Epithelial exposure to hypoxia modulates neutrophil transepithelial migration
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Updated: Nov 2, 2026
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Published on: January 6, 2014
S P Colgan1, A L Dzus, C A Parkos
1Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women's Hospital, Boston, Massachusetts, USA.
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 tMLP. In response to hypoxia/reoxygenation (H/R), transmigration into, and across epithelial monolayer was increased in a dose- (EC50 approximately 7% O2) and time-dependent fashion (3.5 +/- 0.3-fold increase at 2% O2 for 48 h, 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 beta 2 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, nearly 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 IL-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.
Insights
Hypoxia/reoxygenation significantly increases polymorphonuclear leukocyte (PMN) migration across intestinal epithelial cells. This process, mediated by IL-8 and integrins, highlights new crosstalk in intestinal disease pathogenesis.
Area of Science:
- Gastroenterology
- Immunology
- Cell Biology
Background:
- Polymorphonuclear leukocytes (PMN) are implicated in intestinal diseases like necrotizing enterocolitis and reperfusion injury.
- Tissue hypoxia is a suspected mediator in these PMN-induced intestinal pathologies.
- The interaction between intestinal epithelia and PMNs under hypoxic conditions is not well understood.
Purpose of the Study:
- To investigate the effect of hypoxia/reoxygenation (H/R) on PMN transepithelial migration across intestinal epithelial cells.
- To elucidate the molecular mechanisms underlying H/R-modulated PMN-epithelial interactions.
Main Methods:
- T84 human intestinal crypt cell monolayers were exposed to varying degrees of hypoxia (2-21% O2) for up to 72 hours.
- PMN transmigration across monolayers was assessed under H/R conditions, driven by a chemotactic peptide gradient.
- Involvement of protein synthesis, beta 2 integrin CD11b/18, CD47, and interleukin-8 (IL-8) was evaluated using inhibitors, antibodies, and antisense oligonucleotides.
Main Results:
- H/R significantly increased PMN transmigration in a dose- and time-dependent manner (up to 3.5-fold increase at 2% O2 for 48h).
- H/R did not induce epithelial toxicity or compromise barrier function.
- Increased PMN transmigration was dependent on protein synthesis and inhibited by antibodies against CD11b/18 and CD47.
- Hypoxia stimulated epithelial IL-8 production and release, which was associated with the epithelial matrix.
- IL-8 antisense oligonucleotides reduced PMN association with the monolayer but not transmigration, suggesting a role in PMN recruitment.
Conclusions:
- Hypoxia/reoxygenation is a potent stimulus that enhances PMN migration across intestinal epithelia.
- This H/R-induced effect involves protein synthesis, epithelial IL-8 release, and PMN adhesion molecules CD11b/18 and CD47.
- Novel biochemical crosstalk between epithelia and PMNs is established under H/R conditions, relevant to intestinal disease pathogenesis.
