Enhanced interleukin-8 production by cultured Chang liver cells subjected to ethanol exposure and subsequent stimulation with tumor necrosis factor-alpha.
We investigated the production of interleukin-8 (IL-8) and chemotactic activity released from Chang liver cells subjected to long-term treatment with ethanol (Et) and subsequent stimulation with tumor necrosis factor-alpha (TNF-alpha). Chang liver cells were cultured in the presence of 5, 50 or 100 mmol/l Et for 4 weeks and then treated with recombinant TNF-alpha (1, 10, 100 U/ml). The culture supernatants were assayed for IL-8 using a sandwich ELISA and chemotactic activity was measured using a chemotactic chamber. Total RNA was also extracted from these 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-alpha for 48 h rose significantly with increasing concentrations of Et and values obtained were as follows: 4,918 +/- 244.4 pg/ml at 0 mmol/l Et, 5,335 +/- 266.2 pg/ml at 5 mmol/l Et, 8,726 +/- 873.4 pg/ml at 50 mmol/l Et and 9,134 +/- 866.0 pg/ml at 100 mmol/l Et. The chemotactic activity also increased with increasing concentrations of Et and was almost completely suppressed by anti-IL-8 antibody. Using semiquantitative analysis of radioactivity of IL-8 mRNA using a 32P gamma ATP-labeled 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 liver cells stimulated with TNF-alpha may enhance transcription of the IL-8 gene with up-regulation of the TNF receptor.
Ohira
H
H
Department of Internal Medicine II, Fukushima Medical College, Japan.
Obara
K
K
Kuroda
M
M
Takagi
T
T
Miyata
M
M
Tojo
J
J
Ochiai
H
H
Kokubun
M
M
Nishimaki
T
T
Kasukawa
R
R
D000906:Antibodies / Q000032:analysis; D002478:Cells, Cultured; D002633:Chemotaxis / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins; D012997:Solvents / Q000494:pharmacology*; D013268:Stimulation, Chemical; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
D000906:Antibodies; D017931:DNA Primers; D016209:Interleukin-8; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D012997:Solvents; D014409:Tumor Necrosis Factor-alpha; D000431:Ethanol