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 Cδ (PKCδ) 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 PKCδ 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.


CONCLUSIONS
The present results suggest that the interaction between CCL5 and CCR5 increases IL-6 production in human synovial fibroblasts via the PKCδ/c-Src/c-Jun and AP-1 signaling pathways.
Tang
Chih-Hsin
CH
China Medical University and China Medical University Hospital, Taichung, Taiwan. chtang@mail.cmu.edu.tw
Hsu
Chin-Jung
CJ
Fong
Yi-Chin
YC
D045784:Abietanes; D000098:Acetophenones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D000081247:CSK Tyrosine-Protein Kinase; D002478:Cells, Cultured; D018946:Chemokine CCL5 / Q000378:metabolism* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D010616:Phenanthrenes / Q000494:pharmacology; D051745:Protein Kinase C-delta / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011743:Pyrimidines / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D019061:src-Family Kinases
C412373:AG 1879; D045784:Abietanes; D000098:Acetophenones; D001578:Benzopyrans; D018946:Chemokine CCL5; D015850:Interleukin-6; D010616:Phenanthrenes; D016755:Proto-Oncogene Proteins c-jun; D011743:Pyrimidines; D019713:Receptors, CCR5; D018808:Transcription Factor AP-1; C021751:tanshinone; C085746:rottlerin; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D051745:Protein Kinase C-delta