p38 MAPK controls IL-17 production by Th17 cells in vivo. (A) Mononuclear cells were isolated from the CNS of 2× MOG_35-55-CFA immunized mice on day 35 after immunization, stimulated with PMA/ionomycin for 4 hours in the presence of brefeldin A, stained, and analyzed by flow cytometry. (Left panel) Cells were gated on CD45 and the frequency of CD4⁺TCRβ⁺ cells was determined. (Right panels) Cells were gated on CD4 and TCRβ and the frequency of IL-17⁺ and IFNγ⁺ cells was determined. (B) Spleen and draining lymph node (DLN) cells from 2× MOG_35-55-CFA immunized mice treated with either carrier (n = 7) or the SB203580 (n = 8) were harvested on day 21 after immunization and stimulated with MOG_35-55 (50 μg/mL) for 72 hours and IL-17 and IFNγ in the supernatants was quantified by ELISA. (C) Spleen and DLN cells from 2× MOG_35-55-CFA immunized mice (n = 10) were harvested on day 10 after immunization and stimulated with MOG_35-55 in the presence or absence of SB203580 (SB; 5μM) for 72 hours. IL-17 production was quantified by ELISA. (D) Cells obtained as in panel C were stimulated with the indicated amounts of MOG_35-55 in the presence or the absence of SB203580 and proliferation was assessed by [³H]-thymidine incorporation. The significance of differences observed in panels A through C was determined using the Student t test (* ≤ 0.05; *** ≤ 0.001), and by 2-way ANOVA for panel D (SB, P = .31; MOG_35-55, P = .003; and interaction, P = .95).