A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the downregulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, pr oinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the mu rine IL-10 promoter in the RAW264,7 macrophage line activated with LPS, A c omprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analy sis localized this element to nucleotides -89 to -78, and gel shift experim ents revealed that it represents a nonconsensus binding site for Sp1. The f unctional relevance of Sp1 was supported by the high affinity of the intera ction, the close correlation between the nucleotides required for Sp1 bindi ng and promoter function, and the ability of an Sp1 consensus sequence to s ubstitute for the -89/-78 promoter sequence. Evidence that Spl may be a tar get of signaling pathways involved in IL-10 induction was provided by the e xclusive requirement for the Sp1 binding site, by the ability of the Spl si te to confer induction to a heterologous promoter, and by the delineation o f an Sp1 domain that can mediate induction. No relevant contribution from R eI, C/EBP (CCAAT/ enhancer-binding protein), or AP-1 binding sites, which r egulate most proinflammatory cytokine promoters, was observed. Together, th ese results demonstrate that IL-10 gene regulation is distinct from the reg ulation of proinflammatory cytokine genes, and suggest that Sp1 may be a ce ntral mediator of IL-10 induction.