Date of Award

2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Cell Biology, Neurobiology and Anatomy

Abstract

Extensive evidence indicates that alcohol (ethanol) consumption affects human health by altering normal physiological functions of the immune system. This study investigated the effect of a single in vivo exposure of macrophages to clinically relevant levels of ethanol (1.2 and 2.2 g/kg). Following 3 hour exposure, both doses of ethanol decreased ex vivo TNFα production by splenic and alveolar macrophages (AMs). Interestingly, the higher dose of ethanol resulted in sustained suppression of LPS-induced TNFα production at 3 and 6 hours post ethanol administration, as well as decreased IL-6 and IL-12 production after 6 hours, compared control treated groups. LPS or E. coli-stimulated production of TNFα and IL-6 by AMs were reduced at 3 hours after ethanol administration, compared to the saline treated animals. Additionally, acute in vivo (2.2 g/kg) or in vitro (50 mM) ethanol exposure transiently decreased AMs and RAW264.7 murine macrophage phagocytosis of Pseudomonas aeruginosa as early as 3 hours after initial exposure, with functional recovery by 24 hours. To investigate a single receptor pathway, we focused on Fcgamma-receptor (FcgammaR) mediated phagocytosis. In ethanol treated murine AMs and the RAW264.7 cells, phagocytosis of IgG-coated beads demonstrated a 40 and 50% reduction in actin recruitment to the site of the phagosome compared control conditions. Prior to actin polymerization, the phagocytic response requires recruitment of the adhesion molecules paxillin and vinculin, forming a structure we term "phagosomal adhesion". Ethanol did not alter paxillin phosphorylation during phagocytosis via the FcgammaR-pathway but suppressed vinculin phosphorylation, suggesting a defect in phagosomal adhesion maturation. Small GTPases are known to regulate normal actin polymerization. RAW264.7 cells exposed to NSC23766 (a Rac1 inhibitor) show 45% suppression in actin polymerization to the phagosome, regardless of ethanol exposure. This underscores the importance of normal Rac activity during FcgammaR-mediated phagocytosis. Following 15 minutes of IgG-coated bead phagocytosis, Rac activation increases by only 50% in ethanol exposed cells, compared to 175% in control conditions. Taken together, we conclude that the suppressive effects of clinically observed levels of ethanol are dose dependent, have effects that last after clearance of the ethanol from circulation, and can affect multiple macrophage functions.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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