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.     

Pristane-induced arthritis in mice selected for maximal or minimal acute inflammatory reaction

The role of inflammatory and specific immune responses in pristane-induced arthritis (PIA) was investigated in mouse lines produced by bi-directional selective breedings for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reaction, comparing the outcome of PIA and the humoral and cellular response to hsp65. Symptoms of arthritis were detected in 50 % AIRmax mice 120 days after pristane injection, reaching a maximal incidence of 65 %, whereas only 7 % of AIRmin mice developed arthritis within an observation period of 200 days. The production of IgG antibody against hsp65 was found to be similar on both lines, although the IgG1 isotype was predominant in AIRmax, and IgG2a in AIRmin line. In vitro T cell proliferation to hsp65 was similar in the two lines, however, ELISPOT assays carried out soon after pristane treatment, demonstrated higher numbers of IL-6-, TNF-alpha- and IL-4-secreting cells in the spleen of AIRmax than in AIRmin mice, while higher numbers of IFN-gamma-producing cells were found in AIRmin mice. These results suggest a major participation of acute inflammatory mechanisms in the susceptibility to PIA. The genetic background which determines high or low AIR favors a Th2-like response in susceptible AIRmax and Th1-like response in resistant AIRmin mice at the initial phase of arthritis induction.     

Immunological Involvement in the Pathogenesis of Pristane-Induced Arthritis

Evidence is presented that the development of arthritis induced in mice by 2,6,10,14-tetramethylpentadecane (pristane) involves the immune response. Mice irradiated (500 rad) before injection of pristane failed to develop arthritis. By contrast, irradiated mice given lymphoid cells from normal donors and challenged with pristane developed arthritis. Other experiments showed that lymphoid cells from irradiated mice given pristane suppressed the development of arthritis in recipients challenged with pristane. Finally, the incidence of arthritis was significantly higher in CBA/Igb mice given pristane than in the allotypic congenic strain CBA/H, suggesting that a gene linked to the heavy chain immunoglobulin locus controls the development of arthritis.     

Differential effects of immunisation with mycobacterial 65 kD heat shock protein on two models of autoimmunity.

The effects of preimmunisation with the 65 kD mycobacterial heat shock protein (hsp65) on 2 murine models of autoimmunity were compared. Experimental autoimmune haemolytic anaemia (AIHA) can be provoked in mice by repeated injection with rat red blood cells (RBC). In this model, preimmunisation with hsp65 10 days before induction of disease resulted in a partial, but significant, reduction in RBC-bound autoantibody levels measured by Coombs' test. However, preimmunisation with human IgG (hIgG) was associated with a similar suppressive effect. Administration of neither hsp65 nor hIgG affected the direct or indirect anti-rat agglutinin titres of mice subsequently injected with rat RBC. Injection of hsp65 or hIgG prior to induction of AIHA elicited the production of IgG antibodies against the respective immunogen, as judged by enzyme-linked immunosorbent assays. In contrast to the results in experimental AIHA, pristane-induced arthritis (PIA) was effectively prevented by preimmunisation with hsp65, but not with hIgG. It is considered that, whilst hsp65 injection may slightly reduce subsequent anti-RBC autoantibody production in AIHA by antigenic competition, such a mechanism cannot account for the substantial protection against PIA afforded by hsp65 preimmunisation. We suggest that the high, sustained production of anti-hsp65 antibodies observed in mice given hsp65 and pristane may play a role in specifically suppressing arthritogenic immune responses in PIA.     

Comparison between the protective effects of mycobacterial 65-kD heat shock protein and ovomucoid in pristane-induced arthritis: relationship with agalactosyl IgG.

The IgG of patients with rheumatoid arthritis and mice with pristane induced arthritis (PIA) tends to lack the terminal galactose normally on the conserved N-acetylglucosamine linked beta 1-2 to mannose in IgG. The terminal N-acetylglucosamine (GlcNAc) residues of oligosaccharides on agalactosyl IgG may be an important component of the action of these glycoforms. Here, administration of ovomucoid, a glycoprotein rich in terminal GlcNAc, before pristane injection was found to reduce the incidence of PIA. This observation is the second report of an intraperitoneally administered antigen that reduces the incidence of PIA, mycobacterial 65-kD heat shock protein (hsp65) being the first. The suppressive effect of ovomucoid was not transferred from protected to naive recipients by spleen cells at the dose tested. By contrast, transfer of spleen cells from hsp65-protected mice to naive recipients conferred protection and this protection may be antibody-mediated. It is considered that ovomucoid and hsp65 protect against the development of PIA by different mechanisms.     

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.     

The role of oil and agalactosyl IgG in the induction of arthritis in rodent models

The proportion of agalactosyl IgG [Gal(O)] is raised in human rheumatoid arthritis and tuberculosis. We report here that injection of pristane into the peritoneal cavities of mice on days 0 and 50, which is known to induce plasmacytomas and arthritis, also induced a rise in the proportion of Gal(O), correlating with a simultaneous rise in the level of IgG antibody binding to the 65-kDa heat-shock protein of Mycobacterium bovis (hsp65). Arthritis developed in a proportion of those CBA/Igb mice with the highest percentage of Gal(O). Pretreatment with 50 micrograms of recombinant mycobacterial hsp65 intraperitoneal (i.p.) on day -10, or with 500 rad irradiation on day -2 before the first of the two injections of pristane reduced the incidence of arthritis from 24% in control animals, to 5.3% and 0.4%, respectively. The reduced incidence of disease correlated with smaller rises in the % Gal(O) at 50-75 days, although levels at 150-200 days were not affected. The arthritogenic effect of oil was not confined to the pristane model, since a single i.p. injection of oil 21 days before immunizing DBA/1 mice with type II collagen reduced the mean day of onset of this arthritis, [which we have previously shown to correlate with raised % Gal(O)], from 38 to 15 days (p less than 0.001). One interpretation is that an autoimmunogenic stimulus, given when % Gal(O) is raised, is more likely to evoke disease. Since oil granulomata are known to secrete interleukin 6, which has B cell-regulatory properties and is secreted by rheumatoid synovial cells, we tested sera from interleukin 6-transgenic mice, and found a strikingly raised percentage of Gal(O). We suggest, therefore, that the role of oil in the induction of arthritis is the dysregulation of cytokine release of which a raised percentage of Gal(O) may be a direct or indirect consequence, associated with an increased susceptibility to autoimmunogenic stimuli.     

The route of administration of an immunodominant peptide derived from heat-shock protein 65 dramatically affects disease outcome in pristane-induced arthritis

Previous studies have shown that immunization of mice with an immunodominant epitope from heat-shock protein 65 (hsp 65) (amino acids 261-271) can protect from the development of pristane-induced arthritis (PIA) and this protection is mediated by an antigen-specific T helper type 2 (Th2) cytokine response. Here we confirm these findings and show that frequent intranasal administration of this peptide exacerbates disease. In naive mice given peptide intranasally an antigen-specific T-cell population is systemically activated similar to that induced by peptide immunization in incomplete Freund's adjuvant. Thus, a paradox exists whereby apparently similar peptide-specific populations are either associated with protection from, or exacerbation of, PIA. However, comparison of cytokine profiles reveals differences between these two cell populations. Peptide inhalation induces the production of Th1-type cytokines (interferon-gamma) whereas intraperitoneal immunization leads to the production of Th2-type cytokines (interleukin-4, interleukin-5 and interleukin-10) by splenic T cells upon stimulation with peptide. Thus, for the application of nasal 'tolerance' in clinical medicine, it is important to identify antigens and dosing regimes that counteract but do not activate adverse immune responses.     

Role of the Bowel Flora for Development of Immunity to hsp 65 and Arthritis in Three Experimental Models

An infectious aetiology in rheumatoid arthritis (RA) has for long been suggested, although no conclusive evidence for this is at present available. Lately a large interest has been devoted to the involvement of heat shock proteins (hsps) in autoimmune disorders due to their conserved structure and immunogenic properties. Immunity to hsps has been observed both in human autoimmune conditions and in experimental models of autoimmune disease. We have studied the role of the bacterial flora and hsp immunity in the arthritic response in three experimental models of arthritis; type II collagen arthritis (CIA), adjuvant arthritis (AA) and oil induced arthritis (OIA); by using germ free and conventional DA rats.
A high incidence of severe arthritis developed in all the models evaluated irrespectively of whether the animals were in the conventional or germ free state. This confirms earlier results which show a minor effect of the bacterial flora in CIA and AA in high responder strains. These results also show that a severe OIA can develop in germ free animals. Despite the severe arthritic response induced, no serum antibody levels to hsp 65 could be detected in the germ free animals, which was in contrast to the conventional animals where a positive anti-hsp 65 serum response was seen in 35–80% of the animals with CIA, AA or OIA.
These results show that development of a humoral response to hsp 65 in these models of arthritis is dependent on the presence of a bacterial flora. Further, the lack of humoral immunity in germ free animals despite a severe arthritic response indicates that humoral immunity to hsp 65 is not involved in development of disease in these three models of experimental arthritis.     

Cellular and humoral reactivity pattern to the mycobacterial heat shock protein HSP65 in adjuvant arthritis susceptible and resistant Wistar rats.

We have analyzed the cellular and humoral immunity to the mycobacterial 65 KDa heat shock protein (hsp65) in a group of Freund's Adjuvant-immunized rats with a limited susceptibility to Adjuvant arthritis. According to the arthritis indices during the period of study (35 days), two different groups of rats could be distinguished; a) autoimmune Adjuvant arthritic rats (AA), and b) Non-arthritic animals (NA), including both rats which did not display any disease symptoms and rats suffering mild transient inflammation. The cellular response to the immunizing agent (Mycobacterium tuberculosis) or the mitogen Concanavalin A was comparable between both groups of rats. However, we detected an impaired cellular response to the individual hsp65 antigen in the animals that did not develop the disease. On the contrary, the level of hsp65-specific antibodies was much higher in NA animals than in AA rats suggesting a protective role for the hsp65 specific antibodies.     

Protection against tuberculosis by passive transfer with T-cell clones recognizing mycobacterial heat-shock protein 65.

We have previously shown that mice vaccinated by injection with J774 macrophage-like tumour cells that expressed Mycobacterium leprae heat-shock protein (hsp) 65 as a transgene had acquired a remarkably high degree of protection against subsequent challenge with virulent M. tuberculosis. We show here that antigen-specific T cells cloned from spleens of such vaccinated animals can transfer a high level of protection to non-vaccinated recipients. The most efficient cells were of T-cell receptor (TCR) alpha beta+ and CD4- CD8+ type and specifically lysed mycobacteria-infected macrophages. These findings are consistent with the importance for protective immunity of engaging the endogenous antigen-presenting pathway to bias the immune response towards a cytolytic action against a mycobacterial antigen that is expressed at the surface of infected macrophages. TCR gamma delta+ and TCR alpha beta+ cells interacted synergistically.     

Autoreactive T cells from normal mice recognize mycobacterial 65 kd heat-shock protein from Mycobacterium bovis.

We established a TCR V beta 6+ CD4+ autoreactive T cell line from lymph node cells of unprimed BALB/c mice bred in a specific pathogen-free condition. The T cell line proliferated in the presence of irradiated spleen cells expressing MHC class II I-Ad and/or I-Ed molecules. The proliferative response of the autoreactive T cell line was significantly enhanced in response to 65 kd heat-shock protein (hsp) from Mycobacterium bovis, a member of the hsp60 family, which is highly conserved in both prokaryotes and eukaryotes. Our results suggest that the autoreactive T cells in normal mice may recognize endogenous peptides of self-hsp60 bound to MHC class II gene products and are cross-reactive to mycobacterial 65 kd hsp. The autoreactive hsp-specific T cells may therefore play an important role in the immune surveillance against invasion of various pathogens and tumors by recognizing infected and transformed autologous cells that express high levels of endogenous hsp60.     

Effect of T-helper cytokine environment on specificity of T-cell responses to mycobacterial 65,000 MW heat-shock protein.

The purpose of this work was to determine if the fine specificity of T cells differed between mice immunized with an antigen in a T helper 1 (Th1) cytokine-dominated environment as compared with a T helper 2 (Th2) cytokine-dominated environment. It was found that splenic T cells from mice immunized with mycobacterial heat-shock protein (hsp 65) and interleukin-12 (IL-12) produced less interleukin-4 (IL-4) and more interferon-gamma (IFN-gamma) in response to stimulation with hsp 65 in vitro than did T cells from mice immunized with hsp 65 alone. The T-cell proliferative response to hsp 65 did not differ between the two groups of mice, although the responses were higher than those of T cells from non-immunized mice. Strikingly, T cells from mice given hsp 65 and IL-12 gave significantly higher responses to six peptides (corresponding to the sequence of hsp 65) to which T cells from mice immunized with hsp 65 alone did not respond. It is considered that different epitopes are presented to T cells (possibly owing to changes in antigen processing) if the environment is shifted, by IL-12, from Th2 towards Th1 cytokines.     

Arthritis induced in rats with non-immunogenic adjuvants as models for rheumatoid arthritis

Summary:
Rat models are useful for studies of the pathogenesis of rheumatoid arthritis (RA) since rats are extraordinarily sensitive to induction of arthritis with adjuvants. Injection of not only the classical complete Freund's adjuvant but also mineral oil without mycobacteria and pure adjuvants such as pristane and squalene, induce severe arthritis in many rat strains. Models like pristane-induced arthritis in rats are optimal models for RA since they fulfill the RA criteria including a chronic relapsing disease course. Arthritogenic adjuvants like pristane, avridine, squalene and mineral oil are not immunogenic since they do not contain major histocompatibility complex (MHC) binding peptides. Nevertheless, the diseases are MHC-associated and dependent on the activation of αβTCR (T-cell receptor)-expressing T cells. However, it has not been possible to link the immune response to joint antigens or other endogenous components although immunization with various cartilage proteins induce arthritis but with different pathogeneses. To unravel the mechanisms behind adjuvant-induced arthritis, a disease-oriented genetic approach is optimal. Several loci that control onset of arthritis, severity and chronicity of the disease have been identified in genetic crosses and most of these have been confirmed in congenic strains. In addition, many of these loci are found in other autoimmune models in the rat as well as associated with arthritis in mice and humans.     

Influence of Microbial Stimulation on Hypergammaglobulinemia and Autoantibody Production in Pristane-Induced Lupus

Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after ip pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.     

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.     

Isolation and characterization of a cartilage-specific membrane antigen (CH65): comparison with cytokeratins and heat-shock proteins.

We report the isolation and characterization of a 65,000 MW chondrocyte autoantigen (CH65) which may be involved in rheumatoid arthritis. This chondrocyte-specific antigen reacted with sera from patients with rheumatoid arthritis (RA). CH65 did not cross-react with a polyclonal antibody raised against microbial heat-shock protein (hsp) 65, anti-human hsp 65 monoclonal antibodies (mAb) (LK1 and LK2), anti-microbial hsp 65 mAb (IA10, IIC8 and WTB-78H1) and anti-cytokeratin 8, 18, 19 mAb (NCL5D3MAb). CH65 could be purified from chicken chondrocyte membranes by ammonium sulphate precipitation and a novel electro-gel-filtration method. The amino acid analysis yielded an unusually high degree of glycine, serine and asparagine residues. The internal amino acid sequence obtained by tryptic digestion revealed homologies with the cytokeratin family. Despite these homologies, CH65 lacked immunological cross-reactivity with commercial anti-cytokeratin antibodies. Mice mAb generated against the purified CH65 (C6) were used to identify the protein as a tissue-specific constitutive protein membrane from chondrocytes. Sera from patients with RA cross-reacted with purified CH65. The stress or heat-shock protein (hsp 65), implicated in the development of experimental and clinical arthritis, showed no immunological cross-reactivity with CH65 in Western blots. These findings suggest that CH65 may represent an interesting cartilage-specific new antigen in RA. The availability of this antigen in purified form and specific mAb may offer useful tools in arthritis research.     

Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein.

Adjuvant arthritis in Lewis rats is a model of T cell-mediated autoimmune arthritis resembling human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium bovis BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-terminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cell-specific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.     

T-helper 1 dominated responses to erythrocyte Band 3 in NZB mice.

Band 3, the red blood cell (RBC) anion channel protein, is the target autoantigen for the pathogenic RBC autoantibodies and T-helper (Th) cells in New Zealand Black (NZB) mice with autoimmune haemolytic anaemia (AIHA). To determine the subpopulation of these Th cells, they were stimulated with Band 3 and the profile of the cytokines elaborated by the responding cells was measured. NZB T cells stimulated with Band 3 produced high levels of the Th1 cytokine, interferon-gamma (IFN-gamma), but little or no interleukin-4 (IL-4), IL-5 or IL-10. Similar patterns were produced by NZB T cells responding to a spectrin preparation from the RBC membrane skeleton, or to mycobacterial heat-shock protein (hsp) 65 following immunization of mice with hsp 65 in incomplete adjuvant. By contrast, T cells from CBA mice similarly immunized with hsp 65 produced high levels of IL-4 and IL-5 in response to hsp 65. Examination of the isotype of the RBC-bound immunoglobulins in NZB mice revealed that immunoglobulin G2a (IgG2a) autoantibodies were the first to be detected in most mice and that later in the disease, IgG3 autoantibodies were often prominent. It is concluded that, contrary to expectation, the development of RBC autoantibodies in NZB mice is associated with Th1 cytokine-dominated responses.     

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.