Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT‐29

Abstract

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. 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. Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 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. 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. 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 106 cells respectively. 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. 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. 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. 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.

Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells

“…In conclusion, our data support the idea of a regulation of polyamine synthesis by NO in HT-29 cells, suggesting the possibility of interference between two metabolic pathways derived from the common precursor L-arginine. NO synthase activity is low in HT-29 cells [7] but recent data suggest that the production of NO increases in HT-29 cells by a combination of cytokines [31]. Further studies are required to determine the effect of cellular NO overproduction on cell growth.…”

Suppressed Induction of Proinflammatory Cytokines by a Unique Metabolite Produced by Vibrio cholerae O1 El Tor Biotype in Cultured Host Cells

“…When HT29 cells were stimulated with these two cytokines, they were reported to produce NO through a distinct pathway known as the inducible nitric oxide synthase (iNOS) pathway (26,33). As shown in Fig.…”

Lactobacillus plantarum Lipoteichoic Acid Alleviates TNF-α-Induced Inflammation in the HT-29 Intestinal Epithelial Cell Line

“…To examine the inhibitory effect of pLTA on the production of iNOS and NO, HT-29 cells were pretreated with pLTA. They were then re-treated with a cytokine cocktail including recombinant human TNF-α, IFN-γ, and IL-1α for 24 to 48 h (Kolios et al, 1995). The levels of iNOS mRNA and NO production were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and a nitro oxide detection kit, respectively.…”