FIGURE 5.

The role of IKK for IL-22 expression as detected in T/A-stimulated Jurkat T cells. A, Jurkat T cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for 4 h. Where indicated, cells were preincubated for 1 h with IKKVII (10 μm) before T/A addition. After 5 h, nuclear c-Rel and HDAC1 content was evaluated by Western blot analysis. One representative of three independently performed experiments is shown. B, Jurkat T cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for 4 h. Where specified, cells were preincubated for 1 h with the indicated concentrations of IKKVII before T/A addition. After 5 h, IL-22 mRNA was assessed by real time PCR. IL-22 mRNA was normalized to that of GAPDH and is shown as mean fold-induction compared with unstimulated control ± S.D. (n = 5); **, p < 0.01 compared with unstimulated control; ##, p < 0.01 compared with T/A alone; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. C, Jurkat T cells were transfected for 5 h with either pGL3-Prom3 or pGL3-Prom3 displaying the aforementioned CRE/NF-ATS4 double mutation (DM) together with Renilla luciferase as described under “Experimental Procedures.” After 15 h of rest, cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for another 8 h. Where indicated, cells were preincubated for 1 h with IKKVII (5 μm) before T/A addition. Thereafter, cells were harvested and luciferase assays were performed. Means of luciferase activity are shown as fold-induction compared with unstimulated control ± S.D. (n = 5); **, p < 0.01 compared with unstimulated control (transfected with the same plasmid); #, p < 0.05 compared with T/A-activated cells (without IKKVII and transfected with the same plasmid); raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. A–C, all cultures were adjusted to a final concentration of 0.2% DMSO (vehicle for T/A, IKKVII). D, PBMC were either kept as unstimulated control or stimulated with αCD3 (7 μg/ml) for 24 h. Where indicated, cells were preincubated for 1 h with IKKVII (5 μm) before αCD3 addition. All cultures were adjusted to a final concentration of 0.05% DMSO (vehicle for IKKVII). After 25 h, IL-22 release was determined by ELISA. Data are expressed as mean ± S.E. (n = 6); **, p < 0.01 compared with unstimulated control; ##, p < 0.01 compared with stimulation with αCD3 alone; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. E and F, silencing of IKKα/β in T/A-activated (4 h) Jurkat cells was performed as described under “Experimental Procedures.” E, after the transfection/stimulation period, expression of IKKα/β under the influence of control siRNA (siRNAc) or siRNA-IKKα (upper panel) or siRNA-IKKβ (lower panel) was verified by immunoblot analysis. One representative experiment for either IKKα or IKKβ silencing is shown (n = 9 for each IKK). F, expression of IL-22 mRNA under the influence of siRNA-IKKα is shown. mRNA expression was assessed by real time PCR in the same set of experiments. IL-22 mRNA was normalized to that of GAPDH and is displayed as absolute values of fold-induction (n = 9); **, p < 0.01 for siRNAc compared with siRNA-IKKα; raw data were analyzed by paired Student's t test. E and F, all cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for T/A).