Abstract
The epithelial lining of mucosal surfaces acts as a barrier to regulate the entry of antigen and pathogens. Nowhere is this function of the contiguous epithelium more important than in the gut, which is continually exposed to a huge antigenic load and, in the colon, an immense commensal microbiota. We assessed the intracellular signaling events that underlie interferon (IFN) γ-induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFNγ (20 ng/ml), and barrier function was assessed 48 h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of 51Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of noninvasive, nonpathogenic Escherichia coli (strain HB101). Exposure to IFNγ decreased barrier function as assessed by all four markers. The phosphatidylinositol 3′-kinase (PI-3K) inhibitors, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] and wortmannin, did not affect baseline permeability characteristics but completely blocked the drop in TER, increased fluxes of 51Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFNγ. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 μM), but not rottlerin (blocks PKCδ), partially ameliorated the drop in TER and inhibited increased E. coli transcytosis. Addition of the PI-3K and PKC inhibitors to epithelia 6 h after IFNγ exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFNγ-induced increases in epithelial paracellular and transcellular permeability are critically dependent on PI-3K activity, which may represent an epithelial-specific target to treat immune-mediated loss of barrier function.
Footnotes
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This work was supported by the Canadian Institutes of Health Research to DMM (Grant MT-13421 to D.M.M.). D.M.M. holds a Canada Research Chair (Tier 1) in Intestinal Immunophysiology in Health and Disease and is an Alberta Heritage Foundation for Medical Research scientist. J.L.W. was a recipient of a Canadian Institutes of Health Research studentship.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.113639.
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ABBREVIATIONS: IFN, interferon; STAT, signal transducer and activator of transcription; PI-3K, phosphatidylinositol 3′-kinase; PKC, protein kinase C; IL, interleukin; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; EGCG, (-)-epigallacatechin gallate; l-NAME, Nω-nitro-l-arginine methyl ester; NO, nitric oxide; NOS, nitric-oxide synthase; iNOS, inducible NOS; BIM, bisindolylmalemide I; l-NMMA, NG-monomethyl-l-arginine; l-NIL, N6-(1-iminoethyl)-lysine HCl; EPEC, enteropathogenic E. coli; LB, Luria Bertani broth; eGFP, enhanced green fluorescent protein; TER, transepithelial electrical resistance; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; IRF, interferon-regulated factor; iNOS, inducible nitric oxide synthase; EMSA, electrophoretic mobility shift assay; LY, LY294002; PDK, phosphoinositide-dependent kinase; MAPK, mitogen-activated protein kinase; PIA, phosphatidylinositol analog; Akt, protein kinase B; SH5, Akt inhibitor II; API, Akt inhibitor V, Tricirbine; bp, base pair.
- Received September 8, 2006.
- Accepted December 15, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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