The T cells specific for the carboxyl-terminal determinants of self (rat) heat-shock protein 65 escape tolerance induction and are involved in regulation of autoimmune arthritis

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.     

Antibody responses to mycobacterial and self heat shock protein 65 in autoimmune arthritis: epitope specificity and implication in pathogenesis

Many autoimmune diseases are believed to involve primarily T cell-mediated effector mechanisms. There is increasing realization, however, that Abs may also play a vital role in the propagation of T cell-driven disorders. In this study, on the rat adjuvant-induced arthritis (AA) model of human rheumatoid arthritis, we examined the characteristics of serum Ab response to mycobacterial heat shock protein (hsp) 65 (Bhsp65), self (rat) hsp65 (Rhsp65), and linear peptides spanning these two molecules. The AA-resistant WKY (RT.1(l)) rat responded to the heat-killed Mycobacterium tuberculosis immunization with a rapid burst of Abs to both Bhsp65 and Rhsp65. These Abs reacted with numerous peptide epitopes; however, this response was reduced to a few epitopes with time. On the contrary, the susceptible Lewis (RT.1(l)) rat developed a relatively lower Ab response to Bhsp65, and Abs to Rhsp65 did not appear until the recovery from the disease. The Ab response in Lewis rats diversified with progression of AA, and there was an intriguing overlap between the repertoire of Bhsp65-reactive B and T cells during the recovery phase of AA. Nonetheless, subsets of the repertoire of the late Abs in both rat strains became focused on the same epitope regions of Bhsp65 and Rhsp65. The functional relevance of these Abs was evident from the results showing that sera from recovery phase Lewis or WKY rats, but not that of naive rats, afforded protection against subsequent AA. These results are of significance in further understanding of the role of humoral immunity in the pathogenesis of autoimmune arthritis.     

Environmental modulation of autoimmune arthritis involves the spontaneous microbial induction of T cell responses to regulatory determinants within heat shock protein 65

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.     

Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis

Dimethyl dioctadecyl ammonium bromide (DDA) (C(38)H(80)NBr) is a nonantigenic lipoid material. DDA-induced arthritis (DIA) in the Lewis (LEW) (RT.1(l)) rat is a new experimental model for human rheumatoid arthritis (RA). DIA is a T cell-mediated autoimmune disease. However, the precise self/foreign Ags associated with the disease process in DIA are not yet known. We observed that LEW rats with DIA spontaneously raised a vigorous T cell response both to 65-kDa self (rat) heat shock protein (Rhsp65) and mycobacterial hsp65 (Bhsp65), but not to another arthritis-related Ag, bovine collagen type II. The T cell response to Rhsp65 was focused predominantly on determinant regions 120-134 and 213-227 of the self protein. Interestingly, pretreatment of adult LEW rats using either a mixture of peptides 120-134 and 213-227 of Rhsp65 or a low nonarthritogenic dose of DDA induced protection against subsequent DIA. Intriguingly, the protection induced by the latter was associated with spontaneous priming of T cells specific for peptide 213-227 of Rhsp65. Similarly, LEW rats neonatally tolerized against either Rhsp65 or Bhsp65 were significantly protected from subsequently induced DIA at adult stage, showing the disease-modulating attribute of the hsp65-specific T cells. Taken together, the above findings demonstrate that the hsp65-directed T cell repertoire is of significance in the pathogenesis of autoimmune arthritis induced by nonantigenic DDA. Like other animal models of RA involving hsp65, these first insights into the disease-associated Ags in the DIA model would pave the way for further understanding of the immunological aspects of induction and regulation of RA.     

Resistance to adjuvant arthritis is due to protective antibodies against heat shock protein surface epitopes and the induction of IL-10 secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.     

<Emphasis Type="Italic">Celastrus aculeatus</Emphasis>Merr. suppresses the induction and progression of autoimmune arthritis by modulating immune response to heat-shock protein 65

Complementary and alternative medicine products are increasingly being used for the treatment of autoimmune diseases. However, the mechanisms of action of these agents are not fully defined. Using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis, we determined whether the ethanol extract of Celastrus aculeatus Merr. (Celastrus), a Chinese herb, can down-modulate the severity of AA, and also examined the Celastrus-induced changes in immune responses to the disease-related antigen mycobacterial heat-shock protein 65 (Bhsp65). AA was induced in the Lewis (LEW; RT.1^l) rat by immunization subcutaneously with heat-killed M. tuberculosis H37Ra (Mtb). Celastrus was fed to LEW rats by gavage daily, beginning either before Mtb challenge (preventive regimen) or after the onset of AA (therapeutic regimen). An additional group of rats was given methotrexate for comparison. All rats were graded regularly for the signs of arthritis. In parallel, the draining lymph node cells of Celastrus-treated rats were tested for proliferative and cytokine responses, whereas their sera were tested for the inflammatory mediator nitric oxide. Celastrus feeding suppressed both the induction as well as the progression of AA, and the latter effect was comparable to that of methotrexate. Celastrus treatment induced relative deviation of the cytokine response to anti-inflammatory type and enhanced the production of anti-Bhsp65 antibodies, which are known to be protective against AA. Celastrus feeding also reduced the levels of nitric oxide. On the basis of our results, we suggest further systematic exploration of Celastrus as an adjunct therapeutic modality for rheumatoid arthritis.     

The self-directed T cell repertoire against mouse lysozyme reflects the influence of the hierarchy of its own determinants and can be engaged by a foreign lysozyme.

The T cell repertoire is shaped by the processes of positive and negative selection. We have previously shown that mice are tolerant to a native self-Ag, mouse lysozyme (ML), but they respond vigorously when challenged with different ML peptides ("cryptic" self-determinants). In this study, we have addressed the issue of the physiological significance of both the hierarchy (dominance/crypticity) of self-determinants within ML and the anti-cryptic, self (ML)-directed T cell repertoire. Our results demonstrate that there are several ML peptides that bind well to MHC but are totally nonimmunogenic when tested for proliferative T cell response and cytokine secretion: a subset of these peptides presumably represent the originally dominant self-determinants of ML, which have rendered the T cells tolerant during thymic selection. Other ML peptides, which bind well to MHC and are immunogenic, correspond to the cryptic determinants of ML: T cells against cryptic ML determinants escape tolerance induction. Thus, the mature T cell repertoire against ML bears the direct imprint of the hierarchy of self (ML)-determinants. Interestingly, hen egg white lysozyme could prime T cells in vivo that were cross-reactive with certain cryptic ML determinants, and vice versa, without requiring any coimmunization with the foreign lysozyme and ML peptide(s). Moreover, repeated, deliberate priming and expansion of T cells by hen egg white lysozyme immunization concomitantly enhanced T cell response to such cross-reactive ML determinants. This reciprocal self-foreign determinant cross-reactivity may play a previously unrecognized, but crucial, role in the expansion and diversification of self-reactive clones in the autoimmune response.     

DNA fragments of the human 60-kDa heat shock protein (HSP60) vaccinate against adjuvant arthritis: identification of a regulatory HSP60 peptide

Adjuvant arthritis (AA) is induced by immunizing Lewis rats with Mycobacterium tuberculosis suspended in adjuvant. The mycobacterial 65-kDa heat shock protein (HSP65) contains at least one epitope associated with the pathogenesis of AA: T cell clones that recognize an epitope formed by aa 180-188 of HSP65 react with self-cartilage and can adoptively transfer AA. Nevertheless, vaccination with HSP65 or some of its T cell epitopes can prevent AA by a mechanism that seems to involve cross-reactivity with the self-60-kDa HSP60. We recently demonstrated that DNA vaccination with the human hsp60 gene can inhibit AA. In the present work, we searched for regulatory epitopes using DNA vaccination with HSP60 gene fragments. We now report that specific HSP60 DNA fragments can serve as effective vaccines. Using overlapping HSP60 peptides, we identified a regulatory peptide (Hu3) that was specifically recognized by the T cells of DNA-vaccinated rats. Vaccination with Hu3, or transfer of splenocytes from Hu3-vaccinated rats, inhibited the development of AA. Vaccination with the mycobacterial homologue of Hu3 had no effect. Effective DNA or peptide vaccination was associated with enhanced T cell proliferation to a variety of disease-associated Ags, along with a Th2/3-like shift (down-regulation of IFN-gamma secretion and enhanced secretion of IL-10 and/or tumor growth factor beta1) in response to peptide Mt176-190 (the 180-188 epitope of HSP65). The regulatory response to HSP60 or its Hu3 epitope included both Th1 (IFN-gamma) and Th2/3 (IL-10/tumor growth factor beta1) secretors. These results show that regulatory mechanisms can be activated by immunization with relevant self-HSP60 epitopes.     

Insertion of the dibasic motif in the flanking region of a cryptic self-determinant leads to activation of the epitope-specific T cells

Efficient induction of self tolerance is critical for avoiding autoimmunity. The T cells specific for the well-processed and -presented (dominant) determinants of a native self protein are generally tolerized in the thymus, whereas those potentially directed against the inefficiently processed and presented (cryptic) self epitopes escape tolerance induction. We examined whether the crypticity of certain determinants of mouse lysozyme-M (ML-M) could be attributed to the nonavailability of a proteolytic site, and whether it could be reversed to immunodominance by engraftment of a novel cleavage site in the flanking region of the epitope. Using site-directed mutagenesis, we created the dibasic motif (RR or RK; R = arginine, K = lysine), a target of intracellular proteases, in the region adjoining one of the three cryptic epitopes (46-61, 66-79, or 105-119) of ML-M. Interestingly, the mutated lysozyme proteins, but not unmutated ML-M, were immunogenic in mice. The T cell response to the altered lysozyme was attributable to the efficient processing and presentation of the previously cryptic epitope, and this response was both epitope and MHC haplotype specific. In addition, the anti-self T cell response was associated with the generation of autoantibodies against self lysozyme. However, the results using one of three mutated lysozymes suggested that the naturally processed, dibasic motif-marked epitope may not always correspond precisely to the cryptic determinant within a synthetic peptide. This is the first report describing the circumvention of self tolerance owing to the targeted reversal of crypticity to dominance in vivo of a specific epitope within a native self Ag.     

Suppressive effect of glutamic acid decarboxylase 65-specific autoimmune B lymphocytes on processing of T cell determinants located within the antibody epitope

Type 1 diabetes is a T cell-mediated disease in which B cells serve critical Ag-presenting functions. In >95% of type 1 diabetic patients the B cell response to the glutamic acid decarboxylase 65 (GAD65) autoantigen is exclusively directed at conformational epitopes residing on the surface of the native molecule. We have examined how the epitope specificity of Ag-presenting autoimmune B cell lines, derived from a type 1 diabetic patient, affects the repertoire of peptides presented to DRB1*0401-restricted T cell hybridomas. The general effect of GAD65-specific B cells was to enhance Ag capture and therefore Ag presentation. The enhancing effect was, however, restricted to T cell determinants located outside the B cell epitope region, because processing/presentation of T cell epitopes located within the autoimmune B cell epitope were suppressed in a dominant fashion. A similar effect was observed when soluble Abs formed immune complexes with GAD65 before uptake and processing by splenocytes. Thus, GAD65-specific B cells and the Abs they secrete appear to modulate the autoimmune T cell repertoire by down-regulating T cell epitopes in an immunodominant area while boosting epitopes in distant or cryptic regions.     

Clonotypic heterogeneity of Lewis rat T cells specific for the encephalitogenic 68-86 region of myelin basic protein

Experimental autoimmune encephalomyelitis was induced in a Lewis rat by sensitization with synthetic peptide GP68-86, representing the 68-86 sequence of guinea pig myelin basic protein (GPMBP). To delineate T cell determinants of GP68-86, lymph node cells from this rat were activated in culture with GP68-86 and were fused with cells of the mouse thymoma BW5147. The resultant hybrids were cloned by limiting dilution and screened for GP68-86-evoked secretion of IL2 in the presence of rat splenocytes. Twelve T cell hybrids derived in this manner were tested for reactivity to different heterologous species of MBP as well as to substituted or truncated analogs of GP68-86. The hybrids generally exhibited potent reactivity to GPMBP but differed markedly in their reactivity to autologous rat MBP (RMBP). A few exceptional hybrids exhibited crossreactivity with peptides in which native serine75 or serine80 residues of GPMBP were substituted with either alanine75 (A75) or proline80 (P80) residues. These cross-reactive hybrids also possessed high levels of anti-RMBP reactivity. The remaining hybrids were unresponsive to the A75 and P80 substituted peptides and, with one exception, had relatively low levels of anti-RMBP reactivity. Unique reactivity patterns were also revealed by hybrid responses to peptides having modified C-terminal 84-86 residues. In summary, the contrasting fine specificities of different hybrids indicated that several distinct clones of T cells mediate the immune response of Lewis rats against the 68-86 region of GPMBP. Furthermore, heterogeneity in the hybrid response to "self" RMBP may reflect substantial differences in encephalitogenic potency of the T cell clones from which these hybrids were derived.     

Treatment of adjuvant arthritis with granulocyte-colony stimulating factor and peptide derived from heat shock protein 65

Adjuvant arthritis in Lewis rats is induced by the subcutaneous injection of Mycobacterium tuberculosis in mineral oil, and the predominant T cell immune reactivity is against the heat shock protein 65 derived peptide 176-190. We treated Lewis rats with human recombinant G-CSF followed by (i.v) administration of peptide 176-190 after induction of adjuvant arthritis (AA), and observed decreased disease severity, joint destruction, new bone formation and joint ankylosis. Treatment with G-CSF alone was also effective, but to a lesser extent. In addition, we found that splenocytes from rats treated with G-CSF had reduced antigen presenting capacity compared with splenocytes from vehicle treated rats. Primed lymph node cells from G-CSF plus peptide treated rats showed a marked reduction in proliferation and secretion of IFN-gamma after stimulation with the heat shock protein peptide in vitro as compared to controls.     

Specificity of the T cell immune response to acetylcholine receptor in experimental autoimmune myasthenia gravis. Response to subunits and synthetic peptides

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are T cell-dependent diseases mediated by antibodies against acetylcholine receptor (AChR) on skeletal muscle. Most of the antibodies are directed toward conformation-dependent epitopes on the AChR, whereas T cells recognize denatured AChR. In search of T cell epitopes in EAMG, we tested 24 synthetic peptides covering 62% of the alpha-subunit sequence of Torpedo californica electric organ AChR in the T cell proliferation assay with lymph node cells from rats immunized with AChR. In Lewis rats, 2 of these peptides, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90, strongly stimulated T cells and, of these, [Tyr 100]alpha 100-116 was much more potent; 4 other peptides were weakly mitogenic and 18 were ineffective. None of the 24 synthetic peptides alone stimulated anti-AChR production and, when added to cultures along with AChR, [Tyr 100]alpha 100-116 and [Gly 89, Tyr 90]alpha 73-90 suppressed antibody production. Of twelve cloned T cell lines specific to AChR, 4 responded to [Tyr 100]alpha 100-116, indicating the importance of the epitope in alpha 101-116 in Lewis rats. In three other strains of rats whose responses to AChR and its subunits were similar to those in the Lewis rat, neither [Tyr 100]alpha 100-116 nor [Gly 89, Tyr 90]alpha 73-90 was stimulatory. Instead, completely different sets of peptides stimulated their T cells. When peptides were used as immunogens, each strain (except Lewis rats) responded only to the peptides that stimulated AChR-immune T cells from the same strain. Genetically restricted T cell recognition of AChR peptides in rats suggests that T cells from MG patients with different major histocompatibility haplotypes may recognize different AChR peptides.     

Mouse lysozyme-M knockout mice reveal how the self-determinant hierarchy shapes the T cell repertoire against this circulating self antigen in wild-type mice

We have studied T cell tolerance to defined determinants within ML-M using wild-type (WT; ML-M(+/+)) and LysMcre (ML-M(-/-)) C3H (H-2(k)) mice to determine the relative contribution of ML-M-derived epitopes vs those from other self Ags in selection of the ML-M-specific T cell repertoire. ML-M was totally nonimmunogenic in WT mice, but was rendered immunogenic in LysMcre mice. Most of the response to ML-M in LysMcre mice was directed to the immunodominant determinant region 105-119. This determinant is spontaneously displayed (without adding exogenous ML-M) by macrophages of WT, but not LysMcre, mice and is stimulatory for peptide 105-119 (p105-119)-primed T cells. Moreover, neonatal tolerization of LysMcre mice with p105-119 or ML-M abrogated the T cell response to subsequent challenge with ML-M or p105-119. Furthermore, p95-109 and p110-125 of ML-M were immunogenic in LysMcre mice, but not in WT mice, thereby representing subdominant, tolerance-inducing epitopes of ML-M. As expected, the T cell repertoire to cryptic ML determinants in WT mice was also intact in LysMcre mice. Furthermore, the pattern of response to the related homologue of ML-M, hen eggwhite lysozyme, was similar in these two groups of mice. Thus, several codominant T cell determinants within ML-M contribute significantly to tolerance induction, and the anti-cryptic T cell repertoire to ML-M was positively selected on non-ML-M self ligands. These results reveal that the induction of self tolerance to a multideterminant protein follows the quantitative hierarchy of self-determinant expression and are of relevance in understanding the pathogenesis of autoimmunity.     

T cells that recognize peptide sequences of self MHC class II molecules exist in syngeneic mice.

T cell reactivity toward self MHC class II molecules has been recognized in syngeneic MLR in a number of studies, where the T cells are believed to recognize the combination of self/nonself peptide and self MHC molecule. We investigated the stimulation of T cell proliferation by synthetic peptides of sequences corresponding to the first polymorphic amino terminal domain of alpha- and beta-chains of self I-A molecules. Both unprimed and primed T cells responded to a number of peptides of alpha 1 and beta 1 domains of self I-Ad molecules. The response was dependent on the presentation of I-Ad peptides by syngeneic APC and was blocked by anti-class II MHC mAb. Upon further investigation it was observed that I-Ad peptides could inhibit the stimulation of Ag-specific MHC class II-restricted T cell hybridoma due to self presentation of peptides rather than to direct binding of free peptides to the TCR, further supporting their affinity/interaction with intact self MHC class II molecules. The peptide I-A beta d 62-78 showed high affinity toward intact self MHC II molecule as determined by the inhibition of Ag-specific T cell stimulation and yet was nonstimulatory for syngeneic T cells, therefore representing an MHC determinant that may have induced self tolerance. Thus we have shown that strong T cell proliferative responses can be generated in normal mice against the peptides derived from self MHC class II molecules and these cells are part of the normal T cell repertoire. Therefore complete tolerance toward potentially powerful immunodominant but cryptic determinants of self Ag may not be necessary to prevent autoimmune diseases.     

Autoimmunity in Coxsackievirus B3 induced myocarditis: role of estrogen in suppressing autoimmunity

Picornaviruses are small, non-enveloped, single stranded, positive sense RNA viruses which cause multiple diseases including myocarditis/dilated cardiomyopathy, type 1 diabetes, encephalitis, myositis, orchitis and hepatitis. Although picornaviruses directly kill cells, tissue injury primarily results from autoimmunity to self antigens. Viruses induce autoimmunity by: aborting deletion of self-reactive T cells during T cell ontogeny; reversing anergy of peripheral autoimmune T cells; eliminating T regulatory cells; stimulating self-reactive T cells through antigenic mimicry or cryptic epitopes; and acting as an adjuvant for self molecules released during virus infection. Most autoimmune diseases (SLE, rheumatoid arthritis, Grave's disease) predominate in females, but diseases associated with picornavirus infections predominate in males. T regulatory cells are activated in infected females because of the combined effects of estrogen and innate immunity.     

Arthritis protective regulatory potential of self-heat shock protein cross-reactive T cells

Immunization 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 immunization 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 hsp60 and later for mycobacterial hsp70. 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. Preimmunization 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 hsp600 specific T cells in response to self hsp60. 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.     

Streptococcal cell wall-induced arthritis and adjuvant arthritis in F344----Lewis and in Lewis----F344 bone marrow chimeras

Streptococcal cell wall (SCW)-induced arthritis and adjuvant arthritis (AA) are rat models for chronic, erosive polyarthritis. Both models can be induced in susceptible Lewis rats, whereas F344 rats are resistant. In AA as well as in SCW arthritis, antigen-specific T lymphocytes have been demonstrated to be crucial for chronic disease. In this communication we describe our studies to probe the cellular mechanism responsible for the difference in susceptibility of Lewis and F344, using bone marrow chimeras. By transplanting bone marrow cells from F344 into lethally irradiated Lewis recipients, Lewis rats were rendered resistant to SCW arthritis induction. F344 rats reconstituted with Lewis bone marrow, i.e., Lewis----F344 chimeras, develop an arthritis upon SCW injection. For AA comparable results were obtained. These data suggest that both resistance and susceptibility to bacterium-induced chronic arthritis are mediated by hemopoietic/immune cells and that the recipiental environment does not influence the susceptibility to chronic joint inflammation.     

Nasal administration of arthritis-related T cell epitopes of heat shock protein 60 as a promising way for immunotherapy in chronic arthritis

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 B¹ and an increased capacity to inhibit the proliferative A2b responsein 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 B¹. 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.     

A novel angiogenesis inhibitor suppresses rat adjuvant arthritis

Rat adjuvant arthritis (AA) is an animal model of rheumatoid arthritis in which pannus formation and destruction of joints occur after immunization with complete Freund's adjuvant. Neovascularization is present within the synovium and may be critical for pannus growth. In this study the effects of a novel angiogenesis inhibitor, AGM-1470, on AA were evaluated. Lewis rats were immunized with CFA to induce arthritis. AGM-1470 treatment was initiated prior to arthritis onset (preventative protocol) or administered to rats with established disease (suppressive protocol). The severity of synovitis and the immunologic status of all rats were then evaluated. Using clinical and radiographic criteria, AGM-1470 significantly reduced arthritis incidence (preventative protocol) (P < 0.01) and disease severity (both protocols, P < 0.001, compared to controls) without affecting T cell function in vitro or phenotype in vivo. Additionally, histologic sections from control rats revealed marked pannus formation, destruction of bone/cartilage, and neovascularization. These findings were absent in AGM-1470-treated rats. AGM-1470 may offer a new treatment option for rheumatoid arthritis and other angiogenesis-dependent diseases.