类风湿关节炎患者瓜氨酸化Ⅱ型胶原的T细胞增殖反应和抗体生成

T cell proliferative response and antibody formation to citrullinated collagen type Ⅱ in patients with rheumatoid arthritis

目的研究类风湿关节炎（rtA）患者对瓜氨酸化Ⅱ型胶原（Cit—CⅡ）的T细胞增殖反应和抗体生成，探讨Cit—CⅡ在RA发病机制中的作用。方法体外用肽基精氨酸脱亚胺酶（PAD）催化牛CⅡ中的精氨酸转变为瓜氨酸，生成Cit—CⅡ，用1H掺入法检测34例RA患者和18例对照者（包括6例骨关节炎患者和12名健康志愿者）的外周血单个核细胞（PBMC）对CⅡ和Cit—CⅡ的增殖反应，用酶联免疫吸附试验测定患者和对照者血清抗CⅡ抗体及抗Cit—CⅡ抗体水平。结果RA患者的PBMC对CⅡ和Cit—CⅡ的增殖反应阳性率分别为35．3％（12／34）和32．4％（11／34），均分别高于对照组11．1％和0（均P〈0．05）。RA患者的PBMC对Cit—CⅡ增殖反应阳性率与对CⅡ增殖反应阳性率比较，差异无统计学意义（P〉0．05）。RA患者抗Cit—CⅡ抗体阳性率（52．9％，18／34）高于抗CⅡ抗体阳性率（32．4％，11／34）。CⅡ增殖反应阳性组抗CⅡ抗体的阳性率（58．3％，7／12）高于增殖反应阴性组（18．2％，4／22），Cit—CⅡ增殖反应阳性组抗Cit—CⅡ抗体的阳性率（72．7％，8／11）高于增殖反应阴性组（43．5％，10／23），差异均有统计学意义（均P〈0．05）。结论抗Cit—CⅡ抗体在RA中有很高的特异性；RA患者抗CⅡ抗体的生成可能与CⅡ特异性T细胞介导的B细胞活化有关。但是Cit—CⅡ并没有比原型CⅡ引起RA患者更强的外周血T细胞增殖反应，Cit—CⅡ在RA细胞免疫发病中的作用仍需进一步研究。

Objective To investigate the T cell proliferative response and antibody formation to citrullinated collagen type Ⅱ （ Cit-C Ⅱ ） in patients with rheumatoid arthritis （RA） , and explore whether autoreactive T cells responding to Cit-CⅡ plays a role in the pathogenesis of RA. Methods Arginine residues of bovine collagen type Ⅱ （CⅡ ） were converted to citrulline residues by peptidylarginine deiminase （PAD）. Peripheral blood samples were collected from 34 RA patients, and 18 sex- and age- matched controls, including 6 osteoarthritis patients and 12 healthy blood donors. Peripheral blood mononuclear cells （PBMCs） were isolated and cultured with Cit-C Ⅱ , C Ⅱ , or fetal bovine serum RPMI 1640 fluid, phytohemagglutinin （PHA）, or citrullinated buffer as controls. Cellular reactivity levels against Cit-C Ⅱ and CⅡ were investigated by measuring the proliferation of PBMCs to calculate the stimulation index （SI）. ELISA was used to detect the presence of antibodies against Cit-CⅡ and C Ⅱ. Results The positive rate of T cell proliferative response to Cit-C Ⅱ of the RA group was 32.4% （ 11/34）, significantly higher than that of the control group （0 ,P 〈 0.05 ）. The positive rate of T cell proliferative response to CⅡ of the RA group was 35.3% （12/34）, significantly higher than that of the control group （11.1%, P 〈 0.05 ）. Interestingly, Cit-CⅡ could not elicit a better T cell proliferative response than CⅡ did. In the RA patients, the anti-Cit-C Ⅱ antibody positive rate was 52.9% （18/34）, significantly higher than that of the anti-C Ⅱantibody positive rate [ 32.4% （ 11/34）, P 〈 0.05 ）. The serum IgG anti-C Ⅱ antibody positive rate of the patients with positive T cell responses to CⅡ was 58.3% （7/12）, significantly higher than that of the patients with negative Y cell responses to C Ⅱ[ 18.2% （4/22） ,P 〈0.05]. The serum IgG anti-C Ⅱ antibody positive rate of the patients with positive T cell responses to Cit-C Ⅱ was 72.7% （ 8/11 ）, significantly higher than that of the patients with negative T cell responses to Cit-C 11 [43.5% （ 10/23 ） ,P 〈 0.05 ]. Conclusion The recognition of Cit-C 1I by circulating IgG antibodies is a RA-specific serological phenomenon. The formation of C Ⅱ antibody in the RA patients may be related to B cell activation mediated by C Ⅱ specific T cells. The role of Cit-CⅡ in RA cellular immune need be further studied.