Neutrophil Interactions With ICAM-1 at the Luminal Surface of the Intestinal Epithelium Enhance Neutrophil Survival and Alter Epithelial Barrier Function

Abstract

Neutrophil transepithelial migration (TEM) is a hallmark of inflammatory bowel disease (IBD), and is associated with epithelial injury and impaired barrier function. While TEM has been extensively modeled, little is known about neutrophil interactions with ligands expressed on the luminal surface of the intestinal epithelium. ICAM-1 is a well-recognized signaling receptor and endothelial ligand for migrating neutrophils. Its expression is also induced on the luminal (apical) membrane of intestinal epithelial cells (IEC) exposed to interferon γ (IFNγ), and in the colonic mucosa of individuals with active IBD. However, the role of ICAM-1 in mediating functional effects of neutrophil interactions with intestinal epithelial cells is undefined. ICAM-1 expression in cultured IECs and mouse intestinal epithelium was induced by exposure to IFNγ (100U/mL, 24h) and IFNγ+TNFα (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. We confirmed upregulation of ICAM-1 expression in IFNγ treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNγ and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority (˜60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNγ. Neutrophil interactions with apical epithelial membrane of IFNγ pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran (˜2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran (˜1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD.