American Association of Immunologists | 2012-04-15, Pages 3648-3657
Article | Post-print: After Peer Review
Abstract:
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.
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