Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression

Abstract

The regulation of secretory interleukin (IL)-1 receptor antagonist (sIL-1Ra) in response to IL-10 is unique. In contrast to most cytokines, the lipopolysaccharide (LPS)-induced expression of the sIL-1Ra gene is enhanced by concomitant treatment with IL-10. Cotreatment of RAW 264.7 cells with IL-10 + LPS resulted in at least a twofold increase in sIL-1Ra promoter activity and mRNA expression compared with LPS alone; IL-10 alone had no effect on promoter activity or mRNA expression. Examination of sIL-1Ra mRNA expression in bone marrow-derived macrophages (BMDM) resulted in identical results. Transfection of RAW 264.7 cells with the sIL-1Ra/luc reporter and a dominant-negative signal transducer and activator of transcription (STAT)3 (Y705A) expression plasmid inhibited the enhanced response induced by exogenous IL-10 in the presence of LPS. The presence of a functional STAT3-binding site within the proximal sIL-1Ra promoter was demonstrated. As IL-10 is produced by LPS-stimulated macrophages, a role for endogenously produced IL-10 in the response of the sIL-1Ra gene to LPS was suggested. This was confirmed in IL-10-deficient BMDM, which when compared with normal BMDM, had significantly decreased LPS-induced sIL-1Ra mRNA levels that could be restored by exogenously provided IL-10, which induced a fivefold increase of LPS-induced IL-1Ra mRNA in cells from IL-10-/- BMDM. Western blot analysis of phosphorylated STAT3 from wild-type and IL-10-/- BMDM and IL-10 neutralization experiments demonstrated a role for endogenously produced IL-10 in the LPS-induced STAT3 activity. Together, these results demonstrate that endogenously produced IL-10 plays a significant role in LPS-induced sIL-1Ra gene expression via the activation of STAT3.