Clinical and pathogenic significance and its therapeutic application of anti-calpastatin antibodies in rheumatoid arthritis

我们在类风湿性关节炎（RA）患者中发现了针对钙依赖性半胱氨酸蛋白酶（calpain）的天然抑制剂钙pastatin的新型自身抗体。由于钙蛋白酶被认为是一种可能参与关节破坏和炎症过程的中性蛋白酶，其抑制剂钙pastatin的自身抗体的产生可能与RA的致病机制有关。在这项研究中，我们打算利用编码人calpastatin的全长cDNA表达产物来检验1)自身抗体的结构域反应性，2)患者自身抗体对calpastatin的抑制活性，以及3)calpain抑制剂对关节炎模型动物的作用。患者血清中calpastatin的5个结构域（结构域L、I、II、III、IV和V）表现出多种反应性，且反应模式倾向于与疾病相关。RA血清主要反应结构域I和II。81%的RA患者的血清至少有一个结构域反应，而SLE、硬皮病和肌炎患者的血清分别有46%、32%和43%的反应。RA患者血清中含有抗calpastatin抗体的IgG呈剂量依赖性抑制calpastatin结构域融合蛋白的calpain抑制活性。来自RA血清的11个IgG中有5个（45%）恢复了至少一个结构域的超过40%的钙蛋白酶活性，而来自SLE和正常对照的IgG没有抑制钙蛋白酶结构域的功能。在各种钙蛋白酶抑制剂中，calpeptin显著抑制II型胶原诱导的大鼠关节炎的发展。与未处理的大鼠相比，calpep肽处理的大鼠滑膜浸润和软骨破坏较少。这些结果提示抗calpastatin抗体可能是一种新的RA标志物，并且可能与RA的发病机制密切相关。此外，我们的结果导致了一种新的治疗策略。

We found novel autoantibodies to calpastatin, natural inhibitor for calcium-dependent cycteine proteinase (calpain), in patients with rheumatoid arthritis (RA). Since calpain is thought to be one of neutral proteinases that may be involved in joint destruction and inflammation process, the production of autoantibodies to its inhibitor protein, calpastatin, might be associated with pathogenic mechanisms of RA. In this study, we intended to examine 1) the domain reactivity of autoantibodies using the expression products from the full-length cDNA encoding for human calpastatin, 2) the inhibitory activity of calpastatin by patient autoantibodies, and 3) the effect of calpain inhibitors for arthritis model animals.Patient sera showed a variety of reactivity among the five domains of calpastatin (domains L, I, II, III, IV and V), and the reactive patterns tended to correlate to the diseases. RA sera predominantly reacted domains I and II. 81% of sera from RA patients reacted at least one domain, whereas 46%, 32% and 43% were reacted in sera from SLE, scleroderma and myositis, respectively.IgG from RA patient sera containing anti-calpastatin antibodies suppressed the calpain inhibitory activity of calpastatin domain fusion proteins in dose-dependent manner. 5 of 11 (45%) IgG from RA sera recovered more than 40% of calpain activity of at least one domain, whereas IgG from SLE and normal controls did not inhibited the function of calpastatin domains.Among the various calpain inhibitors, calpeptin suppressed significantly the development of type II collagen-induced rat arthritis. Calpeptin-treated rats showed less synovial infiltration and less cartilage destraction than untreated rats.These results suggest that anti-calpastatin antibodies may be useful as a novel marker of RA, and may be closely associated with pathogenic mechanisms of RA. Moreover, our results lead to a new strategy of treatment.