RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3
RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3
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DOI：
10.1002/art.21273
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发表时间：
2005-09-01
影响因子：
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通讯作者：
Kyburz, D
中科院分区：
文献类型：
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作者：
Brentano, F;Schorr, O;Kyburz, D
目标。目的检测类风湿关节炎(RA)和骨关节炎(OA)患者滑膜组织和培养的滑膜成纤维细胞中TLR-3蛋白的表达，探讨TLR-3配体刺激培养的滑膜成纤维细胞的作用。免疫组织化学和免疫荧光法检测TLR-3在滑膜组织中的表达，荧光激活细胞分选和实时聚合酶链式反应技术检测培养的RA滑膜成纤维细胞(RASF)中TLR-3的表达。通过将RASF与聚(I-C)、脂多糖、PAimitoyl-3-半胱氨酸-丝氨酸-赖氨酸-4或RA患者坏死性滑液细胞在存在或不存在羟氯喹或Benzonase的情况下孵育来评估TLR-3信号转导。用酶联免疫吸附分析法检测培养上清液中干扰素-β、CXCL10、CCL5和白介素6的蛋白产量。TLR-3在RA滑膜组织中的表达高于在OA滑膜组织中的表达。TLR-3主要定位于滑膜衬里，大多数表达TLR-3的细胞共表达成纤维细胞标志物。用TLR-3配体聚(I-C)刺激培养的RASF可产生高水平的干扰素β、CXCL10、CCL5和IL-6蛋白。类似地，RASF与RA患者的坏死性滑液细胞共同孵育后，这些细胞因子和趋化因子的上调依赖于TLR-3。我们的研究结果证实了TLR-3在RA滑膜组织中的表达，以及TLR-3配体Poly(I-C)和坏死性RA滑液细胞在体外对RASF的激活作用，提示从坏死细胞释放的RNA可能作为内源性TLR-3配体刺激RASF中促炎基因的表达。
Objective. To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands.Methods. TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, paimitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFN beta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays.Results. TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFN beta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner.Conclusion. Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.