. Increased susceptibility to infections after acute alcohol use is associated with decreased monocyte production of tumor necrosis factor, (interleukin-1 and interleukin-6), cytokines regulated by transcription factor NF-kB. In contrast with enhanced nuclear translocation of the activating p65/p50 NF-kB heterodimer following LPS stimulation, the investigator's preliminary data show that acute alcohol treatment uniquely enhances nuclear binding of the p50/p50 inhibitory homodimer of the NF-kB/Rel family. Thus, they hypothesize that clinically relevant concentrations of alcohol disrupt NF-kB signaling in monocytes by inducing nuclear translocation of the inhibitory p50/p50 homodimer. Unique activation of p50/p50 homodimers by acute alcohol ingestion would result in inhibition rather than activation of NF-kB regulated inflammatory cytokine genes. Moreover, preferential induction of the p50/p50 homodimers by ethanol would desensitize monocytes to additional stimuli of the NF-kB pathway similar to that described for LPS tolerance, which utilizes p50/p50 mediated inhibition of p65/p50-induced genes.
The specific aims of this application are to: (1). investigate the effects of in vivo acute ethanol on monocyte NF-kB/Rel activation, and the mechanisms by which ethanol regulates nuclear translocation and DNA-binding of NF-kB/Rel complexes by evaluating: (a) the kinetics and reversibility of NF-kB/Rel (p50, p65, RelB) and precursor (p105, p100) induction by in vivo acute ethanol in human monocytes; (b) synthesis, nuclear translocation and binding of different NF-kB/Rel dimers in northern (RNA), western blots (protein), gelshift and supershift experiments; (c) role of oxygen radicals. (2) Evaluate the significance of p65- and p50-specific inhibitory kB (IkB) proteins in ethanol-induced changes in NF-kB/Rel activation: Increased stabilization of the IkB molecules that bind p65 will be assessed by investigating the effect of ethanol on IkB tyrosine phosphorylation, degradation, and re-synthesis. The role of alcohol-induced decreases in nuclear levels of Bcl-3 or cytoplasmic levels of IKB (p50-specific IkB molecules) in increased nuclear binding of p50/p50 homodimer will be tested. (3) Evaluate the role of increased DNA-binding of the inhibitory p50/p50 homodimers in ethanol-induced desensitization of monocytes to subsequent stimulation: (a) test whether in vivo acute ethanol exposure desensitizes monocytes to subsequent induction through the NF-kB pathway, (b) study amelioration of ethanol induced monocyte desensitization after IFN priming. These studies should delineate the effect of in vivo acute alcohol on NF-kB signaling in monocytes relevant to inflammation, cirrhosis, atherosclerosis, and HIV infection.
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