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 lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production by whole blood and total mononuclear cells from healthy subjects as measured by bioassay. In the current study, we further examined the effect of alcohol on LPS-induced TNF-alpha gene expression by semiquantitative solution PCR and in situ reverse transcriptase PCR (RT-PCR) hybridization methods. Peripheral blood mononuclear cells were cultured with LPS (10 micrograms/ml) for 4 to 8 h with or without different concentrations of ethanol (0.1, 0.2, and 0.3% [vol/vol]). Total RNA from treated and untreated cultures was extracted and used for solution PCR analysis. Treated and untreated cells were subjected to both conventional in situ hybridization and RT-PCR in situ hybridization. In solution RT-PCR in vitro analysis, alcohol significantly suppressed TNF-specific message. In conventional in situ hybridization, the effect of alcohol on TNF-alpha gene expression was poorly detected. However, when cells were subjected to RT-PCR prior to in situ hybridization, cells treated with alcohol significantly suppressed expression of the message for TNF-alpha. These studies confirm our earlier finding that alcohol suppressed the production of TNF-alpha by LPS-induced whole blood cells and peripheral blood mononuclear cells. Furthermore, these studies also demonstrate that the RT-PCR in situ technique is a powerful tool for detecting and amplifying specific genes in whole cells when limited numbers of cells are available for RNA extraction.
Nair
M P
MP
Department of Medicine, State University of New York at Buffalo, Buffalo General Hospital 14203, USA.
Kumar
N M
NM
Kronfol
Z A
ZA
Greden
J F
JF
Lwebuga-Mukasa
J S
JS
Schwartz
S A
SA
D000431:Ethanol / Q000633:toxicity*; D015870:Gene Expression / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000187:drug effects* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors*; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000235:genetics*
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol; D012194:RNA-Directed DNA Polymerase