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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Phosphatidylinositol 3'-Kinase Is a Critical Mediator of Interferon--Induced Increases in Enteric Epithelial Permeability

Intestinal Disease Research Programme (D.M.M., J.L.W., A.W., J.C., D.P., P.M.J.C., J.L.), Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and Department of Surgical and Gastroenterological Sciences, University Hospital Padova, Italy (V.D.L.)

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) -induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFN (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 IFN 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 IFN. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 µM), but not rottlerin (blocks PKC), 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 IFN exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFN-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.

Address correspondence to: Dr. Derek M. McKay, Gastrointestinal Research Group, Department of Physiology and Biophysics, 1877 HSc, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1. E-mail: dmckay{at}ucalgary.ca

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