| BackgroundRheumatoid arthritis (RA) is a typical and chronic, progressive autoimmune diseases, which is driven by antigens and mediated by T cells. Its characteristic is that lots of autoantibodies against autoantigens and variety of antigen-specific T/B lymphocytes can be detected in the peripheral circulation. RA has a complex etiology and unclear pathogenesis, but currently, there is no effective means of early diagnosis for RA. The disease mainly involved peripheral small joints, and may ultimately lead to progressive joint destruction, deformity, or loss of function in late phase of disease. Therefore, The early diagnosis means is extremely necessary and important for RA. Collagen induced arthritis (CIA) is an animal model of immune articular inflammation induced by collagen type II (CⅡ). CIA has many similarities in symptoms and joint pathology with RA, so it has been considered to be an unique animal model of human RA, and used more widely in RA mechanism research.ObjectiveTo establish new serological test system of RA diagnosis, search for new autoantibodies marker of RA disease, examine the proliferative effect of synthetic cyclic citrullinated peptide (CCP) and CⅡpeptide (CⅡ260-272) on T cells of CIA rats, and detect the levels of interferon-γ(IFN-γ), interleukin-4(IL-4) and interleukin-17(IL-17) secreted by those two antigen specific T lymphocytes mentioned above. Aim to explore the role of those two antigens in occurrence and development of CIA disease, and provide theoretical and experimental basis for further understanding the role of those antigens in RA.MethodsWe established the rat model of CIA. Bovine type II collagen (bCⅡ) was modified and citrullinated by PAD enzyme, meanwhile, CⅡpeptide (CⅡ260-272), CCP and PAD enzyme were selected as immobilized antigens, enzyme-linked immunosorbent assay (ELISA) for autoantibodies against CⅡCit-CⅡ, CⅡpeptide, PAD and CCP antigens were established. The T lymphocytes from CIA rats were isolated and incubated with CCP and CⅡpeptide respectively, and the proliferation of T cells was determined by cell counting kit-8.The levels of IFN-y, IL-4 and IL-17 secreted by those two antigen specific T lymphocytes mentioned above were detected by ELISA, furthermore, the relationship of which with immunity time, cell proliferation and other serological index, articular inflammation index were also analyzed and discussed.Results1. The rats model achieved swelling peak in 10d after the first immunization, and with the extension of time, joint inflammation index continues to rise, the second articular inflammation peak appeared in 16d. Meanwhile, the vascular synovial inflammation and pannus formation have been showed obviously in 2w since the first immunization, although those symptoms relieved later, it still could be observed at later stage (8w). Pathology results showed that articular synovial of CIA rats were hyperplasia, synovial layer was thicken, arranging disorderly, and the inflammatory cells such as lymphocytes, plasma cells, and macrophages, neutrophils infiltrated into synovial, pannus formation were significantly increased. The level of cartilage oligomeric protein (COMP) in CIA rats was significantly increased compared with control group(P<0.01). There were a significant correlation between the expression level of COMP and joint inflammation index (P<0.05).2. The autoantibodies against PAD, CCP, CⅡpeptide, bC II, Cit- bCⅡshowed different degrees of increase, especially anti-C II peptide antibodies and anti-CCP antibodies.The anti-C II peptide antibodies significantly correlated with joint inflammation, synovial phlogistic and pannus formation (P<0.05 or P<0.01). Meanwhile, an obvious correlation among those antibodies was also found(P<0.05 or P<0.01).3.2～3 weeks after the first immunization, T cell proliferation response to CCP and CⅡpeptide was apparent, significantly higher than to FLAK peptide group and control group(P<0.01 or P<0.05). Besides, We found that CCP specific T cell proliferation response and CIA course, joint inflammation and anti-CCP antibody, anti-CⅡpeptide antibody were significantly related (P<0.01 or P<0.05), but there were not conclusive correlations between the CⅡpeptide specific T cell proliferation reaction and anti-CCP antibody (P>0.05).4. In the early stage, the levels of IFN-y and IL-17 in serum of CIA groups improved significantly in comparison with the control group(P<0.01), and IL-4 level remained low all the time.4 weeks later, IFN-y levels began to decrease(P<0.01). The expression levels of IFN-y and IL-17 in the cell culture stimulated by two antigen peptides was also up-regulation, but differently, IL-4 concentration almost maintained a high level. Meanwhile, the analysis indicated that IFN-y and IL-17 produced by CCP stimulation and CCP - AST cells and CⅡpeptide specific T cell proliferation were positively correlated (P<0.01), IL-4 level was related with CCP-AST cell proliferation (P<0.05), but not CⅡpeptide specific T cell.ConclusionWe established the rat model of the collagen-induced arthritis(CIA) successfully, and estimated it. Specific humoral immunity disorder and cellular immunity abnormity were existed widely in development of CIA. Anti-PAD antibody and anti-CCP antibody were detected, in addition to the anti-C II antibodies. The proliferative response of T cell to CCP and CⅡ260-272 antigens in vitro was found in the early stage of CIA rats, these antigen may induce cytokines. secretion and result in disequilibrium of Thl/Th2/Th17, suggesting that the citrullinated antigen, CⅡpeptide and a series related autoantibodies play an important role in the pathogenesis of early RA. |
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