An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen.

Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259-273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.

Autocrine regulation of collagenase 3 (matrix metalloproteinase 13) during osteoarthritis.

OBJECTIVE: To correlate the increased collagenase production previously seen in chondrocytes isolated from osteoarthritic (OA) lesions and the expression of cytokines and cytokine receptors. METHODS: Chondrocytes were isolated from OA cartilage and characterized for synthesis of collagenases, cytokines, and cytokine receptors by Northern and Western blot analyses, RNA protection assay, and flow cytometry. RESULTS: Chondrocytes located in cartilage proximal to the macroscopic OA lesions bound more tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) compared with chondrocytes isolated from morphologically normal cartilage from the same joint. In response to TNFalpha stimulation, messenger RNA (mRNA) levels for the IL-1 receptor I (IL-1RI), IL-1RII, TNF receptor II (TNFR II), and IL-6 receptor as well as the level of proinflammatory cytokines, such as IL-1alpha, IL-1beta, lymphotoxin beta, TNFalpha, and IL-6, also increased. In contrast, treatment with transforming growth factor beta1 (TGFbeta1) resulted in down-regulation of matrix metalloproteinase 1 (MMP-1) and MMP-13 concomitant with a reduction in the levels of mRNA for IL-1RI, IL-1RII, TNFRI, and TNFRII and proinflammatory cytokine levels. In contrast, the levels of mRNA for TGFbeta receptor I, TGFbeta1, and TGFbeta3 were up-regulated. CONCLUSION: These data show that TGFbeta1 has antagonistic effects upon OA chondrocytes, in contrast to the effects seen with TNFalpha. The cyclical course of OA, where a period of active disease is followed by a period of remission, can be explained by a sequential pattern of cytokine stimulation followed by a feedback inhibition of autocrine cytokine production and cytokine receptor expression, thus affecting collagenase synthesis.

Regulation of cartilage collagenase by doxycycline.

OBJECTIVE: To investigate the ability of doxycycline to modulate collagenases, cytokines, and cytokine receptors in chondrocytes from osteoarthritic (OA) cartilage. METHODS: Chondrocytes were isolated from human OA cartilage and treated with doxycycline. Synthesis of collagenases, cytokines, and cytokine receptors was quantified by Northern and Western blot analysis and RNase protection assay. RESULTS: We observed significant inhibition of matrix metalloproteinases (MMP-1) and MMP-13 mRNA and protein production by chondrocytes, isolated from OA cartilage, after treatment with doxycycline. The decrease in collagenase protein level paralleled a decrease in mRNA for these enzymes, suggesting a transcriptional/posttranscriptional level of control. In addition, treatment with 10 microg/ml doxycycline resulted in 2.2-fold upregulation of transforming growth factor (TGF-beta3) and a significant decrease of interleukin 1alpha (IL-1alpha), IL-1beta, and IL-6 mRNA. Upregulation of TGF-beta RI and TGF-beta RII was also detected. These cytokines are known to affect collagenase expression and could contribute to inhibition of MMP-1 and MMP-13 production by OA chondrocytes. A decrease in IL-1alpha, IL-1beta, and IL-6 would reduce stimulation of MMP production, while an increase in TGF-83 would lead to downregulation of local proinflammatory cytokine production as well as of the collagenases themselves. CONCLUSION: Our findings show that a decrease in MMP-1 and MMP-13 collagenase production by articular chondrocytes in response to treatment with doxycycline can be explained by a regulatory effect of doxycycline on the production of cytokine and cytokine receptors.

Intravenous tolerization with type II collagen induces interleukin-4-and interleukin-10-producing CD4+ T cells.

Intravenous (i.v.) administration of type II collagen (CII) is an effective way to induce tolerance and suppress disease in the collagen-induced arthritis (CIA) model. In this study, we demonstrated that a single i.v. dose of CII (as low as 0.1 mg/mouse) completely prevented the development of CIA. This suppression was accompanied by decreases in levels of antibody specific for the immunogen, bovine CII and autoantigen, mouse CII. Splenocytes obtained from CII-tolerized mice and stimulated with CII in vitro produced predominantly the T helper 2 (Th2)-type cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10). In contrast, cells obtained from mice immunized with CII produced predominantly interferon-gamma (IFN-gamma). Two-colour flow cytometric analysis of cytokine expression and T-cell phenotype demonstrated that CD4+ cells and not CD8+ or gammadelta+ cells were the predominant regulatory cells producing IL-4 and IL-10. Transgenic mice bearing a T-cell receptor (TCR) specific for CII had a greater increase in the number of IL-4-secreting CD4+ cells, as well as a marked increase of IL-4 in culture supernatants. This cytokine was produced by transgene-bearing T cells. Elucidation of mechanisms for the induction of tolerance in mature T cells is an important line of study in autoimmune models because of the potential application for treating organ-specific autoimmune disease.

Molecular definition and characterization of recombinant bovine CB8 and CB10: immunogenicity and arthritogenicity.

Theoretically, the ability to produce recombinant type II collagen (CII) peptide fragments in a prokaryotic expression system would be extremely useful for preparing adequate amounts of CII peptides suitable for therapeutic uses. Bacteria do not contain the enzymes involved in the extensive posttranslational modifications that occur during the biosynthesis of CII, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. As these posttranslational modifications may play a role in the immune and arthritogenic response to CII, it was unclear whether collagen expressed in Escherichia coli would be immunologically comparable to tissue-derived CII. Therefore, we prepared recombinant proteins for CB8 and CB10 by cloning CB8 (CII 403-551) and CB10 (CII 552-897) genes from bovine chondrocytes by RT-PCR technique and expressing them in an E. coli expression system. Characterization of these recombinant proteins revealed that both rCB8 and rCB10 stimulated T cell proliferation in a T cell determinant-specific manner. The T cells from mice immunized with rCB8 respond specifically to a synthetic peptide, CII 445-453, the CB8 T cell determinant. Conversely, rCB10-primed T cells respond strongly to CII 610-618, the CB10 T cell determinant. Recombinant CB8-induced autoantibodies that bound to mouse CB8 as effectively and in the same topographic distribution as tissue-derived CB8. Finally, when rCB8 and rCB10 proteins were used to immunize B10.RIII (H-2(r)) mice, rCB8 induced arthritis in 33% of the mice, very similar to the incidence induced by tissue-derived CB8 peptide. As was found to be the case with tissue-derived CB10, rCB10 was completely ineffective in inducing arthritis. Pathological changes of arthritic joints in the mice immunized with rCB8 were similar to those observed in mice immunized with tissue-derived CB8. Thus, these recombinant CII peptides expressed in E. coli can induce an effective immunologic response and suggest that functionally useful CII peptides can be generated by the prokaryotic expression system.