THE INTERLEUKIN-12 P40 GENE PROMOTER IS PRIMED BY INTERFERON-GAMMA INMONOCYTIC CELLS

Interleukin (IL) 12 is a proinflammatory cytokine produced by phagocyt ic cells, B cells, and other antigen-presenting cells that modulates a daptive immune responses by favoring the generation of T helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) gamma production by T and natu ral killer cells. IFN-gamma enhances the ability of the phagocytic cel ls to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-i nduced IFN-gamma acts in a positive feedback loop that represents an i mportant amplifying mechanism in the inflammatory response to infectio ns. We show here that IFN-gamma enhances IL-12 production mostly by pr iming phagocytic cells for lipopolysaccharide (LPS)induced transcripti on of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 h eterodimer; furthermore, IFN-gamma directly induces transcription of t he IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcri ption. The priming effect of IFN-gamma on the LPS-induced p40 gene tra nscription requires preincubation of the cells with IFN-gamma for at l east 8 h to obtain a maximal effect. The priming effect of IFN-gamma f or IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of pos ttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell Lines recapitulate d the tissue specificity of the endogenous gene, being silent in two h uman T cell lines, constitutively active in two human Epstein-Barr vir us-positive B lymphoblastoid cell lines, and LPS inducible in the huma n THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p 40 gene in response to IFN-gamma or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p 40 promoter. A requirement for the region between -222 and -204 in bot h LPS responsiveness and IFN-gamma priming was established. This regio n contains an ets consensus sequence that was shown to mediate activat ion of the promoter by IFN-gamma and LPS, as well as by a cotransfecte d ets-2. The -222 construct was also regulated in a tissue-specific ma nner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not r esult in decreased response to IFN-gamma and LPS stimulation.