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

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

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

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

Anti-Hsp65 antibodies recognize M proteins of group A streptococci.

Group A streptococcal M protein and the mycobacterial heat shock protein, hsp65, are strong bacterial immunogens that have been linked to arthritis and autoimmunity. Recent evidence has shown that streptococcal arthritis and adjuvant arthritis may be related to epitopes shared between group A streptococci and hsp65. We investigated the possibility that immunological similarities were shared between streptococcal M protein and hsp65. Antibodies against the 65-kDa heat shock protein of Mycobacterium tuberculosis were tested for reactivity with group A streptococci and purified recombinant M proteins (rM5 and rM6). Rabbit polyclonal anti-hsp65 serum was highly reactive with M type 5 Streptococcus pyogenes and rM5 and rM6 proteins in an enzyme-linked immunosorbent assay (ELISA). A mouse anti-hsp65 monoclonal antibody (MAb), IIC8, reacted with streptococcal M types 5, 6, 19, 24, and 49 in an ELISA but showed no reactivity with an isogenic streptococcal mutant which did not express M protein. Anti-hsp65 MAb IIC8 recognized rM5 and rM6 proteins in the ELISA, and MAbs IIC8 and IIH9 reacted strongly with rM6 protein in Western immunoblots. The binding of M protein by anti-hsp65 MAbs was shown to be inhibited by both hsp65 and M protein. These data show that anti-hsp65 antibodies recognize streptococcal M proteins.     

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.     

Antibody Response of Restricted Isotype to Heat Shock Proteins in Juvenile Chronic Arthritis

Synovial fluid and peripheral blood mononuclear cells from juvenile chronic arthritis (JCA) patients have previously been shown to exhibit substantial proliferative responses to both human and mycobacterial heat shock protein (hsp) 65. We investigated the nature of the antibody response to mycobacterial and E. coli hsp 65 and human and E. coli hsp 70 in 56 JCA patients using an ELISA. Elevated levels of antibodies to both human and E. coli hsp 70 were demonstrated. With hsp 65, raised levels of antibodies to the mycobacterial but not the E. coli protein were detected. Overall, 48% of patient serum samples contained antibodies of at least one isotype to mycobacterial hsp 65. These antibodies were predominantly of IgG and IgM isotype, a finding in contrast to adult rheumatoid arthritis, where IgA and IgG isotypes are most often detected.     

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

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

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

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

Lactoferrin Triggers In Vitro Proliferation of T Cells of Lewis Rats Submitted to Mycobacteria-Induced Adjuvant Arthritis

We have recently reported antigenic (B-ccll) cross-reactivity between the mycobacterial 65 kDa heat shock protein (hsp65) and human lactoferrin (LF) and we suggested that this cross-reactivity might have a role in mycobacteria-associated autoimmune disease. Here, we have searched for anti-LFT-cell reactivity in Lewis rats submitted to a mycobacteria-triggered autoaggressive disorder (adjuvant arthritis, A A), an autoimmune disorder characterized by high anti-hsp65 reactivity. We have quantified the in vitro proliferate response to LF of lymph node and spleen cells of Lewis rats killed 9, 14 and 21 days after the immunization with the AA-triggering, mycobacteria-containing adjuvant (complete Freund's adjuvant. CFA). We found that LF induced significant proliferation of lymph node T cells of rats undergoing AA. This T-cell proliferation was not as marked as the one provoked by hsp65; it was, nevertheless, significantly higher (P < 0.05) than that produced by a non-arthritogenic antigen (i.e. albumin). T cells from naive or mineral oil (incomplete Frcund's adjuvant, IFA) injected rats did not respond to LF or hsp65. These data indicate that LF may work as an accessory stimulatory factor of the T-cell autoreactivity associated with mycobacteria-induced arthritis.     

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

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

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

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

A synthetic 10-kD heat shock protein (hsp10) from Mycobacterium tuberculosis modulates adjuvant arthritis

The heat shock protein, hsp10, is an abundant protein in Mycobacterium tuberculosis (Mtb), its nucleotide sequence encoding a protein of 99 amino acids with a molecular mass of 10±7kD. This sequence is phylogenetically conserved, being represented by the GroES homologue of Escherichia coli. Hsp 10 and GroES are members of the chaperonin 10 family of molecular chaperones, and GroES is necessary for the optimal activity of GroEL, a member of the chaperonin 60 family and the E coli homologue of mycobacterial hsp65. Since hsp65 has been implicated in both experimental and human rheumatoid arthritis, we aimed to assess the immunomodulatory effects of its co-chaperonin, hsp10, in experimental arthritis. Our results show that an aqueous solution of a mycobacterial hsp10 delayed the onset and severity of adjuvant-induced arthritis in rodents when administered after disease induction but before joint involvement occurred. This biological activity was specific for the hsp10 of Mtb, since neither GroES nor the rat homologue was effective. Using synthetic hsp10 fragments, the activity was localized to the N-terminal region of the molecule. Assessment of circulating antibody levels to mycobacterial hsp10 and hsp65 indicated that all arthritic rats had increased litres to both hsp10 and hsp65: hsp10-treated rats showed further elevation of this humoral response not only to hsp10 but also to hsp65 when compared with the untreated arthritic control. This is the first report of the immunomodulatory activity of mycobacterial hsp10 in experimental arthritis, and exhibits a potential role for this co-chaperonin in pathophysiological situations.     

Diversification of response to hsp65 during the course of autoimmune arthritis is regulatory rather than pathogenic

Summary: Determinant spreading has been implicated in the pathogenesis of certain autoimmune diseases in animal models. We have observed that during the course of adjuvant arthritis (AA) in the Lewis rat, there is 'diversification' of response to the bacterial 65-kDa heat shock protein (Bhsp65) towards its carboxy-terminal determinants (BCTD). Strikingly, pretreatment of naive Lewis rats with BCTD affords significant protection from AA. Our preliminary studies indicate that the diversification of response to BCTD in the Lewis rat is probably triggered in vivo by the induction and enhanced processing of self(rat) hsp65. Thus, the self hsp65-directed T-cell responses appear to be involved in mediating natural remission from acute inflammatory arthritis induced by a foreign antigen, Myco-bacterium tuberculosis. This the first report describing that the new T-cell specificities arising during the course of an autoimmune disease are regulatory/protective rather than pathogenic. Moreover, our results suggest that a final common mechanism involving BCTD might be recruited by other rac strains which either are resistant to AA (WKY rats) or whose susceptibility to AA is modulated significantly by microbial flora (Fisher rats). The results of this study would contribute significantly to understanding of the pathogenesis of human rheumatoid arthritis, and in devising new therapeutic strategies for this disease.     

Inflammation activates self hsp60-specific T cells

Injection of incomplete Freund's adjuvant (IFA) into the footpads of BALB/c mice induced an acute inflammation. Draining popliteal lymph nodes showed major histocompatibility complex (MHC) class II-restricted proliferation when challenged in vitro with recombinant Mycobacterium bovis 65-kDa heat shock protein (hsp65). αβ Tcell receptor-positive, CD4⁺, hsp65-specific T cell lines and clones were generated from these lymph nodes, and 87% of clones responded to a P galactosidase fusion protein containing residues 238–573 of human hsp60. Seventy percent of these hsp60-responsive clones also responded to a synthetic peptide corresponding to residues 412–423 of the mouse hsp60. This peptide also induced significant responses in IFA-primed lymph node cells but not in lymphoid cells from unimmunized mice. These results demonstrate that T cells specific for epitopes in self hsp60 are activated during inflammatory responses induced in the absence of exogenous bacterial hsp65. The findings of this study may provide a basis for understanding the often reported isolation of mycobacterial hsp65-responsive T cells from inflammatory sites of arthritis patients, and the protective effects of preimmunization with hsp65 in experimental models of arthritis.     

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

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

The immune response to mycobacterial heat shock proteins

Several prominent protein antigens involved in antibody and T cell responses to mycobacteria have been identified as members of highly conserved heat shock protein families. The immunogenicity of heat shock proteins in a broad range of infections, along with the extensive sequence identity between corresponding host and pathogen homologues, and the potential for localized accumulation in response to stress stimuli, makes these antigens attractive candidates for theories of autoimmunity based on "molecular mimicry". The 65 kilodalton (kD) heat shock protein of mycobacteria has indeed been shown to modulate the course of autoimmune disease in experimental animals, and immune responses to this and other heat shock proteins have been demonstrated in association with human autoimmune diseases. This paper provides a brief review of the immune response to mycobacterial heat shock proteins during infection and autoimmunity.     

The mode of action of treatment by IgG of haemolytic anaemia induced by an anti-erythrocyte monoclonal antibody

Tolerization of pathogenic antigens is one of the experimental strategies that has been proposed to prevent autoimmune disease. We have investigated here whether neonatal intraperitoneal infection of Lewis rats with Mycobacterium bovis-BCG has any effect on the expression of adjuvant arthritis (AA), an autoimmune disease that is produced by immunization of the rats with dead mycobacteria in mineral oil (i.e. Freund's complete adjuvant (FCA)). We found that neonatal infection with 10⁸ viable BCG bacilli rendered all Lewis rats resistant to the expression of AA after FCA immunization. This BCG-induced protection from reactive arthritis was not seen in Lewis rats infected with smaller inocula (10⁶ BCG bacilli) or if the infection was performed after the neonatal period (e.g. at 3 weeks of age). Neonatal administration of 65-kD mycobacterial heat shock protein (hsp65, a key antigen in the etiopathogenesis of AA) failed to protect Lewis rats from AA; injection of lactoferrin (an autoantigen that may be involved in the physiopathology of autoimmune arthritis) to newborn Lewis rats decreased the severity of AA observed after FCA immunization of the animals. Western blotting revealed that Lewis rats that had acquired resistance to AA also showed changes in their repertoire of antibody specificities; among these alterations was decreased anti-hsp65 reactivity. We conclude that neonatal infection with BCG, but not hsp65 injection, renders Lewis rats resistant to AA and that the phenomenon is associated with change in the repertoire of specificities of circulating antibodies.     

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

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

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.     

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.