Endotoxin inhibits enterocyte migration by increasing integrin function through PI3 kinase

Abstract

Purpose: Experimental necrotizing enterocolitis (NEC) is characterized by circulating endotoxin and impaired enterocyte migration. Because migration requires the detachment of cells from the underlying matrix and the inhibition of surface integrins, we hypothesized that impaired enterocyte migration is due to increased integrin function. Methods: NEC was induced in newborn rats after formula gavage and hypoxia, from which terminal-ileal mucosal scrapings were prepared. IEC-6 cells were treated with LPS (50 ug/ml,12 h) +/− PI3K inhibitor LY(25 uM). Integrin expression was determined by confocal microscopy or SDS-PAGE of cells sub-fractionated by centrifugation at 100,000 g. Integrin activity was determined by adherence of 15um fibronectin coated latex beads to IEC-6 monolayers. Migration was measured by time-lapse microscopy of IEC-6 cells moving into a scraped wound. Results: Expression of α3 and β1 integrins was increased 3-fold in ileal mucosal scrapings from NEC-rats vs. ctrls (n = 3, p < 0.05), and 4-fold in LPS-treated IEC-6 cells vs. ctrls (n = 5, p < 0.05). LPS caused a significant shift of α3 and β1 integrins from the cytosolic to plasma membrane fraction by SDS-PAGE and confocal microscopy (see Figure, arrows point to β1 expression), which was reversed by LY. Increased integrin expression was associated with a PI3K-dependent increase in integrin function determined by bead adhesion (beads/cell: ctrl: 56 ± 4, LPS: 77 ± 4, LPS ± LY: 35 ± 3, LY 45 ± 5, n = 3 experiments, p < 0.001). Finally, maintenance of internalized integrins reversed the LPS-induced inhibition of migration (ctrl: 7 ± 2 um/h, LPS: 0.5 ± 1 um/h, LY 4 ± 2 um/hour, p < 0.05). Conclusion: Enterocyte migration is inhibited by LPS through increased surface expression of integrins in a PI3K dependent manner. Modulation of enterocyte migration via integrin expression may provide novel insights into the pathogenesis of NEC.