Brown LAS, Ping X-D, Harris FL, Gauthier TW. Glutathione availability modulates alveolar macrophage function in the chronic ethanol-fed rat. Am J Physiol Lung Cell Mol Physiol 292: L824-L832, 2007. First published November 22, 2006; doi:10.1152/ajplung.00346.2006.-We have previously demonstrated that chronic alcohol exposure decreases glutathione in the alveolar space. Although alcohol use is associated with decreased alveolar macrophage function, the mechanism by which alcohol impairs macrophage phagocytosis is unknown. In the current study, we examined the possibility that ethanol-induced alveolar macrophage dysfunction was secondary to decreased glutathione and subsequent chronic oxidative stress in the alveolar space. After 6 wk of ethanol ingestion, oxidant stress in the alveolar macrophages was evidenced by a 30-mV oxidation of the GSH/GSSG redox potential (P Յ 0.05). For control macrophages, ϳ80% internalized fluorescent Staphylococcus aureus were added in vitro. In contrast, only 20% of the macrophages from the ethanol-fed rats were able to bind and internalize fluorescent S. aureus. This ethanol-induced decreased capacity for phagocytosis was paralleled by increased apoptosis. When added to the ethanol diet, the glutathione precursors procysteine or N-acetyl cysteine normalized glutathione and oxidant stress in the epithelial lining fluid as well as the alveolar macrophages to control values. This attenuation of oxidant stress was associated with normalization of macrophage phagocytosis and viability. These results suggested that decreased glutathione availability in the alcoholic lung contribute to alveolar macrophage dysfunction via oxidative stress, resulting in not only decreased function but decreased viability. oxidative stress; apoptosis; lung; antioxidants A HISTORY OF CHRONIC ALCOHOL abuse increases the risk for infection, particularly in the lung (2). Although many mechanisms are undoubtedly involved, the increased risk of respiratory infections by alcoholics is partially due to an impaired immune response of the resident alveolar inflammatory cell, the alveolar macrophage. This impaired response is due in part to decreased ability of alveolar macrophages to phagocytose and clear infectious particles from the airways (3, 11). Equally important is impaired release of proinflammatory cytokines, chemokines, and oxidant radicals required for microbial killing (28).In the epithelial lining fluid (ELF) of the alveoli, the antioxidant glutathione (GSH) is essential for the detoxification of endogenous and exogenous oxidant radicals and protection of cells residing in the airway and alveolus. Under stressed conditions such as hyperoxia, the alveolar macrophage rely on the ELF pool of GSH to provide amino acids for de novo GSH synthesis (9), to protect themselves from oxidant injury (20) and maintain membrane integrity during their respiratory burst (30). Thus availability of extracellular GSH or its precursor amino acids is essential to maintain intracellular macrophage GSH homeostasis necessary for o...

BACKGROUND Chronic alcohol abuse is a comorbid variable of Acute Respiratory Distress Syndrome (ARDS). Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Nox) are the main source of reactive oxygen species (ROS) in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2 and Nox4 expression. METHODS AMs were isolated from male C57BL/6J mice, aged 8-10 weeks, which were treated ± ethanol in drinking water (20% w/v, 12 weeks). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox siRNAs (20-35 nM), prior to ethanol exposure. Human AMs were isolated from alcoholic and control patients’ bronchoalveolar lavage fluid. Nox mRNA levels (qRT-PCR), protein levels (western blot and immunostaining), oxidative stress (DCFH-DA and Amplex Red analysis), and phagocytosis (S. aureus internalization) were measured. RESULTS Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox siRNAs 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 alcoholics compared with controls. CONCLUSIONS Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.

In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome (ARDS). Because alveolar type II cell viability is critical for epithelial repair, our objective was to determine if chronic ethanol ingestion increased the sensitivity of type II cells to the inflammatory mediators upregulated during sepsis. In rats chronically fed ethanol, type II cell mitochondrial GSH was depleted, and tumor necrosis factor-alpha (TNF-alpha)-induced generation of mitochondrial reactive oxygen species (ROS) and apoptosis were potentiated. When added to the ethanol diet, the GSH precursor (-)-2-oxo-4-thiazolidinecarboxylic acid (Procysteine; Pro) but not N-acetylcysteine (NAC) normalized type II cell mitochondrial GSH. Likewise, Pro but not NAC normalized TNF-alpha-induced mitochondrial ROS and apoptosis. This suggested that chronic ethanol ingestion potentiated TNF-alpha-induced apoptosis in type II cells via mitochondrial GSH depletion. This may be particularly relevant in ARDS when type II cell viability is critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of ARDS patients with a history of alcohol abuse.

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These results suggested that chronic ethanol ingestion resulted in a progressive depletion of mitochondrial GSH and sensitization of type II cells to inflammatory mediator-induced apoptosis and necrosis. These effects may be particularly relevant during acute stress when proliferation and differentiation of these cells are critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of acute respiratory distress syndrome in patients with a history of alcohol abuse.