title: Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT-29.
abstract: 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-1 alpha (IL-1 alpha), tumour necrosisfactor-alpha (TNF-alpha), interferon gamma (IFN-gamma) 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 micrograms 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-1 alpha/IFN-gamma 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-1 alpha/IFN-gamma 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-1 alpha/IFN-gamma 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-1 alpha (0.3-10 ng ml-1) and IFN-gamma (10-300 u ml-1) in combination induced a concentration-dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL-1 alpha/IFN-gamma stimulation reduced nitrite levels to basal values. These data suggest that the IL-1 alpha/IFN-gamma-induced nitrite production by HT-29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF-alpha produced a significant (P < 0.001) 3 fold increase of IL-1 alpha/IFN-gamma-induced nitrite generation. In marked contrast the presence of TNF-alpha had no effect on IL-1 alpha/IFN-gamma-induced iNOS mRNA expression by HT-29 cells. These findings suggest that the up-regulation by TNF-alpha of IL-1 alpha/IFN-gamma-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.
authors.affiliation: Department of Pharmacology, University of Bath, Claverton Down.
authors.forename: G
authors.initials: G
authors.lastname: Kolios
authors.forename: Z
authors.initials: Z
authors.lastname: Brown
authors.forename: R L
authors.initials: RL
authors.lastname: Robson
authors.forename: D A
authors.initials: DA
authors.lastname: Robertson
authors.forename: J
authors.initials: J
authors.lastname: Westwick
mesh_terms: D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004790:Enzyme Induction; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D007375:Interleukin-1; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase