Phosphatidylinositol 3′-Kinase Is a Critical Mediator of Interferon-γ-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 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.

Interferon-γ-induced increases in intestinal epithelial macromolecular permeability requires the Src kinase Fyn

“…IFNg-induced increased HRP transcytosis across monolayers of Caco2 cells was also significantly reduced by PP1 (Figure 1c and d). In contrast, the drop in TER elicited by IFNg was not affected by Src kinase inhibition (Figure 1e), whereas consistent with earlier studies, 10,12 inhibition of PI3K activity did reduce, in part, the drop in TER.…”

“…Much remains to be done to precisely elucidate the IFNg signalling events that control epithelial barrier function, and whereas others have focused on defining TJ protein and cytoskeletal changes, [5][6][7][8] the present investigation is in accordance with IFNg modulation of epithelial endocytosis and transcellular permeability. 10 The delay in PI3K activity relative to initial STAT phosphorylation is intriguing and suggests mobilization of temporally and spatially distinct signalling pathways, a hypothesis supported by data showing that in T84 cells, neither Src kinase nor PI3K inhibition affected STAT1-dependent immune activities (IRF-1 expression and IP-10 secretion). It is also noteworthy that STAT1 and STAT5 activation is slower and reduced in magnitude in epithelia compared with macrophages ( Figure 4b) and this could contribute to a delayed accumulation of other intracellular signals in the enterocyte, and ultimately to the 24-48 h lag period observed between IFNg treatment and increases in T84 epithelial monolayer permeability.…”

“…However, ablation of STAT1 in epithelial cells would have a huge impact on their function, and thus we sought to explore a tangential pathway that would link the IFNgR to the PI3K pathway, which has been shown to mediate IFNg-induced increases in epithelial permeability. 10,12,32 Our data presented in this study suggest a novel mechanism for IFNg-mediated activation of PI3K, involving interactions between STAT5b, Gab2 and the Src kinase Fyn (summarized in Figure 9). Whereas STAT5b can be phosphorylated in monocyte and T-cell lines in response to IFNg, 33 here we show activation of STAT5, a previously undocumented activity of IFNg in intestinal epithelial cells.…”

“…11 Pharmacological inhibition of STAT1 tyrosine phosphorylation (ie, activation) failed to ameliorate IFNg-induced reduction in transepithelial electrical resistance (TER, a marker of paracellular permeability), and subsequently a requirement for activation of PI3K during IFNg-induced increases in epithelial permeability across monolayers of human colon-derived epithelial cell lines was shown. 10,12 However, there is no indication that PI3K can bind the IFNg receptor, 13 and hence intermediate structural or kinase molecules are needed to convert IFNg/IFNg receptor (IFNgR) interaction into PI3K activation.…”

“…8 We, and others, have shown that IFNg can promote the transcytosis of bacteria across epithelial monolayers. 9,10 Consequently, the elucidation of IFNgmediated intracellular signalling pathways can be pursued with the ambition of identifying epithelial target molecules, the inhibition of which would ablate IFNg-induced increases in gut permeability while not interfering with its ability to activate immune cells to eliminate bacteria that gain access to the mucosa.…”