Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-κB pathway
Andrew T. Gewirtz, Anjali S. Rao, Peter O. Simon Jr., Didier Merlin, Denice Carnes, James L. Madara, Andrew S. Neish
Andrew T. Gewirtz, Anjali S. Rao, Peter O. Simon Jr., Didier Merlin, Denice Carnes, James L. Madara, Andrew S. Neish
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Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-κB pathway

Abstract

Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules. This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia. IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-α and S. typhimurium was NF-κB dependent. However, NF-κB activation and IL-8 secretion induced by S. typhimurium, but not by TNF-α, was preceded by and required an increase in intracellular [Ca2+]. Additionally, agonists that increased intracellular [Ca2+] by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion. Furthermore, the ability of S. typhimurium mutants to induce IκB-α degradation, NF-κB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular [Ca2+] increase in model intestinal epithelia, but not with their ability to invade these cells. Finally, S. typhimurium, but not TNF-α, induced a Ca2+-dependent phosphorylation of IκB-α. These results indicate that S. typhimurium–induced activation of NF-κB–dependent epithelial inflammatory responses proceeds by a Ca2+-mediated activation of an IκB-α kinase. These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event.

Authors

Andrew T. Gewirtz, Anjali S. Rao, Peter O. Simon Jr., Didier Merlin, Denice Carnes, James L. Madara, Andrew S. Neish

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S. typhimurium, but not TNF-α, induce Ca2+ mobilization that is required...
S. typhimurium, but not TNF-α, induce Ca2+ mobilization that is required for NF-κB activation and IL-8 secretion. (a, b) Fluorometric assay of intracellular calcium. Model intestinal epithelia were loaded with the Ca2+ indicator Fura-2 and placed in a thermostated spectrofluorometer and intracellular Ca2+ was measured as described in Methods. TNF-α was added basolaterally or S. typhimurium was added apically as indicated. In b, intracellular [Ca2+] was measured in response to S. typhimurium in the absence or presence of intracellular BAPTA, which was simultaneously loaded with Fura-2. (c) IL-8 ELISA. Model intestinal epithelia were stimulated basolaterally with TNF-α or infected apically with S. typhimurium in the presence (filled bars) or absence (open bars) of 15 μM BAPTA. Basolateral IL-8 secretion was measured 5 hours later. (d) EMSA with IL-8 probe. Model intestinal epithelia were treated as in c. After stimulation (30 minutes for TNF-α; 1 hour for S. typhimurium), nuclear extracts were isolated and NF-κB translocation assessed as described in Methods.