The 65-kDa heat-shock protein in the pathogenesis, prevention and therapy of autoimmune arthritis and diabetes mellitus in rats and mice

Conclusions hsp are molecules which are highly conserved from procaryotes to eukaryotes. At a first glance the immune system should treat these molecules as self. However, strong immune reactions to bacterial hsp are observed during infection in mammals.hsp65 plays a role in several autoimmune diseases in animal models. In AA in Lewis rats the involvement of hsp65 has been revealed by T cell clones which induce disease in naive recipients, or by T cell vaccination experiments. T cell clones which show in vivo activity have been used as tools in vitro to define epitopes involved in the disease process. In this manner mycobacterial hsp65 and its epitope peptide 180–188 were deduced for AA in Lewis rats. Similarily the epitope p277 was defined for diabetes in NOD mice.The role of hsp65 in several other autoimmune diseases was seen when animals were pretreated with hsp65 and found to be protected from subsequent induction of autoimmune disease. From the involvement of hsp65 in several different autoimmune diseases, it would appear that hsp65 is somehow a key factor in natural autoimmunity. At a fist glance this is surprising since mycobacterial hsp65 shows 50% amino acid homology with human hsp65, in other words it is half-self.Peptide epitopes, peptide 180–188 in AA in Lewis rats and p277 in IDDM in NOD mice, have been used for peptide vaccination, which represents another possibility for prevention of autoimmune disease. The immunological mechanism which leads to resistance from autoimmune disease involves hsp65 immunity and appears not to be associated with tolerance or non-responsiveness to hsp65, but seems to be due rather to modulation of naturally existing networks of idiotype-anti-idiotype T cells organized around hsp65 as the target antigen.     

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.     

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring

The 65-kD hsp from Mycobacterium tuberculosis has been reported to induce an autopathogenic subset of T cells in at least two animal models of autoimmune disease. Reports of increased expression of human hsp60 in the inflamed synovial tissue of rheumatoid arthritis (RA) patients, increased proliferation of RA synovial fluid T cells to mycobacterial hsp65, and increased levels of anti-mycobacterial hsp65 antibody in synovial fluid, have suggested that the highly homologous human (hu) hsp60 may be recognized as an autoantigen in RA patients. In the present study, we have examined by ELISA the serum IgG antibody levels to mycobacterial hsp65 and hu hsp60, as well as to the Escherichia coli hsp60, groEL, in patients with RA, systemic lupus erythematosus (SLE), Reiter's syndrome, active tuberculosis, and normal controls. In all these groups, the levels of anti-groEL and anti-hu hsp60 were significantly higher than the anti-mycobacterial hsp65. Anti-hu hsp60 was positively correlated with anti-groEL, but not with anti-mycobacterial hsp65. Anti-hu hsp60 was competitively inhibited by either soluble groEL or hu hsp60, but little or none by mycobacterial hsp65. Reiter's sera were found to have somewhat higher levels of anti-groEL and anti-hu hsp60 than did normal controls. We conclude that IgG anti-hu hsp60 autoantibodies arise primarily as a consequence of the humoral immune response to E. coli groEL through the recognition of cross-reactive epitopes.     

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.     

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, 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 hsp 60-stimulated cells produce T-helper 2 type cytokines.     

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.     

Autoimmune reactions to heat-shock proteins in pristane-induced arthritis

The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil, 2,6,10,14-tetramethylpentadecane (pristane), was shown to depend on an intact immune response possibly to a heat-shock protein (hsp) in the synovium. Initial experiments suggested that some crucial event in the development of arthritis takes place early after pristane injection. First, irradiated pristane-treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors within 25 days of irradiation. Second, mice irradiated up to 50 days after pristane injection, but not later, did not develop arthritis. Evidence for the involvement of an immune response to heat-shock protein (hsp) comes from the finding that mice injected with mycobacterial 65-kDa hsp prior to pristane challenge had a reduced incidence of arthritis in contrast to animals pre-immunized with the E. coli hsp equivalent GroEL or with bovine serum albumin. Other experiments revealed that T cells from mice with gross morphologically defined arthritis proliferated strongly to hsp65 and to normal joint antigens, whereas T cells from animals treated with pristane which did not develop arthritis gave much smaller responses. Mice which developed arthritis also had elevated levels of anti-hsp65 IgG in comparison with non-arthritic animals. These findings strongly suggest that autoimmune reactions to an antigen which cross-reacts with hsp65 are generated in pristane-induced arthritis. It is considered that the autoimmune response is directed to a synovial antigen and that pre-immunization with hsp65 protects the animals from the development of pristane-induced arthritis by altering the specificity or quality of the immune response to this antigen.     

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.     

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.     

Modulation of pristane-induced arthritis by mycobacterial antigens.

Several prominent mycobacterial protein antigens involved in antibody and T cell responses have been identified as members of highly conserved heat shock protein families. In particular, immune responses to the mycobacterial 65 kD heat shock protein (hsp65) have been implicated in the pathogenesis of autoimmune diseases both in experimental animal models and in man. Additionally, hsp65 has been shown to modulate the course of autoimmune disease in such experimental animal systems. In this report, we have examined the synthesis of heat shock proteins by a fast growing mycobacterial strain, M. vaccae, in heat stressed cultures and used the pristane induced arthritis model to investigate the immunoprophylactic and immunotherapeutic potential of heat killed M. vaccae. Heat shock of M. vaccae cultures at 48 degrees C demonstrated a 43-fold increase in hsp65 over that expressed at 37 degrees C. It is therefore suggested that heat killed M. vaccae contains sufficient hsp that can be presented in the context of appropriate adjuvant properties for use as an effective immunomodulatory agent. Immunisation experiments with M. vaccae revealed that protection or exacerbation of pristane induced arthritis was dependent on the dose (given in an oil or aqueous suspension), route and time of immunisation. In addition, it was demonstrated that the development of arthritis correlated with high levels of agalactosyl IgG and that "protected" animals had significantly depressed levels.     

Mycobacterial 65-kD heat shock protein induces release of proinflammatory cytokines from human monocytic cells.

Monocytes having phagocytosed mycobacteria are known to present the bacterial 65-kD heat shock protein (hsp) on their cell surface to alpha beta and gamma delta T lymphocytes. Cytotoxic CD4+ cells may then lyse monocytes expressing mycobacterial 65-kD hsp. However, it is not known whether 65-kD hsp directly stimulates monocyte functions other than antigen presentation. This study has demonstrated that following extraction of bacterial lipopolysaccharide, purified recombinant mycobacterial 65-kD hsp may directly activate THP-1 cells, a human monocytic line, to accumulate mRNA for and secrete tumour necrosis factor (TNF), a cytokine important in granuloma formation, the characteristic host immune response to mycobacterial infection. TNF gene expression and secretion following stimulation by hsp was dose-dependent and abolished by heat-induced proteolysis. Subsequently, THP-1 cells secreted IL-6 and IL-8, cytokines involved in recruitment and differentiation of T lymphocytes. The data indicate that secretion of proinflammatory cytokines from monocytes activated by mycobacterial 65-kD hsp may be important in the host immune response and in the development of antigen-specific T cell-mediated immunity.     

Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stress proteins.

Structural homology between microbial and human stress proteins has been postulated to be a basis for autoimmunization in chronic inflammatory diseases. Therefore, we estimated by ELISA titration the antibody levels to mycobacterial (M) and human (H) recombinant hsp70 and M-hsp65 heat-shock proteins in sera of patients with Crohn's disease (n = 29), ulcerative colitis (n = 20) and nontuberculous mycobacterial disease of the lungs (n = 20). Antibodies to H-hsp60, separated by two-dimensional gel electrophoresis, were tested in six sera of each group of patients. In Crohn's disease, antibody titres to the M-hsp65 antigen without detectable H-hsp60 binding were significantly elevated in 52% of the patients. In contrast titres to both M-hsp70 and H-hsp70 were demonstrable and correlated, but increased over control values only in four (14%) patients. The antibody pattern in ulcerative colitis was found to be quite different: anti-H-hsp60 binding was demonstrable in most patients, although anti-M-hsp65 titres were not elevated. Furthermore, 25% of patients had significantly elevated titres to M-hsp70, but not to H-hsp70. In non-tuberculous mycobacterial pulmonary disease, about 50% of patients had elevated titres to both hsp65 and hsp71 mycobacterial antigens but not to the corresponding human proteins; patients with Mycobacterium xenopi infection had the highest titres in this group. These results demonstrate the existence of distinct disease-associated patterns in the human antibody response to stress protein antigens. However, these data are not sufficient to imply sensitization with mycobacteria in patients with inflammatory bowel diseases, since certain epitopes of heat-shock proteins are shared by several bacterial genera.     

Elevated IgG Antibody Levels to the Mycobacterial 65-kDa Heat Shock Protein Are Characteristic of Patients with Rheumatoid Arthritis

We have previously demonstrated raised levels of IgG and IgA antibody to the mycobacterial 65-kDa heat shock protein (hsp) in the sera of patients with rheumatoid arthritis (RA). We have now attempted to determine whether this phenomenon is specific for RA, and whether it is seen only with the mycobacterial homologue of this particular hsp gene family. We therefore screened antibody levels to the mycobacterial and Escherichia coli hsp 65, and the mycobacterial, E. coli, and human hsp70, in sera from RA, systemic lupus erythematosus (SLE), tuberculosis (TB), ankylosing spondylitis (AS), Crohn's disease, and control donors. RA sera show the greatest increase in IgA binding to the mycobacterial hsp65, but no increase in IgA binding to the E. coli homologue. Similarly, only RA and TB sera show increased IgG binding to the mycobacterial hsp65, and we have shown previously that the titre is greater in RA. In contrast, the use of mycobacterial and E. coli hsp70 preparations as control bacterial hsp gene products has shown that RA patients do not differ from TB or SLE patients in their antibody binding to these proteins. Moreover, neither IgA nor IgG antibody to the human hsp70 in RA sera were higher than in TB, and the IgA binding was not higher than in SLE. These findings suggest that elevated IgG antibody levels to the mycobacterial hsp65 shows some disease specificity, and further studies with the human homologue and at the T-cell level are required.     

Regulation of autoimmune arthritis by the pro-inflammatory cytokine interferon-gamma

The pathogenesis of T cell-mediated diseases like rheumatoid arthritis (RA) has typically been explained in the context of the Th1-Th2 paradigm: the initiation/propagation by pro-inflammatory cytokines, and downregulation by Th2 cytokines. However, in our study based on the adjuvant-induced arthritis (AA) model of RA, we observed that Lewis (LEW) (RT.1(l)) rats at the recovery phase of AA showed the highest level of IFN-gamma in recall response to mycobacterial heat-shock protein 65 (Bhsp65), whereas AA-resistant Wistar-Kyoto (WKY) (RT.1(l)) rats secreted high levels of IFN-gamma much earlier following disease induction. However, no significant secretion of IL-10 or TGF-beta was observed in either strain. Furthermore, pre-treatment of LEW rats with a peptide of self (rat) hsp65 (R465), which induced T cells secreting predominantly IFN-gamma, afforded protection against AA and decreased IL-17 expression by the arthritogenic epitope-restimulated T cells. These results provide a novel perspective on the pathogenesis of autoimmune arthritis.     

热休克蛋白65与免疫关系研究进展

原核生物的Hsp65与免疫的关系非常密切.它既具有免疫原性又具有反应原性,可作为一种保护性抗原,而且与人的Hsp60高度同源,与动脉粥样硬化、佐剂性关节炎、糖尿病等自身免疫病有关,在上述疾病的早期可诱导免疫反应,后期可产生保护作用.更为重要的是,它形成的二聚体结构可导致某些疏水区暴露在表面,有利于非特异性地结合抗原肽 作为分子佐剂,与其他抗原构建的复合物具有交叉递呈功能,尤其能激活细胞毒性T细胞,可用于肿瘤或病毒病的预防和治疗免疫.有研究者将.其应用于开发与其相关的预防或治疗分枝杆菌病、肿瘤或病毒性疾病的疫苗中,并取得了初步成效.     

Stimulation of synovial fluid mononuclear cells with the human 65-kD heat shock protein or with live enterobacteria leads to preferential expansion of TCR-gamma delta+ lymphocytes.

T lymphocyte responses to heterologous or self 65-kD heat shock protein (hsp) have been implicated in the pathogenesis of various forms of arthritis. To delineate the relationship of 65-kD hsp to different synovial fluid (SF) T cell subsets, we stimulated synovial fluid (SFMC) and peripheral blood mononuclear cells (PBMC) from patients with different inflammatory rheumatic diseases and from healthy controls with human or mycobacterial 65-kD hsp, tetanus toxoid (TT), heat-killed or live Yersinia enterocolitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-gamma delta+ lymphocytes in the 65-kD hsp-stimulated SF-derived lines. This expansion of TCR-gamma delta+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-gamma delta+ cells was also shown after SFMC stimulation with live, but not with heat-killed Yersinia or with TT. We conclude that a common mechanism is involved in the selective expansion of TCR-gamma delta+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-gamma delta+ T lymphocyte clones (TLC) derived from a human 65-kD hsp-stimulated line, only a minority of TLC proliferated weakly upon restimulation with this antigen in the presence of autologous monocytes, whereas TCR-alpha beta+ TLC responded vigorously to the human 65-kD hsp and in some cases also cross-recognized the mycobacterial hsp homologue and/or heat-killed Yersinia. This implies that additional factors or cells may be present in the milieu of SFMC cultures that propagate the expansion of TCR-gamma delta+ cells in response to 65-kD hsp or live bacteria.     

Experimental immunization with anti-rheumatic bacterial extract OM-89 induces T cell responses to heat shock protein (hsp)60 and hsp70; modulation of peripheral immunological tolerance as its possible mode of action in the treatment of rheumatoid arthritis (RA)

OM-89 is a bacterial (Escherichia coli) extract used for oral administration in the treatment of RA. Given the evidence that immunity to bacterial heat shock antigens plays a critical role in the immunomodulation of arthritis and possibly inflammation in general, the purpose of the present studies was to evaluate the presence and immunogenicity of hsp in OM-89. Furthermore, we studied the effects of OM-89 in an experimental arthritis, where hsp are known to have a critical significance in disease development. In rats immunization with OM-89 was found to lead to proliferative T cell responses to hsp60 and hsp70 of both E. coli and mycobacterial origin. Conversely, immunization with hsp antigens was also found to induce T cell reactivity specific for OM-89. Based on this and the antigen specificity analysis of specific T cell lines, hsp70 (DnaK) turned out to be one of the major immunogenic constituents of OM-89. Parenteral immunization with OM-89 was found to reduce resistance to adjuvant arthritis (AA), whereas oral administration was found to protect against AA. Given the arthritis-inhibitory effect of oral OM-89 in AA, it is possible that peripheral tolerance is induced at the level of regulatory T cells with specificity for hsp. This may also constitute a mode of action for OM-89 as an arthritis-suppressive oral drug.     

Cellular and humoral reactivity pattern to the mycobacterial heat shock protein hsp65 in pristane induced arthritis susceptible and hsp65 protected DBA/1 mice.

We have analysed the cellular and humoral immunity to the mycobacterial 65 kD heat shock protein (hsp65) in groups of DBA/1 mice with arthritis induced by intraperitoneal injection of the mineral oil pristane. Here we confirm that DBA/1 mice are highly susceptible to pristane induced arthritis (PIA) and demonstrate that the incidence of arthritis can be modulated by either pretreatment with low dose irradiation or by preimmunisation with recombinant hsp65. Global cellular responses to antigens such as BSA or type II collagen were not enhanced or impaired within groups of arthritic (A) or non-arthritic (NA) mice. However, the cellular response to hsp65 in arthritic animals preimmunised with the 65 kD antigen was significantly elevated in comparison to hsp65 preimmunised mice that were resistant to the induction of disease. On the contrary, the level of hsp65 specific antibodies was much high in NA animals than in PIA mice. CBA/Igb mice are partially susceptible to the induction of PIA. We have previously reported that arthritic CBA/Igb mice have both elevated cellular and humoral reactivity to hsp65. Although a central pivotal role for hsp65 has been postulated in autoimmune diseases these results indicate that there is no simple relationship between the pathogenesis of PIA and immune responses to hsp65.     

Proliferative response of synovial fluid and peripheral blood mononuclear cells to arthritogenic and non-arthritogenic microbial antigens and to the 65-kDa mycobacterial heat-shock protein

Cellular immune responses to microbial antigens have been implicated in the pathogenesis of some forms of arthritis including reactive arthritis, Reiter's syndrome, ankylosing spondylitis and rheumatoid arthritis. We investigated the proliferative T cell responses of paired peripheral blood (PB) and synovial fluid (SF) mononuclear cells (MC) to so-called arthritogenic bacteria (Yersinia entero-colitica and Salmonella typhimurium), to control antigens, such as Candida albicans, mumps virus and purified protein derivative, to the recombinant mycobacterial 65-kDa heat-shock protein (hsp 65) and the mitogen phytohemagglutinin (PHA) in 16 patients with different inflammatory rheumatic diseases. The [³H]thymidine uptake of unstimulated cells (medium control) as well as the proliferative response to the different antigens tested was markedly increased in SFMC irrespective of the underlying rheumatic disease. In contrast, mitogenic stimulation was decreased in SFMC. The proliferative response to the hsp 65 correlated significantly with the responses to Yersinia, Salmonella and Candida. These results may reflect an enhanced function of SF antigen-presenting cells, different functional properties and subset distributions of PB and SF T cells with a preferetial accumulation of helper-inducer/memory T cells or a maintenance of an ongoing immune response by T cells cross-recognizing self epitopes such as epitopes located on the hsp 65. Thus, care should be taken in the interpretation of SF T cell responses to microbial antigens as diagnostic tools in arthritis.     

Cross-reactive mycobacterial and self hsp60 epitope recognition in I-A(g7) expressing NOD, NOD-asp and Biozzi AB/H mice

The highly conserved 60 kD endogenous heat shock protein (hsp60) has been suggested to be a target for T cell recognition in autoimmune diseases such as type I diabetes. We previously reported cross-recognition of both mycobacterial hsp60 (Mt60) and self hsp60 (m60) by Mt60 immunized NOD mice. To identify the epitopes involved, we generated T cell lines against m60 or its mycobacterial counterpart and tested these lines for recognition of complete sets of overlapping peptides spanning either hsp60 sequence. T cell lines responded to identical regions in the hsp60 proteins, regardless of their degree of conservation or I-A(g7) binding-affinity. Additionally, we determined whether a protective genetic background would affect the presence of hsp60 cross-reactive T cells in the peripheral repertoire by comparing epitope recognition in I-A(g7) expressing NOD, NOD-asp and Biozzi AB/H mice. Two out of five immunodominant murine peptides were able to induce proliferation in NOD and NOD-asp Mt60 T cell lines, but not in Biozzi AB/H T cell lines. Our results point out that Mt60 immunization not necessarily leads to proliferative T cells responding to endogenous hsp60 peptides in the context of diabetes-predisposing I-A(g7). Moreover, the capacity of T cells to respond to self hsp60 is not influenced by the presence of protective I-A(g7asp). Yet, proliferation of hsp60 autoreactive T cells is solely measured in combination with insulitis and as such serves as a surrogate marker for islet inflammation.