Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells

doi:10.1016/S0016-5085(98)70637-7

Gastroenterology

Volume 114, Issue 1, January 1998, Pages 93-102

https://doi.org/10.1016/S0016-5085(98)70637-7 Get rights and content

Abstract

Background & Aims: Enterocytes play a major role in the mucosa as a source of proinflammatory cytokines and cytotoxins. We tested the hypothesis that bacteria induce expression of the inducible nitric oxide synthase (iNOS) in cultured human enterocytes. Methods: DLD-1 and Caco-2BBe cell monolayers exposed to Salmonella dublin were analyzed for iNOS up-regulation and nitric oxide production (NO_x) in the presence of various proinflammatory cytokines. Results: S. dublin augmented NO_x in interferon gamma (IFN-γ)-primed cells but had no independent effect on iNOS expression. S. dublin–induced NO_x was not mediated by endotoxin and was augmented by an enteroinvasive phenotype. In DLD-1 cells, S. dublin–mediated NO_x was blocked by inhibitors of nuclear factor kappa B (NF-κB) and tyrosine kinase activation and was steroid resistant. Cis-acting elements in the human iNOS promoter responsive to endotoxin and S. dublin stimulation of IFN-γ–treated DLD-1 cells were identified between 10.9 and 8.7 kilobases upstream of the transcription initiation site. Conclusions: S. dublin alters the regulation of iNOS messenger RNA in IFN-γ–treated intestinal epithelial cells via a steroid-resistant pathway involving NF-κB and tyrosine kinase activity. Because bacterial interaction with cytokine-primed epithelial cells induces the synthesis of NO, an endogenous antimicrobial agent, these findings may have implications for the regulation of mucosal immunity.

GASTROENTEROLOGY 1998;114:93-102

Section snippets

Cell culture

DLD-1 cells and Caco-2BBe cells (American Type Culture Collection, Rockville, MD) were grown at 37°C in 5% CO₂ in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 4 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. DLD-1 cells, between passages 5 and 15, were seeded at a density of 50,000 cells/cm² in 24- and 96-well plates and allowed to grow for 72-96 hours to confluence before use. Caco-2BBe cells, between passages 5 and 15, were seeded at a

LPS or S. dublin alone do not induce NO synthesis by DLD-1 cells

Untreated DLD-1 cells and cells exposed to LPS (10 μg/mL) have no detectable NO production. To determine whether intact microorganisms would induce iNOS expression, we selected for study a Salmonella sp., which is a pathogenic gram-negative species capable of invading the intestinal epithelium and eliciting a vigorous inflammatory response.⁸ In preliminary experiments, we observed that coincubation of S. dublin with DLD-1 cells for 18 hours resulted in a loss of viability of the enterocytes.

Discussion

The major new finding of this study is that bacteria have the capacity to induce expression of the human iNOS gene in epithelial cells. Although the regulation of iNOS expression by bacteria has been reported in “professional” immune cells, such as macrophages and neutrophils, this is the first report to our knowledge showing that epithelial cells can respond to bacteria by inducing NO production. This response is independent of the LPS content of the bacteria and is augmented by bacterial

Acknowledgements

The authors thank Jennifer Giles and Vivian Xue for expert technical assistance with tissue culture and analytical assays.

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^☆: Address requests for reprints to: Andrew L. Salzman, M.D., Division of Critical Care Medicine, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229. e-mail: [email protected]; fax: (513) 636-4267.

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Alimentary TractBacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells☆

Abstract

Section snippets

Cell culture

LPS or S. dublin alone do not induce NO synthesis by DLD-1 cells

Discussion

Acknowledgements

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Alimentary Tract
Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells☆