Figure 3

Interleukin (IL)-32 induces the production of IL-17 in human CD4⁺ T cells. (A) CD4⁺ T cells from healthy donor human peripheral blood mononuclear cells (PBMCs) were cultured with membrane-bound anti-CD3 antibody and with/without recombinant human IL-32 (5 ng/ml) for 3 days. IL-17 production was measured by sandwich ELISA. **P < 0.01 (compared with anti-CD3). (B) Flow cytometry analysis of the expression of IL-17A by CD4⁺ T cells treated with membrane-bound anti-CD3 antibody (0.5 μg/ml) and with/without IL-32 (5 ng/ml) for 3 days. These cells were stained with anti-CD4-Percp cy7.7 and anti-IL-17-FITC, to determine the percentage of IL-17⁺ cells in the CD4⁺ gated population. *** P < 0.001 (compared with anti-CD3). (C) IL-17 and RORγt mRNA levels were measured by real-time PCR in stimulated CD4⁺ T cells with a membrane-bound anti-CD3 antibody with/without IL-32 (5 ng/ml). * P < 0.05 (compared with anti-CD3), *** P < 0.001 (compared with anti-CD3). (D) CD4⁺ T cells from healthy donors were cultured with membrane-bound anti-CD3 antibody (0.5 μg/ml), anti-CD28 (1 μg/ml), anti-IL-4 (2 μg/ml), anti-IFN-γ (2 μg/ml), IL-1β (20 μg/ml) and IL-6 (20 ng/ml) to induce Th17 polarization, and with/without recombinant human IL-32 (5 ng/ml) for 3 days. *** P < 0.001 (compared with Th17) (A), (B, left panel) and (D) are representative of three experiments with similar results. (B, right panel) and (C) represent means ± SD of three separate experiments.