Brightbill H D, Plevy S E, Modlin R L, Smale S T
Department of Microbiology, Division of Dermatology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.
J Immunol. 2000 Feb 15;164(4):1940-51. doi: 10.4049/jimmunol.164.4.1940.
IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.
白细胞介素-10（IL-10）是一种由活化的巨噬细胞和Th2细胞分泌的抗炎细胞因子。IL-10的分泌促进促炎细胞因子合成的下调以及Th2反应的发展。在巨噬细胞中，促炎细胞因子似乎由相似的机制诱导产生，但IL-10的诱导机制尚未得到研究。我们分析了用脂多糖（LPS）激活的RAW264.7巨噬细胞系中的小鼠IL-10启动子。全面的突变分析表明，核心启动子上游只有一个元件对启动子诱导至关重要。进一步的精细突变分析将该元件定位到核苷酸-89至-78，凝胶迁移实验表明它代表Sp1的一个非共有结合位点。Sp1的功能相关性得到了以下方面的支持：相互作用的高亲和力、Sp1结合所需核苷酸与启动子功能之间的密切相关性，以及Sp1共有序列替代-89/-78启动子序列的能力。Sp1结合位点对IL-10诱导的唯一需求、Sp1位点赋予异源启动子诱导能力以及对可介导诱导的Sp1结构域的描绘，都提供了证据表明Sp1可能是参与IL-10诱导的信号通路的靶点。未观察到调控大多数促炎细胞因子启动子的Rel、C/EBP（CCAAT/增强子结合蛋白）或AP-1结合位点有相关作用。总之，这些结果表明IL-10基因调控与促炎细胞因子基因的调控不同，并提示Sp1可能是IL-10诱导的核心介导因子。
