Study on signal transduction pathway via integrins in autoimmune T cells

现在人们已经认识到整合素及其配体在炎症的第一步中起着关键作用，如白细胞与内皮细胞之间的粘连。在这里，我们用共聚焦图像分析仪显示的数据显示，在RA滑膜细胞的血管周围和下层区域观察到了抗Beta1和抗磷酸酪氨酸的双重染色信号，而在OA中没有检测到，这提出了一种可能性，即黏附相互作用不仅介导了黏附，而且将信号传递到细胞内，有助于维持RA滑膜的炎症。为了探讨其机制，我们在体外培养体系中检测了滑膜细胞的酪氨酸磷酸化。同型酪氨酸免疫印迹显示RA滑膜细胞中有5种主要蛋白，而在RA滑膜细胞中仅检测到少量微弱条带。由于pp125在RA滑膜细胞中最为显著，因此我们接下来检测pp125是否是FAK。抗pp125FAK的免疫沉淀表明该条带本身就是pp125FAK，并且pp125FAK的酪氨酸磷酸化水平显著升高。此外，激光共聚焦显微镜显示，在巨噬细胞样滑膜细胞中大量检测到抗磷酸酪氨酸和抗pp125FAK的信号。

It is now well recognized that integrins and their ligands are pivotal role in the first step of inflammation such as the adhesion between the leukocytes and the endothelial cells. Here, we showed data by a confocal image analyzer that the double stainig signals by anti-beta1 and anti-phosphotyrosine were observed in the perivascular and sublining areas in RA synovial cells, while it was not detected in OA,raising a possibility that the adhesive interaction not only mediates adhesion, but delivers the signals into the cell, contributing to maintain inflammation in RA synovium. To explore the mechanism, we examined the tyrosine phosphorylation of synovial cells in vitro culture system. The immunoblot for hospho-tyrosine demonstrated the five major proteins form the synovial cells in RA,while few faint bands were detected in OA.Since the pp125, among five major bands, was most prominent in RA synovial cells, we next examined whether pp125 is FAK or not.Immunoprecipitation with anti-pp125FAK has demonstrated that the band was pp125FAK itself and the tyrosine phosphorylation of pp125FAK was significantly elevated in RA synovial cells. Furthermore, confocal laser microscopy showed that the signals by anti-phosphotyrosine and anti-pp125FAK were abundantly detected in macrophage-like synovial cells.