Take Yasuhiro, Nakata Ken, Hashimoto Jun, Tsuboi Hideki, Nishimoto Norihiro, Ochi Takahiro, Yoshikawa Hideki
Department of Orthopaedics, Osaka University Graduate School of Medicine, and Osaka Rosai Hospital, Osaka, Japan.
Arthritis Rheum. 2009 Dec;60(12):3591-601. doi: 10.1002/art.25020.
Osteopontin (OPN) is expressed by fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA), but its pathologic role is still obscure. The present study was undertaken to analyze the role of OPN in RA by focusing on its effects on cell-cell interactions between FLS and B lymphocytes.
FLS obtained from 10 patients with RA and 10 non-RA subjects and a B lymphocyte cell line were studied. The characteristics of OPN expression by FLS were analyzed by Western blotting, immunoprecipitation, and immunofluorescence studies. In cocultures of FLS and B lymphocytes, the effects of OPN on adhesion of B lymphocytes to FLS and the consequent production of interleukin-6 (IL-6) were analyzed in experiments involving overexpression and knockdown of OPN and inhibitory studies with an OPN-blocking antibody. In vivo, the expression of OPN in RA synovium was examined by immunohistochemistry.
A specifically modified 75-kd form of OPN was predominantly expressed in RA FLS, and this was associated with expression of >200-kd thrombin-cleaved OPN that was crosslinked with fibronectin and localized on the surface of the FLS. In FLS-B lymphocyte cocultures, 75-kd OPN-positive FLS produced a significantly higher amount of IL-6 than did 75-kd OPN-negative FLS. When the FLS were separated from B lymphocytes or cultured alone, the production of IL-6 was low and was not significantly different between these 2 culture conditions. Moreover, OPN overexpression enhanced production of IL-6 in 75-kd OPN-positive FLS-B lymphocyte cocultures. Addition of the OPN-blocking antibody inhibited the adhesion of B lymphocytes to FLS. Immunohistochemical analyses revealed that localization of IL-6-positive cells coincided with the sites at which OPN and B lymphocytes were colocalized.
Specifically modified 75-kd OPN was expressed by RA FLS. This form of OPN affected FLS-B lymphocyte interactions by supporting the adhesion of B lymphocytes to FLS and enhancing the production of IL-6.
骨桥蛋白（OPN）由类风湿关节炎（RA）中的成纤维样滑膜细胞（FLS）表达，但其病理作用仍不清楚。本研究旨在通过关注OPN对FLS与B淋巴细胞之间细胞间相互作用的影响来分析OPN在RA中的作用。
研究了从10例RA患者和10例非RA受试者获得的FLS以及一种B淋巴细胞系。通过蛋白质免疫印迹法、免疫沉淀法和免疫荧光研究分析了FLS中OPN表达的特征。在FLS与B淋巴细胞的共培养中，在涉及OPN过表达和敲低的实验以及使用OPN阻断抗体的抑制研究中，分析了OPN对B淋巴细胞与FLS黏附以及随后白细胞介素-6（IL-6）产生的影响。在体内，通过免疫组织化学检查RA滑膜中OPN的表达。
一种特异性修饰的75-kd形式的OPN主要在RA FLS中表达，这与>200-kd凝血酶切割的OPN的表达相关，该OPN与纤连蛋白交联并定位在FLS表面。在FLS-B淋巴细胞共培养中，75-kd OPN阳性的FLS产生的IL-6量明显高于75-kd OPN阴性的FLS。当FLS与B淋巴细胞分离或单独培养时，IL-6的产生较低，并且在这两种培养条件之间没有显著差异。此外，OPN过表达增强了75-kd OPN阳性的FLS-B淋巴细胞共培养中IL-6的产生。添加OPN阻断抗体可抑制B淋巴细胞与FLS的黏附。免疫组织化学分析显示，IL-6阳性细胞的定位与OPN和B淋巴细胞共定位的部位一致。
RA FLS表达特异性修饰的75-kd OPN。这种形式的OPN通过支持B淋巴细胞与FLS的黏附并增强IL-6的产生来影响FLS-B淋巴细胞相互作用。
