Chronic Ethanol Ingestion in Rats Decreases Granulocyte-Macrophage Colony-Stimulating Factor Receptor Expression and Downstream Signaling in the Alveolar Macrophage

Abstract

Although it is well recognized that alcohol abuse impairs alveolar macrophage immune function and renders patients susceptible to pneumonia, the mechanisms are incompletely understood. Alveolar macrophage maturation and function requires priming by GM-CSF, which is produced and secreted into the alveolar space by the alveolar epithelium. In this study, we determined that although chronic ethanol ingestion (6 wk) in rats had no effect on GM-CSF expression within the alveolar space, it significantly decreased membrane expression of the GM-CSF receptor in alveolar macrophages. In parallel, ethanol ingestion decreased cellular expression and nuclear binding of PU.1, the master transcription factor that activates GM-CSF-dependent macrophage functions. Furthermore, treatment of ethanol-fed rats in vivo with rGM-CSF via the upper airway restored GM-CSF receptor membrane expression as well as PU.1 protein expression and nuclear binding in alveolar macrophages. Importantly, GM-CSF treatment also restored alveolar macrophage function in ethanol-fed rats, as reflected by endotoxin-stimulated release of TNF-α and bacterial phagocytosis. We conclude that ethanol ingestion dampens alveolar macrophage immune function by decreasing GM-CSF receptor expression and downstream PU.1 nuclear binding and that these chronic defects can be reversed relatively quickly with rGM-CSF treatment in vivo.

Zinc Deficiency Mediates Alcohol-Induced Alveolar Epithelial and Macrophage Dysfunction in Rats

“…Our research group has determined that chronic alcohol ingestion in experimental animals as well as in otherwise healthy individuals has profound effects on airway epithelial and macrophage function (4)(5)(6)(7)(8)(9). Specifically, the epithelial barrier within the small gas-exchanging airways, the alveoli, is compromised.…”

“…For example, we recently determined that chronic alcohol ingestion in an experimental model decreases GM-CSF receptor expression in both the alveolar epithelium and the alveolar macrophage (6,10) and, in parallel, decreases GM-CSF-dependent barrier formation in the alveolar epithelium (10) and bacterial phagocytic function in the alveolar macrophage (6). Importantly, treating alcohol-fed rats with recombinant GM-CSF via the upper airway normalizes lung epithelial barrier function and decreases endotoxin-induced lung injury (11), and restores alveolar macrophage innate immune functions (6). More recently, we determined that chronic alcohol ingestion significantly alters the expression and/or membrane localization of several key proteins that form the tight junctions between the alveolar epithelial cells and limit paracellular permeability, and that GM-CSF treatment has salutary effects on tight junction protein expression (12).…”

“…Cells were stained for 1 hour at room temperature and resulting fluorescence was measured using FACScan flow cytometer (Becton Dickinson, San Jose, CA). To correlate cellular function with zinc levels, GM-CSF receptor expression and phagocytosis were measured in the alveolar macrophages as we have published previously (6).…”

“…Adult Male Sprague-Dawley rats (initial weights 150-200 g; Charles River Laboratory, Wilmington, MA) were fed the Lieber-DeCarli liquid diet (Research Diets, New Brunswick, NJ) containing either alcohol (ethanol; 36% of total calories) or an isocaloric substitution with maltindextrin ad lib for 6 weeks as previously published (6,10,18). Zinc acetate (10, 100, or 500 mg/L) was added to the alcohol diets of some rats; each mg of zinc acetate provides approximately 0.3 mg of elemental zinc.…”

“…Membrane and intracellular expression of GM-CSF receptors on alveolar macrophages were measured by an established protocol we have published previously (6). Briefly, cells were incubated for 30 minutes at room temperature with rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) to either the rat GM-CSF receptor a or b subunit, or to an isotype-matched control antibody.…”