Figure 4. ASK1 activity in sensitized CD4⁺ T cells is required for IL-17 production.

(A) DNBS-induced activation of ASK1 is inhibited by 1Na-PP1 in LN cells from sensitized ASK1^ASKA mice. LN cells isolated from mice sensitized with DNFB were pretreated with (+) or without (−) 1 µM 1Na-PP1 and then stimulated with DNBS for the indicated time periods. Cells were lysed, and cell lysates were subjected to immunoblot analysis using the indicated antibodies. (B, C) DNBS-induced production of IL-17, but not IFN-γ, is inhibited in 1Na-PP1-treated LN cells from sensitized ASK1^ASKA mice. LN cells isolated from DNFB-sensitized mice were pretreated with (+) or without (−) 1 µM 1Na-PP1 and then stimulated with DNBS for 3 days. The amounts of IFN-γ (B) and IL-17 (C) in the culture medium were measured by ELISA. Data are shown as the mean ± SEM (n = 6) and are representative of four experiments. (D) Proliferation of LN cells from sensitized ASK1^ASKA mice is not affected by 1Na-PP1. Relative numbers of LN cells isolated and treated as shown in (B, C) were measured as relative fluorescence intensity using a Cell Counting Kit-F. Data are represented as the mean ± SEM (n = 3). (E, F, G) ASK1 activity in DNFB-sensitized CD4⁺ T cells is required for DNBS-dependent IL-17 production. CD4⁺ and CD8⁺ T cells were isolated separately from regional LNs of DNFB-sensitized WT or ASK1^ASKA mice. CD11c⁺ DCs were isolated from spleens of DNFB-sensitized mice. CD4⁺ T cells (E, F) or CD8⁺ T cells (G) and CD11c⁺ DCs were co-cultured in the presence (+) or absence (−) of 1 µM 1Na-PP1. Thirty minutes after the treatment with 1Na-PP1, cells were stimulated with DNFB. After 24 hr of co-culture, the amounts of IL-17 in the culture medium were measured by ELISA. Data are shown as the mean ± SEM (n = 7) and are representative of five experiments.