ABSTRACT
BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) influences myeloid cell function. In this study we examined the role of myeloid cell HIF-1alpha on wound healing in vivo using a cell-specific knockout (KO) mouse model. MATERIALS AND METHODS: HIF-1alpha KO mice and wild-type (WT) controls received 8 mm full thickness dorsal dermal wounds. Wound dimensions were measured until full closure. Tissue was obtained from 3-day-old wounds for (immuno-)histochemical analysis. Production of interleukin-1beta (IL-1beta) and nitric oxide (NO) in response to lipopolysaccharide (LPS) and/or desferrioxamine (DFX) was examined in vitro. RESULTS: Early wound closure occurred significantly faster in HIF-1alpha KO mice than in WT mice. Wounds of KO mice contained similar numbers of neutrophils and macrophages, but more activated keratinocytes, consistent with accelerated re-epithelialization. Interestingly, while LPS and LPS+DFX elicited a similar IL-1beta response in macrophages from the 2 mouse types, NO production was blunted in HIF-1alpha KO macrophages. CONCLUSION: Absence of HIF-1alpha in myeloid cells accelerates the early phase of secondary intention wound healing in vivo. This may be associated with a deficient ability of myeloid cells to initiate an appropriate NO production response. Pharmacologic modulators of HIF-1alpha should be explored in situations with abnormal wound healing.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/deficiency , Macrophages/metabolism , Skin/metabolism , Wound Healing/physiology , Animals , Cells, Cultured , Deferoxamine/pharmacology , Drug Combinations , Female , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Interleukin-1beta/metabolism , Keratinocytes/metabolism , Keratinocytes/pathology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/metabolism , Skin/injuries , Skin/pathology , Wound Healing/drug effectsABSTRACT
The butyric acid derivative, 2-(4-morpholynl) ethyl butyrate hydrochloride (MEB), has been reported to induce antigen-specific T cell unresponsiveness and to block T cell-mediated graft-versus-host disease. As a potential therapeutic agent, it was important to determine the effects of MEB on other cells that contribute to immunopathology. Accordingly, we tested the effects of MEB on macrophage functions. MEB did not affect macrophage viability, phagocytic activity, or the activation-induced up-regulation of molecules associated with antigen presentation: MHC-II, CD86, CD40, or ICAM-1. However, MEB potently inhibited activation-induced production of inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha), IL-6, chemokine CCL2 and nitric oxide (NO). MEB inhibited the induction of NO synthase (NOS2), which is necessary for inducible NO, and inhibited nuclear translocation of NFkappaB, suggesting that MEB interferes with the signaling pathway involved in NOS2 induction. Thus, while inducing specific T cell unresponsiveness, MEB also exerts anti-inflammatory activity by acting on macrophages to suppress production of cytokines and NO.
Subject(s)
Antigens/immunology , Butyrates/pharmacology , Histamine Antagonists/pharmacology , Inflammation Mediators/metabolism , Macrophages/metabolism , Morpholines/pharmacology , T-Lymphocytes/drug effects , Animals , Antigens, CD/biosynthesis , B7-2 Antigen , Blotting, Western , CD40 Antigens/biosynthesis , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , Female , Flow Cytometry , Genes, MHC Class II/drug effects , Intercellular Adhesion Molecule-1/biosynthesis , Lymphocyte Activation/immunology , Lymphocyte Activation/physiology , Macrophages/drug effects , Membrane Glycoproteins/biosynthesis , Mice , Mice, Inbred C57BL , NF-kappa B/drug effects , Nitric Oxide/metabolism , Phagocytosis/drug effectsABSTRACT
We previously reported that inhaled isobutyl nitrite inhibited macrophage tumoricidal activity by inhibiting inducible nitric oxide (NO) production. In the present study, a much shorter inhalant exposure regimen (five daily exposures) inhibited inducible NO and the NO synthase (NOS2). One of the ways in which NO and NOS2 are regulated is by ubiquitin-dependent NOS2 degradation. Immunoprecipitated NOS2 showed increased poly-ubiquitination, following exposure to the inhalant. In addition, Western blots of macrophage nuclear extracts for the NFkappaB subunit, p65, showed that exposure to the inhalant inhibited NFkappaB signaling, necessary for induction of NOS2. The inhalant blocked phosphorylation of the NFkappaB inhibitor, IkappaBalpha. The inhibition of NFkappaB signaling following inhalant exposure was confirmed using mice transgenic for the kappaB-dependent promoter of the HIV 5' LTR linked to luciferase. The data suggested that inhalant exposure likely inhibited macrophage NO production by blocking NFkappaB-mediated activation signaling and promoting poly ubiquitination of NOS2.
Subject(s)
Butanes/administration & dosage , Macrophages, Peritoneal/drug effects , NF-kappa B/antagonists & inhibitors , Nitrates/administration & dosage , Nitric Oxide Synthase/metabolism , Signal Transduction/drug effects , Administration, Inhalation , Animals , Blotting, Western , Female , I-kappa B Proteins/metabolism , Immunoprecipitation , Macrophages, Peritoneal/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-KappaB Inhibitor alpha , NF-kappa B/physiology , Nitric Oxide/antagonists & inhibitors , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase Type II , Phosphorylation , Ubiquitin/metabolismABSTRACT
Nitrite inhalant abuse has been correlated with HIV and Kaposi's sarcoma. Mouse models of inhalant exposure show immunosuppression and loss of immune cells. In the present study, isobutyl nitrite caused a dose-dependent loss of viability of a macrophage cell line. In the absence of cells, isobutyl nitrite reacted with hydrogen peroxide to form peroxynitrite. However, assays of mitochondrial respiration and nitration that detect peroxynitrite indicated that very little was present in cell cultures following exposure to the inhalants. Isobutyl, isoamyl, and butyl nitrites inhibited mitochondrial respiration, but only at high concentrations. Similarly, the nitrating activity of isobutyl nitrite occurred only at high concentrations and was not affected by the presence of hydrogen peroxide. Western blots showed that the inhalant did not increase nitrotyrosine formation in RAW cells or in peritoneal exudate macrophages (PEM) from exposed mice. Thus, the toxicity induced by isobutyl nitrite was probably not due to peroxynitrite formation.
