of pemphigus patients directed against keratinocyte cell surface by direct immunofluorescence (IF) and indirect IF. Thereafter, it was shown that these autoantibodies are pathogenic and can induce the formation of a blister [2, 3]. Almost two decades later, in 1982, Anhalt and his colleagues [4] demonstrated the formation of blisters and erosions in neonatal mice following the passive transfer of pemphigus patients' IgG. The pemphigus antigen were defined as desmoglein 3 and 1, two transmembrane glycoprotein compound of the desmosome. The clinical hallmark of pemphigus is the presence of intraepithelial blisters and erosions of the skin and the mucous membranes. Its three major variants, pemphigus vulgaris, pemphigus foliaceus, and paraneoplastic pemphigus, are characterized histologically by cell-to-cell detachment of epidermal and mucosal epithelial cells (acantholysis) caused by IgG autoantibodies directed against desmosomal adhesion molecules of affected epithe-
lium. These autoantibodies can be visualized using the direct IF technique. Pemphigus vulgaris is the most common form of pemphigus in North America and Europe. Similar to many other autoimmune diseases, the etiology is unknown, although the pathophysiology is well defined. The stimulus for the initiation of the autoimmune process in pemphigus is still unknown, but it is suspected that like other autoimmune diseases, pemphigus may develop as a result of exogenous factor like infectious agents and drugs in a genetically susceptible individual. Studies have shown a higher prevalence in Jews of Eastern European origin, and a predominant allele, DRB 1"0402 (HLA-DR4), has been identified. In addition, rarely have penicillamine and other drugs been reported to trigger or even induce pemphigus vulgaris. The lesions of pemphigus vulgaris typically occur first in the oropharyngeal mucosa and subsequently in the skin. Other mucosa such as the genitalia and conjunctiva may be involved as well. The primary skin lesion consists of flaccid bullae that break to form a large painful erosion which usually fails to heal in the absence of specific intervention. The lesions may occur in all parts of the body, though the scalp, face and trunk are the most common sites affected. Pemphigus foliaceus is further divided into three clinical groups: Brazilian pemphigus (endemic pemphigus, fogo selvagem) has an unknown etiology, but epidemiological data suggest an environmental factor found in certain regions of Brazil, inducing the autoimmunity; pemphigus erythematosus (Senear-Usher syndrome) has features of
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both pemphigus foliaceus and systemic lupus erythematosus; and drug-induced pemphigus foliaceus, which can be induced by the use of d-penicillamine and captopril. Two forms of drug-related pemphigus exist: the more frequent one is the drug-induced pemphigus. In this form, unlike more common drug eruptions, drug-induced pemphigus persists even after withdrawal of the culprit drug because the drug induces a true autoimmune disease. The less frequent form is the pemphigus-like drug eruption that will eventually resolve after withdrawal of the causative drug. The target antigen in PF is desmoglein 1. Pemphigus foliaceus has a clinical presentation that is distinctive from PV. Mucosal involvement is extremely unusual, and mainly "seborrheic" areas, such as the scalp, face and upper trunk, are involved. Blisters are not always present because of the superficial level of epidermal separation. Pemphigus foliaceus has the least morbidity of the three forms of pemphigus. Paraneoplastic pemphigus was first described in 1990 [5]. It is nearly always associated with an underlying neoplasm: a preexisting malignancy in two-thirds of cases (usually non-Hodgkin's lymphoma, chronic lymphocytic lymphoma, or Castleman's disease), but is a marker for an occult malignancy in one-third of cases. The autoantibodies that are characteristically present in this syndrome include anti-desmoplakin 1 and 2, envoplakin, periplakin and the 170kD yet undefined protein, and occasionally autoantibodies against desmoglein 1 and 3 as well as plectin [6]. The typical clinical picture consists of a polymorphous eruption on the skin (pemphigus-like blisters and erythema multiformelike eruption) along with intractable stomatitis.
2. CLINICAL EVIDENCE OF AN INFECTIOUS AGENT
2.1. Viruses and Pemphigus Only rarely has pemphigus been described with association to infectious agents, though many studies performed in the past were aimed at isolating a virus from the lesions of pemphigus or documenting a clear serological evidence for a recent viral infection. Individual reports documented the ini-
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tiation, co-incidence, or relapse of pemphigus following infection with varicella zoster virus (VZV) [7], Epstein Bar virus (EBV) [8], HIV [9, 10] and cytomegalovirus (CMV) [ 11 ]. The majority of cases of viral induced pemphigus were reported with herpes simplex virus (HSV) infection. Krain [12] was the first to describe two cases among 59 cases of PV associated with severe HSV infection. In 1996 Ruocco and his colleagues [13] summarized the known data in the literature regarding the association between a viral infection and pemphigus. They found 13 cases of viral-associated pemphigus. Among the cases described in this study, one was related to CMV, one to EBV, two to VZV and 10 to HSV (one case was related to both VZV and HSV). In this interesting series it is noteworthy that 7 cases were proven by viral culture and 5 by serology. In the past, inoculation of animals with materials from pemphigus patients such as fluid from blisters, skin, blood and urine was used to demonstrate the presence of viruses in some of the patients [ 14-18]. Later, tissue culture experiments were performed with inconsistent results [ 19-21]. In more recent reports, other serologic studies such as complement fixation were used to try and prove the viral involvement in the disease but with very disappointing results [22]. In 1999, Ruocco et al [23] used the polymerase chain reaction (PCR) to study peripheral blood mononuclear cells and skin biopsies from pemphigus patients. They showed the presence of DNA sequence of HSV (type 1 and 2) in 50% of mononuclear cells and in 71% of skin biopsies (of 20 pemphigus patients); in all control cases the results were negative. They concluded that the inability to detect HSV in all cases may suggest that viral infection may be an occasional trigger of pemphigus. The association of pemphigus with human herpes virus type 8 (HHV8) has been demonstrated in 1997 by Memar and his colleagues [24]. Tissue-extracted DNA was tested using PCR, Southern blot hybridization, and automated sequencing of the PCR products for the presence of HHV-8 DNA. The researchers tested 12 patients (6 with PV and 6 with PF) and 12 controls (two with Kaposi sarcoma and 10 healthy individuals). Interestingly, lesional skin from 4 of the 6 patients with PV, all 6 of the patients with PF, and both positive controls (with kaposi sarcoma) tested positive
for HHV-8 DNA. Furthermore, the HHV-8 DNA sequences differed between all 6 DNA specimens from PF, while 3 of the 4 DNA specimens from PV were identical. However, HHV-8 DNA was absent in all normal human skin analyzed. The researchers concluded that it is possible that HHV-8 might have trophism for pemphigus lesions.
2.2. Bacteria and Pemphigus The possible role of bacteria or other micro-organisms has been suggested in the past but no solid data has supported these theories. A recent interesting study by Amagai [25] demonstrated the role of Staphylococcus aureus toxin in the mechanism of cell-ceU detachment in Staphylococcal Scalded Skin Syndrome (SSSS). This bacterial-induced syndrome is characterized by a transient generalized and superficial exfoliative disease. The researchers found that the exfoliative toxin produced by Staphylococcus aureus specifically binds and cleaves desmoglein 1, the target antigen in PF. However, there is no evidence that such a mechanism may initiate acantholysis in pemphigus, and to the best of our knowledge PF was not reported in association with infection by Staphylococcus aureus.
2.3. Vaccination and Pemphigus Very few case reports have documented the association of vaccination with pemphigus. We found only two reports relating the onset of pemphigus to a vaccine. The first, reported by Mignogna et al [26] described the occurrence of a new onset of oral PV appearing 1 month after an influenza vaccination. The second case that was recently reported by Cozzani et al [27] described a sevenyear-old girl who developed pemphigus, probably foliaceus, 7 days after an intramuscular vaccination against tetanus and diphtheria. In this specific case, the authors recognized that the relationship might be causal, but they also raised the theory that the vehicle of the vaccine which contained a thiol group may be the culprit. Korang et al [28] reported an extremely unusual case of PF exacerbation after tetanus vaccination accompanied by production of autoantibodies directed against paraneoplastic pemphigus antigens [28].
3. POSSIBLE PATHOGENESIS The possible role of herpes or other infectious agents might be explained in several mechanisms: 1) It has been suggested that a viral infection may stimulate the immune system and cause a nonspecific reaction leading to an upregulation and activation of the cellular and humoral response. In the setup of a genetically predisposed individual this activation of the immune system may cause the production and secretion of interferons and interleukins which are known to stimulate the immune system. More specifically, it is known that interferon gamma induces the expression of HLA class II on keratinocyte cell membranes, thus making the structural site of pemphigus antigen immunologically active. In this aspect it is possible that the chronic viral infection causes an over-production of IL-4, the IL-4 is responsible for a shifting from Thl to Th2 immune response, that further enhances the production of antibodies [29]. 2) The second possible mechanism is the induction of structural changes in the proteins of the keratinocyte host cell membrane by the virus. It has been shown that viruses can alter the structure of cell proteins not only after the infection of the cell but also in nearby cells without actual penetration to the cell [30]. These new antigens may interact and form complexes with the histocompatibility system to produce immunogenic antigens. 3) Infection of a keratinocyte by a virus may cause a destruction of intracellular proteins that are important for self-tolerance, thus exposing antigens to the immune system. 4) It is possible that viruses cross-react proteins of the keratinocyte cell membrane, thus inducing an autoimmune response [31]. 5) Another possible mechanism is that the virus initiates an autoimmune response by inducing anti-idiotype antibodies that are directed against monoclonal antibodies directed against a specific virus. For example, it has been shown that anti-idiotype antibodies that were made against monoclonal antibodies, produced against the antigen of Coxsackie B4 virus, also reacts with surface antigens of other cells [19]. 6) The last suggested mechanism is that viruses
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interact directly with lymphocytes and alter the normal immune mechanism. This alteration can occur in several levels: infection of T lymphocytes may cause the activation of the lymphocyte and may stimulate the production of autoreactive B lymphocytes. Infection of suppressor T lymphocytes may cause their destruction and prevent their defensive abilities against autoimmune process. And last, the infection of B lymphocytes may cause activation of the cell and production of autoimmune antibodies.
4. T H E R A P E U T I C S ASPECTS Up until now, no therapeutic implications have been reported regarding the possible role of viruses in pemphigus. Several researchers have tried to administer antiviral medications to pemphigus patients but without positive results. Naturally, due to the possible role of HSV in the initiation of the autoimmune process in pemphigus, anti-herpetic agents such as acyclovir were given to patients. However, it has not been shown that this therapeutic intervention has influenced the natural course of the disease. In 50% of pemphigus patients the initial clinical presentation is of erosive stomatitis, therefore, it is not surprising that many of them are treated with antiviral agents arising from misdiagnosis. However, such treatment has no influence on the course of the disease. In summary, although there is no solid evidence indicating a possible role of infectious agent in the etiopathogenesis of pemphigus, such a role cannot be excluded. Prospective studies utilizing novel techniques for detection of viral and/or bacterial infections at the initial phase of pemphigus are needed.
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Beutner EH, Jordon RE. Demonstration of skin antibodies in sera of pemphigus vulgaris patients by indirect immunofluorescent staining. Proc Soc Exp Biol Med 1964;117:505-10. Schiltz JR, Michel B. Production of epidermal acantholysis in normal human skin in vitro by the IgG fraction from pemphigus serum. J Invest Dermatol 1976;67:
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254-60. Farb RM, Dykes R, Lazarus GS. Anti-epidermal-cellsurface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase. Proc Natl Acad Sci USA 1978;75:459-63. Anhalt GJ, Labib RS, Voorhees JJ, Beals TF, Diaz LA. Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease. N Engl J Med 1982;306:1189-96. Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M, Izumi H, Ratrie H 3rd, Mutasim D, ArissAbdo L e t al. Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990;323:1729-35. Mimouni D, Anhalt GJ, Lazarova Z, Aho S, Kazerounian S, Kouba DJ, Mascaro JM Jr, Nousari HC. Paraneoplastic pemphigus in children and adolescents. Br J Dermatol 2002; 147:725-32. Goon AT, Tay YK, Tan SH. Pemphigus vulgaris following vaxicella infection. Clin Exp Dermato12001 ;26: 661-3. Markitziu A, Pisanty S. Pemphigus vulgaris after infection by Epstein-Barr virus. Int J Dermatol 1993;32: 917-8. Splaver A, Silos S, Lowell B, Valenzuela R, Kirsner RS. Case report: pemphigus vulgaris in a patient infected with HIV. AIDS Patient Care STDS 2000;14:295-6. Hodgson TA, Fidler SJ, Speight PM, Weber JN, Porter SR. Oral pemphigus vulgaris associated with HIV infection. J Am Acad Dermato12003;49:313-5. Ruocco V, Rossi A, Satriano RA, Sacerdoti G, Astarita C, Pisani M. Pemphigus foliaceus in a haemophilic child: cytomegalovirus induction? Acta Derm Venereol 1982;62:534-7. Krain LS. Pemphigus. Epidemiologic and survival characteristics of 59 patients, 1955-1973. Arch Dermatol 1974;110:862-5. Ruocco V, Wolf R, Ruocco E, Baroni A. Viruses in pemphigus: a casual or causal relationship? Int J Dermatol 1996;35:782-4. Grace AW, Suskind FH. An agent, transmissible to mice, obtained during a study of pemphigus vulgaris. Proc Soc Exp Biol Med 1937;37:324--6. Grace AW, Suskind FH. An investigation of the etiology of pemphigus vulgaris: the isolation of transmissible agent from a fourth case of the disease. J Invest Dermatol 1939;2:1-13. Grace AW. The etiologic agent of pemphigus vulgaris. Bull NY Acad Med 1946;22:480-1. Dostrovsky A, Gurevitch I, Ungar H. On the question of the aetiology of pemphigus vulgaris and dermatitis herpetiformis (Duhring's disease): a clinical experimental study. Br J Dermatol 1938;50:412-35.
18. Werth J. Beitrage zur virusatiologie des pemphigus vulgaris. Archiv Dermatol Syphilis 1938;176:382-90. 19. Ahmed AR, Rosen GB. Viruses in pemphigus. Int J Dermatol 1989;28:209-17. 20. Siegl G, Hahn EE. A paramyxovirus-like virus isolated from pemphigus-disease in man. Arch Gesamte Virusforsch 1969;28:41-50. 21. Angulo JJ. Attempts to isolate a virus from pemphigus foliaceus cases. Arch Dermatol Syphilology 1954;69: 472-4. 22. Dahl MV, Katz SI, Scott RM et al. Viral studies in pemphigus. J Invest Dermatol 1974;62:96-9. 23. Tufano MA, Baroni A, Buommino E, Ruocco E, Lombardi ML, Ruocco V. Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction. Br J Dermatol 1999;141:1033-9. 24. Memar OM, Rady PL, Goldblum RM, Yen A, Tyring SK. Arch Dermatol 1997;133:1247-51. 25. Amagai M, Matsuyoshi N, Wang ZH, Andl C, Stanley JR. Toxin in bullous impetigo and staphylococcal
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scalded-skin syndrome targets desmoglein 1. Nat Med 2000;6:1275-7. Mignogna MD, Muzio LL. Pemphigus induction by influenza vaccination. Int J Dermato12000;39:795. Cozzani E, Cacciapuoti M, Parodi A, Rebora A. Pemphigus following tetanus and diphtheria vaccination. Br J Dermatol 2002; 147:188-9. Korang K, Ghohestani R, Krieg T, Uitto J, Hunzelmann N. Exacerbation of pemphigus foliaceus after tetanus vaccination accompanied by synthesis of auto-antibodies against paraneoplastic pemphigus antigens. Acta Derm Venereo12002;82:482-3. Vercelli D, Jabara HH, Lauener RP, Geha RS. IL-4 inhibits the synthesis of IFN-gamma and induces the synthesis of IgE in human mixed lymphocyte cultures. J Immunol. Johnson RT. The possible viral etiology of multiple sclerosis. Adv Neurol 1975;13:1--46. Isacson P. Myxoviruses and autoimmunity. Prog Allergy 1967; 10:256-92.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection, Autoimmunity and Autoimmune Liver Diseases Xiao-Song He and M. Eric Gershwin
Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis, Davis, CA, USA
1. INTRODUCTION: GENETIC VS. ENVIRONMENTAL FACTORS Several liver diseases, including autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC), are considered autoimmune diseases. While the etiology of these diseases remains to be defined, each is characterized by an abnormal immune response, involving antibody response and T cell response targeted at self antigens. Genetically inherited factors are thought to play a role in the development of these diseases. For example, the prevalence of PBC has been shown to be greatly increased in family members of PBC patients [1]. Although there is no strong association with MHC class I or class II a|leles, the relative risk of a family member of a first-degree relative within a family having a member with PBC is a hundredfold that of the general population [2]. PSC has been associated to certain MHC class I and II molecules [3]. On the other hand, studies with identical twins clearly demonstrates that genetic background is not the only factor that determines the development of PBC [4]. The results of epidemiological studies suggest that environmental factors also play a role in triggering or exacerbating autoimmune liver diseases [5-7]. For example, several evidences support an infectious etiology for PBC. These include the clustering of disease cases in unrelated individuals at certain locations and increased incidence of PBC in immigrants moving from areas of low prevalence to high prevalence [8]. PBC recurred in up to 20% of patients receiving liver transplantation 10 years
ago [9] and the rate of recurrence correlated with increased immunosuppression [10]. This mimics the recurrence of hepatitis C virus (HCV) infection after liver transplantation for end stage hepatitis C [11], suggesting that a persistent infection in the host may be responsible for the development of PBC. Therefore, a combination of environmental factors, including viral or bacterial infection, and a susceptible genetic predisposition is likely to be the cause for these relatively rare diseases. Murine models have been used to study mechanisms for autoimmunity because key components of the immune system can be easily manipulated in genetically defined inbred strains of mice. The availability of murine strains in which select genes have either been deleted (gene knock-out mice) or introduced (transgenic mice) facilitates the analysis of individual gene products for their role in the autoimmune response. In some cases, the experimentally manipulated immune system generates a pathology similar to that of human diseases, especially systemic autoimmune diseases like lupus, in which autoantibodies are directed against components of normal cell nuclei [12, 13]. In addition to the experimental approach using murine models, searching for environmental factors contributing to autoimmune diseases often relies heavily on a different approach, which is to identify potential risk factors by traditional epidemiological study [2, 14], followed by evaluating the cellular and molecular impacts of each potential risk factor against the phenotypes manifested in the disease. Although abnormal antibody and T cell response against autoantigens have been identified in autoimmune liver diseases, it is still not clear whether these
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Table 1. Infectious agents implicated in autoimmune liver diseases
Disease
Infectiousagent
Epidemiological linkage
Immunological linkage (molecular mimicry)
AIH
HCV
LKM autoantibody D450-specific CD4§ cells LKM autoantibody
autoantibody cross-reactivity CD8+T cell cross-reactivity
HDV HSV PBC
E. coli
autoantibody cross-reactivity CD4+T cell cross-reactivity CD4§ cell cross-reactivity CD4+T cell cross-reactivity CD8§ cell cross-reactivity
UTI
Azotobacter vinelandii Pseudomonas putida Pseudomonas aeruginosa
retrovirus PSC
Helicobacter species
retrovirus
Seroreactivity, detection of viral nucleic acid sequence Detection of bacterial DNA Seroreactivity
abnormalities are the initiating cause or the secondary consequence of the respective disease. Despite this obvious difficulty, the identification of overlaps between the full spectrum of immune abnormalities in the disease and the known effects of each risk factor may still serve as a useful guide for establishing a potential link between the risk factor and the disease. In this chapter we will first review the current literature on the abnormal autoreactive B and T cell responses in autoimmune liver diseases. Within this context, we will then discuss the potential link between these abnormalities and viral or bacterial infections as well as the hypothetical mechanisms underlying the induction of autoimmunity by infections. Table 1 summarizes several infectious agents that have been implicated in autoimmune liver diseases, through either epidemiological or immunological linkage.
2. AUTOANTIBODIES IN A U T O I M M U N E L I V E R DISEASES
The three major types of autoimmune liver diseases are manifested by distinct but overlapping sets of antibodies against self-antigens. Little is known at present about the mechanism that underlies the induction of these autoantibody responses. Based on the autoantibody profiles, two major subgroups
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of AIH have been described. AIH-1 is characterized with autoantibodies to fiver kidney microsomes (LKMs), which include different isozymes of the cytochrome P450 family, or CYPs, and those of UDP-glucurononosyl-transferases, or UGTs. AIH2 is characterized with antibodies against a variety of nuclear and muscle autoantigens (ANA, SMA, pANCA and SLA/LP) [15]. In PBC, autoreative antibodies against mitochondria antigens (AMA) are directed predominantly to the E2 subunit of the three enzymes of the 2-OADC family [ 16]. The major autoantibody in PSC is antineutrophil nuclear antibody (ANNA), which can also be found in AIH patients. Many of the autoantigens in these diseases are metabolic enzymes catalyzing the detoxification reactions of a broad array of endobiotic and exobiotic substrates, including reactive oxygen products of bioactivation, carcinogens, therapeutic drugs and other xenobiotics. The liver is the major organ for these metabolic pathways and, as such, the anatomic site where these genes are expressed. These gene products are highly diversified, with many gene families and subtypes that ensure their extremely broad substrate specificity. Although considered the humoral hallmark of autoimmune liver diseases, autoantibody production is not unique to such diseases. Actually autoantobodies can be frequently detected in patients with infectious liver diseases. For example, autoantibodies to CYPs and UGTs can also be detected in
patients with chronic viral hepatitis [ 17]. Anti-UGT and anti-CYP antibodies can be detected in up to 14% of patients chronically infected with hepatitis delta virus (HDV) [18-21 ]. Autoantibodies to members of the cytochrome P450 family (various CYP isozymes) are frequently detected in patients with chronic hepatitis C [22-24]. ANNA, the hallmark of PSC, is found in the majority of patients with ulcerative colitis (UC) [25, 26]. Although UC is also considered an autoimmune disease, portal bacteraemia has been described in patients with UC [27], suggesting a link between the hepatic infection and intestinal disorders. These observations raise the possibility that the production of autoantibodies is not induced by a mechanism unique to autoimmune liver diseases, but rather the consequence of liver cell damage caused by distinct etiologies, for example known or unknown hepatitis viruses or intestinal bacteria that translocate to the liver. It has also been hypothesized that immune responses against virus-infected cells, mediated by NK cells and/or virus specific MHC restricted cytotoxic T lymphocytes (CTL), lead to the lysis of target cells with the release of cryptic proteins that otherwise have never been seen by the immune system. These proteins are thus recognized as foreign, hence initiates the autoimmunity. Alternatively, autoreactive antibody response could be induced by viral antigens. Support for this theory is provided by the finding that LKM antibodies specific for P450 2D6 cross-react with HCV and herpes simplex virus proteins [28]. This suggests that the autoantibodies could have been induced by a B cell response directed at the viral antigens, again implicating viral infection in the induction of autoimmunity. This phenomenon has been termed "molecular mimicry".
3. T CELL RESPONSE IN AUTOIMMUNE LIVER DISEASES The histological hallmark of the autoimmune liver diseases is tissue infiltrating T lymphocytes. Analysis of TCR repertoire and cytokine production in livers of PBC patients has been reported [29]. Such analysis revealed limited usage of V[~ gene family members and conserved amino acid residues in the CDR3 junction sequences, suggesting that the int-
rahepatic T cell responses were driven by specific antigens, resulting in clonal expansion of antigenspecific T cells. In the same study mRNAs for IL-2 and IFN- T but not IL-4 were detected in PBC liver, indicating a Thl-dominant cytokine pattern in the PBC liver. These data suggest that an antigen-specific Thl and CTL response might be important in the immunopathogenesis of PBC and other autoimmune liver diseases. CD4 + T cells specific for autoantigens have been identified and their MHC II-restricted target epitopes mapped in patients with different autoimmune liver diseases. In each case the target protein is the same as those of autoantibodies: P450 in AIH patients with LKM1 antibodies [30, 31] and PDCE2 in PBC [32, 33]. Of note, P450-specific CD4 § T cell clones have also be isolated from a small fraction of HCV-infected patients [31], which again, from a different perspective, raises the possibility that a virus is actually involved in the initiation of autoimmunity. Shimoda et al have assessed the frequency of CD4 § T cells recognizing the HLA DRB4*0101-restricted PDC-E2163_176peptide [33] and found a disease-specific enrichment of 100- to 150-fold of such cells in the hilar lymph nodes and the liver than in periphery. CTLs are thought to be directly involved in the tissue injury in autoimmune liver diseases. CD8 + T cell clones with cytolytic activity specific for autologous B cell lines have been isolated from AIH patients, although their target proteins were not defined [30]. Kita et al have characterized the first MHC class I-restricted epitope in PBC patients, the PDC-E2159_167peptide [34]. Using tetramer technology, a 10-fold increase in the frequency of CTLs specific for this epitope was found in the liver as compared to the blood in PBC patients [35]. Interestingly, the dominant autoreactive B cell epitope [33] and the class I and class H-restricted T cell epitopes (peptides 159-167 and 163-176) appear to localize to the same inner lipoyl domain of the autoantigen PDC-E2.
4. CAUSE OR CONSEQUENCE? Although it is generally believed that autoreactive T cells are involved in immunity-mediated cell lysis and liver destruction, the precise role of the B and T cell responses in the autoimmune fiver diseases is
539
largely unknown. However, it is helpful to consider this role based on current knowledge about B cell and T cell responses so that any potential environmental risk factors can be examined by their effect on either arm of the immune response that is relevant to the disease. It has been suggested recently that antibodies have the intrinsic capacity to destroy antigens by catalyzing production of hydrogen peroxide from molecular oxygen [36]. Alternatively, autoantibodies may cause destruction of the autoantigen by more classical mechanisms, such as activation of the complement cascade. Since the major targets of autoantibodies such as P450s and UGTs are mainly expressed intracellularly within the liver, a role for extracellular antibody in the pathology has been difficult to understand. Nonetheless, direct damage by autoantibodies is an attractive hypothetical etiology for the autoimmune liver diseases. PBC patients are characterized with high titers of AMA antibodies including IgA, which may gain intracellular access by transcytosis through bile duct cells. In fact, PDC-E2 specific IgA has been detected in bile from PBC patients [ 16]. It remains to be proven whether intracellular IgA reacts directly with the target antigens to cause the damage. The production of autoantibodies may be a consequence of liver inflammation and tissue destruction, the common characteristics of autoimmune liver diseases and viral hepatitis. The liver cell damage in these diseases, potentially caused by CTLs, may result in exposure of cellular components. It has been found that HCV core protein induces production of reactive oxygen species (ROS), which can damage cellular proteins and is thought to contribute to the cytopathic effect of HCV [37]. Thus the direct cytopathic effect of HCV may cause modification of autoantigens to generate neoantigens. Recently it was reported that when yeast cells were exposed to stress, several metabolic enzymes including PDC-E2 were oxidatively damaged, resulting in modified proteins [38]. This suggests that stress caused by environmental factors, such as viral infection, may lead to production of neoantigens which is more immunogenic than the autoantigen. Along this line of argument, it appears to be plausible that the autoantigen-specific T cells rather than antibodies are directly responsible for the destruction of hepatocytes in AIH, as well as the bile ducts in PBC.
540
Regarding the issue of how an infection actually breaks the first ring and initiates the cascade of events leading to the collapse of self-tolerance, two studies in transgenic mice are intriguing. In the first study, an HBV transgenic mouse model was utilized. In this model HBV is expressed in the liver and appears to be tolerated by the host. Repeated immunization of these animals with HBsAg or recombinant vaccinia vector expressing HBsAg, however, led to the production of T cell-dependent anti-HB autoantibodies that cleared HBsAg from the serum, but not to the activation of HBsAg-specific CTLs. The immunized transgenic mice showed no signs of autoimmune liver disease, suggesting that autoantibodies alone were not sufficient to induce an autoimmune disease [39]. In another study, transgenic mice expressing an MHC 1-restricted epitope of LCMV GP33 in the liver was used. When CD8 § T cells from TCR-transgenic mice specific for the GP33 epitope were adoptively transferred into these mice, they ignored the GP33 transgene expressed in hepatocytes; however when these mice were infected with LCMV, tolerance to GP33 was broken and the mice developed hepatitis. These results suggest that viral infection may break tolerance at the CD8 § T cell level, leading to autoimmune hepatitis [40]. Together, these transgenic mouse studies favor the hypothesis that T cells (especially CD8 § T cells) but not antibodies specific for autoantigens are directly responsible for the induction of AIH. Nevertherless, it has been reported that autoantigen-specific B cells, once activated by antigen-specific helper T cells, become highly efficient APCs for the antigens captured by their surface Ig. The activated B cells can internalize, process and present their specific antigen to naive T cells much more efficiently than professional APCs [41]. Furthermore, it has been shown recently in PBC that soluble PDC-E2 complexed with autoantibodies against PDC-E2 is much more efficient than soluble antigen alone in inducing PDC-E2-specific CTL lines [34]. These results indicate that autoreactive CD8 T cell response may be facilitated by the autoreactive B cell response, which defines a new role for B cell response in the pathogenesis of autoimmune diseases.
5. M O L E C U L A R MIMICRY
Molecular mimicry, or the resemblance of pathogen- and host antigen-derived epitopes recognized by immune cells, has been suggested to be a mechanism for the induction of autoimmune diseases [42, 43]. This mechanism has been implicated in systemic autoimmune diseases such as multiple sclerosis [44, 45] and rheumatoid arthritis [46]. However, more evidence is needed than simply identifying similar epitopes, either conformational epitopes recognized by antibodies or homologous peptide sequences cross-recognized by epitopespecific T cells. The homology between the pathogen and host epitopes p e r se does not explain the apparent paradox that the autoantigen cannot elicit a primary response, but can be recognized as the target for effector T cells induced by the pathogenic epitope. In order to break the intrinsic tolerance to the autoantigen, an epitope mimic, or mimeotope, has to induce activation and proliferation rather than anergy or apoptosis of the naive T cells, and the expanded epitope-specific T cells must recognize the autoantigen presented by the host cells in the respective tissue to cause autoimmune disease. The activation of na'fve T cells requires them to be stimulated by the peptide epitopes presented by MHC molecules, along with a second signal, or costimulating signal, which is usually provided by professional APCs but not the local tissue cells expressing the autoantigen. When an infection induces a fullscale immune response against the pathogen in an inflammatory environment, the mimeotope carded by the infecting pathogen may have a better chance of being presented by APCs, in concert with the interactions between the co-stimulatory molecules and their natural ligands on the T cells, to elicit a primary T cell response. Once such response is established, the epitope-primed specific T cells may have a lower threshold of activation and may recognize the autoantigen-expressing tissue cells to causing tissue damage. This is supported by the fact that a recall antigen response is usually much more vigorous than the primary response. Virus-induced molecular mimicry that leads to autoimmunity has been demonstrated in a mouse model of multiple sclerosis [47]. In this work, a virus carrying a native or mimic sequence of the immunodominant myelin epitope initiated autoepitope-specific CD4 + T cell
response in the virus-infected mice, resulting in development of organ-specific autoimmune demyelination [48]. The apparent paradox (that the autoantigen p e r se does not elicit a primary response but can be recognized as the target for immune T cells specific for the mimeotope) can also be explained in the context of altered peptide ligands (APL) [49]. According to this theory, distinct but related peptide sequences recognized by the same TCR may trigger different responses when they engage the TCR. Several recent studies have provided support for this view. In one of the first studies, T cell lines specific for an immunodominant T cell epitope on myelin basic protein, the major target of autoimmunity in multiple sclerosis, were generated by in vitro stimulation with the peptide epitope or its APL. It was found that stimulation with an APL made with one amino acid change at a TCR contact residue induced T cell lines that responded better to the native peptide [50], suggesting that an APL may be more immunogenic than the native peptide in terms of inducing a T cell response against the autoantigen. A second study demonstrated that a known HLA A2-restricted epitope of HCV, core178-187, is homologous to two human CYP peptides P450 2A6 and P450 2A7 with 2 different amino acids in each case. A primary CTL response was induced in vitro using PBMC of healthy donors with the HCV peptide, but not with the autologous CYP peptides; however the HCV peptide-specific CTL clones recognized and lysed target cells loaded with the CYP peptides or those presenting endogenously processed P450 protein [51]. These results suggest that intrinsic tolerance to an autoantigen can be broken by a more immunogenic foreign antigen that is distinct from but related to the autoantigen. This scenario may occur during an unknown viral or bacterial infection. Immune responses, including antibodies or T cells against the foreign antigen, may cross-react with the autoantigen to cause the autoimmunity. The infecting virus or bacterium could persist as a known or unknown pathogen; or they may be cleared, leaving no trail behind except for the virus- or bacteriumspecific immune cells. The task of tracing back to the original immunogen from the existing autoreactive T cells can be even more difficult. The TCR repertoire in a single epitope-specific response can be very heterogene-
541
ous in terms of V~ gene usage. TCRs with different VI3 fragments may differ subtly in their fine specificity for variant epitope sequences. This has been found in murine CTL responses against an epitope of HbsAg [52], as well as human CTL responses against an HCV NS3 epitope. It is conceivable that within the primary T cell repertoire against the mimeotope carried by the infecting agent, a minority of the immune T cells may have a better affinity to the autoantigen peptide. These cells will be selectively amplified upon contact with the autoantigen, resulting in an autoreactive T cell response with characteristics significantly different from the primary response against the mimic peptide. Similar scenario may also apply to the antibody response. The finding of molecular mimicry between HCV and CYPs at the CD8 § T cell level, in addition to the detection of CYP-reactive antibodies and CD4 § T cells in HCV infected patients as described above, again suggests that HCV may be involved in the induction of AIH, although more data is needed to validate this hypothesis. Findings in PBC also support the molecular mimicry theory. CD4 + T cells specific for PBC-E2, the target protein of autoimmunity in PBC, have been identified in patients with PBC and were found to recognize an MHC class II restricted epitope, peptide 163-176 of PDC-E2. The amino acid motif ExDK in the peptide is essential for its recognition by the specific T cells. The same motif was identified in homologous peptides derived from proteins of E. coli, Azotobacter vinelandii and Pseudomonas putida, which can also be recognized by CD4 § T cell clones specific for the PDC-E2163_176 peptide. Interestingly, some of the T cell clones were activated more vigorously by bacterial mimicry peptides than by the native peptide [33, 53]. Of note, epidemiological studies have indicated that female PBC patients have a higher incidence of recurrent urinary tract infections (UTI) [54-56], which is frequently caused by E. coli. On the other hand, it has been reported that AMA, the major autoantibody in PBC, were detected frequently in patients with recurrent UTI [57]. Both of these results are consistent with the hypothesis that E. coli infection may initiate the PDC-E2-specific autoimmunity in PBC. Recently, a peptide derived from Pseudomonas aeruginosa has been found to be partially homologous to PDC-E2159_167, the HLA-A*0201-restricted epitope on PDC-E2 rec-
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ognized by autoreactive CTLs in PBC. This bacterial peptide showed a higher binding affinity to the HLA-A*0201 molecule than the prototype epitope on PDC-E2 and was recognized by CTLs specific for the prototype epitope [58]. These findings support the thesis that molecular mimicry at the levels of MHC I- or MHC II-restricted epitope may be implicated in the initiation of autoreactive T cell responses in autoreactive liver diseases.
6. INFECTIOUS DISEASE OR A U T O I M M U N E DISEASE? Infection by a microorganism may trigger a vigorous response in the host immune system, which intersects at many different points with the signaling network that maintains tolerance to autoantigens, resulting in abnormal immune responses against self proteins, or breakdown of self tolerance and initiation of autoimmunity. An infectious agent carrying a potential mimeotope of a liver autoantigen could be much more immunogenic than the native epitope expressed in the liver. For an epitope to induce activation rather than anergy or apoptosis of T cells, it must be presented by class I or II MHC molecules along with proper costimulating signals, like CD80/ 86. However, liver cells express markedly low levels of MHC and costimulating molecules, which has been reasoned as a mechanism for liver tolerance. Conceivably, proteins expressed in the liver environment are not very immunogenic. However, when the same or similar antigenic determinant is introduced through a different route into a different environment, its immunogenicity can be enhanced and established tolerance can be broken. For example, HBV or HBsAg transgenic mice produce high levels of HBsAg in the liver and are tolerant to the protein. However immunizing these mice with purified HBsAg in CFA, with a DNA vaccine carrying the HBsAg gene or a recombinant vaccinia virus expressing the same protein all result in humoral and T cell immune response to the autoantigen [39]. A similar effect has also been reported in chronic HBV carriers, who also produce HBsAg and are tolerant to it [59]. When the immune system first detects a foreign antigen, be it a virus or a bacterium, a full spectrum of innate immune response are mounted to create
a local environment favoring an adaptive immune response. This involves the mobilization of NK cells that kill virus-infected host cells, and professional APCs expressing cytokines and costimulatory molecules that activate na'fve T cells. A mimeotope carried by this invader may become immunogenic under these favorable circumstances. The antigenspecific T cells generated from a primary response may have a lower threshold of activation and therefore may recognize and exert effector function on the autologous antigen presented in a less optimal fashion. Even if an infecting pathogen does not carry any epitope mimic of an autoantigen, its invasion may still markedly change the tolerogenic environment of the liver. NK cells and CTLs may attack virus-infected liver cells, resulting in release of cellular proteins from the apoptotic or necrotic liver cells. These may be internalized and processed by APCs and presented by MHC class II to CD4 + T cells, or presented by MHC class I to CD8 § T cells (cross-presentation), resulting in activation of autoreactive CD4 + and CD8 § T cells. Bacterial pathogens have another feature that may affect the immune system. Some bacteria contain superantigens, such as enterotoxins, that are capable of activating large numbers of T cells irrespective of their antigenic specificity [60, 61 ], which may contribute to the induction of autoimmunity. Based on these discussions, it is plausible to assume that at least some of the autoimmune liver diseases may be induced by a past viral or bacterial infection, which may or may not carry a mimeotope to an autoantigen. These diseases are considered autoimmune diseases by nature because the original infection has been cleared and the immune responses against the autoantigens are believed to be directly responsible for the disease pathogenesis. On the other hand, it is also possible that some of the autoimmune liver diseases are actually caused by an unknown persistent infection with all the clinical symptoms directly related to the infection, while the autoreactive antibody or T cell responses are actually consequences of the tissue cell damage resulted from the infection. For example, Chronic HCV and HDV infections are known to be associated with a variety of autoimmunity manifestations, including LKM autoantibodies [62]. Immunoblotting studies have indicated that seroreactivity with retroviral proteins, including p24 of HIV and pro-
teins of human intracisternal retroviral particle type I, can be frequently detected in patients with PBC and PSC [63]. These antibodies may be attributed to an immune response to uncharacterized viral proteins that share antigenic determinants with these known retroviruses [63]. Considering the heterogeneous nature of the diseases currently categorized as autoimmune liver diseases, perhaps some of them will eventually be considered as infectious diseases instead. It is possible that a virus other than those causing hepatitis A, B, C, D and E may be discovered in the future. While an animal model is not available for PBC, Mason et al established a tissue culture model by coculturing normal biliary epithelial cells (BEC) with perihilar lymph nodes from patients with chronic liver diseases. BECs cocultured with PBC lymph nodes developed a specific phenotype of PBC with immunohistochemical evidence of antimitochondrial antibody reactivity [64]. Furthermore, the supernatants from the PBC cocultures also induced the similar phenotype in fresh BECs [65], suggesting a transmissible agent is involved in PBC. The same researchers have cloned a retroviral sequence directly from biliary epithelium extracted from PBC livers [65]. Pilot studies using antiretroviral treatment for patients with PBC also supported the involvement of a retroviras in the disease process [66]. These results, being preliminary and need to be confirmed in more patients, point to a possible viral etiology as well as a new direction of therapy for PBC.
7. BACTERIAL I N F E C T I O N AND AUTOIMMUNE LIVER DISEASES In addition to E. coli-caused UTI (see above), other bacteria have been linked to autoimmune liver diseases, especially PBC and PSC that involve pathogenesis in the bile tract. Leptospira species are known to infect the fiver and cause hepatitis [67]. Although the bile tract is normally bacteriumfree, it may be infected by various bile-tolerant microbe strains, including Enterococcus species, Haemophilus influenzae, E. coli, and related enteric bacteria [68]. More than 20 Helicobacter species have been described, and at least 13 of them colonize the
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lower gastrointestinal tract of animals. Some of these species may have a zoonotic potential [69]. In addition to the well-known H. pylori, several other species may also cause human gastric infection [70]. Intestinal Helicobacter species can enter the bloodstream, particularly in immunocompromised individuals, and thus it would be expected that these organisms could enter the liver. In murine models, the intestinal helicobacters have been observed to translocate to the liver where viable infection may be associated with inflammation and/or neoplastic change [71, 72]. In a recent study in Rhesus monkey, it was suggested a Helicabacter species could translocate from the intestine to the liver, resulting in hepatitis [73]. Although it has not been successful to directly isolate these bacteria in culture from human hepatic and biliary tissue due to their fastidious growing conditions, other strategies have been used to detect these organisms. By immunohistopathology and transmission electron microscopy, H. pylori was observed in the liver of a patient with PSC [74]. PCR has been used to detect helicobacter DNA based on the specific sequence of 16s ribosomal RNA gene. With this technique, helicobacter DNA was detected in a majority of primary hepatic and biliary cancers [75, 76]. However, similar PCR studies in PBC patients yielded conflicting results [77-79]. Although antibody to H. pylori was frequently detected in patients with chronic liver diseases, a recent case-control study failed to detect a difference in the prevalence of H. pyroli exposure between AIH patients and healthy controls [80]. Despite these discrepancies, the hypothesis that helicobacters infect the human intestine, liver, or biliary tree and may be responsible for some of the autoimmune liver diseases and other unexplained human diseases is certainly very attractive and need to be further explored with various strategies.
8. C O N C L U S I O N S Considering the relative rarity of autoimmune liver diseases, they are likely to be the consequence of multiple events, each of them resulted from the interaction of certain environmental factors and the genetic background of the host. Viral and bacterial infections are some of the environmental factors that have been implicated in this complex process. Iden-
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tification of the infectious etiology for autoimmune liver diseases will point to new preventive or therapeutic interventions. For the acute infection with the potential of initiating autoimmunity, antiviral or antibiotic treatments may prevent the induction of autoimmune diseases. For the persistent infection involving in the pathogenesis of autoimmune liver diseases, such treatments may decelerate the disease progression and may even provide a cure for some of these devastating diseases.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infections and Vasculitis Jan Willem Cohen Tervaert' and Coen A. Stegeman 2
tDepartment of Clinical and Experimental Immunology, University Hospital Maastricht, Maastricht, The Netherlands; 2Departmentof Nephrology, University Hospital Groningen, Groningen, The Netherlands
1. I N T R O D U C T I O N Atherosclerosis and vasculitis are both inflammatory vascular disorders. The possibility of an association between vascular disorders and infectious diseases was already proposed in the beginning of the 20th century. A number of infectious agents and conditions have been associated with atherosclerosis and many potential mechanisms have been hypothesized to result in atherothrombogenesis. Infectious agents that are especially associated with atherosclerosis are cytomegalovirus (CMV), chlamydia pneumoniae and helicobacter pylori. Furthermore, gingivitis has been associated with atherosclerosis. The evidence that infectious agents may be involved in atherogenesis do not yet prove a causal relationship. For vasculitis, the evidence for an infectious agent in the pathophysiology is much stronger. But also here the evidence is restricted to few patients and in most of the patients with vasculitis no clear infectious agent can be convincingly demonstrated. In this review, the evidence for a role of infectious agents in vasculitis will be reviewed.
2. CLASSIFICATION OF VASCULITIS Vasculitis is a condition characterized by inflammation of blood vessels. Its clinical manifestations are dependent on the localization and size of the involved vessels as well as on the nature of the inflammatory process. Vasculitis can be secondary to other conditions or constitute a primary disorder. A wide variety of infections may give rise to vasculitis. Other underlying conditions of second-
Table 1. Primary vasculitides
9 Large vessel vasculitis Giant cell (temporal) arteritis Takayasu' s arteritis 9 Medium-sized vessel vasculitis Polyarteritis nodosa Kawasaki disease 9 Small vessel vasculitis Wegener' s granulomatosis a Churg Strauss syndromea Microscopic polyangiitisa Henoch-Schtinlein purpura Essential cryoglobulinemic vasculitis Cuataneous leukocytoclastic angiitis aassociated with anti-neutrophil cytoplasmic autoantibodies (ANCA).
ary vasculitides are connective tissue diseases, sarcoidosis, malignancies, hypersensitivity to drugs and substance abuse. In many of these secondary forms immune deposits can be demonstrated in the involved vessel wall by direct immunofluorescence of biopsy material. The primary vasculitides are idiopathic systemic diseases. In many of them there is, albeit indirect, evidence that microbes could play a role in the pathophysiology. Definite proof for such a role is, however, lacking. Primary forms of vasculitis are classified based on the size of the vessels involved, the histopathology of the lesions and the presence of characteristic clinical symptoms. A classification scheme was proposed in 1993 by an international consensus group [1, Table 1]. The definitions are not intended to be used as diagnostic criteria and
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in clinical practice many patients do not fulfill exactly the definitions. To classify patients with histologically or angiographically proven vasculitis, the American College of Rheumatology (ACR) has developed sets of criteria for the various vasculitides that are based on clinical signs and symptoms [2]. These criteria, however, also have limitations with respect to sensitivity and specificity.
2.1. Vasculitis Secondary to Infection
A. Bacteria, fungi and parasites. Vasculitis secondary to bacteria or fungi are usually the result of direct invasion of blood vessels. Certain microorganisms have a propensity to invade vessel walls leading to destruction and thrombosis. In the lungs, examples of bacteria that cause necrotizing vasculitis are Pseudomonas aeruginosa and Legionella pneumophila. In addition, the obligate anaerobic Fusobacterium necrophorum may cause thrombophlebitis of the internal jugular vein(s) with subsequent spread to other predominant venous vascular beds following pharyngitis or other infections of the ENT region [3]. Furthermore, necrotizing granulomatous vasculitis like lesions can be found during mycobacterial infections and during infections with fungi such as Aspergillus or Mucor. Infective vasculitis may also be a result of hematogenous dissemination of microorganisms. This may be caused by septic embolization or by microbial invasion of a previously damaged vessel wall during bacteriaemia or fungemia. The resulting lesion is called "mycotic aneurysm". Sites that are predisposed to this mycotic aneurysm formation are arterial bifurcations, arteriovenous fistulae, coarctations and atherosclerotic vessels. Most frequently these infective lesions arise in the aorta, intracranial, superior mesenteric, or femoral arteries [4, 5]. Many of these mycotic aneurysms occur in the setting of infective endocarditis. Mycotic aneurysms,however, may also be associated with other conditions than infective endocarditis, especially in elderly patients or intravenous drug users. In these cases, most of the microorganisms are Staphylococcus Aureus and salmonella species. In addition to mycotic aneurysms of larger vessels, hematogenous dissemination from the primary infectious source or the mycotic aneurysms may lead to small-vessel vasculitis involving the skin and other organs.
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Patients with infective arteritis usually have leukocytosis, an elevated erythrocyte sedimentation rate (ESR) and high C reactive protein levels. Blood cultures are positive in more than 50% of the cases. Also, staining and culture of biopsied purpuric skin lesions, if present, may be positive for bacterial microorganisms. If the source of septic emboli is not endocarditis or an infected vascular prothesis, the primary focus (e.g. osteomyelitis) can be found in less than 50% of the cases. The mortality rate for patients with infected aneurysms is high. Successful treatment generally requires the combination of surgery and antibiotic therapy. Surgical therapy is crucial and should not be delayed, as the risks for possible fatal bleeding and serious ischemic complications is high. Other infections: Before the introduction of penicillin, syphilitic vasculitis was very common. Nowadays aortitis and cerebrovascular disease due to the spirochete Treponema pallidum is rare. There is, however, a rise in the number of cases of syphilis primarily due to the epidemic of HIV. The diagnosis of syphilitic vasculitis can be made serologically. T. pallidum hemagglutination assay (TPHA) is positive in most of the cases. In cases of cerebrovascular syphilis the VDRL-test is positive in CSF in about 50% of the cases. Furthermore, TPHA in CSF is positive. Syphilitic vasculitis is still being treated with high doses of penicillin G. Another spirochete, i.e., Borrelia burgdorferi, is an infrequent cause of CNS vasculitis. The diagnosis of Lyme disease is usually made by the recognition of the typical clinical picture in combination with serological confirmation of infection with B.
burgdorferi. Other microorganisms that are typically causing vasculitis are Rickettsiaceae, a group of obligate intracellular bacteria with tropism for endothelial cells. In Rocky Mountain spotted fever, non-specific symptoms such as malaise, myalgia, nausea and vomiting are followed by a rash characterized by a maculopapular rash and fever. Untreated, patients develop widespread vasculitis leading to renal insufficiency and/or pulmonary edema. Other Rickettsiae infections such as boutonneuse fever and epidemic typhus are also associated with a systemic small-vessel-vasculitis. Upon clinical suspicion patients have to be treated with doxycycline 100 mg 2x per day. Serological evidence occurs too late for
an early diagnosis. Thus, therapy must be instituted based on clinical findings. Finally, localized granulomatous vasculitis can be due to parasitic infections such as Dirofilaria immitis and Wuchereria species. Cases of more disseminated vasculitis associated with infections with parasites such as Toxocara, Schistosoma species, Angylostrongylus, Filariae have been described [reviewed in 6]. Whether these associated vasculitis manifestations are the result of direct infestation of vascular structures by the parasite or result from serum sickness like disease caused by chronic production of parasitic antigens in the presence of an immune response, is unclear.
B. Vasculitis secondary to virus infections. Already in the early seventies initial observations were made that hepatitis B virus (HBV) infection was associated with systemic vasculitis. Since then an increasing number of viruses have been associated with vasculitis. Viruses generally cause vasculitides involving small- or medium-sized vessels. Whether viruses can also induce vasculitis of the large vessels is not known. Occasionally, however, DNA of viruses can be found in biopsies of patients with large vessel vasculitis. Finally, specific syndromes such as retinal vasculitis or central nervous system vasculitis have also be related to certain viruses. Well-known examples are retinal vasculitis due to cytomegalovirus and CNS vasculitis due to varicelia zoster virus. a. Hepatitis B virus associated vasculitis HBV is associated with 2 types of vasculitic syndromes. Firstly, an immune complex mediated small-vessel vasculitis, effecting mainly the skin, and secondly, a polyarteritis nodosa like vasculitis with multisystem involvement [7]. The first form of vasculitis typically occurs in around 10% of the patients prior to the onset of jaundice or other symptoms of hepatitis. Clinical findings are fever, arthralgias, and rash due to vasculitis. Skin biopsies usually show leukocytoclastic vasculitis or a lymphocytic venulitis. By immunofluorescence IgG, IgM, rarely IgA, C3 and HBsAg can be demonstrated. In the circulation, HBsAg, HBeAg and HBV DNA are detected. IgM anti HBc may not yet be present. Complement levels (C3 and C4) are
usually decreased and circulating immune complexes can be detected [7]. The outcome of the syndrome is favorable and no specific therapy is required. In a few cases, however, chronic HBV infection is present and in those patients antiviral therapy is needed. The second form of vasculitis that is associated with HBV is polyarteritis nodosa (PAN). This form of vasculitis usually develops within the first year following HBV infection [8]. Patients present with weight loss, fever, arthralgias, myalgias, mononeuritis multiplex, hypertension, and ischemic abdominal pain. Importantly, in about 20-25% of the cases signs and symptoms of small vessel vasculitis (purpura, glomerulonephritis) are present as well [8]. Typically HBV associated PAN is a 'one shot' disease. Relapses occur only very infrequently. Complement levels are usually decreased. Otherwise non-specific findings such as an elevated ESR and an elevated C-reactive protein are present. In about 20% of the cases, eosinophilia is present. Serologically patients are HBsAg positive and HBeAg positive. Circulating HBV DNA is also present in about 80% of the cases [8]. IgM anti HBC is often not detected. Antineutrophil cytoplasmic antibodies (ANCA) are generally not detected. By immunofluorescence, HBsAg, complement, and immunoglobulins are found in the vessel walls. Biopsies of muscles and nerves can demonstrate vasculitis of small- and medium-sized vessels. Abdominal or renal angiograms can be positive for microaneurysms and/or multiple stenoses. Liver biopsies show in most of the cases chronic hepatitis. Treatment consists of a short course of corticosteroids, plasma exchange, and antiviral therapy such as lamivudine, interferon alpha and/or vidarabine [9]. Based on this regimen, Guillevin et al reported a 10 year survival of 83% and a total clearance of HBV in 24% of the cases with HBV related PAN.
b. Hepatitis C virus has been more recently associated with so-called mixed essential cryoglobulinemia (MEC). In certain areas of the world, nearly all patients with MEC seem to be associated with this virus. Indeed, in about 35-55% of patients with HCV infection circulating cryoglobulins can be detected [ 10]. The incidence of
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HCV associated cryoglobulinemic vasculitis is, however, much lower: around 2-3% of patients with HCV. The most common clinical manifestation of HCV associated cryoglobulinemic vasculitis is skin vasculitis, presenting as purpura, leg ulcers, livedo reticularis, urticaria, nodular lesions or Raynaud's phenomenon. Biopsy of the skin shows leukocytoclastic vasculitis and immunofluorescence findings show IgM, IgG and C3 deposits. Furthermore, HCV has been demonstrated in lesions by in situ hybridization [ll]. Other frequently occurring clinical findings are a distal symmetric polyneuropathy due to vasculitis, arthralgias and in rare cases arthritis. Furthermore, liver involvement is present in about 40% of the cases and in about 30% of the cases a membranoproliferative glomerulonephritis is present. Relatively frequently vasculitis of smalland medium-sized renal arteries is present in the kidney biopsy. Sometimes this arteritis is even present in the absence of glomerular involvement [12]. The diagnosis of HCV associated MEC is made by the detection of both HCV antigen and circulating cryoglobulins. The cryoglobulins in HCV are generally type II cryoglobulins, containing a mixture of polyclonal IgG and monoclonal IgM with rheumatoid factor reactivity. Cryoprecipitates may contain both HCV and specific antibodies to HCV. Other laboratory findings are anemia, low complement levels and high rheumatoid factor levels. The endpoint of treatment should be complete clearance of both HCV and cryoglobulins. Standard interferon alpha therapy (3 • 3 million units per week) is often unsuccessful. Therefore, daily continuous administration or pegylated interferon alpha is recommended. Otherwise, a combination of interferon alpha with ribavirin is advised [13]. In patients with severe vasculitic symptoms, plasma exchange or cryofiltration with a cooling unit is indicated. In addition, low doses steroids and/or immunosuppressives are sometimes needed although these are relatively contraindicated because they may worsen HCV viraemia. c. Vasculitis associated with other viruses Human immunodeficiency virus type I (HIV) is
only occasionally associated with vasculitis [14,
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15]. Since HIV infected individuals are commonly also co-infected with other pathogens it is difficult to estimate the relative importance of the HIV virus in the pathophysiology of the vasculitis. A wide variety of vascular inflammatory diseases is being reported in HIV. The most common vasculitic syndromes are a PAN-like syndrome and CNS vasculitis. A variety of other clinical pathological syndromes have, however, been reported including cryoglobulinemia, Henoch-Sch6nlein purpura, Churg-Strauss syndrome and different forms of angiocentric lymphomas. Since corticosteroids and immunosuppressives have to be avoided as much as possible in HIV, therapy consists of anti-retroviral therapy in combination with either plasma exchange or intravenous immunoglobulins. Human cytomegalovirus (HCMV) has been associated with human atherosclerosis, rapidly progressive coronary vasculopathy in cardiac transplant recipients and to a lesser extent with chronic renal transplant failure. These associations rely mostly on serological data [ 16, 17]. In addition, HCMV has been detected in inflammatory arteritis of the aorta [18]. However, as HCMV and other human herpes virus material can be found in normal as well as affected parts of the aorta it is unclear whether HCMV has any role in the pathogenesis of these disorders [19]. However, severe HCMV disease in hosts immune compromized by HIV infection or immunosuppressive medication can result in necrotizing small vessel vasculitis with thrombosis, bleeding and perforation of gastrointestinal organs [15, 20]. Another herpes virus that is not infrequently associated with vasculitis is variceUa zoster virus (VZV). Vascular inflammatory disease due to VZV is also heterogeneous. The forms that are most commonly detected are an ipsilateral large vessel vasculitis following herpes zoster ophtalmicus and a more disseminated CNS smallvessel vasculitis. Vasculitis following herpes zoster ophtalmicus is a predominant large vessel vasculitis that develops infrequently within 6 weeks after cutaneous VZV infection of the head or neck and usually occurs in immune competent patients. Typically, the vascular involvement is ipsilateral to the side of skin involvement and
leads to focal neurologic deficits [21]. Clinical manifestations are contralateral hemiparesis, aphasia or hemianesthesia. In less than 50% of the cases mental changes are observed. Cranial nerve palsy occurs in 40-50% of the cases. MRI scans and CT scans of the brain are usually abnormal. Cerebral angiography shows signs of vasculitis. In the cerebrospinal fluid antibodies to VZV are detected; polymerase chain reaction for VZV can be, however, negative [22]. In most cases the disease has a favorable outcome; in patients with severe or progressive disease, however, a combination of corticosteroids and antiviral drugs such as aciclovir is warranted. In immune compromized individuals a more diffuse and slowly progressive CNS small-vessel vasculitis can be seen. In these cases the interval between the dermal zoster and the cerebral vasculitis can be very variable, and a preceding dermal zoster may be absent [21]. Other viruses that are associated with vasculitides are parvo-
virus B19, Epstein-Barr virus, herpes simplex virus and others. Generally, these viruses cause a small vessel vasculitis of the skin. In some cases, however, there is suspicion that these viruses induce primary forms of vasculitis (vide infra).
2.2. Primary Vasculitides 2.2.1. Vasculitis involving large vessels Giant cell arteritis and Takayasu's arteritis have no clearly established association with an infection. Previous exposure to mycobacterial, streptococcal, spirochaetal, and parainfluenza viral infection have been mentioned in both giant cell and Takayasu's arteritis [23]. In giant cell arteritis, parvovirus B19 and Chlamydia pneumoniae DNA have been demonstrated in temporal artery specimens [24, 25]. These findings, however, could not be confirmed by others [26, 27].
2.2.2. Vasculitis involving medium- and smallsized arteries Kawasaki disease, also known as mucocutaneous lymph node syndrome, is a form of systemic vasculitis of unknown cause that primarily affects infants and young children. Clinical features include acute
fever; cervical lympadenopathy; conjunctival injection; redness of the lips, tongue, or oral mucosa; erythema of the palms and soles; edema of the hands and feet; a polymorphous cutaneous rash; desquamation of the skin; and cardiac abnormalities in about 20-30% of the patients. Arteritis of the coronary, iliac, or other systemic arteries can be found on histologic examination. The syndrome can occur sporadically, but sometimes clear outbreaks are observed. Several infectious agents have been suspected to be responsible for the syndrome such as streptococci, staphylococci, Epstein-Barr virus (EBV), retrovirus, or parvovirus B19 [28]. In few cases a clear pathophysiological role for toxic shock syndrome toxin- 1 (TSST-1) positive Staphylococcus aureus has been documented [29]. In polyarteritis nodosa (PAN), immune-complex and complement deposits are often found in involved tissues. An infectious agent has to be searched for and is often found. In children, ~-hemolytic group A streptococci are especially associated with PAl',/. In adults, hepatitis B virus (HBV) infection has a firm link with PAN. During recent years, however, a decline in HBV-related PAN has been documented in France and at present less than 10% of PAN cases are HBV positive [8]. PAN-like disease, has been found increasingly often, however, in patients that are infected with HIV [ 16]. Whether PAN in these cases is a result of a direct effect of HIV infection of blood vessels, a result of the immune activation that accompanies HIV infection, or a result of accompanying drug hypersensitivity or complicating infections with viruses such as CMV, HBV, or hepatitis C virus, is at present unknown [ 16]. Finally, many other incidental microbial associations with PANlike vasculitis have been described [reviewed in 281.
2.2.3. Vasculitis involving small-sized vessels In a few children with Wegener's granulomatosis (WG), a disease characterized by chronic inflammation of the respiratory tract, glomerulonephritis, and vasculitis, chronic parvovirus B19 has been suspected [ 16]. In adults, however, this virus is not found. A more important infectious association in WG is chronic nasal carriage of Staphylococcus aureus [30]. Previously, we found that 60-70% of our patients were chronic nasal carriers of S. Aureus
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and that those patients that were chronically carrying S. Aureus relapsed nearly eight times more frequently than non-carriers [31]. Recently, we found that 40-50% of the S. Aureus strains that are found in these nasal cultures are positive for staphylococcal superantigens [29, 30, 32]. Superantigens, that were most frequently found were TSST-1, staphylocococcal enterotoxin A and C, and exfoliative toxin A. Importantly, in a long-term observational study, we found that TSST-1 positive S. Aureus strains but not strains that were positive for other superantigens increased the risk for a relapse of WG [32]. Another observation that links WG with bacterial infection and/or colonisation is the finding that patients with WG in which the disease is restricted to the respiratory tract can be successfully treated with co-trimoxazole as monotherapy [33]. Furthermore, we previously demonstrated in a placebo-controlled trial that co-trimoxazole maintenance therapy in patients with WG not only reduced the infection rate but also reduced the risk to develop a relapse by more than 60% [34]. It has been postulated that cotrimoxazole may exert this effect by eradication of chronic nasal S. Aureus carriage. In a recent study, however, we were unable to demonstrate such an effect since nasal S. Aureus carriage was terminated in a minority of WG patients during co-trimoxazole maintenance therapy. This finding suggest that co-trimoxazole exerts its benefecial effect in WG through a different mechanism, possibly an anti-inflammatory action. Importantly, it has been demonstrated in in vitro studies that co-trimoxazole inhibits myeloperoxidase-mediated halogenation of proteins. In the other two forms of anti-neutrophil cyto-
plasmic antibody (ANCA)-associated vasculitides, i.e., microscopic polyangiitis and the Churg-Strauss syndrome (CSS) the role of chronic nasal S. Aureus carriage is less clear. Cases have been descibed in which CSS was linked to infection with ascaris, aspergillus fumigatus, and/or H/V [28]. In addition, in several CSS cases a link with immunostimulation due to vaccination and/or desensitization is suspected (but never proven). In Henoch-Schrnlein purpura and isolated leukocytoclastic vasculitis of the skin, a precipitating microbial agent is often found. Bacteria, viruses, and sometimes parasites, such as Ascaris, have been associated with these forms of small-vessel vascu-
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litis [28]. Microorganisms that are most frequently involved are Streptococci, Staphylococci, Neisse-
riae, Cytomegalovirus, Parvovirus B19, HI~, HBV, and hepatitis C virus. Vasculitic lesions in these patients are assumed to be the result of immunecomplex deposition initiated by microbial antigens. In some cases, however, other mechanisms may be operative such as in Ricketsiae infections, in which infections primarily infect and damage endothelial cells resulting in vasculitis. During the past decade, it became clear that there is a firm link between hepatitis C virus infection (HCV) and mixed essential ctqyoglobulinaemia (MEC) [10]. Whereas in Mediterrean countries more than 80% of the patients are positive for HCV RNA as detected by polymerase chain reaction, it is assumed that MEC in Northern European countries is less often associated with HCV (see above).
3. MECHANISMS BY W H I C H INFECTIOUS AGENTS T R I G G E R VASCULITIS Different mechanisms may be operative in the induction of vasculitis by infectious agents [29]. Three mechanisms are most likely to be involved: a. Direct microbial invasion of endothelial cells; b. Participation in immune-complex mediated damage of vessel walls; and c. Stimulation of (autoreactive) B and/or T lymphocytes. 3.1. Direct Microbial Invasion
Rickettsiae primarily infect the endothelium of the microvasculature and later on also endothelial cells of small arteries and veins. Another example of microbial invasion of endothelial cells is S. Aureus. It has been demonstrated that S. Aureus binds more readily to endothelial cells than most other bacteria. Following binding, the bacteria are internalized and can persist in phagosome-like vacuoles as a small colony variant. The interaction between S. Aureus and endothelial cells may result in activation of the endothelial cells resulting in enhanced expression of adhesion molecules such as P-selectin and ICAM-1 and in the production of cytokines and chemoattractants such as IL-8 and MCP-1. Furthermore, endothelial cells may be damaged following internalization of alpha-toxin producing strains.
Herpes viruses can also be found in vascular and endothelial tissue and may results in inflammation. Intriguing is the finding in animal models of vascular herpes virus infection that the elastic media of larger vessels seems to be an immunoprivileged site. Hence, large vessels may be chronically infested by the virus and lesions with similarities to both atherosclerosis and Takayasu's arteritis may develop. Especially when infected at a young age or in interferon-), deficient animals severe vascular disease may develop [35, 36].
3.2. Immune-Complex Mediated Damage of Vessel Wafts In biopsies of patients with vasculitis deposits of immunoglobulins and complement components are often found. The nature of the antigen is, however, in most cases unknown, although associations between infections and vasculitis as described in this chapter suggest that antigens may be of microbiological origin. In skin biopsies with vasculitis due to MEC, HCV has been identified [11] and HBV has been identified in some cases of HBV associated vasculitis [37, 38]. Since electrical charge is an important factor for antigen deposition, we studied the possibility that cationic staphylococcal antigens may be involved in immune-complex formation in WG. We found that one of these proteins, staphylococcal acid phosphatase (SAcP), had in vitro high affinity for endothelial cells and that renal perfusion of SAcP in S AcP-immunized rats resulted in a severe crescentic glomerulonephritis. Furthermore, antibodies to SAcP were frequently detected in patients with WG and SAcP was present in 3 of 19 renal biopsies from patients with WG [39]. From these studies, we hypothesized that in WG immune complexes play a role in the initiation of the disease and that staphylococcal antigens are likely candidate antigens.
3.3. Stimulation of (Autoreactive) B and/or T Lymphocytes Infections may stimulate auto-immune responses by different mechanisms [40]. These include shared epitopes between pathogens and host, upregulation of heat shock proteins, and stimulation of lymphocytes by factors such as peptidoglycan, protein A, CpG motifs in bacterial DNA and superantigens.
Superantigens are extremely potent activators of lymphocytes. Stimulation of T cells is dependent on the presence of MHC class II molecules on antigenpresenting cells. In contrast to classical T cell antigens, processing of the superantigens is not needed. Superantigens bind to MHC class II molecules on antigen presenting cells and to conserved regions of T-cell receptor V-beta chains. Virtually all T cells expressing a superantigen-binding V-beta chain proliferate. After proliferation, activated T cells undergo aptoptosis. Furthermore, repetitive stimulation may induce anergy, a process that is possibly dependent on stimulation of CD4+ regulatory T cells. Superantigens may induce autoimmunity by stimulation of autoreactive cytotoxic T cells and/or by T cell dependent activation of antigen-specific B cells. In Kawasaki disease, vasculitic disease activity, TSST-1 producing S. Aureus, and the presence of corresponding V-beta 2+ T cells have been simultaneously documented [29]. In patients with WG, a condition in which T cell expansions and staphylococcal superantigens are frequently found, we failed to show superantigen-related T cell expansions.
4. CONCLUSION Infectious agents have been clearly demonstrated in various vasculitides. Direct evidence of a pathophysiological role of specific microbial agents is, however, scarce. Recently developed molecular approaches such as DNA microarrays may be helpful of studying this issue in the nearby future.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Multiple Sclerosis Samantha S. Soldan and Steven Jacobson
Viral Immunology Section, NIH/NINDS, Bethesda, MD, USA
1. ETIOLOGY OF MULTIPLE SCLEROSIS 1.1. Introduction to MS
Multiple sclerosis (MS) is the most prevalent demyelinating disease of the central nervous system (CNS), affecting approximately 1,100,000 individuals worldwide [1]. In general, a T helper- 1 (Th- 1) mediated autoimmune component driven by myelin protein-specific, pro-inflammatory cytokine secreting T-cells is believed to mediate the pathogenesis of this disorder. The onset of MS typically occurs between the ages of 20 and 40 years and, like most autoimmune disorders, women are affected more often (1.5-2 times) than men. MS was first classified as a discrete entity by Dr. Jean Martin Charcot who called the disorder la sclerose en plaque [2]. In 1868, Charcot gave an accurate account of the clinical symptomatology and pathology of MS and speculated on mechanisms of pathophysiology, disease frequency and clinicopathological correlates [2]. A diverse array of neurological signs and symptoms are associated with MS. These include limb weakness, impaired motor function, sensory symptoms, visual symptoms, eye movement disorders, bladder symptoms, sexual dysfunction, fatigue, ataxia, deafness, spasticity, dementia, and cognitive impairment [3]. Although the clinical progression of MS is variable and unpredictable, there are several distinct clinical courses in which the majority of MS patients can be classified. The most common form of MS is relapsing-remitting MS (RRMS) characterized by disease exacerbations where new symptoms appear or existing symptoms become more severe. These exacerbations last for variable amounts of time and are followed by peri-
ods of total or partial recovery. Disease in RRMS patients may be inactive for months or years. Some individuals who are initially diagnosed with RRMS will develop secondary chronic progressive MS characterized by the accumulation of progressive disability with (relapsing progressive MS;RPMS) or without (secondary progressive MS;SPMS) superimposed relapses. These forms of progressive MS are distinct from the primary chronic progressive form (CPMS), which is characterized by a lack of distinct attacks. These individuals experience a gradual onset of disease with a steady worsening of symptoms. Two additional forms of MS affect a minority of patients. The Marburg variant of MS affects less than 5% of those diagnosed with the disease and is marked by frequently occurring relapses leading to rapidly accumulating disability often resulting in total immobility and death [3]. Benign MS occurs in a small percentage of patients and is characterized by young age at onset with infrequent sensory episodes and full recovery. The etiology of MS is unknown. In part, this is due to the variability of this disease, which suggests that many factors may be involved in the spectrum of clinical syndromes that are defined as MS. It is widely accepted that genetic, immunological, and environmental factors contribute collectively to MS susceptibility. 1.2. Genetic Influences
A strong influence of genetic background on MS disease susceptibility is supported by epidemiological, familial, and molecular studies. The worldwide distribution of MS is skewed with areas of lower prevalence in Asia, Africa and South America and
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areas of high prevalence in North America, Europe, New Zealand, and Australia [4]. The incidence and prevalence of MS follows a north-south gradient in both hemispheres [5-7]. It has been suggested that the north-south gradient observed in the New World reflects the tendency of individuals from Northern Europe to migrate to the northern regions of the United States and Canada and, conversely, those from regions of Europe with a lower incidence of MS to migrate to southern regions of the United States and South America [7, 5]. The importance of genetic background in susceptibility to MS is further supported by epidemiologic studies demonstrating different prevalences of MS among genetically distinct populations living in the same geographic area. For example, the frequency of MS is increased in Hungarians and Bulgarians of Caucasian decent compared to gypsy populations residing in the same regions [8, 9]. A similar situation exists in Australia and New Zealand where those of European descent have a far higher risk of developing MS than the indigenous populations [10, 11]. In the United States, the prevalence of MS among people of Japanese ancestry living on the Pacific Coast (6.7/100,000) is considerably lower than that of Caucasians living in California (30/100,000) [ 12]. However, people of Japanese descent living on the West Coast of the United States have a slightly higher prevalence of MS than those living in Japan (2/100,000) suggesting a significant impact of environmental factors on disease susceptibility [ 12]. A genetic influence in the development of MS has been firmly established by family and twin studies. Adoption studies have demonstrated that biological relatives of patients with MS have a greater likelihood of developing MS than adoptees and, conversely, that family members of adopted individuals with MS do not have an increased risk of developing the disorder [ 13]. The lifetime risk of MS among biological relatives of individuals with MS increases with closer biological relationships. The risk is greatest for siblings of affected individuals, especially sisters, and decreases in second- and third-degree relatives [14, 15]. Additionally, the rate of MS concordance is eight times greater in monozygotic than dizygotic twins. However, the concordance among monozygotic twins is only 20%, which suggests that a susceptible genetic background alone is not sufficient to cause disease
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[16]. The inheritance of MS susceptibility does not follow classic models of inheritance. It is generally believed that MS is a complex, heterogenous disorder, influenced by several genes, each exerting a relatively modest effect. These genes may act independently, or, epistatically on MS pathogenesis. Some genes may be involved in the induction of this complex disorder while others may be involved in disease progression, further confounding genetic studies in MS. Over the years, several genes, most of which are associated with immune function, myelin structure, or mitochondria have been tentatively associated with an increased risk of MS [17-21]. Many of these associations have not been demonstrated consistently in different studies. However, a strong association between MS and the major histocompatibility complex (MHC) class II alleles DR2 and DQwl has been established in several populations [22].
1.3. Immunologic Influences It is widely accepted that an aberrant immune response plays an important role in the etiology of MS. This is based on the association of MS with genes involved with the immune response, the immunopathology of the disease, the clinical response of MS patients to immunomodulatory and immunosuppressive treatments, and similarities with experimental immune-mediated demyelinating diseases in animals. As described above, the MHC class II background of an individual is important in disease susceptibility. Many studies have concentrated on MHC-peptide interactions in order to determine how MHC class II alleles may confer disease susceptibility. It has been determined that the binding affinity of an antigenic peptide to an MHC allele determines T cell immunogenicity and encephalitogenecity [23]. The vast majority of studies concerning MHC-peptide interactions relevant to MS have focused on myelin basic protein (MBP) as an antigenic peptide. MBP-specific T cells have been demonstrated in the peripheral blood of both MS patients and normal individuals. The frequencies of MBP-specific T cells tend to be higher in MS patients than controls. However, similar frequencies of MBP-specific T cells have been demonstrated in affected and unaffected family members thereby
suggesting that the frequency of MBP-specific T cells may be linked to the immunogenetic background of an individual and may be a prerequisite, rather than a consequence, of disease development [24]. Molecular mimicry, a phenomena by which environmental antigens cross react with normal host cell components, may induce an immune response against host proteins such as MBP. Therefore, individuals with higher frequencies of MBP reactive T-cells may be more likely to develop autoreactive T-cell responses as a result of environmental, nonself epitopes mimicking MBP. A complex series of immunological mechanisms associated with events both in the peripheral blood system as well as the central nervous system (CNS) have been proposed [25, 26]. A number of immune abnormalities are frequently observed in MS patients. One of the hallmarks of MS is the intrathecal secretion of oligoclonal antibodies [27]. Olicgoclonal bands (OCB) are found in the CNS tissue and CSF of greater than 90% of MS patients and are used in the diagnosis of the disease. OCB are not specific for MS as they are also found in several other chronic inflammatory CNS conditions of either infectious (such as CNS lyme or chronic viral and bacterial meningitis) or autoimmune (such as CNS lupus erythematosus) origin. Although OCB are not directed against a single antigen, antibody bands specific for viral, bacterial, and self antigens have been desribed [28, 29]. Therefore, it is unclear whether or not the intrathecal synthesis of immunoglobuilns observed in MS results from the presence of cells that are passively recruited into the CNS after the pathogenetically relevant cells crossed the blood brain barrier (BBB). In addition to the presence of OCB, other immunological markers of disease activity have been described in MS. Several proinflammatory cytokines, including TNFtx, INF-y, and osteopontin are upregulated in MS [30]. Treatment of MS patients with IFN-y resulted in a marked increase in exacerbations, supporting the model of MS as a Th-1 mediated autoimmune disease [31]. Furthermore, an increase in TNF-t~ expression proceeds relapses and inflammatory activity as measured by MRI, while the mRNA levels of anti-inflammatory cytokines such as IL-10 and TGF-[3 decline. The overexpression of these cytokines may be involved in disease pathogenesis by causing the upregulation of MHC and adhesion
molecule expression on endothelial and glial cells, activation of macrophages and recruitment of TH1 cells, or by damaging oligrodendroglial cells and myelin sheathes directly [32]. The soluble adhesion molecules ICAM-1 and E-selectin are elevated in MS sera while soluble VCAM-1 and E-selectin are increased in the CSF of MS patients [33]. The utility of immunosuppressive and antiinflammatory therapies in MS also supports an autoimmune component in disease pathogenesis. Although corticosteroid treatment does not alter the long-term course of MS it is used effectively in the treatment of MS exacerbations. It has been demonstrated that the administration of high-dose steroids immediately stops BBB leakage as visualized by gadolinium-enhanced MRI [34]. A number of immunosuppresive and chemotherapeutic drugs including cyclophosphamide, and methotrexate have been used in the treatment of MS with variable success. Currently, two immunomodulatory therapies, namely IFN-I3 and Copolymer-1 (Cop-l), are widely used in the treatment of MS. IFN-15 oposses many of the effects of IFN-y, including the recruitment of inflammatory cells and the up-regulation of MHC and adhesion molecules. Additionally, IFN-~ has been shown to lower the exacerbation rate in MS patients with a relapsing remitting course and inflammatory activity as demonstrated by MRI [35, 36]. COP-1 is a synthetic polypeptide consisting of a random sequence of four amino acids, blocks antigen presentation by competing with antigenic peptides for the MHC binding groove. COP-1 has been demonstrated to be approximately as effective as IFN-I3 in early relapsing remitting MS [37]. Experimental autoimmune encephalomyelitis (EAE) models in various animals have provided great insight into the immunopathogenesis of MS and are indispensable in the development of immunomodulatory therapies for the disease. EAE is an acute or chronic-relapsing inflammatory demyelinating disease of the CNS characterized by inflammation and demyelinating white matter lesions. EAE may be induced in a number of susceptible inbred animal strains by the injection of whole white matter or individual myelin proteins such as proteolipid protein (PLP) or MBP in complete Freund's adjuvant [38]. The ability to transfer EAE from an affected animal to a naYve animal with cellular or humoral components demonstrates that EAE is an
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immune-mediated autoimmune disease. EAE resistant and susceptible strains of mice, rats, and guinea pigs have been bred and, as in MS, are associated with particular MHC-class II backgrounds [38]. EAE has been used to develop treatment modalities that are broad, such as those focusing on the migration of encephalitogenic T cells into the CNS, and specific, such as specific interventions targeting the trimolecular complex. The various EAE models have contributed greatly in our understanding of the immunopathogenesis and immunopathology of MS and will continue to be a great asset in the development of immunomodulatory therapies for this disease. 1.4. Environmental Influences
Infectious agents have been suspected in the etiology of MS for over a century [39]. The first suggestion that infectious agents may be involved in the pathogenesis of MS came from Dr. Pierre Marie, a pupil of Charcot, in 1884. Marie believed that several organisms were involved in the pathogenesis of MS, either alone or in combination, based on the anecdotal association of acute infectious diseases (including typhoid, malaria, pneumonia, and childhood exanthemata) with the onset of disease. Marie hypothesized that MS was triggered by infection, which led to changes in blood vessels ultimately resulting in an inflammatory interstitial reaction of glial cells [40]. Data implicating infectious agents in the pathogenesis of MS include: (1) epidemiological evidence of childhood exposure to infectious agents and an increase in disease exacerbations with viral infection [39, 41] (2) geographic association of disease susceptibility with evidence of MS clustering [42, 4] (3) evidence that migration to and from high risk areas influences the likelihood of developing MS [41] (4) abnormal immune responses to a variety of microbes [43, 44] and (5) analogy with animal models and other human diseases in which viruses can cause diseases with long incubation periods, a relapsing remitting course, and demyelination. As described above, distribution of MS follows a geographic distribution with an increased prevalence in northern latitudes, which may be the result of both genetic and environmental influences. Migration studies have supported an infectious
562
etiology of MS [45-49]. In general, individuals migrating from high risk to low risk areas after the age of 15 tend to take their risk of MS with them. However, individuals who migrate from high risk to low risk areas before the age of 15 acquire a lower risk. These data suggest that exposure to an environmental factor, perhaps an infectious agent, must occur before the age of 15 in order to influence MS susceptibility. Reports of clusters or epidemics of MS also support a role for an infectious agent in MS. In the Faroe Islands off the coast of Denmark, no cases of MS were reported from 1929 to 1943. However, after the occupation of the Faroe Islands by British troops in 1940, twenty islanders developed MS between 1940 and the end of the war. The areas of the Faroe Island MS epidemic were found to correlate with the locations of British troop encampments after 1940 [50, 51]. This initial "outbreak" of MS was followed by additional clusters of the disease each separated by 13 years. It has been proposed that the original cluster of MS was initiated by an infectious agent that only affected the MS susceptibility of individuals between the ages of 11 and 12. The susceptible individuals harbored this microbe in a latent state through adolescence thus accounting for the thirteen-year interval between MS clusters on the Faroe Islands [4]. Other examples of MS epidemics have been described in the Shetland-Orkney Islands, Iceland, Key West Florida, Mossyrock Washington, and Mansfield Massachusetts [52, 53]. 1.4.1. Viruses in demyelinating diseases of animals and man
Viruses have been implicated in a number of demyelinating diseases of the central nervous system in both animal and human subjects. The association of viruses in other demyelinating diseases further supports an infectious influence in the development of MS by demonstrating the capability of infectious agents to induce demyelination that can persist for years in the Central Nervous System (CNS). Viruses invloved in demyelinating diseases of nonhumans include canine distemper virus (CDV), murine coronavirus (JHM strain), Theiler's mouse encephalomyelitis virus (TMEV), and Visna virus [54-57]. TMEV is a mouse enterovirus, belonging to the family of picomaviridae, which is typically found
in the gut. However, TMEV are occasionally able to penetrate the CNS and cause an acute inflammation of the anterior horm cells that resembles poliomyelitis. Pathologically, this disease is characterized by demyelination with the preservation of axons. TMEV is often used as a model for MS because the pathological anomalies are limited to the CNS, infection is latent and persistent, demyelination is mediated by the immune system and occurs after a long incubation period, antibodies to myelin and proteolipid protein can be detected in diseased animals, and there are recurrences of demyelination and remyelination reminiscent of relapsing remitting MS [57]. Virulent and avirulent strains of TMEV have been identified. Of interest, it is the persistent avirlulent strain that causes chronic CNS disease [58]. Susceptibility of mice to TMEV mediated demyelinating disease is associated with MHC class I genes [59]. Murine hepatitis virus (MHV) is a coronavirus that, as the name suggests, primarily infects the liver. However, several murine strains, including the JHM strain, are neurotropic and cause encephalitis with subsequent CNS demyelination. The virus readily infects oligodendrocytes and neurons and kills most animals. However, the virus establishes a persistent infection of astrocytes and those animals that survive acute infection develop a chronic progressive neurologic disease [60] characterized by scattered demyelinating lesions and CNS infiltration of macrophages and lymphocytes [60]. As the disease progresses, lymphocytic infiltration diminishes while demyelination and astrogliosis increase. These lesions resemble the chronic plaques of MS. However, the role, if any, of the immune system in the pathology of MHC induced demyelination is unclear as CNS disease can occur in the absence of B and T cells [61]. CDV is a member of the morbilliviruses and is related to measles and rinderpest viruses. CDV is a common infection of dogs and other canines. Acute infection with CDV may be followed by a demyelinating encephalomyelitis called sub-acute diffuse sclerosing encephalitis. This encephalitis is characterized by tremor, paralysis, and convulsions and may not appear until weeks or months after acute infection Lesions observed show demyelination with sparing of axons and perivascular cuffs of lymphocytes and macrophages [54, 55]. Antibodies
to CDV and CNS myelin are detected in the serum and CSF of affected animals. Canine distemper demyelinating encephalomyelitis strongly resembles subacute sclerosing panencphalitis (SSPE) pathologically, virologically, and immunologically [54, 551. Visna virus is a member of the lentiviruses, which includes human immunodeficiency viruses I and II. Infection of sheep with visna virus results in gait abnormalities followed by paraplegia and total paralysis. The disease course is variable and neurologic signs typically correlate with elevations in CSF protein and pleocytosis. Visna virus has a primary tropism for monocytes and macrophages and is transported to the CNS by infected monocytes that release viral particles when they differentiate into macrophages [62]. Once released into the CNS, the virus infects microglia and leads to the recruitment and proliferation of cytotoxic T lymphocytes (CTL). It is believed that the demyelination lesions observed in this disease my be the result of damage by virus specific CTL and auto-antibodies. Examples of viral-induced demyelinating diseases~of man include progressive multifocal leukoencephalopthy (PML), subacute sclerosing panencephalitis (SSPE), and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/ TSP). PML is a rare subacute demyelinating disease associated with JC virus, a papovirus that is widespread in human populations world wide. Approximately 50-70% of adult humans are seropositive for JC virus 65% of which are infected by the age of 14. Typically, PML occurs in individuals who are immunocompromised or have defective cellular immunity. It has been reported that 3-5% of AIDS patients develop PML [63]. Patients with PML present with variable symptoms depending on the location of CNS lesions [64]. SSPE is a CNS disease of children and young adults that develops as a rare consequence of measles virus infection. The clinical course of SSPE typically begins with subtle mental deterioration followed by lack of coordination and other motor abnormalities [65]. The clinical course of SSPE may last either months or years and ultimately results in coma and death. SSPE patients have high serum and CSF antibodies to all measles structural proteins with the exception of the membrane (M) protein. CNS lesions in SSPE are characterized by perivas-
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cular cuffing with infiltrates of lymphocytes and plasma cells in both gray and white matter. Extensive demyelination and an increase in hypertrophic astrocytes are also observed in this disease. Cowdry type A and B inclusion bodies containing measles virus-specific antigens are found in both neurons and glia. However, intact measles virus particles are absent in brain material of SSPE patients and the mechanism by which measles virus enters the CNS is unknown. The development of SSPE has not been associated with particular strains of measles virus and, therefore, it is likely that other host factors are required for this rare disorder to occur. The human T-lymphotropic virus type I (HTLV-I) is associated with a chronic, progressive neurologic disease known as HTLV-I associated myelopathy/ tropical spastic paraparesis (HAM/TSP). The clinical hallmark of HAM/TSP is a gradual onset of lower extremity weakness, bowel and bladder dysfunction, fecal incontinence, Babinski sign, variable sensory loss [66-69]. The onset of HAM/TSP is gradual in most patients and the disease is clinically indistinguishable from the chronic progressive form of MS. Cerebrospinal fluid (CSF) analysis~ HAMI TSP is remarkable for a oligoclonal bands some of which are directed against HTLV-I [70]. Magnetic resonance imaging (MRI) has demonstrated demyelinating lesions in both the white matter and the paraventricular regions of HAM/TSP brains and swelling or atrophy in the spinal cord [71]. Although the distinct plaques characteristic of MS are not observed in HAM/TSP, loss of myelin with some preservation of axons has been described. The incubation period between infection with HTLV-I and the development of HAM/TSP is typically long and, as will be described subsequently, only occurs in a minority of those infected.
2. INFECTIOUS AGENTS IN MS
2.1. Infectious Agents Associated with MS Infectious agents have long been suspected in the etiology of MS [39, 40]. Over the years, several viruses and bacteria have been associated with MS based primarily on elevated antibody titers or the isolation of a particular virus/bacterium from MS material (Table 1). Despite the extensive efforts
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to associate MS with a particular pathogen, none of these viruses have been definitively associated with the disease. Elevated antibody titers to several viruses including influenza C, herpes simplex 1 & 2, measles, varicella-zoster, rubeola, vaccinia, Epstein-Barr, mumps, SV5, and human herpes virus 6 (HHV-6) have been reported in patients with MS in comparison with healthy controls [93, 87, 94-97]. Most of these reported agents have been discounted from consideration in the pathogenesis of MS. However, a few remain candidate viruses. Several bacteria have also been identified as potential etiologic agents MS. More than 80 years ago, spirochetes were believed to cause MS based on the fact that syphilis can cause a relapsing/remitting inflammatory disease of the CNS with OCB present in the CSF [98]. Other associations of bacteria and MS have been suggested based on the observation of increased antibody titers in MS patients compared to controls [99, 100, 29, 101]. It is unknown whether elevated antibodies to infectious agents found in the CNS of MS patients represent local production of antibody in the CNS as a result of resident lymphocytes or if they are a consequence of "spill-over" of circulating serum antibodies as a consequence of a damaged blood-brain barrier. Of the many viruses from disparate families (Table 1) that have been associated with MS, measles virus has received the greatest consideration. Mealses virus can establish persistence, both in tissue culture and in vivo, as is the case in SSPE, a chronic progressive demyelinating disease of the CNS. In addition; both humoral and cellular immune responses to measles virus differs in MS patients compared to healthy controls [44]. Intrathecal synthesis of measles specific antibodies has been demonstrated in the CSF of MS patients and, paradoxically, decreased measles specific CTL are found in MS patients compared to healthy individuals [44]. Cytoplasmic tubular structures resembling measles nucleocapsids have been found in the astrocytes of one MS patient. Of interest, an explant of this patient's brain tissue developed a cytopathic effect, which was blocked by pretreatment with an anti-measles serum [102]. Additionally, in one study, measles virus-specific RNA has been detected by in situ hybridization in brain material of patients [86]. However, subsequent studies have not confirmed these results [ 103, 104].
Table 1. Partial list of infectious agents associated with MS Agent
Evidence for association
Reference
Bacteria B. burgdorferi M. tuberculosis C. pneumoniae
Increased seropositivity in MS Increased CSF T cell responses Increased culture positive in MS
[72] [73] [29]
Coronavirus Coronavirus
Isolation from mice inoculated with MS brain
[741
Isolated in T cells from MS patient brains Increased CSF antibody Titers in MS Isolated from CSF of MS patients Geographical distribution Geographical distribution Isolated from chimpanzee inoculated with MS brain Higher prevalence of EBV infection in MS Detection of DNA and viral protein in MS brain
[75] [761 [77] [78] [79] [80] [81] [82]
Flaviviruses Rubella Tick borne encephalitis
Increased antibody titers in MS Isolated from mice inoculated with MS blood
[83] [84]
Parainfluenza viruses Parainfluenza virus I
Isolation in tissue culture after cell fusion of brain cells from MS patients
[85]
Measles RNA detected in MS brain tissue Impaired CTL response in MS Increased intrathecal antibody synthesis in MS Increased antibody titers in MS Development of SV5 CPE in T-cells after inoculation with bone marrow from an MS patient
[86] [44] [76] [87] [88]
Retroviruses HTLV-I MSRV Retrovirus/EBV
Detection of retrovirus from T cells of MS Detection of retrovirus RNA in MS CSF EBV activation of retroviral like particles in MS CSF
[89]
Rhabdovirus Rabies virus
Isolation from blood and CSF of two MS patients
[92]
Undefined viral agents Scrapie agent Bone marrow agent
Development of scrapie in sheep after inoculation with MS brain Development of CPE in tissue culture after CSF inoculation
[88] [88]
Herpesviruses HSV
VZV MDV CMV EBV HHV-6
Paramyxoviruses Measles virus
Mumps Simian virus 5
Although an association between the human retrovirus HTLV-I and MS has not been supported [89, 105-108], the possibility of a retroviral etiology of MS has not been excluded. In the absence of evidence for an exogenous retrovirus associated with
[90] [911
MS, it has been suggested that human endogenous retroviruses (HERV) could be involved [109, 110]. HERV comprise up to 1% of human D N A and have been implicated as "triggers" in a variety of autoimmune disorders [ 111-113]. The proposed patho-
565
genic role for HERV is based on the correlation of superantigen expression from the endogenous retrovirus termed IDDMK~,222 and insulin-dependent diabetes mellitus and the presence of autoantibodies that cross-react with HERV proteins in patients with systemic lupus eryternatosus and Sjtigren's syndrome [ 114-116]. A putative endogenous retrovirus, known as the multiple sclerosis retrovirus (MSRV), has been tentatively associated with MS [90]. An extensive characterization of a new family of human endogenous retroviruses, which has been designated as human endogenous retrovirus-W (HERV-W), classifies MSRV as a member of the HERV-W family [ 117, 118]. MSRV pol (polymerase gene encoding retroviral reverse transcriptase) sequences were isolated from retroviral particles released by leptomeningeal cells (LM7) cultured from the CSF of an MS patient [90]. Additionally, pol sequences were isolated from the serum of a significantly higher percentage of MS patients than controls [ 119]. Of interest, proteins from HSV-I have been demonstrated to transactivate MSRV in vitro [90]. This observation may be consistent with the correlation between MS exacerbations and viral infections [ 120]. Sequence analysis of the MSRV pol gene indicates that it is virtually identical to the pol gene of the endogenous retrovirus-9 family which is expressed in MS and control human tissues [121]. A recent study has suggested that MSRV pol sequences are transcriptionally active and expressed in lymphocytes from both MS patients and controls [122]. Notably, in this initial study, MSRV pol was expressed more frequently in lymphocytes and serum from MS patients compared to controls [122]. Endogenous retroviruses may influence immune regulation through direct effects on gene products due to integration sites, interactions with exogenous viruses, or as autoantigens [123]. The possible association of endogenous retroviruses and MS remains controversial and warrants further investigation.
2.2. Mechanisms of Virus-Induced Demyelination in MS There are a number of models of virus-induced demyelination in MS that attempt to explain the complex series of events that ultimately result in the MS lesion. The molecular mimicry model suggests
566
that an immune response against viral antigens that cross-reacts with normal host cell components may contribute to the pathogenesis of MS. Several viral sequences contain part of the human MBP sequence [ 124]. Two relevant examples of molecular mimicry include the cross-reactivity of antibodies against proteins of herpes simplex and measles virus with human intermediate filaments [ 125]. Rabbits immunized with a synthetic peptide containing sequences of the hepatitis B virus polymerase develop EAE lesions. Rabbits that developed EAE as a consequence of immunization with this synthetic peptide generated a humoral and cell-mediated immune response to both myelin and hepatits B polymerase [126]. Additionally, cross-reactivity between a monoclonal antibody to the VP1 protein of Theiler's murine virus and oligodendrocytes has been demonstrated [127]. Demyelinating disease was observed in mice who were administered the VPI monoclonal antibody. More recently, amino acid homologies between immunogenic epitopes of semliki forest virus (SFV) and myelin autoantigens, myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were identified [128]. Immunization of B6 mice with SFV proteins induced significant lymphocyte proliferation to the SFV E2 peptide as well as MOG peptide, 18-32, but not to MBP or PLP peptides. Immunization with both MOG 18-32 and E2 115129, induced a later-onset chronic EAE-like disease [128]. These examples of molecular mimicry support the possibility that immunological recognition of viral peptides of sufficient structural similarity to the immunodominant MBP peptide may lead to clonal expansion of MBP-reactive T cells in MS. Therefore, viruses which are known to cause latent or persistent infections such as herpes viruses, may lead to a chronic antigenic stimulation of autoreactive T cell clones [129]. A second possible mechanism of virus-induced demyelination is that of a "non-specific bystander" effect resulting from the reaction of lymphocytes or macrophages to diverse antigens [ 130]. In this case, oligodendrocytes or myelin sheaths could be damaged by lymphokines or proteases release by activated macrophages and immune cells in response to viral infection. The induction of inflammatory cytokines alone, such as TNF-ct, has been shown to induce demyelination. This mechanism of viral
stimulation of immunocompetent cells that nonspecifically attack the myelin sheath could explain demyelination in the number of infections with diverse viruses. This mechanism for virus-induced demyelination is also proposed in the pathogenesis of HAM/TSP. It has been suggested that the recognition of HTLV-I gene products in the CNS results in the lysis of glial cells and cytokine release [ 131 ]. This model is based on the observation that HTLV-I specific CTL restricted to immunodominant epitopes of HTLV-I gene products can be demonstrated in the PBL and CSF of HAM/TSP patients and that the frequency of HTLV-I specific CTL is lower or absent in HTLV-I asymptomatic carriers. The target of the HTLV-I specific CTLs in the CNS could be either a resident glial cell (oligodendrocytes, astrocytes, or resident microglia) infected with HTLV-I or an infiltrating CD4+ cell. HLA class I and II are not normally expressed in the CNS which would prevent antigen presentation necessary for CTL activity. However, class I and class II expression are upregulated by several cytokines including IFN-~, and TNF-t~ which can be induced by HTLV-I and are known to be upregulated in HAM/TSP patients. The release of cytokine and chemokine production by HTLV-I is potentially destructive to cells of the CNS. A similar mechanism could explain the virus induction of demyelinating disease in MS. Demyelination may also develop as a consequence of virus-induced autoimmune reaction against brain antigens. Indirect evidence in support of this theory comes from EAE in animals and parainfectious encephalomyelitis in humans where virus specific CD4+ lymphocytes proliferate in response to MBP [39, 132]. It is unknown how viruses break immune tolerance and force the host to mount a strong cell-mediated immune response to brain antigens. As the virus replicates, it may incorporate host antigens into its envelope and inserts, modifies, or coats itself cellular antigens on the cell surface. It is biologically possible that these newly exposed antigens may be recognize and treated by the host as foreign [133]. The presence of CD 13 has been demonstrated in the envelope of human cytomegalovirus (HCMV). CD13 becomes associated with HCMV upon budding in the Golgiderived vacuoles during early egress [134]. CD13 specific antibodies were detected in the majority of patients with HCMV viremia or disease after bone
marrow transplantation [ 134, 135]. These antibodies were found to cross-react with structures in normal skin biopsies [134, 135]. Alternatively, lymphotropic viruses might interact with the immune regulatory system by destroying some populations of lymphocytes or stimulating the generation of autoreactive lymphocytes clones. Many lymphotropic virus are capable of transforming infected cells and rendering them immortal. It has been demonstrated in vitro that cells immortalized by viruses, such as EBV, are capable of secreting autoantibodies [ 136]. 2.3. HHV-6 and MS
The human herpesvirus 6 (HHV-6) is one of the latest infectious agents postulated to play a role in the pathogenesis of MS. HHV-6 is a beta herpesvirus for which seroprevalence rates vary from 72% to 100% in healthy adults worldwide [137, 138]. The virion architecture of HHV-6 is similar to that of other herpesviruses and consists of a core containing a linear double-stranded DNA, an icosadeltahdral capsid, a tegument surrounding the capsid, and an envelope spiked with viral glycoproteins on its surface. Although HHV-6 replicates primarily in T-lymphocytes, it is a pleiotropic virus which can either productively or nonproductively infect cells from several lineages including B-cells, microglia, oligodendrocytes, and astrocytes [139-142]. Two variants of HHV-6 (HHV-6A and HHV-6B) have been described based on genomic, antigenic, and biological differences [143]. The genomes of HHV6A and HHV-6B range in length from 160-170 kbp and encode approximately 100 proteins [ 144]. The overall nucleotide sequence identity between HHV6A and HHV-6B is approximately 90% [145]. Of the genes with less than 70% identity between the two variants, all but one (U47) were found in the immediate-early region [145]. Eighteen open reading frames are unique to either HHV-6A or HHV6B. Due to the close sequence identities between the two viruses and the absence of serological assays that can easily discriminate between the two variants, HHV-6A and HHV-6B are often treated as a single virus. However, it has been argued that differences in the biology, cellular tropisms, and restriction endonuclease profiles of the two variants are sufficient to classify them as distinct herpesviruses rather than as vairiants of the same virus [ 145,
567
144]. The HHV-6B variant has been identified as the causative agent of exanthem subitum and accounts for the majority of symptomatic HHV-6 infections in infants. However, the HHV-6A variant has yet to be clearly associated with a particular disease [ 146]. An increased neurovirulenCe of the HHV-6A variant compared to the B variant has been suggested based on a greater detection of the HHV-6A variant than the HHV-6 B variant in the CSF of children and adults [ 147]. Additionally, the HHV-6A variant has been isolated from the CNS of AIDS patients with areas of demyelination [ 148]. HHV-6 is an attractive candidate as a possible etiologic agent in MS for several reasons. 1) Primary infection with HHV-6 usually occurs during the first few years of life and the involvement of HHV-6 with MS is consistent with epidemiological evidence in MS suggesting exposure to an etiologic agent before puberty [149, 4]. 2) HHV-6, particularly the HHV-6A variant, is highly neurotropic [ 147]. Primary infection with HHV-6 occasionally results in neurologic complications including meningitis and menigo-encephalitis and febrile seizures [150--152]. HHV-6 has been demonstrated to cause fatal encephalitis in AIDS patients and in individuals immunosuppressed as a consequence of bone marrow transplantation [148, 153-155]. Furthermore, a neuropathogenic role for HHV-6 has been suggested based on the development of a variety of disorders associated with active HHV-6 infection including fulminant demyelinating encephalomyelitis, subacute leukoencephalitis, necrotizing encephalitis, progressive multfocal leukoencephalopathy, and chronic myelopathy [156-159] 3) One of the fundamental properties of herpesviruses is their tendency to reactivate. The same factors that often lead to herpesvirus reactivation, such as stress and infection with another agent, have also been associated with MS exacerbations. Unfortunately, the mechanisms by which HHV-6 achieves latency and reactivation are poorly understood [ 160, 161 ]. 4) Herpesviruses are typically latent in nervous tissue and can not be structurally identified in a latent state. Therefore, herpesviruses are not likely to be found by electron microscopy. 5) HHV-6 is pleiotrophic and infects cells of both lymphoid and non-lymphoid origin. The pleiotropism of HHV-6 could explain abnormalities observed in both the immune and nervous systems of patients with MS.
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In 1995, Challoner and colleagues suggested a potential role for HHV-6 in MS based on an unbiased search for non-human DNA by representational difference analysis (RDA) [82]. This technique is based on successive rounds of subtractive hybridization and PCR amplification, enriched for DNA sequences present in DNA preparations from MS disease material and control PBMCs. In this study, over 70 DNA fragments were analyzed. One of these fragments was found to be homologous to the MDBP gene of the HHV-6B variant Z29. HHV-6 DNA was found in 78% of MS brains and 74% of control brains. However, monoclonal antibodies against the HHV-6 101 K protein and the DNA binding protein p41 were detected in the brain tissues of MS patients and not in controls. In MS brains, nuclear staining was found in oligodenderocytes surrounding MS plaques more frequently than in uninvolved white matter [82]. While this study did not establish a causal link between HHV6 and MS, it was the first study to suggest an association between a virus and MS using an unbiased technology. The association of HHV-6 with MS has also been supported by immunological and molecular studies. Significantly higher antibody titers against HHV-6 whole virus preparations in MS patients compared to normal controls and an increase in HHV-6 DNA in the PBMCs of MS patients by PCR have been reported [162]. While these preliminary studies were intriguing, they were based on methodologies that do not discriminate between latent and active infection of HHV-6. In order to distinguish between these stages of HHV-6 infection, early antibody responses to the HHV-6 p41/38 early antigen and the presence of HHV-6 serum DNA by nested PCR were examined [96]. It has been demonstrated that HHV-6 serum DNA correlates with active HHV-6 infection and is not found in healthy individuals [163]. In this original report, a significant increase in IgM response to the p41/38 early antigen was demonstrated in patients with the relapsing remitting form of MS in comparison to healthy controls and individuals with other neurologic disease. An increased IgM response to the p41/38 early antigen was also observed in a group of patients with other inflammatory diseases. Additional studies have confirmed the presence of increased IgM responses to HHV-6 in patients with MS [ 164, 165] while no cor-
relation was demonstrated in another report [ 166]. Additionally, HHV-6 serum DNA was detected in 30% (15/50) of MS patients and in 0% of controls (0/47) consisting of healthy individuals, patients with other inflammatory diseases and patients with other neurologic diseases [96]. This NIH cohort has been expanded to include a total of 167 MS patients and 70 controls [ 167]. Our group has continued to demonstrate the presence of HHV-6 DNA in the serum of approximately 23% of MS patients and 0% of controls [ 168, 96, 167]. Subsequent studies by a number of other groups have reexamined the presence of HHV-6 serum DNA in MS patients. Overall the results from these studies have been equivocal [168, 96, 169-173]. It has been suggested that discrepancies in these reports may be attributable to differences in patient selection, techniques, and reagents used [169, 174]. To extend the observation of cell free serum HHV-6 DNA in MS patients, a longitudinal study consisting of 215 samples obtained from 59 MS patients followed prospectively for a 5 month period of time was conducted [167]. Serum HHV-6 DNA was detected from significantly fewer sera obtained during periods of clinical remission. While data from previous studies represent single time points, this study analyzed a large group of MS patients over time and suggests that there is a statistically greater likelihood of detecting HHV-6 DNA in the serum of an MS patient during an exacerbation [167]. Therefore, the time of serum sampling is likely to affect the detection of HHV-6 DNA in the serum of MS patients. Additionally, this report further supports a role for HHV-6 in the pathogenesis of MS by suggesting that the presence of serum HHV-6 DNA, similar to the presence of gadolinium enhancing lesions, coincides with clinical worsening in a subset of patients [ 167]. Cellular immune response to HHV-6 have been compared in MS patients and controls [175, 173, 176, 177]. In a report examining the T-cell lymphoproliferative responses of healthy controls and patients with MS to both variants of HHV-6 and HHV-7 using whole virus lysates [175] it was demonstrated that there was no difference in either the frequency or magnitude of proliferative responses between healthy controls and patients with MS to either the HHV-6B variant or HHV-7. However, a significantly higher percentage of patients with
MS had proliferative responses to the HHV-6A variant (66%) compared to healthy controls (33%). It is, at present, unknown whether the increased frequency of lymphoproliferatve response to the HHV-6A lysate in patients with MS is the result of a higher seroprevalence of the HHV-6A variant in MS patients or in an altered host immune response [175]. Moreover, subsequent studies have demonstrated the amplification of the HHV-6 A variant in PBMC, serum, and urine of MS patients, but not in controls [168, 178]. Collectively, the description of an increased lymphoproliferative response to the HHV-6A variant and the unique amplification of HHV-6A DNA in the PBMC, serum, and urine of MS patients further supports the association of HHV-6 with MS. Further, these studies suggest that the highly neurotropic A variant rather than the B variant may play a role in this disease [ 175]. Therefore, future studies concerning the putative association of HHV-6 with MS must consider variant specific tropisms and immunology. In a study examining cellular immune responses to HHV-6, T-cell responses to recombinant HHV-6 101K protein were described in MS patients and controls [173]. The 101K protein used for this study was cloned from the HHV-6 B variant and has 81% homology with HHV-6A [173]. A lower precursor frequency of 101K specific T-cells was observed in MS patients compared to controls [173]. The impaired T-cell response to the 101K protein of HHV-6 was associated with increased HHV-6 specific IgM responses. Of interest, HHV6 specific T-cell lines derived from MS patients demonstrated Th-1 biased cytokine profiles marked by the inability to produce IL-4 and IL-10 [173]. In a more recent report from Tejada-Simon et al, cross reactivity between MBP (residues 96-102) and HHV-6 U24 (residues 4-10) were examined. Increased precursor frequencies of MBP/HHV-6 cross-reactive T cells were found in MS patients compared to healthy controls [177]. Importantly, this study demonstrates a relationship between HHV-6 and autoreactive immune responses to MBP [177]. This study further supports an association between HHV-6 and MS and suggests a potential role for HHV-6 specific T-cells in the pathogenesis of MS via molecular mimicry. As MS is a CNS disorder, detailed analysis of brain material is paramount in assessing the poten-
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tial association between HHV-6 and MS. HHV-6 is a commensal pathogen of the CNS and HHV-6 DNA can be amplified from 20-70% of brains of MS patients and controls [82, 179, 180]. Recent studies have specifically addressed the frequency of HHV-6 in control and MS brains and identified infected cell phenotypes in pathologically defined tissue [ 181, 182]. Goodman et al used in situ-PCR (ISPCR) to identify individual cells containing the HHV-6 genome from surgical biopsy specimens from MS patients presenting with acute disease who had not received immunomodulatory therapy. This study demonstrated that high frequencies of HHV-6 genome positive neuroglial and inflammatory cells are present in acute-phase lesion tissue and that oligodendrocytes are the predominant cell infected in the acute MS lesion [182]. In a companion study, pathologically defined material from brain autopsies were isolated by laser assisted microdisection prior to HHV-6 DNA amplification; representing a significant advance over PCR amplification of virus DNA from pathologically undefined bulk brain tissue [ 181]. While HHV-6 DNA was amplified from brains of both MS patients and controls, HHV-6 DNA was detected at a significantly higher frequency in MS plaques compared to brain tissue from non-MS neurologic disorders, non-MS inflammatory and normal appearing white matter from MS brains. Collectively, these studies suggest that HHV-6 is present early in the evolution of the MS lesion and may play a significant role in the demyelinative pathogenesis of MS. The relationship between HHV-6 and MS remains controversial and has yet to be clearly defined. Additional serological, cellular immune response, molecular, and clinical studies are necessary to elucidate the role, if any, of HHV-6 in the pathogenesis of MS. 2.4. Chlamydia Pneumoniae in MS
Chlamydia pneumoniae is an obligate intracellular bacterium of the respiratory tract that causes community acquired pneumonia While C. pneumonia primarily infects mucosal surfaces [183, 184], systemic dissemination of the bacterium from the respiratory tract may occur via monocytes and macrophages [185]. Epidemiologic evidence suggests that C. pneumoniae, like HHV-6, is a ubiquitous
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human pathogen [186]. Since its identification as a unique chlamydial species in 1989 [187], C. pneumoniae has been controversially associated with several non-respiratory human pathologies of the cardiovascular system and CNS including atherosclerosis, giant cell arteritis, vasculitis, Alzheimer's disease, encephalitis, HIV associated dementia, and MS [ 188-197]. A relationship between C. pneumoniae and MS was first suggested in a case report that demonstrated the presence of the bacterium in the CSF of an MS patient with rapidly progressive MS [ 197]. Of interest, antibiotic treatment of this patient resulted in marked clinical improvement [197]. This initial report was extended to a larger study that examined the presence of C. pneumoniae in 17 RRMS, 20 and 27 OND controls [29]. In this study approximately 47% of RRMS and 80% of SPMS patients were culture positive for C. Pneumoniae, compared to only 11% of OND controls [29]. In addition, CSF from all relapsing-remitting and 19/20 secondary progressive MS patients were PCR-positive for the of C. pneumoniae outer membrane gene compared to only 5/27 other neurologic disease controls [29]. Of interest, intrathecal antibodies specific for C. pneumoniae were significantly higher in MS patients than in controls with other neurologic diseases [29]. Subsequent studies from this group have demonstrated that oligoclonal bands could be partially absorbed out of CSF from the majority of MS patients using C. Pneumoniae antigens [198] and that the intraperitoneal inoculation of mice with C. pneumoniae, after immunization with neural antigens, increased the severity of EAE [ 199]. PCR studies attempting to reproduce the PCR amplification of C. pneumoniae from the CSF of MS patients have been inconsistent. Several studies reported no C. pneumoniae positive samples by either culture or PCR [200-202]. Others have reported low positivity rates for C. pneumoniae in MS [203, 204], or high frequency of detection of C. pneumoniae in other neurologic disease controls [205]. The difficulties in assessing the potential association of C. pneumoniae in MS are similar to those involved with HHV-6. In both cases, further studies involving multiple MS cohorts are required to determine whether or not there is a meaningful association between these ubiquitous pathogens and the disorder.
2.5. Ubiquity and Disease Considerable focus has been on the identification of a unique virus exclusively associated with MS. However, the search for an "MS-virus" (i.e. a viral infection which invariably results in MS and is not present in disease free individuals) has been unsuccessful [174, 39, 206]. The inability to identify an "MS-virus" could indicate that either no single virus causes MS, the putative "MS-virus" has yet to be identified, or that viruses are not associated with this disease. Alternatively, a new paradigm has emerged that suggests the MS disease process is associated with a common or ubiquitous virus may act as a trigger in genetically or immunologically predisposed individuals [174]. There are several examples of virus infections that lead to disease in only a subset of infected individuals. Some examples of viruses which are associated with multiple disease outcomes in different subsets of individuals include: EBV (Burkitt's lymphoma, nasopharyngeal carcinoma, mononucleosis), measles virus (SSPE), JC virus (PML), and Hepatitis B and C virus (hepatoma) [207-209]. Perhaps the most relevant example of a virus that is common in certain populations but only results in disease in a minority of those infected is that of HTLV-I. Originally identified from a T-lymphoblastoid cell line (HUT 102) of a patient diagnosed with a cutaneous T-cell lymphoma, HTLV-I was the first described human retrovirus [210]. In 1981, HTLV-I was established as the etiologic agent for adult Tcell leukemia (ATL) [211], a hematological malignancy first characterized in Japan. Since the initial description of ATL and the discovery of HTLV-I, the virus has been associated with an inflammatory, chronic, progressive neurologic disease known as HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) and several other inflammatory diseases [212, 66, 213-216]. While between 15-25 million individuals are infected worldwide and seroprevalence rates in endemic areas can exceed 30%, the majority of individuals infected with HTLV-I are clinically asymptomatic [217]. The propensity for certain individuals to develop either HAM/TSP, ATL, or other HTLV-I associated inflammatory diseases while others remain clinically asymptomatic is not fully understood. It has been suggested that host genetics and immune
abnormalities influence an individual's predisposition to HAM/TSP as they are believed to influence the likelihood of developing MS [218]. Therefore, the use of HAM/TSP as a "model" of a chronic progressive neurologic disease that occurs in only a small percentage of infected individuals is particularly germane in examining the possible involvement of a ubiquitous virus in the etiology of MS. The lifetime risk of an HTLV-I infected individual acquiring HAM/TSP over a lifetime is estimated to be 0.25% [68]. In Japan, associations have been made between the likelihood of developing either HAM/TSP or ATL and particular HLA haplotypes [219-221]. HAM/TSP patients of Japanese decent have an increased frequency of certain HLA-Cw7,B7, and DR1 alleles represented by the A26CwB 16DR9DQ3 and A24Cw7B&DR1DQ1 haplotypes. In contrast, Japanese ATL patients have an increased frequency HLA-A26, B 16 and DR19 and decreased frequency of HLA A24 and Cwl compared to controls. The HLA types DRB 1"0901, DQB 1"0303 and DRB 1" 1501 in ATL patients and HLA types DRB 1"0101, DRB 1*0803, DRB 1" 1403 and DRB l ' i n HAM/TSP patients were found to be mutually exclusive [221 ]. The neuropathology of HAM/TSP indicates that immune mediated mechanisms are involved in the progression of this disease. Furthermore, several lines of evidence indicate that the cellular and humoral immune responses of HAM/TSP patients are altered from that of HTLV-I asymptomatic carriers and uninfected controls. The immunologic hallmarks of HAM/TSP include an increase in e x v i v o spontaneous lymphoproliferation in the absence of antigenic stimulation or IL-2 [2221, the presence of HTLV-I specific, CD8+ CTL in the PBL, and an increase in antibodies to HTLV-I in sera and CSF [212]. Natural killer cells tend to be diminished in both number and activity in HAM/TSP. Although the suggestion of disease specific HTLV-I strains has been dismissed as a factor in the determination of disease susceptibility, increased viral load has been implicated in the pathogenesis of HAM/TSP [223]. It has been suggested that increased proviral loads may be a predictor for the progression from the asymptomatic cartier state to HAM/TSP [224]. It has also been suggested that the HLA class I allele A2*01 may confer a protective effect on the development of HAM/TSP by influencing the pro-
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viral load in infected individuals [224]. Of interest, the HLA A2*01 haplotype has also been shown to decrease the overall risk of MS in an HLA allele comparison study from a cohort of Swedish and Norwegian MS patients and healthy controls using PCR-SSCP [225]. Several models for the immunopathogenesis of HAM/TSP have been proposed. All of these models are based on an HTLV-I induced immune-mediated response in the CNS to either specific viral antigens or cross reactive self peptides and none of which are mutually exclusive. The proposed models for the immunopathogenesis of HAM/FSP are similar to those suggested for MS. Therefore, it is hoped that insights into the pathogenesis of HAM/TSP will lead to a better understanding of MS and other neurologic disorders, such as neuro-AIDS, in which virus mediated immunopathogenesis may occur in a subset of infected individuals.
2.6. Multiple Infectious "Triggers" in MS? It is possible that multiple viruses may be involved in the etiology of MS and that particular viruses trigger disease in different subsets of individuals through a common mechanism. A possible mechanism by which HHV-6 and, potentially other viruses could result in MS has recently been suggested by the discovery of the HHV-6 cellular receptor [226]. Santoro and colleagues have clearly demonstrated that CD46, also known as the membrane cofactor protein (MCP) is the cellular receptor for HHV-6. CD46 is a member of family of glycoproteins that function as regulators of complement activation (RCA) and prevent spontaneous activation of complement on autologous cells. CD46 is expressed on all human nucleated cells and soluble forms can be found in plasma tears and seminal fluid of normal individuals [227]. The use of a virtually ubiquitious human molecule as a surface receptor helps to explain the pleiotropism of HHV-6. Of particular interest, CD 46 is also the primate-specific receptor for measles [228, 229]. Notably, HHV-6 and measles Virus, which are from disparate virus families, have been associated with MS and use the same receptor. Additionally, other viruses use various members of the RCA family as cellular receptors. Epstein-Barr Virus, which has also been implicated in MS, uses CD21 while CD55 is used by several echoviruses
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and coxsackieviruses [230, 231]. Could viruses that share a receptor in common such as HHV-6 and measles virus cause MS by a similar mechanism? It is theoretically possible that the engagement of CD46 by one or both of these viruses may result in increased activation of the complement cascade on autologous cells through downregulation of the receptor. This abnormal increase in complement could lead to widespread tissue damage through cytokine dysregulation, cytolysis and nitric oxide production [232, 233]. Alternatively, these viruses and their common receptor could play a role as an MS "trigger" either directly or indirectly through an autoimmune mechanism. As these viruses replicate, they may incorporate host antigens, including their own receptors, into their envelopes as has been demonstrated for CMV, HIV-1, SIV, and HTLV-I [234, 235, 134, 236]. Incorporation of the receptor into the virus envelope could cause the antigen to be recognized and treated by the host as foreign. This phenomenon could account for the increase in CD46 specific auto-antibodies observed in MS [237]. The potential influence of viruses that use members of the RCA family, including HHV-6 and measles virus, on the pathogenesis of MS may, in part, be elucidated by animal studies. Recently, a CD 46 transgenic mouse which can be infected by measles virus was described [229]. Measles virus infection in these transgenic mice was associated with immunosupression and virus replication in the CNS. Measles virus infection was also associated with CNS disease in infected mice [229]. The generation of a CD46 transgenic mouse provides an excellent model for studying the role of measles virus infection in CNS disease. Additionally, the CD46 transgenic mouse may provide a model for studying the neuropathogenesis of HHV-6 infection in the CNS if in fact the virus, which has an extremely limited host range, may productively infect these mice which now express the HHV-6 receptor. Furthermore, a recent study has demonstrated that EAE may be abrogated by the use of a complement inhibitor, which indicates an important role for complement in EAE as well as MS [238]. Importantly, increased levels of the complement regulatory protein CD46 have been demonstrated in both the serum and CSF of MS patients compared to healthy controls and other neurologic disease
patients [239]. Elevated levels of soluble serum CD46 were also found an other inflammatory disease cohort, indicating that an increase in soluble serum CD46 may be a common phenomenon in autoimmune disorders [240, 239, 241]. Therefore, the increased levels of CD46 demonstrated in the serum and CSF of MS patients may be indicative of an increased activation of the completment system in MS, both peripherally and intrathecally. However, a significant correlation was observed between elevated levels of serum soluble CD46 and the detection of serum HHV-6 DNA in serum from MS patients, while no serum HHV-6 DNA was detected in other inflammatory disease controls [239]. Therefore, while an increase in serum soluble CD46 is common in inflammatory diseases in general, immuno-pathogenic mechanisms involving both HHV-6 and its receptor are likely to be unique to MS.
3. C O N C L U S I O N The pathogenesis and etiology of MS have yet to be well defined. Epidemiologic evidence suggests that MS is a multi-factorial disease, which develops as a result of host genetics, immune response and environment. Several lines of evidence, including the documentation of microbes which induce a variety of demyelinating diseases in both humans and animals suggest that a virus may comprise the environmental component in the etiology of this disorder. While many viruses have been proposed as etiologic agents in MS, none of these viruses have been firmly associated with disease pathogenesis. Additionally, mechanism(s) by which virus-host interactions may lead to demyelination are not fully understood. Currently, HHV-6 and C. pneumoniae are receiving much attention as potential MS "triggers". However, the role of these infectious agents in the pathogenesis of MS is unclear. We suggest that multiple agents may induce a virus-specific and/or a cross-reactive autoimmune process resulting in clinical disease in a subset of genetically susceptible individuals. The involvement of multiple infectious agents in MS, as suggested originally by Dr. Pierre Marie over 100 years ago [40], may explain the difficulty in identifying a single agent responsible for this highly variable and chronic disease. Moreo-
ver, we encourage extreme caution in attempts to readily associate viruses in a chronic, progressive neurologic disorder such as MS. As outlined in this review, it is difficult to determine cause from effect particularly when a ubiquitous agent is suggested to play a role in disease pathogenesis. Uniformity in assay design, viral isolation techniques, molecular probes, etc. must be employed by different research groups on a large number of MS cohorts to confirm these virus associations.
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204. Sotgiu S, Piana A, Pugliatti M, Sotgiu A, Deiana GA, Sgaramella E, Muresu E, Rosati G. Chlamydia pneumoniae in the cerebrospinal fluid of patients with multiple sclerosis and neurological controls. Mult Scler 2001;7:371--4. 205. Gieffers J, Pohl D, Treib J, Dittmann R, Stephan C, Klotz K, Hanefeld F, Solbach W, Haass A, Maass M. Presence of Chlamydia pneumoniae DNA in the cerebral spinal fluid is a common phenomenon in a variety of neurological diseases and not restricted to multiple sclerosis. Ann Neuro12001 ;49:585-9. 206. Soldan SS, Jacobson S. Role of viruses in etiology and pathogenesis of multiple sclerosis. Adv Virus Res 2001;56:517-55. 207. Miller DH. Demyelinating, inflammatory and degenerative diseases. Curr Opin Neurol Neurosurg 1990;3: 881-3. 208. Grinnell BW, Martin JD, Padgett BL, Walker DL. Naturally occurring and passage-induced variation in the genome of JC virus. Prog Clin Biol Res 1983;105: 61-77. 209. Szmuness W, Stevens CE, Ikram H, Much MI, Harley EJ, Hollinger B. Prevalence of hepatitis B virus infection and hepatocellular carcinoma in Chinese-Americans. J Infect Dis 1978;137:822-9. 210. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci USA 1980;77:7415-9. 211. Hinuma Y, Nagata K, Hanaoka M, Nakai M, Matsumoto T, Kinoshita KI, Shirakawa S, Miyoshi I. Adult Tcell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci USA 1981;78:6476-80. 212. Gessain A, Barin F, Vernant JC, Gout O, Maurs L, Calender A, de The G. Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet 1985;2:407-10. 213. Mochizuki M, Watanabe T, Yamaguchi K, Tajima K, Yoshimura K, Nakashima S, Shirao M, Araki S, Miyata N, Mori S et al. Uveitis associated with human T lymphotropic virus type I: seroepidemiologic, clinical, virologic studies. J Infect Dis 1992;166:943-4. 214. Nishioka K, Maruyama I, Sato K, Kitajima I, Nakajima Y, Osame M. Chronic inflammatory arthropathy associated with HTLV-I. Lancet 1989; 1:441. 215. Morgan OS, Rodgers-Johnson P, Mora C, Char G. HTLV-1 and polymyositis in Jamaica. Lancet 1989;2: 1184-7. 216. Terada K, Katamine S, Eguchi K, Moriuchi R, Kita M, Shimada H, Yamashita I, Iwata K, Tsuji Y, Nagataki S e t al. Prevalence of serum and salivary antibodies
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infections and Polymyositis-Dermatomyositis Sandra Zampieri, Anna Ghirardello, Luca Iaccarino, Pier Franca Gambari and Andrea Doria
Division of Rheumatology, Department of Medical and Surgical Science, University of Padova, Padova, Italy
1. IDIOPATHIC INFLAMMATORY MYOPATHIES Idiopathic inflammatory myopathies (IIM) are a group of systemic diseases classified within the connective tissue diseases (CTDs). The term inflammatory myopathy is often used interchangeably with the term myositis. They represent a diverse group of diseases of unknown cause with a variety of clinical manifestations, in which the muscle injury results from inflammation [ 1]. Myositis is a relatively rare but increasingly recognized disease. Sometimes it is difficult to early recognize and correctly classify the different subsets of myositis because in the beginning the disease could be clinically undefined. Late diagnosis and mistaken classification of myositis postpone the start of treatment leading to a worse prognosis. 1.1. Classification and Diagnosis From a clinicopathologic perspective, the IIM fall into six major categories [2]: dermatomyositis (DM), polymyositis (PM), inclusion body myositis (IBM), overlap syndromes, cancer-associated myositis, and other forms including rare focal and diffuse variants. The HM can affect individuals at all ages, childhood myositis is defined with onset before the age of 18. The classification scheme generally applied to these diseases was published by Bohan and Peter in 1975 [3]. Inclusion body myositis was rarely recognized
at the time in which these criteria were developed. Since then, other criteria have been proposed for the classification of these diseases: Calabrese et al classification criteria for IBM in 1987 [4] and Tanimoto et al classification criteria for PM and DM in 1995 [5]. Another classification scheme based on clinical, immunopathologic and morphologic features was proposed by Dalakas et al in 1992 [6]. 1.2. Clinical Features The prominent features of myositis are progressive proximal muscle weakness, raised serum muscle enzymes (creatinine kinase, aldolase and lactate dehydrogenase) and characteristic electromyographic abnormalities. Muscle biopsy shows inflammatory infiltrates, necrotic areas, fibrosis and atrophy. Regenerating fibres are also found [7, 8]. Besides muscle abnormalities, patients with myositis may have other clinical features" cutaneous rush, mainly in DM, arthralgia or arthritis, pulmonary, gastrointestinal, cardiac and renal disorders.
2. PATHOGENESIS OF IIM The pathogenesis of myositis is still not known although it has been hypothesised (as in other CTDs), that in genetically susceptible individuals environmental factors (for example viral or bacterial infectious agents) may trigger abnormal autoimmune response. Different pathogenic mechanisms may be involved in the distinct myositis subgroups, but the predominant clinical symptoms of symmetrical
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muscle weakness, raised serum muscle enzymes and electromyographic abnormalities, which are common to all the subsets of myositis, indicate that some pathogenic mechanisms may be shared by inflammatory myopathies [9]. The presence of lymphocytes in muscle tissue biopsy in a majority of myositis patients, and the detection of autoantibodies in patients' serum, imply that the immune mechanisms are involved in the pathogenesis of these disorders, and that T and B cells could have a pathogenic role [9, 10].
In muscle biopsies from myositis patients the macrophage inhibiting protein (MIP)-I alfa is also frequently detected. This chemokine acts as a Tand B-lymphocyte chemoattractant and stimulates the adhesion of CD8+ T cells to extracellular matrix [10]. Several data have been published on the increased expression of these mediators in myositis suggesting that they may play a role, but the exact nature or extent of that role is not sufficiently clear at the moment.
2.1. Cellular Mechanisms
2.2. Humoral Mechanisms
The nature of cellular infiltrates in muscle biopsies from patients affected by IIM provide evidence for immunologic mechanisms of muscle damage. In PM, the inflammatory infiltrates are characterized by a predominance of CD8+ T cells and macrophages and are localized in the endomysial area. They surround and can invade the nonnecrotic muscle fibers. The cellular infiltrates of DM are mainly composed of B cells, CD4+ T cells, and macrophages. The infiltrates are more prominent in the perivascular area and are less present in the perimysial and endomysial areas [11, 12]. The pattern and the distribution of the infiltrates, suggests that the cell-mediated cytotoxicity is an important mechanism of muscle damage in PM, whereas the humoral immune mechanism mediate the damage in DM. Several studies have been published on cytokines and chemokines produced by infiltrating inflammatory cells and it seems that these mediators are involved in the immune response in myositis patients. The molecular pattern of these molecules is very similar between PM and DM. The most frequently reported pro-inflammatory cytokines are interleukin-(IL) 1 alfa and beta, and tumor necrosis factor (TNF) alfa. It has been demonstrated cytokines are produced also by endothelial cells in vessels of muscle tissue and by muscle fibers [ 13]. Another molecule found in almost all muscle biopsies from myositis patients is the trasforming growth factor (TGF)-beta, which has an anti-inflammatory effect and can stimulate fibrosis [ 13, 14].
It is thought that humoral response plays an important role in the development of PM/DM because of the high serum levels of autoantibodies that, in some cases, seem to be associated with specific clinical features (anti-synthetase syndrome). The understanding of the immunology of myositis syndromes has grown with the identification and characterization of autoantibodies associated with PM and DM. In fact, an autoimmune response to nuclear and cytoplasmic autoantigens is found in about 60-80% of these patients. Some of the serum autoantibodies are shared with other autoimmune diseases (myositis-associated antibodies MAAs) and some of them are unique to myositis (myositis-specific antibodies MSAs). MAA are found in 20-50% of the patients'sera; they are commonly encountered in other CTDs. The most important antigenic targets of MAA are the PM/Scl nucleolar antigen, the nuclear Ku antigen (p70/p80), the small nuclear ribonucleoproteins (snRNP) and the cytoplasmic ribonucleoproteins
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(RoRNP). Anti-PM/Scl autoantibodies are generally found in patients affected by myositis, scleroderma or polymyositis overlap with scleroderma [15], antiKu antibodies are found in patients with myositis overlap with other CTDs [16], antibodies directed against snRNP are frequently found in myositis patients and in patients with CTD overlap syndrome whereas antibodies towards Ro/SSA 60kDa, Ro/ SSA 52kDa and La/SSB proteins components of the RoRNP complex are almost exclusively found in patients affected by Sj6gren syndrome and Systemic Lupus Erythematosus (SLE).
The MSA are serological markers of disease. By means of standard techniques (for example immunofluorescence on HEp-2 cells) MSA can be found in about 40% of PM and DM patients. The identified MSA targets include three distinct groups of proteins: the aminoacyl-tRNA synthetases, the nuclear Mi-2 protein and components of the Signal Recognition Particle (SRP). Each MSAs is associated with characteristic clinical features in addition to myositis. Because of a low sensitivity, the absence of these antibodies certainly does not exclude PM or DM, but their presence has strong predictive value for the diagnosis of myositis. A new classification system for myositis using MSAs status was proposed by Targoff et al [ 17] in 1997. The most common MSA that are found in approximately one third of myositis patients, target the aminoacyl tRNA synthetases. Up to now 6 out of 20 aminoacyl-tRNA synthetases have been described, but the most commonly detected one (20-30%) is the anti-histidyl-tRNA synthetase (Jo-1). Patients with anti-tRNA synthetase antibodies, including Jo1, are affected by the so called "anti-synthetase syndrome" which is characterised by myositis, interstitial lung disease, arthritis, Raynaud's phenomenon and hand skin lesions (mechanic's hand). Anti-Mi-2 autoantibodies are considered specific serological markers of DM. They are detected in about 20% of myositis patients and are associated with relatively acute onset, good prognosis and good response to therapy. Patients with anti-SRP antibodies have very acute polymyositis with cardiac involvement, severe prognosis and poor response to therapy. Most MSAs are directed against antigens ubiquitous to all cell types, making organ involvement difficult to explain and even if they are often associated with certain clinical phenotypes, it is not known whether these autoantibodies have a direct role in the pathogenesis of IIM or if they are an epiphenomenon [9].
3. INFECTIONS AS A CAUSE OF AUTOIMMUNITY IN PM/DM During the last years, several studies have been carried out in attempt to analize the complex cor-
relation between infections and immune system alteration in humans, but it is often difficult to verify whether this association is coincidental or whether a pathogenetic link actually exists.
3.1. Epidemiologic Evidence Studies on geographic cluster of myositis and the observation that subsets of patients defined by different MSAs tend to develop disease at different times of the year, have supported the idea that IIM may be initiated by an infectious agent [ 18]. Striking association of environmental factors with myositis onset have not been identified yet. However, seasonal clustering of myositis has been reported, indicating that a common environmental factor such as bacterial or viral infection could trigger disease onset. The published epidemiologic studies on IIM have suggested that the relationship between genetic and environmental risk factors is different in PM and DM [19] and have supplied several data on potential infectious agents that may play a part in disease initiation [2]. If an infectious agent is able to trigger the disease it is possible to recognize a seasonal pattern in the onset of disease which corresponds to the seasonal pattern of the inciting infection. Several studies reported seasonal patterns of PM/ DM as well as a seasonal variation in the frequency of relapses [18-20]. A clear seasonal pattern might be found in groups of patients defined by certain autoantibodies assuming that these antibodies mark the presence of an inciting infection [21 ]. It has been shown that patients with anti-Jo-1 and those with anti-SRP antibodies have different seasonal onset of disease [ 18]. In this study, using weakness as a marker of the onset of disease, it has been shown that the month during which the onset of weakness occurs is not random in patients with anti-Jo-1 antibodies (average month April) and in those with anti-SRP antibodies (average month November). These antibodies are associated with a clinical distinct group of patients which differ also in season and rate of disease onset. In contrast, from this study it emerged that patients classified into traditional categories of myositis do not have recognizable seasonal patterns and do not differ in the rate of onset of disease. Therefore, searches for
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seasonal pattern of disease onset by disease-specific antibodies, may be useful to understand the etiology of the disease.
3.2. Infectious Agents in PM/DM The causative role of infectious agents has been investigated in several autoimmune disorders. For some of them, established associations have been described (for example, Group A streptococcus and rheumatic fever, hepatitis B and C viruses and mixed cryoglobulinemia) and for others the causative infectious factor is suspected, but not definitely demonstrated (for example, mycobacteria and mycoplasmas and rheumatoid arthritis, cytomegalovirus and type I diabetes mellitus) [22]. The etiologic role of infectious agents in IIM has been investigated too. Acute viral infections produce inflammation in the muscle so it is possible that persistent viral infection triggers the chronic process of an idiopathic inflammatory myopathy. Several studies have been carried out searching the viral genome in the muscle tissue from IIM patients, but data are controversial [23-29]. Not all of these studies found direct evidence of the presence of nucleic acids from implicated viruses in muscles from IIM patients. Viral proteins could persist in muscle even if the viral genome does not persist, viral infection could initiate the inflammatory process in the muscle and the process becomes selfsustaining in individuals which cannot mount the counterinflammatory response because of genetic defects and virus can persist in another part of the body and sustain the inflammatory process by way of activated inflammatory cells [23]. Many infectious agents have been proposed as initiating factor, including coxsackie viruses, parvoviruses, enteroviruses, retroviruses, in particular human T-lymphotropic virus (HTLV) and human immunodeficiency virus (HIV), Toxoplasma and Borrelia. Because of their tropism for muscle, coxsackie virus have long been regarded as potential casual agents in the induction of chronic muscle diseases such as PM and DM. The presence of antiviral antibodies in serum and the isolation of virus from patients provide evidence to support this link [2, 24]. Coxsackie viruses have been isolated from few patients with myositis or myocarditis and virus
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RNA has been detected in the muscle of patients with IIM [25, 26]. Searching for parvovirus B19 DNA in muscle tissue demonstrates the absence of the candidate viral genome in biopsies samples and negative immunohistochemistry for viral capsid proteins detection in muscle tissue sections [27]. The same Authors of this study previously reported the first case of DM where B 19 DNA was present in two sequential muscle biopsies [28]. However, the presence of B 19 DNA was not found in the third biopsy performed during a relapse of the disease, suggesting a temporal association between parvovims B 19 infection and active myositis. Enteroviruses have also been identified in the muscle of patients with PM/DM, but their role as causative agents in the development of these diseases is controversial [29, 30]. It has been shown that in patients with HIV, musculoskeletal infections constitute an unusual clinical manifestation. Pyomyositis is an increasingly recognized infection of the striated muscle in HIV-infected patients. It affects almost exclusively males with advanced HIV infection. Most cases are due to Staphylococcus aureus (67%) [31]. Although many cases of PM attributed to HIV have been described, the pathogenesis of HIV-associated PM is unclear. The direct role of HIV in this process remains controversial. By means of sensitive and specific techniques (for example PCR, in situ hybridisation), no study has thus far been able to demonstrate the virus within the muscle fibers. A longitudinal study on HIV-associated PM has recently been published [32]. In this report 13 patients had biopsy-proven PM. Nine of them received immunosuppressive therapy: five patients had complete resolution of myositis, one p a t i e n t died of severe Pneumocystis carinii pneumonia and three had normalization of strength and CK levels. Four patients who had no demonstrable evidence of muscle weakness received no immunosuppressive therapy. The resolution of myositis in patients treated with immunosuppressive therapy reinforces the hypothesis of a immunologic link between HIV infection and development of autoimmune myositis. PM has been shown in correlation also with HTLV-I infection. Epidemiological studies show that the rate of anti-HTLV-1 antibodies in PM patients in the south of Japan where HTLV-1 is
endemic, is significantly higher than that in general population [32, 33] suggesting a possible relation of this virus in the pathogenesis of HTLV-1 seropositive PM patients. Recently the T cell clonotypes in muscle-infiltrating lymphocytes from patients with HTLV-1 PM has been investigated in order to understand the pathogenesis of microbial infection associated HTLV-1 PM. This study showed that certain T cell clones proliferate in the muscle lesion of HTLV-1 PM and may contribute to the pathogenesis of the disease. The Authors conclude that it is more likely that HTLV-1 PM is due not to direct viral infection in the muscle fibers but, rather, to a T cellmediated immunologic process that is initiated by HTLV-1 infection [34]. Elevated titers of antibodies to Toxoplasma and Borrelia have also been reported in IIM, but no convincing evidence for the presence of either of these agents in IIM has been provided.
3.3. Animal Models Animal models have been used in attempt to clarify the possible role of viruses in myositis. One model uses the Tucson strain of Coxsackie virus B 1. In this model, infection of neonatal mice leads to the development of acute viral myositis. In 60% of the mice, the inflammatory process changes from acute to chronic and resembles that found in human polymyositis. In the chronic phase, viral RNA persists in the muscle; this phenomenon may perpetuate the inflammatory response [35]. Encephalomyocarditis virus can also induce an inflammation in mice skeletal muscle that is characterized by mononuclear cell infiltrate, perifascicular atrophy, myofiber degeneration and regeneration [36]. Noteworthy that different inbred mouse strain also show different degrees of susceptibility to the disease, thus reinforcing the idea that the infectious agent is necessary to develop the immune reaction and to perpetuate the inflammatory response, but that host factors are also required for full induction of the disease [37]. Other studies point to production of heart and myosin reactive autoantibodies as mediators of immunopathology [38-40]. Many of these studies used the same strain of virus to infect different mouse strains which develop immunopathic myocarditis by different mechanisms. The heterogeneity
of the immunopathic response is certainly due to differences in the genetic composition of the host. On the contrary, myocardotic and amyocardotic viruses do not appear to change their pathogenicity in different hosts. This implies that certain characteristics of the viruses are essential for expression of the disease. These animal models provide clear evidence that viral infection can cause chronic muscle inflammation and that variety of autoimmune phenomena may be involved in the pathology.
3.4. Hypothesis of Induction of the Autoimmune Response Several hypothesis have been formulated in order to understand the possible mechanism by which infectious agents may induce autoimmune response in genetically susceptible individuals. The possibilities are that the infectious agent 1) interacts in the host with cellular proteins inducing changes in the proteins which are no longer recognized as "self" by the host immune system; 2) makes clustered cellular antigens accessible to the immune system which has never learn to recognize them as self proteins; 3) induces the production of human antibodies carrying pathogenic idiotypes (anti-idiotypic antibodies); 4) carries antigenic sites that "mimic" aminoacid sequences in the normal host proteins (molecular mimicry). The virus could initiates an immune reaction in the muscle which continues after the virus is eliminated. From this point of view, the anti-synthetase antibodies could be "footprints" of previous viral infections [ 10]. It has been suggested that they arise as a result of interaction of a virus with the synthetase during viral replication that breaks tolerance to the immune system in association with the foreign viral protein [10, 41]. It is possible that a group of viruses is involved, each interacting with a different synthetase, thus explaining the fact that only one antisynthetase antibody is found in an individual patient [10]. Using a computer alignment procedure, the aminoacid sequencing of histidyl-tRNA and alanyltRNA synthetases of Escherichia Coli has been compared with those of 3600 proteins tabulated in the National Biomedical Research Fundation pro-
587
tein sequence database [42]. This study was based on the observation that prokaryotic synthetases and proteolytic fragments of mammalian aminoacyltRNA synthetases tend to have similar structures. For histidyl-tRNA synthetase, a match with the hypothetical EC-RF4 protein of Epstein Barr virus was observed. Considerable homology of the alanyltRNA synthetase with an exon associated protein (HA) of adenovirus 2, with a second Epstein Barr virus protein (the hypothetical BPLF1 protein) and with haemoagglutinin molecules of two strains of influenza virus were observed. Interestingly, good homology with tropomyosin of skeletal muscle and epidermal keratine were found, suggesting that antibody against the alanyl-tRNA synthetase would cross-react with these proteins in the muscle if the sequences formed the epitope. In the literature the induction of myositis in mice intramuscularly inoculated with naked cDNA of the human gene for histidyl-tRNA synthetase is reported. The inoculation caused an inflammatory process in the muscle, including mononuclear cell infiltrates within necrotic fibers, and low titer of antibody to the synthetase [43]. No inflammation was found in muscle groups distant from the inoculation site or in the lung, indicating that peripheral tolerance to murine histidyl-tRNA was intact. Moreover, no myositis was induced by intraperitoneal immunization of mice with recombinant human histidyl-tRNA synthetase protein in adjuvant whereas a high titer of antibodies to the recombinant protein resulted from the inoculation. In this study, the local processing and presentation of the antigen to the immune system is crucial to the development of the focal myositis and sustained production of the foreign protein provided an ongoing stimulus for the immune reaction against the muscle. It is well known that human pathogens often express proteins with high antigenic potential with important homologies with human proteins. The phenomenon of molecular mimicry, has been suggested to trigger the development of juvenile dermatomyositis [44]. Sequence of homology between group A streptococcal type 5 M protein and myosine of human skeletal muscle has been found and it has been shown that the shared sequences are the target of immune responses in patients with juvenile dermatomyositis [44]. Molecular mimicry between a
588
major antigen of the bacterium and the target of the disease is the basis for the development of the juvenile dermatomyositis by aberrant immune reactions to streptococcus.
4. INFECTIONS IN PM/DM PATIENTS Patients with PM/DM are a risk category for developing infections. The increased risk of developing infections is the result of immune abnormalities and organ system manifestation associated with these diseases and their treatment [45]. Several types of infections occur in these patients including bacterial, viral and fungal infections. In about 20% of the PM/DM patients aspiration pneumonia produced by Gram-positive and anaerobic bacteria, is the most common infection [46]. Malignancy and pulmonary complications are the main causes of death in PM/DM patients and among these, aspiration pneumonia is one of the most common complications and causes of death [47]. In fact some PM/DM patients have involvement of the striated muscle of the hypopharynx and upper esophagus as well as thoracic muscle myopathy. These clinical involvements create altered swallowing and gastroesophageal reflux and difficulty in handling bronchial secretions [47, 48]. Moreover, the use of immunosuppressive drugs in the treatment of PM/DM could increase the risk of infections. Another predisposing factor is the calcinosis cutis frequently described in juvenile DM. This is a known risk for the development of staphylococcal soft tissue and dermal infections in the area of calcinotic lesion due to S Aureus [46]. Among fungal infections, pneumonia caused by Pneumocistis carinii is often fatal in PM/DM patients and Candidiasis is frequently observed in DM patients, maybe because of the prevalent skin involvement of these patients [45].
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16. Franceschini F, Cavazzana I, Generali D, Quinzanini M, Viardi L, Ghirardello A, Doria A, Cattaneo R. AntiKu antibodies in connective tissue diseases: clinical and serological evaluation of 14 patients. J Rheumatol 2002;29:1393-1397. 17. Targoff IN, Miller FW, Medsger TA, Oddis CV. Classification criteria for the idiopathic inflammatory myopathies. Curr Opin Rheum 1997;9:527-535. 18. Left RL, Burgess SH, Miller FW, Love LA, Targoff IN, Dalakas MC, Joffe MM, Plotz PH. Distinct seasonal patterns in the onset of adult inflammatory myopathy in patients with anti Jo-1 and anti-Signal recognition particle autoantibodies. Arthritis Rheum 1991;34: 1391-1397. 19. Manta P, Kalfakis N, Vassilopoulos D. Evidence for seasonal variation in polymyositis. Neuroepidemiology 1989;8:262-265. 20. Phillips BA, Zilko PJ, Garlepp MJ, Mastaglia FL. Seasonal occurrence of relapses in inflammatory myopathies: a preliminary study. J Neuro12002;249:441-444. 21. Plotz PH. Autoantibodies are anti-idiotype antibodies to viral antibodies. Lancet 1983;II:824--826. 22. Ferri C, Zignego AL. Relation between infection and autoimmunity in mixed cryoglobulinemia. Curr Opin Rheumato12002; 12:53--60. 23. Left RL, Love LA, Miller FW, Greenberg SJ, Klein EA, Dalakas MC, Plotz PH. Viruses in idiopathic inflammatory myopathies: absence of candidate viral genomes in muscle. Lancet 1992;339:1192-1194. 24. Travers RL, Hughes GR, Sewall JR. Coxsackie B neutralization titers in polymyositis/dermatomyositis. Lancet 1977; 1:1268. 25. Bowles NE, Dubowitz V, Sewry CA, Archard LC. Dermatomyositis, polymyositis and coxsackie-B-virus infection. Lancet 1987;I: 1004-1007. 26. Youself GE, Isenberg DA, Mowbray JF. Detection of enterovirus specific RNA sequences in muscle biopsy specimens from patients with adult onset of myositis. Ann Rheum Dis 1990;49:310-315. 27. Chevrel G, Borsotti JP, Miossec P. Lack of evidence for a direct involvement for muscle infection by parovirus B 19 in the pathogenesis of inflammatory myopathies: a follow-up study. Rheumatology 2003;42:349-352. 28. Chevrel G, Calvet A, Belin V, Miossec P. Dermatomyositis associated with the presence of parvorvirus B 19 DNA in muscle. Rheumatology 2000;39:1037-1039. 29. Fox SA, Finklestone E, Robbins PD, Mastaglia FL, Swanson NR. Search for persistent enterovirus infection of muscle in inflammatory myopathies. J Neurol Sci 1994;125:70-76. 30. Jongen PJH, Zoll GJ, Beaumont M, Melchers WJG, van de Putte LBA, Galama JMD. Polymyositis and dermatomyositis: no persistence of enteroviruses or encepha-
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Guillain-Barr
syndrome
N.M. van Sorge ~,2,L.H. van den Berg ~, M.D. Jansen ~,2, J.G.J. van de Winkel 2,3 and W.-L. van der Pol 1
1Department of Neurology and 2Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; 3Genmab, Jenalaan 18d, Utrecht, The Netherlands
I. INTRODUCTION Guillain-Barr6 syndrome (GBS) is a postinfectious polyradiculoneuropathy. Molecular mimicry between microbial structures and glycolipids (gangliosides) on peripheral nervous tissue is thought to elicit a ganglioside-specific immune response, resulting in localized inflammation and tissue damage. Although experimental evidence supports a role for anti-ganglioside antibodies in GBS pathogenesis, the mechanisms, which eventually trigger the production of cross-reactive antibodies in a small percentage of individuals, have remained elusive. This chapter aims at providing an overview of the role of anti-ganglioside antibodies in the pathogenesis of GBS as well as describing possible interaction of pathogens with the immune system, leading to induction of pathogenic antibodies.
2. GANGLIOSIDE STRUCTURE AND FUNCTION
Gangliosides are sialic acid-containing glycolipid structures, consisting of a hydrophobic ceramide moiety, anchored in the cell membrane, linked to externally exposed hydrophilic carbohydrate structures [1]. Nomenclature is based on the number and position of carbohydrate residues and sialic acids (Fig. 1) [2]. Gangliosides are ubiquitously expressed, but are highly concentrated in nervous tissues. Qualitative and quantitative differences exist in ganglioside expression between central (CNS) and peripheral nervous system (PNS) as well as within the PNS itself; GM1 and G D l a are pre-
dominantly expressed in motor nerves and axons [3, 4], GQ lb has been found enriched in cranial nerves [5], and GD lb is highly expressed in sensory nerves [4]. Gangliosides have important physiological functions and are involved in numerous cellular functions, including apoptosis [6], adhesion of cells [7], stimulation and inhibition of nerve regeneration [8, 9] and signal transduction [10, 11]. Gangliosides have furthern-tore been implicated to play a role in molecular processes involved in metastasis and adhesion of tumor cells [12, 13] and adherence of viruses, bacteria and bacterial toxins to eukaryotic cells [14]. Several microorganisms or microbial products use glycosphingolipids as 'docking' receptors. Interactions that were documented include binding of cholera toxin to GM1, Campylobacter jejuni enterotoxin to GM 1, Haemophilus influenzae to GA2/GM 1 and Sendai virus to GD 1a. Since binding of pathogens to multiple saccharides is more efficient than its weak interaction with a single saccharide, 'multivalent' saccharides are being tested as pathogen inhibitors (reviewed in [ 14]). Gangliosides are highly concentrated and developmentally regulated in nervous tissues and may play an important role in the correct development and function of the brain and PNS. Information concerning ganglioside function in vivo has come from studies using ganglioside knock out mice. Mice lacking all complex gangliosides (GM2/GD2 synthase knock out mice, GalNAcT-/-, Fig. 1), but still expressing GM3 and GD3, were generated and viable [15, 16]. Histological analysis of GalNAcT/- mice revealed a normal morphology of all major tissues with the exception of the testis [16, 17].
591
Ceramide (Cer)
Glucosyltransferase
~
Ce~ GIcCer Glucosyltransfera~e !
GD3 s y n t h a s e
c~~
C~r-I~}O LacCer
GalNac Transferase
Cer-~ GD2
GM2
G/~2
Oe'
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C e ~ A AW W Vm
GA1
GDlb
GM1
Cer~ \%7
v GMlb
V
C e r ~ V"7
V
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l CerO '
C e r ~ 11' J& ,d& IW' J& l=r
a-series
7
GTlb
C e r ~ .,...
GTla
GDlc
o-series
/'OO
GQlb
b-series complexgangliosides
Glucose O
Galactose
~I V
N-acetylgalactosamine Sialic acid
Figure 1. General scheme of ganglioside biosynthesis route. Disrupting the gene encoding the enzyme GM2/GD2 synthase (GalNAcT) limits ganglioside synthesis to GM3 and GD3, whereas animals lacking the enzyme GD3 synthase are depleted of all b-series gangliosides. GalNAcT and GD3 synthase double knock out mice only express GM3. GlcCer, glucosylceramide; LacCer, lactosylceramide.
However, GalNAcT-/- mice exhibit an age-related decrease in myelination, as well as axonal degen-
592
eration in both the central and peripheral nervous system [15], which is accompanied by progressive
motor deficits [18]. Hence, complex gangliosides were proposed to be essential for maintainance of a normal neuronal structure and function. GalNAcT and GD3 synthase double knock out mice only express GM3 (Fig. 1). Although brain histology did not show gross abnormalities, these mice display a high mortality rate and are extremely sensitive to sound-induced seizures [19]. It was argued that absence of GD3 may result in malformation of so-called 'lipid rafts' in cell membranes, thus interfering with signal transduction, as GD3 was recently found associated with the Src family kinase Lyn in lipid rafts [20]. This hypothesis is supported by findings in Src family kinase Fyn knock out mice, that have been found sensitive to sound-induced seizures [21]. In humans, one patient has been described to lack GM2/GD2 synthase activity, resulting in a total lack of complex gangliosides, and an 80% reduction in the total amount of brain gangliosides [22, 23]. This patient suffered from seizures, poor motor development, and died shortly after birth. A number of human disorders involve accumulation of gangliosides due to autosomal recessive mutations of genes encoding lysosomal enzymes. [3-hexosaminidase dysfunction (Tay-Sachs disease) leads to accumulation of GM2. Neuronal dysfunction results in mental and motor retardation, the severity of which is inversely related to the residual activity of [5-hexosaminidase [24]. Mutations in the [~-D-galactosidase gene result in a syndrome with a spectrum of clinical phenotypes, known as GM1 gangliosidosis or Morquio disease type B. Severe cases completely lack enzyme activity, resulting in profound neurodegeneration and deterioration of CNS functions, likely attributable to toxic accumulation of GM1 in nervous tissues. Morquio disease type B is characterized by symptoms such as corneal clouding, hearing deficits and skeletal changes, albeit that brain and PNS seem to remain unaffected [25, 26]. Overall, it can be concluded that gangliosides play an important, albeit poorly defined, role in the proper functioning of the CNS and PNS. Gangliosides represent a target for autoimmune responses in patients with Guillain-Barr6 syndrome, which may - thus - be seen as yet another example of a ganglioside-associated disease.
3. GUILLAIN-BARRI~ SYNDROME Guillain-Barr6 syndrome (GBS) is a postinfectious polyradiculoneuropathy, leading .to weakness, sensory loss and respiratory insufficiency in the most severe cases. Guillain, Barr6 and Strohl first described the syndrome in 1916. The initial concept of a clinically homogenous syndrome has been challenged, and the broad spectrum of clinical, immunological and electrophysiological symptoms suggests GBS to be a rather heterogeneous disorder. Yearly incidence is approximately 1.3/100.000/year worldwide, affecting males slightly more frequently than females [27]. About 60% of patients experience an antecedent respiratory or gastrointestinal infection, most commonly caused by Campylobacter jejuni (C. jejuni), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Mycoplasma pneumoniae (M. pneu-
moniae), or Haemophilus influenzae (H. influenzae) [27, 28]. Molecular mimicry between pathogen constituents and gangliosides has been proposed to trigger a pathologic immune response targeted at nerves. Pathologically, GBS is characterized by demyelination or axonal degeneration of motor, sensory or autonomic nerves and nerve roots. GBS is nowadays subdivided in 4 groups based primarily on clinical, electrophysiological, and pathological observations (Table 1) [29, 30]. In acute inflammatory demyelination polyneuropathy (AIDP) myelin sheaths and Schwann cell membranes are predominantly affected by an inflammatory process, resulting in demyelination. AIDP was originally considered to be caused by a T cell and macrophage dominated immune response. However, complement deposits have been detected on myelin and Schwann cell membranes suggesting a contribution of antibodies to AIDP pathogenesis as well [31, 32], albeit that no particular antigen has been identified. Two GBS entities are characterized by destruction of axons; acute motor-axonal neuropathy (AMAN) and acute motor-sensory axonal neuropathy (AMSAN). Both subtypes display similar pathologies, suggesting that they represent entities of a continuous spectrum [33]. Autoantibodies are considered the main effector molecules in the inflammatory response against axons [34], inducing antibody-mediated complement and phagocyte activation. Both AMAN and AMSAN have been
593
Table 1. Characteristics of different GBS subtypes and their putative autoantigens
GBS subtype
Preceding event
Clinical characteristics and electrophysiology (EMG)
Immunological characteristics
Antigen
Acute inflammatory demyelinating polyneuropathy (AIDP)
CMV
Demyelination
M. pneumoniae
T cells, macrophages, complement, antibodies?
GM2 GalCer
Acute motor-axonal neuropathy (AMAN)
C. jejuni H. influenzae
Axonal degeneration of motor nerves
Macrophages,antibodies, complement
GM1,GMlb, GDla, GalNAc-GD1a
Acute motor-sensory axonal neuropathy (AMSAN)
C. jejuni
Axonal degeneration of motor and sensory nerves
Macrophages,antibodies, complement
GM1,GMlb, GD l a
Miller Fisher syndrome (MFS)
C. jejuni
Cranial nerve involvement
Macrophages, antibodies, complement
GQlb, GTla
GalCer, galactocerebroside; CMV, cytomegalovirus; M. pneumoniae, Mycoplasma pneumoniae; C. jejuni, Campylobacter jejuni; H. influenzae, Haemophilus influenzae.
associated with preceding C. jejuni infection, the presence of anti-GM 1, anti-GD 1a and anti-GM lb IgG autoantibodies (Table 1), and more severe disease as compared to AIDP [35-37]. Miller Fisher syndrome (MFS) represents a distinct clinical entity, constitutes approximately 5-10% of all GBS cases and is clinically characterized by ataxia, areflexia and ophthalmoplegia [38]. MFS is strongly associated with preceding gastrointestinal and respiratory tract infections, as well as the presence of anti-GQ lb IgG antibodies in > 90% of patients (Table 1) [38]. "Classical" GBS patients sometimes show low titer anti-GQlb antibodies, a finding that is often associated with signs of ophthalmoplegia [39]. Two clinical observations support a relevant contribution of autoantibodies to GBS pathogenesis: 1) the beneficial effects of plasmapheresis or the infusion of pooled human gammaglobulins (IVIg) [40]. Plasmapheresis and IVIg infusion have been proposed to accelerate removal or neutralization of autoantibodies and other humoral factors, and to inhibit ongoing inflammation [41-43]; 2) The reported flaccid paralysis in a newborn from a mother with GBS [44]. The infant developed GBS 12 days after delivery and sera from both mother and infant were shown to interfere with motor endplate function ex vivo. It was assumed that IgG antibodies were transplacentally transferred.
594
Gangliosides are considered the main target of a pathogenic immune response in GBS and antiGM1 antibodies are present most often. In contrast, antibodies against myelin proteins are infrequently detected [45]. Anti-ganglioside antibodies are detected in plasma of 40% of GBS patients, although the reported prevalence varies considerably, possibly related to methodological differences [46].
4. ANTI-GANGLIOSIDE ANTIBODIES IN GBS PATHOGENESIS
Antibodies against glycolipid structures have first been described in serum from patients with IgM paraprotein associated neuropathy in 1985 [47], but have since been detected in serum from patients with a wide variety of neurological diseases, ranging from inflammatory to neurodegenerative [48-50]. Anti-ganglioside antibodies are most often detected in sera from patients with Guillain-Barr6 syndrome (GBS), although their diagnostic value is limited [50, 51 ]. Anti-ganglioside antibodies have also been detected in small numbers of healthy individuals [52]. Nevertheless, there is abundant evidence for an association between anti-ganglioside antibodies and GBS. Non-protein structures elicit antibody responses
without classical T cell help. Gangliosides are an example of such T cell independent antigens, eliciting at best a rise in anti-GM1 IgM antibodies in humans [53], and experimental animals [54, 55]. Consequently, anti-ganglioside antibodies found in sera from healthy controls are mostly of the IgM class displaying low affinity, and are considered to represent 'natural antibodies' [56]. Such 'natural' anti-GM1 IgM antibodies are unable to induce lysis of GMl-containing liposomes, suggesting they are immunologically inert [57]. Anti-ganglioside antibodies in GBS belong to all three major immunoglobulin (Ig) classes, IgM, IgG and IgA, suggesting a process of class-switching and affinity maturation in vivo. Most ganglioside-specific antibodies in GBS patients are either IgG1 or IgG3 [58-61]. Class-switch of ganglioside-specific B cells might be initiated by antecedent infections. This is supported by observations that isotype and subclasses of ganglioside antibodies were associated with specific preceding infections. GBS or MFS patients with preceding gastrointestinal infection more frequently display anti-GM1 and anti-GQlb IgA antibodies [61, 62]. Anti-GM1 IgG1 in GBS patients and anti-GQlb IgG2 in MFS patients are detected more frequently in sera from patients with preceding diarrhea, in particular with C. jejuni infection, whereas anti-GM1 IgG3 and anti-GQlb IgG3 antibodies are more frequently produced in response to respiratory tract infections [61, 63]. Secondly, GBS-like symptoms and a concomitant rise of anti-GM1 IgM and IgG titers [64, 65] have been observed in patients treated with a ganglioside mixture [66, 67]. Thirdly, clinical GBS symptoms correlate with antibody specificity (Table 1) [50]. Clinical heterogeneity may be attributable to differences in histological expression of gangliosides in peripheral nervous tissue [4], and specificity of the immune response. The best-defined association of antiGQlb IgG antibodies and MFS [68] can likely be explained by very high expression of GQlb in oculomotor nerves [5]. In addition, the presence of anti-GM1 IgG1, but not IgG3 and IgM [63, 69], and anti-GDla IgG are associated with increased risk for axonal degeneration [36, 37] and severe and prolonged motor deficits, whereas anti-GDlb antibodies seem associated with sensory complaints [70-72]. In a recent study using high affin-
ity monoclonal anti-ganglioside antibodies, GDla was indeed primarily detected in motor nerves [4]. Although GM 1 is not enriched in motor neurons [4], biochemical differences between GM1 in sensory and motor nerves have been observed with respect to the length of fatty acid chains and double bonds in the ceramide moiety [73]. GDlb is enriched in sensory nerves in staining, which provides histological support for a GDlb-specific pathological immune response [4]. Finally, immunization of specific rabbit strains with gangliosides results in the development of GBS-like symptoms. GDlb immunizations, eliciting anti-GD lb IgG antibodies, induce ataxic sensory neuropathy [74, 75]. Passive transfer of anti-GDlb positive serum in rabbits induced some degeneration of the spinal cord dorsal columns, although no clinical symptoms were observed [76]. Recently, a rabbit animal model for axonal GBS has become available. Repeated immunization of Japanese White rabbits with GM1 or a ganglioside mixture and adjuvant induces increasing titers of specific anti-GM1 IgM and IgG antibodies and severe motor deficits [77]. Pathologically, peripheral nerves were affected mainly by Wallerian-like degeneration with little or no lymphocyte infiltration, but deposition of IgG on axons and anterior roots was observed [78]. Intraneural injection of immunized rabbits' serum in naive animals produced profound axonal degeneration [79]. Furthermore, animals recovered more rapidly upon administration of polyclonal rabbit IgG, similar to observations in humans upon infusion with IVIg (Yuki N. et al, personal communication).
5. E F F E C T O R FUNCTIONS OF ANTIGANGLIOSIDE ANTIBODIES Anti-ganglioside antibodies may contribute to PNS dysfunction in several ways. First, binding of ganglioside-specific antibodies to axons or myelin sheaths may directly interfere with nerve conduction. GM1 co-regulates cellular calcium levels (reviewed in [11]), which are crucial for proper neuron function and development. Anti-GM1 antibodies have been shown to increase intracellular calcium concentrations by activating calcium channels, or stimulating calcium release from intracellu-
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lar stores [80, 81]. Furthermore, binding of anti-ganglioside antibodies to cell membranes could hamper lipid raft formation and function, a n d - thus - interfere with signal transduction. In addition, several studies demonstrated ganglioside-specific antibodies to block neuromuscular transmission [82-84]. Anti-GQ lb IgG antibodies from MFS patients were demonstrated to block both pre- and postsynaptic neuromuscular transmission in both complementdependent [82, 84] and -independent manner [85]. The complement-dependent neuromuscular interference, designated the ~-latrotoxin-like (o~-LTx) effect, triggers an increase in spontaneous quantal acetylcholine (ACh) release, leading to failure in subsequent ACh release upon physiological stimulation [82]. The complement independent effect is distinct from the ~-LTx effect and may result from blockade of voltage-gated calcium channels or from effects on release of ACh after stimulation [85]. Secondly, antibody binding can trigger complement activation. Anti-GM2 IgM antibodies have been shown to induce complement-mediated cytotoxicity towards neuroblastoma cells expressing GM2 [86]. Deposition of complement components, including C3d and C5-9 complexes (membraneattack complex) has also been demonstrated in nerve biopsies from GBS patients [31, 34, 87]. Such deposits may trigger membrane leakage, disturbance of ionic homeostasis and Schwann cell lysis, thus causing nerve dysfunction [88]. Depletion of complement by cobra venom factor delays disease onset and reduces inflammation and demyelination in the GBS-like animal model experimental allergic neuritis (EAN) [89-91]. Administration of soluble complement receptor 1 reduces clinical and pathological signs of EAN [92, 93], although not to the same extent as treatment with cobra venom factor [92]. Complement regulatory proteins like CD55, CD46 (inhibitors of C3 and C5 convertases), CD59 (inhibits formation of membrane attack complex), and complement receptor 1 (CD35) are present on human Schwann cells, whereas myelin only contains CD59. CD59 expression was found increased on Schwann cells upon demyelination and axonal degeneration in rat EAN [94], which may represent a regulatory mechanism to protect Schwann cells from the cytolytic effects of complement activation. Importantly, unsheathed neurons are known to be extremely sensitive to complement lysis [88,
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95], stressing the importance of CD59 upregulation upon demyelination in EAN. Alternatively, chemotactic properties of complement may trigger accumulation of inflammatory cells. Finally, binding of antibodies to gangliosides may trigger leukocyte effector functions via interaction with immunoglobulin receptors (FcR) on phagocytes. Engagement of FcR by immune complexes triggers leukocyte responses such as cytotoxicity, production of cytokines, phagocytosis and degranulation [96-98]. The vigor of FcR-induced cellular immune responses depends on the efficacy of FcR-Ig interaction, antibody affinity, and antibody specificity. IgG1 and IgG3 anti-ganglioside antibodies represent the dominant IgG subclasses in GBS patients [61, 99, 100] and are capable of interaction with all types of IgG FcR (FcyR). Recently, degranulation and phagocytosis assays were developed in order to assess functionality of antiganglioside IgG in sera from GBS patients [ 101]. Approximately two-thirds of GM1 or GDla-specific IgG-containing sera were capable of inducing either degranulation or phagocytosis. The capacity of ganglioside-specific antibodies to induce leukocyte activation was shown complement-independent, and could be abrogated by addition of blocking leukocyte FcyR with specific monoclonal antibodies. Antibody titers did not correlate with the magnitude of leukocyte effector functions. Other antibody characteristics than titer may - thus - account for the induction of FcyR-mediated functions. In addition to IgG ganglioside-specific antibodies, IgA anti-GM1 antibodies were demonstrated to induce cellular effector functions via the IgA FcR (Fcc~RJ) (van Sorge et al, unpublished data). Anti-ganglioside IgG can be induced in experimental animals by vaccination with ganglioside mixtures. Notably, this seldomly results in the occurrence of GBS-like symptoms in such animals. Recently, it was reported that GBS-like disease could be induced in Japanese white rabbits by repeated and intensive vaccination [77]. Rabbit sera with high ganglioside-specific IgG titers but without clinically overt GBS did not induce degranulation and phagocytosis, which suggests a lack of biological activity [ 101 ]. Previous studies have suggested that GMl-specific rabbit IgG displays a 100-fold lower affinity for GM1 than human equivalents from GBS patients [102]. In contrast, anti-GM1
IgG from rabbits with GBS-like symptoms after ganglioside immunization [77] readily induced leukocyte degranulation (van Sorge et al, unpublished data). No correlation was found between clinical disability scores and magnitude of degranulation responses. Sequentially drawn sera from ganglioside-immunized rabbits were also tested for their capacity to induce leukocyte degranulation in vitro. Sera displayed an increase in functionality one week before onset of clinical symptoms, whereas samples taken before this time only induced moderate leukocyte degranulation. This increase in functionality is not only attributable to an increase in specific antibodies, because sera with identical titers displayed profound functionality differences (van Sorge et al, unpublished data). Taken together, these data suggest that functionality of anti-ganglioside antibodies, measured by in vitro assays, predicts the presence of GBS clinical symptoms in rabbits. Mechanisms contributing to nerve damage or blockade of neuromuscular transmission in vivo not only depend on the antibody (sub-)class, but also on antigen location and conformation. Fatty acid chain length of the gangliosides and the microenvironment in which the ganglioside is expressed both influence binding affinity of anti-ganglioside antibodies [103-105]. More specifically, binding of anti-ganglioside antibodies to their target is enhanced by the presence of longer fatty acid chains. Consequently, binding of specific antibodies to GM1 from sensory nerves was enhanced compared to GM1 isolated from motor nerves [106]. In addition, binding of anti-GM1 IgG was found increased when GM 1 was admixed with phosphatidic acid. Therefore, differences in lipid composition in nervous tissues may well contribute to differential antibody binding and pathogenicity.
6. PRECEDING INFECTION AND M O L E C U L A R MIMICRY GBS is widely considered a postinfectious complication, since it is often preceded by a respiratory or gastro-intestinal infection. C. jejuni, H. influenzae and CMV infections are strongly associated with the occurrence of GBS. Evidence for an association of EBV and M. pneumoniae infection with GBS is less convincing [28, 107, 108]. Infections with H. influ-
enzae and C. jejuni more frequently precede GBS in Northern China and Japan, than in Europe or the United States [107-110], which may be related to an increased percentage of AMAN patients in Asia. Finally, many case studies have reported associations with infection by other pathogens. Gastrointestinal and respiratory pathogens seem to have few characteristics in common, and their tropism suggests that they encounter microenvironments, which differ dramatically. Mucosal B cells in the upper respiratory tract preferentially produce IgA1, and in the gut IgA2 [111]. Although IgG1 is the main subclass produced by mucosal plasma cells, IgG2 is the predominating subclass in the distal gut, and IgG3 in the upper airway mucosa [111]. Interestingly, ganglioside-specific IgG subclass differences correlate with either gastrointestinal or respiratory tract preceding infections in GBS patients [61, 63]. Interactions of C. jejuni with gut epithelia and gut associated immune cells, and CMV with similar cells in the respiratory tract may therefore be distinct, but eventually trigger similar immune effector functions. In order to induce a ganglioside-directed immune response, preceding infections must 1) provide a substrate for the antigenic drive (molecular mimicry), and 2) overcome the regulatory functions of the immune system, which protect the host from autoimmune reactions. Pathogen characteristics, which may aid the occurrence of an autoimmune inflammatory response, have been reviewed by others, and will be concisely summarized below. 6.1. Campylobacter Jejuni
C. jejuni is an encapsulated Gram-negative rod, and a common cause of diarrhea. The molecular mechanisms involved in the interaction of this bacterium with human epithelia and primary immune cells in the gut remain to be elucidated. C. jejuni may cause dysentery symptoms, suggesting entero-invasive potential, but has also been reported to secrete cholera-like toxins [ 112, 113]. Other virulence factors, including presence of flagella, adherent properties and serum resistance, are likely to contribute to C. jejuni pathogenesis [114]. C. jejuni strains expressing lipopolysaccharide (LPS) with ganglioside-like epitopes have been isolated from GBS patients (Fig. 2) [115, 116]. Such
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epitopes may provide the substrate for the antigenic drive of the GBS-associated specific immune response. The presence of ganglioside mimics on LPS from C. jejuni from GBS and MFS patients has been demonstrated using several methods. First, anti-ganglioside antibodies from GBS patient sera cross-react with LPS from GBS- and MFS-associated C. jejuni strains, indicating that they have
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been induced by molecular mimicry [117, 118]. Secondly, GM1 mimicry could be demonstrated by staining C. jejuni LPS with the high affinity GM1 ligand cholera toxin B [115]. Additionally, antiGM1 antibody binding to LPS was inhibited after incubation of LPS with cholera toxin [117, 118]. Thirdly, purified LPS fractions from GBS isolates were examined for their chemical composition and
molecular structure, confirming the presence of different ganglioside mimics [115, 116]. Two observations discard an exclusive association between ganglioside-mimicking C. jejuni strains and GBS. 1) LPS extracted from C. jejuni isolates from enteritis patients also contain ganglioside mimics, although mimicry is more frequent in isolates from GBS and MFS patients [119]; 2) anti-LPS antibodies crossreacting with gangliosides can also be detected in serum from enteritis patients [118, 119]. Interestingly, anti-LPS immune responses differ with regard to the Ig subclass; IgG1 and IgG3 are most often observed among GBS/MFS patients, whereas IgG2 is predominant in most enteritis patients [ 117, 118]. This may indicate other bacterial virulence factors or host characteristics to be implicated in the initial immune response. To establish the relevance of ganglioside mimics on LPS for the production of anti-ganglioside antibodies and subsequent development of GBS, Japanese white rabbits were immunized with purified LPS from a GBS-associated C. jejuni isolate. The procedure elicited ganglioside-specific IgM and IgG antibodies in Japanese white rabbits, and flaccid paralysis (Yuki N e t al, presentation on Peripheral Nerve Society Meeting, Banff, Canada, 2003). Immunization of GalNAcT-/- mice with such LPS elicited antibodies cross-reacting with GM1, which could block neuromuscular transmission in a rat muscle-spindle cord co-culture (Yuki N e t al, presentation on Peripheral Nerve Society Meeting, Banff, Canada, 2003). These experiments underline the importance of GM 1 mimicry of C. jejuni LPS in GBS pathogenesis. How C. jejuni alters immunological homeostasis in the gut is largely unknown. C. jejuni has been demonstrated to invade gut epithelial cells and has the ability to translocate in membrane-bound vacuoles to the basolateral surface for exocytosis [ 120]. Here, they are likely taken up by monocytes/ macrophages, as several studies have documented the capacity of phagocytes to internalize C. jejuni [ 121,122]. Peripheral blood mononuclear cells from GBS patients were shown to contain C. jejuni DNA fragments more often than healthy donors, although no viable bacteria could be recovered [123]. yS-T cells from GBS patients remain unresponsive to stimulation with C. jejuni antigens even years after recovery from disease [124]. Persistence of C. jejuni
in the host cannot be excluded, but in-depth basic studies are needed to clarify the interaction of this bacterium with the human host.
6.2. Cytomegalovirus CMV is a herpes virus, with broad tissue tropism. CMV persists in endothelia and macrophages. CMV infections are very common, but remain unnoticed in the majority of cases. Immunological deficiency (e.g. post-transplantation, AIDS) predisposes to severe clinical complications by CMV, including retinitis, pulmonary and gastrointestinal infections, and graft-versus-host disease after bone marrow transplantation. The commonness of subclinical CMV infection may reflect its ability to evade immune responses [125]. It has been shown to interfere with proper MHC class I expression on its host cell [126, 127], thus preventing lysis of infected host cells by cytotoxic T cells. Antibody-mediated, or complement-mediated cellular cytotoxicity is attenuated by induction of FcTR expression on host cells [128]. These cells bind the Fc-part of IgG, which normally triggers inflammatory effector functions. The capacity of CMV to induce expression of molecules on host cell membrane possibly also provides a substrate for the ganglioside-specific immune response. CMV infection of fibroblasts has been shown to induce expression of GM2-1ike structures [ 129]. Serological evidence of preceding CMV infection in GBS patients has been found associated with GM2-specific IgG antibodies [130-132].
6.3. Epstein-Barr Virus EBV is a herpes virus with a tropism for lymphocytes. Association of preceding EBV and GBS has been reported but percentages are usually low [28, 107, 110]. EBV has strong B cell modulating capacity, which is reflected by its association with (Burkitt) B cell lymphoma and infectious mononucleosis. The molecular interaction of EBV with the human immune system has been reviewed in depth by others, and will only be discussed in brief here. The B cell complement receptor (CD21 or CR2) is used as a 'docking' receptor for EBV glycoprotein (gp) 350/220 [ 133]. Upon additional interaction of another EBV gp, gp42, with HLA-DR, the virus
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is internalized and transported to the nucleus [ 134, 135]. Although this stage of infection is generally regarded as latent, EBV actively manipulates B cell function, and replicates to ensure further spread through the host. Several EBV proteins have potent immune-modulatory capacities. Latent membrane protein 1 (LMP1) expression stimulates the expression of IL-10, amongst others, a potent B cell stimulating cytokine [ 136]. LMP2a contains immunoreceptor tyrosine activation motifs (ITAM), and interferes with Src family protein kinase-dependent signal transduction induced by the B cell receptor [ 137]. Other EBV proteins may mimic IL- 10 function, reducing IFN-~, levels, and thus altering local cellular immune responses [138]. EBV may, furthermore, change the mucosal microenvironment by colonizing epithelia. Unfortunately, it remains unclear whether EBV induces expression of ganglioside-mimicking structures on host cells.
6.4. Mycoplasma Pneumoniae Mycoplasma have acquired an obligate parasitic lifestyle, and lack sophisticated intrinsic biological capacities. M. pneumoniae is a cause of atypic pneumonia, and has been found associated with GBS in some studies [107, 110]. The pathogenic potential of Mycoplasma is well known, although the mechanisms remain enigmatic. Overt clinical infection is mainly reported among immune compromised individuals. However, several findings from microbiological studies may contribute to the induction of a ganglioside-specific inflammatory response. Antibodies against galactocerebroside (GalCer), one of the major glycolipids of peripheral nerves, are often detected in GBS patients preceded by MP [139, 140]. Pathogenic potential of anti-GalCer antibodies was demonstrated by development of a demyelinating neuropathy following repeated immunization of rabbits with GalCer [141 ]. It was recently demonstrated that MP expresses antigens, which display molecular mimicry with GalCer [140]. In addition, Mycoplasma may secrete molecules that are capable of altering local immune responses, including superantigens capable of polyclonally activating B cells.
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7. ORIGIN OF ANTI-GANGLIOSIDE ANTIBODIES The events that trigger the specific immune response in the presence of a microbial ganglioside mimic remain to be identified. Microenvironmental characteristics may play an essential role in the initiation of autoimmunity, as has been demonstrated in an animal model for multiple sclerosis (MS). Incorporation of a peptide mimic in a viral vector readily induced MS-like disease in laboratory animals, whereas repeated immunizations with the same peptide in strong adjuvant did not elicit disease [ 142]. These findings suggest that microbe tropism and local inflammatory reactions are crucial elements in GBS pathogenesis. Some indirect information comes from studies comparing the immune response against C. jejuni in GBS and non-GBS patients [52, 58, 61, 99, 117, 118]. The occurrence of ganglioside-specific IgG1 and IgG3 in serum from GBS patients, in contrast to IgG2 in serum from enteritis patients, may reflect differences in B cell stimulation. These can result from microbial heterogeneity, or inter-individual differences in the innate or specific immune responses.
7.1. Microbial Heterogeneity GBS-associated C. jejuni strains may have characteristics that increase the odds of postinfectious complications. Clinical isolates have been investigated by genetic and serological typing methods in an attempt to identify microbial factors that predispose to GBS. The Penner serotyping method [143] employs the presence of heat-stable antigens now identified as capsular polysaccharides [ 144]. C. jejuni Penner O:19 and O:41 were more frequently detected in GBS patients as compared to controls, especially in Japan, the United States and South Africa [145-148]. However, these findings were not corroborated in other countries [149, 150]. Penner serotypes O: 19 and 0:41 appear to be clonally related [151-153]. GBS-related strains of other Penner serotypes are genetically very diverse and do not appear to be separate genetic lineages when compared to enteritis-associated strains [149, 154]. Genetic studies may contribute substantially to our understanding of microbial characteristics,
which contribute to pathogenesis. Recently, cstlI gene heterogeneity of C. jejuni was suggested to be associated with the occurrence of GBS. Strains bearing LPS with a GQlb-like epitope carded the cstlI gene (which encodes sialic acid transferase) without exception, whereas only half of the GQlbnegative strains were cstlI positive [155]. This finding indicates that this gene may be necessary for synthesis of GQ lb epitopes, and might contribute to GBS/MFS pathogenesis.
7.2. Host Susceptibility GBS-associated pathogens are very common disease causing microorganisms. The annual incidence of C. jejuni-induced diarrhea is approximately 1 per 100 [ 156], and it is estimated that 0.1% of C. jejuni infections is followed by GBS [157]. This suggests differences in GBS susceptibility, although familial GBS cases are rare [158, 159]. Several association studies have been performed in an attempt to identify genetic markers that predispose to GBS. Innate cytokine production is mainly determined by genetic make up, and has been shown a crucial factor for outcome of inflammatory disease [160]. Elevated levels of TNF-a in serum of GBS patients have been reported [ 161-163]. Interestingly, a TNFtx promotor single nucleotide polymorphism (SNP) was found associated with antecedent C. jejuni infection in GBS patients (Table 2) [ 164]. Although the functional relevance of this SNP is uncertain [165], intrinsic TNF-tx production capacity may co-determine susceptibility to C. jejuni-associated GBS. The relevance of HLA heterogeneity for GBS susceptibility has been studied intensively [166173]. Unfortunately, these studies have failed to yield consistent results (Table 2). Associations of AIDP and AMAN with different HLA haplotypes have been reported, and have triggered speculation about the existence of multiple pathogenic pathways [ 169, 172]. Importantly, the absence of unequivocal HLA associations with GBS may be related to the observation that crucial antigenic targets in GBS are not peptides but glycolipids. Genetically determined differences in the effector phase of GBS may contribute to susceptibility and severity as well. Receptors for IgG (FcTR) are important molecules for the initiation of classi-
cal type II (cytotoxicity) and III (Arthus reaction) hypersensitivity reactions [174]. Functional polymorphisms of human FcyRIIA, IIIA and IIIB have been described, and determine the efficacy of antibody-induced leukocyte activation [96, 97]. Two studies with small sample sizes have documented associations of IgG receptor polymorphisms with either susceptibility or severity of GBS (Table 2) [175, 176]. A meta analysis encompassing 350 Western-European GBS patients and 693 controls strongly supports an association of the Fc~RIIIa158F allele, which interacts relatively inefficiently with IgG, with a mild disease course (van Sorge et al, unpublished data). FcTRIIIa is constitutively expressed on macrophages and a subset of monocytes, which have been detected histologically in peripheral nerves from GBS patients.
7.3. T Cell Dependent Mechanisms in AntiGanglioside Antibody Development Glycolipid structures are considered T cell independent antigens, but the presence of IgG1, IgG3 and IgA anti-ganglioside antibodies in GBS patients suggests T cell help. Increased levels of IL-2 and soluble IL-2 receptors have been detected in serum from GBS patients, consistent with T cell activation [177, 178]. Peripheral T cell activation during the acute phase of GBS has been detected, but clonal expansion of peripheral T cells or T cells in sural nerves has not been convincingly shown [179]. However, histological studies documented the presence of CD8+ T cells in peripheral nervous tissue from GBS patients in the subacute phase, suggesting a contribution of these cells to cytotoxic reactions in situ [ 180]. Upon interaction with mucosal antigen presenting cells (APC), constituents of C. jejuni and other GBS-associated microbes, may be presented to naive T cells in secondary lymphoid organs of the respiratory and gastrointestinal tracts. Professional APC are capable of presenting glycolipids, including gangliosides such as GM1, to CDl-restricted tyJ~ and ~,/~ T-cells. CD1 family molecules are non-polymorphic MHC-like structures expressed by dendritic cells, macrophages, and NK-T cells [ 181, 182]. Proliferation of such T cells in mucosaassociated lymphoid tissues may eventually provide the context for proliferation of ganglioside-specific
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A. Afferent phase Gastrointestinal infection by Campylobacterjejuni is the most frequently reported antecedent infection in GBS. Host and bacterial characteristics that contribute to "breakdown in tolerance" may include the following: 1. Direct interaction of GBS-associated C. jejuni strains with B cells, thus circumventing the need for classical T cell help. This could occur after interaction of B cells with superantigens or by crosslinking patterns recognition receptors (PRR) and B cells receptors, inducing both affinity maturation and class switching. 2. Modulation of antigen-presenting route by GBS-associated C. jejuni strains. Sera from GBS patients often contain anti-ganglioside antibodies of the IgG1 and IgG3 subclass, indicating T cell help. This may suggest that professional antigen presenting cells, e.g. dendritic cells (DC), process microbial structures and present them to T cells in secondary lymphoid organs. 3. T cell independent B cell activation in the spleen can be induced by release of DC-derived soluble factors.
B. Effector phase Effector functions of anti-ganglioside antibodies depend on antibody isotype. Both IgM and IgG are able to activate complement and may cause reduction of nerve conduction velocity. IgG and IgA complexes interact with Fc receptors (FcR) on inflammatory cells, inducing potentially harmful leukocyte effector functions, including cytotoxicity and phagocytosis. A = GM1/GMI-like structure.
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B cells, class switching and affinity maturation (Fig. 3). The CD1 family comprises five classes, subdivided in two groups: group I includes CD l a, CD lb, CD lc and CD le, whereas CD ld is the exclusive member of group II [183]. CD1 molecules differ structurally and biochemically, and are differentially expressed in sub-cellular compartments. Interestingly, CD1 antigen loading does not necessarily require intracellular processing by APC [184]. Furthermore, several ligands demonstrated promiscuous binding to CD 1 classes in vitro [ 185]. Intracellular glycolipid processing may lead to the accumulation in specific intracellular compartments, and to preferential loading of specific CD 1 molecules [ 186]. Intracellular processing of glycolipids has not been studied in detail, but may lead to different CDl-glycolipid interaction patterns than the ones observed after addition of gangliosides to APC in vitro. Furthermore, the interaction of microbes with APC may modulate CDl-dependent antigen presentation. Two recent reports suggest a possible contribution of CD 1 to GBS pathogenesis. First, the expression of CD lb on endoneurial macrophages in sural nerves of GBS patients was shown increased as compared to controls [187, 188]. Furthermore, non-protein antigen preparations of GBS-associated C. jejuni strains were shown to induce proliferative T cell responses using peripheral blood mononuclear cells from GBS patients, but not from controls [ 187]. Further research should lead to a better deftnition of the role of CD1 in GBS pathogeiaesis. The endogenous microbial flora of the gut may have an important function in preventing deleterious autoimmune reactions, possibly by maintaining suppressor activity [189]. The tolerogenic nature of the intestine is furthermore retained by the capacity of CD4+CD25+ regulatory T cells (TR) to inhibit both T cell dependent responses as well as controlling innate immune mechanisms [ 190]. Engagement of Toll-like receptors (Tlr) by mi'crobial products like LPS was demonstrated to block TR-mediated suppression, enabling activation of responder T cells [ 191]. The effect of gastrointestinal infection, in particular by C. jejuni, on commensal flora has not been studied, but may alter local immunological homeostasis and favor autoimmune inflammatory responses.
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7.4. Mechanisms of Antibody Production in the Absence of T Cell Help B cell activation, proliferation and class switching can be induced in the absence of T cell help (Fig. 3). Activation of specific B cell subsets, such as B 1 cells and marginal zone B cells (MZB), is largely T cell independent. Among these B cell subsets self-reactive B cell clones are frequently detected, and may well contribute to autoantibody production and autoimmune disease [192]. Recent evidence, however, also implicates a role for 'conventional' B2 cells in T cell independent autoantibody production. B 1 cells are characterized by the expression of CD5 and high surface IgM levels, and have the capacity for self-renewal. These cells produce large amounts of natural IgM antibodies recognizing both bacterial and self-antigens with broad specificity and low affinity [193]. These antibodies are coined 'natural' since they are produced in the absence of environmental stimuli. Plasma cells in the lamina propria of the gut are derived from B 1 cells and produce large amounts of IgA directed against cell wall components of commensal bacteria [194]. T cell help or the presence of secondary lymphoid tissue are not required for the production of natural IgM and IgA [ 194, 195]. Interestingly, the production of IgA in the gut requires the presence of commensal flora. As discussed above, IgA specific anti-ganglioside responses have been detected in GBS patients, and gastrointestinal infections frequently precede GBS. Marginal zone (MZ) B cells are derived from 'conventional' B2 cells and are characterized by high surface density of complement receptor 2 (CD21) and IgM [196]. These cells are especially implicated in rapid IgM and IgG responses to T cell independent type 2 antigens (TI-2), like bacterial polysaccharides [196]. Localization of MZ B cells in the spleen provides them with excellent opportunities to interact with blood-borne antigens as blood flow is strongly reduced by splenic architecture [197]. Interactions that may eventually cause ganglioside-specific B cell proliferation may include the following: 1) GBS-associated pathogens may alter the immunological microenvironment by secretion of molecules with cytokine-like activity. The role of
EBV-associated proteins has been discussed previously. Interestingly, EBV proteins may stimulate IL- 10 secretion [ 136], or mimic its function [138]. This cytokine is a potent stimulus for B cells, and excess production has been shown associated with B cell autoimmune disease such as systemic lupus erythematosus (SLE) [ 198]. 2) Microbes produce antigens with specific lymphocyte stimulatory potential. 'Superantigens' share the capacity of activating B cells and T cells, interacting ectopically with either T or B cell receptors while retaining strong stimulatory capacity. Superantigens induce aspecific clonal lymphocyte proliferation, which might cause 'bystander' activation of ganglioside-specific B cells. Both bacteria and viruses (including Mycoplasma) are known to produce superantigens [199, 200]. The preferential use of V 1]15 by activated T cells in AIDP patients was suggested to indicate a superantigen-induced immune response [201]. However, there is little evidence of polyclonal lymphocyte activation in GBS, which argues against contribution of superantigens in GBS pathogenesis. 3) Heterotypic crosslinking of specific and innate leukocyte receptors may represent yet another mechanism of T cell independent B cell activation and subsequent ganglioside-specific antibody production. a. Simultaneous engagement of a BCR specific for the constant part of IgG and Toll-like receptor (TLR) 9 by chromatin IgG complexes, was shown to activate antigen-specific B cells in the absence of additional T cell stimulation [202]. GBS-associated pathogens may express structures, which are recognized by TLR; simultaneous binding of ganglioside mimics on C. jejuni LPS by the BCR may trigger proliferation of ganglioside-specific B cells. b. MZ B cells express high levels of CD21, which possibly endows them with the capacity to respond rapidly to TI-2 antigens. CD21 is part of the CD19 complex, which is essential for B cell responses. High CD19 expression was shown to substantially lower the threshold needed for B cell activation[203]. Increased CD 19 expression i s - thus - associated with progressive breakdown in peripheral toler-
ance, increasing production of autoantibodies in several mouse models [204]. Interestingly, B cells from patients with the antibody-mediated autoimmune disease systemic sclerosis demonstrate a 20% higher CD19 and CD21 expression compared to healthy controls [205]. Expression of CD 19/CD21 has not been studied on B cells of GBS patients. However, phagocytes have been shown to internalize C. jejuni [121], and DNA fragments have been detected in peripheral blood mononuclear cells from GBS patients [ 123]. Differentiation of MZ B cells into plasma cells in response to blood-derived antigens was recently shown to depend on capture and transport of antigen by professional antigen presenting cells to the spleen, and the secretion of DC soluble factors, such as BAFF (B cell Activating Factor of the TNF Family) and APRIL (A Proliferation-Inducing Ligand) [206]. Interestingly, in serum from patients with antibody-mediated autoimmune disease, like SLE and Sjrgren's syndrome, increased levels of DC-derived soluble factors have been detected [207, 208]. It is tempting to speculate that C. jejuni, or its constituents, are transported to the spleen and eventually trigger activation of MZ B cells. 4) Finally, T cell independent differentiation and class switching of conventional B2 cells by LPS has recently been described. Mice producing hen egg lysozyme (HEL)-specific B cells with the intrinsic capacity of class switching [209] were crossed with soluble HEL transgenic mice, causing anergy of most HEL (self)-reactive B cells. Strikingly, anergic B cells could still proliferate and class switch to IgG when activated independently of the BCR with T cell-independent stimuli such as LPS [209]. Ganglioside-specific B cells may reside in a similar anergic state, due to selfreactive properties. Hence, C. jejuni LPS might activate ganglioside-specific anergic B cells, causing proliferation and class switching.
8. CONCLUSION Guillain-Barr6 syndrome (GBS) is a rare complication of infection with common pathogens. Increasing experimental evidence supports a role for
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anti-ganglioside antibodies in G B S pathogenesis. A l t h o u g h there is convincing evidence that molecular mimicry b e t w e e n microbial molecules and host structures contributes to G B S development, the specific m o l e c u l a r m e c h a n i s m s , which trigger the ganglioside-specific i m m u n e response, remain elusive. Host and p a t h o g e n characteristics, which contribute to the d e v e l o p m e n t o f this m o n o p h a s i c a u t o i m m u n e disorder, remain to be identified.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Systemic Sclerosis S. Guiducci ~, R. Giacomelli 1,2, A. TyndalP ,3 and M. Matucci Cerinic ~
IDepartment of Rheumatology, University of Florence, Italy; 2Departmentof Internal Medicine, University of L'Aquila, Italy; 3Departmentof Rheumatology, University of Basle, Switzerland
Systemic Sclerosis (SSc) is a connective tissue disease characterised by skin and visceral organ involvement [1]. The disease affects the immune, the vascular and the connective tissues leading eventually to fibrosis. Cellular immunity in SSc is characterized by an increase of CD4+ T-helper and y/8-TCR+ lymphocytes, and a decrease of CD8+ cytotoxic T cells in peripheral blood. After T-cell activation, infiltration into the skin and internal organs is an early event in SSc. This activation is a selective process that appears to be influenced by antigen. Oligoclonal CD8+ T cells can be detected in the lungs with alveolitis, showing evidence of antigen-driven selection; similarly, V~51+~,/8-T lymphocytes increased in blood, lung and skin show evidence of selection by antigen. The importance of a particular T cell subpopulation may depend upon the organ involved and the stage of disease [2]. The humoral aspect of SSc is characterized by the generation of self-reactive antibodies such as, antitopoisomerase, anticentromere, antifibrillin-1 and antiendothelial antibodies. Immune abnormalities observed in SSc can cause both microvascular damage and collagen overproduction by fibroblasts which are the pathophysiological hallmarks of the disease [2]. Vascular damage is characterized by both microvascular endothelial cell activation (increased expression of adhesion molecules such as E-selectin and intracellular adhesion molecule1), and injury (endothelial cell apoptosis) [3]. In addition, the fibrotic process is characterized by abnormal fibroblast/myofibroblast cell proliferation and the deposition of excessive extracellular matrix proteins in affected organs. The cause of SSc remains unclear. Numerous
environmental and infectious agents have been suggested as possible triggering factors of SSc. The exposure to exogenous agents, such as inhaled or ingested chemicals or infectious agents (bacterial and viral), have been put forward as potential environmental triggers for SSc [1, 2]. Increased antibody titers (DNA topoisomerase I, centromeric proteins, and RNA polymerases I and III) suggest that target antigens are presented to the immune system as native molecules or even as a multiunit complex. The homology between viruses and autoantibody targets suggests indeed that molecular mimicry may have a role in initiating antibody response in SSc [2]. This suggestion seems corroborated by the evidence of diversity and plasticity of the anti-DNA topoisomerase I autoantibody response in SSc [4]. This molecular mimicry, inducing autoimmune responses, may be a mechanism by which infectious agents may contribute to the development and progression of SSc. The main aim of the immune system is to respond to microrganisms but in most autoimmune diseases, the epidemiology does not suggest any link with previous infection. This means that if environmental organisms are involved in the genesis of these diseases, they might be organisms that are ubiquitous [5]. This also means that other determinants make some subjects more susceptible than others to autoimmunity. It is very likely that all autoimmune diseases including SSc, are significantly influenced by multiple microbial agents. Nevertheless, the number of agents involved is probably limited, and their understanding may provide useful perspectives for prevention or therapy [5]. In this review, we analyse the literature pertain-
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ing to the link between SSc and microorganisms.
1. VIRUSES The possible pathogenic role of two viruses, namely human cytomegalovirus (HCMV) and human parvovirus B19 (B 19), has been recently proposed [2, 6-10]. 1.1. Parvovirus B19 B 19 has been proposed as a causative agent in rheumatoid arthritis and systemic vasculitides [ 11 ]. B 19 genome has been detected in serum, bone marrow, skin and cultured skin fibroblasts from SSc patients [7, 9]. The prevalence of serum B 19-related markers has been detected in SSc: viremia was found in 4% of SSc patients, a very high rate in comparison with that of healthy blood donors, which does not exceed 0.6% [12, 13]. B 19 genomic sequences were demonstrated in 57% (12/21 patients) bone marrow biopsies from SSc patients and were never detected in the control group [7]. It has been suggested that bone marrow may represent a reservoir from which the B19 virus could spread to SSc target tissues [14]. Indeed, antibodies to the B19 IgG-type and against the non-structural viral protein NS1, both possible markers of persistent B 19 infection, were more frequently found in SSc patients (33%) than in controls ( 13 %) [7]. B19 genome was detected in fibroblasts from SSc skin and its persistence in several passages in culture was also observed [ 15]. This led the authors to propose that B 19 may persistently infect skin SSc fibroblasts, being thus responsible for fibroblasts alteration [15]. Healthy human synovial fibroblasts infected by parvovirus B 19 change phenotype in vitro and express invasive properties similar to that observed in rheumatoid arthritis [16]. Moreover, the presence of B 19 RNA sequences within fibroblasts may exert a direct cytotoxic effect and directly and/ or through B 19 driven immune-mediated reaction [17]. This may trigger a molecular mimicry mechanism involving specific target antigens implicated in SSc and consequent production of autoantibodies directed against SSc fibroblasts and their products [16, 18].
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1.2. Cytomegalovirus Notably, latent HCMV infection has been suggested as an accelerating factor in autoimmune vasculopathy, allograft rejection, and re-stenosis [19]. Pandey and LeRoy [19] proposed that latent HCMV, or human herpes virus 5, infection and its downstream effects on immune, vascular, and repair mechanisms could serve as an accelerating factor in SSc. The most direct link between HCMV and SSc is the high-titer antibodies to the polyglycine motifs of HCMV observed in a comparative study of Epstein-Barr virus and HCMV in the sera of patients with several rheumatic and connective tissue diseases [3]. The appearance of SSc shortly after an acute episode of viral infection suggested a possible triggering link for HCMV in SSc [8]. One possible mechanism involved in HCMV contribution to SSc pathogenesis might be the presence of HCMV sequences within endothelial and epithelial cells. This may be responsible for the viral lytic effect, directly and/or through HCMV driven autoimmune reaction [20]. HCMV infection may play a part in the pathogenesis of the SSc through the infection of endothelial and others cells, and through the upregulation of fibrogenic cytokines and induction of immune disregulation [ 19]. HCMV infection of the endothelium is characterized by latency, reactivation, and downstream shedding of the virus [21]. In both rat and mouse models [2 l, 22], it was shown that HCMV infection leads to intimal lesions, with an increased neointima to media (N/M) ratio of the injured vessel of 41% in the CMV-infected rat model [21]. Moreover, infection of endothelial cells by HCMV selectively alters the expression of integrins, downregulating t~5131 and t~2~1 and upregulating ot6~ 1 and ct3~ 1 integrins [23]. The presence of HCMV sequences within endothelial cells may be responsible for the viral lytic effect, directly and-or through HCMV driven autoimmune reaction. Recently, circulating IgG antibodies binding HCMV late protein UL94 and inducing apoptosis of endothelial cells have been shown in SSc [6, 24]. These antibodies induced endothelial cell apoptosis through specific interaction with cell surface integrin-NAG-2, a protein highly expressed on human endothelial cells, forming complexes with
integrins a6131 and o~3131 [6, 25]. Thus, it may be hypothesised that HCMV triggers a host antiviral response generating specific autoantibodies cross reacting with endothelial antigens [6]. In addition, cross reactivity of anti-topoisomerase I (Scl-70) antibodies, which are characteristic of the diffuse type of SSc, with a peptide sequence of the UL70 protein of HCMV [26] may suggest molecular mimicry as a mechanism initiating the autoantibody response [4]. The further role of HCMV in SSc pathogenesis seems also supported by the fact that the gene products of HCMV major immediate early locus are involved the induction of the fibrotic program in human dermal fibroblasts [ 19]. In addition, in SSc patients indirect evidence of HCMV role includes an association between increased circulating levels of HCMV-specific antibodies and the prevalence of SSc related autoantibodies [20, 27]. Although these studies provide important information linking CMV infection to SSc, a direct causal association between CMV infection and SSc, are still missing.
1.3. Epstein-Barr (EBV) The attention has also been focused on another member of the herpesvirus, EBV, whose DNA was detected in SSc lungs [28]. The interes~ in EBV stems from the observation that an antibody against an hnRNP autoantigen called p542 [29] may develop in mononucleosis, and that IgG anti-p542 are found in SSc [30, 31 ]. Since p542 is a hnRNP, it falls into the same general class of nuclear antigens that are known targets in SSc. In these patients, autoantibodies against a 60-62 kDa cellular protein which cross react with the glycine/alanine repeat in EBNA-1 may be detected [20]. The EBNA-1 antigen of EBV was the presumed primary target of the autoantibody reacting with the strongly antigenic gly/ala repeat of EBNA-1, as well as with a gly22alal-ser5 repeat in p542-HCMV has also a gly-rich antigenic epitope, glyl7-ala2-ser5 occurring in an early antigen [32, 33]. Indeed, we still do not know if anti-p542 or the anti-60-62 kDa protein in SSc is due to immunization by EBV. The alternative might be that HCMV, which has an antigenic glycine-rich
epitope in its early antigen, theoretically could act similarly to the glycine/alanine repeat in EBV. In SSc these viral products could synergize with other factors in the microenvironment predisposing to SSc development.
1.4. Endogenous Retroviruses (ERV) Frequently, ERV were implicated in autoimmunity [34], and as possible causative agents in SSc [35-37]. ERV may lead to autoimmunity directly, by encoding autoantigens, or indirectly, by affecting the expression of genes regulating immune responses and tolerance [38]. ERV are likely targets of cross-reactivity for virally induced immune responses. Such cross-reactivity, i.e. molecular mimicry between self-antigens and viral proteins, has been proposed as a trigger of autoimmunity [38-42]. Several autoantigens, such as U1 small nuclear ribonucleoprotein [43] and topoisomerase I [44], share cross-reactive epitopes with murine retroviral gag proteins. This raised the possibility that the natural targets of human T-cell lymphotropic virus (HTLV-I)-reactive antibodies of patients with autoimmune disease may correspond to endogenous retroviral sequences (ERSs). In 115 SSc sera, the presence of an antibody against the retroviral p24gag and pl7gag protein of human immunodeficiency virus type 1 (HIV-1) was detected by Western blot analysis [35]. Antibody to p24gag was present in 16.5% of SSc patients, and in 25.9% of the diffuse and 7.0% of the limited cutaneous subset. Instead, antibody to pl7gag was identified in only 3.8% of the whole group of SSc patients [35]. Molecular mimicry is again one possible explanation for the presence of these viral antibodies in SSc. It has been reported that patients with diffuse SSc have antibodies that bind to a synthetic peptide corresponding to a common sequence found in DNA topoisomerase I and several mammalian p30gag proteins [36]. This may eventually suggest that these peptide-binding antibodies represent an antigenic cross-reactivity. HRES- 1 (HTLV-related endogenous sequence 1) is a human endogenous retroviral sequence capable of protein expression, it encodes in fact, a 28kDa nuclear protein (HRES-1/p28) in T cells [38, 39, 45]. Significantly higher HRES-1 peptide binding activity in 23% SSc sera was found [45]. These data indicate that HRES-1/p28 may serve as an autoan-
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tigen eliciting autoantibodies cross-reactive with HTLV-I gag antigens [45]. There may be two possible mechanisms for generation of HRES-1-specific autoantibodies: molecular mimicry may trigger HRES-1 antibodies [46], or abnormal immune presentation may lead to autoimmune response against HRES- 1. HRES-1 has been mapped to a common fragile site of chromosome 1 at lq42 [47] and this suggests the potential involvement of HRES-1 in the fragility of lq42. Chromosomal instability at lq42 may be associated with aberrant activation and HRES-1 autoantibodies [48]. This possibility is interesting because of the increased chromosomal fragility [48] and the presence of HRES-1 autoantibodies observed in SSc.
infection in SSc is associated with an increased concentration of antimycobacterial hsp65. It is now known that gastric infection with HP is correlated to the risk of coronary heart disease [49] and seems associated to Raynaud's phenomenon (RP) [58]. In 26 women with primary RP, 81% incidence of HP infection was found [58]. In a study of patients with primary RP, eradication of HP infection with a triple antibiotic regimen was associated with complete disappearance of RP in 17% of those treated and a reduction in symptoms in an additional 72% of patients [59]. The preferential occurrence of HP infection in SSc may be explained by an increased prevalence of HP infection, favored by the well known disturbed gastrointestinal motility [23].
2. BACTERIA
3. PATHOGENETIC HYPOTHESES
Recent research on the involvement of bacterial infections in the pathogenesis of SSc has been focused on Helicobacter pylori (HP), a gastric bacterium implicated in coronary artery disease [49]. Increased seroprevalence of HP in SSc has been reported by several groups [50-53]. HP sheds extracellular products that elicit local and systemic immune response that could be responsible for tissue damage [54]. Among these, a 60-kDa protein, belonging to the heat-shock proteins (hsp), seems to play an important role. Since microbial hsp have a high level sequence h o m o l ogy with their mammalian counterparts, and some antigenic determinants may be represented in both agent and host proteins, it has been postulated that immune response to HP hsp could be involved in the pathogenesis of systemic and organ specific autoimmune diseases [54, 55]. HP-associated, human hsp60 and mycobacterial hsp65 are members of the 60-kDa family of heat shock proteins, and are believed to share both high sequence homology. Thus, a cross-reaction of antibodies against these proteins may exist [55, 56]. Several recent observations indicate that the titer of antibodies against HP hsp60 is significantly higher in HP infected than in non infected subjects [54, 56]. Higher prevalence of HP infection has been found in SSc (78%) associated with elevated levels of anti-hsp65 but not of anti-hsp60 [57]. These findings indicate that HP
3.1. Molecular Mimicry
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The clear evidence that molecular mimicry of a host protein by a pathogen can induce an autoimmune disease led to the hypothesis that viruses are involved in the development and/or acceleration of the pathologic features of SSc [60-63]. HCMV data [6] indicate that in SSc, infection with HCMV is able to generate a host antiviral response that has the ability to be self-reactive toward autoantigens and endothelial cells. Such self-reactive antibodies against the virus induce endothelial cell apoptosis through interaction with the integrin o~3~1- and c~6[31-NAG-2 protein complex [25]. These results provide a pathogenetic mechanism by which HCMV infection may contribute to vascular damage in SSc. Autoantibodies against intracellular antigens are associated with endothelial cell damage [64, 65], considered the primary event in the pathogenesis of the disease. This molecular mimicry mechanism has been also suggested for different disorders characterized by diffuse vascular disease, including SSc [ 19]. HCMV driven autoimmunity may be crucial in the cascade of events leading to typical SSc alterations, namely endothelial cell injury and consequent up-regulation of fibrogenic cytokines. Thus, latent HCMV infection may contribute to SSc progression through its ability to infect
endothelial cells [19]. In conclusion, molecular mimicry is one of the mechanisms that may account for the link between infection and autoimmunity [66---68]. However, in SSc a direct link between autoantibodies, HCMV infection and endothelial cell damage is still lacking.
3.2. Endothelial Cell Damage Endothelial cell is considered the largest immunologically active surface of the body with its ability of cell-cell recognition and adhesion and in the immune defense. No immune reaction can occur and no inflammatory process is possible without the mediation of the endothelium. It is clear that endothelial injury occurs in SSc [69] and that the presence of Raynaud's phenomenon and diffuse microangiopathy suggest that endothelial injury may represent one of the first steps in the pathogenesis of SSc [69]. The relationship between the autoimmune response and endothelial cell damage in SSc is matter of debate, in particular whether immune alterations follow or preceed endothelial changes [25]. Endothelial cell may be infected by viruses [70] that play a particular role in inducing vasculitis. It is possible that the internalization of the bacterial agents by endothelial cell, or the infection of endothelial cell by microrganism, may be the starting event, followed by the inflammatory response. Several infectious agents lead to vascular pathology [71], the most important of which are: viral-induced cytophatic changes; cell-mediated immune responses directed at endothelial cell infected by viruses, leading to endothelium activation via upregulation of the expression of adhesion molecules; immune complexes-mediated vasculitis. In the early phases, viral antigens are found in endothelial cell, and after few days of viral infection, endothelial abnormalities are followed by necrosis. A similar mechanism of endothelial cell injury is seen in HCMV infection [6, 21 ]. This leads to leukocyte recruitment and adhesion to the vascular endothelium while antibodies against endothelial cells (AECA) induce both upregulation of adhesion molecules with consequent mononuclear cell adhesion [65] and endothelial cell apoptosis [64]. Cytokines secreted in response to infection either locally or at distant sites may lead to endothelial
activation and damage. Viral or bacterial expression of self-like proteins or epitopes lead to the production of autoreactive cellular and humoral immune responses (molecular mimicry). Thus, induction of repair mechanisms is characterized by extracellular matrix protein deposition and the recruitment and proliferation of myofibroblast cells, as a response to infection-triggered endothelial injury [72]. In conclusion, a microorganism infecting an endothelial cell may lead to endothelial injury and a downstream cascade of events involving endothelial cell itself as well as immune cells and other neighbouring cells such as fibroblasts. 3.3. Superantigens Superantigens are proteins that are expressed endogenously in the organism or that are derived exogenously by bacteria [73]. Microbial superantigens are thought to initiate an immediate T cell response by direct binding to the T cell receptor V region, bypassing the MHC complex and the need for antigen processing and presentation. This could be an initiating step of autoimmunity. Friedman et al [74] suggested that B cell may bind to microbial superantigens to surface class II MHC molecules and become a target of T-helper lymphocytes with TCRVI3 segments. This model could be applied to endothelial cell, in particular considering the fact that the endothelium may work as antigen presenting cells, triggering, together with superantigens, the immune response.
3.4. Microchimerism A possible mechanism that may explain sporadic development of SSc in response to CMV infection and the 8:1 ratio of SSc-affected women to SScaffected men is the microchimerism hypothesis. The term microchimerism refers to one individual harboring DNA or cells at a low level that derive from another individual. Chronic graft-versus-host disease (cGvHD) shares similarities with some autoimmune diseases and is a iatrogenic form of chimerism, occurring as a complication of hematopoietic stem cell transplantation. The HLA genes of the donor and the host are known to be of central importance to the development of cGvHD. When also considered in light of the female predilection
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to autoimmunity, these series of observations led to the hypothesis that microchimerism and HLA genes of host and non-host cells are involved in some autoimmune diseases, such as SSc [75]. Microchimeric T cells of male fetal origin were verified 27 years after giving birth in one woman and have been shown to be more common and more numerous in women with SSc than in healthy, age matched controis [76]. Cellular microchimerism was also found in 3 patients with SSc but not in controls. In fact, the absolute amount of male DNA was higher in the patients, and the in vitro addition to blood mononuclear cells of an anti-CD28 costimulatory signal acted as a powerful amplification of microchimeric cells in SSc [77]. Recent studies have demonstrated the presence of microchimeric cells in 82.9% of peripheral blood and skin lesions SSc of patients compared to 63.6% of controls [78-80]. Circulating levels of CD4+ and CD8+ T cells were found to be significantly higher in SSc patients than in controls [79]. Furthermore, patients with diffuse SSc had significantly more CD4+ microchimeric T cells than controls [79]. The engraftment and survival of these cells are dependent on the complex relationships between the tissue antigens of the mother and offspring (or host and donor) and are highly variable as a result [81]. Still today the pathogenic potential of microchimeric cells is unknown. The suggestion that CMV may induce the proliferation of microchimeric cell derives from in vitro studies that show that CMV-infected graft endothelial cells can initiate a host T-cell activation cascade, and that cytokines, produced as a consequence enhance graft endothelial alloimmunogenicity [82, 83]. Waldman WJ et al [84, 82] indicate endothelial cell as a mediator of CMV response in the transplant recipient [82] and demonstrate that endothelial cells can stimulate autologous T cell responses to CMV in the absence of accessory APC and suggest potentially novel mechanisms of immune activation [84]. Therefore, in people with circulating microchimeric T cells, the endothelium represents an allotypic stimulus to those cells. However, if the endothelium is infected with CMV, proliferation and cytokines expression may be amplified, thus triggering a cascade characterised by endothelial activation, vascular inflammation, and neointimal formation. This may mimic the same pathway trans-
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planted T cells follow in graft-versus-host disease. In SSc, studies of microchimerism and graft-versushost reactions have put forward the role of allogenic rejection. In these vascular reactions, the presence of latent HCMV infection may increase the intensity of allograft rejection, and may accelerate dramatically organ failure [19]. For instance, this may happen in diffuse cutaneous systemic sclerosis, with dramatic kidney, heart, and lung failure in similar fashion as to that occurring in transplantation, and in coronary artery bypass grafting [19]. However, another hypothesis is that infection by any microorganism may trigger the intervention of 1,/8 T cells, found in significant amount in SSc skin [85]. Usually, the meeting of these cells with microorganisms shift from a Th2 tolerogenic to a Thl cytotoxic pattern. The 1,/8 T cells may encounter resident microchimeric cells inducing a cross reaction against "non-self' cells igniting automatically a GVHDlike reaction [86]. Therefore, if microchimerism plays a role in SSc, it may be considered one step in a multistep process.
4. CONCLUSIONS To date, scarce evidence has been provided to support the hypothesis that microorganisms are involved directly in the pathogenesis of SSc. However, some models, such as molecular mimicry, microchimerism and others, are potentially interesting to explain pathogenetic mechanisms for vascular injury, fibroblast activation and tissue remodelling. The evidence suggests that we are still far away from understanding the real involvement of microorganism in the etiopathogenesis of SSc.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Tuberculosis and SLE-Like Symptoms as a Complication of Biological Anti-TNF Therapy Milan Buc ~and Jozef Rovensk)~2
;Department of Immunology, School of Medicine, Bratislava, Slovak Republic; 2National Institute for Rheumatic Diseases, Piegt'any, Slovak Republic
Pathogenesis of autoimmune diseases is rather complex. However, progress in the characterisation of cytokines has led to the realisation that one cytokine, tumour necrosis factor (Box 1), has a paramount role in their pathogenesis, esp. in rheumatoid arthritis and M. Crohn [1, 2]. This discovery started a new era of development of biological drugs to treat them. Anti-TNF monoclonal antibodies and TNF-binding receptors were developed and approved by US FDA and the European Union's Commission for the therapy of rheumatoid arthritis, Crohn's disease, later for juvenile idiopathic arthritis of the polyarticular type, ankylosing spondylitis, and psoriatic arthritis. The blocking agents licensed for use are infliximab (Remicade), a humanised, mouse-derived, genetically engineered, monoclonal antibody to TNE adalimumab (Humira), a highaffinity, full-length human IgG1 monoclonal antibody, etanercept (Enbrel), a recombinant human, soluble fusion protein of TNF type II receptor and IgG 1, which competitively inhibits TNF [2--4]. The efficacy of anti-TNF therapy has been validated by numerous studies (for review see [5]). The analysis of cohorts of rheumatoid arthritis patients who underwent anti-TNF therapy has shown the diminution of inflammatory process, diminished damage to both cartilage and bone, reduction of angiogenesis, serum concentration of matrix metaloproteinases MMP-1 and MMP-3, levels of rheumatoid factor, fibrinogen, platelets etc. 60-80% of patients respond clinically to the blockade of TNF [2, 3]. The analysis of a cohorts of M. Crohn's is patients has shown that only infliximab is effective, etanercept is not, despite promising first results [6, 7]. An
Box 1. Tumour necrosis factor and its basic biologic ~ctions Tumour necrosis factor (TNF) is the principal mediator of an inflammatory response. Its major cellular source is activated mononuclear phagocyte, although antigenstimulated T cells, activated NK cells, and activated mast cells can also secrete this cytokine. TNF is initially synthesised as a non-glycosylated trans-membrane type II protein of M r approximately 25,000. Membrane TNF assembles as a homotrimer. A Mr 17,000 fragment of each subunit can be proteolytically cleaved off the plasma membrane to produce the "secreted" form, which circulates a stable homotrimer of M r 51,000. TNF binds two distinct receptors, the Mr 55,000 (TNF-RI) and the Mr 75,000 (TNF-RII); most biological effects are mediated through TNF-RI. The principle biologic actions of TNF at low concentrations are: (1) TNF causes vascular endothelial cells and leucocytes to express or up-regulate adhesion molecules that contribute to accumulation of leucocytes at local sites. (2) TNF stimulates mononuclear phagocytes and other cells to secrete chemokines. (3) TNF activates inflammatory leucocytes to kill microbes. (4) TNF acts both as an angiogenesis factor and as fibroblast growth factor. If the stimulus for TNF production is sufficiently strong TNF acts as and endogenous pyrogen, acts on mononuclear phagocytes to stimulate secretion of IL-1 and IL-6, supports production of the acute phase proteins and has some other activities. ,
,
explanation for the different efficacies of infliximab and enanercept, respectively, can be inferred from different means by which TNF is produced in both diseases. In Crohn's disease, the inflammatory process is driven by TNF produced mostly by mucosal CD4 § T cells, contrary to rheumatoid arthritis where
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monocytes are the principal source of the soluble TNF. Accordingly, infliximab, which binds and removes both soluble and membrane bound TNF is successful in rheumatoid arthritis and Crohn's disease, respectively. However, etanercept, which binds and removes soluble TNF alone, is efficient in rheumatoid arthritis only. Additionally, in Crohn's disease, infliximab promotes apoptosis of effector T cells by binding the membrane form of TNF, which serves as the primary mechanism of its clinical effects [8]. Anti-TNF therapy has been shown to have good efficacy for the treatment of the afore mentioned diseases. However, interfering with the immune system it is responsible for some complications (Table 1), esp. the reactivation of M. tuberculosis infections. There are reports that infliximab therapy is associated with an appropriate 4-fold increase in tuberculosis incidence among rheumatoid arthritis patients compared with those without such treatment [9-11 ]. Individuals suffering from active pulmonary tuberculosis expel droplets containing minute numbers of bacilli. Alveolar macrophages engulf these droplets, but do not kill the p a t h o g e n - M. tuberculosis arrests the phagosome at an early stage of maturation and prevents its fusion with lysosomes. Specific T cells are stimulated in the draining lymph nodes and induce bacterial containment in small granulomatous lesions of the lung, but fail to achieve complete microbial eradication. So, a dynamic balance between bacterial persistence and host defence develops. This balance might be life-long, so that the individual is infected but does not suffer from an overt clinical disease. The bacilli remain dormant until the host immune defence mechanisms are suppressed as in HIV infection, cancer etc. [ 12, 13]. An effective immune response against tuberculosis involves activation of T cells, macrophages, and granuloma formation. Both CD4 + and CD8 + T cells have a role in fighting M. tuberculosis infection. Residence of M. tuberculosis within the phagosome ensures that antigens have a ready access to the MHC class II antigen-processing machinery. Mycobacteria-specific CD4 + T cells are typically of the TH1 type; they are potent IFN-y producers. IFN-y is a central factor in the activation of anti-mycobacterial activities of macrophages, it stimulates anti-bacterial mechanisms in macrophages, notably reactive
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Table 1. Adverse effects associated with anti-TNF treatment Adverse effect
Approximate frequency
Infusion reactions
Mild to moderate (6-17%) Severe ( 1-4 %)
Congestive heart failure Infections
Drug induced SLE Malignancy CNS demyelination
Upper respiratory infections (10-40%) Tuberculosis Pneumocystis carinii pneumonia Listeriosis Histoplasmosis <1% Rare
oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) [13-15]. Despite its residence within phagosomes, M. tuberculosis is also capable of stimulating MHCclass-I-restricted CD8 + T cells. Engulfment of antigen-loaded vesicles by bystander dendritic cells has been shown to induce MHC-class-I-restricted CD8 + T-cell responses. Vesicles are released, in particular, from apoptotic cells that are abundant in caseous lesions of tuberculosis patients. It is therefore probable that certain mycobacterial antigens are transferred to dendritic cells (DC) and by cross-presentation presented through MHC class I molecules to CD8 + T cells. CD8 + T cells, similar to CD4 + T cells, can produce IFN-y, but their main function is target cell killing. Granulysin is responsible for bacterial killing and presumably gains access to M. tuberculosis that resides within macrophages through pores formed by perforin [16, 17]. It was also reported that V ~ / 8 2 T lymphocytes kill macrophages harbouring live mycobacteria by granulysing-dependent mechanisms [ 18]. Group 1 CD1 molecules typically present glycolipids that are abundant in the mycobacterial cell wall, such as phosphatidylinositol mannosides, lipoarabinomanan, mycolic acids and hexosyl1-phosphoisoprenoids. Group 1 CD1 molecules are abundantly expressed on DC, but are virtually absent on macrophages. The transfer of glycolipids from infected macrophages to bystander DC can
therefore constitute another important mechanism for promoting CD1 presentation and T cell activation. Generally, CDl-glycolipid-specific T cells produce IFN-y and express cytolytic activity [15, 191. Although the central role of T cells and IFN- 7 in the control of M. tuberculosis is beyond doubt, granuloma formation, in which activated macrophages restrict the mycobacterial growth, is an important defence mechanism, too. The formation and maintenance of granulomas depends on the activity of tumour-necrosis factor (TNF) and lymphotoxin [20-22]. TNF, in collaboration with IFNy, increases the phagocytic and cytocidial activity of macrophages and induces apoptosis of permissive macrophages. On the other hand, TNF by means of stimulating chemokine production (CCL-2, -3, -4, -5, -8) as well as the expression of endothelial cell adhesion molecules (e.g. CD54) is crucial in a recruitment and accumulation of mononuclear cells [13, 20]. TNF and TNF-receptor p55-chain (TNFRp55) deficient mice showy a breakdown of granuloma formation causing the death of mycobacteria infected mice. In addition TNFRp55 signalling is required for modulation of the T cell response as in its absence a hyper-inflammatory T cell mediated tissue destruction becomes evident [23]. Because both antibacterial mechanisms and demarcation are seriously impaired in the absence of TNF, disorganised, diffuse necrotising infiltrations are present in TNF and TNFR deficient mice [20-22]. Granulomatous inflammation is a highly dynamic process and continuous recruitment of inflammatory cells into the lesions is necessary to maintain antimycobacterial defence. Therefore, even during the chronic phase of infection, when compact granulomas have already been established, neutralising TNF can no longer contain mycobacterial growth within the lesions and granuloma breakdown will be followed by dissemination of bacteria; the result is a clinical form of tuberculosis, usually extra-pulmonary or disseminated. It is more frequently seen in rheumatoid arthritis than in M. Crohn patients on infliximab [5, 20]. Reactivation of tuberculosis in patients who were undergoing anti-TNF therapy underlines the importance of TNF in containing M. tuberculosis. Current estimates place the number of individuals infected with M. tuberculosis at 1-2 billion globally.
Approximately 16 million of these individuals have active disease, and the rest are presumed to harbour the infection in a latent form [2, 24]. It is therefore highly probable that a great deal of patients suffering from rheumatoid arthritis is also at risk of M. tuberculosis reactivation and clinical vigilance is needed to minimise the risk when biological drugs are intended to use for their treatment. In patients with risk factors for tuberculosis, screening for the infection, and perhaps prophylactic anti-tuberculosis therapy, may thus be justified before TNF-blockers are given. Infliximab has a half-life of about 9 days and therefore currently it is given as an intravenous infusion on days 1 and 15 of the treatment, followed by maintenance infusions every 6-8 weeks. Etanercept has a half-life of about 3-5 days and is given twice a week as a subcutaneous injection [5, 25]. Thus infliximab treatment probably results in a sustained and complete neutralisation of TNF activity, whereas etanercept may only restrict the peaks of TNF concentration. It can be therefore supposed that the infliximab treatment will be more likely to be associated with tuberculosis activation as etanercept. The prevailing impressions among clinicians seem to support this theoretical expectation, however no comprehensive review on the topic is available at the moment [20, 25, 26]. Blocking activity of IL-1 by its natural antagonist, IL-1Ra (anakinra) has started to be used in the treatment of rheumatoid m'thritis with a success comparable with that obtained by etanercept. During the first year of approval of IL-IRa, the daily administration in over 20,000 patients with rheumatoid arthritis has not resulted in the reports of opportunistic infections. So far, there are also no reports of reactivation tuberculosis, even in a European population with high risk of tuberculosis [5, 27]. Drug induced systemic lupus erythematosus (SLE) was first reported more than 50 years ago. To the list of drugs that are able to induce SLE like symptoms and autoantibodies characteristic for SLE infliximab and etanercept, respectively, can be included, too. In the phase III of ATTRACT (Anti-Tumour Necrosis factor Trial in Rheumatoid Arthritis with Concomitant Therapy) study, 16% of infliximab treated patients developed doublestranded DNA (dsDNA) antibodies compared with none in the placebo-treated group [28]. In a 1-year
625
randomised, masked, controlled study comparing the efficacy of infliximab plus methotrexate with that of methotrexate alone in 428 rheumatoid arthritis patients, antinuclear antibodies developed in 53--68% of infliximab patients but in only 26% of controls and anti-dsDNA in 7-10% of infliximab patients but none of the controls [29, 30]. Charles et al [31] reported similar results. However in the study of De Rycke et al [32], no anti-dsDNA antibodies were observed and the development of anti-nuclesome, anti-histone or anti-ENA antibodies was not statistically significant in rheumatoid arthritis and spondylarthropathy patients treated by infliximab. Similar results were reported with the etanercept therapy. During the 6-month clinical-trials in the frame of the regulatory approval process for infliximab and etanercept, 11% of 323 etanercept treated patients became antinuclear-antibody positive and 15% of these were anti-dsDNA positive [33]. Although autoantibody production appears to be a relatively frequent phenomenon in the setting of infliximab or etanercept therapy, drug-induced SLE seems to be less common. In the ATTRACT trial with more than 300 infliximab-treated rheumatoid arthritis patients, one developed drug induced SLE [28]. In the study out of 771 infliximab-treated patients three developed features of SLE including rash, pleuropericarditis, and coexistent autoantiboidies [34]. Similarly, out of 880 patients included into infliximab trials, three developed SLE with rash and pleuropericardial manifestations, which resolved in 6 weeks to 6 months with prednisone treatment [35]. Recently Shakoor et al [33] also described four female patients who developed SLE 6 weeks to 14 months after the start of therapy with etanercept. All four had antinuclear antibodies, antibodies to dsDNA, and Smith antibodies (anti-Sm), which are more commonly associated with SLE than with drug-induced SLE. Three of the four had antibodies to histones, which are associated with drug-induced SLE. After discontinuation of etanercept, SLE-related symptoms resolved [35]. Anti-dsDNA antibodies may be involved in the pathogenesis of SLE. In vitro studies disclosed that anti-dsDNA antibodies enhance the release of proinflammatory cytokines (IL-1, IL-8, TNF) from monocytes and polarise the immune reaction towards TH2 pathway [36]. Anti-dsDNA antibodies
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may therefore play a role in lupus pathogenesis by augmenting inflammatory reactions and promotion of autoantibody production that are commonly found in patients with active SLE. The induction of SLE would be a very serious complication as is connected with infections that remain a source of mortality [37, 38].
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avium. Infect Immunity 1999;67:3571-3579. 24. Hinglez-Wilson SM, Sambadamurthy VK, Jacobs WR. Survival perspectives from the world's most successful pathogen, Mycobacterium tuberculosis. Nature Immuno12003;4:949-955. 25. Mikuls TR, Moreland LW. TNF blockade in the treatment of rheumatoid arthritis: infliximab versus etanercept. Expert Opin Pharmacother 2001 ;2:75-84. 26. Dinarello ChA. Setting the cytokine trap for autoimmunity. Nature Med 2003;9:20-22. 27. Cohen S, Hurd E, Cush J, Wieinblatt ME, Moreland LW, Kremer J, Bear MB, Rich WJ, McCabe D. Treatment of rheumatoid arthritis with anakinra, a recombinant human interleukin 1 receptor antagonist, in combination with methotrexate: results of a twenty-four-weak, multicenter, randomised, double-blind, placebo-controlled trial. Arthritis Rheum 2002;46:614-624. 28. Maini R, St Clair E, Breedveld F, Furst D, Kalden J, Weisman M, Smolen J, Harriman G, Feldman M, Lipsky P. Infliximab (chimeric anti-tumour necrosis factor ot monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomised phase III trial. The Lancet 1999;354:1932-1939. 29. Lipsky PE, van der Heijde EW, St Clair EW Infliximab and methotrexate in the treatment of rheumatoid arthritis; Anti-tumor necrosis trial in rheumatoid arthritis with concomitant therapy study group. N Engl J Med 2000;343:15904-1602. 30. Day R. Adverse reactions to TNF-o~ inhibitors in rheumatoid arthritis. The Lancet 2002;359:540-541. 31. Charles P, Smeenk R, DeJong J, Feldman M, Maini RN. Assessment of antibodies to double-stranded DNA induced in rheumatoid arthritis patients following treatment with infliximab, a monoclonal antibody to tumour necrosis factor cz. Arthritis Rheum 2001;44: 1977-1983. 32. DeRycke L, Kruithof E, Van Damme N, Hoffman IE, Van den Bosse N, Van den Bosch F, Veyes EM, De Keyser E Antinuclear antibodies following infliximab treatment in patients with rheumatoid arthritis or spondylarthropathy. Arthritis Rheum 2003;48:1015-1023. 33. Shakoor N, Michlaska M, Harris ChA, Block, JA. Druginduced systemic lupus erythematosus associated with etanercept therapy. The Lancet 2002;359:579-580. 34. Physicinas' Desk Reference. 55th Ed. Montvale: Medical Economis Company, 2001. 35. Markham A, Lamb HM. Infliximab: a review of its use in the management of rheumatoid arthritis. Drugs 2000;59:1341-1359. 36. Sun KH, Yu CL, Tang SJ, Sun GH. Monoclonal antidouble-stranded DNA autoantibody stimulates the expression and release of IL-lbeta, IL-6, IL-8, IL-10
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9 2004 Elsevier B. V. All rights reserved. Irffection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Behqet's Syndrome Giilen Hatemi and Hasan Yazici
Istanbul University, Cerrahpasa Medical School Department of Internal Medicine, Division of Rheumatology, Istanbul, Turkey
Behqet's syndrome (BS) is a systemic vasculitis with mucocutaneous, ocular, arthritic, vascular and central nervous system involvement. It is most commonly seen in countries around the Mediterranean sea, in the Middle East and the Far East. The prevalence is reported as around 1:300,000 in Northern Europe, 1:10,000 in Japan and 8-38: 10,000 in Turkey [ 1]. The age of onset is generally in the third or fourth decade. BS is associated with HLA B51, especially in patients from Japan and the Mediterranean countries and particularly with the severe forms of disease [2]. The syndrome has certain features different from other autoimmune disorders such as the rarity of associated Sjrgren's syndrome [3], lack of increase in autoantibodies including normal levels of CD5+ B cells [4] and more severe disease expression among males [5]. On the other hand there is also abundant evidence for autoimmune aberration such as increased levels of soluble interleukin-2 receptors [6], and the presence of autoantibodies like antiendothelial cell [7] and antilymphocyte autoantibodies. Oral mucosa may be important in the etiopathogenesis of BS since oral aphtae are present in almost all BS patients and these lesions are present for years before the appearance of other manifestations in many patients. Demonstration of increased proportion of certain streptococcal strains in the oral flora of BS patients as well as the isolation of a virus in certain BS lesions [8-10] has led to extensive search on the role of infections in the etiopathogenesis of BS.
1. VIRUSES 1.1. Herpes Simplex Virus Behqet, himself had proposed a viral etiology upon observation of intracellular inclusion like forms in smears from the hypopyon of the anterior chamber and aphtae [11]. Later, a virus was isolated from ocular fluid [9] and subsequently from the eye and brain tissues of a patient who died with neurological involvement [10]. However these observations did not later meet the more vigorous standards of modem virology. During the 1980's RNA complementary to Herpes Simplex Virus type 1 (HSV- 1) and later HSV- 1 DNA was detected in peripheral blood mononuclear cells of BS patients. Eglin detected RNA complementary to HSV-1, using in situ hybridization, in peripheral blood mononuclear cells of BS patients, especially those with ocular and arthritic involvement [12]. HSV-1 DNA was detected by dot blotting technique in whole blood of BS patients, more frequently than both rheumatoid arthritis patients and healthy controls [ 13]. Studd used polymerase chain reaction (PCR) to detect HSV-1 DNA in peripheral blood leucocytes [14]. Polymerase chain reaction (PCR) was also used to demonstrate HSV DNA in saliva [15], biopsies of gastrointestinal ulcers [16] and genital ulcers [ 17] of BS patients. HSV DNA was detected more frequently in all these specimens from patients with BS compared to similar specimens from healthy controls, patients with Crohn's disease and in the episiotomy tissue of normal pregnancies. In con-
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trast, other workers could not detect HSV DNA in biopsies from oral ulcers [ 14], in leukocytes or oral smears of BS patients [18]. A BS animal model was developed by inoculation of the earlobe of ICR mice with HSV [ 19]. This induced mucocutaneous symptoms including oral, genital and skin ulcers, eye symptoms including uveitis, iridocyclitis, conjunctivitis and hypopyon, gastrointestinal ulcers, arthritis, neural involvement and skin crusting. Famciclovir was administered to these mice and was shown to ameliorate some of the symptoms [20]. To determine whether this amelioration correlated with the expression of inflammatory mediators, RT-PCR was performed on the spleens of improved and relapsed mice after administration of famciclovir. IL-2 was expressed in non-responsive or relapsed mice, but not in improved mice. IFN-y was always expressed and was not related to improvement of ulceration. IL-4 and IL-10 were never expressed. As there are contraversies regarding the presence of HSV DNA in certain lesions of BS, there are also contraversies among reports about response to antiviral agents. There are case reports on the effectiveness of acyclovir in BS [21, 22], but a placebo controlled study in 22 BS patients failed to demonstrate a significant decrease in the frequency of oral and genital ulcers with the use of acyclovir [23]. In another study where acyclovir was used in addition to plasma exchange to remove circulating immune complexes and restore cellular immunity in 7 BS patients with severe ocular involvement, no effect was observed [24]. This lack of response to antiviral agents may indeed be taken as evidence against a viral etiology in BS. On the other hand a more likely indication is that the association of HSV infection with BS represents an altered immune response to HSV in BS patients rather than active infection itself. Supportive evidence for this is the fact that the CD4+ cells of patients with BS produce low proliferative responses when stimulated with HSV- 1 [25].
1.2. Hepatitis Viruses The relationship of BS with Hepatitis A, B and C viruses known to be associated with certain autoimmune diseases such as Sj6gren's syndrome and vasculitis such as polyarteritis nodosa, mixed cryoglob-
630
ulinemia and Henoch-Schoenlein's purpura [26-28] were also studied. Several groups searched for antiHCV antibodies, HBsAg, Anti-HBs, Anti-HAV Ig G and Ig M in BS patients [29-32]. None showed a higher frequency in BS patients than observed among the healthy controls. Only one study showed a somewhat higher frequency of HBV DNA and GBV-C RNA in BS patients compared to what was seen among a group of blood donors [33].
1.3. Human lmmunodeficiency Virus There are case reports about patients with HIV infection who had symptoms of BS [34-36]. A scenario for a causal relationship rather than coincidence can be made since one of these patients was from Uganda and the other from Zimbabwe, countries where BS is apperently quite rare [37]. However much more likely is the explanation that HIV infection and its complications can cause clinical findings similar to BS.
1.4. Parvovirus B19 Parvovirus B 19 antibodies have been reported to be associated with other vasculitides and connective tissue diseases [38, 39]. They were also sought for in BS. Anti-B 19 Ig M antibodies were detected in serum samples of 6 of 41 BS patients while none of the 40 healthy controls had these antibodies [40]. It remains to be seen whether there is an etiologic association between BS and parvoviral antibodies, the findings were coincidental or what was observed was due to an opportunistic infection.
2. BACTERIA
2.1. Clinical Evidence of Bacterial Etiology It has been observed that many BS patients have a history of a chronic focus of infection with a higher incidence of chronic tonsillitis and dental caries [41]. These seem to be related to streptococcal infections. Furthermore aggrevation of BS symptoms after treatment of dental caries and surgery for chronic tonsillitis were reported [42]. BS patients born in the United Kingdom were reported to have a higher rate of tonsillectomy and cold sores in their
history, a late birth order, travel to countries with a high incidence of BS and earlier age at first sexual intercourse. These are all suggestive of an infectious trigger during childhood or adolescence [43]. In addition to chronic infections, exacerbations of BS symptoms may also occur after acute episodes of infection with Strep. agalactiae vaginitis [44] or gingival infections with methicillin-resistant S. aureus [45]. Recently we have shown that pustular acneiform lesions in BS were not sterile [46]. Their microbiology was rather different from that of acne vulgaris. Staphylococcus aureus was grown in 58% of pustules in BS patients, while it was grown in 29% of pustules from acne vulgaris patients. Pustules on unusual acne sites such as arms and legs seemed to be mainly responsible for this difference since S. aureus was grown in 86% of pustules on unusual acne sites. Prevotella sp. bacteria which grow on secondarily infected skin lesions such as psoriasis and eczema rather than in acne vulgaris were grown in 17 of 70 (24%) BS pustules, whereas it was not grown in any of the 37 pustular acne vulgaris lesions. It is not clear, however, if the pustular lesions we studied were secondarily infected or the bacteria we grew had etiologic importance. Did, from our group had shown that papulopustular lesions were more common in BS patients with arthritis [47]. Also a factor analysis of the clinical manifestations of BS among a totally different population of patients confirmed the finding that pustular lesions and arthritis were indeed associated [48]. These observetions suggest that arthritis in BS can perhaps be related to acneassociated, reactive arthritis. Furthermore the effect of penicillin on mucocutaneous lesions and arthritis in BS had been looked at in two different studies. It was shown that prophylactic penicillin treatment was beneficial for both the mucocutaneous lesions [49] and the arthritis episodes [50]. A similar effect was observed with minocycline which reduced both the frequency of clinical symptoms and the production of pro-inflammatory cytokines by peripheral blood mononuclear cells stimulated by streptococcal antigens [51 ].
2.2. Streptococci Streptococci are the bacteria which have most frequently been studied in BS since they are the prominent bacteria of oral flora and dental infections. Their presence seems to precede disease exacerbations as noted above [42]. In one study, biopsies of oral aphtae showed components of Strep. sanguis and an increased proportion of Strep. saffguis and especially of uncommon serotypes of Strep [52]. Recently it was demonstrated that these uncommon strains of Strep. sanguis were not different from Strep. oralis in their biochemical and serological properties [53]. Serum antibody titers to Strep. sanguis strains 113-20, 114-23 and 118-1 were higher in BS patients than in healthy controls [54]. Oral streptococci that penetrate the oral mucosa by their own enzymes such as Ig A1 protease and neuraminidase are thought to contribute to the development of hypersensitivity to streptococci among the BS patients [55]. In another study, BS symptoms were induced by streptococcal antigen skin test [51]. Delayed skin reactions to whole cells and cell walls of certain strains of streptococci after intradermal injections was observed in BS patients. Also systemic BS symptoms were observed following steptococcus skin test in 15 of 85 BS patients [56]. It was also shown that cutaneous injections of Strep. pyogenes, Strep. viridans, Strep. haemolyticus, and Strep. faecalis caused strongly flared and erythematous pustular reactions, while injections of Proteus vulgaris, Pseudomonas aeruginosa, and E. Coli resulted in mildly flared reactions [51 ]. Furthermore intracutaneous injection of the cell wall of Strep. salivarius resulted in a positive skin reaction and a prick test with the cell wall of Strep. salivarius to the oral mucosa resulted in oral aphtous ulceration like lesions [51 ]. T cell responses were studied to explain the hypersensitivity to streptococci observed among BS patients. Peripheral ~,8+ CD8+ T cells of BS patients showed a significantly proliferative response to Strep. sanguis strain KTH-1 [57]. T cells of BS patients stimulated with KTH-1 antigens produced greater amounts of IL-6 and both KTH-1 antigens and E. Coli derived antigens cause increased IFN- 7 production by T cells of BS patients [58]. T cells from BS patients produced IFN-~, when stimulated
631
with staphylococcal superantigens SEB and SEC1 even at very low concentrations, that do not stimulate T-cells of healthy or diseased controls [59]. The same study showed that T cells of BS patients did not show overrepresentation of VI35 and VI312 specificities which are preferentially stimulated by SEB and SEC1. Thus the authors suggest that the hypersensitivity of T cells from BS patients might be due to intrinsic abnormalities in their T cells. 2.3. Other Bacteria
Due to a proposed similarity of clinical signs between BS and certain enteric and sexually transmitted diseases, antibodies against Yersinia, Campylobacter, Salmonella and Chlamydia were studied and an increase was not demonstrated [60]. Infections caused by other bacteria such as systemic nocardiosis [61, 62] and Pneumocystis carinii pneumonia [63] have been reported. It is most likely these represented opportunistic infections.
3. HEAT SHOCK PROTEINS
Lehner and collegues suggested that, since different species of streptococci seemed to play a role in the etiopathogenesis of BS, a common antigen such as a stress protein might be involved in pathobgenesis [64]. The first study investigating the role of HSP in BS was reported in 1991 [64]. A significant increase of Ig A antibodies to mycobacterial 65 k-DA HSP in the serum of BS patients was shown. Antibodies against Strep. sanguis and pyogenes were detected at 65 k-DA bands, similar to responses against m-HSP 65 of oral mucosal extracts. Direskeneli observed increased levels of IgA and IgG antibodies to 111-125, 154-172 and 311-325 m-HSP peptides and human homologus HSP 136-150 and 336-351 peptides in BS patients [65]. Four peptides (111-125, 154--172, 219-233 and 311-325) of mycobacterial HSP were shown to stimulate significant lymphoproliferative responses in BS patients. When corresponding human HSP peptides were compared, similar or higher lymphoproliferative responses were observed [66]. Hasan and colleagues showed that "f8 T lymphocytes preferentially responded to these peptides, especially in clinically active patients. He suggested that since
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HSP peptides had a high specificity for BS, their assay could be used in even diagnosing BS [67]. Administration of these mycobacterial and homologus human HSP T cell peptide determinants to Lewis rats induced an uveitis [68]. Furthermore the rats which developed uveitis following immunisation with HSP peptides showed significantly higher serum IgG and IgA levels to specific peptides when compared to the rats without uveitis [69]. Local responses to HSP were also studied. Upregulation of HSP expression in mucocutaneous lesion biopsies of BS patients and increased 78 T cell counts were demonstrated [70]. Examination of the cerebrospinal fluid of BS patients with cerebral parenchymal involvement showed significantly higher anti-m-HSP 65 IgG, IgM or IgA responses when compared to patients with multiple sclerosis or non-inflammatory CNS disorders [71 ]. Due to the significant homology between mammalian and microbial HSP's it is suggested that recurrent exposure to HSP may cause bacterial HSP responsive T cells to stimulate autoreactive T cells by cross-reactivity mechanisms. In turn these T cells might produce Th-1 like proinflammatory and/ or inflammatory cytokines leading to tissue injury by delayed-type hypersensitivity reaction and macrophage and neutrophil activation. This molecular mimicry mechanism may be responsible for triggering of BS in genetically susceptible individuals.
4. CONCLUSIONS The evidence for infectious agents playing a role in the pathogenesis of BS remains, at best, speculative. An interesting possibility is that more than one pathological mechanism is involved [72] and this is why perhaps the designation "syndrome" is more approriate than the "disease". More clinical and basic research is obviously needed. Finally, a question, rightfully often asked by the patients is whether BS is infective. Again there are no formal studies addressing this issue. On the other hand it might be worth mentioning here that during our 25 years of experience with close to 6000 patients, we have only seen one husband and wife pair, concommitant for BS.
ACKNOWLEDGEMENT This work has partially been sponsored by Turkish A c a d e m y of Sciences.
13.
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685-90. 54. Yokota K, Hayashi S, Furjii N, Yoshikawa K, Kotake S, Isogai E, Ohno S, Araki Y, Oguma K. Antibody response to oral streptococci in Behqets disease. Microbiol Immunol 1992;36(8):815-22. 55. Yokota K, Oguma K. IgA protease produced by Streptococcus sanguis and antibody production against IgA protease in patients with Behqet's disease. Microbiol Immunol 1997;41:925-31. 56. The Beht;et's disease research committee of Japan. Skin hypersensitivity to streptococcal antigens and the induction of systemic symptoms by the antigens in Behqet's disease- a multicenter study. J Rheumatol 1989;16(4):506-11. 57. Mochizuki N, Suzuki N, Takeno M, Nagafuchi H, Harada T, Kaneoka H. Fine antigen specificity of human gamma delta T cell lines (V gamma 9+) established by repetitive stimulation with a serotype (KTH1) o f a gram-positive bacterium, Streptococcus sanguis. Eur J Immunol 1994;24:1536-43. 58. Hirohata S, Oka H, Mizushima Y. Streptococcal related antigens stimulate production of IL-6 and interferongamma by T cells from patients with Behqet's disease. Cell Immunol 1992; 140:410-19. 59. Hirohata S, Hashimoto T. Abnormal T cell responses to bacterial superantigens in Behqet's disease. Clin Exp Immunol 1998;112:17-24. 60. Toivanen A, Lahesmaa-Rantala R, Meurman O, Sappinen O, Kosunen T, Yazici H, Yurdakul S, Ozbakir F, Muftuoglu A. Antibodies against Yersinia, Campylobacter, Salmonella, and Chlamydia in patients with Behqet's disease. Arthritis Rheum 1987;30(11): 1315-7. 61. Korkmaz C, Aydinli A, Erol N, Yildirim N, Akgtun Y, Inci R, Boiron P. Widespread nocardiosis in two patients with Behqet's disease. Clin Exp Rheumatol 2001;19(4):459-62. 62. Pamuk GE, Pamuk ON, Tabak F, Mert A, Ozturk R, Aktuglu Y. Systemic Nocardia infection in a patient with Behqet's disease. Rheumatology (Oxf) 2001 ;40(5): 597-9. 63. Raychaudhuri SP, Siu S. Pneumocystis carinii pneumonia in patients receiving immunosuppressive drugs for
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infections and Immune Thrombocytopenic Purpura Alexander J. Chou and James Bussel
Weill Medical College of Cornell University, New York, NY, USA
1. INTRODUCTION Immune thrombocytopenic purpura (ITP) is an idiopathic disease defined by a low platelet count in the absence of other causes of thrombocytopenia such as malignancy, disseminated intravascular coagulation, or medication-induced thrombocytopenia. It is an (auto) immune-mediated disorder that affects both adults and children although their clinical manifestations can be very different. In children, greater than 50% of the cases present with bleeding manifestations during or 1-4 weeks after a viral illness. Fortunately, in approximately 80% of pediatric cases, the disease resolves within 6 months. For the other 20% of children, their clinical course is nonremitting, mirroring that seen in adult ITP [ 1, 2]. Adult ITP, in contrast to pediatric ITP, is a more chronic, persistent disease of varying severity which often does not resolve despite aggressive treatment. The disease usually has a slow onset (months to years) as evidenced by the fact that in a recent study, 50% of patients were diagnosed on a routine count, not on one obtained because of signs or symptoms of bleeding [3]. The average platelet count in these patients is 50,000-75,000/ktL but in most cases the platelet count then falls slowly [1]. The signs and symptoms of the disease usually depend upon the nadir platelet count and poorly characterized issues connected with platelet vascular function. Surprisingly, even occasional patients with platelet counts as low as 5,000/l.tL, can be relatively asymptomatic (although some of these cases turn out to be those in which the platelet count has been underestimated by standard counting techniques) [1]. Diagnosis of ITP relies upon clinical criteria. Attempts at using "more specific" tests such
as anti-platelet antibodies have not yet been consistently fruitful. Treatment "up front" consists of corticosteroids, intravenous immunoglobulin (IVIG), and/or anti-D, followed as needed by splenectomy, and immunosuppressants such as azathioprine, mycophenolate mofetil, cyclophosphamide, and, more recently, rituximab [1, 2, 4]. The clinical course of the disease as well as its treatment have been the subject of many studies. However, the exact pathophysiologic mechanisms involved in ITP still remain elusive. As indicated above in children ITP is often post-viral. Kahane in 1981 showed that sera from patients with serological diagnoses of CMV, varicella, HSV, and EBV showed anti-platelet antibodies while those from normal healthy controls did not [5]. One fascinating feature is that any one of a large number of viruses have been known to be followed by ITP rather than this phenomenon being restricted to a small number of viruses. This illustrates the close association between viral infections and ITP. The current discussion will focus on the role of infections in the pathophysiology of ITP.
2. MECHANISMS OF DISEASE 2.1. Antibody Formation What we believe happens in ITP is that autoantibodies present in the peripheral circulation cause accelerated destruction of opsonized platelets [6, 7]. A "humoral" causative factor was first described by William Harrington in the 1950s. He infused himself, and later other healthy volunteers, with sera from a patient with active ITP and was able to repro-
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Table 1.
Proposed Mechanisms of Disease Infectious Agent
Strength of link with ITP'
Molecular Mimicry
Direct Infection of Megakaryocyte
HIV VZV EBV2 CMV
+++ + + +
+ + ? ?
+ + ? +
H. pylori
++
+
c
Hepatitis C
++
+
aBased primarily on eradication / suppression of virus increasing the platelet counts. blmpact of EBV stimulation of B cells in the pathophysiology of EBV associated ITP is unknown. CSo far, there is no evidence of infection playing a role in the pathophysiology.
duce not only the petechiae and purpura characteristic of acute ITP, but more significantly the profound (fortunately transient) thrombocytopenia [8]. IgG was subsequently identified as the "humoral factor" important in the manifestations of ITP in a series of studies and confirmed by the finding of transient neonatal thrombocytopenia, a result of transplacental passage of IgG. The platelet glycoproteins containing antigens to which autoantibodies have been demonstrated include: GPIIb/IIIa, GPIb/IX, GPIb, GPIIIa, GPIa/lla, GPIV, GPV, the HPA system most of which are on GPIIB/IIIA, and even HLA-DR antigens [6, 7, 9, 10]. After a triggering event, such as a viral or bacterial infection, antibodies are made as part of the "normal" immune response. In the case of ITP, some of those antibodies attach themselves to the circulating platelets. Cells of the reticuloendothelial system (RES), especially the spleen and the liver, then clear the antibody-coated platelets from the circulation, causing the characteristic thrombocytopenia and associated clinical hemorrhagic symptoms [1]. In addition, complement-mediated and complement-independent lysis of the antibody coated platelets may also contribute to the immune thrombocytopenia seen in ITP [1, 11, 12]. Immune complexes formed by platelet associated antibodies which bind to platelets also aid in the peripheral destruction of platelets v i a the complement cascade. The question then becomes why the body forms these auto-antibodies in the first place. This topic will be discussed in greater detail in a number of
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other chapters in this volume. Molecular mimicry (Fig. 1) occurs when host antigens resemble those on viruses. The prototype of molecular mimicry causing disease is rheumatic heart disease, where cardiac epitopes / antigens are recognized by the immune system in the course of a streptococcal infection. During the course of an infection, the immune system vigorously produces antibodies against certain microbial or viral determinants. These antibodies may also recognize host antigens [13]. In the case of ITP, those antibodies are usually made against platelet surface antigens such as GPIIb/IIIa. (This has already been demonstrated in cases of HIV-related ITP where anti-HIV antibodies cross-react with platelet membrane glycoproteins GPIIIa; we will go into further detail regarding the pathophysiology of HIV-related ITP later on in the chapter.) Not only are antibodies made against platelets, there have also been studies which show that antibodies are made against the megakaryocytes themselves [ 14]. It stands to reason that if a chronic infection or latent virus can cause continued stimulation of the immune system against a part of itself for which there is molecular mimicry, then this infection could be responsible for sustaining the activity of chronic ITP. It is unclear at this time what the role of infections is in the majority of cases. It has been difficult to directly link particular infectious agents to the pathophysiology of primary ITP but until recently H. p y l o r i driven cases of ITP would have been considered "primary" or "idiopathic". While some
Figure 1. MolecularMimicry. During the course of an infection, immune cells produce antibodies against certain viral antigens. However, these same antibodies can be cross-reactive to antigens found on platelets and megakaryocytes, thus mediating their destruction.
viruses such as HTLV-1 have not been associated with ITP, subsequent sections of this chapter will touch upon specific infections (HIV, VZV, EBV, CMV, H. pylori, and hepatitis C) and the evidence that links them to the pathophysiology of ITP. The chronicity of ITP can also be explained in part by the idea of epitope spreading (Fig. 2). After the initial insult, antibody-bound platelets are destroyed piecemeal by the RES or by complement. In the process, self-antigens become exposed which are novel to the host. Antigen presenting cells (APCs) then pick up these "foreign" antigens and present them to T cells. Thus new sets of autoantibodies may continue to be produced against other parts of the platelet membrane and even against cytoplasmic antigens. A vicious cycle is propagated where new antigens are exposed and the immune system hyperstimulated [13]. This is believed to be what causes the detection of platelet antibodies to be non-specific and unhelpful in the diagnosis of ITP.
This mechanism allows for epitope spreading to occur even in cases which are not initially of immunologic origin. Multiple antibodies are detected in cases of acute and chronic ITP. However, it is often unclear which of the many platelet antibodies is the one originally responsible for the disease or whether the additional antibodies contribute substantially to its persistence. In addition, new antigens can be made through this mechanism of epitope spreading. As the RES clears the antibody-coated platelets, different parts of the platelet are endocytosed and re-presented on APCs. These proteins may become novel / cryptic proteins towards which the body can then make new antibodies [13]. Unfortunately, these antibodies cross-react enough with normal platelet antigens that they attach to normal platelets and cause their destruction through the mechanisms discussed previously. A related hypothesis, postulated by Semple in
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Figure 2. Epitope Spreading. After the initial insult, antibody-bound platelets are destroyed piecemeal by the reticuloendothelial system or by complement. In the process, self-antigens become exposed which are novel to the host, and against which the immune system can now make new antibodies. In addition, new antibodies are made as new epitopes are produced by the piecemeal destruction of platelets and their surface antigens. Toronto as well as others, is that of determinant spreading in the pathophysiology of ITE During the development of the immune system, a certain level of self-determinants is necessary for negative selection of autoreactive cells to occur in the thymus [4, 13]. However if these antigens are not present at a certain ttweshold level, these auto-reactive immune cells are not deleted and may be released into the peripheral circulation. The self-reactive cells remain dormant until stimulated by exposure to the self antigen, i.e., during the process of platelet destruction, or by exposure to inflammatory cytokines which activate the cells. In addition, the self-determinants recognized by these immature auto-reactive T-cells may resemble those found on infecting viruses or other microbes. Then during the course of an infection, the inflammatory cascade produced may lead to activation of these auto-reactive cells [4]. With their proliferation and increased activity, platelets are destroyed. Recent work by Wardemann et al lends support for this hypothesis. These investigators found that 55-75% of all antibodies expressed by early immature B cells are self-reactive. However, most of these self-reactive antibodies were removed at
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two discrete checkpoints in B cell development: the immature B cell stage in the bone marrow and the transition between new emigrant and mature B cells in the periphery [ 15].
2.2. The Role of Cytokines Cytokines which regulate the activity of the immune system have also been implicated in the pathophysiology of ITP. Several studies have suggested that Thl factors (IL-2, IFN-7) are up-regulated in ITP while Th2 factors (IL-10) are down-regulated [2, 16-18]. Thl cells are proinflammatory and secrete cytokines such as IL-2, and other pro-inflammatory factors. Th2 cells instead secrete cytokines which suppress cell-mediated immune responses (i.e., IL4). A Th0 response is thought to be generated by cells more immature than those of the T h l or the Th2 type, and thus many of the cytokines seen in Th 1 or Th2 responses are also present in a Th0 type of response. An imbalance in the cytokine milieu, or in T cell subpopulations, in the host may favor Thl responses over Th2 responses. This then leads the individual to become more proinflammatory and
thus more prone to persistent, autoimmune phenomenon such as ITP. A study done by Semple et al showed that a majority of the patients with chronic ITP had elevated serum levels of IL-2, ILl0, and IFN-gamma, consistent with activation of Thl responses. None of the patients had detectable levels of IL-4 or IL-6 (Th2-associated cytok_ines) [16]. Nugent also confirmed the lack of IL-4 and postulated that it, and possibly other Th2 cytokines, could be used as treatment [ 19]. In addition, studies in children by Mouzaki et al showed that patients with acute ITP presented with a Thl, Th0/Thl, or Th0 in vivo cytokine gene expression. However, those patients who maintained stable remissions tended to be associated with a Th2 gene expression pattern. Also a Th0 or Th2 pattern after IVIG treatment was associated with good prognosis [20]. In addition to these cytokines, other substances released in the inflammatory state are present in cases of ITP. Plasma levels of both macrophage-colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) are increased in ITP. Products released from activated or destroyed platelets such as platelet-activating factor (PAF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-[~) can serve as chemokines for many immune cells [ 17]. The presence of these powerful immunemodulating substances can create a local cytokine milieu that is conducive for further immune mediated destruction of platelets. Many years ago, it was hypothesized that platelets were the original white blood cells. By serving as an immunologically inert but sticky substance in the blood, platelets were thought to clear substances in the blood such as bacteria [21 ]. Nugent in Orange County has taken this one step further by studying the role of platelets as APCs [2]. Activated platelets have been shown to express CD40L and CD69, both involved in the activation of T and B cells. There are several pathways described in which IL-1 is stimulated. In proximity to B cells, platelets are able to stimulate them to produce pro-inflammatory cytokines such as IL-1 [22]. In the presence of PHA-stimulated mononuclear cells, platelets seem to drive the production of IL-1 by B cells and monocytes [2]. It can therefore be theorized that following a viral infection, platelets can stimulate autoantibody production by increasing the produc-
tion of IL-1. However and if this happens in vivo, it represents another pathway by which a viral infection could stimulate production of autoantibodies. 2.3. Direct Effects on Megakaryocytes
Not only can autoantibodies affect platelet number (and function), autoantibodies have also been shown to have a direct effect on megakaryocytes in the pathophysiology of ITP. Alimardani et al showed that antibodies to GPlb can potentially inhibit platelet release from megakaryocytes by enhancing the interaction between newly formed platelets and their parent megakaryocytes [23]. Chang et al assessed the effect of plasma from ITP patients as well as purified ITP monoclonal autoantibodies on the function of megakaryocytes in vitro. What they found was that both sera isolated from patients with acute ITP and monoclonal autoantibodies specific to GPIIb heavy chain as well as a neoantigen on GP Ilia are able to inhibit megakaryocytopoiesis in vitro. These effects did not seem to be mediated by enhanced apoptosis [ 14]. However the exact mechanism through which this occurs is not yet known. Recent studies, published primarily in abstract form, have sUggested that there is a resolution of the quandary of the marrow in ITP: how can there be increased megakaryocytes yet decreased platelet production. In this case, apoptosis may be important. Especially in chronic ITP, there is a growing body of evidence implicating the role of chronic infections in the suppression of platelet production which could explain the relative ineffectiveness of therapy targeted only at interrupting the peripheral destruction of platelets. These events may occur similarly to ones described above with involvement of molecular mimicry, epitope spreading and direct infection of megakaryocytes. The underlying immune defect may in fact result in decreased platelet production as a consequence of viral infection of the megakaryocytes themselves. This is further evidenced by the fact that treating the infection often improves the thrombocytopenia.
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3. SPECIFIC INFECTIONS 3.1. Human Immunodeficiency Virus
HIV-related thrombocytopenic purpura is a welldescribed phenomenon occurring at any one time in 10% of HIV positive individuals and about 30% of AIDS patients. The role of HIV in the genesis of thrombocytopenia in these patients is made clearer by the effect of treatment of the virus. In 1988, Hymes et al observed that seven HIV-positive patients with initial platelet counts of 100,000-150,000/ktL who received zidovudine (AZT) had "positive responses", i.e., increases in platelet count to greater than 150,000/ktL with concomitant decreases in hematocrit, within two weeks of starting the medication [24]. Since then several studies have been performed which confirmed these findings [25-27]. More recently combination highly active anti-retroviral therapy (HAART) has also been shown in small series to have similar effects on patients with HIV-associated thrombocytopenia [28]. While it is assumed that reduction of the viral load in the peripheral circulation is "required" for a positive effect of antiviral therapy on the platelet count, this has not been formally demonstrated in a series of patients. Furthermore whether any treatment combination that suppresses the HIV virus will be effective in increasing the platelet count or specific agents, such as AZT, are particularly effective remains unknown. The thrombocytopenia in HIV is largely the result of two mechanisms: 1) peripheral destruction of autoantibody-coated platelets via complement mediated and non-complement mediated mechanisms; 2) decreased platelet production caused: a) directly by HIV infection of the megakaryocytes; b) indirectly by secreted cytokines and/or chemokines; and/or c) by the same mechanisms which result in platelet destruction. These mechanisms seem to coexist in patients to a greater or lesser extent in all HIV-infected patients. Although the pathogenesis of the "autoantibodies", proposed to be binding to platelets or immune complexes (consisting of antibodies to HIV and anti-idiotypic antibodies to these antibodies), remains uncertain, considerable work is ongoing on this topic. Viral infection of the megakaryocyte can lead to expression of viral proteins on the meg-
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akaryocyte surface as well as their released platelets. In addition, non-specific adsorption of viral particles on to the platelet surface can occur due to the "stickiness" of the platelets themselves. There may also be cross-reactivity between platelet proteins and HIV proteins, i.e. molecular mimicry. Immune complexes may also form consisting of anti-HIV and anti-anti-HIV antibodies (the so-called anti-idiotypic antibodies). These immune complexes bind to platelets due to viral antigens on the platelet surface, or as a result of "locking in" by FcR_II binding. As a result of these and perhaps other mechanisms, anti-HIV antibodies and/or immune complexes are deposited onto the platelet surface. These antibody coated platelets are destroyed through clearance by the RES, classical complement mediated lysis, and recently, evidence of non-complement mediated lysis. Nardi et al showed in 2001 that autoantibodies can induce a complement-independent lysis of antibody coated platelets. Using in vitro and in vivo studies in mice, he demonstrated that purified anti-GPIIa induced platelets fragmentation via generation of H202 without the help of complement proteins [ 11 ]. Studies have shown that HIV does in fact infect megakaryocytes, particularly early in their development, i.e., at the CFU-Mk stage. Lymphotropic strains of HIV utilize the CXCR4 receptor as a coreceptor, in addition to CD4, for infection. CXCR4 appears to be expressed and functional in the megakaryocytic lineage [28]. Viral particles as well as viral transcripts have been demonstrated within the megakaryocytes themselves [29]. Infected megakaryocytes exhibit typical morphologic changes seen in HIV infection (denuded megakaryocytes with absence of cytoplasm, ballooning of the peripheral zones) [30]. Increased apoptosis has also been seen in GPIIb/IIIa positive cells from the bone marrow of HIV-infected patients [31]. As seen in other viral infections, HIV infected megakaryocytes may have HIV-encoded proteins on their surface. As a result, antibody and/or immune complex deposition on megakaryocytes can lead to immune mediated destruction of the megakaryocyte, or interference of the normal effective release of platelets from the megakaryocyte. These findings all suggest that megakaryocytes and their precursors are infected by HIV; linking this infection to the thrombocytopenia however is inferential based on the high prevalence
of thrombocytopenia in HIV-infected patients and the platelet response to anti-retroviral therapy.
3.2. VariceHa Zoster Virus (VZV) Varicella Zoster Virus is a common infection that has frequently been associated with ITP although the exact prevalence of VZV-associated ITP is unknown. The ITP associated with VZV typically is post-viral. Occasionally the ITP begins before the VZV has completely cleared or the VZV occurs in a patient with pre-existing ITP. Co-existence of the infection with ITP can be very serious because the VZV can become hemorrhagic. Several mechanisms have been put forth regarding VZV in particular. Wright et al looked at platelet reactive antibodies in two children with acute ITP and varicella infection. They found that antibodies reactive to VZV were cross-reactive to certain platelet specific antigens. In this study however the platelet antigens were not identified [32]. Other investigators have shown VZV cross-reactive antibodies against platelet antigen GPIV and GPV [33]. It has also been shown that VZV can infect megakaryocytes, as in HIV. In cases of hemorrhagic varicella, megakaryocytes may undergo nuclear degeneration and vacuolization [34]. This may contribute to the severe thrombocytopenia and the blackish hemorrhagic lesions by decreasing the production of platelets at a time when there is ongoing consumption.
3.3. Epstein-Barr Virus (EBV) Epstein-Barr Virus is also a virus that has been specifically linked to ITP. In several small studies, VZV and EBV together account for less than 10% of the cases of virus-associated ITP. A retrospective study from Taiwan showed that 32% of children prese~ing with ITP were also acutely infected with EBV. However, EBV is known to be endemic in Taiwan. The authors concluded that there is indeed a close association between acute EBV and ITP but the role of EBV in the pathogenesis of ITP needed further elucidation [35]. One possibility is that the mechanisms by which other viruses have been shown to cause "post-viral" ITP may very well be the same in EBV associated ITP. Alternatively EBV has certain unusual properties and can be associated with ITP
by other mechanisms. First it can "immortalize" B cells (at least in vitro) and thereby one can speculate that it might lead to "perpetual" anti-platelet antibody synthesis [36]. This could prevent the acute ITP from resolving in the way in which it normally might. Second, it is associated with the small percentage of acute ITP in which there is hepatosplenomegaly [37]. This may represent an activation of the immune system quantitatively different from that seen with other viruses. Furthermore it may lead to greater efficiency of destruction of opsonized platelets simply by virtue of increased phagocytic capacity and a larger percentage of blood flow going to an enlarged spleen. Data is clear, as summarized above, regarding the association of ITP and EBV infection. Data however is not clear, despite the mechanisms discussed above, regarding an increased chronicity of ITP in children who acquire their ITP as a result of EBV.
3.4. Cytomegalovirus (CMV) CMV has been shown to be lymphotropic and can infect as well as activate T-cells. Once inside the cell, it has the capacity to alter the normal physiology of the cell by interactions with normal host proteins. For example, a CMV-encoded protein binds to p53 and thus may alter the normal function of the p53 protein [38]. Here one could speculate that this interaction might interfere with apoptosis of autoreactive cells. Other CMV-encoded proteins have been shown to alter antigen processing and presentation in APCs. Thus after infection with CMV, host antigens that are normally protected from the immune system can become immunogenic and ones that might have "escaped" recognition will become more notorious. For example, CD13 is a cell surface molecule expressed on lymphocytes, granulocytes, monocytes, and is important in the infectious capacity of CMV. CMV can only infect cells expressing CD13; cells resistant to CMV become susceptible once transfected with the gene for CD13. During the formation of the CMV viral particle, CD 13 is selectively absorbed onto the viral envelope [38]. When a host antigen like CD13 is present on a viral particle, it then can become immunogenic as the viral particles are processed and presented by APCs, and T-cell immune responses are activated. As reported
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in Blood by Crapnell et al in 2000, CMV can infect megakaryocytes in vitro [39]. Like in the CD13 story, one can postulate that CMV infection does something similar with a megakaryocyte molecule, making it immunogenic. A study of more than 100 patients with ITP failed to identify, by urine culture, an increased carriage of CMV in these patients [unpublished data, Levy and Bussel]. 3.5. Helicobacter pylori
Helicobacterpylori is a common gram-positive bacterium known to be pathogenic in cases of gastric and duodenal ulcers. It has also been well-described to play an important role in the pathogenesis of B cell lymphomas, so-called "MALT lymphomas". Certain of these otherwise typical lymphomas have been able to be treated solely by H Pylori eradication with the lymphoma regressing with this treatment alone [40, 41]. Recently, H. pylori has been implicated in autoimmune diseases such as megaloblastic anemia, Sj/Sgren's syndrome, and ITP [42, 43]. However, the role of H. pylori in ITP has been widely studied and surprisingly controversial. Initial studies by Gasbarrini et al in 1998 showed that 8 of 8 patients with ITP and documented H. pylori infection, evaluation of which was initiated because of GI symptoms possibly related to steroid use for their ITP, increased their platelet counts after eradication of H. pylori infection [44]. Since then several series, primarily from Japan and Italy, reported similar findings, i.e., an unexpectedly high incidence of H. pylori in patients with ITP (although not well controlled for age), and also improvement in the platelet count with eradication of the H. pylori infection [45, 46]. However, recent studies from Spain, France, as well as the United States have contradicted these earlier studies [42, 47, 48]. Jarque et al conducted a prospective study where 56 adults with ITP were enrolled on the study. Forty patients were diagnosed with H. pylori infection by [~3C]urea breath test and were treated with a combination of omeprazole, clarithromycin and amoxicillin for 7-10 days. Resolution of the infection was defined as a negative [~3C]-urea breath test after 2 months. Platelet counts were assessed at 3 months after H. pylori treatment. Of the 32 assessable patients, eradication of H. pylori was achieved in 23 (72%).
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Of those only 3 had a platelet increase of greater than 30,000/~tL. When compared to the non-treated group, the response rate was not significantly different [47]. Michel et al found similar results. In 74 patients with chronic ITP and a platelet count of below 60,000/l.tL, 22% (sixteen) were found to have H. pylori on a urea breath test. The bacterium was eradicated in fourteen of the fifteen treated patients. After three months, only one patient had a platelet count greater than 50,000/ktL and a doubling of the initial count [48]. The mechanism of the putative link between H. pylori and ITP remains poorly understood. Recent studies have implied that H. pylori proteins may be involved in the relationship of the von Willebrand multimers and GPIB. This would imply a consumptive thrombocytopenia that could be ameliorated [49]. Other studies have suggested molecular mimicry between certain strains of H. pylori and platelets. Depending upon the variability of the mechanisms, the strains of H. pylori, and the "susceptibility" of individual patients and patient populations, this presumably would explain the conflict among published studies.
3.6. Hepatitis C Hepatitis C is the least well-defined of the chronic infections which are related to thrombocytopenia. There may be a higher than expected prevalence of ITP in Hepatitis C Virus (HCV) infected individuals but this remains unsettled since the US average infection rate is approximately 1.5% and certain, especially inner city populations, may have an even higher rate [50]. Hence if a population of ITP patients had a 3% infection rate of hepatitis C, this might not reflect an increased co-existence. Hepatitis C has been increasingly shown to cause multiple autoimmune phenomena. These include ITP, mixed cryoglobulinemia, lichen planus, polyarteritis nodosa, and the development of multiple autoantibodies (i.e., anti-nuclear antibodies, antismooth muscle antibodies, and anti-thyroid antibodies) [51, 52]. These clear settings of autoimmunity strengthen the proposed tie of ITP and hepatitis C. Thrombocytopenia in hepatitis C infection has multiple causes including hepatic and splenic sequestration as a result of worsening liver damage and "hypersplenism". Treatment of HCV with inter-
feron-alpha has also been shown to induce the production of auto-antibodies in patients and, possibly by its anti-proliferative effects, to cause thrombocytopenia [53]. Finally, while poorly demonstrated, it is hypothesized that with sufficiently severe liver disease such that the synthetic capacity of the liver is impaired, thrombopoietin levels may be low enough to result in thrombocytopenia [54]. From the point of view of the infection itself, the hepatitis C virus has been shown to infect lymphocytes and cause non-specific B-cell proliferation [55, 56]. This in turn could lead to immune dysregulation and the formation of multiple auto-antibodies. Pockros in 2002 reported that patients with HCV have detectable anti-platelet antibodies, specifically to GPIIb/IIIa, and GPIb/IX. In this series, treatment with some form of immunosuppression led to resolution or improvement in the ITP [51 ]. Interestingly, HCV-RNA has been isolated from washed platelets of patients with HCV, and HCV has been shown to be able to replicate within megakaryocytes [51]. Primary treatment of HCV-ITP remains the optimal strategy for management of the thrombocytopenia. However, no compelling data exists to support this approach, which may not be valid if the infection is coincidental instead of etiologic. Furthermore, the anti-proliferative effects of interferon have resulted in serious hemorrhage in at least two patients by causing an immediate lowering of the platelet count [57]. Therefore we have pursued the philosophy of treating the HCV (with interferon-cz and ribavirin) but giving another treatment, usually anti-D, several days prior to beginning the antihepatitis C combination. This typically elevates the platelet count sufficiently that the combination has time to reduce the hepatitis C viral load and begin to ameliorate the platelet count before the effect of the anti-D disappears. The ability to eventually improve the platelet count with treatment of the hepatitis C is consistent with the suspected pathogenic role of the virus in perpetuating the autoimmune disease.
4. FUTURE DIRECTIONS / CLOSING THOUGHTS Our understanding of the pathophysiology of ITP has advanced dramatically since the first in vivo experiments by Dr. Harfington more than 50 years
ago. Yet much still needs to be learned about this interestingly diverse disease. Though we know that auto-antibodies are made against platelet antigens, it is still unclear as to the initial trigger for antibody production. Other issues that warrant further investigation include the mechanisms through which viruses initiate or maintain the disease process in ITP; this would have to take into account individual susceptibility factors (i.e., genes). Other areas to be further elucidated include the role of inflammatory and anti-inflammatory cytokines, and mechanisms through which autoreactive immune cells escape negative selection by the developing immune system. The interaction between the immune system and infectious agents in the pathophysiology of ITP will remain a critical point of investigation. Further understanding of the pathophysiology underlying ITP should ultimately lead to more specific diagnostic tests as well as novel treatments which may allow improved management of at least a fraction of cases.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Inflammatory Bowel Disease Joel V. Weinstock
University of Iowa Hospitals and Clinics, Division of Gastroenterology, Iowa City, IA, USA
1. INTRODUCTION Ulcerative colitis (UC) and Crohn's disease (CD) are two medical conditions collectively called inflammatory bowel disease (IBD). Although the line between UC and CD can be blurry, they are classified as separate diseases because each has distinguishing clinical and pathological features (Table 1). UC and CD occur at about equal frequency without gender bias. Symptoms of disease usually appear in the second or third decade of life. With UC, there is a second peak of disease onset much later in life. Most patients with UC or CD spontaneously cycle between periods of disease activity and remission. Medical reports from the 18th and 19th century describe patients with chronic intestinal disorders suggestive of UC and CD implying that these conditions have been with us for some time. CD received wide recognition as a disorder distinct from enteric tuberculosis because of a series of publications in the early 20th Century [1-3]. CD carries the name of a physician who was one of the first to help characterize the disease [3]. It is likely that neither UC nor CD are distinct diseases resulting from a single cause. Rather, they are several conditions with shared clinical and histological features. The pathology perhaps reflects an individual's propensity to immune dysregulation due to the constellation of several distinct genetic traits and is triggered by various poorly defined environmental factors. The objective of this chapter is to review our changing concepts related to the immunopathology and cause of IBD.
Table 1. Clinical and pathological features helping to distinguish UC from CD UC
CD
Macroscopicfeatures Rectal involvement Continuous involvement Skip regions (excluding cecal patch) Terminal ileal ulceration Stricturing Aphthous ulcerations Fistulization and/or anal fissuring Longitudinal ulcerations
Microscopicfeatures Crytitis, crypt branching and mucin depletion Uninterrupted disease (not patchy) Paneth cell hyperplasia Patchy involvement Transmural inflammation Granulomas Neuronal hypertrophy
2. PATHOLOGICAL CHARACTERISTICS OF DISEASE 2.1. UC
UC presents as chronic diarrhea frequently containing blood. Patients often experience fatigue, lower abdominal cramping and sore joints. The condition must be distinguished from common infectious dis-
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Figure 1. Colonoscopic appearance of ulcerative colitis showing mild, moderate or severe activity. A) Normal colonic mucosa with the usual visible blood vessels. B) Mild colitis. The normal mucosal blood vessels are no longer visible because of edema. C) Moderately active colitis with edema, surface exudate and bleeding when touched with the colonoscope. D) Severe colitis. Widespread ulceration (white areas) with only small islands of inflamed mucosal remaining.
eases and from chronic diarrhea due to dietary factors, stress or gastrointestinal motility disturbances. Other causes of intestinal inflammation like celiac disease, ischemic colitis, diverticulitis, radiation colitis, collagenous colitis, lymphocytic colitis and graft versus host disease are easily differentiated from IBD. In UC, there is mucosal inflammation that always involves the rectum. The condition is called "ulcerative proctitis" when disease is limited to this region of the colon. About half the patients will never have disease beyond the distal half of the colon except perhaps for a patch of inflammation in the cecum ("cecal patch"). The disease can extend proximally to involve the entire colon and even the distal ileum ("backwash ileitis"). This ileitis, however, is different from CD in that the ileum does not ulcerate or stricture. While the name of the condition implies
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colonic mucosal ulceration, this feature is only evident in severe disease. The inflammation induces mucosal edema, hyperemia and bleeding. With more severe disease, there is exudate and ulceration (Fig. 1). Colonic ulcers can coalesce leaving only small islands of inflamed mucosa. These islands can hypertrophy after the ulcers heal forming "pseudopolyps" usually no more than 1 cm in size. UC rarely causes intestinal fibrosis, fistulization or obstruction. Unless there is fulminant disease, the inflammation remains limited to the mucosa and submucosa of the intestinal wall. There are increased numbers of plasma cells, lymphocytes, macrophages and eosinophils that infiltrate the gut. Neutrophils can invade the intestinal epithelium forming "crypt abscess". The epithelial lining undergoes changes particularly with more chronic disease. There is
atrophy and distortion of epithelial crypt morphology ("crypt branching"), loss of goblet cells and Paneth cell hypertrophy. In some patients, the mucosa undergoes dysplastic changes, which is the precursor to neoplasia (adenocarcinoma). No one feature described above is pathognomonic for UC. The constellation of chronic recurring symptoms, and suggestive clinical and pathological findings presenting in the absence of pathogenic organisms permits the diagnosis. 2.2. CD
The initial symptoms of CD can be indistinguishable from that of UC. However, the early manifestations of CD tend to be more indolent and variable, often delaying the diagnosis for several years. Patients can present with diarrhea and fatigue. They also can exhibit singularly or in combination intermittent crampy abdominal pain, intestinal soreness, anemia, growth retardation, perianal fistulization, anal fissuring, "appendicitis", bowel obstruction, sore joints and/or weight loss. Patients often give a history of recurrent oral aphthous ulcerations. Intestinal inflammation of CD can affect any region of the gut. Most patients (about 70%) have small bowel involvement with a predilection for the terminal ileum. Colonic involvement, which develops in about 50% of CD patients, frequently spares the rectum. A characteristic feature of CD is its propensity to affect several regions of the intestine simultaneously while leaving healthy the segments of bowel that connect areas of disease ("skip lesions"). The inflammation extends deep into the abdominal wall and out to the serosal lining. This can result in intestinal fibrosis, stricture formation and intestinal obstruction. Deep fissuring of the mucosal lining can extend through the bowel wall resulting in intraabdominal and perianal abscesses. Transmural inflammation also can lead to abnormal channels (fistulization) between loops of bowel, the urinary bladder and the skin. These allow the passage of intestinal contents into the other structure. Fistulization is most common in the perianal area, developing in more than 10% of the patients. Stool leaking into the bladder or out the skin can cause serious infection and severe symptomatology. Bowel to bowel (enteroenteric) fistulation contributes to diarrhea
Figure 2. Aphthous ulcers of the colon (arrows). These shallow 1 mm ulcers with erythematous edges are lesions characteristic of early Crohn's disease.
and malabsorption. These various complications of CD can necessitate surgical intervention. Early macroscopic evidence of CD can include aphthous ulceration of the mucosal lining (Fig. 2). These shallow, distinctive 1-2 mm ulcers can coalesce leaving islands of mucosa forming a "cobblestone" appearance. The larger ulcers frequently have a stellate configuration or run linear to the long axis of the intestine (rake ulcers). Colonoscopic and radiographic evidence of strictures, rectal sparing, skip regions, terminal ileal disease and fistulization favor the diagnosis of CD over UC. Radiological demonstration of a long stricturing lesion of the terminal ileum is called the "string sign". The microscopic features of CD include a patchy lymphocytic infiltrate often extending transmurally. Other features can include enhanced numbers of lymphoid aggregates, fissuring, neuronal hypertrophy and granuloma formation. CD is a Thl-type granulomatous disease. Noncaseating granulomas are the histological signature lesion of CD [4]. However, granulomas are found in only about 60% of surgical or biopsy specimens. This may reflect the stage of disease activity or perhaps signify fundamental differences in disease pathogenesis among subgroups of patients. They are seen more frequently in children than adults. Some reports suggest that they cluster near blood vessels [5] and lymphatics [6]. Intestinal granulomas can develop in diseases like intestinal tuberculosis and sarcoidosis. However, in the fight clinical setting,
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granulomas in intestinal biopsies or surgical resections from patients living in modem industrialized societies usually signify CD. The granulomas of CD have one of several morphological appearances [4]. They can develop in any region of the intestinal wall and in mesenteric lymph nodes. These lesions rarely cluster, do not caseate, contain few giant cells and are few in number. This is in contrast to the granulomas of intestinal tuberculosis. The differentiation of CD from enteric tuberculosis can prove problematic in patients riving in less developed countries where intestinal tuberculosis is relatively common. Circumferential, rather than linear mucosal ulceration, a shrunken cecum and other morphological features can signify enteric tuberculosis [7]. RNA analysis using a polymerase chain reaction assay can help to differentiate tuberculosis from CD [8].
3. EXTRAINTESTINAL PATHOLOGY OF IBD In addition to inflammation of the intestines, 5-10% of patients with either UC or CD develop inflammation of other organs [9]. This can involve the eyes (uveitis, episcleritis), skin (erythema nodosum, pyoderma gangrenosum) [10], biliary system (primary sclerosing cholangitis), ears and joints. Primary sclerosing cholangitis, a disease usually associated with UC rather than CD, leads to fibrous strictures of the bile ducts [11]. This stricturing causes duct obstruction and liver damage often requiting liver transplant. Patients also may have painful joints (arthralgia) that are particularly troublesome in the morning. One form of arthralgia involves larger joints asymmetrically, is migratory and does not lead to deformity or swelling [ 12]. Still other patients develop a second type of persistent, nondestructive arthropathy that involves peripheral joints [13]. A third form of joint disease, which is damaging, is ankylosing spondylitis. This condition affects predominantly the spine and hips. It is 10 times more prevalent in IBD patients than in the general population [14]. Unlike the other extraintestinal clinical manifestations of IBD, ankylosing spondylitis follows a clinical course independent of the IBD activity and occurs in patients having the HLA-B27 genotype.
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CD involvement of the duodenum and ampulla can injury the pancreatic duct causing pancreatitis [ 15]. Pancreatitis also can result from primary sclerosing cholangitis [ 15] or the use of medications like prednisone, metronidazole [16] and azathioprine or its related metabolite 6 mercaptopurine [ 17] that are commonly used to control disease activity. Other conditions resulting from bowel damage, chronic intestinal inflammation and/or disease activity include gallstones (cholelithiasis) [18], kidney stones (nephrolithiasis), iron or B12 deficiency anemia, osteoporosis, and GI cancers (colon, small bowel and biliary). Patients with CD are prone to gallstones because of bile salt malabsorption. Calcium malabsorption as well as chronic dehydration can lead to development of calcium oxylate or calcium phosphate kidney stones. Damage to or resection of the terminal ileum impairs B 12 absorption, and chronic blood loss causes excess iron loss. These can lead to B 12 and iron deficiency, which result in anemia. Osteoporosis can result from patients consuming diets poor in calcium and vitamin D, immobility as well as from the use of corticosteroids to treat disease [19]. Chronic inflammation in any region of the gut predisposes to cancer. In particular, patients with UC with extensive colon involvement are subject to a progressively increasing risk for colon cancer after 8 years of disease [20, 21 ]. There is substantial risk of cholangiocarcinoma in primary sclerosing cholangitis and some risk for small bowel or colon cancer in CD [21, 22].
4. SUSCEPTIBILITY TO IBD-GENETICS VS ENVIRONMENT 4.1. The Case for Genetics
UC and CD are disorders of complex derivation caused by the interplay of poorly defined environmental exposures and, at least in some instances, the inheritance of susceptibility genes. However, neither UC nor CD predictably results from any one genetic defect or mutation. It is likely that various genes influence risk for disease, therapeutic response, disease location, disease virulence, complications (eg. stricturing in CD) and extraintestinal manifestations. About 25% of patients with UC or CD have a
history of IBD in first or second-degree relatives [23, 24]. In the United States, the prevalence of IBD is lower for Hispanics and Asians than for Whites and African Americans [25]. The Jewish population carries a substantially greater risk for IBD than the non-Jewish population in the United States, Europe and Israel [26-28]. This heterogeneous risk for IBD among various segments of the population could signify disease predisposition based on either genetic makeup or environmental exposure. Studies involving twin pairs separated at birth have shown a higher concordance rate for CD among monozygotic as opposed to dizygotic twins supporting a genetic basis for mostly CD [29]. Published reports from several genomic screening studies using IBD sibling-pair families report genes linked to disease susceptibility on various chromosomes. Most of these observations, however, have not been confirmed. One definite susceptibility gene associated with CD is NOD2 (also called CARD15) located on chromosome 16 [30, 31]. In macrophages and monocytes, NOD2 produces an intracellular NFkB signaling protein that recognizes muramyl dipeptide, which derives from bacterial peptidoglycan [32, 33]. About 5% of the population has one of several missense mutations in the NOD2 gene rendering NOD2 dysfunctional. Patients bearing some variants of the NOD2 defect are 2-3 fold more susceptible to CD. Those with two mutant genes are more likely to develop CD at a younger age, and their disease has a predilection for the terminal ileum and stricturing [34--36]. Since NOD2 defects increase susceptibility to CD, it is reasonable to speculate that normal NOD2 signaling in response to innate sensing of intestinal bacteria protects from CD. However, most patients with CD have no mutations in their NOD2 gene and, conversely, most persons with NOD2 defects never develop CD. Various other genetic risk factors have been proposed for IBD [37]. Among those for both UC and CD include polymorphisms of the TNFtx gene and its receptors [38]. Other candidate genes related to UC include polymorphisms in the promoter region of the gene for CD 14, which is a component of the LPS receptor [39]. These and many other published associations with gene mutation and IBD risk [4042] require further substantiation.
4.2. The Case for Environment
Geographic and ethnic variations in IBD frequency suggest that environmental factors not yet identified greatly affect the risk of disease [43]. The prevalence of IBD has increased steadily in North America and Western Europe since the 1950s [44], and now perhaps up to 1 in 250 people has IBD in some areas of these affluent industrialized regions [45, 46]. CD was originally limited to white populations in the United States, but now is routinely diagnosed in people of African-American and Hispanic origin. The US and Europe display a North-South gradient in disease frequency with IBD being more common in the North than in the South [47]. The magnitude of this gradient recently diminished in Europe [47]. US military veterans are at low risk for this disease if they were raised in the rural South [48]. History of prisoner-of-war status or combat in tropical regions lower the risk [49]. CD and UC are rare in South America [50], Central America, Africa [51, 52] and Asia [53] with the white population of South Africa being the exception [54]. Migration studies show that children of people from regions of low CD or UC frequency acquire a greater disease risk if they move to areas of high disease prevalence [55-57]. IBD is more common in urban verses rural areas [58]. Occupation influences prevalence of IBD also. Jobs that expose people to dirt and heavy physical exercise appear protective [59]. There is a higher than expect concurrence of disease in spouses [60]. Patients with CD are more apt to live in more hygienic environments with hot water, indoor plumbing and other conveniences [61, 62]. The prevalence of IBD is higher among Ashkenazi Jews who live in the US, Canada and Western Europe. Except for South Africa [54, 63], Jews living in countries near the equator have substantially lower rates of disease. In Israel, early studies suggested that Jewish immigrants from Middle Eastern and Mediterranean countries had low rates of disease compared to people from Europe and North America [64]. However, living in Israel may have modified the risk. The various Jewish ethnic groups living in Israel presently develop IBD according to the prevalence expected in Israel, rather than that anticipated according to their country of origin [65, 66]. IBD is rare in the Arab community in Israeli [67]. The frequency of
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the NOD2 defect, the only gene definitely associated with a risk for CD, is no higher in Jewish than non-Jewish populations.
4.3. Possible Environmental Factors Affecting Risk for Disease IBD usually begins in the second to third decade of life. This could signify that exposure or lack of exposure to environmental factors during childhood predisposes to disease.
4.3.1. Biological agents 4.3.1.1. Commensal enteric bacteria- the bad guys? There is evidence that microbial organisms and their products play a role in the pathogenesis of IBD. UC and CD occur most readily in regions of the intestine with highest bacterial colonization. Also, diversion of the fecal stream improves CD in distal bowel segments [68], and normal intestinal flora is necessary for expression of disease in mouse models of IBD [69]. A leading theory for the pathogenesis of IBD suggests that there is an abnormal host immune response to normal commensal enteric bacteria leading to loss of immune tolerance. Still others suggest that IBD results from a defect in mucosal barrier function allowing excessive exposure to bacteria and their products. There is experimental evidence supportive of both hypotheses. Regardless of the validity of either theory, it is likely that the amount, composition and function of enteric bacteria affect these diseases. 4.3.1.2. Helminths - the good guys ? Extremely hygienic life styles and underexposure to certain organisms like helminths may predispose to disease. Helminths are parasitic animals, some of which can live in locations like the intestinal lumen, blood stream or muscles of the host. These organisms inhabit more than 1/3 of the world population. Helminth colonization is most common in children living in warm climates and subject to poor sanitation. The infective forms of these organisms are spread through contact with contaminated soil, food or water. Before the 1940s, many children and adults in the United States carried helminths. Worm carriage was particularly common in rural
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areas of the South and in indigent populations of major cities [44]. In the United States, helminth colonization has steadily declined except in recent immigrants from less developed countries [70] and in indigenous populations riving in underserved regions [71]. Many helminths induce within their host Th2 responses, production of powerful immunomodulatory molecules like ILl0 and TGFI3, and "regulatory" T cells. People bearing helminths display dampened immune responses to unrelated concurrent antigenic exposures [44, 72]. These changes in immune responsiveness can persist long after elimination of these helminthic exposures [73, 74]. Thus, it is proposed that people harboring helminths are less likely to develop immunological diseases. Substantial epidemiological data and animal experimentation support the link between helminths and protection from immunological diseases [75-78].
4.3.1.3. The role of pathogens IBD does not appear to result from exposure to any one particular pathogen. The vast majority of patients develop IBD without any apparent proceeding or concurrent infection. Yet, there are many reports suggesting that the immune response to various pathogens can precede disease onset. Such infections perhaps trigger inflammation that exacerbates subclinical, pre-existent IBD or serve as true primary triggers for IBD activation. Infectious exposures also can reactivate known quiescent disease and enhance ongoing IBD activity. Clostridium difficile is an enteric bacterium that, at times, induces colitis and chronic diarrhea. This organism can live in the intestine without causing harm. However, it can produce toxins (A and B) that cause disease when antibiotics or other factors disrupt normal intestinal flora. The clinical and histologic findings of mild C. difficile colitis can mimic IBD. More severe disease is an easily recognizable syndrome presenting with characteristic pseudomembranes on the colonic mucosal surface and distinctive histological findings. Early studies suggested that IBD patients were at high risk for acquiring toxin-producing C. difficile [79]. Still other studies found that IBD patients are no more likely to harbor toxin-producing C. difficile then other chronically ill patients receiving antibiotics intermittently [80]. Regardless, toxin production concurrent with active IBD can worsen
ongoing disease and hinder IBD treatment. Such patients frequently improve substantially with metronidazole therapy to eradicate toxin-producing C. difficile. It is possible the magnitude of the problem is somewhat under-recognized, since the convenient and low cost modem day tests for C. difficile stool toxin are not highly sensitive and thus more subject to false negativity. There are other organisms also associated with IBD. Cytomegalovirus infection both in immunosuppressed and immunocompetent people can exacerbate IBD, make it unresponsive to therapy [81-83] and occasionally is linked with onset of new disease. Infection with Entamoeba histolytica, a protozoan, can induce chronic colitis that does not resolve after the primary infections clears ("post amebic colitis"). Some postulate that IBD is a chronic inflammatory disease caused by unrecognized intestinal infection. The pathology of IBD can mimic that of several known infectious diseases. This hypothesis, although attractive, remains extremely controversial. Many organisms were proposed and dismissed as etiologic agents for IBD over the last 50 years. Two of the more recently proposed candidates include Mycobacterium paratuberculosis and measles virus. There are little consistent molecular, immunological or epidemiological data to support either association [84, 85]. The similarity of CD to intestinal tuberculosis makes the chronic Mycobacteria infection hypothesis attractive. M. paratuberculosis causes an enteritis in cattle call Johne's disease. The organism resides in the soil and can contaminate milk. However, patients with CD demonstrate no specific humoral or cellular immune response to mycobacterial antigens. Also, non-consanguineous people exposed to patients with CD (ie. physicians and nurses treating CD) or cattle with Johne's disease (ie. veterinarians treating animals with M. paratuberculosis, farmers with infected herds) do not acquire the disease. Laboratories have failed to confirm early reports of M. paratuberculosis DNA in CD tissue. Also, immunosuppressive therapies known to promote dissemination of Mycobacterium infections (ie. prednisone, anti-TNF~x) are beneficial in patients with CD [86]. Yet, new reports of a possible association with patients with CD keeps the hypothesis viable [87].
4.3.2. Smoking Cigarette smoking is a define risk factor for development of CD [88]. Continued tobacco use worsens the course of ongoing CD [89] and promotes disease recurrence after surgical resection [90]. It also appears to make the disease more resistant to therapy. In all situations, smoking cessation appears beneficial [91 ]. The relationship between smoking and UC is less well defined. Some studies suggest that smoking decreases the risk for UC [88]. Former smokers may be at increased risk. Cigarette smoke contains many chemicals that may affect immune function and intestinal permeability. Tobacco also can induce vascular spasm, vasculitis and a hypercoagulable state, which could prove detrimental for CD [5].
4.3.3. Appendectomy Appendectomy under the age of 20 decreases the incidence of UC [92-94]. However, only appendectomy for management of appendicitis affords this protection. Incidental appendectomy confers no benefit. The explanation for this observation is elusive. It is tempting to speculate that peritonitis in response to enteric organisms at a young age induce formation of regulatory T cells that prove protective later in life. There is no definite link between appendectomy and CD, although a recent study now suggests that this increases the risk [95].
4.3.4. Medications Several medications are believed to exacerbate IBD or can provoke an IBD-like syndrome. Patients with UC and CD are discouraged from using nonsteroidal anti-inflammatory drugs because they are widely thought to worsen IBD [96-98]. They also can cause small bowel ulceration, bleeding, stricture and perforation in apparently normal people. There is experimental evidence in IBD animal models suggesting that blockade of arachadonic acid metabolic pathways that lead to the production of various prostaglandins and leukotrienes can exacerbate or precipitate colitis [99]. Gold therapy can incite an allergic-like reaction featuring eosinophilic colitis [ 100]. There may be a slightly increased risk of IBD
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with oral contraceptive use [ 101].
4.3.5. Other factors Other risk factors have been proposed. There is no clear relationship between diet and development of IBD. Also, studying large IBD populations have revealed inconsistent findings regarding seasonal variations in disease activity. A recent prospective study, however, suggests an increased risk for UC during the winter [102]. Others report higher CD relapse rates in the Spring and Fall [ 103]. Emotional stress probably can exacerbate, but does not cause disease. There is no psychosomatic component to IBD, and IBD patients display no consistent psychopathology.
5. THE I M M U N O L O G Y OF IBD 5.1. Lessons from Animal Models of IBD
Genetic engineering and some serendipity have produced many new animal models of IBD. The actual specific causes of IBD remain unknown. Animal models of IBD thus can only mirror certain aspects of human disease. Yet, they are providing insight into the elements of mucosal immunoregulation and barrier function needed to maintain peaceful coexistence between us and the intraluminal intestinal antigens derived from food and microorganisms. These models show that various defects in immune regulation, immune effector function and in the mucosal barrier can lead to disease. They also suggest that loss of tolerance to our normal commensal intestinal flora is sufficient to drive the inflammation. Please see Strober et al [104] for an additional in-depth review of this topic.
5. I. 1. The critical role of T cells Disturbances in aspects of T cell function can cause intestinal inflammation. However, mice totally deficient in T cells and/or B cells do not develop colitis while housed in standard specific pathogen-free facilities. This shows that severe immunodeficiency does not lead to IBD and that innate immunity is sufficient for control of normal intestinal flora. In the resting gut, T cell function is severely curtained
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through the processes of cellular deletion, anergy induction and regulatory T cell production. The CD4+ T cell subset appears to play the most decisive role for development of the disease in most of the animal models of IBD [ 105-108]. T cells also have regulatory as well as effector functions. There are regulatory T cells (called Trl, Th3, or CD25+) that produce immunomodulatory factors like TGF[3 and/or ILl0 and which inhibit inflammation via cytokine secretion and/or by cell-cell contact. T- and B-cell deficient mice (SCID or RAG) reconstituted with CD45R rn T cells [ 109], and transgenic mice with T cells that can not express TGF[3 RII [110] develop colitis because of defective regulatory T cell function. The protective T cells in the SCID-transfer model are CD25+, and they require TGF[~, ILl0 and CD28-CTLA4 interactions [111-114]. Three additional colitis models featuring regulatory T cell dysfunction are the IL2-/- mouse [115, 116], IL2ct or [3 chain -/- mouse [ 117, 118], and the bone marrow-reconstituted Tge26 mouse [ 119]. All of them develop IL12-dependent, Thl-type intestinal inflammation requiting CD40L-CD40 interactions. The pathology in these models appears to result from abnormal thymic function.
5.1.2. Importance of cytokine dysregulation It is common to classify murine (and human) inflammation into one of two broad categories called T helper cell type 1 (Thl) or Th2, respectively. Thl responses are strong cellular immune reactions characterized by the production of IFNy, TNFt~ and IL2. These inflammations are rich in lymphocytes and produce IgG2a. Th2 responses make IL4, IL5, IL9 and ILl3. IgE and IgG1 production, and eosinophil infiltration characterize Th2 inflammation. Th2 inflammations include allergic reactions and responses to helminths. Nearly all murine models of enteritis are Thl responses. The notable exceptions are the colitis of the TCRtx chain deficient mouse [120] and oxazolone-induced colitis [ 121]. Various molecular defects engineered to cause IFN7 and ILl2 overproduction [122, 123] predispose to colitis attesting to the importance of Thl cytokines in disease pathogenesis. IFN 7, usually derived from CD4+ T cells, is an important effector molecule that enhances
macrophage and dendritic cell function, among other things. Over-production of TNFo~ (TNF ARE mouse) also leads to disease [124]. In this latter IL12-dependent Thl model, the pathology is mostly dependent on CD8+ T cells. Over-production of TNFc~ from any one of several cellular sources is sufficient to drive disease. Murine IBD can result from defective production of regulatory cytokines. I L l 0 - / - mice spontaneously develop a Thl-type colitis [125] dependent on the presence of intestinal flora [126]. Blockade of ILl2 prevents and partially reverses ongoing disease. Mice with an IL 10 receptor defect [ 127] or a defect in the signaling pathway for ILl0 (Stat3) [ 128] also can develop IBD. ILl0 is a major immunomodulatory molecule that, for instance, suppresses IL12 and TNF~ production, and the expression of costimulatory molecules. In the SCID or RAG colitis model of IBD induced by CD45RB Fa T cell transfer, only ILl 0-producing regulatory CD4+ T cells can prevent disease. CD4+ T cells that make IL 10 also protect RAG-IL 10KO T cell reconstituted mice from colitis induced by NSAID administration. However, in the T cell receptor o~ IBD model, B cell ILl0 also may have an important regulatory role [ 129, 130]. TGF[~ is an important regulatory cytokine with pleiotropic functions that, for instance, affects cellular proliferation, cytokine secretion and collagen deposition. Defects in TGF~ production or signaling [110] lead to enteritis in several models suggesting an important role for this cytokine in murine IBD. 5.1.3. Models of epithelial injury or dysfunction The surface epithelium of the intestine allows absorption of dietary substances, electrolytes and water while excluding intestinal microorganisms and hannfial antigens. Many studies suggest that these epithelial cells express nonclassical MHC molecules (CDld) [129], produce cytokines and interact with intraepithelial lymphocytes. Through these processes, they may regulate intraepithelial lymphocyte cytotoxicity [131] and perform other functions [132] that modulate mucosal immunoreactivity. Primary defects in intestinal epithelial cell function can cause disease. Two notable examples of
models illustrating this concept are the N-cadherinand the multiple drug resistant (mdr)la-deficient mouse [133, 134]. Cadherins are transmembrane glycoproteins important for cellular adherence. Mdrla mice have a defect in epithelial transport function. Both develop Thl intestinal inflammation probably seconding to impaired exclusion of bacterial products. Oral administration of dextran sulfate to rodents can induce colitis by injuring the epithelial bartier. This model has been used extensively in IBD research. Deficiency in expression of the prostanoid receptor EP4 predisposes mice to dextran sulfateinduced colitis [135]. The EP4 receptor helps maintain mucosal integrity and down regulates immune responses. Both nitric oxide and reactive oxygen radicals also have a critical role in this model of IBD [135]. Trefoil peptides promote epithelial restitution. In trefoil-deficient mice, oral administration of dextran sulfate sodium induces severe colitis presumably because of a failure in epithelial repair [136]. 5.1.4. The role of intestinal flora Where examined, most mouse models of IBD maintained in a germ-free environment are highly resistant to acquiring disease [126, 137, 138]. Antibiotic therapy also ameliorates animal IBD [139]. Studies using both the C3H/HeJBir [140] and SCID-reconstituted colitis models [122] show that antigens in the flora can induce the mucosal inflammation. The various bacterial species comprising normal intestinal flora have unequal capacity to provoke mucosal inflammation [141]. Normally, regulatory T cells may drive non-responsiveness to commensal enteric bacteria [ 142] 5.1.5. Other models of lBD and other misc. mechanisms There are several other Thl models of IBD (ie C3H/ HeJ Bir mouse [143], SAMP1/Yit mouse [106], glial cell-deleted mouse [144, 145]) in which the pathophysiology is less well defined. The SAMP1/ Yit mouse is particularly noteworthy because it develops ileitis, mucosal granulomas and, at times, fistulas reminiscent of CD. Transgenic mice with disrupted enteric glial cell networks develop Thl-
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type enteritis [144, 145]. Thus, the nervous system is essential for maintaining bowel integrity. Proposed is a critical role for the IgE FceR1 receptor [146] in a model of Thl colitis. Also emphasized is the importance of selective expression of adhesion and homing receptors using the CD45m-SCID transfer model [ 147, 148].
5.1.6. Models of natural protection It is proposed that our natural exposure to helminths protects ,. people from developing IBD through induction of IL4, ILl0, TGFI3, regulatory T cells and by other means [44, 75]. Helminths (eg. intestinal worms, schistosome eggs) protect mice from TNBS-induced colitis [ 149] and other immunological diseases [ 150]. They also reverse ongoing colitis in IL10KO animals via the induction of regulatory T cells. Probiotics are living micro-organisms that, when taken in appropriate amounts, improve health. There are some animal data suggesting that probiotics will be therapeutically beneficial in IBD [ 151 ].
5.2. Human Investigation Both UC and CD are destructive inflammatory disorders of the intestines. Except for the NOD2 gene defect, which is expressed in about 10-20% of CD patients, there are no other distinct immunoregulatory defects or abnormal inflammatory pathways identified to date associated with either form of IBD. In these disorders, the pathology leads to a breach of the mucosal barrier that allows penetration of large numbers of luminal antigens leading to immune stimulation. It is difficult to identify primary immunological events underlying disease pathogenesis in such a complex setting. Patients frequently display nutritional deficiencies and receive strong immunosuppressants to control disease, which are additional factors confounding investigation of immune dysregulation. The following are some of the immunological phenomenology associated with these diseases. However, the significance of most of these observations for the most part are yet undefined.
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5.2.1. Immunoglobulins and autoimmunity The inflamed mucosa of both UC and CD has increased numbers of plasma cells that produce various immunoglobulin subtypes. UC and CD differ in the ratio of IgG1 and IgG2-producing B cells in the intestinal wall. UC generates relatively more IgG1 and CD more IgG2 [152]. This could signify that the inflammation of UC is somewhat more Th2 and CD more Thl. Since 1959 [153], there have been numerous reports of autoantibodies in IBD. Described are antibodies against colonic extracts as well as intestinal and biliary epithelial cells [154-158]. Other autoantibodies reported in IBD include anti-DNA, anti-thyroglobulin, anti-smooth muscle, anti-gastric parietal cell, anti-reticulin, anti-pancreatic, anti-goblet cell and others [159-162]. One series suggested that 68% of patients with UC and 32% with CD make at least one of these autoantibodies [163]. These autoantibodies do not appear to contribute to intestinal injury and thus may simply be epiphenomena. Mouse models of IBD suggest that T cells, rather than B cells underlie the pathogenesis of IBD. However, some of these autoantibodies (ie, anti-goblet cell [159], anti-nuclear [161], antiendothelial cell [ 162] are highly prevalent in apparently healthy first-degree relatives of IBD patients, which may signify that IBD families have a proclivity for immunodysregulation.
5.2.2. The ANCA and ASCA phenomena Many patients with IBD, vasculitides, primary sclerosing cholangitis and autoimmune hepatitis develop anfi-neutrophil cytoplasmic autoantibodies (ANCA) directed against cytoplasmic granules. Its association with UC was first reported in 1983 [164]. A perinuclear staining pattern, called p-ANCA, is characteristic for IBD and primary sclerosing cholangitis [165]. The latter observation is not surprising, since 50% of patients with primary sclerosing cholangitis have UC. 50-80% of patients with UC produce p-ANCA, while patients with CD express p-ANCA much less frequently [166]. ANCA does not appear to contribute to disease activity and has no known relationship to the pathogenesis of IBD. It does occur in family members of patients with UC at higher than expect frequency for
that of general population [ 167]. Many patients with CD (40-75%), particularly those with small bowel involvement, produce anfi-Saccharomyces cerevisiae antibodies (ASCA) directed against oligomannosidic epitopes of this yeast. So do some of their related family members who bear no apparent disease [ 168, 169]. The first report of this association was in 1988 [ 170]. ASCA is less common in UC, but its presence does not distinguish CD from UC with certainty [166]. The significance of ASCA with regard to the pathogenesis of IBD remains unknown. Also true for ANCA, occurrence of ASCA in family members perhaps suggests a genetic propensity to over-react to a common environmental antigen. During the initial diagnosis of IBD, it is not possible to differentiate UC from CD in about 10% of patients ("indeterminate colitis"). This can confound therapeutic decisions. About 50% of such patients express either p-ANCA or ASCA, which often, but not definitely predicts the ultimate classification of the disease [ 171 ].
gen-specific T cells multiply to increase their numbers and orchestrate a more specialized immune reaction involving antibodies and other factors. This also leads to immune memory. The expansion and activation of T cells must remain controlled to limit immune reactivity. Unwanted T cells can be induced to die, a process called apoptosis, through engagement of specific receptors like Fas. In both UC and CD, mucosal T cells are resistant to Fas-mediated apoptosis mediated by CD2 and other pathways [183-187]. This relative resistance to apoptosis with insufficient elimination of activated T cells could have pathogenic significance [ 188]. 5.2.4. Eosinophils
UC and to a lesser degree CD contains increased numbers of eosinophils in the inflamed intestinal tissue [ 189, 190]. This is most evident in very active rather than chronic IBD. The significance of this observation is unknown. 5.2.5. Cytokines
5.2.3. T cells
T cells infiltrate the mucosa of patients with UC and CD. There are increased numbers of both CD4+ and CD8+ T cells expressing mostly the ct~l T cell receptor (TCR). T cells expressing TSTCR are less evident [172]. The mucosal T cells bear surface antigens suggesting a state of activation [ 173, 174]. In CD and UC, CD40 appears to be hyper-expressed [173, 175] on the T cells which in turn could lead to over-activation of CD40 ligand expressing B cells, dendritic cells and macrophages. TCR usage suggests that the infiltrating T cells recognize a broad range of regional antigens not unique to IBD [ 176-178]. However, the earliest mucosal lesions of CD (aphthous ulcers) may express some lesion-specific, dominant T cell clones [ 179]. Lamina propria lymphocytes from IBD patients are not cytotoxic in allogenic mixed lymphocyte reactions [180] and display minimal antibody-dependent cellular cytotoxicity against epithelial and other targets [181]. They react normally to bacterial antigens, mitogens and alloantigens [ 182]. T cells are an essential cellular component of the process called adaptive immunity. In adaptive immunity, T cells recognize antigens. These anti-
5.2.5.1. Thl vs Th2 In CD, the Thl pathway of inflammation is highly active. ILl2, in conjunction with ILl8, are major inducers of IFN T production and Thl cell development. ILl2 can act through a dimeric receptor that ~,ontains a 131 and a ~2 chain. Engagement of this receptor stimulates the STAT4 signal transduction pathway to induce T cell differentiation. IL12R~2 RNA transcripts and phosphorylated STAT4 proteins are increased in the mucosa of active CD [ 191 ]. This is not the case for UC or inactive CD. The lamina propria mononuclear cells also make ILl2, IFNy and TNFct, but little IL4 [191-194]. To allow full development of Thl effector cells, IFNy, STAT1 and perhaps other factors most activate the T cell transcription factor T-bet. GATA-3 is a transcription factor that inhibits Th 1 and promotes Th2 cell expansion. In CD, lamina propria T cells display increased expression of T-bet and down regulation of GATA3 [123]. Thus, it is reasonable to assume that activated Thl cells have a role in CD pathogenesis. The inflammation of UC does not fit readily into the Thl/Th2 paradigm. The colitis of UC frequently features neutrophils infiltrating crypt epithelium,
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lymphocytes and eosinophils. The colonic microscopic pathology of UC and CD can be indistinguishable. In UC, there is increased IL5 and IFN), production, but relatively low expression of IL4 [191, 1951.
5.2.5.2. TNFot About 60% of patients with CD improve with antiTNFct therapy (infliximab) inferring an important role for TNFct or T cells expressing membranebound TNFt~. However, many patients become resistant to treatment in less than 1 year. This could signify that TNFot is important, but not central to CD pathogenesis. However, some patients develop antibodies to the anti-TNFtx reagent, which could explain some of this acquired resistance [196]. TNFt~ is not a critical cytokine in UC, since most UC patients do not respond to this therapy. The role of TNFtx in human CD remains controversial. TNFct is produced at the site of inflammation [ 197, 198], and TNFct-R2 expression is upregulated on lamina propria and peripheral blood T cells [ 198, 199]. TNFt~ can stimulate lamina propria mononuclear cells to make IFN'f [200]. This could infer an important role for TNFot in regulating Thl cell function. However, the anti-TNFt~ therapeutic agent is a complementfixing antibody. Thus, an alternative mechanism of action could be through complement lysis of effector T cells beating membrane-bound TNFtx. 5.2.5.3. Other proinflammatory cytokines (ILl, IL2, IL6, ILl5, ILl8) The mucosa of patients with CD produces increased amounts of ILl and IL6. Unlike resting mucosal macrophages from normal colon, those from IBD express active ILI~ converting enzyme allowing secretion of active forms of ILI~ [201]. IL6 can rescue mucosal T cells from apoptosis. Thus, excess production of IL6 may prolong T cell survival and contribute to chronic intestinal inflammation [202]. Also in CD, there is increased production of ILl 8 [203,204] and ILl 8 binding protein [205], IL2 [193], ILl5 [206] and ILl6. ILl8 frequently works in conjunction with IL 12 to enhance Th 1 cell formation, but it also can promote Th2 development. IL2 and IL 15 share many immunoregulatory properties. They protect T cells from apoptosis. They also induce T cells to proliferate and to make proinflammatory cytokines. CD8+ T cells, CD4+ T cells and
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mast cells all make ILl6 [207].
5.2.5.4. Regulatory cytokines Animal models of IBD show the importance of ILl0 and TGF~ for maintenance of mucosal tolerance to intestinal flora. Other than their presence in mucosal inflammation [208, 209], there are little data regarding their potential importance in human IBD. Initial clinical trials with recombinant ILl0 have shown little promise. 5.2.6. Chemokines and endothelial cell ligands Chemokines are proteins that attract cells into sites of inflammation. Many cell types, including stressed intestinal epithelial cells [210], produce these products, and leukocytes display chemokine receptors. In IBD, it is likely that various chemokines help to selectively recruit leukocyte subsets to regions of the gut and promote their activation [211-214]. For instance, it is reported that in CD selective expression of chemokine CCL25 attracts T cells to the small bowel [215]. Leukocytes adherence to vascular endothelial cells is an important early step in their migration into sites of inflammation. There are various complementary adhesion molecules that mediate this interaction. Endothelial cells from patients with CD and UC bind leukocytes readily [216]. In the inflamed intestine of IBD, inducer/memory T cells selectively express the 4C8-1igand [217]. Thus, selective blockade of either chemokine or endothelial cell ligands could prove useful avenues for therapeutic intervention. Natalizumab, which blocks the adhesion molecule ct4 integrin, has shown some clinical utility in CD [218].
5.2.7. Eicosanoids Eicosanoids, which are products of arachidonic acid metabolism, include the prostaglandins and leukotrienes. The inflamed mucosa generates these products. They are important in IBD, since agents that block both pathways like 5-aminosalicylates can benefit both CD and UC [219-221]. However, pathway-selective antagonists like aspirin may worsen IBD by inhibiting production of protective prostanoids like prostaglandin E2 [222].
5.2.8. Neuropeptides
The intestine is heavily innervated. There are more neuronal elements in the gut than in the spinal cord. Nerves and various leukocyte subsets produce neuropeptides like vasoactive intestinal polypeptide, substance P and somatostatin. In the gut, these peptides regulate epithelial cell function, vascular blood flow and intestinal motility. They also modulate mucosal immune reactivity. IBD can remit and relapse spontaneously suggesting that mucosal immune homeostasis is subject to ongoing control. Understanding the processes that naturally control spontaneous remission could lead to cure of these diseases. Anxiety and stress can precipitate reactivation of IBD suggesting that nerves and/or neuropeptides could be some of these controlling factors [223]. For instance, in human IBD (CD and UC), there is increased substance P mRNA expression [224] with disproportionate over-representation of NK-1R on human mucosal CD4+ T cells [225]. Substance P and its receptor are important enhancers of Thl cell development and IFN2(secretion [226]. Experiments using various murine IBD models showed that a NK-1R antagonist used in vivo can suppress colitis [227, 226] and intestinal IFN), production [226].
can process and present antigen to surrounding lymphocytes either promoting or inhibiting reactivity [232-235]. Moreover, they are a potential source of chemokines and other mediators of inflammation that could aggravate IBD. 5.2.11. Fibrosis
A feature most notable in CD versus UC is intestinal fibrosis. This often leads to intestinal obstruction. Not all patients with CD develop severe intestinal fibrosis. This may reflect differences among patients in the intensity and nature of their inflammation. Also, it could signify that some dgatients have a greater genetic predisposition to scar formation in response some types of chronic intestinal injury. It remains uncertain if fibroblasts, myofibroblasts or smooth muscle cells are the major source of the excess intestinal collagen in CD. The collagen is composed of several types (I, III, IV and V). Various inflammatory mediators promote collagen production and resorption [236, 237]. In IBD, there is little known regarding the complex processes that regulate collagen deposition, remodeling and dissolution [238].
6. LESSONS FROM TREATMENT 5.2.9. Reactive oxidants
Mucosal inflammation produces many reactive oxidants like H202 and nitric oxide [228], which can injure tissue. Such molecules may contribute to epithelial cell injury in IBD [229]. 5.2.10. Mucosal epithelial barrier
The first-encountered, natural cellular barrier to luminal antigens and bacteria is the intestinal epithelial lining. They gate intestinal absorption and permeability. Some patients with CD have increased intestinal permeability [230]. UC patients also have a barrier defect [231]. Some postulate that this is a primary defect that allows greater systemic exposure to luminal antigens. It is probable, however, that the permeability defect is a result rather than the primary cause of the inflammatory injury. It also is proposed that intestinal epithelial cells
Therapeutic success and failure perhaps affords additional insight into the pathogenic mechanisms underlying IBD. Anti-inflammatory agents and powerful immunosuppressants frequently improve CD and UC. This observation favors the hypothesis that IBD results from immune dysregulation, rather from exposure to invasive infectious pathogens. For instance, infliximab (anti-TNFo0 therapy is beneficial for CD, but reactivates latent tuberculosis [86]. In CD and UC, aminosalicylates improve disease in about 60% of patients [239]. They block production of various prostaglandins and leukotrienes attesting to the importance of these inflammatory mediators in the disease process. They also are potent free radical scavengers. However, they have limited efficacy for maintenance of remission in CD [2401. There are several immunosuppressants in common use. Corticosteroids with all their immunological effects provide only short-term benefit
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[241-243]. Azathioprine and its major metabolite, 6-mercaptopurine, affect T lymphocyte development and induce long-term improvement in many patients with either UC or CD [241, 244, 245]. Methotrexate, another powerful immunosuppressant, affords long-term benefit at least in CD [246, 247]. Cyclosporine can induce remission particularly in severe UC. Anti-TNFct therapy with infliximab is initially affective in 60% of patients with CD [248, 249], but is of low efficacy in patients with UC. This suggests that TNFct and/or the cells bearing surface TNFt~ may have special importance in CD. This selective clinical response also infers that there is a fundamental difference in immune pathogenesis between classical CD and UC. However, for many CD patients, this medication looses its effectiveness in less than 1 year [250]. Other anti-TNFt~ medications are much less effective than infliximab in the control of CD suggesting that infliximab has another mechanism of action besides neutralization of TNFct activity. Antibiotic therapy (metronidazole [251, 252] and perhaps ciprofloxacin [253, 254]) can improve CD and perhaps UC. Diverting the fecal stream can improve CD in distal bowel segments [68]. However, UC is much less responsive to fecal diversion. These observations could signify the importance of the intestinal flora in disease pathogenesis. Paradoxically, some patients develop diversion colitis resulting from intestinal mucosa deprivation of essential nutrients. Intervenous feeding or oral elemental diet can induce long-term remission in patients with CD, but not UC [255,256]. Disuse of the intestine leads to mucosal atrophy, reduction in intestinal flora and changes in flora composition, which could be the underlying mechanisms of action. Patients with classic UC who require colectomy and ileostomy usually do not get recurrence of disease. However, patients with CD almost universally suffer disease relapse in previously healthy regions of intestine after resection of diseased segments [257]. This again attests that UC and CD have significant, fundamental differences in mechanisms of disease. To avoid ostomies, most younger patients with UC requiting colonic resection request ileoanal pouch anastomosis, which preserves continence [258]. This involves making from a segment of
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small intestine an intraabdominal pouch to store waste. Such pouches are prone to developing inflammation (pouchitis) [259]. This problem is not encountered in patients receiving colectomy and ileoanal pouch anastomosis for the non-inflammatory disease called hereditary familial polyposis. This suggests that patients with UC have an underlying predisposition for mucosal immune over-reactivity. Space does not permit an in-depth discussion of the various therapeutic considerations in disease management. Please see the following references for further discussion [260-263].
7. CONCLUSION The last 10 years have brought a substantial change in our conceptual understanding of the pathogenic mechanisms leading to IBD. It is now appreciated that the constellation of clinical and pathological features called UC and CD likely are not single diseases. They are common clinical manifestations of perhaps several unrelated or partly related disorders. A popular hypothesis of the 1960s considered IBD a psychosomatically induced, autoimmune disease. We now believe that immune dysregulation and subsequent aberrant interactions with normal intestinal flora probably are important processes in disease pathogenesis. Also, it appears that at least in CD, Thl immunoregulatory pathways have a role in the pathology. Another prominent belief 10 years ago was that IBD was a genetically inherited disease, since it has the propensity to develop in select population subgroups and a proclivity for multiple occurrences in families of affected individuals. There now is a better appreciation that poorly identified environmental factors and life-style, as well as genetic makeup promote disease. Also, "excess" hygiene and lack of exposure to some common environmental organisms during childhood may predispose to IBD. However, we must remain receptive to new ideas and concepts, since the actual, basic underlying defects that lead to IBD remain unidentified.
ACKNOWLEDGEMENTS Grant support: the National Institutes of Health (DK38327, DK58755), the Crohn's and Colitis
Foundation of America, Inc. and the Veterans Administration.
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9 2004 Elsevier B. V.. All rights reserved. Infection and A u t o i m m u n i t y Y. Shoenfeld and N.R. Rose, editors
Infection and Spondyloarthropathies J. Alcocer-Varela and J.C. Crispin Acufia Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Mddicas y Nutrici6n Salvador Zubirtin, Mexico City, Mexico
1. I N T R O D U C T I O N Spondyloarthropathies (SPA) are a heterogeneous group of related chronic autoimmune diseases of the joints with common clinical, radiological, and genetic characteristics. Diseases included in this group are ankylosing spondylitis, reactive arthritis, psoriatic arthritis, undifferentiated SpA, juvenile chronic arthritis, acute anterior uveitis, and arthritis with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Their clinical spectrum goes from acute reactive arthritis (ReA) of short duration (1-3 months) to chronic spondyloarthropathy, with ankylosing spondylitis (AS), the most chronic presentation [ 1]. The pathogenesis and clinical manifestations of SpA are influenced by genetic and infectious factors. The strong association between AS and HLAB27 exemplifies the former and ReA exemplifies the infectious model of the disease. The observation that in some patients ReA and undifferentiated SpA may evolve into AS suggests that these diseases have a common pathway in their pathogenesis [2]. The association of HLA-B27 with ankylosing spondylitis (AS) discovered more than 30 years ago was a breakthrough in our understanding of this rheumatic disease. This association remains the strongest HLA association with a common inflammatory condition, yet there is considerable evidence that susceptibility to AS is more complex than was initially thought. Only a small fraction of B27-positive individuals develop spondyloarthropathy, but what determines which B27 carriers will develop AS is unknown. First-degree relatives of AS patients have a higher risk of developing
Table 1. Genes other than HLA-B27 in SpA Increase susceptibility to A S -
HLA-B60
-
HLA-DR
Influence disease susceptibility or disease phenotypes - TNFo~ -- Transporter of Ag Peptides (TAP) - Cytochrome P-2D6 IL-1 receptor antagonist - MICA Heat Shock Protein 70 -
-
Microsatellite markers with possible links - Chromosomes lp, 2q, 6p 9q, 10q and 19q -
the disease themselves than do B27-positive individuals without affected relatives, suggesting the presence of shared factors other than B27. The study of twins has demonstrated that almost all of the population variance for AS is determined by genetic rather than random environmental factors: genetic factors other than B27 determine which B27-positive individuals develop arthritis, and the environmental trigger is probably ubiquitous. Even in B27-transgenic rat models of the disease, which have provided evidence for the direct involvement of B27, the background strain of the rat influences the penetrance of the transgene. The contribution of B27 to the genetic susceptibility to AS has been estimated in family and twin studies to be 20-50% of the total [3-4]. There are other genes related to this disease, as is shown in Table 1. One of the most
675
interesting association is related to tumor necrosis factor t~ (TNFt~) promoter alleles with ankylosing spondylitis. It has been shown that allelic variations in the TNFt~ promoter influence disease susceptibility in B27 positive individuals. This protective effect of variant promoter alleles could be related to differences in TNFt~ production or could reflect the association of different B27 haplotypes with ankylosing spondylitis [4]. Numerous studies have proven an intimate involvement of intestinal inflammation in the pathogenesis of these disorders. This evidence includes a) triggering of B27-associated reactive arthritis by certain enteric pathogens, b) increased prevalence of ankylosing spondylitis and peripheral arthritis in patients with inflammatory bowel disease, c) increased prevalence of inflammatory bowel disease in family members of patients with ankylosing spondylitis, d) acute intestinal inflammation occurring in the setting of reactive arthritis arising from genitourinary infection, e) the presence of microscopic bowel inflammation in a high proportion of patients with B27-associated rheumatic disease, f) antigenic and structural crossreactivity between the HLA-B27 molecule and components of enteric bacteria, g) diminished internalization of enteric bacteria by cultured fibroblasts expressing HLAB27, and h) triggering of inflammatory arthritis in animals by experimental injection of peptidoglycan from normal bowel flora [5-7]. The first evidence of intestinal involvement in SpA was observed in ReA patients. These patients develop a chronic spondyloarthropathy after a gastrointestinal infection with enterobacteria such as Yersinia enterocolitica, Salmonella typhimurium and enteritidis, Shigella flexneri, and Campylobacterjejuni. This relationship between gut inflammation and arthritis was confirmed in HLA-B27 transgenic rats. Recent studies confirm that an important number of patients with inflammatory bowel disease present musculoskeletal symptoms compatible with SpA (30-40%),with inflammatory low back pain in 30%, synovio/tendonitis in 15% and sacroiliitis in 25%. The clinical expression in these patients seems to be dependent on the HLA genotype and are possibly related to a loss of tolerance to enteric bacteria [8].
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2. ANTIGENS IN THE PATHOGENESIS OF B27-ASSOCIATED DISEASES Infectious agents can induce autoimmune diseases throught different mechanisms such as molecular mimicry, and increase in the immunogenicity of autoantigens derived from the inflammatory process that occurs in the target tissue. Infectious agents also stimulate the production of regulatory cells whose effects extend beyond the responses to the invading microbe (bystander suppression mechanism). Recent data show that IL-10 and TGF-[3, produced by T regulatory cells (CD4+,CD25 +) can inhibit both TH1 and TH2 responses and are mediators of this regulatory mechanism [9-10]. Attempts to identify bacterial structures in affected tissues have been carried out using several methods with different results. Using the polymerase chain reaction reaction (PCR), several studies have detected DNA from Chlamydia trachomatis, Borrelia burgdorferi. Campylobacter sp., Shigella sp., Sahnonella sp., Yersinia sp and Mycobacterium sp in synovial fluid cells or synovium from adults with recent onset undifferentiated oligoarthritis or ReA. Studies have also been able to dentifiy RNA transcripts of C. trachomatis and B. burgdorferi. However, the search for bacterial DNA in juvenile onset SpA has apparently produced negative results. Because the patients in such studies belonged to population where exposure to arthritogenic bacteria seems low in comparison with that reported from developing countries, an explanation for those findings would be the inclusion of patients with low risk for infection. Thus, the study of populations where infections constitute a major health problem seems attractive. The fact that the bacteria most frequently isolated from children with diarrhoea in Mexico are the same bacteria that trigger ReA, and that SpA in Mexico frequently starts in childhood is interesting [11-12]. Hypothetically, the onset and relapses of SpA in our population could be the result of recurrent infections by arthritogenic bacteria at an early age.
3. INFECTIONS IN THE PATHOGENESIS OF B27-ASSOCIATED DISEASES
Klebsiella pneumoniae has been proposed to be the underlying pathogen causing ankylosing spondylitis. Different studies have shown that the antiKlebsiella antibodies are higher in patients with AS compared to controls. An abnormal T lymphocyte response to Klebsiella infection has been reported, although these studies are difficult to interpret since considerable overlap exists between the response observed among patients and healthy individuals. Actually, data do not support K. pneumoniae or any other infectious agent as a causative pathogen in AS. In the other hand, bacteria clearly have an important role in the development of reactive arthritis. Epidemiological research has demonstrated that in its postenteric form, Shigella, Salmonella, Yersinia, and Campylobacter species are arthritogenic [13-14]. Different components of bacteria can occasionally be detected in joints suggesting that reactive arthritis is caused by an infection inside the joint. Using the polymerase chain reaction (PCR) to detect such bacteria, analysis of synovial fluid has shown that the finding of C. trachomatis is nonspecific for reactive arthritis, since Chlamydia nucleofides can be detected in patients with other rheumatic conditions such as rheumatoid arthritis, psoriatic arthritis, osteoarthritis and inclusive normal subjects. Similar results have been obtained detecting 16S rRNA (a highly conserved sequence found in all bacteria) and nucleotide sequences characteristic of C. trachomatis or B. burgdorferi
[~5]. A general conclusion from these studies is that deposition of bacteria in joints may be a common pathway, and is not unique to patients with reactive arthritis. The lack of specificity of these studies in human synovial fluid suggests that other factors are involved in precipitating and perpetuating disease. It has been proposed the existence of a defect of the innate immune response as a condition responsible for bacterial localization to the joints. Supporting this is the finding of a decreased scavenger receptor in synovial tissue from patients with spondyloarthropathy [ 16]. In order to know if B27 modifies the host response to infection with arthritogenic bacteria, different experiments with cell lines transfected
with B27 have shown that these cells are more resistant to invasion by arthritis-causing bacteria and also are able to survive at a higher rate during several days in culture [ 17]. A conclusion from these studies is that B27 participates and modifies the signal transduction events of host cells. Although the risk of acquiring a bacterial infection does not appear to be significantly increased by the presence of B27, this allele may modify the cellular response in addition to altering the degree of invasion and/or the replication of intracellular bacteria. However, this hypothesis is based upon data derived predominantly from invitro assays, and a better knowledge of these effects in humans is necessary. Different animal models suggest that the HLAB27 gene, and not a closely linked gene, is the arthritis-causing genetic component. However, the presence of HLA-B27 alone is also not sufficient for the development of spondyloarthropathy. A pathogen is required since transgenic animals do not develop arthritis if they are grown in a pathogen-free environment and arthritis-free animals develop arthritis when changed to an environment with infectious agents [ 18]. Genes other than HLA-B27. Although the evidence in humans and in animal models clearly indicate that the HLA-B27 gene is an arthritis-causing gene, its is also evident that other genes such as the presented on Table 1 have a role in the susceptibility to AS [4]. Studies conducted in large populations with different ethnic background will be important to understand their relevance in determining disease susceptibility and severity. Also, some genetic factors may have a protective role against development of spondyloarthropathy or reactive arthritis as is exemplified by the finding of a microsatellite marker close to the gene for intefleukin-10 that is associated with a low risk of development of reactive arthritis [19].
4. ROLE OF T CELLS IN THE PATHOGENESIS OF B27-ASSOCIATED DISEASES The strong association between HLA-B27 and spondyloarthropathies, along with the fact that HLA-
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Table 2. Evidence against the involvement of CD8§ T cells in spondyloarthropathies Experimental evidence
Reference
B27§ mice lacking 132m(with extremely low levels of CD8 T cells) develop inflammatory disease
Khare et al, 1995 (Ref. [20])
TAP-1 knockout mice that express human HLA-B27/132mdevelop inflammatory disease
Khare et al, 2001 (Ref. [21])
CD4§ T cells are more efficient than CD8§T cells in transferring inflammatory disease in adoptive transfer studies
Breban et al, 1996 (Ref. [22])
Synovial fluid T cell clones specific against bacteria (from patients with reactive arthritis) are mostly CD4§
Hermann et al, 1989 (Ref. [23]) Hermann et al, 1990 (Ref. [24])
The proliferative response of synovial fluid T cell clones (from patients with reactive arthritis) specific for bacterium is class H-dependent
Hermann et al, 1992 (Ref. [25])
A very small percentage of synovial fluid CD8§ cells from patients with reactive arthritis recognize bacterium-derived peptides
Kuon et al, 2001 (Ref. [15])
Depletion of CD8ctl3§T cells through thymectomy and anti-CD8 treatment did not reduce arthritis in HLA-B27 transgenic rats
Rheman et al, 2000 (Ref. [26])
B27 is a class I molecule of the MHC (expected to present antigens to CD8 § T cells), has obviously biased the works that deal with the pathogenesis of spondyloarthropathies and other B27-associated diseases. Accordingly, efforts have focused primarily on the theory that postulates that B27 presents bacterium-derived arthritogenic peptides to CD8 + T cells that, as a consequence of molecular mimicry, set up a pathological immune response against self tissues. It is an appealing model (the information that supports it was discussed in earlier sections), nevertheless, solid data granting CD8 § T cells a causal role in B27-related diseases is still lacking. In contrast, information that argues against a pivotal role of CD8 § T cells in the pathogenesis of spondyloarthropathies exists (Table 2). Several murine models in which the B27 heavy chain is expressed in situations that hamper its function have been developed [22]. For example, mice transgenic for B27, but devoid of 152m (~lzm-/-), express the B27 heavy chain, but are unable to assemble B27-[52m-peptide heterotrimers. Thus, they exhibit B27 molecules that, although antigenic, are non-functional, at least for peptide presentation [20]. Likewise, when tapasin or TAP-1 knockout mice are made transgenic for B27, they display empty (lacking the peptide) B27-~2m heterodimers, or B27 homodimers, not capable of presenting endogenous peptides [21]. Even though these ani-
mals exhibit a non-functional B27 molecule, they spontaneously develop a systemic inflammatory disease. This suggests that although HLA-B27 is related (probably causally related) to B27-associated diseases, its mere presence, and not its ordinary function (endogenous peptide presenter to CD8 § T cells), is what contributes to disease development (Table 3). Hermann and coworkers cloned synovial fluid T cells from patients with reactive arthritis secondary to infection with Yersinia enterocolitica, Chlamydia, and Salmonella typhimurium. The bacterium-reactive clones were CD4 +, and antigen-induced T cell proliferation could be inhibited by a monoclonal antibody to HLA class II [23-25]. In a quite different approach, Kuon et al used two biomathematical computer programs, to search for Chlamydia-derived peptides capable of binding to HLA-B27. They found twelve that were able to stimulate synovial fluid-derived CD8 + T cells from B27 § transgenic mice and/or patients with reactive arthritis. Surprisingly however, only 0.1% of synovial fluid CD8 + T cells from patients with .Chlamydia-induced reactive arthritis exhibited the mentioned specificities [15, 30]. Experiments in which T cells or fractions of T cells (namely CD4 + and CD8 § from disease-prone rats (33-3) are transferred into athymic rats (33-3 rnu/rnu) have demonstrated that the CD4 § T cell
Table 3. Evidence that favors a pathogenic role for B27 .
.
.
.
.
.
.
.
.
.
.
Experimental evidence
Reference
B27§transgenic rats and mice develop inflammatory disease
Taurog et al, 1993 (Ref. [32]) Khare et al, 1995 (Ref. [20])
B27/~2m transgenic rats with low level of B27 expression are resistant to the disease development
Breban et al, 1996 (Ref. [22])
TAP-1 knockout mice that express human HLA-B27/~2m develop inflammatory disease
Khare et al, 2001 (Ref. [21])
Disease in B27§ 132m-~-transgenic mice (which express free B27 heavy chains not associated with I]2m)is ameliorated by in vivo treatment with anti-B27 antibody
Khare et al, 1996 (Ref. [36]) Khare et al, 1998 (Ref. [28])
Empty B27 heterodimers (not containing a peptide) are present on the surface of C1R cells transfected with HLA-B*2705
Benjamin et al, 1991 (Ref. [29])
subset is more efficient in transferring inflammatory disease than the CD8 § counterpart (vide infra). Accordingly, CD8 § T cell depletion had no effect in the course of arthritis in HLA-B27 transgenic rats [26]. Interestingly though, when depletion included a thymus-independent CD8+ subset (CD8o~cz T cells), arthritis was ameliorated [31]. In conclusion, there is enough available evidence to consider that, in murine models of B27-associated disease (and probably so in human disease), conventional CD8 § T cells do not play a primary pathogenic role (Table 2). Maxime Breban and coworkers dissected the role of T cells and bone marrow-derived non-T cells in the pathogenesis of the B27/l~2m transgenic rat disease [22]. They used lines of double-transgenic rats that express HLA-B27 and human 132-microglobulin in lymphoid tissue. The rats spontaneously develop a systemic inflammatory disease that shares many clinical characteristics with human B27-associated diseases: arthritis, colitis, enteritis, psoriasiform skin and nail lesions, and genital inflammation. Interestingly, only transgenic lines with a high expression of the B27/l]2m transgenes develop the disease [32, 33]. Breban et al produced B27/132m transgenic nude (rnu/rnu) rats. The resultant athymic transgenic rats remained healthy and disease expression was abrogated (Table 4). By contrast, when bone marrow from transgenic nude rats was transferred to healthy, non-transgenic, syngenic animals, disease was induced. This suggested that HLA-B27 tissue expression is not necessary for
the induction of the disease. In order to evaluate the relative contribution of different subsets of T cells, they reconstituted athymic, double transgenic (B27/human 132m)rats with either CD4- lymph node derived cells, CD8- lymph node derived cells, or non-fracfioned lymph node and spleen derived cells. T cell transfer, whether comprised of T cell subsets or non-fractioned T cells, induced the disease. However, when only CD4- T cells were transferred, disease was milder and appeared later. These experiments, along with the fact that disease expression is abolished in nude rats implied an important role for T cells, especially CD4 § T cells in the pathogenesis of the disease. Nevertheless, the influence of other bone marrow-derived cells had not been clearly established. In order to rule out the possible influence of the recipient's T cells, in the next set of experiments, they transferred transgenic bone marrow cells into thymectomized, lethally irradiated, non-transgenic animals. As expected, disease was induced. However, when they reconstituted the thymectomized, irradiated, non-transgenic recipient with transgenic lymph node cells and non-transgenic bone marrow cells, all the animals remained healthy. This elegant design showed that disease induction is absolutely dependent in B27 expression on non-T, bone marrow-derived cells. Besides, it demonstrated that, although T cells are a facilitating factor for disease generation, their presence is not entirely necessary. This concept was reaffirmed when they transferred T cell depleted transgenic bone marrow into thymectomized, T cell depleted
Table 4. Bone Marrow Transfer Experiments in B27/132mtransgenic nude rats
This table summarizes the experiments described in Ref. [3]. See text for details. Abbreviations: B27/132mTg rat, HLA-B27/~-microglobulin double transgenic rat; BM, bone marrow; 33-3 rnu/rnu, athymic, double transgenic rats; LN, lymph node cells; FL, fetal liver cells.
(with anti-CD5 and anti-ot~-TCR antibody treatment), non-transgenic, syngenic rats. Even though the animals were devoid of T cells (both B27 § and B27-), disease appeared. Moreover, its presentation was earlier and more severe, suggesting that the T cell depletion eliminated a regulatory T cell population. These experiments are summarized in Table 4. The last trials are not congruent with the fact that disease is abrogated in transgenic nude rats. There is no evident explanation for such discrepancy. The number of transferred T cells is an aspect that should be cautiously considered, because when T cells are
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transferred into a lymphopenic host, homeostatic regulatory processes are altered and inflammatory and even autoimmune phenomena may emerge as a consequence of T cell expansion. Thus, the different sizes of the resulting T cell pools in the recipients might be an influencing factor in the different experimental designs [34]. Another drawback in the preceding animal models is that the rat lines do not express HLA-B27 in non-lymphoid cells [32]. Thus, the importance of its expression in affected tissues (e.g. synovial tissue, colon epithelium) cannot be evaluated. An additional limitation is the
lack of information regarding HLA-B27 expression in the thymus and therefore, its contribution in the T cell repertoire selection process (namely positive and negative selection). In spite of the outlined limitations, the work of Breban and coworkers implies that in B27-associated diseases, the presence of B27 in bone marrow-derived cells is of outmost importance, and that T cells, particularly CD4 § probably play an important role. Furthermore, it suggests that other bone marrow non-T cells (e.g. monocytes and/or dendritic cells) probably participate in the pathogenesis of these conditions. The information outlined suggests that B27 § bone marrow derived non-T cells and CD4 § T cells are the most important immunological factors involved in the pathogenesis of B27-associated diseases. However, there is no straightforward link between CD4 § T cells and B27, a class I MHC molecule. Boyle et al investigated this issue in an interesting work, and demonstrated that CD4 § T lymphocytes derived from three B27 § ankylosing spondylitis patients, were able to recognize HLAB27 expressed in different HLA-B27 transgenic cell lines. The recognition was associated with a proliferative response. It could be blocked with antibodies against HLA class I, B27, and CD4, but not by antibodies against HLA class II molecules [35]. Interestingly, B27-reactive CD4 § T cells did not recognize B27 § Epstein Barr virus-transformed lymphoblastoid cell lines, nor U937-B27 cells. The difference between the cells that were and were not recognized by the B27-reactive CD4 § T cells was that none of the former were able to process and display endogenous peptides associated with the B27 molecule (i.e. they displayed either empty B27 heterodimers, or free B27 heavy chains expressed as either monomers or homodimers). Thus, it is possible that CD4 § T cells recognize empty B27 molecules in antigen presenting cells. This is an important matter because cell surface expression of free B27 heavy chains has been demonstrated in B27 transgenic rats [36]. Furthermore, Allen and coworkers found that B27 forms homodimers, and that their assembly is dependent on disulfide bonding through a cysteine residue in position 67. Moreover, the heavy chain complexes appeared to be able bind peptides [37]. Thus, it is feasible to suggest that in vivo CD4 § lymphocytes recognize B27 heavy chain homodimers (either empty or loaded with extracel-
lular-derived peptides).
5. B A C T E ~ A AS TRIGGER OF AN AUTOIMMUNE RESPONSE IN B27ASSOCIATED CONDITIONS The relation between infection and spondyloarthropathies, predominantly reactive arthritis, is well established. In fact, recent infection is considered in the preliminacr classification criteria of the European Spondyloarthropathy Study Group [38]. In B27 transgenic rats, disease is prevented when animals are kept in germ-free conditions. However, when they are moved into conventional facilities, they develop joint and gut inflammation [ 18]. Thus, at least in experimental conditions, the immune system must meet bacteria as a prerequisite in order for the inflammatory disease to ensue. In humans, evidence of previous infection is detectable in approximately 60% of patients with acute reactive arthritides [5]. In one study, evidence of causative pathogens (mostly Yersinia, Salmonella, Chlamydia trachomatis, Borrelia) was identified in 47 to 69% of patients with non-specific oligoarthritis [39]. Nevertheless, in reports of single-source epidemics of reactive arthritis (associated with
Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Shigella flexneri, and Salmonella enterica) the frequency of the condition has been found significantly lower than in hospital series (between 1 and 21%), but the frequency of HLA-B27 is also considerably lower than in hospital series [5]. Likewise, community-based series show that, at the population level, reactive arthritis is mild and has a good prognosis; additionally, its association with HLA-B27 is low [13]. These data suggest that when a group of subjects is exposed to a potentially arthritogenic bacterium, only those who are in some way predisposed (either by HLA-B27 or other factors) develop reactive arthritis. Several bacteria have been associated to reactive arthritis. However, Gram-negative bacteria in the intestinal (Shigella flexneri, Salmonella, Yersinia, Campylobacter jejuni) and urogenital (Chlamydia trachomatis) tracts are the classical triggering infections [5, 12]. Bacterial antigens (from Chlamydia, Yersinia, Salmonella, and Shigella) and DNA (from Yers-
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Table 5. Hypothesis proposed to explain the role of enterobacteria in B27-associated diseases
Hypothesis
Reference
Molecular mimicry of HLA-B27 with enterobacteria
Schwimmbeck et al, 1987 (Ref. [41]) Ringrose, 1999 (Ref. [8])
Presentation of arthritogenic peptides from environmental antigens by HLA-B27 to CD8§T cells
Benjamin et al, 1990 (Ref. [42])
Presentation of HLA-B27-derived peptides by class II molecules
Parham, 1997 (Ref. [44])
Polymorphisms of genes of molecules involved in antigen processing and presentation (Tap-1/Tap-2, proteosomes)
Powis et al, 1992 (Ref. [45]) Pearce et al, 1993 (Ref. [46]) Maksymowych et al, 1994 (Ref. [47])
inia and Chlamydia) have been found in synovial fluid and tissue of patients with reactive arthritis [5, 11]. Likewise, in patients with juvenile onset spondyloarthropathy presence of microbial DNA
(Salmonella, Shigella, Campylobacter, Mycobacterium tuberculosis) has been detected [14]. Further, there is evidence that some patients with ankylosing spondylitis may have synovial fluid cells carrying bacterial antigens [40]. Even so, it is difficult to ascertain the meaning of such findings, because the presence of bacterial products has also been described in other inflammatory arthritides (such as Rheumatoid Arthritis) in which infection lacks a predominant pathogenic role [6]. The mechanisms that lead from gastrointestinal or urogenital infection to the development of an autoimmune response against the joints are largely unknown. Several hypothesis have been proposed (Table 5), but none has completely fulfilled the conceptual and experimental challenges. The information obtained from animal models and from the epidemiological studies mentioned before suggests that infection is a facilitating factor, in some cases a trigger, for the development of B27-associated diseases. A number of studies have demonstrated antigenic similarities between bacteria-derived peptides and the HLA-B27 molecule. For example, Klebsiella nitrogenase enzyme shares hexamer amino acid homology with HLA-B27 [48]. Accordingly, there is cross-reactivity between Klebsiella and HLA-B27 [49]. In an interesting work, Popov et al immunized normal mice with either a cDNA encoding HLAB27, or with HLA-B27 + splenocytes. Subsequently, they obtained immunized splenocytes and tested
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their response against Chlamydia. Response was augmented in lymphocytes from immunized mice, showing that the alloreactive immune response against HLA-B27 resulted in a reduced threshold for CTL-mediated reactivity against Chlamydia [50]. The original model of molecular mimicry proposed that HLA-B27 presents bacterium-derived arthritogenic peptides that activate T cells that attack selftissues. Nevertheless, the evidence discussed before suggests that an immune response against certain bacteria may activate T lymphocyte capable of recognizing HLA-B27 itself. This would explain why CD4 + T cells are more important than CD8 + T cells in the pathogenesis of spondyloarthropathies. Actually, data exist that indirectly support such notion. For example, patients with active AS have increased titers of anti-Klebsiella antibodies [49]. On the other hand, Husby and coworkers have shown that rat antisera against Klebsiella nitrogenase enzyme react with synovial biopsies of HLA-B27-positive patients with AS [51 ].
6. RELATIONSHIP B E T W E E N INTESTINAL I N F L A M M A T I O N AND J O I N T PATHOLOGY Even though in human B27-associated diseases the relation between gut and joint inflammation is not as manifest as in B27 transgenic rodents, it exists, and is probably more significant than usually considered. An important number of patients with inflammatory bowel disease develop musculoskeletal manifestations compatible with spondyloarthropathy (30-40%): inflammatory back pain
in 30%, synovitis or tendinitis in 15%, and sacroiliitis in 20 to 25% [6, 7]. In addition, in one work, endoscopic studies revealed chronic subclinical gut inflammation in 25 to 75% of patients with different subtypes of spondyloarthropathies [52]. The risk for psoriasis is seven times higher in persons with Crohn's disease than in the general population. Likewise, arthritis will develop in as many as 50% of B27 § individuals affected by Crohn disease [12]. The basis for these relations and their link with HLA-B27 are unknown. Nevertheless, there are similarities in the gut mucosa between patients with Crohn disease and spondyloarthropathies: the expression of the adhesion molecule E-cadherin is upregulated, and there is an increased number of CD163 § macrophages [6, 52, 53]. Interestingly, these CD163 § macrophages are also increased in spondyloarthropathy synovia. They express higher levels of HLA-DR than CD163- macrophages. Besides, they produce TNFa, but not IL-10 upon LPS stimulation [54]. Another interesting link between gut and joint immune system is exhibited by the fact that gut-derived lymphocytes from patients with inflammatory bowel disease exhibit abnormal patterns of ~7 integrin expression and are able to bind to synovial vessels using multiple homing receptors [ 16, 55]. Even though it is indirect, the information outlined suggests an underlying defect in the immune system of the gut mucosa of patients with B27associated diseases. It is feasible to hypothesize that the intestinal and urogenital mucosa are the sites were pathogenic T cells are primed, probably during immune reactions against Gram-negative arthritogenic bacteria. Then, owing to the molecular similarities mentioned above, T cells home into the joints and other tissues were they cause the inflammation that leads to the clinically evident disease.
diseases. This assumption holds true also in rats, as shown by breeding experiments. Several of the rat backgrounds tested (Lewis, Fisher, PVG) are permissive to disease expression, whereas one of them (Dark Agouti) confers disease resistance by a mechanism that is independent from MHC [56]. Heat shock protein 70 is polymorphic and is located near to HLA-B. ,am association between two genotypes and the presence of spondyloarthropathy and ankylosing spondylitis has been found in Mexican patients. The association was independent of HLAB27 [56]. Interestingly, in ankylosing spondylitis, disease expression is familial and heritable, yet seems to be independent of factors determining disease susceptibility [57].
8. CONCLUSIONS 9 B27-associated diseases result from a complex interplay of genetic and environmental factors. 9 HLA-B27 is the most important genetic predisposition factor. Its mere presence may be causally related to the disease development. 9 Infectious diseases (mostly involving genitourinary and gastrointestinal tracts) are probably the main environmental triggers of the disease. 9 In summary, the available information supports a model in which B27 + bone marrow derived cells (presumably antigen presenting cells) interact with CD4 § T cells and stimulate an autoimmune response. It is possible that CD4 + lymphocytes recognize B27 heavy chain homodimers, that may be empty, or perhaps loaded with an extracellular-derived peptide. Possibly, CD4 § T cell priming happens in the gut, and somehow the immune response is directed into the joints.
REFERENCES 7. G E N E T I C FACTORS IN B27-ASSOCIATED DISEASES Whereas inbred rats from disease-prone B27 transgenic lines uniformly develop rat-spondyloarthropathy, only a small proportion of human HLA-B27 carriers ever suffer from these conditions. Thus, additional genes and environmental factors are likely to be involved in the human B27-associated
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18. Taurog JD, Richardson JA, Croft JT et al. The germ free state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats. J Exp Med 1994; 180:2359-2364. 19. Kaluza W, Leirisalo-Repo M, Marker-Hermann E et al. IL10.G microsatellites mark promoter haplotypes associated with protection against the development of reactive arthritis in Finnish patients. Arthritis Rheum 2001;44:1209-12. 20. Khare SD, Luthra HS, David CS. Spontaneous inflammatory arthritis in HLA-B27 transgenic mice lacking 132-microglobulin: a model of human spondyloarthropathies. J Exp Med 1995;182:1153-1158. 21. Khare SD, Lee S, Bull MJ, Hanson J, Luthra HS, David CS. Spontaneous inflammatory disease in HLA-B27 transgenic mice does not require transporter of antigenic peptides. Clin Immuno12001;98:364-369. 22. Breban M, Fern~indez-Sueiro JL, Richardson JA et al. T cells, but not thymic exposure to HLA-B27, are required for the inflammatory disease of HLA-B27 transgenic rats. J Immunol 1996;156:794-803. 23. Hermann E, Fleischer B, Mayet WJ, Poralla T, Meyer zum Buschenfelde KH. Response of synovial fluid T cell clones to Yersinia enterocolitica antigens in patients with reactive Yersinia arthritis. Clin Exp Immunol 1989;75:365-370. 24. Hermann E, Mayet WJ, Poralla T, Meyer zum Buschenfelde KH, Fleischer B. Salmonella-reactive synovial fluid T-cell clones in a patient with post-infectious Salmonella arthritis. Scand J Rheumatol 1990;19: 350-355. 25. Hermann E, Mayet WJ, Thomssen H, Sieper J, Poralla T, Meyer zum Buschenfelde KH, Fleischer B. HLA-DP restricted Chlamydia trachomatis specific synovial fluid T cell clones in Chlamydia induced Reiter's disease. J Rheumatol 1992; 19:1243-1246. 26. Rheman MI, Dorris ML, Kaushik P, Satumtira N, Taurog JD. Depletion of CD8 T cells does not prevent the development of spondyloarthropathy (SPA) in HLA-B27 transgenic rats. Arthritis Rheum 2000;43: $395. 27. Sartor RB. Colitis in HLA-B27/132 microglobulin transgenic rats. Int Rev Immunol 2000;19:39-50. 28. Khare SD, Bull MJ, Hanson J, Luthra HS, David CS. Spontaneous inflammatory disease in HLA-B27 transgenic mice is independent of MHC class II molecules: a direct role for B27 heavy chains and not B27-derived peptides. J Immunol 1998;160:101-106. 29. Benjamin RJ, Madrigal JA, Parham P. Peptide binding to empty HLA-B27 molecules of viable human cells. Nature 1991;351:74-77. 30. Turner MJ, Colbert RA. HLA-B27 and pathogenesis of spondyloarthropathies. Curr Opin Rheumatol 2002; 14:
367-372. 31. May E, Satumira N, Dorris ML, Iqbal I, Lightfoot E, Taurog JD. Depletion of all CD8aa+ cells, but not CD8a+I3+ cells alone, attenuates arthritis in HLA-B27 transgenic rats. Arthritis Rheum 2001;44:S 160. 32. Taurog JD, Maika SD, Simmons WA, Breban M, Hammer RE. Susceptibility to inflammatory disease in HLA-B27 transgenic rat lines correlates with the level of B27 expression. J Immunol 1993; 150:4168-4178. 33. Breban M, Hammer RE, Richardson JA, Taurog JD. Passive transfer of the inflammatory disease of HLAB27 transgenic rats by bone marrow engraftment. J Exp Med 1993;178:1607-1616. 34. Stockinger B. T lymphocyte tolerance: from thymic deletion to peripheral control mechanisms. Adv Immunol 1999;71:229-65. 35. Boyle LH, Goodall JC, Opat SS et al. The recognition of HLA-B27 by human CD4 § lymphocytes. J Immunol 2001; 167:2619-2624. 36. Khare SD, Hansen J, Luthra HS, David CS. HLA-B27 heavy chains contribute to spontaneous inflammatory disease in B27/human [~2-microglobulin (132m) double transgenic mice with disrupted mouse 132m. J Clin Invest 1996;98:2746-2755. 37. Allen RL, O'Callaghan CA, McMichael AJ et al. HLAB27 can form a novel 132-microglobulin-free heavy chain homodimer structure. J Immunol 1999;162: 5045-5048. 38. Dougados M, van der LS, Juhlin R et al. The European Spondyloarthropathy Study Group preliminary criteria for the classification of spondyloarthropathy. Arthritis Rheum 1991;34:1218-1227. 39. Weyand CM, Goronzy JJ. Clinically silent infections in patients with oligoarthritis: results of a prospective study. Ann Rheum Dis 1992;51:253-258. 40. Granfors K, Jalkanen S, von Essen R et al. Yersinia antigens in synovial-fiuid cells from patients with reactive arthritis. N Engl J Med 1989;320:216-221. 41. Schwimmbeck PL, Yu DTY, Oldstone MB. Autoantibodies to HLA B27 in the sera of HLA B27 patients with ankylosing spondylitis and Reiter's syndrome: molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease. J Exp Med 1987;166:173-181. 42. Benjamin R, Parham P. Guilt by association: HLA-B27 and ankylosing spondylitis. Immunol Today 1990;11: 137-142. 43. Hermann E, Meyer zum Buschenfelde K-H,Wildner G. HLA-B27-derived peptides as autoantigens for T lymphocytes in Ankylosing Spondylitis. Arthritis Rheum 1997;40:2047-54. 44. Parham P. Presentation of HLA class I-derived peptides: potential involvement in allorecognition and HLA-B27-
associated arthritis. Immunol Rev 1997; 154:137-154. 45. Powis SJ, Deverson EV, Coadwell WJ et al. Effect of polymorphism of a MHC-linked transporter on the peptides assembled in a class I molecule. Nature 1992;357: 211-215. 46. Pearce RB, Trigler L, Svaasand EK, Peterson CM. Polymorphism in the mouse Tap-1 gene. Association with abnormal CD8+ T cell development in the nonobese diabetic mouse. J Immunol 1993; 151:5338-5347. 47. Maksymowych WP, Wessler A, Schmitt-Egenolf M et al. Polymorphism in an HLA linked proteasome gene influences phenotypic expression of disease in HLA-B27 positive individuals. J Rheumatol 1994;21: 665-669. 48. Scofield RH, Warren WL, Koelsch G, Harley JB. A hypothesis for the HLA-B27 immune dysregulation in spondyloarthropathy: contributions from enteric organisms, B27 structure peptides bound by B27, and convergent evolution. Proc Nat Acad Sci USA 1993;90: 9330-9334. 49. Ebringer A. Ankylosing spondylitis is caused by Klebsiella: evidence from immunogenetic, microbiologic, and serologic studies. Rheum Dis Clin North Am 1992;18:105-121. 50. Popov I, De la Cruz C, Barber Bet al. The effect of an anti-HLA-B27 immune response on CTL recognition of Chlamydia. J Immuno12001;167:3375-3382. 51. Husby G, Tsuchiya N, Schwimmbeck PL et al. Cross-reactive epitope with Klebsiella pneumoniae nitrogenase in articular tissue of HLA-B27+ patients with ankylosing spondylitis. Arthritis Rheum 1989;32: 437-445. 52. Mielants H, Veys EM, Cuvelier C et al. Ileocolonoscopic findings in seronegative spondyloarthropathies. Br J Rheumatol 1988;27:$95-S 105. 53. Demetter P, Baeten D, De Keyser F et al. Subclinical gut inflammation in spondyloarthropathy patients is associated with upregulation of the E-cadherin/catenin complex. Ann Rheum Dis 2000;59:211-216. 54. Demetter P, Baeten D, De Keyser F et al. Increased number of macrophages in non-inflammed colon of patients with spondyloarthropathy, a human model of early Crohn's disease. Gastroenterology 2000;118: A342. 55. Van Damme N, Elewaut D, Baeten D et al. Gut mucosal T cell lines from patients with ankylosing spondylitis are enriched with a t~EI]7 integrin. Clin Exp Rheumatol 2001; 19:681-687. 56. Salmi M, Jalkanen S. Human leukocyte subpopulations from inflammed gut bind to joint vasculature using distinct sets of adhesion molecules. J Immunol 2001;166: 4650-4657. 57. Breban M, May E. Treatment of the SpA-like disease
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Celiac Disease and Infection Shimon ReiP and Aaron Lemer 2
~Division of Pediatric Gastroenterology, Dana Children s Hospital Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv; 2Department of Pediatrics, Carmel Medical Center, Rappaport Faculty of Medicine, Technion Israel Institute of Technology, Haifa, Israel
1. I N T R O D U C T I O N Gluten-sensitive enteropathy, otherwise known as celiac sprue, is characterized by an abnormal proximal small intestinal mucosa arising as a result of an inappropriate inflammatory response to ingested gluten antigens present in wheat in genetically susceptible individuals. This immune response was recently found to be directed to a 33mer peptide of the gliadin component of gluten. Elimination of gluten, the cornerstone of treatment for CD, may dramatically influence the course of the disease and prevent complications, among them osteopenia, malignancy, and miscarriage [1]. With the recent introduction of newer and more precise serologic tests, such as antibodies against tissue transglutaminase (tTG), significant progress has been achieved, not only in exploring the genetic basis, clinical course and epidemiology of the disease but also in understanding its pathogenesis. Although CD is considered an immunologic disease, infection may play role in several aspects of CD. This chapter will elaborate about the possible role of infection in the pathogenesis of CD [2, 3].
2. E V I D E N C E F R O M CLINICAL PRESENTATION In the past, CD had been considered a disease of childhood that was usually diagnosed before the age of 2 years and presented with symptoms of diarrhea, malabsorption and weight loss following the introduction of cereals into the infant diet. Adults and
adolescents often present atypically, with vague and non-specific symptoms or problems that at first glance appear unconnected to an intestinal pathology. The variety of symptoms, signs, and severity of CD has originated the concept of the celiac iceberg with those patients with typical complaints, usually relating to the gastrointestinal tract, floating above the surface. Many more as yet undiagnosed patients with minimal or no symptoms latent CD, or with atypical ones make up a silent CD, unseen majority hidden below the surface [4]. The most common presentation is diarrhea, although constipation may occur. Hematologic abnormalities such as macrocytosis, combinations of iron, folate, and vitamin B12, Howell-Jolly bodies and other features of hyposplenism, prolonged prothrombin time, and the appearance of ecchymoses are not uncommon. Another group of patients presents with general lethargy, weight loss, and glossitis. Metabolic or endocrine problems may predominant and include short stature, relative infertility affecting both sexes, recurrent miscarriage, delayed puberty and altered menopause, as well as osteoporosis and osteomalacia. Less common are mental health disturbances such as depression, or even psychosis. In general, no increase rate of infection or predisposition to infectious diseases was reported in CD. However, in it can occur as a consequence of malabsorption due to hypoproteinemia, or to secondary immune deficiency, as well as related to hyposplenism or IgA deficiency associated with CD [5].
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3. EVIDENCE FROM ALTERATION IN THE INCIDENCE OF CD
CD was believed to affect 1:1500 individuals throughout Europe, with a higher prevalence (1: 150) in Ireland, and lower in USA and northern Europe [6]. Recent serologic screening of normal populations, however, has yielded prevalence between 1:100 1:300 with widespread occurrence of CD worldwide [7 9]. The increased rate of detecting CD may be attributed to the more sensitive and accurate newer serologic tests such as antigliadin, antiendomysial and antitransglutaminae antibodies. However, this huge epidemiologic drift in the incidence of CD led to the concept that apart from gluten sensitivity in genetically predisposed persons, there is also interaction with environmental factors in CD. Breast feeding, and the timing of the commencement of gluten ingestion [10], viral infection that promote the secretion of interferon [11], and smoking [12] are some factors that might contribute to disease occurrence. Finally, the seasonal variation in the incidence of CD also indicates a role of infectious agent in the triggering of CD [13].
4. EVIDENCE FROM THE PATHOGENESIS OF CD
The currently accepted theory of the pathogenesis of CD is that susceptible people, e.g., HLA type DQAl*0501 and DQB 1"0201, exhibit an aberrant response to dietary gluten, and that the resulting small intestinal damage is caused by locally activated CD4 § T-lymphocytes. Glutensensitive T lymphocytes recognize gluten-derived peptides epitopes when presented in association with DQ2. Activation of these normally silent CD4 § T-lymphocytes triggers a T-helper type 1 pattern of cytokine production, including the release of IFN- and leading to mucosal damage [3]. Glutamine is the most abundant amino acid in gliadin, making up 35% of its composition, and it may be central to the toxic effect of these proteins in celiac patients. The tTG enzyme selectively deamidates gluten protein glutamine to glutamic acid. The introduction of negative charges results in peptides that bind with relatively high affinity
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to the disease-associated HLA-DQ2 or HLA-DQ8 molecules [ 14, 15]. In addition to this process, tTG increases their stimulatory effect on gluten-sensitive intestinal T lymphocytes and exposes neoepitopes in wheat proteins. Alternatively, when presented in association with DQ2, it can stimulate peripheral blood T-lymphocytes. In an attempt to find the precise structure of the toxic element of gluten, a single epitope of gliadin was recently recognized as the dominant epitope recognized by Tlymphocytes in patients with CD [16]. This 33-mer peptide is stable in-vivo and in-vitro to breakdown by all gastric, pancreatic and intestinal brush-border membrane peptidase, and it reacts with tTG by deamination of specific glutamine residues within this sequence which is essential for HLA-DQ2 binding and subsequent T lymphocyte stimulation [ 14, 15]. Interestingly, the primary sequence of the 33-mer gliadin peptide also had homologues among a few nongluten proteins. Among the strongest homology was internal sequence from pertactin (a highly immunogenic protein from Bardotella pertusis). This homology to pertactin could have a biologic relevance. Thus, the region in pertactin that is homologous to the 33-mer gliadin peptide is known to be part of the immuno-dominant segment of the protein and it elicits a vigorous antibody response because it reacts with tTGase on the extracellular surface of antigen presenting cells to produce a long-lived intermediate that is internalized via endocytosis and presented to the immune system via the class II MHC-mediated pathway. Therefore, tTGase-mediated endocytosis might be a highly effective mechanism for oral tolerance with the use of immunogenic peptide epitopes such as pertactin as long as they are resistant to intestinal peptidase breakdown [ 16]. Another implication of the pathogenesis of CD focused on gluten-reactive T cell lines or clones and the destructive T helper cells (TH1) reaction induced by immunodominant gluten peptides. Still we poorly understand mechanism of T cells anergy or active suppression that are crucial to distinguish between beneficial nutritional and microbial antigens and detrimental antigens in the gut. Key immune regulators are dendritic cells potent antigens-presenting cells that can prime T cells not only for destruction, but also for tolerance or anergy. In addition, the rout and sequence of antigen supply
determine whether T cell response will be destructive or suppressive, as illustrated by experimental autoimmune neuritis. This autoimmune disease can be induced in mice by subcutaneous vaccination with an oligomeric immunodominant P2 peptide, in contrast the same peptide after intravenous or oral application prevents or even reverses established disease. Besides dendritic cells and suppressor T cells biology in CD, the small intestinal microbial milieu, which may alter epithelial permeability, admitting an influx of gluten and other antigens may play a role [ 14]. Thus, celiac disease is considered a T-cell mediated chronic inflammatory bowel disorder with an autoimmune component. Loss of tolerance to gluten is a possible cause. Patients with CD develop anti gluten T cell response in the intestine [17]. The reason for this occurrence is obscure; however, changes in intestinal permeability secondary to alteration in intercellular tight junctions or in the processing of gluten are potential mechanism [17, 18]. It is plausible to speculate that infection by changing intestinal permeability can be the first event initiating the inflammatory response in genetically predisposed individuals.
5. CELIAC AS AN AUTOIMMUNE DISEASE AND THE ROLE OF INFECTION Whether CD is an inflammatory disorder with secondary autoimmune reactions or whether it is a primary autoimmune disease induced by an exogenous factor such as an infection remains unclear. Autoimmune disorders arise ten times more often in patients with CD than they do in the general population. Such disorders include, Type I diabetes, thyroid disease, Addison s disease, autoimmune chronic hepatitis, neurologic disorders, IgA deficiency [19 22]. The association of autoimmune disorders and CD is thought to be related to a shared genetic tendency (HLA alleles) and a common immunologic mechanism in addition to the presence of CD itself. Results from several studies suggest that autoimmune diseases might be prevented by early diagnosis and treatment of CD, and that in those with established autoimmune disorders, gluten free-diet could offer a chance of improvement in symptoms [21].
The pathogenesis or the trigger for onset of an autoimmune disease is obscure. It can be speculated that in CD, as in other autoimmune diseases, a trigger of the immune system to develop CD may result from an event occurring when the immature system is vulnerable, both tolerance and intolerance to various antigens may be induced. Viruses may initiate the autoimmune process either by direct mimicry of antigens or by affecting the immunoregulatory system [23]. Viruses that primary replicate in gut associated lymphoid tissue may be considered as pathogenic in CD [24].
6. SPECIFIC INFECTIOUS AGENTS RELATED TO CD The association between of CD with adenovirous (serotype 12) is particular interesting. One of the virus s proteins, Elb protein, demonstrates similar antigenic sequencing of amino acid shared with gluten peptides [25]. However, no evidence of persistent virus DNA at least in duodenal mucosa was found by molecular biology techniques [26, 27]. Infection with adenovirus and subsequent exposure to gliadin could trigger the development of CD resulting from cross-reacting immune reactions. This observation is only experimental and is far from providing substantial conclusion. Intrauterine virus infection has been suggested in immune mediated diseases. For example, intrauterine rubella virus and enterovirus infection have been reported to increase the risk of type I diabetes in the offspring [28, 29]. In particular, viruses that replicate in the gut-associated lymphoid tissue (like enteroviruses) might alter the cytokine milieu and favor immunization to dietary gluten. To explore this possibility, mothers of children who developed CD were studied for an increased enterovirus exposure during pregnancy. No such correlation could be determined [30].
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intolerance in celiac sprue. Science 2002;297:2275 9. 17. Sollid LM, Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunoll 2002;2:647 645. 18. Fassano A, Not T, Wang W. Zonulin, a newly discovered modulator of intestinal permeability, and its expression in celiac disease. Lancet 2000;335:1518 1519. 19. Counsell CE, Taha A, Ruddel WS. Coeliac disease and autoimmune thyroid disease. Gut 1994;35:844 846. 20. Ventura A, Magazuu G, Greoc L. Duration of exposure to gluten and risk of autoimmune disease in patients with celiac disease. Gastroenterology 1999;117:297 303. 21. Ventura A, Neff E, Ughi C, Leopaldi A, Citta A, Nott T. Gluten dependent diabetes-related and thyroid-related autoantibodies in patients with celiac disease. J Pediatr 2000;137:263 265. 22. Lerner A, Blank M, Lahat N, Shoenfeld Y. Increased prevalence of autoantibodies in celiac disease. Dige Dis Sci 1998;43:723 726. 23. Kagnoff MF, Austin RK, Hubert HK, Kasarda DD. Possible role for a human adenovirus in the pathogenesis of celiac disease. J Exp Med 1984;160:1544 1557. 24. Lahdeaho ML, Parkonen P, Renuala T, Maki M, Lehtinen M. Antibodies to E1 b protein derived peptides of enteric adenovirus type 40 are associated with celiac disease and dermatitis herpetiformis. Clin Immunol Immunolpathol 1993;69:300 305. 25. Kagnoff MF, Paterson YJ, Kumar PJ. Evidence of the role of a human intestinal adenovi_nas in the pathogenesis of celiac disease. Gut 1987;28:995 1000. 26. Lawler M, Humpheries P, O Farrelly C. Adenovirus 12 E1A gene detection by polymerase chain reaction in both normal and celiac duodenum. Gut 1994;35:1226. 27. Carter MJ, Wilcocks MM, Mitchison HC, Record OC, Madeley CR. Is a persistent adenovirus infection involved in Coeliac disease. Gut 1989;30:1563 1567. 28. Forrest JM, Menser MA, Burgess JA, High frequency of diabetes mellitus in young adults with congenital rubella. Lancet 1972;11332 334. 29. Dahlquist GG, Ivarsson S, Lindberg B, Forsgeran M. Maternal enteroviral infectionduring pregnancy as arisk factor for childhood IDDM. Diabetes 1995;44:408 413. 30. Carlsosn AK, Lindberg BA, Brendberg ACA, Hyotoy H, Ivarsson SA. Enterovirus infection during pregnancy is not a risk factor for celiac disease in the offspring. J Pediatr Gastroenterol Nutr 2002;35:649 652.
9 2004 Elsevier B. V. All rights reserved.
Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infections Associated with Retinal Autoimmunity John J. Hooks ~, Barbara Detrick z and Robert Nussenblatt ~
1Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD, USA; 2Department of Pathology, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA
1. INFECTIONS & AUTOIMMUNITY IN THE RETINA The topic of immune-mediated vision loss, with an emphasis on autoimmune reactivity and autoimmune disease in the eye, is a rapidly expanding area of research and therapy. Numerous studies in other body sites have clearly identified links between infections and autoimmunity and autoimmune disease [4, 5]. Only a limited number of studies have been reported in which retinal disorders have been evaluated to study this relationship. We will begin this chapter with a brief overview of infection and autoimmunity in the eye. This will be followed by specific examples of infections and autoimmunity in the retina. We will highlight two human diseases triggered by Onchocerca volvulus or Toxoplasma gondii and an experimental model referred to as experimental coronavirus retinopathy (ECOR), triggered by the murine coronavims, mouse hepatitis virus (MHV).
2. THE EYE: I N F E C T I O N AND AUTOIMMUNITY
The visual axis is a precious sense. The eye is an organ that is known to have immunologic processes that are both infectious and non-infectiously driven. The eye is unique in that it lacks lymphatics and still enjoys an intimate relationship with the immune system. An inflammatory process in the eye is termed an uveitis, and it in no way reflects the origin of the inflammatory process nor where it is located in the eye. While there are many descrip-
tions of inflammatory processes in the eye, there are three major presentations of these conditions. If the inflammatory condition is centered in the front of the eye, the process is termed an anterior uveitis. If upon examination the dominant part of the inflammation is centered in the vitreous of the eye, it is termed an intermediate uveitis. Finally, if inflammation occurs in the back of the eye, that is centered in the retina or the choroid of the eye, it is termed a posterior uveitis. Clearly inflammatory conditions may involve several parts of the eye, and if all anatomic of the eye are involved it is termed a panuveitis. Most of the cormqaents of this chapter will address disorders of the back of the eye, i.e., those involving the retina. Eye specialists have the great advantage of being able to visualize directly the parts of the eye that can be involved in an inflammatory process. In addition to simple visualization, many additional tools can be readily applied. Electrophysiologic testing is easily and frequently performed, This is an excellent way to evaluate the retina's ability to react to a light stimulus. Fluorescein angiography, the use of dye injected into an arm vein and then rapid photographs are taken of the back of the eye. This approach helps to visualize the vascular system and the integrity of the retina. The severity of the inflammatory response can be graded by direct visualization of the inflammatory response in the eye. Most i n f l a m m a t o r y p r o c e s s that w e r e c o g n i z e w i l l h a v e a
cellular response associated with it. We also know that antibody mediated pathology, as seen in such entities as cancer associated retinopathy, can occur, but appears to be the distinct minority of cases. The eye is a complex organ from the point of
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view of the immune system. It is known that antigen placed into the anterior chamber of the eye will induce a deviated immune response, with a marked decrease in cell mediated responses, but an intact cytotoxic and B-cell response is seen [ 1]. Addtionally, the retina is a complex structure with several layers needed to turn the light stimulus into a chemical signal ultimately sent to the brain. At the photoreceptor level and the single layer just below it, the retinal pigment epithelium, a number of uveitogenic antigens have been identified and characterized [2]. Two antigens in particular, the retinal S-antigen and the interphotoreceptor retinal binding protein (IRBP), have been used to develop a model of autoimmune ocular disease which is termed experimental autoimmune uveoretinitis (EAU) [3]. This model has many qualities of the disease seen in humans and has helped to better understand the underlying mechanisms that lead to disease. One major difference between this model and the human disease is of course that it is not spontaneous. It is not clear what triggers the human disease. This chapter will explore one such trigger, that of ocular infection. Several entities, some based on animal models, other seen in the clinic, will be discussed to elucidate the possible role between infection and autoimmunity.
3. EXPERIMENTAL CORONAVIRUS RETINOPATHY Experimental coronavirus retinopathy (ECOR) is an animal model system that we generated in the 1990's to demonstrate that a virus can trigger a progressive retinal degenerative disease [6]. Studies during the past 12 years have identified that this degenerative eye disease is composed of three basic components; a virus component, a genetic component and an immunologic component [7, 8]. In our system, we selected a naturally occurring neurotrophic strain (JHM) of a mouse hepatitis virus that infects and persists within the retina. The virus causes an acute infection, marked by virus replication in distinct retinal cells, neutralizing antibody and the production of cytokines, namely IFN-gamma. This disease also has a genetic component. That is, different strains of mice behave differently after virus infection. Two strains of mice, BABL/c and CD-1, were extensively
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studied after coronavirus infection. During the early phase of the disease (day 1-8) the virus infects and replicates within the retina of both BALB/c and CD- 1 mouse stains [9]. However, on days 10 to 140, only the BALB/c mice experience a late phase of the disease which is marked by a retinal degeneration. The CD-1 mouse does not undergo the retinal degenerative phase but rather, the retina returns to a normal architecture within 20 days. Finally, the immune component of this disease is characterized by the presence of autoantibodies, specifically, anti-retinal and anti-RPE auto-antibodies. The presence of these antibodies are observed only in the retinal degenerative susceptible BALB/c mice. These auto-antibodies are absent in the retinal degeneration resistant CD-1 mice. In summary, ECOR is a virus triggered retinal degenerative disease that is influenced by both genetics and the immune response. In this session we will discuss in detail the virologic, pathologic, immunologic, genetic and autoimmune factors involved in this model system.
3.1. Virologic Component of ECOR Coronaviruses are large, enveloped, positive strand RNA viruses that cause significant diseases in a number of animal species and humans. In animals, coronaviruses are responsible for important diseases of livestock, poultry and laboratory rodents. Until recently, man was known to be infected with two strains of coronavirus. Either of these strains are responsible for approximately 50% of the common colds. A new human coronavirus has been identified as the causative agent for severe acute respiratory syndrome (SARS) [12]. One of the closest relatives to the human S ARS-Coronavirus is the murine coronavirus, mouse hepatitis virus (MHV). The JHM strain of MHV is the most thoroughly studied neurotropic coronavirus. It causes both acute and chronic central nervous system effects in mice and rats. Acute encephalomyelitis and chronic CNS disease have been observed in mice. In rats an autoimmune disease known as subacute demyelinating encephalomyelitis, has been described. Initial studies in the ECOR system, showed that inoculation of this JHM strain into the vitreous or anterior chamber of BALB/c mice resulted in retinal tissue damage [7, 8]. Infectious virus could be
detected within the retina between 1 and 6 days post inoculation (PI), reaching a peak level of 10 4.5 pfu/ ml at day 3 [13]. Virus antigen was also identified within the retina between day 2 and 6 PI [8]. Virus antigen was first detected within the RPE cell and the ciliary body epithelial cell at day 2 and this virus replication intensified at day 3 and 4. Between day 3 and 6 virus antigen was also detected in Mullerlike cells that span the multiple layers of the neural retina. Occasionly, virus antigen was also observed within the ganglion cells. After day 7, infectious virus and viral antigen could not be detected within the retina. However, in situ hybridization studies identified that the viral RNA persisted within the retina until 60 days PI [14]. Anti-virus neutralizing antibodies were first noted at day 7 PI [ 13] and coincided with the disappearance of infectious virus and viral antigen.
3.2. Retinal Pathology in ECOR After inoculation with JHM virus, two distinct patterns of retinal pathology were noted in the BALB/c mice [8]. The early phase of the disease, day 1 to 8, was characterized by retinal vasculitis and perivasculitis. The late phase of the disease, after day 10, was characterized by retinal degenerative changes. The retinal layers revealed disorganization with large areas of outer and inner segment loss. In addition, the RPE cells were morphologically abnormal with focal RPE cell swelling or proliferation, or with focal RPE cell atrophy or loss. Analysis of retinal cell function also revealed dramatic changes [15, 16]. There was a significant decrease of complete loss of electroretinogram (ERG) patterns and the disappearance of an important transport protein in the retina, the interphotoreceptor retinoid-binding protein (IRBP).
3.3. Host Response in ECOR The host immune response to this virus infection was evaluated by tracking the cellular infiltrate and identifying the cytokine profile within the retina [10]. The most prominent infiltrating cell was the macrophage. MAC-1 staining was detected in 100% of the eyes at day 6 and 10 PI, and was occasionally seen on day 20 and beyond. The second most prominent cell was the T cell. CD4 T cells were present
in the retina at days 3 and 6. This was followed by a shift to CD8 T cells which were observed at day 6 and 10 PI. A low number of CD8 T cells were still noted at day 20 PI. B cells and NK cells were not detected. During the course of the disease, cytokine profiles were studied by evaluating retina tissue and sera [10]. Analysis of pooled retinal mRNAs from untreated, mock-injected and virus infected BALB/ c mice revealed the presence of IL-6, IFN-y and TNF-~ mRNAs in virus infected retinas isolated during the acute disease, day 4 and day 8 PI. Gene expression for these cytokines was not detected in retinas from untreated or mock-injected mice. EIA analysis of sera identified the presence of these same cytokine proteins in virus infected mice and not in untreated or mock injected mice. The presence of retinal mRNA for IFN-y was also associated with the upregulation of MHC Class I and II molecules within the retina. MHC class I and II molecules were not identified within the normal or mock injected retinas. It was noted that the first cell to express these MHC molecules was the RPE cell. This cell is also the first cell to express new viral antigens during the infection in vivo and is persistently infected in vitro [ 17]. It is critically important to point out that this RPE cell has been shown to process and present retinal and non-retinal antigens to sensitized T cells and is upregulated to express MHC class II molecules during retinal autoimmune and degenerative processes [ 18, 19].
3.4. Genetic Factors in ECOR The genetic constitution of the host can be a critical factor in determining the outcome of a virus infection [9]. We therefore evaluated the possible role of host genetics in ECOR. We inoculated selected strains of mice and evaluated the retinal disease. BABL/c, C57B1, A/J and CD-1 mice were studied. When C57B1 and AJJ mice were evaluated, we observed a disease pattern similar to that seen in BALB/c mice. However, retinal changes were less severe than those seen in BALB/c mice. Retinal tissue damage induced by JHM virus in CD-1 mice was very different (Table 1). Only the early phase of the disease, consisting of retinal vasculitis, was observed. These CD-1 mice did not develop the retinal degenerative disease. In fact, by day 20 PI,
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Table 1. Retinal inflammation and retinal degenertation in mice inoculated with murine coronavirus (JHM strain)
Retinal Disease
Day
BALB/c Mice
CD- 1 Mice
Positive / tested Inflammation (Vasculitis)
Degeneration
(%)
Positive / tested
0 / 30
0
0 / 20
1-7 10-45
26 / 26 0 / 30
100 0
20 / 20 0 / 20
0 1-7 10--45
0 / 30 0 / 26 30 / 30
0 0 100
0 / 20 0 / 20 0 / 20
0
(%) 0 100 0
Table 2. Anti-retinal antibody production and retinal degeneration in coronavirus inoculated mice
Mouse
Treatment
Autoantibody in Retinal Tissue Positive / Number tested
Retinal Degeneration
BALB/c
untreated Mock injected JHM Virus
0 / 20 0 / 15 22 / 22
0/20 0/15 22 / 22
CD-1
untreated Mock injected JHM Virus
0 / 15 0 / 15 0 / 20
0/15 0/15 0/20
the retina had a normal appearance. These studies underscored the role of genetics in ECOR and showed that the genetics of the host profoundly affected the nature of retinal tissue damage. Since the CD-1 mice did not exhibit the late retinal degenerative phase of the disease, we evaluated a variety of parameters and compared the findings with the data obtained in BALB/c mice. For example, during the acute phase of the disease (days 3 10), virus load in the retina, production of anti-virus antibody, blood-retina barrier breakdown, lymphoid trafficking and MHC Class I and II staining were similar in both mouse strains. Moreover, gene expression for IFN-~, in pooled retinas from CD-1 mice was positive at PI day 4 and 8. Again, this is similar to the pattern observed in BALB/c mice. The disease pattern in the late phase was clearly different in the two mouse strains. BALB/c mice displayed a retinal degeneration with blood-retina barrier breakdown, and CD-1 mice showed a normal retinal architecture. Immuno-staining observed at
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day 20 in CD-1 mice was clearly different from that observed in BALB/c mice, in that CD-1 mice were negative for MHC class I and II expression and CD8 T cells were absent from the retina. 3.5. A u t o i m m u n e C o m p o n e n t of E C O R
In ECOR, the late phase of the disease was associated with the lack of direct evidence for viral replication within the retina. This observation suggested that the continued degenerative process may be associated with alterations directly induced by virus replication during the first few days after infection or it may be associated with additional factors. Inasmuch as viruses are know to trigger an autoimmune phenomena and some human retinopathies may be associated with autoantibody formation, we studied the possible production of antiretinal autoantibodies [ 11]. We found that the retinal degenerative process in BALB/c mice was associated with the presence of antiretinal autoantibodies (Table 2). These
Figure 1. Anti RPE cell autoantibodies. A & B are immunoperoxidase staining showing anti RPE cell autoantibodies. Frozen sections of normal rat eyes were incubated with (A) normal mouse sera or with (B) sera from JHM virus-infected mice (day 15) (1:40 dilution). C & D are immunofluorescent staining showing RPE cell autoantibodies. Cytospin preparations of freshly isolated rat RPE cells were incubated with (C) sera from mock-injected BALB/c mouse (day 10) (1:40 dilution) or with (D) sera from JH]VI virus infected BALB/c mouse (day 10) (1:40 dilution). Arrows indicate areas of positive staining. autoantibodies were not found in sera from normal or mock-injected mice. The presence of antibodies to retinal tissue was evaluated by immunoperoxidase staining on frozen sections of normal rat eyes. Two patterns of staining were observed, reactivity in the neural retinal and reactivity in the retinal pigment epithelium (RPE) (Fig. 1). The antiretinal autoantibodies first appeared as IgM class antibodies. This was later replaced by IgG class autoantibodies. The
anti-RPE cell autoantiboedies were predominantly of the IgG class. As stated above, JHM virus infected CD-1 mice developed a retinal disease that is different than the retinal disease observed in BALB/c mice. In CD-1 mice, only the early stage, consisting of retinal vasculitis was seen. The CD-1 mice recovered and were not susceptible to the later phase of the disease, the retinal degenerative disease. We therefore, evaluated
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the development of a retinal degenerative disease and the development of antiretinal autoantibodies in these two strains of mice after inoculation with JHM virus [11]. The data summarized in Table 2 shows that all of the BABL/c mice developed antiretinal antibodies and developed pathologic changes consistent with retinal degenerative processes. In contrast, none of the CD-1 mice developed antiretinal autoantibodies. That is, in those mice that failed to develop antiretinal autoantibodies, they also failed to develop a retinal degeneration. These findings suggest a role for autoimmunity in the pathogenesis of ECOR.
4. TOXOPLASMOSIS (T. GONDII) Toxoplasmosis is a disorder that has a worldwide distribution. It is caused by the obligate intracellular parasite, Toxoplasma gondii. Over 500 million people are believed to have the disease. In 1908, the organism was first described in the brain of the North African rodent, the gondii, by Nicolle and Manceaux [20] and by Splendore in a rabbit [21 ]. The first connection between this organism and human disease was made by Janku [22]who described the presence of the organism in a child who died of disseminated toxoplasmosis. While suspected for a long period, it was not till the early 1950's that the parasite was shown to cause ocular disease. Helenor Campbell Wilder, working at the Armed Forces Institute of Pathology in Washington, DC, identified the organism in eyes that were believed to have other types of inflammatory processes, particularly tuberculosis [23]. It is interesting to note that a similar observation has been made more recently in Nepal, where many cases of ocular tuberculosis have now been rediagnosed as toxoplasmosis of the eye. The cat (and perhaps related species) appears to be the definitive host. The sexual cycle is one of schizogony and gametogony leading to the the development of toxoplasma oocysts, which are 10-12 ~m in size and are found uniquely in the intestinal mucosa of cats. Two forms of the organism can be found in man, cysts and tachyzoites. The tachyzoites (the proliferative intracellar form) are believed to be the cause of most of the tissue damage in human, though often it is very difficult to demonstrate the presence of the stage of the
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organism. The bradyzoites (the latent form of the organism found in cysts) are found in host cells. Hundreds of bradyzoites (with very slow metabolic rates) have a propensity towards neural tissue such as the eye and brain, but are also found in skeletal muscle and heart. It is assumed that attacks occur with rupture of the cyst, leading to a pouring out of bradyzoites and then the conversion of bradyzoites to tachyzoites. The mechanisms that lead to cyst rupture are still unknown. 4.1. Clinical Features While the hallmark of the disease is changes in the posterior portion of the eye, changes in the front of the eye are also noted. An anterior uveitis can be seen in many patients with this disorder. This is an interesting finding since the organism is not seen in the anterior segment of the eye except possibly in immunocompromised individuals. Additionally, there is a loss of pigment in the iris that can be seen and is associated with changes in the back of the eye [24]. This finding, termed Fuch's heterochromia, is thought to be an autoimmune phenomenon. The classic finding in ocular toxoplasmosis is that of a retinal lesion, which is destructive. It is typically an oval lesion where all layers of the retina and frequently many layers of the choroid have been infected. It is the result of an immune response believed to have occurred against the toxoplasma organism. While there may be only one lesion, often there are multiple lesions surrounding an old large scar, and these are called satellite lesions. In addition to the lesion itself, during the active stage of the disease, evidence of retinal vascular leakage is seen. It has been hypothesized that this vasculitis is due to an immune complex related phenomenon. Typically while stigmata of the disease may be present in both eyes, recurrences of the disorder occur only in one eye. Additionally, while reactivation of the disease is believed due to the breakage of cysts and the presence of tachyzoites, it is rare to see this stage of the organism in the retina. Patients who are immunocompromised, such as those with AIDS, will often have bilateral disease and multiple lesions, suggesting a different mechanism in these patients as compared to the immunocompetant patient.
4.2. Evidence for Autoimmunity Abrahams and Gregerson [25] evaluated five patients with ocular toxoplasmosis. This longitudinal study measured serum antibody responses to the retinal S-antigen, a "P" antigen (thought to contain rhodopsin), and new antigen designated p59 ag, all isolated from bovine retina. They reported that all the patients initially tested showed antibody responses to all three antigens. The anti-S-antigen responses tended to decrease with clinical improvement, while the anti-P antibodies remained high even after the acute attack was over. A more recent report by Whittle and colleagues [26] looked at a larger number of toxoplasmosis patients. In this study a total of 36 patients with toxoplasma retinochoroiditis were evaluated for evidence of anti-retinal antibodies. Thirty-four of these sera showed antibodies directed against the photoreceptor layer of the retina when tested with indirect immunofluorescence. Six of 16 controls showed a similar staining pattern with the p value as reported as < 0.001. Interestingly, using an EIA to measure the presence of anti-S-antigen antibodies, the researchers observed that 27 of 36 sera from toxoplasmosis retinochoroiditis patients were positive, but so were 10 of 16 normals, with a calculated p value of greater than 0.05. The antibodies seen in the two assays did not appear to run in parallel. The author's interpretation of their data was that the extent of anti-retinal antibodies could not be explained by anti-S-antigen findings alone. They argued that these findings probably did not reflect an epiphenomenon since patients with idiopathic retinal vasculitis were also evaluated. In that study the number of sera positive from patients with idiopathic retinal vasculitis was considerably lower than that found in the toxoplasmosis group. Our group has had the chance to evaluate cell mediated responses of lymphocytes from patients with ocular toxoplasmosis. In a very early study [27] in which we looked at proliferative responses from patients with all kinds of uveitic conditions, we reported that a small number of ocular toxoplasmosis patients' lymphocytes did respond to the uveitogenic retinal S-antigen. In a later study, we evaluated the proliferative cell mediated responses in 40 patients with ocular toxoplasmosis. In addition to the retinal S-antigen, we also evaluated the response to crude toxoplasma antigen and to puri-
fled antigens from the parasite [28]. In addition, we performed an EIA to look for anti-S-antigen antibodies and HLA phenotyping to see if a specific HLA type was associated with S-antigen responsiveness. Of the 40 patient's lymphocytes tested, 16 (40%) had proliferative responses with a stimulation index above 2.5 (see Fig. 2). There appeared to be no correlation with this responsiveness and any HLA phenotype. Additionally, we were unable to demonstrate anti-S-antigen antibodies using EIA. The ocular toxoplasmosis patients could be divided by their lymphocytes responsiveness to the various toxoplasma antigens tested. However, no correlation was seen in S-antigen responsiveness and the stimulation index to toxoplasmosis antigens.
5. O N C H O C E R C I A S I S Infection with the nematode parasite Onchocerca volvulus can result in severe eye disease, often referred to as fiver blindness. It is estimated that approximately 18 million people in tropical Africa, the Arabian peninsula and Latin America are infected with the organism and of these, approximately one to two million are blind or have severe visual impairment. Humans are infected with the helminth larvae by the bite of a black fly of the Simulium genus and approximately one year after infection, the adult female worms produce microfilariae. In fact, the adult worm can live for up to 15 years, producing 900 to 1900 microfilariae per day. It is the microfilariae that are able to move through subcutaneous and ocular tissues. When these microfilariae die, they incite an immune response that is associated with clinical symptoms.
5.1. Clinical Features Onchocerciasis is one of the leading causes of blindness in the developing world. Ocular disease occurring in the anterior segment of the eye consists of corneal opacification and sclerosing keratitis, whereas, ocular disease occurring in the posterior pole is characterized by retinal degeneration [29]. Clinical disease activity in the anterior segment is associated with microfilarial load and it is generally believed that ocular pathology is a result of host directed inflammatory responses to the nematode.
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I 0! "TO RETINAL S..ANTIGEN
Figure 2. Proliferative responses of peripheral lymphocytes from 40 ocular toxoplasmosis patients to the retinal S-antigen. Sixteen of these had stimulation indices above 2.5 and were designated as "high responders". This responsiveness was not correlated to either a specific HLA phenotype nor the vigor of the cell mediated response to toxoplasma antigens. (Reprinted with permission.)
In contrast, pathology associated with the retina and optic nerve has not been directly linked to microfilarial load.
5.2. Evidence for Autoimmunity Posterior ocular onchocerciasis is characterized by atrophy of the retinal pigment epithelium and as lesions advance, subretinal fibrosis occurs [30]. A number of studies indicate that this retinal disease process may involve autoimmune responses. In 1987, Chan and associates identified that a majority of onchocerciasis patients had anti-retinal antibodies in their sera and vitreous [31 ]. Using FA assays on human retina tissue, they observed reactivity in the inner retina and photoreceptor layers. During the 1990's, Braun, McKechnie and associates performed a number of studies to elucidate the nature of the autoimmune reactivity [32-35]. They identified a recombinant antigen in O. volvulus that showed immunologic cross-reactivity with a component of the RPE [32, 33]. By western blot analysis, an antibody to a 22,000 mw antigen (OV39) of O. volvulus recognized a 44,000 mw component of the RPE cell. Subsequent studies have shown that hr 44 Ag
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is present in the optic nerve, epithelial layers of iris, ciliary body and RPE. Although OV39 and the hr 44 proteins are not homologus, they did show limited amino acid sequence identity [36]. Immunization of Lewis rats with either OV39 from O. volvulus or hr 44 from human retinal tissue, induced ocular pathology [35]. The retinal disease in the rat was characterized by extensive breakdown of the posterior blood-ocular barrier, iridocyclitis and retinitis and the activation of retinal microglia. These studies indicate that, molecular mimicry between O. volvulus and human RPE protein may contribute to the retinopathy found in patients with onchoceriacis.
6. RETINOPATHIES THAT MAY HAVE INFE CTI OU S/AUTO I M M U N E E T I O L O G I E S (WHITE-DOT SYNDROMES) A large group of clinical entities have been grouped under the title, White dot syndromes. As the name infers, they are all characterized by whitish lesions of varying sizes that are found strewn throughout the fundus. Some have a significant inflammatory
reaction associated with them while others do not. The natural history of some may lead to significant visual handicap while others may not. Some of these disorders seem to progress while others fade away. The disorders that are included in this list include such entities as acute multifocal placoid posterior pigment epitheliopathy (AMPPE), serpiginous choroiditis, the multifocal evanescent white dot syndrome (MEWDS), and multifocal choroiditis. The underlying cause of these diseases is unknown. Many of these disorders seem to be preceded by a viral illness, and one disorder, AMPPE, was hypothesized to be due to the an Epstein-Barr infection [37]. This concept is no longer thought to be the case [38]. However, a few patients have been treated with anti-viral medications, with unclear responses. The most common therapy for all of these conditions is that of immunosuppression and therapy is directed against what is believed to be an autoimmune, or least non-infectious, process in the back of the eye.
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16. Vinores SA, Wang Y, Vinores MA et al. Blood-retinal barrier breakdown in experimental coronavirus retinopathy: association with viral antigen, inflammation, and VEGF in sensitive and resistant strains. J Neuroimmunol 2001; 119:175-82. 17. Wang Y, Detrick B, Hooks JJ. Coronavirus (JHM) replication within the retina: analysis of cell tropism in mouse retinal cell cultures. Virology 1993;193: 124-37. 18. Detrick B, Rodrigues M, Chan CC, Tso MO, Hooks JJ. Expression of HLA-DR antigen on retinal pigment epithelial cells in retinitis pigmentosa. Am J Ophthalmol 1986;101:584-90. 19. Percopo CM, Hooks JJ, Shinohara T, Caspi R, Detrick B. Cytokine-mediated activation of a neuronal retinal resident cell provokes antigen presentation. J Immunol 1990;145:4101-7. 20. Nicolle C, Manceaux L. Sur une infection a corps de Leishman (ou organismes voisins) due Gondii. Compt Rend Acad Sci 1908;147:763-766. 21. Splendore A. Un nuovo protozoa parassita dei conigli: incontrato nell lesioni anatomiche d'une malattia che ricorda in molti punti kala-azar dell'uomo. Rev Soc Sci Sao Paulo 1908;3:109-112. 22. Janku J. Pathogenesis and pathologic anatomy of coloboma of macula lutea in eye of normal dimensions, and in microphthalmic eye, with parasites in the retina. Cas Lek Cesk 1923;62:1021-1027. 23. Holland GN, Lewis KG, O'Connor GR. Ocular toxoplasmosis: a 50th anniversary tribute to the contribution of Helenor Campbell Wilder Foerster. Archives of Ophthalmology 2002; 120:1081-1084. 24. Toledo de Abreu M, Belfort RJ, Hirata PS. Fuchs' heterochromic cyclitis and ocular toxoplasmosis. American Journal of Ophthalmology 1982;93:739-744. 25. Abrahams IW, Gregerson DS. Longitudinal study of serum antibody reponses to retinal antigens in acute ocular toxoplasmosis. American Journal of Ophthalmology 1982;93:224-231. 26. Whittle RM, Wallace GR, Whiston RA, Dumonde DC, Stanford MR. Human antiretinal antibodies in toxoplasma retinochroiditis. British Journal of Ophthalmology 1998;82:1017-1021. 27. Nussenblatt RB, Gery I, Ballintine E et al. Cellular immune responsiveness of uveitis patients to retinal S-
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Infection and Atherosclerosis Johan Frosteg~rd
Department of Medicine, Unit of Rheumatology, Karolinska Hospital Stockholm, Sweden
1. INTRODUCTION During recent years it has become clear that atherosclerosis has many characteristics in common with chronic inflammatory diseases and may indeed be described as an inflammatory disease. This is indicated by several lines of evidence. Firstly, immune competent cells - mainly monocytes/macrophages and T cells, but also some B-cells, eosinophils, and mast c e l l s - are abundant in lesions and many of them are activated and produce cytokines and other pro-inflammatory factors [1-3]. Furthermore, systemic inflammation, as determined by raised levels (albeit marginally) of C-reactive protein, is often present in CVD-patients and can predict a raised risk of clinical events at least at group level [4]. Whether CRP could also function as a risk factor at the individual level is not generally accepted, one reason being that infections by pathogens such as Pneumococciae could cause an increase in CRP levels that is 100 times higher than the minute increase related to atherosclerosis. It is also well known in the clinic that minor infections and trauma could influence CRP levels. A typical feature of the atherosclerotic plaque is the presence of foam cells, which are mainly derived from macrophages. These constitute a large part of the plaques, and are filled with lipids, mainly low density lipoprotein that has undergone oxidation (oxLDL) [3]. It would be a mistake to oppose the concept of atherosclerosis as an inflammatory disease to a more traditional opinion of atherosclerosis as a condition caused by metabolic pertubations and dyslipidemia. For example, the systemic inflammation in systemic lupus erythematosus (SLE) (where the
risk of CVD is exceedingly high) is closely related to dyslipidemia and CVD. A raised activity in the tumor necrosis factor (TNF)-system may be one common denominator [5, 6]. Even though inflammation is a major feature of atherosclerosis, it is far from clear which are the causative agents, and autoimmunity, inflammation and infections could all be non-mutually exclusive contributors of atherosclerosis and CVD [7]. Several non-infectious factors have been much discussed, including Heat shock proteins (HSP), especially HSP 60/65 [8] and oxidized low density lipoprotein (oxLDL)-related compounds [9]. These two candidates may be involved in the chronic inflammation and immune activation typical of atherosclerosis, but interestingly, in both cases, a relation with infections has been discussed [8, 9]. Furthermore, since oxLDL induces HSP 60/65, these two factors may cause immune activation by a related mechanism [ 10]. In principle, atherosclerosis and CVD could be related to infections directly, or indirectly. Direct causes include pathogens that directly cause or at least promote the atherosclerotic process or plaque rupture, leading to CVD. Indirect causes could depend on cross-reactivity between immune reactions to pathogens and components of the vessel wall, or be related to other factors like CRP induced by the infection or the total infectious burden. Both these possibilities will be discussed and it should be noted, they are not mutually exclusive and may even be difficult to separate. An illustration of the complexity is an interesting study, where it was demonstrated that acute myocardial infarction was preceded by an acute respiratory-tract infections, with an increased risk of AMI for a period of about
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2 weeks, which could imply both direct and indirect effects [ 11 ].
2. CHLAMYDIA PNEUMONIAE The pathogen where most evidence in favour of a role in atherosclerosis and CVD has been accumulated is C Pneumoniae. This is a small, intreacellular bacteria that is transmitted by aerosol droplets and typically leads to respiratory tract infections. It is common in the population and a majority of elderly adults have antibodies against C Pneumoniae which is usually taken as an evidence of a history of infection [12]. The association - retrospective - between antibodies against C Pneumoniae and CVD was first reported by P Saikku and coworkers [13] in the late eighties and has been confirmed by many other investigators [ 12]. However, prospective studies performed thereafter did not in general lend support to the notion of a major role played by C Pneumonae and in many cases no association at all was demonstrated [14]. In addition, the fact that a majority of all indiduals at an older age have signs of raised antibody levels against C Pneumoniae further complicates epidemiological studies. However, varying laboratory methodology and types of antibodies studied, lack of thorough analysis of confounding factors and differences in populations studied could play a role. Indeed, recent research by Saikku's group indicates that persistently but not transiently elevated C pneumoniae IC/IgA and Hsp60 IgA antibodies, together with an elevated CRP level, predicted coronary events [ 15], a finding that adds support that C Pneumonia may contribute to atherogenesis in a complex way. Another line of evidence in favour of the C Pneumoniae hypothesis comes from studies demonstrating the presence of this pathogen in atherosclerotic lesions [16, 17]. Even though it could be the case that the pathogen is present in lesions only as a passive bystander, thriving in the lipid-rich environment of atherosclerotic lesions without causing much damage, several lines of evidence indicate mechanisms by which the infection could be atherogenic. The pathogen could elicit or potentiate an inflammatory reaction in the lesions, leading to endothelial cell activation, expression of adhe-
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sion molecules and recruting inflammatory cells including monocytes/macrophages and T cells. C Pneumoniae could in principle also stimulate atherogenesis by increasing uptake of oxidized low density lipoprotein (oxLDL) by macrophages, leading to foam cell formation and plaque growth [18]. Furthermore, C Pneumonae could promote LDLoxidation per se [19]. Another interesting development is the finding that HSP in Chlamydia could contribute to inflammation by eliciting an immune response and promoting an inflammatory reaction that could promote atherogenesis and plaque rupture, e g through binding to Toll-like receptors, or as a consequence of cross reactivity between human and bacterial HSP [20]. Available studies using mice or rabbit models of atherosclerosis are heterogenous and in mice models, both positive and negative studies exist [21-23]. If antibiotics that reduce or eradicate C Pneumonae were proved to be beneficial against CVD, this would provide strong evidence for the infection-hypothesis in atherosclerosis and CVD. However, it could still be more complicated than that. C Pneumonae may resist standard antibiotic therapy, hiding inside monocytes and to establish an effect of tratment may be further ambiguous by the fact that antibiotics like tetracyclines have anti-inflammatory properties [24]. Like so much other research investigating C Pneumonae in CVD, clinical data from treatment studies are not conclusive. Some clinical trials using antibiotics to treat CVD report promising data [24-27], while other studies are negative [28]. Interestingly, use of antibiotics, especially penicillin, was associated with a favourable outcome in stroke incidence in elderly individuals [29]. It is also possible that C Pneumoniae plays a role in plaque rupture without being atherogenic per se, but this interesting possibility is difficult to evaluate in animal models for atherosclerosis, since they typically do not develop similar CVD as humans. Taken together, C Pneumoniae does not seem to be necessary for the development of atherosclerosis, though it is possible that it could promote atherogenesis at least in some groups of patients and this hypothesis has neither been proven, nor disproven.
3. CYTOMEGALOVIRUS (CMV) The history of research on a putative association between CMV and atherosclerosis has similarities with C Pneumoniae. One obvious such similary is that previous infection with both these agents are almost ubiquitous in the age groups most likely to develop CVD. Also for CMV, early positive epidemiological studies have been difficult to reproduce unequivocally [30-33]. Surprisingly, two articles published in Circulation demonstrate contradictory results, one being positive [34] and the other negative [35], of the effect of CMV-infection on endothelial function, an early measure of cardiovascular risk. Studies on the presence of CMV in atherosclerotic lesions are also contradictory, and negative studies have been published [33, 36]. It also appears that CMV, like C Pneumoniae, exerts potentially proatherogenic effects in experimental systems. The most important of these include effects on smooth muscle cell migration and endothelial adhesiveness following infection. It is also possible that CMV infection (and other herpes virus) promote a pro-coagulant state by activation of the coagulation cascade. It is therefore important to remember that although an important role played by CMV in atherogenesis is not supported by available evidence, CMV could still play a role in CVD by increasing the risk of thrombosis [33]. An interesting possibility is that while CMV may not be pivotal in general atherogenesis and CVD, it may still play an important role in post-transplantation or venous graft atherosclerosis [37].
4. ORAL PATHOGENS Periodontitis is a chronic, tissue-destructive inflammation, which degrades the attachment apparatus of the teeth, causing tooth loss and, in its most severe form, edentulousness. The more severe form of the disease is present in approximately 10-15% of an adult population [1], whereas 35% [2] have moderate or mild signs of periodontitis. Several studies have reported epidemiological associations between periodontitis and CVD [38-40]. CVD and periodontitis have risk factors in common, including smoking and diabetes and also socioeconomic status, and an over-estimation of the association between
these two diseases due to insufficient compensation for this has also been implicated [41, 42]. We recently demonstrated that patients with periodontitis differ in risk factor profile as compared to controls, the most striking difference being decreased HDL-levels [42a]. This may suggest that indirect mechanisms also involving chronic inflammation and its relation to dyslipidemia are important. One mechanism could be release of bacteria, bacterial products or pro-inflammatory cytokines from the chronic periodontal lesion into the blood stream. This might lead to a systemic inflammatory response, which resembles a risk factor profile that is consistent with cardiovascular disease. Although DNA from oral bacteria has been found in atherosclerotic plaques, a bacterial contribution to this plaque formation has yet to be demonstrated [43]. It is also possible that immune reactions and cross reactivity, e g against heat shock proteins or phospholipids in oral pathogens play a role. For example, Actinobacillus actinomycetemcomitans binds to the PAF-receptor, which is the case also with oxidized LDL, a central player in atherosclerosis [44, 45].
5. OTHER INFECTIONS
Several other infectious agents have been discussed as putative causes of atherosclerosis, including Helicobacter Pylori, herpes simplex virus, Hepatitis A, Mycoplasma, Influenza virus and other members of the herpes family [46]. Even nanobacteria have been suggested to play a role, especially in the calcification that typically occurs in atherosclerosis [47]. The evidence that these agents play a role in atherosclerosis and CVD appears to be weaker than those discussed previously. For example, data indicating presence of Helicobacter Pylori in plaque are conflicting [48], and epidemiologic data are conflicting [49], with socioeconomic status as one of several possible confounders.
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6. UNSPECIFIC MECHANISMS: MODIFIED LDL, PHOSPHOLIPIDS, INFLAMMATION AND HSP Lipids have been in the focus of atherosclerosis research for a long time. In line with this, an immune response related to LDL rendered immunogenic by oxidation or other forms of modification is present both for B cells [50] and T cells [51, 52]. The most investigated form of modified LDL, OxLDL, has many possible pro-atherogenic and pro-inflammatory properties including foam cell formation in monocytes/macrophages and other cell types and has chemotactic effects on T cells and monocytes. OxLDL promotes monocyte-endothelial interaction, activates and differentiates monocytes/ macrophages, but can be toxic [9]. Many of these effects are caused by platelet activating factor (PAF)-like lipids and/or lysophosphatidylcholine (LPC), which are present in LDL that has undergone oxidation and/or enzymatic modification, one such enzyme being phospholipase A2 [44, 53]. Anti-phospholipid antibodies (aPL) are major risk factors for both arterial and venous thrombosis, especially in connection with autoimmune diseases like SLE. Since aPL cross-react with oxLDL [54] and many aPL recognize oxidized phospholipids [ 14], it appears very plausible that an important part of the immune reactivity to oxLDL is related to the phospholipid fraction. aPL are known to be induced by many different bacterial and viral agents, including mycoplasma and HIV, but it is often thought that antibodies elicited by infections do not have the same prothrombotic properties as autoimmune aPL. However, recently, an novel mechanism relating aPL and bacterial agents was described. According to this, a mechanism of molecular mimicry in experimental APS, demonstrating that bacterial peptides homologous with a major co-factor an likely antigen for antibodies against many phospholipids, especially cardiolipin, b2GPI, induce pathogenic anti-b2GPI antibodies along with APS manifestations [55-59]. Also CMV may induce pathogenic aPL [60]. It is thus possible that some, if not all, aPL generated by infections could cause an increased risk of CVD since thrombosis after plaque rupture is the major underlying factor in CVD like stroke and MI. Another interesting observation suggesting that
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infections can promote atherosclerosis comes from recent findings indicating that acute childhood infections are accompanied by intimal thickening [61].
7. CAN SOME INFECTIONS PROTECT AGAINST ATHEROSCLEROSIS? Immunization with oxLDL in animal models protects against atherosclerosis in parallel with raised antibody levels against OxLDL (aOxLDL) [62]. Furthermore B-cell depletion aggravates atherosclerosis in mice models of the disease [63]. Some aOxLDL may therefore protect against disease in animal models. Even though animal models of atherosclerosis have given important insights into the role of immunity in atherosclerosis, they do not develop similar CVD as humans [64] and it is presently unclear if they are representative of most human atherosclerosis or only of a subfraction of this complex disease. In humans aOxLDL are associated in several studies with the established CVD [9]. In early, asymptomatic disease, however, aOxLDL levels appear to be low. We recently demonstrated that aOxLDL are decreased in borderline hypertension (BHT), which represents a state of early human CVD [65]. Consistent with this, a negative association between IgM aOxLDL and atherosclerosis in healthy individuals was reported [66]. It is therefore possible that some of these antibodies are protective at early stages of disease. At a later stage, they may have an adverse role, especially so with aOxLDL of IgG subclass. It is also possible that both low and high aOxLDL-levels may increase the risk of CVD, in parallel to closely related antiphospholipid antibodies, which at very high levels predispose to arterial disease [67, 68]. In a recent study it was demonstrated that phosphorylcholine, a component of S Pneumoniae, is one major antigen in oxLDL recognized by aOxLDL antibodies that have protective properties. Furthermore, immunization with pneumococcal vaccine in this mice model of atherosclerosis also protected against atherosclerosis [69]. It is therefore possible that some infections, e g by agents with PC as a major antigen, can indeed be protective.
8. OxLDL-RELATED IMMUNEMODULATION Previous research by us and others have indicated that oxLDL and components thereof, can promote T cell activation [51, 52, 70-74]. By use of the sensitive ELISPOT-technique we recently demonstrated that a pro-inflammatory T helper 1 cytokine, IL-12, in itself had a comparable capacity as oxLDL, PAF, and LPC to induce IFN-gamma, a major T cell cytokine. OxLDL could induce IL-12 in immune competent cells isolated from blood or directly from atherosclerotic plaques [75]. Taken together our data supports the notion that phospholipids in oxLDL as LPC and PAF can promote immune activation through a non-specific mechanism, in which IL-12 and the balance between this (and other pro-inflammatory cytokines) and IL-10 (and other anti-inflammatory cytokines) may be important, indicating that oxLDL is not necessarily a T-cell antigen by itself. It could be hypothesized is that some phospholipids with inflammatory properties are present on microbial agents [45, 76, 77] and may modulate immune activation and atherosclerosis in the vessel wall, a possibility that should be studied further.
9. HEAT SHOCK PROTEINS AND IMMUNE REACTIONS IN CVD AND ATHEROSCLEROSIS Heat shock proteins (HSP) are evolutionarily conserved proteins that play a major role as chaperons, protecting cells from damage, but they are also immunogenic and present in bacteria, which has led to speculation that they are related to autoimmunity. HSP have been implicated also in atherosclerosis. An intriguing finding is that immunization using HSP (as opposed to oxLDL) increases atherosclerosis in animal models [78, 79]. In line with this are findings in human studies indicating that antibodies against HSP 60/65 may play an important role in CVD [10, 80-83]. Circulating HSP 60 is raised in early CVD [80]. However, we recently demonstrated that soluble HSPs, especially HSP 70, predict a favorable outcome in atherosclerosis measurements in hypertension [84]. These findings suggest that HSP and other antigens, which are pro-
posed as pro-atherogenic, can under certain circumstances protect against the disease.
10. CONCLUSIONS It is important to note, that according to Koch's Postulate, before a clear causative relationship between a pathogen and a certain disease can be established, three conditions should be fulfilled: 1) the pathogen should be present in individuals with the disease; 2) it should cause the disease when isolated and introduced in a healthy subject, and 3) it should be poss~le to identify the agent in the subject that has contracted the disease. When atherosclerosis is concerned, it is clear that these postulates are not unequivocally fulfilled in atherosclerosis, and infection as a cause of atherosclerosis or CVD is therefore an un proven albeit interesting possibility from a more orthodox point of view. Taken together, several different infectious agents may in principle contribute to atherosclerosis and CVD and for at least C Pneumonae, the evidence is relatively compelling though clear-cut evidence remains to be demonstrated. Likewise, evidence of oral pathogens are also interesting, but for other infectious agents, including CMV, the evidence of any relation to atherosclerosis or CVD is scarce, except that for CMV, there may be an association with transplantation atherosclerosis. Infections may still contribute to atherosclerosis in an indirect way, involvning general systemic inflammation and its relation to established risk factors like dyslipidemia and emerging ones like CRP. However, it does not seem that infections are a prerequisite of this disease process.
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9 2004 Elsevier B. V All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
The Pathogenic Role of Heat Shock Proteins in Atherosclerosis Qingbo Xu ~and Georg Wick 2
tDepartment of Cardiological Sciences, St George's Hospital Medical School, London, UK; 2Institutefor Pathophysiology, University of lnnsbruck Medical School Innsbruck, Austria
1. INTRODUCTION Heat shock proteins (HSPs) or stress proteins are a superfamily of intracellular proteins made by many cell types, whose synthesis is preferentially augmented by exposure to excessive heat or other harmful insults [1]. They fulfill a range of normal functions which includes intracellular assembly, folding and translocation of proteins. Essentially under normal circumstances they act as chaperones making sure that the cellular proteins are the fight shape and in the fight place at the fight time [2]. With regard to translocation of proteins, they are simply providing conveyance of intracellular proteins from one compartment to another. In response to stress stimuli or cellular insults, the cells produced high levels of HSPs to protect themselves against these unfavourable conditions. Indeed, increased production of HSPs has been shown to protect cells against apoptosis induced by oxidative stress, toxins, heat shock, ethanol and cellular damage after ischemia
[3]. Given the high degree of amino acid sequence homology between HSPs of different species, the immune response to HSPs derived from pathogens may cross-react with host HSPs [4]. Thus, HSPs may be autoantigens in some circumstances. The HSP60 family has been shown to be involved in the development of many diseases, such as adjuvant arthritis in rats, rheumatoid arthritis in humans, insulin-dependent diabetes mellitus in mice, and systemic sclerosis in humans [5], whereas HSP47, HSP60 and HSP70 have been identified as being involved in the pathogenesis of atherosclerosis
[6-91.
Atherosclerosis is recognized as the leading source of morbidity, disability and mortality within many parts of the world, including Europe, the United States and much of Asia. Atherosclerosis is seen to be the principal cause of heart attack, stroke and gangrene of the extremities and so is responsible for 50% of all mortality [10]. Atherosclerosis is essentially the stiffening of blood vessels due to lesions localizing in the arterial intima showing mononuclear cell infiltration, lipid deposition, proliferation of smooth muscle cells and extracellular matrix accumulation. Atherosclerosis is the most common form of arteriosclerosis, in which cholesterol-containing plaques deposit in the intimal layers of large and medium sized arteries, eventually causing severe occlusion of the lumens [11]. The effects of narrowed vessels lead to restriction of gaseous exchanges at capillary sites within the body. Recently, it is believed that atherosclerosis is an immuno-inflammatory disease [12], of which HSPs play important roles in mediating the pathogenesis of the disease [9]. This chapter will focus on the mechanistic studies of HSPs in the development of atherosclerosis.
2. ATHEROSCLEROSIS When living tissue is attacked, for example by autoimmune damage, physical, chemical or microbial agents, a series of local processes are initiated in order to contain the offensive agent, to neutralize its effect, to limit spread and thus hopefully eradicating it. Inflammation results in healing and repair of the injured site. Essentially the onset of atherosclerosis
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is in the healing and repair of the vessel, which indirectly causes problems elsewhere [ 13]. The process, in normal circumstances is a protective response to insults to the endothelium and smooth muscle cells of the arterial wall. This consists of the formation of fibrofatty and fibrous lesions, preceded and accompanied by inflammation. There are several theories or concepts on the pathogenesis of atherosclerosis but a consistent theory is yet to be established, although, many authors and scientists largely rely on Ross's [ 10] theory to explain their findings. The earliest recognizable lesion of atherosclerosis is the so-called 'fatty streak', an aggregation of T lymphocytes and lipid rich-macrophages within the innermost layer of the artery wall, the intima [11, 14]. The macrophages and T lymphocytes release several cytokines in the lesion advancing its progress further. The progression of the early lesion is dependant on age, the earliest signs develop in childhood but more severe pathogenesis of the disease is recognizable by middle age, though clear visible signs and effects are seen more so in old age [15]. The lesion progresses from a fatty streak to ~i raised fibrous plaque to a complicated lesion. The aorta is the main artery affected but other frequently affected vessels include the coronary arteries, cerebral arteries, carotid arteries and iliofemoral arteries. The enlargement of the growing lesion is due to accumulation of mononuclear cells and the proliferation of smooth muscle cells. The end result is an advanced complicated plaque lesion [ 11 ]. A growing advanced complicated lesion (thrombus) inevitably ruptures to cause many of the peripheral cardiovascular related diseases, i.e. a blockage in the peripheral arteries. A ruptured lesion releases some of the fatty material (embolism) to head downstream to cause a blood flow blockage inhibiting blood to reach tissues for vital exchange to occur [ 16]. Typical symptoms seen in patients with blockages in their femoral arteries, is intermittent claudication-pain in legs whilst walking. Rupture of the fibrous cap or ulceration of the fibrous cap rapidly leads to the formation of thrombosis. The rupture is due to thinning of the fibrous cap caused by the influx and activation of macrophages and T cells. The macrophages release a range of enzymes which cause degradation of the matrix thus plaque rupture and clinic events [ 16].
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3. THE ROLE OF INFLAMMATORYIMMUNE PROCESSES IN THE DEVELOPMENT OF ATHEROSCLEROSIS An association between inflammatory processes and atherogenesis has been postulated by many authors in the past, but, surprisingly, was ignored for a long time by groups engaged in classical atherosclerosis research or considered to be only a secondary phenomenon [17]. Such changes include, the occurrence of granular deposits of immunoglobulins and co-distributed complement components, increased expression of C3b receptors (CR1) and C3b 1 receptors (CR3) on macrophages within atherosclerotic lesions, but not in unaltered vessels [ 18]. However, B cells are only found in very low numbers in various stages of atherosclerotic lesions, and the site of production for these immunoglobulins must, therefore, be sought elsewhere [19, 20]. Various candidates against which these antibodies could be directed have been discussed, among them oxLDL [21] and, as shown by ourselves, certain stress proteins [22]. Other than these humoral immune phenomena, it is now clear that T cells are among the first cells infiltrating the intima of arteries during the earliest stages of atherosclerosis, most probably before monocytes [23]. In an immunohistological study of vascular arterial specimens of young (<35 years) and old (>65 years) patients who died from non-atherosclerosis-associated diseases, we were able to show that the earliest mononuclear cells infiltrating the intima at sites of the development of atherosclerotic lesions were T cells and macrophages. A majority of these early T cells are CD4 +, HLA-DR + and interleukin-2 receptor+ (IL2R+), i.e. activated [19]. Other authors were able to show that T cells in late atherosclerotic plaques express the low molecular variant of the leukocyte common antigen (CD45RO) and the integrin very late activation antigen-1 (VLA-1) [24, 25]. Hansson et al [25], analyzing the rearrangement of T cell receptor (TCR) genes in these latter cells derived from advanced lesions, showed that they represent a polyclonal population rather than displaying restricted T-cell receptor TCR usage. Mosorin et al [26] have recently confirmed our data in rabbits [27] by showing HSP60 to be the main antigenic candidate against which T cell clones derived from
human atherosclerotic plaques are reacting. Regardless of which antigen these lymphocytes may recognize, it seems unprobable that endothelial cells that aberrantly express major histocompatibility complex (MHC) class II antigens act as primary antigen-presenting cells for T cell sensitization. In our own experiments, we were only able to show MHC class II expression by endothelial cells at sites where T cell accumulations, and thus production of gamma-interferon (IFNy), where present in the intima directly beneath these areas [ 19]. Therefore, we and others concluded that the expression of MHC class II molecules by endothelial cells represents a secondary rather than a primary phenomenon due to the effect of IFN-gamma produced by neighboring T-cells. We originally reasoned that sensitization of T cells takes place at other sites, e.g. the draining lymph nodes. However, as will be detailed below, we have recently made the surprising discovery that a whole network of dendritic cells resembling Langerhans cells in the skin is present in the arterial intima [28], an observation that has completely changed our ideas with respect to the possible in situ T cell sensitization in the vascular system. The large majority of CD3 + in the mononuclear infiltrate in atherosclerotic lesions expresses the TCRtx/[3, but an unexpectedly high proportion also expresses the TCR),/8 [29]. While the latter type of cells only constitutes approximately 1% in peripheral blood, enrichment to 10% and more within early atherosclerotic lesions can be observed. The majority of these latter cells express the TCRy2 chain, i.e. resembles the TCRy/8+ population found in the intestinal mucosa. On the other hand, TCR Vy982+ cells characteristic of circulating TCRy/~5+ cells are not proportionally increased in the intima compared to peripheral blood. Furthermore, we were able to demonstrate considerable accumulations of T cells at various sites of the normal arterial intima of adults and even children and babies, i.e. without concommitant occurrence of atherosclerotic lesions [30]. Together with the above-mentioned accumulations of TCRy/8+ cells, notoriously present in the socalled mucosa-associated lymphoid tissue (MALT) compared to other lymphatic organs, e.g. the spleen, this observation prompted us to put forward the hypothesis of the existence of a "vascular-associated lymphoid tissue" (VALT). We hypothesized that the VALT may fulfill a task similar to MALT, i.e. moni-
toting exogenous or autologous antigenic material that comes into contact with bodily surfaces, in this case that of the vascular system [31, 32]. It later turned out that the observation of an enrichment of TCRy/8+ cells may have special importance, since they are known to preferentially react with certain stress proteins (HSPs), a phenomenon that will be detailed below. Finally, it was possible to demonstrate on the protein- and mRNA-level that endothelial cells as well as leukocytes occurring in atherosclerotic lesions are able to produce a variety of immunological-inflammatory mediators [33]. Among others, these include interleukin- 1 (IL- 1), tumour necrosis factor t~ (TNFtx), lymphotoxin, IL-2, IL-6, IL-8, monocyte-chemotactic peptide-1 (MCP-1) and IFNy [ 17]. Together, these molecules can modulate the local cellular immune response within emerging atherosclerotic lesions [34]. In addition, growth factors, such as platelet-derived growth factor (PDGF), exert a mitogenic effect on mesenchymal cells and stimulate leukocyte migration. Thus, they play an important role in the maintenance of the immunologic-inflammatory reaction. Normal vascular tissues do not display a high content of PDGF-~ receptors, but in the case of arterial diseases associated with an activation of macrophages and T cells, a considerable expression of such receptors can be observed. The immune reaction within the arterial wall, therefore, seems to entail an increased responsivity to PDGF-~ [12]. Based on these data, we performed a large series of studies in experimental animals and humans that are summarized in the next paragraph, and that finally led to the formulation of our new autoimmune hypothesis for the development of atherosclerosis.
4. HSPS
HSPs are multigene families that range in molecular size from 10 to 150 kDa and are found in all major cellular compartments. According to molecular weight they are divided into following families: HSP10, small HSPs, HSP40, HSP60, HSP70, HSP90 and HSPll0. Each family of HSPs contains one or more members (Table 1) [2]. Excellent reviews have been written on the chaperone function of HSPs in general [35, 36] and in the cardiovascu-
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Table 1. Heat shock proteins
Pathological involvement
Family
Members/othern a m e s
Physiological function
HSP10
HSP10, HSP17
Promotes substrate Release with HSP60
Small HSP
HSP22,~A-crystallin, HSP23, ~B-crystallin, HSP27, HSP28
F-actin assembly, Molecular chaperones
HSP40
HSP32, HO- 1, HSP40, Hdj-1, HSP47
Guides protein folding, Binding and transport of collagen
Atherosclerosis
HSP60
HSP58, GroEL, HSP60, HSP65, Grp58
Assemble polypeptides; Translocate proteins across membranes; Accelerate protein folding and unfolding
Adjuvant arthritis, Rheumatoid arthritis, Atherosclerosis, Diabetes mellitus, Systemic sclerosis Schizophrenia
HSP70
HSP68, Dnak., Hsc70, Hsx70, HSP72, HSP73, HSP75, Grp75, HSP78, Grp78
Molecular chaperone: Assembly and transport newly synthesized proteins; Fold or unfold polypeptides; Remove denatured proteins; Bind to specific polypeptides (e.g., p53); ATPase activity
HSP90
HSP83, HptG, HSP87, grp94, HSP90-c~, HSP90-[~
Bind to specific polypeptide, receptors (e.g., glucocorticoid receptor)
HSP110
HSP94,HSP104, HSP105, HSP110, Apg-1, Grpl70
Onlyin yeast (?)
lar system [3, 37]. HSP60 forms a large (970 kDa) hetero-oligomeric protein complex called TCP1 ring complex, containing TCP1 and several other proteins, which is essential for protein assembly. As a chaperone, HSP70 plays a role in both the assembly and transport of newly synthesized proteins within cells, as well as in the removal of denatured proteins [38]. Thus, HSPs appear to be important in preventing damage and in cellular repair processes following injury. Indeed, increased production of HSPs has been shown to protect cells against apoptosis induced by oxidative stress, toxins, heat shock, ethanol, and cellular damage following ischemia or sepsis-induced injury [39-44]. The upregulation of HSP expression thus leads to a generation of an immune response. The HSPs in essence become autoantigens leading to autoimmunity. It seems extracellular HSPs are a powerful way of sending
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Tuberculosis, Leprosy, Filariasis, Atherosclerosis
Schistosomiasis, Systemic lupus erythematosus
a 'danger signal' to the immune system in order to generate a response that can help to get rid of an infection or disease [45].
5. HSP E X P R E S S I O N IN A T H E R O S C L E R O T I C LESIONS
A number of HSP types have been detected in atherosclerotic plaques within human blood vessels. Although, the initial event eliciting enhanced HSP synthesis in the atherosclerotic region remains a subject of discussion [9]. As outlined earlier HSP expression is due to the stressful conditions within the developing plaque. Cell proliferation, inflammation and chronic ischemia all are events taking part in the multifactorial process of atherosclerosis [3]. Detection of HSPs in mammalian arteries was
first reported by Berberian et al [46]. They reported the expression of HSP70 in human and rabbit arteries and how it was distributed in relation to necrosis, lipid accumulation, vascular smooth muscle cells and macrophages. HSP70 has a pathological involvement not only in atherosclerosis but also tuberculosis and leprosy. It was found that HSP70 expression was densely concentrated mostly in the thickened atheromas- consistent with severe necrosis and lipid accumulation. These results indicated that elevated HSPs in plaque cells, particularly macrophages, were more stressed within the depth of the atheroma in association with necrosis. Kleindienst et al [29] from our group detailed the expression of HSP60. This study involved essentially two control groups, vessels with severe atherosclerosis and normal vessels (with no atherosclerotic lesions and mononuclear infiltration). As expected with the severe atherosclerotic vessels, HSP60 was detected on endothelium, smooth muscle cells, and/or mononuclear cells of all carotid and aortic specimens. This is highly indicative as the other control group of normal lesion vessels did not express this particular HSP. From the results, intensity of HSP60 expression correlated positively with the atherosclerotic severity. Clearly, HSPs are directly associated with the pathogenesis of atheroma formation as their absence in normal lesions is highly indicative. Therefore expression of heat shock proteins has a direct influence on the development of non-specific inflammatory and adaptive immune responses in a number of ways. HSP47 acting as a chaperone for procollagen has also been found to be involved in atherosclerosis [47]. Strong focal expression was evident in atherosclerotic, but not normal, arteries and was prevalent in the collagenous regions. All cells expressing type I procollagen also expressed HSP47 [48]. Heat shock and oxLDL stimulated the expression of HSP47 mRNA by smooth muscle cells. These findings identify HSP47 as a novel constituent of human coronary atheroma, and selective upregulation by stress raise the possibility that HSP47 may be a determinant of plaque stability [48].
6. INFECTIONS AND HSP IMMUNITY Although atherosclerosis is very much considered a chronic inflammatory disease, the last ten years of research has found that infections may also have an influence in its pathogenesi [49]. A number of microorganisms, such as Chlamydia pneumoniae and Helicobacter pylori have been shown to have an association with cardiovascular disease [50]. The infecting microorganism Chlamydia pneumoniae's mode of attack is primarily through the body's airways and lungs. The pathogen once inside the lungs can then spread to secondary sites of the body - the arterial wall being one of these sites. Helicobacter pylori have also shown a presence in atherosclerotic plaques [51 ]. Its point of entry to the body is through the gastrointestinal tract and so it infects the gastric epithelial cells then spreads. It is ascertained that Helicobacterpylori does not induce atherosclerosis. Interestingly, Kol et al [52] demonstrated the co-existence of chlamydial and human HSP60s in atherosclerotic lesions. These data support the concept that elevated HSP expression in lesions in these instances may be induced by the pathogen Chlamydiae species. During its normal cycle generating infectious progeny, Chlamydiae express basal levels of HSP. In the presence of interferon-gamma, a product of activated T cells within atheroma, certain Chlamydiae achieve a state of intracellular chronic, persistent infection, in which they remain viable but metabolically quiescent and do not replicate [53]. During such chronic and persistent infections chlamydial HSP60 production is abundant. These findings suggest that chlamydial infections might exert their role in atherogenesis via HSP production. Furthermore, a correlation between endotoxin and atherosclerosis was also found [54]. Endotoxin induces local inflammation and systemic toxicity during Gram-negative infections and results in aortic endothelial injury with or without cell death and replication followed by increased leukocyte adhesion [55, 56]. Seitz et al [57] reported that increased levels of HSP60 were found in aortic endothelial cells of rats in response to endotoxin. These authors also demonstrated endothelial expression of HSP60 with in vitro administration of endotoxin [57]. In addition, virus infections resulted in increased HSP expression in cardiomyocytes [58]. Ultraviolet irra-
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diation of the virus prevents virus replication and failed to elicit liSP production in heart cells [58]. Thus, pathogen infections can lead to substantial HSP expression in the infected ceils, including those of the vessel wall [59], We were able to show that the ability to develop atherosclerosis increases with the infectious load to which an individual has been subjected, The incidence 9of at.heroscterosi:s is n,ot only associated with the presence of serum antibodies :against Chlamydia and high |,evels of the acute-phase C reactive protein (CRP), l~at is further significantly augmemed if res~ piratory infections are simultaneously present [60]. These Observations show that infectious agents in general can stimulate immune responses to HSPr0] 65, and we have demonstrated a several-fold increase of anti-mHSP65 antibody titers during the course of an infection with Gram~negative bacteria. In addition to chlamydiai infections, anti-mHSP65 antibodies were found to be closely correlated with immune reactions to endotoxin in the general population [54], interestingly, bacterial cell wall LPS can concomitantly induce hHSP60 and adhesion molecules on endothelial ceils, a prerequisite for the celtul~ immune response to hHSP60 [61 ]. In the recent paper, we performed a populationbased study (n=826) to determine serum sotubte HSP60 (sHSP60), carotid atherosclerosis and risk factors [62]. We demonstrated that sHSP60 levels were significantly elevated in subjects with prevaten~incident carotid atherosclerosis and correlated to intima-media thickness independent of age, sex-and ~ h e r risk factors, interestingly, sHSP60 was also correlated with anti-LPS anti-Chlamydia and ami--HSPr0 antibodies, inflammation markers and chronic infections.. Conco~t.amly, Pockt.ey et al i[63] meamared sHSPr0 :and sHSP70 in subjects wi~ borderline hypertension. A major novel observation in ~thi.'sreport was findings that sHSP60 was ~esent at a signifi,ca:nfly enhanced level in patients wi:th bo~erline hypertension, which was associated With i~fi:ma-media ~thicl~qess and early atheroscterosis, ,These data provide additional support for the role of sHSPs in the inductio~progression of both hypetlension and atheroscterosis, Where and bow sHS:~ :are released into blood is currently ~ ~ w n . Given that :all types of tissues M'~hly express HSPs in ~esponse ,to ,stress there :are several possibifities. First, infectious agents
may be the major factor contributing to sHSP60 release from the organisms and from human cells. For example, Chlamydiae, during their life cycle, undergo both phases of nonlytic infection, in which they remain viable, but do not replicate, and phases of lytic infection [64]. During the lyric phases, host cells release their own HSP60, produced during a chronic phase 'of infection, and also Chlamydial HSP60, that has been produced by bacteria. Support for this theory is that sHSPr0 levels are significantly correlated with antJ-Chlamydia antibodies [.62] and both Chlamydial and human HSP60s exist at high levels in human atherosclerotic lesions [52]. Second, sHSPr0 could be released from the dying cells of tissues during chronic inflammation and from atheroma because earlier studies have shown the occurrence of cell death within atheroma [65, 66]. Recent data demonstrated that open heart surgery resulted in .the release of sHSP70 into the blood of patients [67], which may be due to cell damage and inflammatory responses. Finally, surface expressed HSPs in the cell undergoing apoptosis may be released into blood via the formalion of micropartictes, which have been identified in the circulating blood of patients with acute coronary syndromes and in nonischemi'c patients :[68]. IImse microparticles generated in vitro from activated platelets or leukocytes stimulate cultured endothelial cells to produce prostacyclin and cytosines and to express adhesion molecules [68-71].. The micropa~cles circulating in the peripheral blood of patients with .acute myocardial infarction affect endothelium-dependent responses in normal b~ood vesse!ls [69]. sHSPs may be present in the mi'cropardctes and serve .as active components exerti'ng their role in these processes. Therefore, HSP60 re|ease into the circulation could be the result of diff~ent pathways.
% BIOMECHANICAL STRESS INDUCED HSP EXPRE: SSION The vessel watl is exposed essemially to two mum types of hemodynamic forces, often referred .to biomechanical stresses. The two types are Shear stress (.the dragging frictional force created by blood flow) and mechanical stretch (a strain stress e r ~ by blood pressure)~[72]. Strain stress restflts in -the
stretching of the vessel wall. It produces elongational stretch. The arterial wall is integrated with the circulatory system. It is a functional component of the system that continuously remodels in response to haemodynamic stress as well as many other stressors [73]. After being exposed to shear stress, maximal HSP60 induction in endothelial cells was observed [74]. In Northern blots, the level of HSP60 mRNA was markedly increased after only 1 hour of shear stress in human umbilical vein endothelial cells compared with static control cells. In vivo investigations in Lewis rats confirmed these in vitro findings: the intima and media of the fight common carotid artery exposed to increased wall shear stress (after ligation of the left common carotid artery) were stained for HSP60. The vessel wall of the left lowshear-stress-exposed side was negative. These findings demonstrate that shear stress results in HSP60 induction in endothelial cells in vivo and in vitro, providing the prerequisite for humoral and cellular reactions to endothelial HSPs in the earliest stages of atherosclerosis. Many factors cause acute systemic hypertension, whi'ch in turn can result in stress to the vessel wall and lead to vascular disease. In previous studies, Udelsman et at [75] demonstrated that restraint, or im..m,obilization stress, results in the induction of HSP70 gene expression in the aorta of adult rat and showed that this response was markedly attenua:ted with age. We have also provided evidence that restraint-induced HSP70 expression occurs secondary to a rise in systemic blood pressure [76]. 'Old rats were unable to mount a significant stress-induced hypertensive response, providing an explanation for the reduced HSP70 response in the old rats. These findings support the conclusion that HSP70 induction occurs as a physiological response to acute hypertension [77]. To clarify the direct role of biomechanical stress o n HSP induction in smooth muscle cells, Xu et al [78] thus carried out a study to examine the possibility of HSPTO being produced because of mechanical stress. We provided evidence that mechanical forces evoked rapid activation of HSP70 expression in smooth muscle cells [78]. Elevated protein levels were preceded by HSP70 mRNA transcription, which was associated with HSF1 phosphorylation and activation stimulated by mechanical
forces. Although mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal protein kinases or stress-activated protein kinases (JNK/SAPK) and p38MAPKs [79-81], were also highly activated in response to cyclic strain stress, inhibition of ERK and p38MAPK activation by their specific inhibitors did not influence HSF1 activation [78]. Interestingly, smooth muscle cell fines stably expressing dominant negative rac (rac N17) abolished HSP protein production and HSF1 activation induced by mechanical forces, while a significant reduction of HSP70 expression was seen in ras N17 transfected-cell lines. Therefore, mechanical stretch induced-HSP70 expression is mediated by HSF1 activation and regulated by rac/ras GTP-binding proteins [78].
8. SIGNAL TRANSDUCTIONS LEADING TO H S P TRANSCRIPTION The regulation of heat shock protein gene expression in eukaryotes is mediated by the conserved heat shock transcription factor (HSF) [82]. An intracellular signalling cascade leads to the activation of HSP gene transcription thus protein synthesis of a specific HSP type. Gene transcription of HSPs is brought about by HSFs but its activation is due to stressors [83]. What underpins the regulation of HSF primarily involves a change in activity rather than a change in its synthesis or stability. Therefore HSF is present in a latent state under normal conditions but becomes activated as a consequence of heat stress. So HSFs activate the transcription of genes that encode the products required for chaperoning protein folding, processing, targeting, degradation and function. Although HSFs have been extensively studied with respect to their role in thermotolerance and the activation of gene expression in response to environmental stress, the involvement of HSFs in response to stresses associated with cell growth and differentiation, and in response to normal physiological processes is becoming a lot clearer [84]. The mechanism by which HSFs work is by their interaction with a specific regulatory element, called the heat shock element (HSE). HSFs are present in the cell in a non-DNA binding state; they are activated in response to various stresses to
717
a DNA-binding form [85]. This activation process is poorly understood. There are various stimuli thought to activate different signal pathways that lead to HSF activation. The HSFs that have been identified for HSP gene transcription are: HSF1, HSF2, HSF3 and HSF4. Much research is yet to be carried out to pinpoint the exact mechanism at the molecular level of the HSFs [7]. The signal transducers or the pathways leading to HSF activation are not fully elucidated, and further studies will be needed to clarify the mechanism of HSP expression at a molecular level. As discussed, HSP synthesis is regulated at the level of transcription by a mediator from a family of HSFs [82]. This claim is only based on research from a number of in vitro studies on cultured cells. Of the many HSFs found, a very distinct HSF has been found to exist only in mammalian species. With regard to HSP in vivo studies on animal models, it is established that there is overexpression of host HSP60, HSP70 and HSP47 [29, 46, 48]. To further scrutinize the molecular mechanisms of HSP expression involving activation of HSFs in atherosclerotic lesions in animal models we recently studied the profile of HSFs in vivo [86]. Severe atherosclerotic lesions developed in the aortas of rabbits 16 weeks after feeding a 0.2% cholesterol diet. When protein extracts from the aortas were subjected to Western blot analysis, the level of HSF1 in proteins from atherosclerotic lesions of hypercholesterolemic rabbits were significantly higher than those of normal vessels. Gel mobility shift assays revealed the formation of protein-heat shock element complexes containing HSF1 in protein extracts from atherosclerotic lesion [86]. Furthermore, triglyceride-rich lipoprotein, oxidized-triglyceride-rich lipoprotein, low-density lipoprotein, and oxidized low-density lipoprotein did not activate HSF1 in cultured smooth muscle cells, whereas HSF1 was highly activated in cells treated with tumour necrosis factor-. Interestingly, mechanical stretching of smooth muscle cells resulted in HSF1 translocation from the cytoplasm to the nucleus and hyperphosphorylation followed by increased HSP70 expression. Thus, our findings provide the first evidence that HSF1 is activated and highly expressed in atherosclerotic lesions and that cytokine stimulation and disturbed mechanical stress to the vessel wall may be responsible for such activation [86].
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9. ANIMAL MODELS SUPPORTING THE AUTOIMMUNITY TO HSPS Our initial experiments attempted to identify the antigen(s) that may incite the cellular and/or humoral immune response at the beginning of the development of atherosclerotic lesions. Among the many candidate antigens, microbial constituents, e.g. confronting the immune system in the course of various infections, and oxLDL emerged as the most prominent. Our experimental and clinical data suggest that the earliest stage of atherogenesis consists of an autoimmune reaction against HSP60 [87]. The first, presumably autoimmune, step of atherosclerosis that can be induced by three immunizations of rabbits at five week intervals with mHSP65 is still reversible, i.e. subsides after an interval of 32 weeks, while the more severe atherosclerotic lesions induced by feeding a cholesterol-rich diet only or following immunization plus feeding a cholesterol-rich diet do not regress during this period of time [88]. Immunosuppression of rabbits by T-cell depletion with anti-CD43 antibodies in combination with prednisolone (to prevent the formation of anti-mouse Ig antibodies) inhibits the development of atherosclerosis induced by immunization with mHSP65 [89]. The mild atherosclerotic aortic lesions that develop in C57BL/6J mice upon feeding a cholesterol-rich diet are aggravated by immunization with mHSP65 [90]. Interestingly, immunization of such mice with chemically-modified LDL prevents the emergence of atherosclerosis [91, 92]. The results of these experiments, which are even more significant when ApoE-/- mice are used, proved that (a) immunity to HSP is pathogenic, and (b) an immune reaction to oxLDL may be beneficial, probably by removing oxLDL via immune complex formation [21]. As expected, the peripheral bloods as well as the arterial lesions induced in rabbits by immunization with mHSP65 are enriched for HSP65-reactive T cells compared to unimmunized controls. It was, however, notable that T cell lines derived from atherosclerotic lesions of unimmunized rabbits fed a cholesterol-rich diet only also showed a significantly increased proportion of HSP65 specificity compared to T cell lines derived from the peripheral blood of the same animals [27], pointing to a prefer-
ential activation of such cells in the lesions. Immunization of normocholesterolemic rabbits with heat-killed mycobacteria or recombinant mHSP led to the emergence of arteriosclerotic lesions, i.e. intimal infiltration by mononuclear cells with a preponderance of activated CD4+ T cells, but no foam cells, at sites of the arterial tree known to be predisposed to disease development [87]. Rats, rabbits and humans express HSP60 in endothelial cells at these sites, providing a prerequisite for interaction of specific T cells and antibodies elicited previously by infection or vaccination. In addition, it should be mentioned that immunization of rabbits with recombinant mHSP65 results in development of atherosclerosis, but not arthritis, while the reverse is true for rats [5]. This phenomenon seems to be due to the recognition of different HSP60 epitopes by the immune system of these two species. Detailed analyses aimed at the identification of the epitopes responsible for sensitizing HSP65/60-reactive T cells found in atherosclerotic lesions of rabbits compared to those known to play a role in the induction of arthritis in rats (peptide AA 180-188 of the 573 AA long HSP65) as well as in human rheumatoid arthritis are still lacking. Given the high sequence homology between bacterial and human HSP60s (65% at the amino acid level) [4], it is naturally conceivable that cross-reactions of antibodies and T cells against HSPs between microbes and humans contribute to the development of atherosclerosis. Whilst recent studies by Lamb et al [93, 94] confirmed that subcutaneous immunisation with BCG (containing HSP65) enhances lesion development, two groups of investigators independently reported that the mucosal administration of small dosages of HSP65 results in the reduction of atherosclerotic lesions in apoE-/- mice [95, 96]. The mechanisms whereby different responses are achieved when administering the antigen via different routes, i.e. mucosal vs subcutaneous immunisation, are speculative at present. It is likely, however, that whilst subcutaneously administered HSP induces atherosclerosis via the activation of TH1 CD4+ T cells, the mucosal administration of HSP65 leads to TH2 CD4+ T cell responses [97]. Thus, it raise a hope that atherosclerosis might be prevented or reduced by vaccination against antigens, e.g. HSPs [97].
10. CLINIC STUDIES D E M O N S T R A T I N G T H E R O L E OF AUTOANTIBODIES TO HSPS IN ATHEROGENESIS In a large study of sera from a group of human volunteers, aged 40-90 years, the Bruneck Study, we showed a correlation of anti-mHSP65 antibody titers with the presence of sonographically-demonstrable atherosclerotic lesions in the A.carotis [98]. Statistical analyses revealed that these antibodies reflected a risk factor independent of classical risk factors for atherogenesis, except age. Furthermore, it was shown that these antibodies not only react with mHSP65, but show strong cross-reactivity with HSP60 of Chlamydia pneumoniae (criSP60), mHSP65, GroEL and, most importantly, hHSP60 [99]. As mentioned, it was then shown that human affinity chromatography-purified anti-criSP60 or anti-GroEL antibodies were also able to lyse stressed human endothelial cells [100] or macrophages [ 101 ] in a complement-dependent fashion or via ADCC. That the mitochondrial protein HSP60 is transported to the cell surface [102] has, in the meantime, been unequivocally corroborated by others [103], although the possible functional role at that location is still elusive. In a recent follow-up study to our work on anti-HSP65/60 antibodies in the sera of the Bruneck Study, we were able to show that this parameter is very robust, and thus a good indicator of morbidity, but it is also an indicator of mortality, since patients who died during the period from 1990 to 1995 had significantly elevated antibody titers [ 104]. The association of high titers of anti-HSP60/65 antibodies with cardiovascular disease is not only valid for carotid, but also for coronary atherosclerosis [105]. Two independent groups demonstrated that more than 70% of the study subjects had antihuman HSP60 antibodies [106, 107]. The prevalence of coronary artery disease was significantly increased in seropositive compared with seronegative patients. Importantly, HSP60 antibodies were related to disease severity, which persisted after adjustment for traditional risk factors ie. age, race, sex, smoking, diabetes, hypercholesterolemia, hypertension, and C-reactive protein levels [106, 107]. Moreover, Huittinen et al [108] reported that human HSP60 IgA or Chlamydial HSP60, antibodies were a significant risk factor for coronary events.
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Table 2. Summary of epidemiological studies on HSP antibodies Author & reference
Cases/controls
Disease
HSPs
Odds ratio & p valuesa
Xu et al [98] Gruber et al[ 109] Hoppichler et al [105] Mukherjee et al [111] Frostegard et al [ 110] Birnie et al [112] Prohaszka et al [ 115] Xu et al [104] Chan et al [108] Zhu et al [ 107] Burian et al [ 106] Gromadzka et al [ 114] Prohaszka et al [ 117] Ciervo et al [116] Huittinen et al [ 119] Veres et al [ 118] Mahdi et al [ 120] Huittinen et al [ 120]
867 107/90 203/76 28/12 66/67 136 74 750 61/21 274/91 276/129 180/64 424/321 179/100 239/239 386/386 250/250 241/241
Carotid AS Vascular diseases CHD, MI CHD, PTCA Hypertension CHD CHD Carotid AS Peripherial AS CAD CAD C ischemia CHD CHD CHD HOPE CAD CHD
HSP65 HSP65 HSP65 HSP65 HSP65 HSP65 HSP60 HSP65 HSP70 HSP60 HSP60 HSP65/70 HSP60 HSP60 HSP60 HSP65 criSP60 HSP60
1.52 (1.09-2.02) p < 0.001 p < 0.05 p = 0.036 p = 0.034 p = 0.012 p < 0.0001 1.42 (1.02-1.98) p = 0.0037 1.86 (1.13-3.04) 2.6 (1.3-5.0) p < 0.0001 p < 0.007 p < 0.05 2.0 (1.1-3.6) 2.1 (1.2-3.9) 3.9, p = 0.005 2.11 (1.08-4.13)
ap values are shown when lacking data of odds ratios in the papers. AS, atherosclerosis; CHD, coronary heart disease; CAD, coronary artery disease; MI, myocardial infarction; HOPE, heart outcomes prevention evaluation.
When an elevated human HSP60 IgA antibody level was present simultaneously with a high C pneumoniae IgA antibody level and an elevated C-reactive protein level, the relative risk was 7.0. In addition, many other groups [105, 109-120] confirmed the elevated levels of HSP antibodies in coronary heart disease, myocardial infarction, stroke, hypertension, and restenosis after angioplasty (Table 2). Therefore, an elevated human HSP60 antibody level may be a risk factor for atherosclerosis and could be a marker of the disease, especially when it is present with C pneumoniae infection and inflammation. Interestingly, myocardial infarction entails decreased titers of these antibodies presumably due to complex formation of endogenously released HSP60 with pre-existing antibodies and removal of immune complexes via the reticular EC system. Cardiac ischemia which leads to the release of endogenous antigenic HSP60, has been demonstrated in a perfused rat heart model [ 121]. Initial attempts to identify mHSP65/HSP60, cross-reactive epitopes recognized by human anti-
720
bodies using overlapping peptides spanning the whole mHSP65 molecule, revealed three linear epitopes, i.e. at the N-terminus AA 91-105, the C-terminus AA 501-515, and a less well defined AA 171-185 stretch in between [ 122]. Since antibodies, in contrast to T cells, generally recognize conformational rather than linear epitopes, it is of interest that computer modelling of hHSP60 based on the known structure of GroEL showed that the N- and C-terminal linear epitopes seemed to associate to form a conformational epitope in the native hHSP60 molecule. Very recently, we purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified [123]. Antisera specific for these cross-reactive epitopes were produced
by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease. Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis [ 123].
11. IN VITRO STUDIES SUPPORTING T H E ROLE OF HUMORAL AND CELLULAR I M M U N I T Y IN E N D O T H E L I A L DAMAGE
In vitro, the same stressors lead to the simultaneous expression of HSP60 and adhesion molecules (intercellular adhesion molecule- 1 - ICAM- 1; endothelial leukocyte adhesion molecule-1 - ELAM-1; vascular cell adhesion molecule- 1 - VCAM- 1) by endothelial cells at the mRNA and protein levels [61]. However, arterial endothelial cells seem to be more susceptible than venous endothelial cells to the action of various stressors (notably oxLDL), probably based on a lower threshold for the latter due to the pre-stressing effect of life-long exposure to the higher arterial blood pressure. It is known that venous bypasses of occluded arteries often undergo severe restenosis and "venosclerosis" subsequent to being subjected to the higher arterial blood pressure conditions. We have recently developed a mouse model [124, 125] for carotid bypasses that allows for an in depth study of this issue using appropriate donor (e.g. cytokine or adhesion molecule knock out mice) and recipient combinations, as well as various therapeutic (drugs, antisense oligonucleotide, etc.) interventions [ 126, 127].
12. T H E A U T O I M M U N E HYPOTHESIS OF ATHEROGENESIS The immunologic hypothesis obviously may encompass the response-to-injury as well as the altered lipoprotein hypotheses. The influence of stress on endothelial cells is a primary response that entails an inflammatory reaction mediated by HSP65/60-
reactive T cells and HSP60/65-specific antibodies, finally resulting in the development of the classical atherosclerotic lesions when additional risk factors come into play. B iochemically-altered lipoproteins, such as oxLDL, may first act as stressors on EC and only later lead to the transformation into foam cells of blood-born macrophages and SMC immigrating from the media into the intima. Moreover, it is not yet clear where the sensitization of HSP60-reactive lymphocytes occurs, and whether we are dealing with a cross-reaction between microbial HSP60 and hHSP60 triggered by an exogenous antigen only, e.g. during infection, or are witnessing a bona fide autoimmune reaction against chemically altered autologous hHSP6o. However, we have solid data that all individuals studied so far in our laboratory (aged one month to 80 years) show inflammatory foci in the arterial intima at areas subjected to major haemodynamic stress. Fig. 1 summarizes our concept of the autoimmune pathogenesis of atherosclerosis. This concept is based on the fact that most individuals possess antibodies and T cells recognizing HSP60 epitopes that may result in cross-reactivity between microbial (bacterial, parasitic-viral) antigens and hHSP60. In addition, non-HSP microbial epitopes are, of course, also recognized by the human immune system. Together, these immune mechanisms confer protection to the individual, who "pays" for this protective immunity when arterial EC are maltreated by atherogenic risk factors, e.g. hypertension, smoking, oxygen radicals, infections, etc. In this case, the anti-microbial HSP60 response will recognize autologous HSP60 and lead to the first inflammatory stage of atherosclerosis, which may be followed by the development of more severe lesions, i.e. plaques, including the formation of foam cells, ECM and extracellular lipid depositon, exulceration and even calcification. The specificity of the immune response for cross-reactive atherogenic HSP60 epitopes depends on the individual T and B cell repertoire and the association of the relevant peptides with appropriate MHC class II and class I molecules. Thus far, we do not know whether cellular or humoral effector mechanisms are the first to mediate vascular damage. From immunohistological data in humans and early observations in mice, we presently favour the concept that atherosclerosis is initiated by CD4+/TH1 cells, and that humoral
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I Risk factors, e.g. oxLDL and infections 1 .
l Toxins e.g. LPS
.
.
.
.
.
Lipid molecule mimicry
JHSPs
Inflammation I
Autoimmunity
\
/
[ AtherosclerosisI Figure 1. Schematic representation of the potential role of HSP60 in atherogenesis. A variety of stressors, such as oxLDL, infections, biomechanical stress, free radicals, toxins, heat shock and other stress, induce HSP production in the arterial wall. HSPs activate both innate and adaptive immunities resulting in proinflammatory responses and autoimmune reactions, which contribute to atherosclerosis.
antibodies play an accelerating and aggravating role.
ACKNOWLEDGEMENTS We a c k n o w l e d g e all the authors f r o m our collaborating groups w h o s e work is cited in the present review. This w o r k was supported in part by grant from O a k Foundation.
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9 2004 Elsevier B. V. All rights reserved. Infection and Autoimmunity Y. Shoenfeld and N.R. Rose, editors
Interaction between Rheumatoid Arthritis and Infections Luis J. Jara', Gabriela Medina ~, Olga Vera-Lastra 2, Carmen
N a v a r r o 3 and
Juan M. Miranda 4
IClinical Research Unit, 2Internal Medicine Department and 4Rheumatology Department, Hospital de Especialidades, Centro Medico La Raza, IMSS, Mexico City, Mexico; 3Molecular Biology Department, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico
1. INTRODUCTION Rheumatoid arthritis (RA) is a systemic chronic inflammatory disease characterized by symmetric inflammation and destruction of joints. Unfortunately, instead of large efforts and technology, the etiology of RA remains elusive although it appears that genetic, infectious, environmental, and hormonal factors are all involved in complex interrelated ways. In relation with infectious hypothesis, the Koch postulates requires isolation of microorganism inside the infected host, its identification by culture or direct microscopic analysis, and the transfer of the illness to another susceptible host causing similar signs and symptoms. These postulates have not been reproduced by different microorganisms implicated in RA [ 1-2]. However, despite RA did not fulfill those postulates to consider it as an infectious disease, recent evidences support this hypothesis. One of the most exciting areas of investigation has been the application of Polymerase Chain Reaction (PCR). This technique is useful to the detection of bacterial and viral DNA in patients with RA. With PCR technique, foreign antigens of bacteria, virus, and superantigens can be found in the synovial fluid. In addition, the lymphocytes from synovial fluid can develop a local immune response stimulated by microorganisms such as Proteus mirabilis and Epstein-Barr virus. These lymphocyte responses were higher with these microorganisms than with other stimuli. Those data suggest an immune-infectious cause for RA [3--4]. However, the difficulty of separating pathogens from contaminants has hampered these studies. On
the other hand, the presence of foreign antigens is not specific for RA. Therefore, the role of these organisms in initiating and perpetuating inflammation in RA remains unknown, but continues to be actively investigated. This chapter provides an update on various mechanisms in which infectious agents may play a role as inciting or perpetuating factors in the pathogenesis of RA.
2. HOST-INFECTION INTERACTION IN RHEUMATOID ARTHRITIS Several theories have been proposed to explain the biological interaction between microbial antigens, immune system, and RA (Fig. 1). The main theories are: 1. Molecular mimicry (MM) 2. Superantigens (SA) 3. Heat shock proteins (HSP)
2.1. Molecular Mimicry Although the triggering event in most autoimmune diseases is unknown, an infectious cause has long been postulated to explain the development of autoimmunity. The hypothesis of molecular mimicry suggest that infectious agents may trigger an immune response against autoantigens, because the foreign antigen is molecular similar to the host antigens but differ sufficiently to induce an immune response when presented to T cells. Therefore, the tolerance to autoantigens is breakdown, and the pathogen-specific immune response that is gener-
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Figure 1. Molecular mimio3': Foreign antigens (EBV, Proteus mirabilis, etc), are similar to the host antigens but differ sufficiently to induce an immune response when presented to T cells. Superantigen: In RA, bacterial or viral antigens can activate T cells, bypassing the involvementof antigen processing cells, which can lead to proliferation and expansion, anergy, or apoptosis. Heat shock protein: As a consequence of stress factors, HSP from human and bacteria are expressed in synovial tissue of RA patients. HSP is associated with a protective role against pro-inflammatory products.
ated cross-reacts with host structures may cause tissue damage and disease [5]. For many autoimmune diseases, a connection with microbial infection through a mimicry mechanism has been proposed. The classic clinical example for molecular mimicry is the association of Group A streptococcal M protein and cardiac myosin in the development of rheumatic fever [6]. However, direct evidence of molecular mimicry in the pathogenesis of RA is scarce. Albani et al [7], suggested that Escherichia coli heat shock protein dnaJ displayed the QKRAA aminoacid sequence present in the HLA-DRB 1 shared epitope. The synovial cells from patients with early RA had a high response to the antigen, suggesting a cross-reactivity between dnaJ and activated T cells. Proteus mirabilis also carries sequences showing molecular mimicry to the "shared epitope" (EQK/RRAA) present in HLA-DR1/4 molecules and to type XI collagen of hyaline cartilage [8]. Based on analogous sequences of amino acids or on cross-reactions of monoclonal antibodies, numerous examples of such molecular mimicry have been
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reported. There are, however, no clear examples of human RA caused by molecular mimicry [9].
2.2. Superantigens Superantigens are potent immunomodulators derived from microorganisms such as bacteria, viruses and mycoplasma that can activate T cells, B cells, natural killer cells, and monocytes and are known to trigger experimental autoimmune disease. Their effects on immune system are obtained through their binding both to outer portion of binding grooves of an MHC on antigen presenting cells and to non-recognizing structure of hypervariable region of T cell antigen receptor. Superantigens induce not only T/B activation but also immunological tolerance through oligoclonal deletion and/ or anergy [3, 10]. Contributions of superantigens to the pathogenesis of RA has been discussed since a superantigen, mycoplasma arthritidis mitogen (MAM) revealed to be arthritogenic to mice, and the murine arthritis resembled human rheumatoid arthritis in the pathological findings. Recently,
Sawitzke et al [ 11 ], showed the selected elevation of antibodies to MAM in RA, in comparison with sera from patients with systemic lupus erythematosus, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, or healthy controls. This study suggested that MAM or a MAM-like molecule might be associated with RA. Anti-superantigen antibody did not correlate with the presence of rheumatoid factor. No superantigen definitely mediating autoimmune disease in humans has not been yet identified. 2.3. Heat Shock Proteins Patients with RA are confronted with a multitude of stressful events during the course of their disease. Heat is a physical stress factor present in the inflamed synovial membrane. Flares of disease activity based on an increased inflammatory activity lead to hyperemia and the release of pyrogenic substances. In fact, hyperthermia is a frequent and long-known symptom of RA. Heat shock factor's (HSFs) were discovered as signal transducers of heat stress and heat shock proteins (HSP's) as the effectors molecules that allow cellular adaptation to heat. Several multimember families of HSP's, a group of highly conserved microbial proteins, have been discovered and are characterized by their molecular mass and intracellular localization. Other stress factors such as shear stress, oxidative stress and proinflammatory cytokines can also induced certain types of HSP. Despite of the large number of investigations on autoimmunity against HSPs in RA, studies on the expression of HSPs in the synovial membrane have been scarce [ 12]. HSP60 is the human homologue of mycobacterial HSP65 and is expressed in the synovial membrane of patients with RA and juvenile RA; however, the synovial tissues of normal and osteoarthritis patients also express a considerable amount of HSP60.The predominant localizations are the synovial lining, the synovial endothelium, and the cartilage-pannus junction [13, 14]; in addition, synovial lymphocytes also express HSP60 [ 15]. Besides HSP60, the inducible form of HSP70 is also expressed in the synovial membrane of RA patients and it is not found in the synovial membrane of osteoarthritis [16]. In adjuvant arthritis and human arthritis, the expression of HSP60 and HSP70 in the synovial
membrane and the presence of an autoimmune response directed to HSP is associated with a protective role against a variety of toxic conditions such as oxidative stress, TNF t~, apoptotic death, etc, rather by than a pathogenic effect on stressed cells [ 17, 18]. Other protective mechanisms of HSP have been studied. The beta-chains of HLA-DR molecules associated with susceptibility to RA share a common aminoacid sequence with HSP70 DnaK. Using a highly sensitive method, Maier et al showed that peptides of associated and non-associated HLA-DR alleles, bind to HSP70. However, peptides containing the amino-acid sequence DERAA, found in HLA-DR alleles and strongly associated with protection from RA, did not bind any HSP70. This result suggests a possible association of non-binding of HSP70 to HLA-DR molecules and protection from RA [19]. Juvenile idiopathic arthritis (JIA) is in a majority of the cases of self-limiting, and sometimes even a self-remitting disease. A growing amount of data suggests that active T cell regulation determines, at least partly, the clinical outcome of JIA. In this regard, de Kleer et al [20], analyzed the T cell reactivity of peripheral blood and synovial fluid, in response to human HSP60 from oligoarticular and polyarticular JIA. The results show that T cells responding to human HSP60 in oligoarticular JIA patients express CD30 with a high production of IL-10 and a low production of IFN gamma (regulatory phenotype). In contrast T cell from polyarticular JIA patients responded to human HSP with virtually no expression of CD30 and a low IL-10: IFN gamma ratio. Taken together, these data imply that immune modulation with HSP can be an effective way to restore natural occurring T cell responses, and, thus, treat JIA and RA [21]. Other study showed that RA patients, whose HSP60-stimulated T cells produced IL-4 and/or ILl0, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by HSP 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose HSP60-stimulated cells produce T-helper 2 type cytokines [22].
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Table 1. Infectious agents and rheumatoid arthritis
Epitope
Autoantigen
PCR
Antibody
Reference
Parvovirus B 19
B 19-VP1/VP2
[27, 33]
Anti-EBV
[46-48]
Cytomegalovirus HTLV-I/II
Gp 110 QKRAA Gp65 protein Gp21, p24, p29
B 19 DNA, VP1/VP2 EBV-DNA
Anti-B 19
Epstein-Barr virus
CMV-DNA HTLVl-p24, p19
Anti-CMV Anti-HTLV1
[52, 53] [57-60]
HTLV-5
HRV-5 DNA
HLA-DRB1 Collagen II HLA-DRB1 Fibroblast CD8+ T cells HOXD9 Fibroblast ?
HRV-5 DNA
?
[62]
Infectious Agent Virus
Bacteria
Mycobacteria
Mycobacterial DNA
Mycoplasma
P107, P48, P29, MAM (superantigen) ESRRAL EQKRAA
Proteus mirabilis
Escherichia coli
HSP dnaJ QKRAA Bacterial components Muramicacid (peptidoglycan)
Mycobacterial DNA Mf HLA-DRB1 EQKRAA, Collagen II HLA-DRB1 Q RAA 9
3. WHAT IS THE ROLE OF VIRUSES IN T H E R H E U M A T O I D ARTHRITIS D I S E A S E PROCESS?
From a long time it has been proposed than certain viruses can be responsible of immune alteration in RA. The main viruses are: parvovirus B 19, retrovirus as HTLV I and II, Epstein-Barr virus, cytomegalovirus and herpes virus between some others (Table 1) [23]. 3.1. Parvovirus B I9 and RA
Parvovirus B 19, discovered in 1975 by Yvonne Cossart et al [24] is composed of a small non-enveloped particle enclosing a single-stranded DNA-genome of approximately 5500 nucleotides in length. The capsid consists of two structural proteins, VP1 (83 kDa) and VP2 (58 kDa); VP2 is the major structural protein (96% of total capsid proteins) [25]. Parvovirus B19 infectious has been associated with erythema infectious in children. In adults,
732
Anti-HSP65, 70 Anti-Mf, Anti-MAM ESRRAL peptide Anti-Proteus mirabilis
[65, 66]
HSP dnaJ"
Anti-E. coli
[7]
Pan-bacterial DNA
?
[86, 88]
[11, 74, 75] [78, 79, 81]
acute infection is often accompanied by a selflimited polyarthritis. Rheumatoid factor may be present, and the arthritis occasionally can become chronic. Parvovirus B 19 is also associated with a variety of autoimmune diseases such as lupus-like disease, vasculitis, dermatomyositis, autoimmune neutropenia, immune thrombocytopenia, hemolytic autoimmune anemia, etc. [26]. The possible role of this virus in the etiology of RA is suggested by epidemiological, clinical and experimental studies. Murai et al [27] report a group of patients with chronic arthritis very similar to RA after parvovirus infection with erosive changes in bone, rheumatoid nodules, and rheumatoid factor. Human parvovirus B 19 was detected in the synovial tissue in 30 of 39 patients with RA and HLA-DR4, and infrequently in those with osteoarthritis and traumatic joints with evidence of in vivo viral activity [28]. In contrast, Hokyner et al [29], studied the synovium of 30 healthy adult subjects undergoing arthroscopy after trauma. They found serological evidence of previous parvovirus B19 infection in 60% of patients,
and 67% had parvovirus DNA in their synovium. No patients had a history of chronic arthritis. Several groups have studied the incidence of parvovirus B19 infection in patients with early RA and they have found anti-B 19 IgM antibodies between 2 and 6% of patients [30, 31]. In a cohort study, 147 newly ascertained subjects with inflammatory polyarthritis with a disease duration of less than 16 weeks were followed up for three years. Only 4 (2.7%) patients had evidence of recent B19 infection (anti-B19 IgM positive) and 3 of them developed RA. Similar results were found in patients with recent parvovirus infection followed for a mean of 5 years. None of patients had chronic arthritis [32]. The following evidences support the etiological role of B 19 DNA: 1. Parvovirus B 19 chronic arthritis can be undistinguished from RA. 2. The association with HLA-DR4 and rheumatoid factor production. 3. The presence of B 19 DNA in the synovial membrane of RA patients. 4. The demonstration of parvovirus B 19 capsid proteins in lymphocyte cells in synovial tissue of RA patients [33]. 5. The development of RA after a parvovirus B 19 infection. 6. Fibroblasts activation incubated with B 19 DNA. 7. Cross reactivity of anti-B 19 antibodies with type II collagen. Parvovirus B 19 infection is undoubtedly associated with short-lived arthropathy, with similar characteristic to RA. There are evidences that B 19 infection, could only to be implicated in a very small proportion of RA, if any. However, serological evidence of human parvovirus infection was very infrequent in a large cohort of patients with acute arthritis, and persistence of B 19 DNA in synovium cannot be reliably linked to RA [29, 32]. A recent study, compared the clinical outcome of remission during 6 months follow up in patients with arthritis of less than 3 months' duration (21% with RA), with and without signs of prior infection. In this population, 45% of the patients presenting with a new-onset arthritis had had a prior infection, but there were only a few cases preceded by infections by parvovirus B 19 infections. Remission during first 6 months
was observed in patients with and without prior infection [34].
3.2. Epstein-Barr Virus (EBV) and RA From a long time ago it has been postulated the etiological roll of Epstein-Barr virus in RA. EBV is a gamma herpes virus that infects more than 90% of population. The virus is transmitted by oral way; it replicates in oropharynx and then infects B lymphocytes where it stays latent. EBV is a potent stimulator of polyclonal B cell proliferation, and can induce the proliferation of autoantibody-producing B cells, including those producing rheumatoid factors. Serological studies from the1970 are also suggested that autoantibodies found in patients with RA might cross-react with EBV nuclear antigen. The cellular immune response plays an important role to control the EBV infection [35-36]. In 1976, Alspaugh and Tan [37] were the first in suggest an etiopathogenic role of EBV in RA. These researchers found antibodies against lymphocytes infected by EBV in the sera from RA patients. The antigen was called RANA (rheumatoid arthritis associated nuclear antigen) and the activity anti-RANA was directed trough the nuclear antigen of EBV. (EBNA1) [38, 39]. Other studies showed a cross reactivity between anti-EBV antibodies with self antigens such as collagen [40]. However, the importance of these findings was recently established due to the finding of specific T-lymphocytes clones against EBV antigens in RA patients. An analysis of cell receptors suggests that specific T-lymphocytes were cloned into the articular space from the donor [41]. A recent molecular analysis showed that DNA and RNA that codifies for EBV are more frequent in the synovial tissue than in normal controls, especially in HLA-DRB1 patients (shared epitopes). Therefore EBV may be an environmental risk factor for RA particularly in patients with HLA-DRB 1 alleles [4]. The most important evidence for the hypothesis that rheumatoid synoviocytes could be a target for EBV infection results from isolation of an EBV-carrying fibroblast cell line from RA synovial tissue [42]. Other study, using the PCR technique showed the presence of EBV DNA and proteins involved with viral replication in the synovial tissue of RA patients [43]. However, using similar techniques, other studies found little evidence of EBV infection
733
in synovial tissue [44, 45]. Further recent study has suggested that RA is associated with a decreased T cell response to Epstein-Barr glycoprotein 110. This protein is important in the control of the viral replication, suggesting that RA is associated with persistence of Epstein-Barr infection because gp110 EBV viral protein contains a sequence of aminoacids (QKRAA) that corresponds to the third hypervariable region of HLA-DRB 1 alleles associated with RA risk [46]. Recently, Balandraud et al [47] demonstrated that in patients with RA the EBV DNA load in peripheral blood mononuclear cells is increased in almost 10-fold compared with that in normal controls. The EBV load was stable over time and is not obviously influenced by disease modifying antirheumatic drugs or by HLA-DR. The concept that EBV plays a role in disease triggering is particularly appealing in oligoarticular juvenile idiopathic arthritis (JIA) since in the majority of cases the onset of JIA occurs before 6 years of age, and in the majority of people the encounter with the virus occurs during the first years of life. In this regard, T cell cytotoxic activation with production of proinflammatory cytokines in response to stimulation with self HLA-derived peptides or homologous peptides derived from EBV proteins, were found only in patients with oligoarticular JIA, and not in controls. These findings suggest that molecular mimicry may be part of the pathogenesis of the RA and JIA, and could be the basis of one of the likely multiple candidates for antigen-specific immunotherapy approaches in the future [48]. The frequency EBV proteins into synovial tissue is variable. Therefore, it seems unlikely that EBV infection has a primary etiologic role in RA, but may be a co-factor involved in defective immunodysregulation or molecular mimicry. EBV may have a role in the progression or exacerbation of inflammatory responses within the RA joint. Recently it has been shown in vitro that retinoids can limit the proliferation and differentiation features of EBV. If treatments can be developed which limit or avoid reactivation of EBV, these may be beneficial in RA [49-51].
3.3. Cytomegalovirus (CMV) and RA Similar to EBV the initial studies showed antiCMV antibodies in patients with RA. Next studies
734
detected CMV DNA and this virus was isolated in the synovial tissue from RA patients [52, 53]. An interesting finding was that RA patients and antiCMV antibodies also had higher fitters rheumatoid factor. It has been suggested that immuno-supressive effect of methotrexate treatment may be responsible in part of the association between cytomegalovirus and RA [54].
3.4. Retrovirus and RA Due to the retroviruses are capable of induce autoimmune phenomena transforming its RNA in DNA and its further integration to the DNA of the host; they are strong candidates to play an etiological roll in RA. The most studied retroviruses in RA are HTLV-I and II. HTLV-I infection has been associated with chronic arthritis, particularly in Japanese subjects. The first studies demonstrated a higher prevalence of anti-HTLV-I antibodies in Japanese patients with RA than in controls [55, 56]. There was also found in RA patients' serological reactivity against p21 glycoprotein, a component from HTLV-I [57]. The site of infection in HTLV-1 infection is the synovial fibroblast, which becomes transformed in a tumor-like fashion. Fugisawa et al [58] showed that T-lymphocytes infiltrating joints recognize Env or Tax proteins from HTLV-I suggesting than these cells can precipitate the process of chronic articular inflammation. In the synovial membrane of these patients were found p24 and p19 antigens from HTLV-I. However these findings have only found in oriental patients but not in other patients [2]. The observation that about 8% of healthy American blood donors carry HTLV-1 Tax in their lymphocytes prompted studies to determine whether Tax positivity is more prevalent among patients with RA. In this regard, the prevalence of HTLV-1 Tax positive among patients with RA is 3 times higher than among healthy blood donors [59]. Persistent hyperplasia of synovial lining cells in RA is one of the major pathological changes that contributes to the progression of synovitis and destruction of bone and cartilage. Recently, Khoa et al [60] demonstrated that homeobox (HOXD9) gen, one of the major classes of transcription factors, was highly expressed in RA synoviocytes and HTLV-1, was also able to promoter and activate HOXD9
transcription. These observations have led to a search for other retrovirus in RA, particularly as retrovirus-like particles have been observed in RA synovium by electron microscopy. Recently, a novel retrovirus has been identified in humans. Human retrovirus-5 (HRV-5), first isolated in salivary gland tissue of a patient with Sj0gren syndrome, has been explored for its role in inducing autoimmunity [61]. Subsequent PCR studies have revealed HRV-5 proviral DNA in 53% of RA synovial samples, and in 10% of peripheral blood samples from patients with RA. The virus was found in a similar proportion of peripheral blood samples from patients with systemic lupus erythematosus, but not in blood or tissue from other rheumatic diseases [62]. This finding, however, could not be replicated by Gaudin et al [63] in a similar study. The differences may be related to geographic variations between the samples but as yet, a possible causal role of HRV-5 in the induction of RA cannot make. A recent study no support involvement of infectious retrovirus in actived RA synovial fibroblasts, but neither do they exclude a role of retroviruses in macrophages and T cells [64].
4. RHEUMATOID ARTHRITIS AND BACTERIA Over 70 years ago the histological similarity between rheumatoid synovitis and tuberculosis led to speculate that RA may be a mycobacterial infection, and many forms of mycobacteria are known to be arthritogenic [65]. Studies have shown elevated antibodies to mycobacterial heat shock proteins in sera of RA patients, suggesting the possibility of a cross-reactive epitope [66], however, other studies have not been capable to confirm these findings [67]. In addition, experimental studies have failed to show any evidence of mycobacterial infection by culture and/or molecular techniques, and trials of anti-mycobacterial therapy have failed to produce consistent improvement in disease [68-70]. Mycoplasmas cause erosive arthritis with rheumatoid factor in some animals species and can be chronic. In humans, most, but not all studies report an increased frequency (5-30%) of mycoplasma infection in RA compared to controls. Similar prev-
alence for infection is also reported in other chronic inflammatory rheumatic diseases [71-73]. These data do not provide strong support for any obligate role for mycoplasma in RA. However, the finding that both mycoplasma fermentans (Mf) and specific antibodies to Mf were present in the synovial fluid of RA patients suggests that in some RA patients Mf may play a role in initiating or perpetuing synovitis [74]. Mycoplasma produces a superantigen (MAM) and patients with RA had antibodies anti-MAM [ 11 ]. In support of the role of mycoplasma in RA, Ribero-Dias et al [75], showed in murine macrophages, that MAM induces nitric oxide release that is dependent on signaling through MHC class II molecules and Ir'-N gamma but independent of TLR4 expression. Clinical trials that demonstrate a partial response to tetracycline treatment in RA may not support a role for mycoplasma. The mechanism of action of this class of antibiotics might due to the anticytokine action or the inactivation of metalloproteinase [76]. Reactive arthritis of Lyme disease, produced by Borrelia burgdorferi spirochetal, has renewed interest about search of bacteria or bacterial fragments in patients with RA. In this regard, molecular biological techniques have allowed detect fragments of latent microorganism in synovial tissue. These techniques permitted to identify bacterial DNA and peptidoglycan in synovial tissue from RA patients. These results suggest that DNA bacterial wall components can stay in articulations from some RA patients and may increase the synovial inflammation [77]. Current areas of research include the investigation of the role of other bacteria in onset and exacerbation of RA, particularly Proteus mirabilis. Studies have found a correlation between high titers of antibodies to Proteus mirabilis and active RA [78, 79]. It still remains to be determined whether this increase in antibodies is due to a generalized inflammatory response or whether these antibodies represent a true increase in infection and carriage of Proteus, particularly in the upper urinary tract of patients with RA [80]. Recent investigation of molecular mimicry between P. mirabilis, collagen type II, and HLA-DRB1 has support the role of this infection in RA In this regard, Tiwana et al [81 ], have reported on a degree of sequence homology between P. mirabilis hemolysin ESRRAL
735
peptide and the RA DRB 1 susceptibility sequence EQKRAA No convincing evidence has yet been presented, however, to show a link between persistent carriage of P. mirabilis and RA [82]. More recently, Bartonella spp has been investigate in patients with RA [83]. Unfortunately, bacterial species identified by PCR frequently have had equal occurrence in control populations [84]. Other study, suggested that Escherichia coli heat shock protein dnaJ displayed the QKRAA aminoacid sequence present in the HLA-DRB 1 shared epitope [7]. Indeed, synovial cells from patients with early RA exhibited a marked response to the antigen, supporting cross-reactivity between that and activated T cells. The same group showed that IgG monoclonal anti-DNA antibody in a RA patient had homology with antibodies developed after staphylococcal and Hemophilus infection [85]. DNA from a wide variety of bacteria was isolated from synovial fluid from RA patients. However, this is perhaps most consistent with inflamed synovium acting as a nonspecific trap for bacterial fragments with perpetuation of the disease. The skeleton of bacterial cell walls mainly consists of polysacchafide, peptidoglycan, and cell wall-associated proteins. Peptidoglycan is the major constituent of the gram-positive bacterial cell wall, whereas lipopolysaccharide (LPS) is the dominant component of the gram-negative cell wall. Muramic acid is a component of the peptidoglycan moiety of the bacterial cell wall. In a recent study, Chen et al [86] detected muramic acid in the joints of some RA patients, suggesting persistence of bacterial cell wall components, rather than bacterial DNA, in the joints of chronic RA. However, the presence of bacterial cell wall is not specific for RA, since they were also found in joint tissues of osteoarthritis patients. Several studies have implicated bacterial cell wall components having multiple immunologic activities that may contribute to joint inflammation: The Toll-like receptors (TLR) of the first line of host defense recognize bacterial peptidoglycan. This component of bacterial wall produce proinflammatory cytokines and increase the expression of co-stimulatory molecules CD80 and CD86 from synovial macrophages. All of these evidences suggest that the bacterial cell wall components are potential arthritogenic agents [87]. Bacterial products such as peptidoglycans acting as adjuvants, are able to activate synovial
736
fibroblast of patients with RA, at least partially via TLR. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction [88]. Peptide p135, of the human cartilage proteoglycan, which contain the shared epitope QKRAA, a sequence that is over represented in bacterial HSP and HLA-DR4 alleles, is immunogenic/arthfitogenic in BALB/c mice [89]. The role of these bacteria in RA continues to investigate, but the pathogenic role for any one bacterium remains to be determined (Table 1).
5. CONCLUSIONS The hypothesis of chronic infection continues to be investigated as a pathogenic mechanism in RA. However, no firm evidence to date has clearly defined the role of any specific infectious agent in RA. Persistence in joint tissue has not been shown for any specific microbe or virus. Studies suggest that multiple different infectious agents have the capability of triggering the events that lead to chronic arthritis. One or more viruses can be detected in the synovial specimens of patients with early arthritis, irrespective of the clinical diagnosis [90]. This observation might be explained by migration of inflammatory cells harboring viral DNA into the inflamed joints. Both CD4+ and CD8+ T cells are likely to be recruited to other sites of inflammation including skin and lungs. In this regard, clonal population of CD8 + T cells specific for epitopes from common viruses are present in synovial fluid from patients with inflammatory arthritis [91]. It is possible that microbial products found in joint tissue are an epiphenomenon or innocent bystander. However, there are evidence that bacterial peptidoglycans act as adjuvant, activating synovial fibroblast. Finally, future studies will produce more valid results, but should not close our eyes to the possibility that RA is unrelated to infection.
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human T lymphotrophic virus type I/II envelope glycoprotein p21. Clin Immunol Immunopathol 1996;79: 182-8. Fujisawa K, Okomoto K, Asahara H. Evidence for autoantigens of Env/Tax proteins in human T cell leukemia virus type I env-px transgenic mice. Arthritis Rheum 1998;41:101-9. Zucker-Franklin D, Pancake BA, Brown WH. Prevalence of HTLV-1 Tax in a subset of patients with rheumatoid arthritis. Clin Exp Rheumatol 2002;20:161-9. Khoa DN, Nakasawa M, Hasunuma T et al. Potential role of HOXD9 in synoviocyte proliferation. Arthritis Rheum 2001 ;44:1013-21. Griffiths DJ, Venables PJ, Weiss RA et al. A novel retrovirus sequence identified in humans. J Virol 1997;71: 2866-72. Griffiths DJ, Cooke SP, Herve C et al. Detection of HRV5 in patients with arthritis and SLE. Arthritis Rheum 1999;42:448-54. Gaudin P, Moutet F, Tuke PW et al. Absence of human retrovirus 5 in French patients with rheumatoid arthritis. Arthritis Rheum 1999;42:2492-94. Seemayer C, Kolb S, Neidhart M e t al. Abscence of inducible retrovirus from synovial fibroblasts and synovial fluid cells of patients with rheumatoid arthritis. Arthritis Rheum 2002;46:2811-6. Rook GA, Lydyard PM, Stanford JL. Reappraisal of the evidence that rheumatoid arthritis and several other idiopathic diseases are slow bacterial infections. Ann Rheum Dis 1993;52(Suppl 1):$30--8. Bahr GM, Rook GAW, A1-Saffar M, van Embden J, Stanford JL, Behbehani K. Antibody levels to mycobateria in relation to HLA type: evidence for non-HLAlinked high levels of antibody to the 65 kD heat shock protein of M. bovis in rheumatoid arthritis. Clin Exp Immunol 1988;74:211-5. Worthington J, Ribgy AS, MacGregor A Silman AJ, Carthy D, Ollier W. Lack of association of increased antibody levels to mycobacterial hsp65 with rheumatoid arthritis. Ann Rheum Dis 1993;52:542-4. Pras E, Schumacher HR, Kasten DL, Wilder RL. Lack of evidence of mycobacteria in synovial tissue from patients with rheumatoid arthritis. Arthritis Rheum 1996;39:2080-1. Borg AA, Davis MJ, Fowler PD, Shadfor MF, Dawes PT. Rifampicin in early rheumatoid arthritis. Scan J Rheumatol 1993;22:39--42. Van der Heijden I, Wilbrink B, Schouls LM, van Embden JD, Breedveld FC, Tak PP. Detection of mycobacteria in joint samples from patients with arthritis using a genus-specific polymerase chain reaction and sequence analysis. Rheumatology Oxf 1999;38: 547-53.
71. Schaeverbeke T, Gilroy CB, Bebear C et al. Detection of mycoplasma fermentans, but not M. penetrans, by PCR assays in synovial samples from patients with RA and other rheumatic disorders. J Clin Pathol 1996;49: 824-8. 72. Haier J, Nasralla M, Franco AR, Nicolson GL. Detection of mycoplasmas infections in blood of patients with rheumatoid arthritis. Rheumatology Oxf 1999;38: 504-9. 73. Schaeverbeke T, Renaudin H, Clerk M e t al. Systematic detection of mycoplasmas by culture and PCR in 209 synovial fluid samples. Br J Rheumatol 1997;36: 310-4. 74. Horowitz S, Evinson B, Borer A, Horowitz J. Mycoplasma fermentans in rheumatoid arthritis and other inflammatory arthritides. J Rheumatol 2000;27:274753. 75. Ribero-Dias F, Shio MT, Timenetsky J et al. Mycoplasma arthritidis superantigen (MAM)-induced macrophage nitric oxide release is MHC class II restricted, interferon gamma dependent, and toll-like receptor 4 independent. Exp Cell Res 2003;286:345-54. 76. Tilley BC, Alarcon GC, Heyse SP et al. Minocycline in RA: a 48 week double-blind placebo controlled trial. Ann Intern Med 1995;122:81-9. 77. Van der Heijden IM, Wilbrink B, Tchetverikov I e t al. Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides. Arthritis Rheum 2000;43:593-8. 78. Subair H, Tiwana H, Fielder M e t al. Elevation in antiProteus antibodies in patients with rheumatoid arthritis from Bermuda and England. J Rheumatol 1995;22: 1825-8. 79. Rashid T, Darlington G, Kjeldsen-Kragh Jet al. Proteus IgG antibodies and C-reactive protein in English, Norwegian and Spanish patients with rheumatoid arthritis. Clin Rheumatol 1999;18:190-5. 80. Senior BW, Anderson GA, Morley KD et al. Evidence that patients with rheumatoid arthritis have asymptomatic "non-significant" Proteus mirabilis bacteriuria more frequently than healthy controls. J Infect 1999;38: 99-106. 81. Tiwana H, Wilson C, Alvarez A et al. Cross-reactivity between the rheumatoid arthritis-associated motif EQKRAA and structurally related sequences found in Proteus mirabilis. Infect Immun 1999;67:2769-75. 82. Gaston JSH. Proteus - Is it a likely aetiological factor in chronic polyarthritis? Ann Rheum Dis 1995;54:157-8. 83. Tsukahara M, Tsuneoka H, Tateishi H et al. Bartonella infection associated with systemic juvenile rheumatoid arthritis. Clin Infect Dis 2001;32:E22-E23. 84. Dillon B, Cagney M, Manolios Net al. Failure to detect Bartonella henselae infection in synovial fluid from
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sufferers of chronic arthritis. Rheumatol Int 2000;27: 2747-53. Chukwuocha RU, Zhang B, Lai CJ et al. Isolation of an IgG monoclonal anti-dnaJ antibody from an immunoglobulin combinatorial library from a patient with rheumatoid arthritis. J Rheumatol 1999;26:1439-35. Chen T, Rimpilainen M, Luukkainen R et al. Bacterial components in the synovial tissue of patients with advanced rheumatoid arthritis or osteoarthritis:analysis with gas chromatography-mass spectrometry and panbacterial polymerase chain reaction. Arthritis Rheum (Arthritis Care Research) 2003;49:328-34. Zhang X, Pacheco-Tena C, Inman RD. Microbe hunting in the joints. Arthritis Rheum (Arthritis Care Research) 2003;4:479-82. Kyburz D, Rhetage G, Seibl R et al. Bacterial pepti-
doglycans but not CpG oligodeoxinucleotides activate synovial fibroblast by toll-like receptor signaling. Arthritis Rheum 2003;48:642-650. 89. Hanyecz A, Barrios T, Berlo S E e t al. Induction of arthritis in SCID mice by T cells specific for the "shared epitope" sequence in the G3 domain of human cartilage proteoglycan. Arthritis Rheum 2003;48:2959-73. 90. Stahl HD, Hubner B, Seidel B e t al. Detection of multiple viral DNA species in synovial tissue and fluid of patients with early arthritis. Ann Rheum Dis 2000;59: 342-6. 91. Fazou C, Yang H, McMichel AJ, Callan MFC. Epitope specific of clonaUy expanded populations of CD8 T cells found within the joints of patients with inflammatory arthritis. Arthritis Rheum 2001;44:2038--45.
Subject Index [~l-adrenoreceptor 300 132-glycoprotein-I (~2GPI) 3, 185, 474 ]32GPI. See 132-glycoprotein-I 3,8 T cells 304, 631,632 Acantholysis 531 Acinetobacter 383 ACL. See IgG anti-cardiopilpin Active vaccination 105 Acute inflammatory demyelination polyneuropathy (AIDP) 593 Acute motor-axonal neuropathy (AMAN) 593 Acute motor-sensory axonal neuropathy (AMSAN) 593 Acute reactive arthritis (ReA) 675 Acyclovir 630 Adenine nucleotide translocator 299 Adjuvants 87 Adjuvant effect 311 Adult ITP 637 AECA. See Antiendothelial cell antibodies Affinity maturation 595 AIDP. See Acute inflammatory demyelination polyneuropathy AIDS 171,642 AIG. See Autoimmune gastritis AIN. See Primary autoimmune neutropenia AITD. See Autoimmune thyroid diseases ALB1-GP33 mice 158 Allergic reaction 60 A M A N . See Acute motor-axonal neuropathy A M S A N . See Acute motor-sensory axonal neuropathy ANA. See Antinuclear antibodies Anakinra 625 ANCA. See Antineutrophilic cytoplasmic antibodies Animal models 295, 718 Ankylosing spondylitis (AS) 384, 468, 675 Anti-70 kDa U 1-RNP 172 Anti-alpha myosin 176 Anti-calreticulin reactivity 442 Anti-cardiolipin antibodies 173 Anti-dsDNA antibodies 626 Anti-EPO 176 Anti-erythropoietin antibodies 176 Anti-ganglioside antibodies 591,594 Anti-HIV antibodies 638 Anti-idiotypic antibodies 642
Anti-myosin autoantibodies 176 Anti-phosphotidyl choline 174 Anti-phosphotidyl inositol 174 Anti-phosphotidyl serine 174 Anti-platelet antibodies 637 Anti-retinal antibodies 697 Anti-thyroglobulin antibodies 175, 523 Anti-thyroid peroxidase antibodies (anti-TPO) 175, 176 Anti-TPO. See Anti-thyroid peroxidase antibodies Anti-TSHR 176 Anti-[~2GPI antibodies 173, 476 Antiendothelial cell antibodies (AECA) 75 Antigen presenting cells 291 Antimicrobial chemotherapy 59 Antimicrobial treatment 57 Antineutrophilic cytoplasmic antibodies (ANCA) 174, 185 Antinuclear antibodies (ANA) 290 Antiphospholipid antibody (aPL) 474 Antiphospholipid syndrome (APS) 1,172, 184, 473 Anti HSP72 antibodies 366 aPL. See Antiphospholipid antibody Aplastic crisis 182 Apoptosis 78, 641 APS. See Antiphospholipid syndrome Arteritis 160 Arthritis 24, 106, 118 AS. See Ankylosing spondylitis Atherosclerosis 549, 701, 711 Atrophic gastritis 346 Autoantibodies 87, 158, 221,280, 290, 452, 629 Autoantigens 23, 711 Autoimmune cytopenia 185 Autoimmune epithelitis 289 Autoimmune gastritis (AIG) 346 Autoimmune hepatitis 167,221 Autoimmune liver disease 167 Autoimmune myocarditis 3 Autoimmune response 583 Autoimmune thrombocytopenia 172, 174 Autoimmune thyroiditis 166 Autoimmune thyroid diseases (AITD) 3, 363, 515 Autoreactive cytotoxic T cells 439 Autoreactive T cells 350 Avascular necrosis (AVN) 173 AVN. See Avascular necrosis Azathioprine 637
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B-cell activation 290 B-cell lymphoma 166 B27-associated diseases 676 Bacillus Calmette-Gurrin (BCG) 117 Bacterium 541,630 BCG. See Bacillus Calmette-Gurrin BCG vaccine 107 BD. See Behqet's disease Behqet's disease (BD) 172, 468 Borrelia burgdorferi 395, 480 Bovine spongiform encephalopathy (BSE) 383 Branched-chain ct-ketoacid dehydrogenase 299 BSE. See Bovine spongiform encephalopathy Bystander effect 156 Bystander tissue destruction 252 B cells 65, 164, 290, 305,641 C-reactive protein 701 Campylobacter 632 Campylobacter jejuni 108,591 Canine distemper virus (CDV) 563 CAR. See Coxsackie-adenovirus receptor Cardiac-specific antibodies 300 Cardiac autoimmunity 176 Cardiac dysfunction 309 Cardiac myosin (CM) 299 Cardiovascular immunopathology 159 Castleman's disease 60 Cataract 59 Catastrophic APS 475 CB3. See Coxsackievirus B3 CCP. See Complement control proteins CD. See Crohn's disease CD4 171,173,237, 238, 305 CD4+CD25+ cell 111 CD4-322 173 CD46 572 CD5 65,629 CD5 expression 66 CD8 § T cells 48, 238, 305 CDV. See Canine distemper virus Celiac disease 468, 687 Central nervous system (CNS) 251 Cerebrospinal fluid 632 CFA. See Complete Freund's adjuvant Chagas' disease 3,439, 444 Chemokine receptor expression 60 Childhood 334 Chlamydia 632 Chlamydia pneumoniae 570, 702 Chronic Chagas' disease cardiomyopathy 449 Churg-Strauss syndrome (CSS) 554 CIE. See Counter immunoelectrophoresis
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Class-switching 595 CM. See Cardiac myosin CMV. See Cytomegalovirus CNS. See Central nervous system CNS vasculitis 174 Complement 303, 593 Complement control proteins (CCP) 481 Complete Freund's adjuvant (CFA) 301 Coronavirus 691 Corticosteroids 59, 637 Counter immunoelectrophoresis (CIE) 172 Coxsackie-adenovirus receptor (CAR) 293, 302 Coxsackieviruses 292, 299 Coxsackievirus B3 (CB3) 3, 299 Coxsackievirus B (CVB) 159, 229, 365 Coxsackievirus type A13 295 Coxsackievirus type B4 230, 237, 295 CpG motifs 555 Creutzfeldt-Jakob disease 383 Crohn's disease (CD) 167, 467, 649, 675 Cross-reactive T cells 355 Cryoglobulinemia 174, 189, 201,290 Cryptic epitopes 28 Cryptogenic fibrosing alveolitis 167 CSS. See Churg-Strauss syndrome CTLA-4 363 CVB. See Coxsackievirus B CVD-patients 701 Cyclophosphamide 637 Cytokines 238, 239, 326, 640, 692 Cytomegalovirus (CMV) 59, 159, 495,593, 599, 614, 637, 643, 703 Cytotoxicity 50 DAF. See Decay-accelerating factor DC. See Dendritic cells DCM. See Dilated cardiomyopathy
Decay-accelerating factor (DAF) 293 Dendritic cells (DC) 159 Dermatomyositis (DM) 175, 583 Desmoglein 532 Diabetes-prone BioBreeding (DP-BB) 238, 241 Diabetes-resistant BioBreeding (DR-BB) 238 Diabetes type I 295 Dilated cardiomyopathy (DCM) 159, 299 Diphtheria 105 DM. See Dermatomyositis DP-BB. See Diabetes-prone BioBreeding DPT (diphtheria-pertussis-tetanus) 107 DR-BB. See Diabetes-resistant BioBreeding DR2 177 E. coli 493
EAE. See Experimental autoimmune encephalomyelitis EAN. See Experimental allergic neuritis EBV. See Epstein-Barr virus EC. See Endothelial cells ECOR. See Experimental coronavirus retinopathy
ELISA 172 EMC. See Encephalomyocarditis Encephalomyocarditis (EMC) virus 236 Endogenous retroviruses (ERV) 615 Endothelial cells (EC) 75, 617 Enteroviruses 292 Environmental factors 87 Environmental influences 562 Epiretinal membranes 59 Epitope spreading 156, 251 Epstein-Barr virus (EBV) 3, 163, 223, 235,593, 615, 637, 643 ERV. See Endogenous retroviruses Erythema infectiosum 182 Erythropoietin 176 Etanercept 623 Etiology 263, 271 Evan's syndrome 185 Experimental allergic neuritis (EAN) 596 Experimental autoimmune encephalomyelitis (EAE) 19 Experimental coronavirus retinopathy (ECOR) 691 Famciclovir 630 FcR. See Immunoglobulin receptors FcRII binding 642 FcctRI 596 FcTR 596 Fibrosis 613 GAD. See Glutamic acid decarboxylase GalNAcT-/- 591 Ganglioside-mimicking 600 Gangliosides 591 Gastric proton pump 348 GBS. See Guillain-Barr6 syndrome GD. See Graves' disease GD3 synthase 593 Genetic background 559 Genetic influence 560 Giant cell arteritis 553 Glutamic acid decarboxylase (GAD) 231 GM-CSE See Granulocyte-macrophage colony stimulating factor GM2/GD2 synthase 591,593 GPIa/IIa 638 GPIb 638 GPIb/IX 638 GPIIb/Illa 638
GPIIIa 638 GPIV 638 GPV 638 Graft rejection 27 Granulocyte-macrophage colony stimulating factor (GMCSF) 641 Graves' disease (GD) 172, 175, 363, 515 Graves patients 522 Guillain-Barr6 syndrome (GBS) 591 HAART. See Highly active antiretroviral treatment Haemophilus influenzae 591 Haemophilus influenza type b (Hib) 111 HAM/TSP. See HTLV-I associated myelopathy/tropical spastic paraparesis Hashimoto's thyroiditis (HT) 363, 515 HBV. See Hepatitis B virus HCMV. See Human cytomegalovirus HCV. See Hepatitis C virus Heart 450 Heart-infiltrating CD4§ T cell 324 Heart failure 299 Heat shock protein (HSP) 23, 555, 632, 701,705, 711,729 Helicobacter pylori 345, 477, 616, 638, 644 Henoch-Schrnlein purpura 554 Hepatitis A virus 222, 630 Hepatitis B vaccine 106 Hepatitis B virus (HBV) 157, 213, 630 Hepatitis B virus associated vasculitis 551 Hepatitis C 638, 644 Hepatitis C infection 189 Hepatitis C virus (HCV) 157, 189, 201,203, 213, 551,630 Herpes simplex virus (HSV) 60, 629, 637 HERV. See Human endogenous retroviruses HHV-6. See Human herpesvirus 6 HHV-6, Cellular immune response to 569 HHV-6A 567 HHV-6B 567 HHV-6 101K 569 HHV-6 p41/38 early antigen 568 Hib. See Haemophilus influenza type b Highly active antiretroviral treatment (HAART) 59 HIV. See Human immunodeficiency virus HLA-A2 subtype 48 HLA-B27 107, 373, 675 HLA-DR4 177 HLA-DR4 haplotype 106 HLA-DR antigens 638 HLA B51 629 HLA class II antigens 60 HLA class I antigens 60 HLA haplotypes 60 HRES-1 274
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HSP. See Heat shock protein HSV. See Herpes simplex virus HT. See Hashimoto's thyroiditis HTLV. See Human T-lymphotropic virus HTLV-I. See Human T-lymphotropic virus type I HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) 563 Human cytomegalovirus (HCMV) 552 Human endogenous retroviruses (HERV) 271,565 Human herpesvirus 6 (HHV-6) 567 Human immunodeficiency virus (HIV) 59, 171, 213, 496, 630, 638 Human immunodeficiency virus type I (HIV) 552 Human T-lymphotropic virus (HTLV) 173 Human T-lymphotropic virus type I (HTLV-I) 564 Hydrops fetalis 182 Hypersplenism 644 IBD. see Inflammatory bowel disease IDDM. See Insulin-dependent diabetes mellitus Idiopathic inflammatory myopathies (IIM) 583 I F - T 306, 624, 630, 640, 641 IFNs. See Type I interferons IgG anti-cardiopilpin (ACL) 440 IgM anti-J32 GPI antibodies 174 Ignition point 58 IIM. See Idiopathic inflammatory myopathies IL-1 306 IL-10 309, 630, 640 IL-12 60, 307 IL-2 630, 640 IL-4 309, 630, 641 IL-6 60, 183,641 IL-8 183 Immune-complex 555 Immune deviation 22 Immune regulation 35 Immune response 373 Immune restoration 58 Immune restoration inflammatory syndromes (IRIS) 57 Immune thrombocytopenic purpura (ITP) 174, 637 Immunoadsorption treatment 300 Immunocompetence 57 Immunodeficiency 57 Immunoglobulin receptors (FcR) 596 Immunotherapy 28 Infection triggered reactive arthritis 408 Infectious agents 586 Inflammation 711 Inflammatory bowel disease (IBD) 3, 167, 649 Infliximab 623 Influenza virus 157 Innate immunity 59, 303,484
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Ins-HA mice 157 Insulin-dependent diabetes mellitus (IDDM) 23, 157 Insulin-producing [5 cells 229 Interferon ot 365 Intravenous immunoglobulin (WIG) 185,594, 595, 637 IRIS. See Immune restoration inflammatory syndromes Isolated leukocytoclastic vasculitis of the skin 554 ITP. See Immune thrombocytopenic purpura IVIG. See Intravenous immunoglobulin JC virus (PML) 571 Kaposi's sarcoma 60 Kawasaki's disease 185,553 Keratoconjuctivitis sica 289 Kilham rat virus (KRV) 238 Klebsiella pneumonia 494 Koch 124 KRV. See Kilham rat virus La(SSB) 290 LAC. See Lupus anticoagulant LCMV. See Leukocytic choriomeningitis virus Leishmania 440 Leishmaniasis 441 Leprosy 58, 77 Leukocytic choriomeningitis virus (LCMV) 157 Leukocytoclastic vasculitis 174 Liebman-Sacks endocarditis 3 Lipid rafts 593 Lipopolysaccharide (LPS) 303, 597 Liver diseases 537 LNH. See Lymphoid nodular hyperplasia LPS. See Lipopolysaccharide Lupus anticoagulant (LAC) 173 Lyme arthritis 395,405 Lyme disease 397, 550 Lymphadenitis 59 Lymphoid nodular hyperplasia (LNH) 109 Lymphoma 205,290 M-CSE See Macrophage-colony stimulating factor M-protein 1 M. Crohn 623 M. tuberculosis 494, 624 M2 muscarinic receptor 300 Macrophage-colony stimulating factor (M-CSF) 641 Macrophages 236, 252 Macular edema 59 Major histocompatibility (MHC) complex 302 Malaria 439 MALT lymphomas 644 Mannose-binding lectin (MBL) 497
MAPL. See MHC-altered peptide ligands MBL. See Mannose-binding lectin MBP. See Myelin basic protein MBP-GP mice 159 MBP-NP mice 159 MCP. See Membrane cofactor protein Measles 105 Measles virus 222, 564 MEC. See Mixed essential cryoglobulinaemia Megakaryocytes 642 Megaloblastic anemia 644 Membrane cofactor protein (MCP) 572 Membranoproliferative glomerulonephritis 190 MFS. See Miller Fisher syndrome MG. See Myasthenia gravis MHC. See Major histocompatibility MHC-altered peptide ligands (MAPL) 45 MHC class II background 560 MHV. See Murine hepatitis virus Microbial antigens 439 Microchimerism 617 Microglial cells 251 Microscopic polyangiitis 554 Migrating arthritis 1 Miller Fisher syndrome (MFS) 594 Mixed cryoglobulinemias 214 Mixed essential cryoglobulinaemia (MEC) 551,554 MM. See Molecular mimicry MMR (measles-mumps-rubella) 107 Molecular mimicry (MM) 9, 26, 46, 110, 156, 238, 251, 275, 311,346, 384, 410, 451,476, 521,591,597, 616, 638, 729 Monoclonal antibodies 500 Mononeuritis multiplex 190 Morquio disease type B 593 MS. See Multiple sclerosis MSAs. See Myositis-specific antibodies MSRV. See Multiple sclerosis retrovirus Multiple sclerosis (MS) 19, 45, 108, 159, 163, 251,263, 386, 559 Multiple sclerosis retrovirus (MSRV) 566 Mumps 105 Mumps virus 234 Murine hepatitis virus (MHV) 563 Murine models 499 Myasthenia gravis (MG) 26 Mycobacteria 59 Mycobacterium tuberculosis 494 Mycophenolate mofetil 637 Mycoplasma pneumoniae 593 Mycotic aneurysm 550 Myelin 251 Myelin basic protein (MBP) 252
Myocarditis 31, 160, 299 Myositis-specific antibodies (MSAs) 584 M protein 321 Natural antibodies 595 Natural killer (NK) cells 304 Neonatal thrombocytopenia 638 Nested RT-PCT 294 Neuromuscular transmission 596 Neutropenia 59 NK. See Natural killer Nocardiosis 632 NOD. See Nonobese diabetic Non-immunocompromized 57 Nonobese diabetic (NOD) 241 OA. See Osteoarthritis Obsessive-compulsive disorder 333 Oligoclonal bands 570 Onchocerca volvulus 445 Onchocerciasis 442, 697 Opportunistic infections 495 OspA. See Outer-surface protein A Osteoarthritis (OA) 406 Outer-surface protein A (OspA) 395 PAN. See Polyarteritis nodosa
Paradoxical deterioration 59 Paradoxical inflammation 57 Parasites 443 Parasitic antigens 444 Parotid gland enlargement 290 Parvovirus 181 Parvovirus B 19 3, 495, 614, 630 Parvovirus B 19 infections 213 Passive vaccination 105 Pathogenesis 583 PCR. See Polymerase chain reaction PDGE See Platelet-derived growth factor Pemphigus 531 Penner serotyping 600 Peptide technology 499 Peptidoglycan 555 Peripheral tolerance 160 Persistent infection 294 Petechiae 638 Phage display technology 499 Plasmapheresis 594 Platelet-derived growth factor (PDGF) 641 PM. See Polymyositis PML. See JC virus; See also Progressive multifocal leuko-~ encephalopthy Pneumocystis carinii 632
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Polyarteritis nodosa (PAN) 185, 213, 551,553 Polymerase chain reaction (PCR) 186 Polymorphisms in cytokine genes 60 Polymyositis-dermatomyositis 583 Polymyositis (PM) 3, 172, 583 Polyprotein 293 Postulates 124 Prevotella 631 Primary autoimmune neutropenia (MN) 186 Primary biliary cirrhosis 167, 172, 175 Pristane-induced lupus 87 Progressive multifocal leukoencephalopthy (PML) 563 Proinflammatory cytokines 306 Proliferation 50 Protein A 555 Proteolipid protein 253 Protozoan disease 439 Pseudomonas aeruginosa 493 Pulmonary infiltrates 59 Pulmonary tuberculosis 624 Punctate keratitis 442 Purpura 638 RA. See Rheumatoid arthritis Radiation 365 Rat insulin promoter (RIP) 157 Raynaud's phenomenon 172, 175,289 RCA. See Regulators of complement activation RCA family 572 ReA. See Acute reactive arthritis; See also Reactive arthritis Reactive arthritis (ReA) 373,404 Recombinant adenovirus 160 Regulators of complement activation (RCA) 572 Regulatory T cell 239 Reiter's syndrome 373 Renal disease 440 Reovirus 240 RES. See Reticuloendothelial system Reticuloendothelial system (RES) 638 Retina 691 Retinal pigment epithelium 692 Retrovirus 132, 235,239 Reversal reaction 57 RF. See Rheumatic fever RHD. See Rheumatic heart disease Rheumatic fever (RF) 1,321,480 Rheumatic heart disease (RHD) 480 Rheumatoid arthritis (RA) 60, 163, 166, 183, 384, 623,729 Rheumatoid factor 106, 290 Rheumatoid polyarthritis 404 RIP. See Rat insulin promoter Rituximab 637
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Ro(SSA) 290 Rocky Mountain spotted fever 550 Rose-Bona criteria 2 Rubella 105 Rubella vaccine 107 Rubella virus 234, 240 SA. See Superantigens Saccharomyces cerevisiae 481 Salivary gland epithelial cell 294 Salivary gland epithelial cell cultures 291 Salmonella 493, 632 Schistosomiasis 441 Scleroderma 166 Scrapie 383 Sialadenitis 292 Sj6gren's syndrome (SS) 3, 164, 177, 289, 644 SLE. See Systemic lupus erythematosus SLEDAI 491 SM-LacZ mice 159 Small nuclear ribonucleoproteins (snRNP) 172 Smoking 365 snRNP. See Small nuclear ribonucleoproteins SpA. See Spondyloarthropathies Splenectomy 637 Spondyloarthritis 373 Spondyloarthropathies (SPA) 675 SS. See Sj6gren's syndrome SSc. See Systemic sclerosis SSPE. See Subacute sclerosing panencephalitis Staphylococcus aureus 493, 553,631 Streptococcal infections 333 Streptococcal pneumonia 493 Streptococci 631 Streptococcus pyogenus 480 Stress 364 Subacute sclerosing panencephalitis (SSPE) 563 Superantigens (SA) 555,600, 617,632, 729 Suppressor/inducer T cell 222 Swine-flu influenza 110 Syphilitic vasculitis 550 Systemic lupus erythematosus (SLE) 25, 60, 87, 105, 164, 171, 184, 271,491,625 Systemic necrotizing vasculitis 174 Systemic sclerosis (SSc) 613 T-cell epitopes 350 TAbs. See Thyroid antibodies Takayasu's arteritis 553 Tattoos 60 Tay-Sachs disease 593 Temporal arteritis 185 TGF-~. See Transforming growth factor-beta
Thl 58 Th2 58 Theiler's murine encephalomyelitis virus (TMEV) 251 Thyroid antibodies (TAbs) 519 Thyroid disease 175 Thyrotoxicosis 166 Thyrotropin receptor autoantibodies (TSHR-Ab) 515 Tic disorder 333 TLR. See Toll-like receptor TMEV. See Theiler's murine encephalomyelitis virus TNF. See Tumor necrosis factor TNF-ot. See Tumor necrosis factor tx TNF-~I. See Tumor necrosis factor I~ Tolerance 28 Toll-like receptor (TLR) 303,484, 505, 604 Toxoplasmosis 696 Transforming growth factor-beta (TGF-~) 641 Trypanosoma cruzi 3 TSH-R 367 TSHR-Ab. See Thyrotropin receptor autoantibodies TSH receptor 515 Tuberculoma 58 Tuberculosis 58 Tumor necrosis factor (TNF) 306, 623 Tumor necrosis factor ct (TNF-ct) 60, 183 Tumor necrosis factor [~ (TNF-I]) 306 Type 1 (insulin-dependent) diabetes meUitus 229 Type I diabetes mellitus 3, 505 Type I interferons (IFNs) 304 Tyrosine kinase signaling pathways 237 T cells 305, 450, 624, 677 T cell receptor crossreactivity 7 T cell repertoire 24 T lymphocytes 110 UC. See Ulcerative colitis
Ulcerative colitis (UC) 167, 649, 675 Uveitis 59, 691 Vaccination 28, 105, 117 Vaccination and autism 109 Vaccination and diabetes 111 Vaccinia virus (VV) 157 Varicella 637 Varicella zoster virus (VZV) 552, 638, 643 Vasculitis 172, 174, 185, 189, 202, 213, 549 VCP. See Virus complement control protein Viral pathogen 266 Virus 539 Virus-induced autoimmune diseases 134 Virus-induced models of autoimmune diseases 144 Viruses 629 Virus complement control protein (VCP) 482 Virus infection 123 Visna virus 563 Vitritis 59 VP1 181 VP2 181 VV. See Vaccinia virus VZV. See Varicella zoster virus Wegener's granulomatosis 185, 553 Xerostomia 289 YE. See Yersinia enterocolitica Yersinia 632 Yersinia enterocolitica (YE) 367 Yersinia lipoproteins 444 Yersinia outermembrane proteins (YOPs) 367 YOPs. See Yersinia outermembrane proteins YOP antibodies 368
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