1-1-diethyl-2-hydroxy-2-nitrosohydrazine and Inflammation

Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

Topics: Anemia, Sickle Cell; Animals; Cell Movement; Cyclic N-Oxides; Disease Models, Animal; Free Radical Scavengers; Hemoglobins; Hemolysis; Humans; Hydrazines; Hydroxyurea; Imidazoles; Inflammation; Leukocytes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Nitric Oxide Donors; Primary Cell Culture; Tumor Necrosis Factor-alpha; Viscosity; Water

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.

Topics: Animals; Apoptosis; Cell Movement; Coculture Techniques; Dose-Response Relationship, Drug; Flow Cytometry; Hydrazines; Immunohistochemistry; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neutrophils; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Peritoneum; Phagocytosis; Staphylococcal Infections; Staphylococcus aureus; Time Factors; Tumor Necrosis Factor-alpha

Although apolipoprotein E3 (apoE3) is known to be atheroprotective, its mechanisms of protection in endothelial cells remain unclear.. Cultured human aortic endothelial cells were stimulated with tumor necrosis factor (TNF)-alpha in the presence of human recombinant apoE3 solubilized in dimyristoyl phosphatidylcholine liposomes. Using flow cytometry and real-time polymerase chain reaction, a significant increase of inflammatory cell adhesion proteins (vascular cell adhesion molecule-1 and E-Selectin), and MCP-1, interleukin-8, and intercellular adhesion molecule-1 gene expression was observed within 5 hours of TNF-alpha exposure, which was markedly attenuated in cells coincubated with apoE3. Treatment with apoE4 resulted in increased inflammatory gene expression relative to either TNF treatment alone or TNF + apoE3 treatment. NO synthase inhibition experiments demonstrated NO to be an active participant in the actions of both TNF and apoE. To clarify the role of NO, dose-response experiments were performed with 0.03 to 300 micromol/L DEA-NONOate. Using flow cytometry and real-time polymerase chain reaction, a modulatory role of NO in TNF-induced endothelial cell activation was observed.. These data suggest a role of vascular wall apoE3 to balance the intracellular redox state in injured endothelial cells via NO-dependent pathways.

Topics: Apolipoprotein E3; Apolipoprotein E4; Arteritis; Cells, Cultured; Chemokine CCL2; E-Selectin; Endothelium, Vascular; Gene Expression Regulation; Humans; Hydrazines; Inflammation; Intercellular Adhesion Molecule-1; Interleukin-8; Nitric Oxide; Nitric Oxide Donors; Oxidation-Reduction; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

The key player for adaptation to reduced oxygen availability is the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of the redox-sensitive HIF-1alpha and the constitutively expressed HIF-1beta subunits. Under normoxic conditions, HIF-1alpha is rapidly degraded, whereas hypoxia, CoCl(2), or desferroxamine promote protein stabilization, thus evoking its transcriptional activity. Because HIF-1 is regulated by reactive oxygen species, investigation of the impact of reactive nitrogen species was intended. By using different nitric oxide (NO) donors, dose- and time-dependent HIF-1alpha accumulation in close correlation with the release of NO from chemically distinct NO donors was established. Intriguingly, small NO concentrations induced a faster but transient HIF-1alpha accumulation than higher doses of the same NO donor. In contrast, NO attenuated up-regulation of HIF-1alpha evoked by CoCl(2) in a concentration- and time-dependent manner, whereas the desferroxamine-elicited HIF-1alpha signal remained unaltered. To demonstrate an autocrine or paracrine signaling function of NO, we overexpressed the inducible NO synthase and used a coculture system of activated macrophages and tubular cells. Expression of the NO synthase induced HIF-1alpha accumulation, which underscored the role of NO as an intracellular activator for HIF-1. In addition, macrophage-derived NO triggered HIF-1alpha up-regulation in LLC-PK(1) target cells, which points to intercellular signaling properties of NO in achieving HIF-1 accumulation. Our results show that NO does not only modulate the HIF-1 response under hypoxic conditions, but it also functions as a HIF-1 inducer. We conclude that accumulation of HIF-1 occurs during hypoxia but also under inflammatory conditions that are characterized by sustained NO formation.

Topics: Animals; Benzoates; Cell Hypoxia; Cell Line; Cobalt; Coculture Techniques; Deferoxamine; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Glutathione; Guanylate Cyclase; Hydrazines; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Inflammation; Kidney Tubules, Proximal; Macrophage Activation; Macrophages; Mice; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrogen Oxides; Nitroso Compounds; Nuclear Proteins; Oxadiazoles; Oxazines; S-Nitrosoglutathione; Spermine; Swine; Transcription Factors