An IFN-γ-Inducible Transcription Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates IL-12 p40 Expression in Macrophages

Abstract

IL-12 is a cytokine that links innate and adaptive immunity. Its subunit p40 is induced in macrophages following IFN-γ/LPS stimulation. Here we studied the role for IFN consensus sequence binding protein (ICSBP), an IFN-γ/LPS-inducible transcription factor of the IFN regulatory factor (IRF) family in IL-12 p40 transcription. Macrophage-like cells established from ICSBP−/− mice did not induce IL-12 p40 transcripts, nor stimulated IL-12 p40 promoter activity after IFN-γ/LPS stimulation, although induction of other inducible genes was normal in these cells. Transfection of ICSBP led to a marked induction of both human and mouse IL-12 p40 promoter activities in ICSBP+/+ and ICSBP−/− cells, even in the absence of IFN-γ/LPS stimulation. Whereas IRF-1 alone was without effect, synergistic enhancement of promoter activity was observed following cotransfection of ICSBP and IRF-1. Deletion analysis of the human promoter indicated that the Ets site, known to be important for activation by IFN-γ/LPS, also plays a role in the ICSBP activation of IL-12 p40. A DNA affinity binding assay revealed that endogenous ICSBP is recruited to the Ets site through protein-protein interaction. Last, transfection of ISCBP alone led to induction of the endogenous IL-12 p40 mRNA in the absence of IFN-γ and LPS. Taken together, our results show that ICSBP induced by IFN-γ/LPS, acts as a principal activator of IL-12p40 transcription in macrophages.

Interferon regulatory factor‐1 gene deletion decreases glomerulonephritis in MRL/lpr mice

“…The critical role of IFN mediation of tissue injury was supported by studies showing that when IFN-c was inhibited, IL-12 alone failed to elicit tissue injury in lupus nephritis [38][39][40]. Fantuzzi et al [41] showed decreased production of the pro-inflammatory molecules IFN-c and IL-18 in IRF-1 -/-mice when challenged with inflammatory stimuli.…”

Identification of IRF-8 and IRF-1 target genes in activated macrophages

“…In addition, it was shown that IRF-8 is an essential factor for proper functioning of mature macrophages. For example, IRF-8 null mice fail to mount Th1-mediated immune response, since they fail to produce the IL-12 p40 subunit (Wang et al 2000). Similarly, IRF-1 -/-are also devoid of Th1 responses due to deficiency of the same IL-12 subunit (Taki et al 1997; Lohoff et al 1997).…”

“…Unlike other IRF members, IRF-8 is capable of binding to its target DNA sequence only following association with either IRF-1 and IRF-2 or non-IRF transcription factors, such as PU.1, an essential factor for hematopoiesis (Sharf et al 1997;Wang et al 2000;Dahl and Simon 2003) The domain essential for these protein-protein associations, termed IRF Association Domain (IAD), is conserved among all other IRF members excluding IRF-1 and IRF-2. The association modules of IRF-1 and IRF-2 with IRF-8 were identified as PEST domains, enriched with proline, glutamic acid, serine, and threonine, originally shown in PU.1 .The specific partner that interacts with IRF-8 dictates not only the DNA binding site but also the transcriptional activity e.g.…”

“…CL-2 macrophage cells, established from IRF-8 KO mice, were previously reported (Wang et al 2000). Cells were maintained in RPMI 1640, supplemented with 40μM β-mercaptoethanol, 10% FCS and antibiotics.…”

Effect of amino acid substitutions in the human IFN-γR2 on IFN-γ responsiveness

“…8 Phosphorylated STAT1 dissociates from the receptor, dimerises and translocates to the cell nucleus, where it regulates gene expression either directly (for example, CD54, CXCL10 and CCL2) [9][10][11] or indirectly via the induction of transcription factors such as IRFs and CIITA (for example, IL12B, B2M and major histocompatibility complex). 12,13 Although STAT1 is the main mediator of IFN-g responses, IFN-g has also been reported to induce STAT3 or STAT5 phosphorylation. 14 Besides the JAK2-binding site, the intracellular domain of IFN-gR2 contains a BAX-inhibiting domain 15 and an internalisation motif 16 (Figure 1).…”