ENHANCED INTERLEUKIN-8 PRODUCTION BY CULTURED CHANG LIVER-CELLS SUBJECTED TO ETHANOL EXPOSURE AND SUBSEQUENT STIMULATION WITH TUMOR-NECROSIS-FACTOR-ALPHA
H. Ohira et al., ENHANCED INTERLEUKIN-8 PRODUCTION BY CULTURED CHANG LIVER-CELLS SUBJECTED TO ETHANOL EXPOSURE AND SUBSEQUENT STIMULATION WITH TUMOR-NECROSIS-FACTOR-ALPHA, Digestion, 58(1), 1997, pp. 72-77
We investigated the production of interleukin-8 (IL-8) and chemotactic
activity released from Chang liver cells subjected to long-term treat
ment with ethanol (Et) and subsequent stimulation with tumor necrosis
factor-alpha (TNF-alpha). Chang liver cells were cultured in the prese
nce of 5, 50 or 100 mmol/l Et for 4 weeks and then treated with recomb
inant TNF-alpha (1, 10, 100 U/ml). The culture supernatants were assay
ed for IL-8 using a sandwich ELISA and chemotactic activity was measur
ed using a chemotactic chamber. Total RNA was also extracted from thes
e cells and IL-8 mRNA was assayed by RT-PCR. In addition, TNF-receptor
expression on the Et-treated cells was analyzed by flow cytometry. IL
-8 levels in supernatants of cells stimulated with 100 U/ml of TNF-alp
ha for 48 h rose significantly with increasing concentrations of Et an
d values obtained were as follows: 4,918 +/- 244.4 pg/ml at 0 mmol/l E
t, 5,335 +/- 266.2 pg/ml at 5 mmol/l Et, 8,726 +/- 873.4 pg/ml at 50 m
mol/l Et and 9,134 +/- 866.0 pg/ml at 100 mmol/l Et. The chemotactic a
ctivity also increased with increasing concentrations of Et and was al
most completely suppressed by anti-IL-8 antibody. Using semiquantitati
ve analysis of radioactivity of IL-8 mRNA using a P-32 gamma ATP-label
ed primer for IL-8 mRNA, Et-treated cells were shown to have markedly
higher levels of radioactivity than untreated cells. In addition, TNF-
receptor expression was significantly higher in cells treated with 100
mmol/l Et. These data suggest that long-term Et treatment of Chang li
ver cells stimulated with TNF-alpha may enhance transcription of the I
L-8 gene with up-regulation of the TNF receptor.