Fineschi Serena, Goffin Laurence, Rezzonico Roger, Cozzi Franco, Dayer Jean-Michel, Meroni Pier Luigi, Chizzolini Carlo
University Hospital and School of Medicine, Geneva, Switzerland.
Arthritis Rheum. 2008 Dec;58(12):3913-23. doi: 10.1002/art.24049.
Previous studies have revealed the presence of IgG antifibroblast antibodies (AFAs) capable of binding to the surface of fibroblasts in systemic sclerosis (SSc) sera. Since chemokines may directly or indirectly affect the development of fibrosis, this study was undertaken to investigate the production of chemokines induced by AFAs in fibroblasts, and to characterize the signaling pathways and surface molecules involved.
AFA-positive and AFA-negative IgG were tested on fibroblasts. Chemokine messenger RNA expression was screened by microarray and quantitative reverse transcription-polymerase chain reaction. Production of CCL2, CXCL8, and CXCL10 proteins was assessed by enzyme-linked immunosorbent assay. Pharmacologic inhibitors were used to study signal transduction, with results assessed by Western blotting and immunofluorescence analysis. Fibroblasts with defective expression of Toll-like receptors (TLRs) and anti-TLR monoclonal antibodies (mAb) were used to assess AFA specificity.
In human fibroblasts, AFA-positive IgG induced the preferential transcription of chemokines with profibrotic and proangiogenic potential, including, but not exclusively, CCL2, CXCL1, CXCL8, CKLF, and ECGF1, which were distinctly different from those induced by interferon-gamma. Levels of CCL2 and CXCL8 proteins were increased in AFA-stimulated fibroblast culture supernatants. AFA binding to fibroblasts resulted in concomitant activation of ERK-1/2, c-Jun, and NF-kappaB. CCL2 production was sensitive to inhibition of both proteasome and JNK, while CXCL8 production was sensitive only to inhibition of proteasome. AFAs failed to up-regulate CCL2 expression in TLR-4-deficient fibroblasts but not in TLR-6- or TLR-2-deficient fibroblasts. Moreover, anti-TLR-4 mAb, but not anti-TLR-2 mAb, partially inhibited the production of CCL2 induced by AFAs in human fibroblasts.
Autoantibodies that bind to the surface of fibroblasts may contribute to the pathogenesis of SSc by up-regulating the fibroblast production of profibrotic and proangiogenic chemokines, in a proteasome- and TLR-4-dependent manner.
既往研究已揭示系统性硬化症（SSc）血清中存在能够结合成纤维细胞表面的IgG抗成纤维细胞抗体（AFA）。由于趋化因子可能直接或间接影响纤维化的发展，因此开展本研究以调查成纤维细胞中AFA诱导的趋化因子产生情况，并确定所涉及的信号通路和表面分子。
在成纤维细胞上检测AFA阳性和AFA阴性IgG。通过微阵列和定量逆转录-聚合酶链反应筛选趋化因子信使核糖核酸表达。采用酶联免疫吸附测定法评估CCL2、CXCL8和CXCL10蛋白的产生。使用药理学抑制剂研究信号转导，并通过蛋白质印迹法和免疫荧光分析评估结果。利用Toll样受体（TLR）表达缺陷的成纤维细胞和抗TLR单克隆抗体（mAb）评估AFA特异性。
在人成纤维细胞中，AFA阳性IgG诱导具有促纤维化和促血管生成潜力的趋化因子优先转录，包括但不限于CCL2、CXCL1、CXCL8、趋化素样因子（CKLF）和内皮细胞生长因子1（ECGF1），这些与γ干扰素诱导的趋化因子明显不同。在AFA刺激的成纤维细胞培养上清液中，CCL2和CXCL8蛋白水平升高。AFA与成纤维细胞结合导致ERK-1/2、c-Jun和核因子κB同时激活。CCL2的产生对蛋白酶体和JNK的抑制敏感，而CXCL8的产生仅对蛋白酶体的抑制敏感。AFA未能上调TLR-4缺陷型成纤维细胞中CCL2的表达，但在TLR-6或TLR-2缺陷型成纤维细胞中则不然。此外，抗TLR-4 mAb而非抗TLR-2 mAb可部分抑制人成纤维细胞中AFA诱导的CCL2产生。
结合成纤维细胞表面的自身抗体可能通过以蛋白酶体和TLR-4依赖的方式上调成纤维细胞产生促纤维化和促血管生成的趋化因子，从而促进SSc的发病机制。
