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Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT‐29
Author(s) -
Kolios George,
Brown Zarin,
Robson Rachel L.,
Robertson Duncan A.F.,
Westwick John
Publication year - 1995
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1995.tb15938.x
Subject(s) - nitrite , nitric oxide synthase , nitric oxide , cycloheximide , cell culture , microbiology and biotechnology , cytokine , biology , chemistry , endocrinology , immunology , nitrate , protein biosynthesis , ecology , genetics
1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 10 6 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml −1 ) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant ( P < 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL‐lα/IFN‐γ combination revealed significant ( P < 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 10 6 cells respectively. 6 Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml −1 ) and IFN‐γ (10–300 u ml −1 ) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF‐α produced a significant ( P < 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level. 9 These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.