Effect of co-stimulation of TNF-α (10 ng/ml) and IFN-γ (0.1 ng/ml) on IL-32 mRNA expression and IL-32 release by RA fibroblast-like synoviocytes (FLSs). (a) FLSs were incubated simultaneously with IFN-γ and TNF-α, and IL-32 mRNA levels were determined after activation for 4 h and 24 h by using quantitative RT-PCR. (b) IL-32 release was determined with ELISA in culture supernatants. (c) IL-32α mRNA levels were determined by using quantitative RT-PCR in FLSs activated with either IFN-γ and TNF-α or medium (c). (d) Intracellular IL-32α expression was determined in IFN-γ + TNF-α- or medium-activated (c) FLSs. Data are expressed as the mean ± SD of three experiments (triplicates) on samples from four different patients with RA. *P < 0.01; **P < 0.001.