| Clin Diagn Lab Immunol. 1996 Jul;3(4):392-8. |
|
Alcohol inhibits lipopolysaccharide-induced
tumor necrosis factor alpha gene expression by peripheral blood mononuclear
cells as measured by reverse transcriptase PCR in
situ hybridization.
Nair MP, Kumar NM, Kronfol ZA,
Greden JF, Lwebuga-Mukasa JS, Schwartz SA.
Department of Medicine, State University of New
York at Buffalo, Buffalo General Hospital
14203,
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.
PMID: 8807202 [PubMed
- indexed for MEDLINE]