PMC:2791889 / 15575-18078 JSONTXT

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Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure 5B).\nWe then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure 5C), a compound that blocks downstream ERK activation. To ensure that only T cell signaling was being affected by U0126, we used an APC-free system in which the T cells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12. As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-γ after 1 week of culture (Figure 5C). Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure 5C). Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain et al., 2007; Mody et al., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain et al., 2007; Mody et al., 2001). PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure 5D and Figure S5A). Upon addition of inhibitors to other signaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3β inhibitor, CT99021 (Bain et al., 2007), no effect on IL-10 production was observed (Figure S5B). Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3β 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