| Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by a chronic inflammation of the synovial membrane which is associated with destruction of cartilage and bone. It is a common human autoimmune disease with a prevalence of about 0.3～1.0%. The pathogenesis is not completely understood.'Oral tolerance'has classically been defined as the specific suppression of cellular and/or humoral immune responses to an antigen by prior administration of the antigen by the oral route. Mucosal tolerance is an attractive approach for treatment of autoimmune and inflammatory diseases because of lack of toxicity, ease of administration over time, and antigen-specific mechanisms of action. Oral tolerance induced by typeⅡcollagen (CⅡ) and synthesis polypeptide 250-270 (syCⅡ250-270) peptide have been demonstrated to be a nondangerous, effective way for preventing harmful inflammatory in animal model of RA. However, it is more difficult to extract CⅡfrom animal cartilage tissue. Furthermore, the source of material is much limited to supply the demand of basal and clinical application for research and treatment of RA and the other diseases. The objective of this paper is to high-performance express the recombinant polymerized gene encoding human collagen typeⅡpolypeptide 250-270 (rhCⅡ250-270). Then, in order to proved that rhCⅡ2 50-270 characterize with predicted immunological characterization, we had explored its ability to stimulate the T cells of PBMC in patients with RA and detected autoantibodies to rhCⅡ250-270 in the sera of rhCⅡ250-270-immunized mice.It has been demonstrated that oral antigen administration can suppress animal models of autoimmune diseases. Unfortunately, although positive results have been observed in phase II trials, no effect was observed in phase III trials of CⅡin rheumatoid arthritis. In order to further investigat the modulation of rhCⅡ250-270 peptide, with immunodominant epitopes on special cellular and humoral immune response in the course of oral administration by in vitro and in vivo experiment, we induced low doses of orally administered rhCⅡ250-270 in early disease in mice CIA. The cellular events that characterize lymphocyte responses to specific stimuli include expression of cell surface activation antigens (CD69 or CD25), cytokine production (IFN-γ, IL-4) and proliferation (BrdU incorporation) were simultaneous detected. The antigen-specific antibody and antibody-forming cells were detected by ELISA and ELISPOT, respectively.Methods1. The human immunodominant peptide of CⅡ250-270 was expressed in E.coli.and purified by affinity chromatography for the first time. Fusion protein was expressed in pGEX-4T-1 and isolated and purified by Glutathione Sepharose? affinity chromatography.2. rhCⅡ2 50-270 was proved to characterize with predicted immunological characterization by flow cytometry analysis of activation and proliferation of antigen specific T cells including cellsurface markers (e.g. CD3, CD69 and CD25) and BrdU incorporation, and by the activity of IgG rhCⅡ2 50-270-specific antibody in sera in mice immunized with rhCⅡ2 50-270.3. DBA/1 mice were immunized with emulsified chicken CⅡor rhCⅡ2 50-270 in complete Freund's adjuvant by intradermal injection to induce the mice model of arthritis; Oral tolerance was induced by oral rhCⅡ2 50-270 or syCⅡ2 50-270 in different doses (5 mg/mL, 1 mg/mL, respectively, twice before the induction phase of CIA).4. The lymphocytes were obtained from spleen of mice killed at 7 days of booster immunization, and the antigen specific reactive T cells were stimulated by CⅡor CⅡ250-270 in vitro. The proliferation response and phenotype were analyzed by BrdU incorporation and fluorescence-activated cell sorter (FACS). Intracellular cytokines (IFN-γ, IL-4) and surface antigen (CD4, CD3, CD69 and CD25 )of splenocytes were assayed by FACS.5. The frequency of IgG anti-CⅡ, anti-rhCⅡ250-270, and anti-syCⅡ( 250-270 antibody-forming spleen cells was measured with ELISPOT; ELISA was used to determine antigen-specific antibodies in sera in mice.6. To observe the effects of oral CⅡ250-270 on joint structure at the histologic level, inflamed joints were stained with hematoxylin and eosin and evaluated after mice was sacrificed at 3 weeks after booster immunization. Results1. rhCⅡ2 50-270 had been successfully expressed and purified. The relative molecular mass(Mr)of expressed product was 43 KD which is in accord with predicted. The purity of purified fusion protein reached 89.3 percent assessed by SDS-PAGE gel thin-layer scanning.2. rhCⅡ( 250-270 in vitro could stimulate the response of specific lymphocyte in RA patients PBMC including the increase of surface activation antigen marker CD69, CD25 expression and DNA synthesis. rhCⅡ2 50-270 in vivo could induced antigen-specific antibody in DBA/1 mice. As well as native CⅡ, rhCⅡ( 250-270 could be recognized as immunogenic antigens by T and B cells.3. We found that CⅡ(250-270 to some extent reduced the severity of CIA through the utilization of immunological regulatory mechanisms induced by feeding rhCⅡ250-270 to CIA. The general condition of the mice was improved.4. Splenocytes from mice fed rhCⅡ250-270 and syCⅡ250-270 stimulated significantly less specific splenocyte activation compare with CIA group. The percentages of CD3+CD25+ cell from mice fed CⅡ250-270 were reduced by at least 18.76% stimulated by rhCⅡ250-270. The percentages of CD3+BrdU+ cell from mice fed CⅡ250-270 after stimulation with antigens in vitro significantly less than CIA group. Those from mice fed rhCⅡ250-270 (5mg/mL),and syCⅡ250-270 (1mg/mL) were 27.67%, 24.79% and stimulated by rhCⅡ250-270, respectively, while in CIA was up to 50.46%. Splenocytes from the fed polypeptide groups of mice that were secreted significantly less IFN-γ, which were 9.86% and 12.89% stimulated by rhCⅡ250-270, respectively, than splenocytes from CIA (24.36%).5. The level of CⅡ- and CⅡ2 50-270 -specific IgG in serum from mice fed with rhCⅡ250-270 were (0.82±0.02) and (0.84±0.04) respectively,and significantly lower than those of collagen-induced arthritis (CIA) control group. The anti-CⅡ250-270 antibody responses were obviously suppressed (P<0.01).The frequency of antibody-forming cells in the spleen from rhCⅡ250-270-fed mice were (158±9 counts/well ) and (181±10 counts/well)respectively, and also significantly were reduced when compared with that in CIA control group (247±16 counts/well ).6. Consistent with the lack of treatment effect, oral rhCⅡ250-270 in doses of 5mg/mL have lower histologic scores for synovial inflammation or pannus invasion, degrees of cartilage damage and bone damage at either the joints.Generally, the human immunodominant peptide of CⅡ250-270 was expressed and purified for the first time in this paper. Then, it was proved to characterize with predicted immunological characterization. Further, present study identification that oral rhCⅡ250-270 could induce specific suppress of cellular and humoral in CIA. These findings and the experiment system, together with a better understanding of the mechanisms of oral tolerance and regulation cellular and humoral immune response in CIA, will help the development of innovative therapeutic intervention for RA.
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