Epithelial exposure to hypoxia modulates neutrophil transepithelial migration

Abstract

Polymorphonuclear leukocytes (PMN) are central to the pathogenesis of a number of intestinal diseases, PMN-induced damage to the protective epithelium occurs in hemorrhagic shock, necrotizing enterocolitis and conditions resulting in intestinal reperfusion injury. In such diseases, tissue hypoxia has been implicated as a pathophysiologic mediator. Thus, we hypothesized that exposure of intestinal epithelia to hypoxia may modulate. PMN-epithelial interactions. In this study, T84 cell monolayers, a human intestinal crypt cell line, and isolated human PMN were used to examine the influence of hypoxia/reoxygenation (H/R) on PMN transepithelial migration. Confluent T84 cell monolayers were exposed to hypoxia (range 2-21‰ O2 for 0-72 h) and reoxygenated with buffer containing PMN. Transmigration of PMN (basolateral to apical orientation) was driven by a transepithelial gradient of the chemotactic peptide fMLP. In response to hypoxia/reoxygenation (11/R), transmigration into, and across epithelial monolayer was increased in a dose- (EC50 ~ 7‰ O2) and time-dependent fashion (3.5 ± 0.3-fold increase at 2‰ O2 for 48h, p < 0.001). Such conditions of H/R were not toxic to epithelia and did not influence epithelial barrier function. The influence of H/R on PMN transmigration was protein synthesis-dependent (>80‰ decreased in the presence of cycloheximide) and could be inhibited by addition of functionally inhibitory antibodies to the PMN β2 integrin CD11b/18 (>80‰ attenuated) and to CD47 (>90‰ decreased compared to control). Hypoxia induced epithelial production and basolateral release of the PMN activating chemokine interleukin-8 (IL-8, nearby sixfold increase over normoxic control) which remained avidly associated with the epithelial matrix. Treatment of epithelial cells with IL-8 antisense oligonucleotides resulted in decreased monolayer-associated PMN but did not influence PMN transmigration suggesting that epithelial 11-8 production may serve as a recruitment signal for PMN to the basal surface of polarized epithelia. The present observations indicate that H/R provides a relevant stimulus for novel biochemical crosstalk between epithelia and PMN.