Effect of TNF-α (10 ng/ml) on IL-32 mRNA expression and IL-32 release by RA fibroblast-like synoviocytes (FLSs). (a) Dose-dependent effect of TNF-α (1, 5, 10, and 20 ng/ml) on IL-32 mRNA expression by RA FLSs activated for 4 h. IL-32 mRNA expression in RA, OA, and normal FLSs activated for 4 h with TNF-α (10 ng/ml). (b) IL-32 α, β, γ, and δ mRNA levels were determined by using quantitative RT-PCR in FLSs activated with TNF-α for 4 h and (c) 24 h. (d) Confocal analysis of IL-32 expression in FLSs activated for 16 h with TNF-α (c) or medium (a, b). (e) IL-32 release was determined after stimulation for 24 and 48 h with TNF-α or medium (c) with ELISA.(f) Intracellular IL-32α expression was determined with a cell-based ELISA in FLSs activated for 16 and 24 h with TNF-α or medium (c). Data are expressed as the mean ± SD of three experiments (triplicates) on samples from four different patients with RA. *P < 0.01.