Alcoholism increases the incidence of lung infection. The mechanism is unknown but may result from an interaction between ethanol, tumor necrosis factor (TNF), corticosterone and nitric oxide (NO). We hypothesize that ethanol-induced changes in NO in lung vascular endothelium (EC), neutrophils (PMN) and alveolar macrophages (AM) mediate, in part, its suppression of pulmonary host defense in a rat model of acute and chronic alcoholism. The specific questions to be answered are (a): Does ethanol upregulate EC production of NO? Free single cell intra- and extracellular NO and NO2 will be measured amperiometrically in the EC of pulmonary artery (PA) and vein (PV) of alcoholic rats (AR) and rats on a non-alcoholic, isocaloric diet (NAI). NO will be measured under basal conditions and during stimulation with TNF, endotoxin (LPS), PMN, bradykinin (BK) and A23187. Constitutive NO synthase (NOS) I and inducible NOS II activity will be assessed in the EC of NAI and AR by quantifying the formation of 14C-citrulline and NO2 from 14C-arginine with HPLC. We will define if ethanol upregulates NOS gene expression or translation by measurement of mRNA for NOS I and II using c-ERT PCR in PA and PV from NAI and AR. Sex differences in ethanol's effect on NO will be tested using male and female rats (day 2 estrous) (n-5/gp/sex). (b): Does up-regulation of EC NO suppress PMN adhesion to EC? PMN, PA and PV obtained from AR and NAI will be co- incubated in Ussing chambers at 37C. Adhesion and migration of PMNs will be assessed using histologic and 51Gr-labelling techniques. EC and PMN changes in NO synthesis, enzyme activity and mRNA and superoxide (SOX) assayed by cytochrome c reductase activity will be evaluated. (c): Does ethanol down-regulate AM and recruited PMN NO to decrease phagocytosis and bacterial killing? AR and NAI will be treated with Klebsiella pneumonia or LPS and AM and PMN assayed for bacterial killing, phagocytosis and changes in the NO and SOX systems as described above. The role of NO in pulmonary host defense will be critically tested using AR and NAI pretreated with modulators of the NO system. (d): Does ethanol deregulated NOS I and II by down-regulating TNFalpha and upregulating corticosteroids?. AT and NAI will be given exogenous TNF, LPS, TNF receptor antagonist or RU38486. PMN adhesion, AM and PMN bacterial killing, phagocytosis, TNF and NO assayed as described above. The role of cAMP, adenosine, Ca2+ and intracellular Fe3 in this process will be tested. This project will define new approaches for preventing and treating ethanol-induced suppression of lung host-defense mechanisms.
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