The CCL5/CCR5 Axis Promotes Interleukin-6 Production in Human Synovial Fibroblasts

Objective. CCL5 (RANTES) was originally identified as a product of activated T cells and plays a crucial role in the inflammatory response. This study was undertaken to investigate the intracellular signaling pathways involved in CCL5-induced interleukin-6 (IL-6) production in human synovial fibroblasts. Methods. CCL5-mediated IL-6 expression was assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action of CCL5 in different signaling pathways were studied using Western blotting. Knockdown of CCR5 and protein kinase C delta (PKC delta) protein was achieved by transfection of small interfering RNA (siRNA). Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the IL-6 promoter. Transient transfection was used to examine IL-6 and activator protein 1 (AP-1) activity. Results. Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCL5 and CCR5, and expression was higher than that in normal synovial fibroblasts. Stimulation of OASFs with CCL5 induced concentration-and time-dependent increases in IL-6 production. CCL5-mediated IL-6 production was attenuated by CCR5 monoclonal antibody, CCR5 inhibitor (Met-RANTES), and CCR5 siRNA. Pretreatment with a PKC delta inhibitor (rottlerin), a c-Src inhibitor (PP2), or an AP-1 inhibitor (tanshinone IIA) also blocked the potentiating action of CCL5. Treatment of OASFs with CCL5 increased the accumulation of phosphorylated c-Jun in the nucleus, AP-1 luciferase activity, and c-Jun binding to the AP-1 element on the IL-6 promoter. CCL5-mediated AP-1 luciferase activity and c-Jun binding to the AP-1 element were inhibited by Met-RANTES, rottlerin, and PP2. Conclusion. The present results suggest that the interaction between CCL5 and CCR5 increases IL-6 production in human synovial fibroblasts via the PKC delta/c-Src/c-Jun and AP-1 signaling pathways.