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Objective— HSP60-specific T cells contribute to the development of the immune responses in atherosclerosis. This can be dampened by regulatory T cells activated via oral tolerance induction, and we explored the effect of oral tolerance induction to HSP60 and the peptide HSP60 (253 to 268) on atherosclerosis. Methods and Results— HSP60 and HSP60 (253 to 268) were administered orally to LDLr −/− mice before induction of atherosclerosis and resulted in a significant 80% reduction in plaque size in the carotid arteries and in a 27% reduction in plaque size at the aortic root. Reduction in plaque size correlated with an increase in CD4 + CD25 + Foxp3 + regulatory T cells in several organs and in an increased expression of Foxp3, CD25, and CTLA-4 in atherosclerotic lesions of HSP60-treated mice. The production of interleukin (IL)-10 and transforming growth factor (TGF)-β by lymph node cells in response to HSP60 was observed after tolerance induction. Conclusion— Oral tolerance induction to HSP60 and a small HSP60-peptide leads to an increase in the number of CD4 + CD25 + Foxp3 + regulatory T cells, resulting in a decrease in plaque size as a consequence of increased production of IL-10 and TGF-β. We conclude that these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) may provide new therapeutic approaches for the treatment of atherosclerosis.

Objective: To obtain more insight in the role of IGF-1 in cardiac remodeling and function after experimental myocardial infarction. We hypothesized that cardiac remodeling is altered in IGF-1 deficient mice, which may affect cardiac function. Methods: A myocardial infarction was induced by surgical coronary artery ligation in heterozygous IGF-1 deficient mice. One week after surgery, left ventricular function was analyzed, and parameters of cardiac remodeling were measured. Results: No significant difference in cardiac function was found between infarcted wildtype and knock-out animals, despite a marked reduction in capillarization and blunting of the hypertrophic response of the interventricular septum in the IGF-1 deficient group. Furthermore, decreased DNA synthesis and increased apoptosis rates were observed in the IGF-1 knock-out mice. Conclusion: IGF-1 deficient mice show preservation of cardiac function 1 week after MI, despite an altered cardiac remodeling process.

Rheumatoid arthritis (RA) is the most common, crippling human autoimmune disease. Using Western blotting and tandem mass spectroscopy, we have identified the endoplasmic reticulum chaperone BiP, a 78-kDa glucose-regulated protein, as a possible autoantigen. It preferentially stimulated increased proliferation of synovial T cells from patients with RA but not from patients with other arthritides. Mice with established collagen- or pristane-induced arthritis developed IgG Abs to BiP. Although BiP injected in CFA failed to induce arthritis in several strains of rats and mice, including HLA-DR4+/−- and HLA-DR1+/+-transgenic animals, it completely inhibited the development of arthritis when given i.v. 1 wk before the injection of type II collagen arthritis. Preimmunization with BiP suppressed the development of adjuvant arthritis in Lewis rats in a similar manner. This is the first report of a mammalian chaperone that is an autoantigen in human RA and in experimental arthritis and that can also prevent the induction of experimental arthritis. These findings may stimulate the development of new immunotherapies for the treatment of RA.

Background —Nebivolol is a β 1 -selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. Methods and Results —Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective β 2 -adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of β 2 - but not β 1 -adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca 2+ concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. Conclusions —In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial β 2 -adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca 2+ ] i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.

Adjuvant Arthritis (AA) can be induced in Lewis rats by immunisation with mycobacterial antigens. The disease can be passively transferred with T cell clone A2b, which recognises the 180-188 amino acid sequence in mycobacterial heat shock protein 60 (hsp60) and which crossreacts with crude cartilage proteoglycans. We succeeded to induce peripheral tolerance to this AA-associated T cell epitope following nasal administration of a peptide containing this epitope (mycobacterial hsp60 176-190). In rats treated nasally with 176-190 and immunised with mycobacterial hsp60, proliferative responses to 176-190 were reduced. AA was inhibited nasally with 176-190 treated rats and not in rats nasally treated with a control mycobacterial hsp60 peptide (211-225). Moreover, nasal 176-190 led to similar arthritis protective effects in a non-microbially induced experimental arthritis (avridine induced arthritis). In a subsequent study we tried to prevent and to treat AA through nasal administration of mycobacterial hsp60 peptide 180-188 and a peptide analogue of 180-188, 180-188(L183-A) (Alanine 183), which has been shown to have an increased MHC-binding affinity for rat RT1 Bl and an increased capacity to inhibit the proliferative A2b response in vitro. We found that nasal administration of 180-188 had a moderate arthritis suppressive effect in AA, whereas its analogue peptide Alanine 183, had a strong suppressive effect. This strong arthritis suppressive effect was only partly due to the higher MHC-binding affinity for rat RT1 Bl. Furthermore, it was possible to passively transfer nasal Alanine 183 induced disease protection. The present findings may in our view offer novel prospects for immunotherapy through nasal administration of (analogue) peptides, with a mimicry relationship with joint specific cartilage proteoglycan epitopes.

SUMMARY 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.

IgA nephropathy (IgAN) is the commonest cause of glomerulonephritis and clinical exacerbation of IgAN is frequently associated with mucosal infection. T-cell receptor gamma delta (TCR gamma delta+) cells are increased in both the circulation and in renal biopsies of patients with progressive IgAN. We examined the hypothesis that specific peptides within the 65,000 MW heat-shock protein (hsp) might stimulate TCR gamma delta cells and play a part in the immunopathogenesis of IgAN. We studied T-cell proliferative responses stimulated by overlapping peptides derived from the sequence of mycobacterial 65,000 MW hsp. Three T-cell epitopes have been identified (peptides 51-65, 71-85 and 281-295). The three peptides have a synergistic effect and they stimulate significantly higher proliferation of T cells in patients with IgAN than in disease or healthy controls. This response was inhibited by monoclonal antibodies (mAb) to TCR gamma delta+ and human leucocyte antigen (HLA) class I, but not by mAb to HLA class II. The involvement of TCR gamma delta+ cells was confirmed by up-regulation of the proportion of TCR gamma delta+ cells when stimulated with the three specific peptides. We suggest that IgAN might be associated with mucosal infection by a variety of micro-organisms and that peptides within the microbial hsp cross-react with the homologous human hsp which may stimulate TCR gamma delta+ cells and play a part in the pathogenesis of IgAN.

Background The mechanisms of the established atheroprotective effects of estrogen have not been entirely clarified. Recent data suggest that agents that hasten the recovery of the endothelium after denuding injury will deter the development of neointimal lesions. Because estrogen has been shown to exert angiogenic effects in vitro and in vivo, we performed a series of experiments to evaluate whether estrogen was capable of accelerating reendothelialization. Methods and Results Ovariectomized Sprague-Dawley rats received estrogen replacement therapy in the form of subcutaneously implanted pellets designed to release 1.5 or 5.0 mg 17β-estradiol over 30 days. Deendothelializing balloon injury was performed 1 week after pellet implantation, and animals were euthanatized after 1 week for evaluation of reendothelialization (Evans blue staining) or 2 weeks for evaluation of reendothelialization and neointimal formation. At both time points, the use of estradiol caused a dose-dependent increase in reendothelialization, which was measured as absolute area and percentage of area that is reendothelialized. Estradiol accelerated functional endothelial recovery, manifested as an increase in nitric oxide production. Neointimal thickening was also shown to be inhibited in a dose-dependent fashion. Conclusions Estrogen accelerates functional endothelial recovery after barotraumatic deendothelializing injury. These findings, along with the recent demonstration of estrogen receptor expression by endothelial cells, suggest that the antiatherogenic action of estrogen may be mediated in part through direct effects on endothelial cells.

Lewis rats are susceptible to several forms of experimental arthritis-induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II-restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell-mediated regulation of inflammation.

Previously we have shown that blood mononuclear cells (MNC) obtained from patients with rheumatoid arthritis (RA) have the capacity to induce depletion of proteoglycans (PG) in human cartilage explants. This was observed especially after stimulating MNC with mycobacterial antigens, rather than with the mitogen Concanavalin A (Con A). We have now co-cultured cartilage explants in the presence of T-cell clone A2b obtained from the rat model of adjuvant arthritis (AA). We show that inhibition of the cartilage PG synthesis is a consequence of antigen-specific T-cell activation and that it is mediated by a humoral factor. This seems to be a cytokine rather than an enzyme. Moreover, at the level of polyclonally responding T cells, inhibition of PG synthesis due to T-cell activation by mycobacterial antigens was shown to depend on prior mycobacterial immunization. Arthritogenic T-cell clone A2b also showed PG synthesis inhibitory effects when co-cultured with cartilage alone. The inhibitory activity was shown to be unrelated to the degree of T-cell proliferation. We conclude that antigen-specific T-cell activation may be one of the initiating events leading to cartilage damage in arthritic processes. The measurement of T-cell-mediated PG synthesis inhibition may be a more sensitive and relevant assay for the detection of pathogenic T cells than T-cell proliferation.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin-dependent diabetes mellitus of NOD mice, we studied the specificities of helper T-cell clones capable of causing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence of the human variant of the 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cell vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes.

The significance of immune responses to certain heat shock proteins (HSPs) that develop in virtually all inflammatory diseases is only now becoming clear. In experimental models, HSPs prevent or arrest inflammatory damage, and initial clinical trials in chronic inflammatory disease have shown HSP peptides to promote production of anti-inflammatory cytokines-indicating immunoregulatory potential. HSPs are ubiquitous self-antigens that are highly expressed in inflamed tissues. The prokaryotic homologous proteins, present in every bacterial species, are dominantly immunogenic. This is striking, especially as these proteins have large areas of sequence homologies with the host (mammalian) counterparts. In several experimental models of autoimmune diseases, immunisation with bacterial HSPs inhibited disease development, as did oral/nasal administration. Based on the experimental evidence so far, it is tempting to speculate that: firstly, exposure to homologues of these self-antigens, as present in, for instance, the bacterial intestinal flora, has a decisive impact on the regulation of self-tolerance at the level of T cells; and secondly, such proteins or their derivative peptides may have a role in an antigen specific immunotherapy approach involving modulation of relevant T cells, without the immediate necessity of defining disease specific autoantigens. Recent findings in experimental asthma and atherosclerosis have indicated that the field of application of such immunotherapy can be broader than just autoimmunity.

Background Anti-heat-shock protein 60 (HSP60) antibody-levels have been linked to carotid atherosclerosis and cardiovascular risk in a variety of studies. The potential role of cellular immune reactions against HSP60 has so far attracted little attention in epidemiological research. Methods and results In vitro T-cell reactivity to various HSP60s and tuberculin was assessed in blood samples from a elderly subpopulation of the Bruneck study (100 men, 50–69 years) and the young participants of the ARMY study (141 men, 17–18 years), and analyzed for a potential association with common carotoid artery intima-media thickness (IMT). In vivo skin reaction against tuberculin was recorded in subjects of the Bruneck study and correlated with the in vitro proliferative response to tuberculin ( P =0.004). T-cells isolated from peripheral blood of all individuals proliferated upon stimulation with HSP60s. In multivariate linear regression analysis adjusted for standard risk factors, T-cell stimulation was significantly related to IMT in the ARMY ( P =0.005 for human HSP60 and P =0.064 for mycobacterial HSP60) but not in the Bruneck study. Conclusions T-cell reactivity against HSP60s correlated with IMT in male youngsters but not in men aged 50 and over, indicating a more prominent role of specific cellular immunity to HSP60s in the young and very early stages of atherosclerosis.

Background—Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis.Methods and Results—Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor−/−mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%;PP+CD25+Foxp3+cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-β production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis.Conclusions—OxLDL-specific T cells, present in LDL receptor−/−mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+CD25+Foxp3+regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Immunisation with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunisation with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp601 and later for mycobacterial hsp70.2,3 Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunisation with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60.4 Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10. References Anderton SM, van der Zee R, Prakken B, Noordzij A, van Eden W. Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J Exp Med. 1995;181:943–52 Tanaka S, Kimura Y, Mitani A, Yamamoto G, Nishimura H, Spallek R, Singh M, Noguchi T, Yoshikai Y. Activation of T cells recognizing an epitope of heat-shock protein 70 can protect against rat adjuvant arthritis. J Immunol. 1999;163:5560–65 Wendling U, Paul L, van der Zee R, Prakken B, Singh M, van Eden W. A conserved mycobacterial heat shock protein (hsp) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the mammalian self-hsp70 homologue. J Immunol. 2000;164:2711–17 Paul AG, van Der Zee R, Taams LS, van Eden W. A self-hsp60 peptide acts as a partial agonist inducing expression of B7-2 on mycobacterial hsp60-specific T cells: a possible mechanism for inhibitory T cell regulation of adjuvant arthritis? Int Immunol. 2000;12:1041–50

SUMMARYT cell-mediated immunity is thought to play an important role in the pathogenesis of WG. In previous studies a minority of WG patients as well as some healthy controls showed in vitro proliferation of their peripheral blood mononuclear cells (PBMC) to PR3, the main autoantigen in WG. The relevant peptides responsible for this in vitro proliferation have not been identified. In order to define immunogenic peptides, PBMC of 13 WG patients in remission and 10 healthy controls were tested for proliferation to linear peptides of PR3 and to whole PR3. Fifty overlapping peptides spanning the whole PR3 sequence were synthesized. Peptides were tested in pools of five peptides and as single peptide. PBMC of two WG patients and one healthy control proliferated to whole PR3 and to peptide pools. In addition, 10 WG patients and eight healthy controls that did not proliferate to whole PR3 did proliferate to pools of PR3 peptides. Although more WG patients tended to react to particular peptide pools, no significant difference was seen between lymphocyte proliferation to PR3 peptides of WG patients and that of healthy controls. The pools of peptides recognized were mainly located at the N- and C-terminus of PR3. No correlation was observed between HLA type and proliferation on particular peptide pools. No proliferation of PBMC was observed to single peptides. In conclusion, T cells of WG patients proliferate in vitro more frequently to PR3 peptides than to the whole PR3 protein. Peptides derived from the signal sequence, the propeptide or peptides located at the C-terminus of PR3 induce highest levels of proliferation. No specific PR3 sequence could be identified that was preferentially recognized by PBMC of WG patients compared with controls.

SUMMARYT cell epitopes of the 65-kD heat shock protein (hsp) were investigated in patients with recurrent oral ulcers (ROU). Peripheral blood mononuclear cells were stimulated with overlapping synthetic peptide (15ers), derived from the sequence of the 65-kD hsp of Mycobacterium tuberculosis. Specific lymphoproliferative responses were stimulated only with peptide 91–105 in ROU, compared with healthy or disease controls (P < 0·01). This was confirmed by studying 760 short term cell lines generated with the 65-kD hsp and then stimulated with the peptides. The frequency of short term cells lines responding to peptide 91–105 in ROU was significantly greater than in healthy (P < 0.0001) or disease controls (P < 0·01). A comparative investigation with the homologous human 60-kD hsp peptide 116–130 also showed significantly greater lymphoproliferative responses in ROU than in healthy (P < 0·01) or disease controls (P < 0·001). The potential involvement of the T cell epitope 91–105 in the pathogenesis of ROU is supported by finding a significant increase in the lymphoproliferative responses stimulated with peptide 91–105 during the stage of ulceration, compared with remission in 9/11 patients studied sequentially (P < 0·05). The results suggest that oral ulceration might be initiated by the microbial hsp peptide 91–105 stimulating the mucosal Langerhans cells, which may generate autoreactive T cell clones primed to the homologous peptide 116–130.

Synovial fluid and peripheral blood mononuclear cell proliferative responses to the 60-kD human heat shock protein (HSP60) were studied in 23 patients with juvenile chronic arthritis (JCA) and 7 non-JCA control patients. All patients showed active arthritis at the time of study. The patients were divided into two groups according to the presence (group A) or absence (group B) of T lymphocyte reactivity to human HSP60. We show that reactivity to human HSP60 is primarily, though not exclusively, occurring in patients with a remitting course of disease, i.e., the subgroup of HLA-B27 negative JCA patients with an oligoarticular onset. Immunohistochemical analysis of HSP expression in synovial membranes showed a significantly higher intensity of staining in JCA patients than in non-JCA controls. The results suggest that, in accordance with the earlier observation made in experimental models, T lymphocyte reactivity to human HSP60 in this subgroup of JCA patients may be part of T cell regulatory mechanisms that control the development of arthritis.

T-T cell interactions have been proposed in postulated network theories of immunoregulation and autoimmunity. Despite previous reports of protection induced by T-cell receptor (TcR)-derived peptides in experimental autoimmunity, no evidence for T-T cell interactions by direct recognition of processed TcRs on native T cells was obtained. Here we report that immunization of rats with overlapping sets of peptides of the TcR alpha or beta chain allowed us to detect immunogenic TcR peptides. Remarkably enough, these TcR peptides appeared to cluster within the hypervariable complementarity-determining regions of the TcR. Immunization of rats with these TcR peptides induced CD4+ TcR peptide-specific T cells, which recognized both rDNA TcR proteins and the original, arthritogenic T cell in a major histocompatibility complex class II-restricted way. These findings indicate that activated T cells can process and present their own TcR in the context of major histocompatibility complex class II molecules and, furthermore, that such peptides can be recognized by TcR variable gene-specific T cells.

Appreciation of the nature of autoiramunity has changed with the advent of techniques enabling the cloning of antigen specific T lymphocytes. In experimental animal models T cells have been cloned and maintained by stimulation with antigens, such as myelin basic protein (MBP), thyroglobulin and many other antigens known to cause experimental autoimmunity upon appropriate immunization. The inevitable conclusion from this was that within the healthy immunological repertoire B cell and T cell self-reactivity was a physiological component. Subsequent demonstration that such cloned T cells were capable of transferring experimental autoimmune diseases, has made the question of how these cells remain safely contained within the normal functioning repertoire into one of the most pressing issues of modern immunology (Cohen, 1986). Along with the studies aimed at furthering our understanding of this point, it has become clear that antigen specific manipulation of autoimmunity may become a reality.

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.

PhoE is a pore-forming protein, abundantly expressed in the Escherichia coli outer membrane. Previous investigations have shown the possibility of inserting antigenic determinants in cell surface-exposed regions of PhoE by recombinant DNA techniques without disturbing the biogenesis and the functioning of the protein. This method proved to be successful for foot-and-mouth disease virus B cell determinants. We have now shown for the first time that PhoE can also be used as a carrier molecule for T cell epitopes. A well-characterized T cell epitope (180-188) of the 65-kDa heat-shock protein (hsp 65) of Mycobacterium tuberculosis was expressed in PhoE and tested for recognition by specific T cell clones. Specific and efficient T cell proliferation was found after stimulation with this protein construct in vitro. Interestingly, paraformaldehyde fixation of antigen-presenting cells did not abrogate T cell recognition. Thus, in contrast to hsp 65 itself, recognition of epitope 180-188 in the context of PhoE appeared to be independent of antigen-processing events. At the level of polyclonal T cell responses the epitope in the context of PhoE is recognized more efficiently than 180-188 as synthetic peptide or in the context of the hsp 65 molecule itself. These findings indicate that PhoE may serve as attractive vaccine carrier not only for B, but also for T cell epitopes. Furthermore, the possibility for expression of PhoE constructs in attenuated Salmonella typhimurium strains offers the exciting prospect of new types of live oral vaccines expressing selected combinations of B and T cell epitopes.

Arthritis - induced experimentally in rats by immunization with mycobacteria has been shown to depend on specific T cell recognition of an epitope present on the mycobacterial 65-kD heat-shock protein. This particular epitope has been observed to have a structural mimicry with a cartilage-associated molecule present in the joints. Since the bacterial heat-shock proteins and the cartilage-associated molecules are of a conserved nature, one might infer from the experimental model that in humans similar mimicry could play a role in the initiation of autoimmune arthritis. Recent findings from the analysis of immunological reactivity to the 65-kD in rheumatoid arthritis patients seem to support such a role for the mycobacterial 65-kD heat-shock protein in human disease.

We report that streptococcal cell wall (SCW)-induced arthritis in rats, a T cell-dependent chronic, erosive polyarthritis, can be prevented by pretreatment of the rats with the mycobacterial 65-kD heat shock protein. This 65-kD protein shows extensive amino acid homology with prokaryotic and eukaryotic 65-kD heat shock proteins and is a ubiquitous bacterial common antigen. Both the clinical and histopathologic manifestations of the arthritis were prevented completely when rats were pretreated with 50 micrograms of 65-kD protein intraperitoneally at 35, 25, 15, or 5 d before administration of SCW. In such protected rats, SCW-specific T cell responses were suppressed, as compared with responses in arthritic rats. Pretreatment with 65-kD protein had no effect on the production of antibodies against SCW, on a nonspecific inflammatory reaction (zymosan-induced arthritis), or on general cellular immunity in vivo (delayed type hypersensitivity reaction to a nonrelated protein antigen). Furthermore, the protection against SCW arthritis was transferable by splenic T cells to naive recipients. Our data show that pretreatment with the 65-kD mycobacterial heat shock protein protects rats against a subsequent bacterium-induced arthritis. This protection is immunologically specific and resides in the lymphoid cell population.

The way the immune system evolved has been commonly thought to have been influenced during evolution through selective pressure exerted by microbial invaders. So, immune recognition and the subsequent immune response have become significant means of the host to combat the attack of exogenous invaders. Since, however, the exogenous microbial world presents itself with a wealth of antigenic variety on each single organism, immune recognition can be selective at the antigen level. Teleologically, an unselected response would be a very inefficient maneuver inevitably leading to jamming of the system. Moreover, one could envisage more pertinent reasons for being selective, among them that responding to certain antigens might jeopardize the maintenance of self integrity. This means that a response directed to such antigens might cause uncontrollable disturbances in the balance of elements that interact in the immunological network and might lead to the ultimate development of immunopathology. This could happen when the exogenous stimulator antigen bears a resemblance to self molecules, a situation called antigenic mimicry.

SUMMARY 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 enterocotitica. Phenotyping of the resulting T cell lines revealed an increase of up to 97% TCR-γδ+ lymphocytes in the 65-kD hsp-stimulatcd SF-derived lines. This expansion of TCR-γδ+ cells was less pronounced with cultures of PBMC. A preferential expansion of TCR-γδ+ 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-γδ+ lymphocytes upon SFMC infection with live Yersinia or upon contact with 65-kD hsp. Out of a panel of TCR-γδ+ 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-αβ+ 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-γδ+ cells in response to 65-kD hsp or live bacteria.

Peptide analogues of disease-associated epitopes were studied for inhibition of experimental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) in Lewis rats. EAE- and AA-associated analogues were selected as competitors because of their in vitro inhibitory activity on proliferation of encephalitogenic and arthritogenic T cells. Although the EAE-associated competitor had a superior major histocompatibility complex (MHC) binding affinity, the AA-associated competitor was a better inhibitor of the in vitro proliferation of arthritogenic T cells. Furthermore, although in vivo EAE was inhibited by both competitors, AA was only inhibited by the AA-associated competitor. Remarkably, in contrast to what was expected of a regular MHC competitor peptide, the AA-associated peptide analogue also prevented AA upon immunization before disease induction and appeared to induce T cell responses that crossreacted with the original disease-associated epitope. Therefore, it is concluded that antigen-specific regulatory mechanisms were involved in synergy with MHC competition. The integration of both qualities into a single "competitor-modulator" analogue peptide may lead to the development of novel, more effective, disease-specific immunomodulatory peptides.

Objective. To investigate whether oral administration of mycobacterial heat-shock protein 65 (HSP65) during adjuvant arthritis (AA) induces regulatory cells and cytokines. Methods. AA was induced in Lewis rats and from the time of disease onset HSP65 in the presence of soya bean trypsin inhibitor (STI) was administered orally every other day. The number of splenic CD4 + CD25 + T cells and antigen-induced cytokine mRNA expression were determined. Results. Oral treatment with HSP65uSTI reduced AA symptoms. After one feeding of HSP65uSTI, the number of CD4 + CD25 + splenic T cells increased and HSP65-specific T cells expressed increased levels of interferon c and interleukin 10. After two feedings, the expression of interleukin-10 mRNA remained increased, whereas there was low expression of interferon c mRNA. The number of CD4 + CD25 + splenic T cells remained increased. Conclusions. Oral treatment with HSP65uSTI after AA onset reduces disease symptoms via dynamic changes in the number of CD4 + CD25 + splenocytes and in antigen-induced cytokine

Heat-shock proteins have been shown to be critical antigens in a number of autoimmune diseases. In human arthritis and in experimentally induced arthritis in animals, disease development was seen to coincide with development of immune reactivity directed against not only bacterial hsp60, but also against its mammalian homologue. We have developed murine monoclonal antibodies after immunization with recombinant human hsp60. Antibodies with unique specificity for mammalian hsp60, not crossreactive with the bacterial counterpart (LK1), and antibodies recognizing both human and bacterial hsp60 (LK2) were selected. Both antibodies recognize epitopes located between amino acid positions 383 and 447 of human hsp60. In immunogold electron microscopy, the mitochondrial localization of hsp60 in HepG2 cells was shown. Furthermore, both LK1 and LK2 showed a raised level of staining in light microscopy immunohistochemistry of synovial membranes in patients with juvenile chronic arthritis. The increased staining for LK1, with a unique specificity for mammalian hsp60, thus unequivocally demonstrates that this is due to a raised level of expression of endogenously produced host hsp60 and not to deposition of bacterial antigens.

SUMMARYMycobacterial and homologous human heat shock protein T cell peptide epitopes specific for T lymphocytes in Behçets disease were investigated for their pathogenicity in Lewis rats. The potential pathogenicity of eight peptides and two controls was assessed by administering the peptides in enriched Freund's adjuvant into the footpads of male Lewis rats. Anterior uveitis which is a major manifestation of Behçet's disease was induced with two out of the four mycobacterial and all four homologous human peptides. The most effective peptides inducing indocyclitis in 64–75% of rats were peptides with amino acids 336–351 and 136–150, derived from the sequence of the human 60-kD heat shock protein. A few of the rats also showed evidence of focal loss of photoreceptors. These results suggest that selected peptides within heat shock protein 60 kD which function as T cell epitopes in Behçet's disease are capable of inducing uveitis in rats. This supports the view that the peptide T cell determinants may be involved in the pathogenesis of Behçet's disease.

The 60-kD heat shock protein (hsp60) has been implicated in the etiology and pathogenesis of both experimental and naturally occurring autoimmune diseases such as juvenile chronic arthritis (JCA). Human hsp60 is expressed in inflamed synovial tissue, and T lymphocytes both from peripheral blood and synovial fluid show reactivity to human hsp60. Because the anti-hsp60 B lymphocyte response has been less well studied, we have determined the occurrence of IgG anti-human hsp60 antibodies in patients with JCA and various other autoimmune diseases of childhood. Serum IgG anti-human hsp60 antibodies in JCA patients were significantly higher compared with control children (358 and 163 U/mL, respectively). Within the group of JCA patients, the highest antibody titers were found in the subgroup with a polyarticular onset of JCA. IgG anti-human hsp60 antibody levels in synovial fluid were 3− to 4-fold higher compared with paired serum samples. Because this difference was not found for total IgG or for irrelevant antibodies (anti-polyribosylribitol phosphate), this suggests local anti-hsp60 antibody production in the synovial compartment. The occurrence of anti-hsp60 antibodies is not specific for JCA but also is found in children with systemic lupus erythematosus and in cystic fibrosis, whereas mixed connective tissue disease and insulin-dependent diabetes are negative in this respect. Whether the anti-human hsp60 antibodies are directed toward species-specific sequences or to conserved sequences of the hsp60 molecule remains to be determined.