Gene-Specific Repression of Proinflammatory Cytokines in Stimulated Human Macrophages by Nuclear IκBα

We have previously shown that increased nuclear accumulation of I kappa B alpha inhibits NF-kappa B activity and induces apoptosis in human leukocytes. In this study, we wanted to explore the possibility that the nucleocytoplasmic distribution of I kappa B alpha can be used as a therapeutic target for the regulation of NF-kappa B-dependent cytokine synthesis. Treatment of LPS-stimulated human U937 macrophages with an inhibitor of chromosome region maintenance 1-dependent nuclear export, leptomycin B, resulted in the increased nuclear accumulation of I kappa B alpha and inhibition of NF-kappa B DNA binding activity, caused by the nuclear I kappa B alpha-p65 NF-kappa B interaction. Surprisingly, however, whereas mRNA expression and cellular release of TNF-alpha, the beta form of pro-IL-1 (IL-1 beta), and IL-6 were inhibited by the leptomycin B-induced nuclear I kappa B alpha, IL-8 mRNA expression and cellular release were not significantly affected. Analysis of in vivo recruitment of p65 NF-kappa B to NF-kappa B-regulated promoters by chromatin immunoprecipitation in U937 cells and human PBMCs indicated that although the p65 recruitment to TNF-alpha, IL-1 beta, and IL-6 promoters was inhibited by the nuclear I kappa B alpha, p65 recruitment to IL-8 promoter was not repressed. Chromatin immunoprecipitation analyses using I kappa B alpha and S536 phosphospecific p65 NF-kappa B Abs demonstrated that although the newly synthesized I kappa B alpha induced by postinduction repression is recruited to TNF-alpha, IL-1 beta, and IL-6 promoters but not to the IL-8 promoter, S536-phosphorylated p65 is recruited to IL-8 promoter, but not to TNF-alpha, IL-1 beta, or IL-6 promoters. Together, these data indicate that the inhibition of NF-kappa B-dependent transcription by nuclear I kappa B alpha in LPS-stimulated macrophages is gene specific and depends on the S536 phosphorylation status of the recruited p65 NF-kappa B. The Journal of Immunology, 2010, 185: 3685-3693.