title: Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases.
abstract: Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.
authors.affiliation: Division of Neonatal-Perinatal Medicine, Department of Pediatrics, Emory University, Atlanta, GA 30322, USA.
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Yeligar
authors.forename: Frank L
authors.initials: FL
authors.lastname: Harris
authors.forename: C Michael
authors.initials: CM
authors.lastname: Hart
authors.forename: Lou Ann S
authors.initials: LA
authors.lastname: Brown
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000328:Adult; D000437:Alcoholism / Q000201:enzymology / Q000235:genetics / Q000276:immunology; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology; D002460:Cell Line; D000431:Ethanol / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology; D008168:Lung / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D008297:Male; D051379:Mice; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology; D018384:Oxidative Stress / Q000187:drug effects; D010587:Phagocytosis / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000276:immunology; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D000098:Acetophenones; D007527:Isoenzymes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000431:Ethanol; C056165:acetovanillone; D019255:NADPH Oxidases