Effect of TLR2, TLR3, and TLR4 ligands on IL-32 mRNA expression and IL-32 release by RA fibroblast-like synoviocytes (FLSs). (a) IL-32 mRNA expression in RA, OA, and normal FLSs activated for 4 h with either 1 μg/ml of lipopolysaccharide LPS, or 1 μg/ml of BLP, or 10 μg/ml of poly I:C. (b) IL-32 α, β, γ, and δ mRNA levels were determined by using quantitative RT-PCR in FLSs activated with either 1 μg/ml of LPS, or 1 μg/ml of BLP, or 10 μg/ml of poly I:C for 4 h, and (c) 24 h. (d) Confocal analysis of IL-32 expression in FLSs activated for 16 h with either LPS (c), BLP (d), poly I:C (e), or medium (a, b). (e) IL-32 release was determined with ELISA after stimulation for 24 and 48 h with either LPS, BLP, poly I:C, or medium (c). (f) Intracellular IL-32α expression was determined with a cell-based ELISA in FLSs activated for 16 h with either LPS, BLP, poly I:C, 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.