The role of Act1, a NF-κB-activating protein, in IL-6 and IL-8 levels induced by IL-17 stimulation in SW982 cells

Abstract

Context: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation in the synovial membrane of affected joints. It has been shown that several kinds of cytokine were increased in synovial fluid, while the underlying mechanism remains poorly understood.Objectives: NF-κB activator 1 (Act1) is a recently identified protein binding to the IκB kinase complex. Our study aimed to investigate the expression of Act1 induced by cytokine IL-17 stimulation in SW982 cells.Materials and methods: The human synovial sarcoma cell line SW982 and primary cultured RA fibroblast-like synovial cells were used. RT-PCR and Western blot assays were selected to investigate the genetic and protein expression of Act1. Additionally, four independent Act1 small interfering RNA (siRNA) oligonucleotides were designed and obtained according to the GenBank cDNA, the sequence of Act1 (Traf3ip2). Finally, enzyme-linked immunosorbent assay (ELISA) double antibody sandwich was used to assay supernatant IL-6 and IL-8 concentrations.Results: The Act1 mRNA expression level increased significantly after stimulation with IL-17 (5–100 ng/ml) in SW982 cells. Additionally, the level of Act1 mRNA expression correlated positively with the concentration of IL-17 (p < 0.01). IL-17 induced IL-6 and IL-8 in SW982 cells was in a concentration- and time-dependent way. Furthermore, ELISA assay revealed that IL-17 (20 ng/ml) significantly increased IL-6 (1927.4 ± 288.77 versus 786.5 ± 172.42 ng/ml, p < 0.01) and IL-8 levels (984.8 ± 95.09 ng/ml versus 307.1 ± 90.83 ng/ml, p < 0.01) compared with control group after stimulation for 24 h. However, transfection of Traf3ip2 siRNA markedly decreased IL-6 (995.9 ± 115.30 ng/ml versus 1816.1 ± 273.27 ng/ml, p < 0.01) and IL-8 levels (575.6 ± 65.96 ng/ml versus 929.4 ± 124.39 ng/ml, p < 0.01) compared to transfection negative control. These findings suggested that IL-6 and IL-8 level induced by IL-17 in SW982 cells could be reversed by down-regulation of Act1 expression level with Traf3ip2 siRNA.Conclusion: Our results suggested that Act1 might play a key role in the pathophysiology and the treatment of RA.