title: Modulation of proinflammatory cytokines by nitric oxide in murine acute lung injury.
abstract: We tested the hypothesis that NO synthase inhibition alters proinflammatory cytokine expression during acute lung injury in mice. Five-week-old CD-1 mice were pretreated with l-NAME or d-NAME and then received an intratracheal injection of endotoxin (or PBS). TNF-alpha and IL-6 ELISAs and RT-PCR were performed on lung homogenates sampled 6 h later. l-NAME increased TNF-alpha and IL-6 protein and mRNA expression in lungs. Immunostaining demonstrated that TNF-alpha was expressed predominantly by macrophages in the lung. l-NAME did not alter pulmonary macrophage concentration. To better understand the effect of NO synthase inhibition, elicited murine peritoneal macrophages were stimulated in vitro with LPS after addition of l-NAME, d-NAME, nitroprusside, or control. Nuclear proteins were extracted 3 h later and electrophoretic mobility shift and supershift assays were performed using radiolabeled NF-kappaB consensus sequence oligonucleotides. Endotoxin increased NF-kappaB p50/p65 heterodimer binding. Binding was further increased by l-NAME and decreased by nitroprusside. The effect of nitroprusside was not blocked by guanylate cyclase inhibition. We conclude that, in endotoxin-induced acute lung injury, NO synthase inhibition increases proinflammatory cytokine protein and mRNA expression in part because NO decreases the amount of NF-kappaB available for binding to the regulatory region of proinflammatory cytokine genes.
authors.affiliation: University of British Columbia Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, British Columbia, Canada. kwalley@prl.pulmonary.ubc.ca
authors.forename: K R
authors.initials: KR
authors.lastname: Walley
authors.forename: T E
authors.initials: TE
authors.lastname: McDonald
authors.forename: Y
authors.initials: Y
authors.lastname: Higashimoto
authors.forename: S
authors.initials: S
authors.lastname: Hayashi
mesh_terms: D000818:Animals; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004731:Endotoxins / Q000633:toxicity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D007124:Immunoenzyme Techniques; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000502:physiology; D009599:Nitroprusside / Q000494:pharmacology; D012128:Respiratory Distress Syndrome / Q000276:immunology* / Q000473:pathology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D016207:Cytokines; D004731:Endotoxins; D004791:Enzyme Inhibitors; D015850:Interleukin-6; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D009599:Nitroprusside; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019331:NG-Nitroarginine Methyl Ester