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Doctorial Thesis from the Department of Immunology The Wenner-Gren Institute, Stockholm University MERCURY-INDUCED AUTOIMMUNITY: GENETICS AND IMMUNOREGULATION MONIKA HANSSON STOCKHOLM 2004 SUMMARY The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercuryinduced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered. © Monika Hansson ISBN 91-7265-813-4 Typografen Text och Bild AB, Stockholm 2004 ORIGINAL PAPERS This thesis is based on the following papers, which will be referred to in the text by their Roman numerals: I. Manuchehr Abedi-Valugerdi, Monika Hansson and Göran Möller. 2001. Genetic control of resistance to mercury-induced immune/autoimmune activation. Scand. J. Immunol. 54: 190-197. II. Monika Hansson and Manuchehr Abedi-Valugerdi. 2003. Xenobiotic metal-induced autoimmunity: mercury and silver differentially induce antinucleolar autoantibody production in susceptible H-2s, H-2q and H-2f mice. Clin. Exp. Immunol. 131: 405414. III. Monika Hansson and Manuchehr Abedi-Valugerdi. Mercuric chloride induces a strong immune activation, but does not accelerate the development of dermal fibrosis in tight-skin 1 (Tsk1) mice. Submitted. IV. Monika Hansson, Mounira Djerbi, Hodjattallah Rabbani, Håkan Mellstedt, Mustapha Hassan, Joseph W DePierre and Manuchehr Abedi-Valugerdi. Interactions between Th1-type of autoimmunity (induced by collagen), and Th2-type of autoimmunity (induced by mercuric chloride) in DBA/1 mice. Submitted. 3 TABLE OF CONTENTS ABBREVIATIONS.....................................................................................................................5 INTRODUCTION ......................................................................................................................6 TOLERANCE.............................................................................................................................8 Central T-cell tolerance ..............................................................................................................8 Peripheral T-cell tolerance...........................................................................................................9 The role of dendritic cells in T-cell tolerance ..................................................................................9 Regulatory T cells and T-cell tolerance ........................................................................................10 B-cell tolerance .......................................................................................................................10 AUTOIMMUNITY AND AUTOIMMUNE DISEASE ...........................................................12 Genetics of autoimmune diseases ...................................................................................................................12 Major histocompatibility complex (MHC) genes and susceptibility to autoimmunity..................................12 The role of non-MHC genes in the development of autoimmunity ...............................................................13 Gene aberration and development of autoimmunity ......................................................................................13 The role of environmental factors in induction and perpetuation of autoimmunity ......................................14 MERCURY-INDUCED AUTOIMMUNITY...........................................................................15 An experimental model to study the induction and development of autoimmunity ......................................15 Description of the model.................................................................................................................................15 Autoantibody production ................................................................................................................................15 Th1/Th2 paradigm in mercury model.............................................................................................................16 The role of IL-12 in regulation of mercury-induced autoimmunity...............................................................17 Genetic predisposition to mercury induced autoimmunity.............................................................................18 THE PRESENT STUDY ..........................................................................................................19 AIMS.........................................................................................................................................19 METHODOLOGICAL REMARKS.........................................................................................20 Mercury and silver treatment (paper I-IV) .....................................................................................................20 Evaluation of mercury-induced autoimmune manifestations (paper I-IV) ....................................................20 H-2 genotyping (paper I-III) ...........................................................................................................................20 Histological examination (paper III and IV) ..................................................................................................20 Detection of antigen-specific IgG1 and IgG2a (papers III and IV) ...............................................................21 Induction of CIA .............................................................................................................................................21 Evaluation of the development of arthritis .....................................................................................................21 SUMMARY OF THE STUDY.................................................................................................22 RESULTS AND DISCUSSION ...............................................................................................22 Paper I. ....................................................................................................................................................................22 Paper II....................................................................................................................................................................24 Paper III...................................................................................................................................................................25 Paper IV. ......................................................................................................................................27 CONCLUDING REMARKS....................................................................................................30 ACKNOLEDGEMENTS..........................................................................................................31 REFERENCES .........................................................................................................................33 APPENDIX: PAPERS I-IV 4 ABBREVIATIONS AFA ANolA APC BCR CII CD CIA CTLA-4 DC EAE H-2 IC IDDM IFN Ig IL MBP MHC MS PD-1 QTL RA RAG SLE T1D TCR TGF Th1, Th2 TNF Anti-fibrillarin antibodies Anti-nucleolar autoantibodies Antigen presenting cell B cell receptor Type II collagen Cluster of differentiation Collagen-induced arthritis Cytotoxic T-lymphocyte associated antigen 4 Dendritic cell Experimental autoimmune encephalomyelitis Histocompatibility locus 2 Immune complex Insulin-dependent diabetes mellitus Interferon Immunoglobulin Interleukin Myelin basic protein Major histocompatibility complex Multiple sclerosis Programmed cell death 1 Quantitative trait locus Rheumatoid arthritis Recombination activating gene Systemic lupus erythematosus Type 1 diabetes T cell receptor Transforming growth factor T helper type 1 and type 2 Tumor necrosis factor 5 INTRODUCTION Central to the immune systems ability to mobilize a response to an invading pathogen is its ability to distinguish self from nonself. To efficiently protect the host from invading pathogens, both adaptive and innate mechanisms have evolved. These mechanisms involve self-nonself discrimination and it has been suggested that this self-nonself distinction has served as a driving force in the evolution of the complicated mechanisms responsible for the construction of T and B cell receptors. How the immune system learns to discriminate between self-antigens and foreign antigens have preoccupied scientists for decades, and different theories have been put forward. In the beginning of 1900, Ehrlich and Morgenroth reported that animals could form antibodies against xenogeneic and allogeneic red cells, but never against autologous cells. To explain these phenomena Ehrlich formulated the theory of horror autotoxicus, which postulated that the organism would not normally mobilize its immunological resources to effect a destructive reaction against its own tissue (reviewed in Silverstein and Rose 1997). Between 1930 to early 1960, the dominant theory was an instruction theory, which offered an explanation for the large repertoire of antibody specificities, by suggesting that the antigen act as a template for the globulin producing machinery (reviewed in Silverstein 2002). In 1955, with the increasing interest in tissue transplantation, immunodeficiency diseases, autoimmunity and tolerance, Jerne put forward the natural selection theory of antibody formation. Now the antigens purpose was to select a matching antibody and transport it to an appropriate cell, which would be stimulated to produce more of the same. This idea did not gain much support at the time, but catalysed the formulation of the clonal selection theory by Burnet 1957 (reviewed in Silverstein 2002). This theory consisted of the necessary and sufficient components of a biological theory of antibody formation: antigenic selection of pre-existing specificities; a cellular dynamic to maintain and expand the response; and a mechanism to explain the development of immunological tolerance. Burnet realized that a division of the world into self and nonself could not be genetically based but rather be acquired. Therefore, he postulated that, during development of the immunological repertoire in ontogeny, any antigen present in utero would eliminate newly formed anti-self clonal precursors. As follows, the result of this clonal elimination an individual’s immune system could only respond to those foreign antigens that had not been encountered during fetal development (Silverstein and Rose 1997). However, over the years this theory has been modified, since our knowledge of the immune system expands. 6 New theories have been put forward in the past decades. Based on the two-signal theories and the finding that neonatal immune system, like adults can be primed to respond to foreign antigens, Matzinger and colleagues suggested that the primary driving force for the immune system is not to recognize self and nonself, but the need to protect against danger, the danger discrimination. Danger signals refer to signals from stressed, damaged or necrotic cells that trigger dendritic cells, resulting in the activation immune system (reviewed in Matzinger 1994). Janeway and colleagues have a different view about danger discrimination. In defending the selfnonself discrimination, they have pointed out that not only central and peripheral tolerance is important. The danger signals that activate the immune system are co-stimulatory molecules and cytokines induced by pathogens that are recognized by the innate immune system, and that this is the evolutionary consequence of discriminating between non-infectious self and infectious-self (reviewed in Janeway et al 1996, Medzhitov and Janeway 1997). Below I will discuss some aspects of our current knowledge of tolerance and autoimmunity. 7 TOLERANCE B and T lymphocyte antigen receptors have a remarkable capacity to bind and recognize numerous diverse foreign antigens. This immunological diversity is the result of random gene rearrangements during the development of these lymphocytes. The danger of rearrangements of B- and Tcell receptor genes is the potential risk of generation of B and T lymphocytes that express receptors capable of recognizing self-antigens, which thereby might result in the development of horror autotoxicus (Silverstein AM, 2001). To circumvent this problem the immune system has developed mechanisms to prevent the generation and/or the activation of B and T lymphocytes specific for self-antigens. Perhaps the most important of these mechanisms is central tolerance, during of which developing lymphocytes that express antigen receptors with high affinity for self antigens are clonally deleted in the primary lymphoid organs, the bone marrow and thymus. Central T-cell tolerance In the thymus, developing T cells go through positive and negative selection events before they are permitted to enter the periphery. These events form the basis for the self non-self discrimination of the immune system and are collectively recognized as central tolerance. During positive selection, thymocytes with a rearranged αβTCR will interact mainly with self-MHC molecules in the cortex. If the interactions are of low avidity the thymocytes will down regulate RAG recombinases and proceed to the next stage of development. If no interaction occurs the cell will continue to express RAG and rearrange the Vα until a successful TCR is generated, but if the cell fails it will die by a mechanism called “death by neglect”. In contrast, if the thymocytes at this cortical stage express a TCR with high affinity for self-antigen it was thought that the cell was deleted (reviewed in Kreuwel and Sherman 2001). However, McGargill et al. have presented evidence that a mechanism of receptor editing exists for immature T cells. They showed that thymocytes with high affinity for antigen presented by cortical epithelial cells, instead of being deleted continued to rearrange the TCRα in attempt to construct a more suitable TCR (McGargill et al. 2000). If the thymocytes survive this first step, they proceed to the medulla were they must pass additional selection criteria, which is also based on the strength of the signal through the TCR. Strong interactions between the TCR and self-peptide-MHC complexes lead to elimination of the thymocytes through apoptosis. This process of clonal deletion eliminates self-reactive T cells (reviewed in Kreuwel and Sherman 2001). However, there are some limitations of central tolerance; one is the requirement for the relevant autoantigens to be present in the thymus. Although there is evidence that many tissue specific antigens are present (Derbinski et al. 2001), many self-antigens may not access the thymus (Lo et al. 1989), whereas others are expressed later in life, after the T cell repertoire has been formed (Matzinger 1994). There is also a substantial possibility that those lymphocytes that express receptors specific for foreign antigens, may cross react with self proteins (Mason 1998). 8 Another consideration in this context is the promiscuity of TCR recognition, as a very stringent negative selection increases the risk of dangerously narrowing the repertoire available to respond to foreign pathogens. It might be more beneficial to let some thymocytes with a degree of selfreactivity to escape thymic negative selection and be regulated elsewhere (reviewed in Walker and Abbas 2002). Peripheral T-cell tolerance It has been shown that normal healthy individuals have self-reactive T cells in the periphery (Lohmann et al. 1996, Semana et al. 1999), and that these T cells are more likely to express a low affinity TCR for self-antigens (Bouneaud 2000). This suggests that there might be regulatory mechanisms operating in the periphery that under normal conditions control these potentially autoreactive T cells. One such mechanism is T cell ignorance of self-antigens because the antigens are sequestered at sites not accessible to the blood/lymph borne immune system (Alferink et al. 1998). Ignorance can also be the result of low amounts of antigen, not reaching the threshold required to trigger a T cell response (Kurts et al. 1998). Interactions of an autoreactive T cell with self-antigens might also lead to a functional inactivation state, called anergy. Anergy was first shown in vitro as the result of TCR ligation in the absence of co-stimulation (Jenkins and Schwartz 1987). Later experiments have implicated that signaling through alternative receptors rather than just lack of co-stimulation induces anergy in T cells in vivo. Indeed, it has been demonstrated that ligation of cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) was required for anergy in vivo. The importance of CTLA-4 in controlling the activation of T cells is evidenced by the lethal lymphoproliferative disorder found in mice lacking this molecule (reviewed in Walker and Abbas 2002). Another molecule that is believed to be involved in the control of T cell unresponsiveness, is PD-1 (programmed cell death 1), which is expressed at high levels in anergic T cells (Lechner et al. 2001). It is believed that PD-1 might function by causing cell cycle arrest or inhibiting cytokine secretion (reviewed in Walker and Abbas 2002). The role of dendritic cells in T-cell tolerance Dendritic cells (DC) are believed to play an important role in establishing tolerance, both in the thymus and in the periphery. In the thymus, they are localized in the medullary regions where they present self-antigens to developing T cells and delete those that express a TCR with high affinity for self-antigens. In the periphery, circulating immature DCs continuously sample the environment, capturing self-antigens as well as innocuous environmental proteins. It has been suggested that proteins captured and processed by DCs in this steady state, meaning in the absence of acute infection and inflammation, are tolerogenic, i.e., the DCs silence the corresponding antigen-specific T cell. Indeed, recent experiments have shown that antigen-loaded immature DCs silence T cells either by deletion or by expanding regulatory T cell populations (reviewed in Steinman and Nussenzweig 2002). 9 Regulatory T cells and T-cell tolerance Another important factor that is involved in the regulation of self-reactive T cells in the periphery is the presence of regulatory T cells. There are two different kinds of these cells: those, which are produced in the thymus and express CD4 and CD25 and those which are generated after tolerogenic encounters with antigen in the periphery. CD4+CD25+ T cells constitute approximately 10% of peripheral murine CD4+ T cells. They express little CD45RB but constitutively express CTLA4. The importance of CTLA-4 is evidenced by the observation that inhibition of CTLA-4, either by using antibodies or CTLA-4 deficient regulatory cells, reduces their suppressor functions significantly. They have a partially anergic phenotype, evidenced by their poor proliferative response upon TCR stimulation. In vitro studies have shown that, rather than proliferating in response to antigen, these cells suppress the proliferation of other T cells. The suppression is mediated through cell-cell contact, independently of cytokines such as IL-4, IL-10 and TGF-β. Although the activation is antigen-specific, once activated, these cells inhibit both CD4+ and CD8+ T cell responses in an antigen-nonspecific manner (reviewed in Maloy and Powrie 2001, and Walker and Abbas 2002). Repetitive stimulation of T cells with alloantigens in the presence of IL-10 has been shown to induce another set of regulatory T cells, termed TR1 cells, which partially rely on the production of IL-10 and TGF-β for their function. There may be multiple mechanisms that the regulatory T cells use to suppress immune responses. Studies in animal models have provided compelling evidence for an important role for cytokines, such as IL-10 and TGF-β, in the effector function of these cells in vivo. However, the cytokines involved vary depending on the model. The immunesuppressive properties of TGF-β and IL-10 are most likely explained by their ability to inhibit APC function and to mediate direct anti-proliferative effects on T cells (reviewed in Maloy and Powrie 2001). B-cell tolerance There are several mechanisms that are important for generating and maintaining B cell tolerance. In the bone marrow, developing B cells expressing a BCR with high affinity for self antigens exhibit developmental arrest during which they continue V(D)J recombination of their Ig genes, especially the light chain genes. This process is called receptor editing and changes the BCR antigen specificity (Nemazee 2000). If the editing is successful and the strong self-reactivity is lost, then the immature B cell can resume maturation and leave the bone marrow, if not, it will be clonally deleted (reviewed in Ohashi and DeFranco 2002). Self reactive B cells that enter the periphery can also be regulated through peripheral tolerance mechanisms. They can either be deleted due to strong interactions with antigen or rendered anergic due to less strong interaction with antigen. B cells with weak self specificity may also exist in a naïve functional state, sometimes referred to as “ignorance”. This state is mainly due to low exposure to antigen and/or lack of T cell help. 10 Studies in transgenic mice using the model antigen hen egg lysozyme (HEL), have demonstrated that B cell anergy is effective in silencing B cells. This is in part due to the poor signaling through the BCR by anergic B cells (Cooke et al. 1994, Eris et al.1994, Benschop et al. 2001), which renders them highly susceptible to Fas-mediated apoptosis when presenting antigen to specific Th cells (Rathmell et al.1995, Rathmell et al. 1996). This is because activated Th cells express CD40L on their surface which induces Fas expression on B cells and thus makes them susceptible to Fas-mediated apoptosis, whereas signaling through BCR protects from apoptosis (Rothstein et al. 1995). However, recent studies of anergic B cells recognizing double stranded (ds) DNA have revealed that provision of T cell help induce production of anti-dsDNA autoantibody in these B cells (reviewed in Ohashi and DeFranco 2002). Why tolerance to dsDNA was broken in these experiments, but not in the experiments with HEL, is not clear. It could reflect experimental differences in the way T cell help was provided to the B cells, for instance it has been shown that high levels of IL-4 protects B cells from Fas-induced apoptosis. Differences in the nature of the antigens as well as variations in the states of anergy might also explain the different outcomes in these experiments (reviewed in Ohashi and DeFranco 2002). It seems as if B cell anergy is not a single homogenous state since different Ig transgenic mice with specificity for dsDNA have anergic B cells with a range of phenotypes. In some cases anergic B cells can be stimulated by LPS to divide and differentiate into antibody secreting cells, whereas in other cases, the anergic B cells are not responsive to LPS (reviewed in Ohashi and DeFranco 2002). 11 AUTOIMMUNITY AND AUTOIMMUNE DISEASE Autoimmune disease is the consequence of an immune response directed against self, leading to destruction or dysfunction of the targeted tissue. Traditionally autoimmune diseases have been divided into organ-specific or systemic diseases. This division is depending on whether the immune response is directed to a self-antigen confined to a particular organ or to a self-antigen widely distributed in the body. Organ-specific diseases are often considered to result from deficiencies in tolerance induction mechanisms, which results in failure to eliminate or deactivate selfreactive lymphocytes. The etiology of most autoimmune diseases is still unknown. However, it has been proposed that multiple factors including genetic, environmental and immunologic factors contribute to the induction and development of autoimmune diseases. Moreover, there is accumulating evidence that T cells play a central role in the development of various autoimmune diseases, especially organ-specific diseases, both in humans and animals (reviewed in Van Parijs and Abbas 1998). Genetics of autoimmune diseases Identifying autoimmunity genes has so far been difficult, one of the major reasons for this is that the risk ratios for developing disease if an individual susceptibility allele is present is probably quite low. The fact that many of these alleles probably interact with other genes in the background, as well as with environmental factors, further complicates the problem (Gregersen 1999, reviewed in Gregersen 2003). In identical twins, the twin concordance rates for autoimmune diseases are generally in the range of 15-30%, depending on the autoimmune disease. These relatively low concordance rates provides evidence for the contribution of non-genetic factors, such as environmental factors or chance events, also contribute to the variability of disease expression in the population (Gregersen 1999). Major histocompatibility complex (MHC) genes and susceptibility to autoimmunity Classical genetic studies on various autoimmune diseases have demonstrated that genetic predisposition plays an important role. The most potent genetic influence on susceptibility to autoimmune diseases is the major histocompatibility complex (MHC) (particularly MHC class II), which has been known for many years to affect susceptibility to different diseases with autoimmune origin. Susceptibility to a number of autoimmune diseases, including rheumatoid arthritis (RA), multiple sclerosis (MS) and type 1 diabetes (T1D), is associated with particular alleles of MHC class II genes such as HLA-DR and/or –DQ genes. This association provides strong evidence for the role of MHC class II antigen presentation in these disease processes (Todd et al. 1987, reviewed in Nepom and Erlich 1991, Wucherpfennig and Strominger 1995). 12 It has been possible to define structural features that determine the interactions of the relevant MHC-class II molecules and peptides in several DR-associated autoimmune diseases. For example, susceptibility to RA is associated with the “shared epitope”, a segment of the DRβchain that is very similar in sequence among the disease –associated subtypes of DR4 (DRB1*0401 and 0404) and DR1 (DRB1*0101). In structural terms, this “shared epitope” primarily defines the shape and charge of the P4 pocket (reviewed in Wucherpfennig 2001). The role of non-MHC genes in the development of autoimmunity Over the years, several non-MHC susceptibility loci for autoimmune disease have been identified in humans and animal models. It has been shown that many of these loci are clustered into 12 nonoverlapping genomic regions that confer much of the susceptibility for systemic lupus erythematosus (SLE), T1D, MS, experimental autoimmune encephalomyelitis (EAE), RA and Crohn´s disease ( reviewed in Morahan and Morel 2002). When interpreting these results one should bear in mind the fact that genes with related functions tend to cluster in the genome, and that many quantitative trait loci (QTLs) correspond to more than one susceptibility gene. For example, in murine autoimmune diseases, fine mapping of the NZM2410 sle loci and several of the NOD Idd loci for insulin dependent type 1 diabetes in NOD mice have revealed that each original locus corresponds to more than one susceptibility gene (Rev in Wanstrat and Wakeland 2001, Morel et al. 2001). Gene aberration and development of autoimmunity More than 40 non-MHC genes have been associated with spontaneous development of lupus-like disease when aberrantly expressed in mice (reviewed in Kono and Theofilopoulos 2000, Wakeland et al. 2001, Mohan 2001, Morahan and Morel 2002). These genes can be classified into at least three functional categories: 1) molecules involved in the clearance of apoptotic cells, which if accumulated may result in nuclear-antigen-driven expansion of autoreactive lymphocytes, (SAP, CRP, Mertk, IgM and C1q). 2) Molecules that compromise lymphocyte apoptosis (FAS, FASL, PTEN, PI3K, Bim and BCL-X), and which may act by inhibiting deletion of self-reactive B and T cells. 3) Molecules that amplify or moderate lymphocyte signaling and expansion (CD19, Lyn, Fyn, Cr2, CD22, CD45, P21, PD1, TAC, Ptp1c and Blys), and which are believed to amplify antiself humoral and cellular immune responses. While some of these molecules primarily affect B or T cells, other may influence the expansion and activation of myeloid cells as well. These observations that molecules with different functional properties all lead to lupus development, suggest that there are multiple checkpoints that control nuclear-antigen-reactive lymphocytes and that defects in anyone of them lead to systemic autoimmunity in mice (reviewed in Raman and Mohan 2003). The findings from forward genetic studies indicate which genes can predispose to lupus, but do not reveal which genes that actually is responsible for spontaneous lupus development in mice and humans. 13 To resolve this issue, several reverse genetic studies have been preformed, both in humans and in mice. In a reverse genetic study the starting point is a well characterized disease and then by working backwards it may be possible to identify the genetic loci responsible for the disease or phenotype. This approach have revealed the existence of more than fifty loci predisposing to different manifestations of lupus, including antinuclear antibodies, glomerulonephritis, splenomegaly etc in mice (reviewed in Kono and Theofilopoulos 2000, Wakeland et al. 2001, Mohan 2001, Morahan and Morel 2002, Raman and Mohan 2003). In the past few years, seven human susceptibility loci have been mapped using genome scans in human-lupus. In addition, several suggestive loci have been implicated in lupus susceptibility in two or more scans. Moreover, loci that confer susceptibility to nephritis, thrombocytopenia, hemolytic anemia, associated with RA, neuropsychiatric manifestations, or anti-dsDNA antibodies, have been found in studies of phenotypically stratified lupus patients. Recent research has also shed some light on how epistatic interactions of several different genetic loci might be required for full-blown lupus to develop (Raman and Mohan 2003). The role of environmental factors in induction and perpetuation of autoimmunity Defining specific pathogenic environmental agents that may trigger the development or progression of autoimmune diseases remains the focus of increasing investigations. A factor that promotes a given autoimmune disease may not be identical to factors that influence the severity or progression of the disease. In autoimmune connective tissue diseases, which include SLE, systemic sclerosis (scleroderma), Sjögren’s syndrome, RA and systemic vasculitis, exposure to several environmental agents such as UV light, drugs, silica, mercuric chloride, hair products and organic solvents have been considered as the risk factor for the development of these diseases (reviewed in D’Cruz 2000, Mayes 1999). The mechanisms by which environmental agents in the context of susceptible genes induce or influence the severity of the autoimmune diseases are not well understood. However, several mechanisms including molecular mimicry, creation of cryptic self-peptides, induction of Th1/Th2 imbalance and interference with thymic selection might be involved. Regarding the last possibility, it has been shown that environmental agents may affect thymic selection of T cells. Administration of procainamide-hydroxylamine (PAHA) into the thymus of normal mice resulted in chromatinreactive T cells and autoantibodies against chromatin, a characteristic feature of human procainamide-induced lupus. In vitro culture of neonatal thymi from TCR-transgenic mice in the presence of PAHA and TCR ligand lead to enhanced positive selection of T cells expressing the transgenic TCR, but did not alter negative selection (Kretz-Rommel et al. 1997, Kretz-Rommel and Rubin 2000). These results suggests that PAHA enhanced the positive selection of otherwise nonselected low-affinity T cells specific for chromatin. 14 MERCURY-INDUCED AUTOIMMUNITY An experimental model to study the induction and development of autoimmunity Experimental models of chemically induced autoimmunity have been used to study the possible associations between the environmental, genetic and immunological factors and the development of autoimmune diseases in humans (Pieters et al. 2002, Druet 1990). Among these models, mercuric chloride (HgCl2)-induced autoimmunity in rodents represents a relevant and very well established model. This is owing to the fact that mercury is an abundant environmental pollutant and that this element has been used for thousands of years in industry and medicine (Pelletier et al. 1994). Description of the model In genetically susceptible mice and rats, subcutaneous injections with subtoxic doses of mercuric chloride (1.0 mg/kg body weight) induce a systemic autoimmune disease, characterized by a T-cell dependent polyclonal B cell activation, increased serum levels of IgG1 and IgE, production of autoantibodies of different specificities and formation of renal IgG deposits (Mathieson 1992, Griem and Gleichmann 1995, Enestrom and Hultman 1995, Goldman et al. 1991). These characteristics appear 7-10 days after the beginning of mercury administration and a single injection may be adequate to induce the syndrome. Although mercury is commonly administered subcutaneously, mercury given intraperitoneally, orally, in drinking water, or aerosolized also effectively induces similar autoimmune manifestations in susceptible animals (Mathieson 1992, Hultman and Enestrom 1992, Hultman and Enestrom 1987). The effects of mercury are dose dependent as the increase in serum IgE levels positively associates with dosage of mercury in susceptible rats (Pelletier et al. 1994) and increase in serum IgG of specific autoantibodies and renal IgG immune complexes correlate with the dosage of mercury in susceptible mice (Hultman and Enestrom 1992). Autoantibody production Production of autoantibodies of various specificities is one of the main characteristic of mercuryinduced autoimmunity in susceptible rats and mice. Mercury-treated rats develop antibodies against both exogenous and endogenous antigens including antibodies against 2,4,6-trinitrophenyl (TNP), glomerular basement membrane, double-stranded DNA (ds-DNA), thyroglobulin, collagen, phospholipids, myeloperoxidase and laminin P1 (Hirsch et al. 1986, Pusey et al. 1990, Druet et al. 1994, Aten et al. 1995), while in susceptible mice, treatment with mercury elicits antibodies, which are against TNP, chromatin/histone, collagen, IgG, phospholipids and nucleolar proteins (Hultman et al. 1989, Reuter et al. 1989, Al-Balaghi et al. 1996). 15 Mercury-induced anti-laminin autoantibodies are uniquely found in rats (Druet et al. 1994, Aten et al. 1995), whereas, anti-nucleolar autoantibodies (ANolA) are exclusively found in certain susceptible mouse strains (Hultman et al. 1989, Reuter et al. 1989, Mirtcheva et al. 1989, Pietch et al. 1989, Hultman et al. 1992). Several studies have shown that mercury-induced ANolAs react with fibrillarin, a 34–36 kDa protein (Hultman et al. 1989, Reuter et al. 1989, Takeuchi et al. 1995), which is associated with the U3, U8, U13, U14, X and Y small nucleolar RNAs in vertebrates (Fournier and Maxwell 1993). Fibrillarin is also a target for autoantibodies in a subset of patients with scleroderma (Takeuchi et al. 1995, Kasturi et al. 1995, Arnett et al. 1996). Interestingly, it has been found that murine mercury-induced and spontaneous human ANolA share several similarities in binding to fibrillarin (Takeuchi et al. 1995). This finding led to the suggestion that both mercury-induced and spontaneously developed ANolA interact with similar evolutionary conserved epitopes in fibrillarin (Takeuchi et al. 1995). A peculiar and interesting feature of mercury-induced autoimmunity in the mouse and rat is that the polyclonal B-cell activation and autoantibody production spontaneously disappear after 2-3 weeks, despite continuous injections of mercury (Pusey et al. 1990, Al-Balaghi et al. 1996, Pietsch et al. 1989, van Vliet et al. 1993, Bowman et al. 1984, Chalopin and Lockwood 1984, Mathieson et al. 1991). The exact mechanism(s) for auto-regulation of mercury-induced autoantibody production is not well understood. However, it has been proposed that in rats, this auto-regulation is mediated by CD8+ T cells and IL-2-producing CD4+ T cells (Bowman et al. 1984, Castedo et al. 1994). It remains to elucidate whether similar types of cells are also responsible for the regulation of mercury-induced antibody production in the mouse. Th1/Th2 paradigm in mercury model Mercury-induced autoimmunity is one of the autoimmune models in which Th1/Th2 imbalance has long been considered to play a critical role (Goldman et al. 1991, Mathieson 1995). The notion from early studies that mercury induced increases in the serum levels of IgG1 and IgE (Hirsch et al. 1982, Pelletier et al. 1988) led to the suggestion that Th2-type T cells play an important role in the pathogenesis of disease, as IL-4 produced by Th2 cells induces class switching to these isotypes (reviewed in Finkelman et al. 1990). Ochel and colleagues addressed the role of this cytokine in murine mercury-induced autoimmunity by treating susceptible A.SW mice (H-2s) with an anti-IL-4 monoclonal antibody (mAb) prior treatment with mercury. It was observed that treatment with anti-IL-4 mAb partially or completely abrogated the increases in IgG1 ANolA and IgE, but it enhanced the production of mercury-induced IgG2a, IgG2b and IgG3 ANolA (Ochel et al. 1991). Similar results were also obtained from mercury-treated IL-4 deficient A.SW mice (H-2s) (Bagenstose et al. 1998). 16 Taken together, these results indicate that IL-4 is required for the class switching to the IgG1 and IgE isotypes, but is not essential for the development of ANolAs. Attempts have been made to block the activation of the Th2-type of response seen in mercuryinduced autoimmunity. IFN-γ secreted by activated Th1 cells has been shown to play an important role in regulating cellular immune responses and down-regulating Th2 activities (Mosmann and Coffman 1989). Accordingly, it was shown that mercury induces IFN-γ production in the spleen cells of mercury-resistant LEW rats (Van der Meide et al. 1995). Furthermore, in resistant B10.D2 mice (H-2d), mercury induces an IFN-γ-dependent suppression of antibody formation to sheep red blood cells (Doth et al. 1997). To address whether IFN-γ can reverse the Th2-mediated immune responses by mercury, recombinant IFN-γ was administered to B10.S mice prior to mercury treatment. This pretreatment led to a reduction in serum IgE levels and anti-SRBC antibody levels, but failed to prevent ANolA production and immune glomerulonephritis in B10.S mice (Doth et al. 1997). Conversely, mAb neutralization of IFN-γ in resistant B10.D2 (H-2d) mice alleviated the mercury-induced immunosuppression, but could not convert the mice to a mercury-susceptible phenotype (Doth et al. 1997). To more directly investigate the possible suppressive roles of the IFN-γ in mercury-induced autoimmunity, Kono and colleagues examined the IFN-γ -deficient H-2s mice for the susceptibility to disease. Surprisingly, they observed that mercury treatment does not elicit ANolA or tissue lesions in these animals (Kono et al. 1998).If confirmed, this novel finding suggests that IFN-γ rather than being suppressive play a hitherto unidentified role in breaking tolerance to nucleolar antigens that takes place in this model. The role of IL-12 in regulation of mercury-induced autoimmunity An alternative approach was pursued to modulate the response to mercury observed in A.SW (H2s) mice to that of a Th1 phenotype by administration of IL-12 prior to treatment with mercury. The IL-12 cytokine induces T cell proliferation and IFN-γ production and is necessary for differentiation of naive T cells into the Th1 subset and thereby is critical for the initiation of inflammatory immune responses (Trinchieri 1993). IL-12 also contributes to certain Th1-mediated autoimmune disorders including multiple sclerosis, insulin-dependent diabetes mellitus (IDDM) and inflammatory bowel disease (Gately et al. 1998). Further, in mice susceptible to Leishmania major infection, a Th2 mediated infectious disease (Reiner and Locksley 1995) IL-12 administration can bias toward a protective Th1 response (Heinzel et al. 1993, Sypek et al. 1993). Thus, IL-12 can serve as a potentially important immunoregulator of disease outcome. In A.SW mice, IL-12 treatment dramatically reduced ANolA titers of all classes and partially inhibited the mercury-induced increase in IgG1, but had no effect on serum levels of IgE and renal Ig deposits (Bagenstose et al. 1998). Taken together, it is conceivable that Th1 and Th2 cytokines indeed play important roles in the B-cell activation seen in mercury-induced autoimmunity. However, the question remains whether Th1 cells participate in both the induction and regulation of this syndrome. 17 Genetic predisposition to mercury induced autoimmunity Like in many other experimentally induced and/or spontaneously developed autoimmune diseases, genetic factors play crucial roles in conferring susceptibility/resistance to mercury-induced autoimmunity in both rats and mice. Genetic analysis on the F1 and backcross hybrids between susceptible and resistant animals have revealed that at least three or four genes or clusters of genes are involved, of which one is within MHC class II region (Sapin et al. 1984, Druet et al. 1982, Sapin et al. 1982). In mercury susceptible BN rats (RT1n), three susceptibility loci on chromosomes 9, 10 and 20 have been identified for Th2-mediated autoimmune responses (Fournie et al. 2001). The susceptibility locus on chromosome 20, which contains the MHC region, controls the kinetics of the IgE response and the autoantibody response to DNA. The other two loci on chromosomes 9 and 10 control the IgE response, the anti-laminin response and the deposition of IgG in glomeruli (Fournie et al. 2001). In mercury susceptible H-2s mice (SJL, A.SW and B10.S mice), susceptibility to mercury-induced ANolA production could be mapped to the I-A loci of H-2 class II genes (Hultman et al. 1992). It has been shown that the other H-2 class II locus (I-E) either suppresses (Mirtcheva et al. 1989) or does not influence the mercury-induced ANolA response (Hultman et al. 1992). Determination of susceptibility to mercury-induced autoimmunity in H-2 congenic mice and in mouse strains carrying identical H-2 genotype, but different non-H-2 background genes have revealed that non-H-2 genes mainly control the susceptibility to and the severity of the B cell activation, IgE production and renal IgG deposition (Hultman et al. 1993, Abedi-Valugerdi and Möller 2000). However, further studies are required to map the chromosomal localization of these genes in the susceptible mice. 18 THE PRESENT STUDY AIMS The general aims of this study were to analyze the genetic susceptibility/resistance to mercuryinduced autoimmune manifestations, and to investigate how mercury-induced autoimmunity is regulated. We specifically aimed at investigating how resistance to murine mercury-induced autoimmunity is inherited, regarding the four major characteristics; formation of IgG1 antibodies, increase in serum IgE levels, production of ANolA and the development of renal IgG1 deposits (Paper I). Furthermore, we applied the “cryptic peptide hypothesis” as a working hypothesis for metalinduced ANolA production, to address whether mercury and silver could induce ANolA responses in a similar fashion (Paper II). We also tested our hypothesis that mercury confers more adverse immunological effects in individuals, who are genetically or immunologically, predisposed to develop spontaneous autoimmune disease in an animal model of Scleroderma (Paper III). Finally, we addressed the Th1/Th2 cross-regulation paradigm by examining whether, in a susceptible mouse strain, simultaneous induction of mercury-induced autoimmunity and collagen-induced arthritis could result in attenuation of one or both of the diseases (Paper IV). 19 METHODOLOGICAL REMARKS The methods used in papers I-IV are described in detail in the “Material and Method” section of each paper. However, I will give a brief description of the methods used below. Mercury and silver treatment (paper I-IV) All mercury-treatments in the experiments were performed according to the same protocol. The experimental mice were injected subcutaneously (s.c.) with 0.1 ml HgCl2, 1.6 mg/kg body weight (b.w.) every third day for 4 weeks. In paper II the silver-treatment was performed in a similar fashion, the experimental mice received 0.1 ml AgNO3, 2.5 mg/kg b.w. s.c. every third day for 4 or 8 weeks. Control mice received sterile 0.9% NaCl s.c. in all experiments. Evaluation of mercury-induced autoimmune manifestations (paper I-IV) At the end of each experiment we analyzed the development of the mercury- and silver-induced characteristics in spleens, kidneys and serum using several methods. To enumerate splenic antibody secreting cells we performed Protein A plaque assay. Detection of renal IgG1 deposits was performed using a direct immunofluorescens method. For detection of serum ANolA an indirect immunofluorescens method was used. Finally, serum IgE levels were measured with a sandwich ELISA method. H-2 genotyping (paper I-III) Genomic DNA samples from were prepared from mouse tail biopsies. H-2 typing was performed by PCR of genomic DNA, using primers, which could discriminate between different H-2 genotypes. All amplified DNA samples were then electrophoresed through an agarose gel mix, and after electrophoresis, the gels were stained with ethidium bromide and photographed. Histological examination (paper III and IV) Fixed skin (paper III) or limb (paper IV) samples were embedded in paraffin and stained with hematoxylin and eosin (H &E) according to routine histological methods. The thickness of the skin was measured vertically from the top of the granular layer to the junction between dermis and subcutaneous fat on H & E stained sections using a light microscope equipped with a computer analyzer. The limbs were examined for histological evidence of inflammation, pannus formation and destruction of cartilage and bone 20 Detection of antigen-specific IgG1 and IgG2a (papers III and IV) Serum levels of antigen-specific IgG1 and IgG2a were determined using a standard ELISA technique, where the antigen of interest was coated to ELISA plates, followed by incubation with serum and subsequent detection with AP-conjugated anti-IgG1 or IgG2a antibody. Induction of CIA Chicken type II collagen (CII) was mixed in equal volume with complete Freund´s adjuvant (CFA). The mice were injected intradermally (i.d.) with 0.1 ml of this emulsion at the base of the tail. 21 days later, the animals were injected intraperitoneally (i.p.) with 100 µg CII in 0.05 M acetic acid. Evaluation of the development of arthritis Following their secondary immunization with CII, the animals were assessed daily for the appearance of arthritis by examining and scoring each of the forepaws and hindpaws. The severity of arthritis in each affected paw was graded according to an established scoring system. The sum of the scores for all four paws in each mouse was employed as an index of the overall severity and progression of arthritis in each animal. 21 SUMMARY OF THE STUDY RESULTS AND DISCUSSION Paper I. Previously it has been shown that mercury-induced autoimmune manifestations in susceptible strains of mice are genetically controlled. The susceptibility to ANolA production has been shown to be under the control of genes within the H-2 loci (Robinson et al. 1986, Mirtcheva et al. 1989, Hultman et al. 1992, Hultman et al. 1993, Hultman et al. 1996, Hanley et al. 1997, Johansson et al. 1998, Abedi-Valugerdi and Möller 2000). It has also been shown that H-2 genes in combination with genes residing outside the H-2 loci could confer susceptibility to polyclonal B cell activation and renal IgG deposition (Hultman et al. 1992, Hultman et al. 1993, Hultman et al. 1996, Hanley et al. 1997, Johansson et al. 1998). The mercury-induced increase in serum levels of IgE seems to be controlled mainly by non-H-2 genes (Abedi-Valugerdi and Möller 2000). In a study of susceptibility to mercury-induced autoimmunity in several mouse strains of different H-2 genotypes it was found that SJL and A.SW mice, both with an H-2s genotype were highly susceptible regarding all characteristics of mercury-induced autoimmunity in mice. The NZB mice were also found to be highly susceptible although they did not develop an ANolA response. Most other strains in this study were classified as intermediate responders, since they were able to develop at least one of the characteristics of mercury-induced autoimmunity. Only one of the tested mouse strains, the DBA/2 qualified for the classification non-responder, (Abedi-Valugerdi and Möller 2000). Since only a few studies in the past had focused on the genetics of resistance to mercury (Hanley et al. 1997) and the existence of a highly mercury resistant mouse strain we decided to further investigate this resistance. In this study, we used F1 hybrid crosses between the resistant DBA/2 (H-2d) mice and mercury susceptible NZB (H-2d), SJL (H-2s), A.CA (H-2f), and DBA/1 (H-2q) as well as backcross hybrids between (DBA/2 x SJL)F1 and SJL mice to examine the genetic control of resistance to mercury-induced autoimmunity regarding four major characteristics, formation of IgG1 antibodies, increase in serum IgE levels, production of ANolA, and development of renal IgG1 deposits. Our first finding was that the development of most characteristics was down-regulated in the F1 hybrids. This implies that dampening genetic factors in the DBA/2 strain can be transmitted to the F1 progeny as dominant traits. This is in contrast to findings in the rat, where susceptibility to mercury-induced increases of IgE and development of IC-mediated glomerulonephritis are inherited as dominant traits in crosses between susceptible and resistant strains of rats (Druet et al. 1977, Sapin et al. 1982, Sapin et al. 1984). 22 To further investigate the inheritance of resistance to mercury-induced autoimmunity we examined mercury-induced responses in backcross hybrids of (DBA/2 x SJL)F1 x SJL. We found that the backcross hybrids could be categorized as non/low, intermediate and high responders, for increases in IgG1, IgE and development of renal IC deposits, with a defined phenotypic ratio for each characteristic. This suggests that resistance to any one of these characteristics is controlled by more than one gene. Based on this suggestion and the finding that the resistance in the F1 hybrids was a dominant trait, we concluded that a complete resistance requires heterozygosity in all genes responsible for the phenotype. The phenotypic ratios obtained in the backcross hybrids, suggests that a two-gene model is valid for the resistance to increases in IgG1 and IgE (Griffiths et al. 1999). H-2 genotyping of the backcross hybrids revealed that genes within the H-2d loci were not involved in this resistance. Thus, two non-MHC loci might be linked to the resistance to these characteristics. However, we did not address the location of the genes conferring resistance but it is possible that polymorphisms in TCR and FcγR genes are associated with resistance to increased IgG1 responses. This suggestion is supported by the findings that in vivo induction of IgG1 is T-cell-dependent (reviewed in Finkelman et al. 1990) and that FcγRs play a pivotal role in the regulation of B-cell-activation (Friedman 1993). Since, B cells require cytokine-secretion, especially of IL-4 and IL-13, and ligation to CD40L expressed by T cells to undergo isotype switching to IgE (Bacharier and Geha 2000), we propose that likely candidate genes involved in the resistance to increase of serum IgE levels, might be found among genes controlling the expression of cytokines and co-stimulatory molecules. Support for the involvement of co-stimulatory molecules in preventing mercuryinduced increases in IgG1 and IgE levels, is the report that administration of anti-B7-1 in combination with anti-B7-2 antibodies prevented increases of both IgG1 and IgE when administered to susceptible H-2s mice (Bagenstose et al. 2002). Evaluation of the phenotypic ratio obtained from the backcross hybrids for the formation of renal IgG1 deposits suggests that three genes are likely to be responsible for the resistance to this characteristic. Again, after genotyping we could conclude that genes within the H-2d loci were not involved. This gains support from a previous study of mercury-treated mouse strains, expressing H-2d on different backgrounds, where it was found that only the DBA/2 mice were resistant to mercury-induced renal IgG1 deposits (Abedi-Valugerdi and Möller 2000). In a study by Kono and co-workers, two genome-wide scans were performed using F2 intercrosses of DBA/2 with either SJL or NZB mice. They reported that a single major QTL on chromosome 1, designated Hmr1, was common to both crosses and encompassed a region containing several lupus susceptibility loci. Hmr1 was only linked to glomerular IC deposits and not autoantibody production (Kono et al. 2001). 23 The phenotypic ratio obtained in the backcross hybrids regarding ANolA synthesis was in agreement with a one-gene model (Griffiths et al. 1999). When correlating the IgG1 ANolA production with H-2 haplotype we found that all of the heterozygous (H-2s/d) were either non- or low responders and that a large proportion of the homozygous (H-2s/s) backcross hybrids were high responders and none of them were low responders. This supported the suggestion that one gene or a cluster of genes within the H-2 loci determines the resistance to IgG1 ANolA production, and that this resistance is mainly conferred by the H-2d haplotype. This is also in agreement with the conclusion made by Hanley and co-workers, that the expression of a resistant H-2 genotype per se is able do down-regulate ANolA responses in otherwise susceptible mice (Hanley et al. 1997). How a mercury-resistant H-2 haplotype, despite being co-expressed with a mercury-susceptible H-2 haplotype, can confer resistance to mercury induced ANolA is poorly understood. However, it has been demonstrated in adoptive transfer experiments, that the absence of ANolA production in F1 mice was not due to a difference in thymic education or the absence of anti-fibrillarin specific T cell help and it was suggested that the resistance was due to an intrinsic property of the haplotype heterozygous B cells (Hanley et al. 1998). This suggestion might be applied for (DBA/2 x SJL)F1 and (DBA/2 x A.CA)F1 hybrids in which the degree of ANolA penetration was low, but it does not explain the high degree of ANolA penetration in the (DBA/2 x DBA/1)F1 hybrids in this study. Therefore, additional studies of how resistant H-2 haplotypes down-regulate mercuryinduced ANolA in H-2 heterozygous hybrids are required. Paper II. It has previously been shown that another metal, silver, can induce ANolA in mercury-susceptible mouse strains which carry the H-2s and H-2q genotypes (Hultman et al. 1994, Hultman et al. 1995, Johansson et al. 1997). Since mercury and silver are able to form strong chemical bonds with organic donors (Frausto da Silva and Williams1991), a general mechanism for induction of ANolA by mercury (Griem and Gleichmann 1995) and silver (Johansson et al. 1997) was put forward. It has been proposed that by having a high affinity for different organic donors, mercury and silver and possibly other heavy metals bind tightly to several protein side-chains, forming stable metal-protein complexes. The formation of metal-protein complexes would lead to an incomplete protein unfolding. Further enzymatic cleavage would then result in the creation of cryptic self-peptides. Presentation of these cryptic self-peptides by MHC class II to autoreactive T cells could subsequently lead to the stimulation of autoreactive B cells and the production of autoantibodies (Griem and Gleichmann 1995, Johansson et al. 1997). In this study we considered this proposition and performed experiments to address the question whether mercury and silver induce comparable ANolA responses in H-2 susceptible mouse strains. The experiments were performed using H-2 congenic of B10 mouse strains, B10.S (H-2s), B10.G (H-2q) and B10.M (H-2f), as well as mouse strains expressing H-2s, H-2q, and H-2f genotypes on different backgrounds (A.SW, FVB/N, and A.CA respectively). 24 We found that similar to mercury, silver was able to induce ANolA/AFA in mice with H-2s and H2q genotypes, irrespective of their non-H-2 genes. This is consistent with findings in a previous study (Hultman et al. 1995), and indicates that these H-2 genotypes per se are able to confer susceptibility to both silver- and mercury-induced ANolA/AFA production. Although mercury and silver showed similarities in the induction of ANolA production in H-2s and H-2q mice, they also exhibited two major differences. First, silver was found to be less potent than mercury in stimulating the ANolA response in H-2s and H-2q mice, as evidenced by consistently lower serum titers of ANolA. Second, and more importantly, in contrast to mercury, silver was not able to induce ANolA responses in mice with H-2f genotype (B10.M and A.CA mice). The unresponsiveness to silver-induced ANolA synthesis in these mice could not be due to suppressive effects exerted by non-H-2 genes, as A.CA mice, which are high responders to mercury, were completely resistant to silver. Analysis of F1 hybrid crosses between silver susceptible A.SW (H-2s) and silver-resistant A.CA (H-2f) mice showed that these F1 hybrids were resistant to silver but not mercury. This finding was novel and surprising, since both H-2 haplotypes are co-expressed in the F1 hybrids (H-2s/f); some degree of ANolA responses was expected in the silver injected animals. Since both H-2s and H-2f haplotypes are permissive for the development of nucleolar/fibrillarin specific T cells, the unresponsiveness to silver observed in the F1 hybrids can not be attributed to lack of specific T cells or specific T cell help. In fact, the mercury-induced ANolA response in the F1 hybrids indicates that T cells capable of activating antinucleolar/ fibrillarin specific B cells are present in these mice. Therefore it seems likely that expression of the silver-resistant H-2f haplotype on APCs confers the resistance property to silver-induced ANolA synthesis in the F1 hybrids. Based on current knowledge about the presentation of cryptic self-determinants and on our findings, we proposed that mercury and silver might use similar pathways to induce ANolA synthesis in susceptible mice. However, they create and/or display dissimilar cryptic self-peptides, which lead to differential production of ANolA in susceptible mouse strains. It is also possible that compared to mercury, silver is less efficient in providing co-stimulating conditions (as it is less toxic than mercury) for ANolA production in H-2 susceptible strains. Paper III. As it has been mentioned in the introduction section of this thesis, genetic predisposition, immune dysregulation and exposure to environmental factors are the major prerequisites for the development of autoimmune diseases. In the context of this statement, we have proposed that mercury as an environmental hazard can exert more adverse immunological effects in individuals who possess genetic or immune dysregulating predisposing factors for the development of autoimmune disease. 25 Investigators in our group have previously tested this proposition in young mice in strains which, are genetically predisposed to develop either a systemic lupus erythematosus like [(NZB x NZW)F1 (H-2d/z) hybrids] or an insulin dependent type1 like [nonobese diabetic (NOD) mice] autoimmune diseases. As a support for our proposition, it was observed that in both strains of mice mercury was able to induce a strong immune activation with autoimmune characteristics (AlBalaghi et al. 1996, Brenden et al. 2001). In this paper we further tested this hypothesis, using type 1 tight-skin mice, a murine model for scleroderma (Green et al. 1976, Saito et al. 1999). We investigated the mercury-induced immunopathological alterations in Tsk1/+ and their non-Tsk (+/+) littermates, F1 hybrids between Tsk1/+ and B10.S (H-2s) mice, as well as in backcross hybrids between (Tsk1/+ x B10.S)F1 and B10.S mice, in order to clarify the role of mercury in the development of scleroderma-like disease. Our first observation was that the number of splenic antibody producing cells in the Tsk1/+ mice was of a significantly higher magnitude than in their non-Tsk(+/+) littermates. This is consistent with findings that Tsk1/+ have elevated serum levels of different Ig isotypes compared to non-Tsk littermates (Saito et al. 2002). This implies that, in addition to a defect in the fibrillin1 gene that causes formation of collagen deposits in the skin and internal organs (Green et al. 1976, Saito et al. 1999, Siracusa et al. 1996), Tsk1/+ mice also exhibit dysfunctional humoral immunity. It has therefore been proposed that B cells in these mice possess a hyperactive phenotype, and that CD19 may play an important role since CD19-deficient Tsk1/+ mice do not exhibit this phenotype (Saito et al. 2002). A striking finding in this study was that after exposure to mercury the B cells of Tsk1/+ mice were hyperactive and a strong immune/autoimmune response was induced, but this could not be observed in their non-Tsk (+/+) littermates. Thus, while Tsk1/+ mice can be categorized as mercury high responders, their non-Tsk littermates can be classified as low responders. The hallmark of mercury-induced autoimmunity in mice is the production of ANolAs that react with fibrillarin (Hultman and Eneström 1989, Robinson et al. 1984, Mirtcheva et al. 1989, Hultman et al. 1992, Hultman et al. 1993, Hanley et al. 1997, Abedi-Valugerdi and Möller 2000). Fibrillarin is also a target for autoantibodies in some patients with scleroderma. These ANolA from scleroderma patients have been shown to share several similarities with mercury-induced ANolA (Takeuchi et al. 1995). We found in this study that despite possessing hyperactive B cells and skin fibrosis, Tsk1/+ mice did non produce ANolA in response to mercury. Even co-expression of the Tsk1/+ phenotype with mercury-susceptible H-2s haplotype could not evoke an ANolA response in mercury-treated (Tsk1/+ x B10.s)F1 hybrids. This suggests that neither presence of hyperresponsive B cells nor the expression of Tsk1 phenotype in Tsk1/+ mice can fulfill the requirements for development of ANolA production. 26 One of the main characteristics of the Tsk 1 mutation is the occurrence of skin fibrosis (Green et al. 1976, Saito et al. 1999, Siracusa et al. 1996). However, we observed that mercury, despite being able to induce strong immune activation in Tsk1/+ mice, could not potentiate the development of skin fibrosis. This observation may reflect two things, either that the skin fibrosis is only weakly associated with the immune dysfunction in the Tsk1/+ mice, or that mercury differentially affects the immune dysfunction and the development of skin fibrosis. The latter is supported by our findings that treatment with mercury resulted in a reduction of skin thickness in the Tsk1/+ mice, and the findings that mercury and other heavy metal ions, such as cadmium and silver, have been shown to suppress the collagen synthesis in human rheumatoid synovial cells and in human lung fibroblasts in vitro (Goldberg et al. 1983, Chambers et al. 1998). Obviously further experiments are required to verify mercury’s suppressive effects on the development of skin fibrosis in Tsk1/+ mice. Paper IV. Native type II collagen (CII) in complete Freund´s adjuvant (CFA) can induce an autoimmune polyarthritis, collagen induced arthritis (CIA), which resembles human rheumatoid arthritis, in susceptible animals of different species, including rats, mice and monkeys (Trentham et al. 1977, Yoo et al. 1988, Courtenay et al. 1980). Genes encoding the H-2 control murine susceptibility to CIA, and only mice expressing H-2q and H-2r genotypes develop polyarthritis after immunization with CII in CFA. It has also been shown that CD4+ T cells play a central role in the development of CIA in mice (Luross and Williams 2001, Holmdahl et al. 2002). Moreover, in CIA, Th1 type of cells appear to play a pathogenic role, whereas effector functions of Th2 type of cells is down-regulated and also seem to protect against the development of inflammatory arthitides (SchulzeKoops and Kalden 2001). In contrast, exposure of the same mice to mercuric chloride induces a Th2-oriented systemic autoimmune disease (described elsewhere in this thesis). The objective of this paper was to address the Th1/Th2 cross-regulation paradigm by examining whether in a susceptible animal, simultaneous mercury-induced autoimmunity and CIA can interact. The consequence of such a reaction might be prevention and/or amelioration of one or both of these diseases. Administration of mercury prior to the induction of CIA caused only augmented production of anti-CII IgG2a antibodies, without affecting any other characteristics of the two diseases. This finding indicates that under certain circumstances, Th2-mediated mercury-induced autoimmunity and Th1-type CIA can coexist without influencing one another. Furthermore, these findings suggest that under inflammatory conditions caused by mercury and CIA together, mercury acts as an additional adjuvant that preferentially enhances the induction of specific IgG2a (a Th1 related isotype) anti-CII antibodies associated with CIA. 27 In agreement with this suggestion, mercury has been shown to exert adjuvant effects in connection with the specific humoral immune responses in rats immunized with ovalbumin (Watzl et al. 1999) and in mice immunized with horse red blood cells (Roether et al. 2002). During the induction phase of CIA, treatment with mercury resulted in an aggravation of CIA, as well as increased mercury-induced ANolA production. This observation implies that the immune/autoimmune activation, which occurs during the course of CIA, is also able to potentiate the humoral immune responses induced specifically by mercury. Although further investigations are required to clarify the exact mechanisms underlying this phenomenon, we propose that the adjuvant properties of CFA may be involved. In agreement with this proposal is the report that CFA increases mercury-induced immune responses in susceptible mice and can even cause a mouse strain that does not otherwise respond to mercury to exhibit a weak response (Hultman and Eneström 1987). A striking finding in our study was that mercury-administration after the onset of CIA exacerbated the severity of CIA. Increased serum levels of IgE and of anti-CII antibodies, but a substantial reduction in ANolA titers accompanied this effect. Interestingly, during the remission phase of CIA, this suppression is abolished and the synthesis of ANolA restored. In combination with the observation described above, this finding suggests that while the induction and remission phases of CIA provide permissive conditions, the effector phase of CIA suppresses the mercury-induced production of ANolA. In the light of differential expression of Th1 cytokines during the course of CIA, it is conceivable that production of high levels of IFN-γ or IL-12 during the onset of the disease exerts a suppressive effect on the mercury-induced production of ANolA. Furthermore, since the synthesis of IFNγ is essential for the development and induction of mercury-induced autoimmunity (Kono et al. 1998, Häggqvist and Hultman 2001), it is unlikely that expression of this cytokine causes the down-regulation of ANolA. Rather, it appears highly likely that IL-12 exerts this suppressive effect. In fact, this proposal receives strong support from the finding that administration of IL-12 to susceptible mice treated with mercury potently down-regulates the production of different isotypes of ANolA without affecting the synthesis of IgE or the deposition of immune complexes in the kidney (Bagenstose et al. 1998). Finally, splenic IFN-γ/IL-4 ratios were increased in CIA mice, irrespective of whether they were injected with mercury or not, but reduced in mice treated with mercury alone. This result further confirms the concept that CIA is a Th1-oriented autoimmune inflammatory disease (Luross and Williams 2001, Schulze-Koops and Kalden 2001), whereas mercury-induced autoimmunity is Th2mediated (Goldman et al. 1991, Eneström and Hultman 1995, Griem and Gleichmann 1995, Mathieson 1995). 28 Moreover, injection of mercury into mice at any stage of CIA, i.e., prior to or during the induction phase or after onset of the disease, did not reverse the increased IFN-γ/IL-4 ratio exhibited by these animals. This finding is in agreement with the clinical symptoms observed in CIA mice treated with mercury and implies that down-regulation of the Th1-mediated autoimmunity elicited by CIA requires a more potent stimulation of Th2 cytokine production than that caused by mercury. Consistent with this proposal is the report that immunization with CII in incomplete Freund’s adjuvant, a potent Th2 adjuvant (Billiau and Matthys 2001) down-regulates the synthesis of IFN-γ, as well as the development of CIA (Mauri et al. 2003). 29 CONCLUDING REMARKS To date, it is well accepted that for the development of autoimmune diseases, integrations of genetic predisposition, environmental factors and immune dysregulation are required. Regarding the genetic markers of susceptibility to autoimmune diseases, certain genes for MHC molecules that both form and adjust the adaptive immune response seem to play a crucial role. Moreover, a number of non-MHC genes that are important in controlling immune responses have also been associated with the development of autoimmune diseases. It is likely that in certain humans and mice that possess autoimmune susceptible genetic make up, exposure to certain environmental agents such as infections and chemicals leads to an immune dysregulation as well as an inappropriate immune response against self-components, which thereby results in organ damage and/or dysfunction. In the studies performed in this thesis, I was able to show that indeed under a certain environmental condition, an immune dysregulation with autoimmune characteristics could occur in genetically susceptible, but not resistant animals. I was also able to show how resistance to this environmentally induced autoimmunity is inherited. Furthermore, we could also comprehend to some extent how two environmentally induced autoimmune diseases having opposing immune dysregulations, regarding their cytokine profiles, interact in a susceptible mouse strain. However, many of the steps involved in the pathogenesis of autoimmune diseases require further studies. Future data will, hopefully, lead to a better understanding of the mechanisms that control the autoimmune phenotype and the development of new and better treatment strategies. 30 ACKNOLEDGEMENTS This work was performed at the Department of Immunology, Wenner-Gren Institute, Stockholm University. I would like to take the opportunity to express my sincere gratitude to family, friends and colleagues who have helped and supported me during the years. I would especially like to thank the following persons: Manuchehr Abedi-Valugerdi, my supervisor, for endless support, patience and encouragements, for always having an open mind and time to discuss science and for teaching me all I know about working with mice. Göran Möller, for accepting me as a student at the department. Marita Troye-Blomberg, for all the help she has provided when I needed it the most. Lena Israelsson, for teaching me the laboratory skills I needed, for being a good friend and for making me feel welcome when I came to the department. Gunilla Tillinger, our former secretary, for all “non-science” discussions, and for social skills out of the ordinary. The seniors, Carmen Fernandez, Klavs Berzins and Eva Sverremark-Ekström, for sharing their knowledge of immunology and other things. Alf Grandien, for keeping me company on the balcony in rain or sunshine, and for emphasizing the importance of participating in the social events at the department. My roommates, Karin Lindroth, always ready to help solve both technical and scientific problems, and for being a good cross-word puzzle partner, Anna Tjärnlund for good times and many laughs and for not kicking me out when I have been to chatty, and Masashi Hayano, for always being nice and forthcoming. Ann Sjölund, Margareta Hagstedt and Gelana Yadeta, for trying to make the department run smoothly and for providing all the help and assistance I could have asked for. 31 All the present students and all the former students at the department, Ariane Rodriguez- Munoz, Anna-Karin Larsson, Qasi Kaleda Rahman, Jacob Minang, Ahmed Bolad, Salah Eldin Farouk, Manijeh Vafa, Nina-Maria Vasconcelos, Halima Balugon, Shiva S. Esfahani, Valentina Screpanti, Gun Jönsson, Eva Nordström, Ben Adu Gyan Cecilia Rietz, Mounira Djerbi, Anki Häglind, Elisabeth Hugosson and Caroline Ekberg, for all the good times we have shared both at the lab and outside “in the real world”. The staff in our animal facilities, Pia Lundell, Eva Nygren and Solveig Sundberg for taking care of my mice. All my friends, and especially Catarina Kvarnmalm for support and encouragements when everything felt impossible and for a memorable night at Elverket, we must do that again sometime. My parents, Hjördis and Sten-Ove, för att ni alltid har trott att jag ska fixa det, även när jag inte har trott det själv och för att ni alltid ställer upp när det behövs. My brother Roland and my sisters Karin and Yvonne and their families, for training me to argue in endless discussions at family dinners. “Silverbågarna”, Inger and Sten, for taking interest in what I do, even though I never worked with rats. Evelina Silverbåge, för att du finns och ser till att jag inte tappar focus på vad som egentligen är viktigt här i livet. And last but not least, Thomas Silverbåge, for love and support, for always trying to understand what I am doing, for solving all my computer problems and for printing this thesis. 32 REFERENCES Abedi-Valugerdi M, Moller G. (2000). Contribution of H-2 and non-H-2 genes in the control of mercury-induced autoimmunity. Int Immunol. 12: 1425-30. al-Balaghi S, Moller E, Moller G, Abedi-Valugerdi M. (1996). 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