ALCOHOL INHIBITS LIPOPOLYSACCHARIDE-INDUCED TUMOR-NECROSIS-FACTOR-ALPHA GENE-EXPRESSION BY PERIPHERAL-BLOOD MONONUCLEAR-CELLS AS MEASURED BY REVERSE-TRANSCRIPTASE PCR IN-SITU HYBRIDIZATION

We recently showed that alcohol significantly suppressed lipopolysacch aride (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as m easured by bioassay. In the current study, we further examined the eff ect of alcohol on LPS-induced TNF-alpha gene expression by semiquantit ative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybr idization methods. Peripheral blood mononuclear cells were cultured wi th LPS (10 mu g/ml) for 4 to 8 h wither without different concentratio ns of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated a nd untreated cultures was extracted and used for solution PCR analysis . Treated and untreated cells were subjected to both conventional in s itu hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific mess age. In conventional in situ hyhridization, the effect of alcohol on T NF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated wit h alcohol significantly suppressed expression of the message for TNF-a lpha. These studies confirm our earlier finding that alcohol suppresse d the production of TNF-alpha by LPS-induced whole blood cells and per ipheral blood mononuclear cells, Furthermore, these studies also demon strate that the RT-PCR in situ technique is a powerful tool for detect ing and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.