ABSTRACT
IFN regulatory factors (IRFs) are a family of transcription factors and include several members that regulate expression of pro- and anti-inflammatory genes. Mice with a targeted mutation in IRF-2 (IRF-2(-/-)) were studied after injection of LPS to evaluate the importance of IRF-2 in the regulation of endotoxicity. IRF-2(-/-) mice were highly refractory to LPS-induced lethality. Although hepatic TNF-alpha mRNA and circulating TNF-alpha were significantly elevated in LPS-challenged IRF-2(-/-) mice, levels of IL-1, IL-12, and IFN-gamma mRNA and protein, as well as IL-6 protein, were significantly lower than levels seen in LPS-challenged IRF-2(+/+) mice. IRF-2(-/-) mice were also more refractory to TNF-alpha challenge than were control mice, which was consistent with their diminished sensitivity to LPS, yet no significant difference in the mRNA expression of TNFRs was observed. IL-12R beta 2 mRNA levels from LPS-challenged IRF-2(-/-) mice were significantly different after 1, 6, and 8 h, suggesting that both diminished IL-12 and altered IL-12R expression contribute to the paucity of IFN-gamma produced. IRF-2 knockout mice also failed to sustain LPS-inducible levels of IRF-1 and IFN consensus sequence binding protein mRNA expression, two transacting factors required for IL-12 transcription, perhaps as a result of diminished IL-1 beta, IL-6, and IFN-gamma levels. Liver sections from IRF-2(+/+) and IRF-2(-/-) mice were analyzed 6 h after a typically lethal injection of LPS. IRF-2(-/-) mice exhibited greater numbers of apoptotic Kupffer cells than did wild-type mice, suggesting a novel anti-apoptotic role for IRF-2. Collectively, these findings reveal a critical role for IRF-2 in endotoxicity, and point to a previously unappreciated role for IRF-2 in the regulation of apoptosis.
Subject(s)
DNA-Binding Proteins/physiology , Lipopolysaccharides/toxicity , Transcription Factors , Animals , Antigens, CD/biosynthesis , Antigens, CD/genetics , Apoptosis/immunology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endotoxemia/genetics , Endotoxemia/immunology , Endotoxemia/mortality , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Injections, Intraperitoneal , Interferon Regulatory Factor-2 , Interferon Regulatory Factors , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/biosynthesis , Interleukin-1/antagonists & inhibitors , Interleukin-1/biosynthesis , Interleukin-10/biosynthesis , Interleukin-10/genetics , Interleukin-12/antagonists & inhibitors , Interleukin-12/biosynthesis , Interleukin-12/metabolism , Interleukin-6/antagonists & inhibitors , Interleukin-6/biosynthesis , Kupffer Cells/cytology , Kupffer Cells/immunology , Lipopolysaccharides/administration & dosage , Liver/immunology , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/biosynthesis , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/genetics , Receptors, Interleukin-12 , Receptors, Tumor Necrosis Factor/biosynthesis , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Recombinant Proteins/administration & dosage , Recombinant Proteins/toxicity , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Tumor Necrosis Factor-alpha/administration & dosage , Tumor Necrosis Factor-alpha/toxicityABSTRACT
The etiology of many end-organ problems associated with hemorrhage has been attributed to the inflammatory response to hemorrhage. In a murine model of nonresuscitated, fixed-volume hemorrhage, we sought to elucidate the role that hemorrhagic insult alone plays in the generation of the early inflammatory cascade. Differences could be appreciated as early as 1 h post-hemorrhage, with consistent differences detected by 3 h in all of the major cytokine genes studied. Significant upregulation of IL-1beta , IL-6, TNF-alpha, and IL-10 mRNA expression was observed in both the liver and lung samples of mice subjected to fixed-volume hemorrhage when compared with sham-hemorrhaged mice. The cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) genes also were upregulated in the livers and lungs of hemorrhaged mice. Finally, expression of the genes that encode the Toll-like receptors (TLR)-2 and -4 was increased by hemorrhage. Taken collectively, these data demonstrate that the initial inflammatory cascade associated with hemorrhage occurs within hours after the initial hemorrhagic event, and can be associated with significant modulation of expression of key pro- and anti-inflammatory cytokine, enzyme, and TLR genes, suggesting that these may be possible new therapeutic targets.
Subject(s)
Drosophila Proteins , Hemorrhage/complications , Hemorrhage/genetics , Inflammation/etiology , Inflammation/genetics , Animals , Base Sequence , Cyclooxygenase 2 , Cytokines/genetics , DNA, Complementary/genetics , Disease Models, Animal , Female , Gene Expression , Isoenzymes/genetics , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Prostaglandin-Endoperoxide Synthases/genetics , Receptors, Cell Surface/genetics , Time Factors , Toll-Like Receptor 2 , Toll-Like ReceptorsABSTRACT
Cyclooxygenase (COX) exists as two isoforms: COX-1, which is constitutively expressed in most cell types; and COX-2, which is inducible by lipopolysaccharide (LPS) and cytokines in a variety of cell types. Although previous studies have implicated two DNA binding proteins, interferon regulatory factor (IRF)-1 and IRF-2, in the regulation of LPS- and IFN-gamma-induced COX-2, their effects in vivo and in vitro are not well-defined. Using real-time PCR, COX-2 gene expression in the livers and lungs of mice challenged in vivo and in macrophages stimulated with LPS in vitro was investigated in wild-type and in IRF-1 and IRF-2 knockout mice. In response to 35 mg/kg LPS, IRF-1-, but not IRF-2-deficient mice, exhibited much poorer induction of COX-2 gene expression in both the livers and lungs. In vitro, COX-2 mRNA levels were also poorly induced in IRF-1-deficient macrophages, while IRF-2- deficient macrophages exhibited higher levels than in normal macrophages. IRF-1 and IRF-2 were confirmed to activate and repress expression of the COX-2 promoter, respectively, in a transient transfection system and the role of specific DNA binding sites confirmed by site-specific mutagenesis. Collectively, these data provide evidence for an important role for IRF-1 in vivo and in vitro and for IRF-2 in vitro in the regulation of COX-2 expression by LPS.
