Presence of Human 65 kD Heat Shock Protein (hsp) in Inflamed Joints and Subcutaneous Nodules of RA Patients

Monoclonal antibodies to the human homologue of the bacterial 65 kD heat shock protein (hsp) were used to investigate the tissue distribution of endogenous hsp 65 in normal versus rheumatoid synovial tissue, in subcutaneous nodules of patients with rheumatoid arthritis (RA) and in several instances of non-rheumatoid inflammation. A strong reactivity of the anti-hsp antibody was found in the cartilage-pannus junction in rheumatoid joints and in rheumatoid nodules, but not in normal joints or in normal or inflamed kidney or liver (irreversible graft rejection, chronic glomerulonephritis or primary biliary cirrhosis). The findings provide a new hypothetical explanation for a role of T cells reactive with the 65 kD hsp in the generation of both articular and extra-articular lesions in chronic rheumatoid arthritis.     

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.     

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.     

A Monoclonal Antibody to the Mycobacterial 65kDa Heat Shock Protein (ML 30) Binds to Cells in Normal and Arthritic Joints of Rats

A monoclonal antibody reactive with the mycobacterial 65 kDa heat shock protein (ML 30) was investigated for reactivity with biopsies from normal rat joints and with inflamed joints due to adjuvant arthritis (AA) or collagen induced arthritis (CIA). Immunohistochemical stainings with the anti-hsp 65 antibody on paraffin sections from normal rat joints revealed a weak but exclusive staining of cells within the synovial lining. Also normal chondrocytes and bone marrow cells showed occasional staining. In biopsies from inflamed joints obtained from rats suffering from AA or CIA, an intense staining with ML 30 was seen within the cartilage-pannus junction as well as sites of bone erosion. An increased staining, compared with the normal, was also seen in chondrocytes of the eroded cartilage and in some bone marrow cells. No staining with ML 30 was seen in biopsies from inflammatory lesions due to delayed type hypersensitivity reactions in the skin of rats. Reactivity of ML 30 was also seen in a Western blot assay performed on lysates from inflamed synovia from rats with CIA, preferentially with a component slightly below 60 kDa in molecular weight. The demonstration of epitopes cross-reactive with hsp 65 of mycobacteria in normal and, in higher quantity, in arthritic rat joints, suggests, together with our preliminary biochemical findings, that a recently identified mammalian counterpart to bacterial hsp 65 is both preferentially expressed in normal joints and subject to increased expression in arthritis of different aetiologies.     

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.     

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.     

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.     

Natural antibodies to 65 kD mycobacterial heat shock protein in rats do not correlate with susceptibility for Mycobacterium tuberculosis induced adjuvant arthritis

Natural antibodies to 65 kD heat shock protein (hsp65) of Mycobacterium bovis were found in the sera of Lewis rats. The levels of these natural hsp65 antibodies differed substantially between the individual rats. Each rat was subsequently tested for its susceptibility to develop arthritis following injection of M. tuberculosis in incomplete Freund adjuvant. It was found that the incidence and severity of the induced arthritis did not differ between groups of Lewis rats with relatively high and relatively low natural antibody levels to hsp65. Inoculation of rats without natural antibodies to hsp65 with intestinal contents did not induce hsp65 antibodies, although the rats were able to respond to the antigen.     

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

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

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by hsp 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60-stimulated cells produce T-helper 2 type cytokines.     

The Dynamics of Articular Leukocyte Trafficking and the Immune Response to Self Heat-Shock Protein 65 Influence Arthritis Susceptibility

INTRODUCTION: Adjuvant arthritis (AA) shares several features with human rheumatoid arthritis, and it can be induced in the Lewis (LEW) rat but not the Wistar Kyoto (WKY) rat (both RT.1(l)) by immunization with heat-killed Mycobacterium tuberculosis (Mtb). We set out to unravel the mechanisms underlying the differential susceptibility to AA of these MHC-compatible rat strains. MATERIALS AND METHODS: We compared the levels of T-cell proliferative and cytokine response to the immunoregulatory self (rat) hsp65 (Rhsp65) after an arthritogenic (Mtb) challenge and the kinetics of migration of adoptively transferred, (111)Indium-labeled, Mtb-primed leukocytes into the hind paw joints of recipient rats. RESULTS AND DISCUSSION: The WKY rats raised a significantly higher level of T-cell proliferative response coupled with a temporally opposite cytokine profile against the disease-regulating Rhsp65 compared to that of LEW rats. Moreover, the arthritogenic leukocytes accumulated into the joints of WKY rats at significantly lower numbers than that in LEW rats. CONCLUSIONS: These results offer novel insights into the immune events influencing the pathogenesis of autoimmune arthritis.     

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.     

Limiting dilution analysis of proliferative T cell responses to mycobacterial 65-kDa heat-shock protein fails to show significant frequency differences between synovial fluid and peripheral blood of patients with rheumatoid arthritis.

Recent evidence has pointed to the mycobacterial 65-kDa heat-shock protein (hsp 65) as an antigen that may be important in the pathogenesis of rheumatoid arthritis (RA). Using limiting dilution analysis the frequency of purified protein derivative of tuberculin (PPD) and hsp 65-responsive T cells was measured in paired peripheral blood and synovial fluid samples of patients with RA. There was no increase in the anti-PPD or anti-hsp 65 frequency in synovial fluid compared with peripheral blood. In addition, no difference was found between peripheral blood of RA patients and healthy controls. These results do not support the idea of an important pathogenic role of T cells responding to hsp 65, or a cross-reacting antigen, in RA.     

Detection and partial characterization of human B cell colony stimulating activity in synovial fluids of patients with rheumatoid arthritis.

The joint fluids of 37 patients with rheumatoid arthritis, eight patients with traumatic injuries to their joints, two patients with Reiter's syndrome and three patients with psoriatic arthritis were tested for the presence of B cell colony stimulating activity (B cell CSA). B cell CSA was found in all of the joint fluids from the patients with rheumatoid arthritis but in none of the joint fluids from patients with traumatic injuries to their joints or in the joint fluids from the patients with Reiter's syndrome. A trace of B cell CSA was found in the joint fluid of one of the three patients with psoriatic arthritis. There was a positive correlation (r = 0.796) between the amount of rheumatoid factor present in the joint fluids and the titre of B cell CSA. This correlation was highly significant (P less than 0.001). The B cell CSA was localized to component(s) with molecular weight ranges 115-129 kD and 64-72 kD and an isoelectric point of 6.8. Its activity was sensitive to reduction with 2-mercaptoethanol and to the oxidising action of potassium periodate.     

Rheumatoid arthritis with diffuse pulmonary rheumatoid nodules

Rheumatoid nodules in dermal or subcutaneous tissues, while indicative of rheumatoid arthritis, are very rare. It is even less common to identify these rheumatoid nodules by biopsy as well as in autopsy materials from lung tissue. These nodules may be single or multiple, which seldom cause respiratory symptoms. Here, a patient with diffuse pulmonary rheumatoid nodules and interstitial fibrosis throughout both lungs, is described. The patient, with articular symptoms and seropositivity, exhibited a rapid clinical course and died of respiratory failure 3 months after the appearance of dyspnea. Chest radiography indicated interstitial pneumonitis with bilateral diffuse peripheral shadows. At autopsy, numerous rheumatoid nodules and interstitial fibrosis had destroyed both lungs, such that no residual normal pulmonary tissue remained. It is believed that this was an extremely rare case exhibiting large numbers of rheumatoid nodules throughout the lungs. Findings with this patient indicate that, in patients with rheumatoid arthritis, clinical interstitial pneumonitis confirmed radiologically does not exclude the existence of rheumatoid lung nodules.     

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.     

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.     

Preferential Binding withEscherichia colihsp60 of Antibodies Prevalent in Sera from Patients with Rheumatoid Arthritis

One hundred thirty-two patients with various connective tissue disorders, including 60 with rheumatoid arthritis (RA), had antibodies against human as well asEscherichia colihsp60 in titers significantly higher than those of normal controls. There was a correlation between titers of antibody to human hsp60 and those toE. colihsp60. Levels of antibodies against human andE. colihsp60 were lower in joint fluids than in sera, indicating little production of antibodies in the joint. Antibodies affinity-purified withE. colihsp60 bound strongly with the homologous hsp60, but weakly with human hsp60. However, antibodies affinity-purified with human hsp60 bound comparably with bothE. colihsp60 and human hsp60. Antibodies affinity-purified withMycobacterium tuberculosishsp65 bound to human hsp60 with a reactivity similar to the reactivity of those affinity-purified with human hsp60. The reactivity to the three hsp60 species was lost when sera were absorbed withE. colihsp60, while the reactivity toE. colihsp60 remained after extensive absorption withM. tuberculosishsp65 or human hsp60. These results indicate that anti-hsp60 antibodies in patients with RA and other connective tissue disorders are raised by infection with intestinal microorganisms such asE. coli.They may represent another example of autoimmune responses triggered by antigenic mimicry of host proteins to microbes and suggest that the reactivity of antibodies from RA patients withM. tuberculosishsp65 might have been a cross-reaction with theE. colihomologue.     

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.     

A case of Weber-Christian disease with later development of rheumatoid arthritis

Weber-Christian disease (WCD) is a syndrome characterized by recurrent subcutaneous nodules, fever, occasional lipoatrophy, fatigue, arthralgia, and myalgia. We report a case of WCD associated with rheumatoid arthritis. A 65-year-old woman consulted our outpatient clinic because of bilateral hand swelling. The patient had presented with fever and subcutaneous nodules in her trunk and upper and lower extremities in 1983. At that time, the dermatology department diagnosed this patient as having WCD after biopsy of the nodules demonstrated lobular panniculitis. She has been treated with corticosteroid (5-15 mg/day) since then. The patient continued to have recurrent episodes of transient inflammatory arthritis in the small joints of the fingers and fever, and was initially assessed at our institution in October 2007. Finally, in November 2007, she was diagnosed as having both WCD and rheumatoid arthritis (RA) and treated with corticosteroid (5 mg/day) and methotrexate (MTX) (7.5 mg/week). Thereafter, her clinical symptoms gradually improved. This is the second case of WCD showing the subsequent development of RA, successfully treated with MTX, in the English literature. This case may provide clinical insight into WCD and RA.