Molecular mechanisms in down-regulation of IL-10 gene expression by IFN-gamma
Interleukin-10 (IL-10) is a suppressive cytokine that down-regulates immune response and inflammation. It was shown previously that IL-10 expression is up-regulated after stimulation with LPS and that IFNgamma is able to down-regulate LPS-induced IL-10 expression.;In the present study, the cis- and trans-elements involved in the IFNgamma-mediated down-regulation of LPS-induced IL-10 expression in the human B-cell line RPMI 8226.1 were analysed. The inhibition of IL-10 trans-activation activity by IFNgamma was time-dependent. A stronger effect was seen when cells were primed with IFNgamma for 2 hours previous to stimulation with LPS. The presence of the STAT-binding site in the IL-10 promoter was sufficient to mediate the down-regulation of LPS-induced IL-10 expression. Of note, stimulation with IFNgamma alone had a slight increasing effect on IL-10 promoter activity in some constructs tested.;In the nucleus, tyrosine-phosphorylated STAT1 and STAT3 were detected in response to IFNgamma and LPS, respectively. After stimulation with both IFNgamma and LPS, binding of the respective STAT1 and STAT3 binding factors to the STAT site of the IL-10 promoter was demonstrated by EMS and supershift assays.;Chromatin immunoprecipitation showed that binding of STAT3 to the IL-10 promoter in response to LPS does occur in intact cells and that this is inhibited by IFNgamma in vivo, possibly by the recruitment of STAT1.;In addition, IFNgamma inhibited the expression of LPS-induced IL-10 mRNA in cells of the RPMI 8226.1 cell line and in human blood mononuclear cells (PBMC). Finally, the expression of IL-10 protein in response to LPS was also down-regulated by IFNgamma.;In conclusion, the present study shows that IFNgamma down-regulates the LPS-induced expression of IL-10 at the promoter, mRNA and protein level by inhibiting binding of STAT3 to the IL-10 promoter.