Received for publication September 11, 2006.
Revised December 14, 2006.
Accepted for publication December 15, 2006.

PI-3K is a critical mediator of interferon--induced increases in enteric epithelial permeability

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 signalling events that underlie interferon- (IFN)-induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semi-permeable supports were treated with IFN (20ng/ml), and barrier function assessed 48h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of ⁵¹Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of non-invasive, non-pathogenic Escherichia coli (strain HB101). Exposure to IFN decreased barrier function as assessed by all 4 markers. The PI-3K inhibitors, LY294002 and wortmannin, did not affect baseline permeability characteristics, but completely blocked the drop in TER and increased fluxes of ⁵¹Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFN. Also, use of the pan-PKC inhibitor, bisindolylmalemide I (5µM), 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 6h 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.

Key words: Signal transduction, bacterial transcytosis, human, intestine, protein kinase C, tight junction

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