The interleukin 12 p40 gene promoter is primed by interferon gamma in monocytic cells.

Abstract

SumlTlaryInterleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting ceils that modulates adaptive immune responses by favoring the generation ofT helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) y production by T and natural killer cells. IFN-y enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-y acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-~/ enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-y directly induces transcription of the 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 transcription. The priming effect of IFN-y on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-~/for at least 8 h to obtain a maximal effect. The priming effect of IFN-~/for 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 posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-y or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-'y priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-y and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. 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 result in decreased response to IFN-y and LPS stimulation.

New insights into IL-12-mediated tumor suppression

“…2 Thus, IL-12 is secreted as an early pro-inflammatory cytokine in response to infections. 3 Additional amplifying signals such as interferon-g (IFN-g), 4 IL-15 5 or cluster of differentiation (CD)40L-CD40 cell-cell interactions 6 are necessary for the optimal production of biologically active IL-12. Conversely, IL-12 is negatively regulated through cytokines such as IL-10 and transforming growth factor-b1 (TGF-b1).…”

Nod1 and Nod2 induce CCL5/RANTES through the NF‐κB pathway

“…Transient transfections were performed by electroporation essentially as previously described [34]. Briefly, murine macrophages of cell line RAW264.7 were grown to confluence (10 6 /mL) in RPMI 1640 medium supplemented with 10% FCS.…”

Regulation of cytokine production during phagocytosis of apoptotic cells

“…Understanding this delicate balance is essential to appreciate the complexity of macrophage biology. A large number of studies have been devoted to the dissection of the molecular mechanisms involved in the regulation of proinflammatory cytokine gene expression, which unveiled several important transcription factor families that mediate inflammatory response of macrophages such as NF-kB, NF-IL6, C/EBP, and interferon regulatory factors [89][90][91][92][93][94][95][96]. In contrast, much less is known about the regulation of anti-inflammatory cytokines.…”