T cell reactivity to an epitope of the mycobacterial 65-kDa heat-shock protein (hsp 65) corresponds with arthritis susceptibility in rats and is regulated by hsp 65-specific cellular responses.

Adjuvant arthritis (AA) can be induced in genetically susceptible rats by immunization with heat-killed mycobacteria suspended in mineral oil. From our analysis of arthritogenic T cell clone A2b, obtained from an arthritic Lewis rat and specific for the 180-188 epitope of mycobacterial 65-kDa heat-shock protein (hsp 65), the possible origin of AA was explained by the existence of a molecular mimicry of the 180-188 epitope with a cartilage-associated self antigen. We now have shown that Lewis rats respond to the 180-188 epitope after Mycobacterium tuberculosis immunization and that arthritis-resistant Fisher and (Lewis x Fisher)F1 rats, although major histocompatibility complex class II identical with Lewis, do not respond to this epitope. However, in rare cases of arthritis in Fisher rats, responses to the epitope were seen. We obtained no evidence for a defect at the level of antigen processing and presentation or for suppression in Fisher rats. Thus, non-responsiveness in Fisher rats was likely due to a difference at the level of the T cell repertoire. Previously, we have reported that pretreatment with hsp 65 in experimental arthritis, and not only in AA, caused resistance to arthritis induction. We now present evidence that immunization with hsp 65 or in vitro stimulation with hsp 65 may lead to inhibition of responses specific for epitope 180-188. Thus the hsp 65-induced resistance to arthritis is probably caused by the induction of regulatory control specifically targeted at the 180-188 epitope. Especially in rats that tend to focus their responses on the critical 180-188 sequence, such as Lewis, regulation seems to develop following immunization with hsp 65. Since recent evidence suggests that hsp 65 and also the 180-188 epitope have a role in human arthritic conditions, the present findings are expected to contribute to further experimentation directed at exploiting hsp 65 or its epitopes for the development of new therapeutical approaches in humans.     

Searching for the Cartilage-associated Mimicry Epitope in Adjuvant Arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis ( Mt ) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein ( Mt. hsp65 178-186 ). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65 178-186 specific T cells.     

Searching for the cartilage-associated mimicry epitope in adjuvant arthritis

Adjuvant arthritis (AA) is a T cell mediated disease which can be induced in genetically susceptible rats by immunization with heat-killed Mycobacterium tuberculosis (Mt) suspended in incomplete Freund's adjuvant. The critical mycobacterial T cell epitope for the induction of AA was previously identified as residues 178-186 of the mycobacterial 65 kDa heat shock protein (Mt. hsp65(178-186)). It was suggested that the development of AA was due to molecular mimicry between a mycobacterial epitope and a cartilage-associated self-antigen. However, until now such cartilage-associated mimicry epitope has not been identified. In this study we designed a computer search profile to predict mimicry self-epitopes, and investigated whether one or more of these self-epitopes could serve as mimicry epitopes in AA. Although several of these self-epitopes were recognized by arthritogenic T cells, no cross-reactivity was found between T cells specific for these self-epitopes and Mt. hsp65(178-186) specific T cells.     

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.     

Generation and characterization of a clonotypic antibody specific for the T cell receptor of an arthritogenic T cell clone--studies in adjuvant arthritis

Adjuvant Arthritis (AA) can be induced by passive transfer of a T cell clone (A2b) derived from arthritic rats, specific for Heat Shock Protein 60, HSP60 176-190. Furthermore, a crucial role for T cells with HSP60 176-190 specificity in AA was shown by induction of tolerance using HSP60 176-190 or by immunization with an altered peptide ligand based on the same sequence. To study clonal expansion of A2b-like T cells during AA and to determine their role in AA induction, we generated a clonotypic antibody, 16C4, specific for the TCR of the A2b T cell clone (TCR AV11S1/BV18). This antibody stained A2b T cells in flow cytometry experiments, induced proliferation of A2b cells when fixed on a solid support, and inhibited antigen-induced A2b proliferation when added in solution. A2b-like T cells were detected in a low frequency in lymphoid organs of arthritic rats. Thus, as in vivo administration of 16C4 did not inhibit AA, cells containing the determinant recognized by 16C4 are possibly not the sole contributors to AA development. Furthermore, epitope specific interventions by antigen administration may be possible even in cases where the epitope specific T cell clonotype is of low frequency.     

Suppression of adjuvant arthritis in rats by induction of oral tolerance to mycobacterial 65-kDa heat shock protein

Oral administration of mycobacterial 65-kDa heat shock protein (HSP) given daily for 5 days prior to immunization with Mycobacterium tuberculosis (Mt) suppressed the development of adjuvant arthritis (AA) in rats. AA was significantly suppressed by 30 and 300 μg HSP, and variably by 0.3, 3 μg or 1 mg. Histological analysis of joint samples obtained from control and test rats confirmed the suppression of AA in the fed group. Feeding Mt or hen egg lysozyme (HEL) failed to affect AA, indicating that the suppression was HSP specific. The oral administration of 30 μg HSP decreased both delayed-type hypersensitivity (DTH) reactions and proliferative responses to HSP and Mt. In addition, the proliferation of lymph node cells (LNC) from Mt-sensitized rats was inhibited by the addition of spleen cells (SPC) from HSP-fed animals, possibly by the secretion of transforming growth factor (TGF)-β. Spleen cells obtained from tolerized donors were capable of transferring the tolerance to naive recipients. These results demonstrate that feeding HSP is an effective way to suppress AA and that the suppression of AA may be mediated by regulatory T cells generated following oral administration of mycobacterial 65-kDa HSP.     

Efficient recognition by rat T cell clones of an epitope of mycobacterial hsp 65 inserted in Escherichia coli outer membrane protein PhoE.

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.     

Juvenile rheumatoid arthritis patients manifest immune reactivity to the mycobacterial 65-kDa heat shock protein, to its 180-188 peptide, and to a partially homologous peptide of the proteoglycan link protein.

Immune reactivity to the 65-kDa mycobacterial heat shock protein (hsp65) has been associated with arthritis in rats and humans. In this report we evaluated patients with juvenile rheumatoid arthritis for such immunity. A high proportion of affected children showed both antibody and T lymphocyte responses to hsp65 and to two related peptides: the nonapeptide 180-188 sequence of hsp65 and a partially homologous peptide of the cartilage proteoglycan link protein. The titer of circulating antibodies was generally higher in patients with clinically active disease. In contrast to the juvenile rheumatoid arthritis patients, patients with adult rheumatoid arthritis tended to have lower responses of their peripheral blood T lymphocytes to the whole hsp65 molecule. Moreover, the adult rheumatoid arthritis patients did not respond to the peptides. Thus, there appear to be immunological differences between juvenile and adult forms of rheumatoid arthritis related to hsp65 reactivity.     

Modulation of experimental autoimmunity: treatment of adjuvant arthritis by immunization with a recombinant vaccinia virus.

Live recombinant vaccinia viruses, expressing antigens from pathogenic microorganisms, are studied for their use as vaccines designed for the protection against infectious diseases. Infections with these vaccinia virus recombinants, expressing proteins or epitopes from viruses, parasites, or bacteria, have resulted in the development of specific neutralizing antibodies or cytotoxic T lymphocytes. Here, we describe the generation of a recombinant vaccinia virus expressing the mycobacterial 65-kDa heat shock protein (HSP65). A vaccinia recombinant virus was constructed by placing the gene for the Mycobacterium bovis BCG HSP65 under control of a vaccinia virus promoter and inserting this mycobacterial gene in the thymidine kinase locus of the vaccinia virus genome. Mycobacterial HSP65 is a critical antigen in the autoimmune model of adjuvant arthritis induced in Lewis rats by the immunization with Mycobacterium tuberculosis. We report the induction of immunity directed to this mycobacterial HSP65 by testing for the presence of specific antibodies and T-cell proliferation. Furthermore, induction of such immunity resulted in a reduction of arthritis severity when given to rats before or, even more interestingly, during development of arthritis. Disease reduction was not found after administration of HSP65 in the absence of vaccinia virus as a vector when given during arthritis development. Therefore, recombinant vaccinia virus may offer new prospectives for specific intervention in autoimmunity.     

Heat-shock protein T-cell epitopes trigger a spreading regulatory control in a diversified arthritogenic T-cell response

Summary: Adjuvant arthritis (AA) in Lewis rats is T-cell mediated and seems to depend on T cells recognising the 180–188 epitope of mycobacterial heat-shock protein (hsp) 60. Analysis of arthritogenic T-cell clone A2b has revealed a mimicry of this particular epitope with an articular cartilage-associated target T-cell epitope. Nasal administration of synthetic peptides covering this 180–188 sequence led to epitope-.specific tolerance and resistance to AA. Since this tolerisation protocol also inhibited avridine arthritis, one may conclude that this form of epitope-specific tolerance had effectuated a spreading tolerisation at the level of target antigens that included a diverse set of possible arthritis -associated antigens. In vitro anergised T cells exhibited suppressive activity in a co-culture system. As in this case - depending on the presence of the antigen of the anergic T cell – such T cells suppressed responder T cells of a different antigenic specificity, we postulated that anergic T cells may be responsible for a spreading of tolerance. It seemed that such spreading of tolerance was channelled through the antigen-presenting cells (APC) and was dependent on direct cell-cell contact. This and additional forms of spreading of tolerance could be responsible for specific nasal tolerance, causing inhibition of the development of an arthritogenic inflammatory response. This can be similarly che case for the arthritis protection that resulted from immunisation with hsps. Analysis of T-cell responses following hsp immunisations revealed that the arthritis inhibitory activity resided in T cells with specificity for a conserved part of microbial hsp60. The same T cells cross-responded to rat self-hsp60. Low level expression of the latter molecule on non-professional APC could possibly have induced a suppressive anergic state in these autoreactive cells. Thus, immunisation with microbial hsp would have led to an expansion of such T cells, leading to raised disease-suppressive potential when selectively trapped and activated in the inflamed self-hsp-overexpressing joint. Alternatively the cross-recognised self-hsp epitope could have the regulatory qualities of an altered peptide ligand or a partial agonist for T cells that see the microbial homologue as the full agonist.     

Differential rat T cell recognition of cathepsin D-released fragments of mycobacterial 65 kDa heat-shock protein after immunization with either the recombinant protein or whole mycobacteria

T cells specific for the mycobacterial 65 kDa heat-shock protein(hsp65) play a pivotal role in the development of adjuvant arthritis(AA) in Lewis rats. Upon adoptive transfer, CD4⁺ T cells recognizinga particular hsp65 epitope trigger the onset of disease. Activationof hsp65-reactlve T cells can be achieved by immunization withheat-killed mycobacteria in mineral oil—complete Freund'sadjuvant (CFA)—or with purified recombinant hsp65. Arthritis,however, will only develop after immunization with CFA. In fact,prelmmunlzatlon with hsp65 protects against any subsequent attemptto induce AA. In this study, we examined polyclonal lymph nodecell responses in Lewis rats, Immunized with either CFA or purifiedrecombinant hsp65 in incomplete Freund's adjuvant, to a setof hsp65 fragments generated by a mild digestion with cathepsinD. Prollferatlve responses to several hsp65 fragments variedwith the type of antigen used for immunization. A cathepsinD-released fragment, Identified as residues 376–408, preferentiallytriggered proliferation of rat T cells after hsp65 Immunization.Prelmmunlzatlon of Lewis rats with this peptlde delayed theonset and reduced the severity of AA. Prelmmunlzatlon with anotherfragment which was preferentially recognized after CFA immunization,representing residues 40–60, did not have such a protectiveeffect. Our findings suggest the presence of mycobacterial hsp65determinants that selectively trigger AA-regulatlng T cellsand illustrate that cathepsin D may be used as an experimentaltool to generate such determinants.     

Modulation of adjuvant arthritis in Lewis rats by recombinant vaccinia virus expressing the human 60-kilodalton heat shock protein.

The immune response to the mycobacterial 65-kDa heat shock protein (hsp65) is considered an important event in the induction of adjuvant arthritis (AA) in rats; this induction probably occurs through a molecular mimicry mechanism involving cross-reactivity against the rat homolog hsp60. To analyze the role of mammalian molecule hsp60 in arthritis, we generated a recombinant vaccinia virus (hsp60-VV) carrying the human hsp60 gene inserted into the thymidine kinase locus under the control of the 7.5k vaccinia virus promoter. Human hsp60 is almost identical to its rat homolog (97.4% linear amino acid homology) and shares about 50% of amino acid positions with Mycobacterium tuberculosis hsp65. The latter supposedly carries a critical epitope for AA induction that is not present in human hsp60. Infections with hsp60-VV of monkey cell cultures led to the expression of the human hsp60 molecule, as evidenced by immunoblotting analysis with specific monoclonal antibodies. Also, Lewis rats infected with hsp60-VV produced specific antibodies, demonstrating the in vivo expression of human hsp60 in the infected animals. Therefore, we used hsp60-VV to analyze whether the delivery of hsp60 could affect the induction of AA in Lewis rats. hsp60-VV clearly reduced and retarded arthritic symptoms when administered to rats at day 7 after AA induction. In contrast, inoculation of rats with a control recombinant vaccinia virus did not affect the course of the disease. The improvement in AA with hsp60-VV administration was associated with a specific immune response, as determined by the presence of antibodies to hsp60 in the sera and the proliferation induced by hsp60 of T cells from popliteal lymph nodes. These results support a critical role for immunity to heat shock proteins in AA. Since the protective construct is virtually identical to rat homolog hsp60, we conclude that immunity directed to conserved areas of this family of proteins is directly involved in the pathogenesis of AA.     

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

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

Ubiquitin-fusion degradation pathway plays an indispensable role in naked DNA vaccination with a chimeric gene encoding a syngeneic cytotoxic T lymphocyte epitope of melanocyte and green fluorescent protein

Antitumour immunity against murine melanoma B16 was achieved by genetic immunization with a naked chimeric DNA encoding a fusion protein linking green fluorescent protein (GFP) to the N-terminus of a major CD8(+) cytotoxic T lymphocyte (CTL) epitope of tyrosinase-related protein 2 (TRP-2(181-188)) of murine melanoma, designated as pGFP-TRP-2. Tumour growth was profoundly suppressed in C57BL/6 mice immunized with pGFP-TRP-2, while mice vaccinated with pTRP-2 showed rapid tumour growth and died within 40 days after tumour challenge. Splenocytes of mice immunized with pGFP-TRP-2 showed high CTL activity specific for TRP-2(181-188). GFP-TRP-2 expressed in COS-7 cells was rapidly degradated in vitro and the degradation was almost completely prevented by adding a proteasome inhibitor, MG-132, in the culture. Furthermore, the antimelanoma immunity induced by genetic immunization with pGFP-TRP-2 was completely cancelled in mice deficient in proteasome activator PA28alpha/beta. Taken together, GFP-TRP-2 processed by cytosolic proteasome played a central role in breaking peripheral tolerance to a melanoma/melanocyte antigen, TRP-2(181-188), by activating CD8(+) CTL specific for TRP-2(181-188). TRP-2(181-188) fused to GFP may be readily cut off from GFP by the ubiquitin-fusion degradation (UFD) pathway and efficiently presented to major histocompatibility complex class I molecules, resulting in effective induction of CD8(+) T cells specific for the CTL epitope. Furthermore, CD4(+) T cells specific for GFP were shown to play a crucial role in the antimelanoma immunity, probably potentiating activity of TRP-2-specific CTL and/or the "ubiquitin-proteasome pathway". It is noteworthy to document that genetic immunization with pGFP plus pTRP-2(181-188) failed to exert the antitumour immunity.     

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.     

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

In a previous study we have shown that Lewis rats were completely protected from adjuvant arthritis by pretreatment with a nonapeptide (residues 180-188) of the 65-kD mycobacterial heat shock protein. Here we address questions of specificity and mechanism(s) of protection. We demonstrate that complete protection against adjuvant arthritis can only be achieved by pre-immunization with the nonapeptide, while pretreatment with either the octapeptide (residues 181-188) of the 65-kD heat shock protein or unrelated immunogenic peptides failed to affect adjuvant arthritis. Interestingly, pretreatment with the nonapeptide of the 65-kD heat shock protein did not protect Lewis rats from type II collagen-induced arthritis. These results demonstrate that protection is both epitope and disease specific. Co-injection of the nonapeptide with mycobacterial antigen even at a weight ratio of 5:1 (nonapeptide:mycobacteria) failed to influence the disease, suggesting that the role of the nonapeptide is not as a 'blocking peptide'. T cells from rats immunized with nonapeptide respond to the nonapeptide as well as to mycobacteria in vitro, and adoptively transfer protection to naive recipients. The data indicate that the nonapeptide-induced protection may result from a T cell-mediated specific suppression.     

Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human arthritis

Heat shock proteins (hsp) are highly conserved, immune-dominant microbial proteins, whose expression is increased at sites of inflammation. In the experimental model of adjuvant arthritis (AA) immune responses to hsp determine the outcome of disease. AA can be transferred with a single T cell clone specific for a sequence of mycobacterial hsp65 (Mhsp65). Immunization with whole Mhsp65 on the other hand, protects in virtually all forms of experimental arthritis, including AA. This protective effect seems the consequence of the induction of a T cell response directed against self-hsp60. A similar protective effect of self-hsp60-specific T cells seems present in patients with a spontaneous remitting form of juvenile idiopathic arthritis. Next to hsp60, other hsp have similar protective effects in arthritis, while other conserved microbial proteins lack such capacity. Nasal administration of hsp60 peptides induces IL-10-driven regulatory T cells that are highly effective in suppressing arthritis. Thus hsp60, or peptides derived from hsp60, are suitable candidates for immune therapy in chronic arthritis.     

Suppression of hen egg lysozyme-induced arthritis by intravenous antigen administration: no role in this for antigen-driven bystander suppression.

The induction of tolerance, particularly by intervention before established immunity, is widely accepted. We studied the effects of intravenous (i.v.) administration of hen egg lysozyme (HEL), before as well as after immunization, on a HEL-induced arthritis. Arthritis and also cartilage destruction were almost completely suppressed when 100 micrograms HEL was injected before immunization. Antigen-specific proliferative T cell responses and IL-2 production in vitro were inhibited. Antigen-specific immunoglobulin and IgG1 titres were equal in control and tolerized mice, in contrast to lowered IgG2a titres in tolerized animals. Detailed histological studies showed that the immune complex-dependent polymorphonuclear cell phase (< 24 h after arthritis induction) was equal for control and HEL-injected mice. Only in the T cell-dependent phase of the arthritis (> 24 h), did suppression become pronounced in tolerized mice. I.v. administration of 100 micrograms HEL after immunization could only marginally reduce infiltrate and exudate, and no reduction of cartilage destruction was seen. An elegant way to interfere in an established immunity can be offered by creation of bystander suppression. We show that i.v. administration of HEL followed by triggering with HEL, at the moment either of immunization or of arthritis induction, does not reduce a methylated bovine serum albumin (BSA)-arthritis. We conclude that arthritis can be suppressed almost totally when HEL is injected intravenously before immunization. Treatment after immunization is less effective. The i.v. induced suppression is T cell-mediated and and antigen-specific: no bystander suppression circuit can be generated.     

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.     

Reversal of tolerance induced by transplantation of skin expressing the immunodominant T cell epitope of rat type II collagen entitles development of collagen-induced arthritis but not graft rejection

Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a na?ve immune system would lead to T cell priming and graft rejection or instead to tolerance and arthritis protection. Interestingly, TSC grafts were accepted and not even immunization of recipient mice with CII in adjuvant induced graft rejection. Instead, TSC skin recipients displayed a reduced T and B cell response to CII and were also protected from arthritis. However, additional priming could break arthritis protection and was accompanied by an increased T cell response to the grafted epitope. Strikingly, despite the regained T cell response, development of arthritis was not accompanied by graft rejection, showing that these immune-mediated inflammatory responses involve different mechanisms.