Effective host defense against bacterial infection is most dependent upon the vigorous recruitment and activation of neutrophils and macrophages. Chronic alcohol consumption can significantly alter the ability to generate inflammatory responses, predisposing the host to a vast array of local and systemic infections, particularly bacterial infections of the lung. Specific cellular mechanisms by which alcohol attenuates pulmonary host defense against bacterial challenge have not been completely characterized. In this application, we will assess the effect of alcohol on the in vitro and in vivo expression of the important activating and chemotactic cytokines tumor necrosis factor-alpha (TNF), macrophage inflammatory protein-2 (MIP-2), and macrophage inflammatory protein-1 alpha (MIP-1alpha), and the anti-inflammatory cytokine interleukin-10. We have chosen to focus our studies on these cytokines, as our preliminary studies have indicated that: 1) TNF, MIP-2, and MIP-1alpha are important murine leukocyte activating and chemotactic cytokines expressed during the evolution of Klebsiella pneumonia; 2) the alcohol-induced suppression of these cytokines in vivo correlates with functional alterations in leukocyte recruitment and bacterial clearance; and 3) the inhibitory effects of alcohol on cytokine expression, leukocyte recruitment, and bacterial clearance are reversed by passive immunization with neutralizing anti-IL-10 serum. It is the hypothesis of this proposal that recruited neutrophils and macrophages represent important phagocytic cells involved in effective lung antibacterial host defense. Alcohol alters lung leukocyte recruitment and bacterial clearance by inhibiting the in vivo production of the leukocyte activating and chemotactic cytokines, TNF, MIP-2, and MIP- 1alpha. The inhibitory effect of alcohol is due, in part, to the relative overexpression of IL-10, resulting in a chronic state of immunosuppression. A murine model of Klebsiella pneumonia has been developed in our laboratory to achieve the following specific aims: 1) to assess the effect of alcohol on the molecular and cellular regulation of TNF, MIP-2, and MIP1alpha gene expression from cultured murine alveolar macrophages; 2) to determine the in vivo effect of alcohol on the generation of inflammation (leukocyte infiltration and tissue injury), bacterial clearance, and mortality utilizing a murine model of Klebsiella pneumonia; 3) to determine the in vivo effect of alcohol on the compartmentalized expression of TNF, MIP-2, and MIP-1alpha during the evolution of Klebsiella pneumonia; 4) to determine the contribution of TNF, MIP-2, and MIP-1alpha to inflammatory cell recruitment, bacterial clearance, and mortality during the evolution of Klebsiella pneumonia by passive immunization with specific neutralizing antibodies in non-alcohol fed mice, and by reconstituting cytokines intratracheally in alcohol-fed mice; and 5) to determine the contribution of IL-10 to the alcohol-induced suppression of cytokine production in vitro and in vivo. Elucidation of specific molecular mechanisms mediating alcohol-induced suppression of pulmonary host defense will provide insights into the development of novel treatment strategies to be employed in alcoholics with serious bacterial infections of the lung.
