Figure 1.

Type I IFN signaling dominates late LPS-stimulated IL-10 expression and hinders a synergistic effect of cAMP. (A) Silencing of the type I IFN receptor enables synergistic IL-10 expression by LPS and isoproterenol also at the late phase. RAW264.7 macrophages were transfected with a full mouse IL-10 promoter reporter construct, and with a plasmid encoding shRNA against either IFNαR1 or a control sequence. The cells were incubated with LPS (10 ng/ml) ± isoproterenol (Iso, 1 μM) for the indicated time. Luciferase reporter data represent three independent experiments and are expressed as mean ± SD of values normalized against renilla luciferase activity; ****p < 0.0001 compared to cells treated with LPS alone (two-way ANOVA followed by Tukey's multiple comparison test). The experiment was carried out three times with similar results. (B) Isoproterenol and IFNα exclusively synergize with LPS in IL-10 expression. Mouse macrophage RAW264.7 cells were incubated with LPS (10 ng/ml) and/or isoproterenol (Iso, 1 μM) and/or IFNα (1,000 units/ml) for 3 h. IL-10 secretion to the medium was measured by ELISA and data representing six independent experiments are expressed as mean ± SD; ****p < 0.0001 (two-way ANOVA followed by Tukey's post-test). IL-10 secretion from cells that were not treated with LPS was undetectable (<40 pg/ml). The experiment was carried out 3 times with similar results. Right panel—fold-increase by isoproterenol, calculated from the data presented in the left panel.