Phosphatidylinositol 3'-kinase is a critical mediator of interferon-gamma-induced increases in enteric epithelial permeability.
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) gamma-induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFNgamma (20 ng/ml), and barrier function was assessed 48 h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of (51)Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of noninvasive, nonpathogenic Escherichia coli (strain HB101). Exposure to IFNgamma 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 (51)Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFNgamma. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 muM), but not rottlerin (blocks PKCdelta), 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 IFNgamma exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFNgamma-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.
McKay
Derek M
DM
Intestinal Disease Research Programme, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada. dmckay@ucalgary.ca
Watson
James L
JL
Wang
Arthur
A
Caldwell
Jackie
J
Prescott
David
D
Ceponis
Peter M J
PM
Di Leo
Vincenza
V
Lu
Jun
J
D018988:Bacterial Translocation / Q000187:drug effects*; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000276:immunology; D003106:Colon / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D004926:Escherichia coli / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology / Q000494:pharmacology*; D007408:Intestinal Absorption / Q000187:drug effects* / Q000276:immunology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology / Q000276:immunology / Q000382:microbiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors
D004791:Enzyme Inhibitors; D007371:Interferon-gamma; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors