Significance Of Citrullinated Type â…¡ Collagen And Its Antibodies In The Pathogenesis Of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an antigens drove and T cell mediated autoimmune disease. There has been considerable interest in recent years in the observation that a very high proportion of patients with RA have IgG antibodies to citrulline-containing proteins. Recent data indicated that citrullinated proteins might be involved in the pathogenesis of RA. Because type II collagen (CII) is a major and specific molecule in joints, we investigated whether this protein was citrullinated and whether autoantibodies react with citrullinated type II collagen in sera of RA patients. We also study the T cell proliferative response to type II collagen in patients with RA and regulation of tumor necrosis factor (TNF)-αto peptidylarginine deiminase (PAD)-2,4 mRNA expression.The study consists of three sections:Section I : Detection of anti-citrullinated type II collagen (Cit-CII) antibodies in RA patientsArginine residues of bovine CII were converted to citrulline residues by PAD in vitro. Then we detected anti-CitCII antibodies in sera from patients with RA by enzyme-linked immunosorbent assay (ELISA) and western blotting.1.Preparation of citrullinated type II collagenType II collagen was citrullinated by incubation with PAD at a concentration of 10U/mg protein for 12h at 37.0℃. Citrullination of type II collagen was confirmed by western blotting with anti-modified citrulline (anti-MC) antibody. SDS-PAGE showed that Cit-CII had slightly slower mobility than non-citrullinated CII and could be detected by anti-MC antibody. This indicated that the bovine CII was citrullinated by PAD in vitro.2.Assay for anti-CitCII antibodies and anti-CII antibodiesSerum samples were obtained from patients including 175 RA, 112 SLE and 37 OA. Sera from 160 blood donors were used as healthy controls. Anti-CitCII antibodies and anti-CII antibodies were measured by ELISA. The positive rate of anti-CitCII antibodies in RA patients sera (50.3%,88/175) were significantly higher than those in SLE patient sera (17.9%,20/112), OA patient sera (18.9%,7/37) and healthy control sera (2.5%,4/160). The positive rate of anti-CitCII antibodies were significantly higher than the positive rate of anti-CII antibodies (33.7%,59/175) in RA patient sera. The levels of anti-CitCII and anti-CII antibodies in SLE were not statistically different from that in OA patients. Anti-CitCII antibodies showed a sensitivity of 50.3% and a specificity of 90.0% in RA, and the sensitivity and specificity of anti-CII antibodies were 33.7% and 80.3% respectively. Both the sensitivity and specificity of anti-CitCII antibodies in RA were higher than those of anti-CII antibodies, which suggested that the recognition of the citrullinated CII by circulating IgG antibodies was a RA-specific serological phenomenon.3. Associations between anti Cit-CII antibodies and clinical features in RAThere was a positive correlation between anti-CitCII and anti-CCP antibodies. CRP, ESR, RF, X ray imaging, course of disease and age were not associated with anti-CitCII antibodies by spearman correlation analysis.Section II: T cell proliferative response to Cit-CII and CII in patients with RAWe assayed T cell proliferative responses to Cit-CII and CII in peripheral blood mononuclear cells (PBMC) from RA patients and explore whether autoreactive T cells responding to Cit-CII could play a role in the pathogenesis of RA.1.T cell proliferative response to Cit-CII or CII in PBMCCellular reactivity against Cit-CII or CII was investigated by measuring proliferation of PBMC obtained from 34 RA patients and 18 controls, including 6 patients with OA and 12 healthy subjects. Positive responses to Cit-CII were observed in 32.4% of the RA patients and in 0% of the controls. Positive responses to CII were observed in 35.3% of the RA patients and in 11.1% of the controls. These difference were highly significant (P<0.05). In RA patients mean SI of CII or Cit-CII were both significant higher than that of controls (P<0.05).These findings suggested that autoimmune T cell responses to bovine CII or Cit-CII may play a role in the pathogenesis of RA. Interestingly, Cit-CII could not elicit a better T cell response.2.Correlation of T cell response with antibody response to CIIIgG anti-CII were measured in 34 sera from patients with RA. Eleven (32.4%) of 34 patients with RA tested positive for circulating IgG anti-CII. In the sera, patients with positive T cell responses to bovine CII (n=12) had a higher frequency of IgG anti-CII positivity (58.3% versus 18.2%; P<0.05) than did patients with negative T cell responses (n=22).3.Correlation of T cell response with antibody response to Cit-CIIIgG anti-CitCII were measured in 34 sera from patients with RA. Eighteen (52.9%) of 34 patients with RA tested positive for circulating IgG anti-CitCII. In the sera, patients with positive T cell responses to bovine CitCII (n=11) had a higher frequency of IgG anti-CitCII positivity (72.7% versus 43.5%; P<0.05) than did patients with negative T cell responses (n=23).Section III:Regulation of TNF-αto expression of peptidylargine deiminase (PAD)-2, 4 mRNATo explore the regulation mechanism of PAD mRNA expression in vivo, we study the expression changes of PAD mRNA in differentiating HL-60 cells stimulated by proinflammatory cytokine TNF-α.Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to granulocyte by exposure to dimethyl sulfoxide (DMSO). Differentiation of the HL-60 cells into granulocytes was confirmed by Wright-Giemsa stain and netroblue tetrazalium (NBT) reduction test. Differentiating HL-60 cells were stimulated by TNF-αwith different concentration and different time. The results suggested that PAD2 and PAD4 mRNA expression was TNF-αdose-dependent and time-dependent. PAD2 and PAD4 mRNA levels were both increased by TNF-αexposure after 2-4 h and then increased more slowly to a maximum amount by 8 h. PAD2 and PAD4 mRNA levels were both increased to maximum amount by TNF-αconcentration with 25ng/ml.The innovation of this study is: (1) Bovine CII was citrullinated by PAD in vitro for the first time. The sensitivity and specificity of anti-CitCII antibodies in RA were both higher than those of anti-CII antibodies, which suggested that the recognition of the Cit-CII by circulating IgG antibodies was a RA-specific serological phenomenon and anti-CitCII antibodies may be a new marker for evaluation of patients with RA. (2) We assayed T cell proliferative responses to Cit-CII and CII in peripheral blood mononuclear cells (PBMC) from RA patients. It indicated that Cit-CII could not elicit a better T cell response than CII, which suggested that Cit-CII played a role on RA cellular immune and would be further studied. (3) We study the regulation of proinflammatory cytokine TNF-αto expression of peptidylargine deiminase (PAD)-2,4 mRNA and found PAD-2,4 mRNA expression were up-regulated by TNF-α.