Interleukin (IL)-32 induces IL-17 production in an autoimmune arthritis mouse model. (A) CD4+ T cells were isolated from the spleens of collagen-induced arthritis (CIA) mice, an autoimmune arthritis model. The cells were cultured with membrane-bound anti-CD3 (0.5 μg/ml) and with/without IL-32 (5 ng/ml) for 3 days. IL-17 production was measured by sandwich ELISA. ** P < 0.01 (compared with anti-CD3). (B-D) Expression of IL-17A by CD4+ T cells cultured under Th17-polarizing conditions for 3 days: anti-CD3 (0.5 μg/ml), anti-CD28 (1 μg/ml), anti-IFN-gamma (2 μg/ml), anti-IL-4 (2 μg/ml), anti-IL-2 (2 μg/ml), IL-6 (20 ng/ml), TGF-beta1 (2 ng/ml) with/without IL-32 (5 ng/ml) and/or type ll collagen with irradiated antigen-presenting cells, and then stimulated for 4 h with PMA and ionomycin, followed by intracellular cytokine staining. The percentage of IL-17+ cells in the CD4+ gated population (B), *** P < 0.001 (compared with Th17) and cytokine level (C) were measured by sandwich ELISA, *** P < 0.001 (compared with Th17) and mRNA levels (D) were measured by real-time PCR, * P < 0.05 (compared with Th17). B-D are representative of three experiment with similar results. (E) Expressions of IL-17, IL-32 and tartrate-resistant acid phosphatase (TRAP) in the synovium of the CIA and IL-1Ra-knock-out (KO) mice, two autoimmune arthritis mouse models. The results shown are representative of five experiments with similar results.