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Updated: Aug 4, 2026
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Published on: January 6, 2014
Neutrophil migration across model intestinal epithelia: monolayer disruption and subsequent events in epithelial
A Nusrat1, C A Parkos, T W Liang
1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Background & Aims:
Acute inflammation of the intestine is associated with transepithelial migration of polymorphonuclear leukocytes (PMNs) and epithelial wounds that rapidly reseal. The aim of this study was to determine mechanisms by which such PMN-induced epithelial wounds reseal.
Methods:
Epithelial wound closure was modeled in vitro using T84 intestinal epithelial cells and PMNs. Wound closure was analyzed by confocal microscopy and by determination of barrier function. Wounds were highlighted by apical labeling with antibody to a basolaterally restricted ligand, beta1-integrin.
Results:
High-density PMN transepithelial migration for 70-110 minutes produced multifocal epithelial wounds that were 1-120 microm in diameter and markedly diminished epithelial barrier function that returned to baseline within 12-20 hours. Large wound closure was initiated by cell flattening and extension of F-actin/vinculin/paxillin-enriched lamellipodia at the leading edge. As wounds became small (approximately <30 microm), epithelial cells at the wound edges assumed columnar phenotype with poorly formed or absent lamellipodia. Apical localized circumferential, dense F-actin/myosin II rings were found to encircle such wounds, suggesting final closure by a sphincter-like contraction.
Conclusions:
These data model mucosal repair in acute inflammatory conditions and, for the first time, show sequential early and late mechanisms by which epithelial discontinuities repair.
Insights
Polymorphonuclear leukocytes (PMNs) induce intestinal epithelial wounds that reseal through distinct early and late repair mechanisms. Cell flattening and lamellipodia extension initiate closure, followed by sphincter-like contractions for final sealing.
Area of Science:
- Gastroenterology
- Cell Biology
- Immunology
Background:
- Intestinal inflammation involves polymorphonuclear leukocyte (PMN) migration and epithelial wound formation.
- Rapid resealing of these epithelial wounds is crucial for mucosal integrity.
Purpose of the Study:
- To elucidate the mechanisms by which PMN-induced intestinal epithelial wounds reseal.
- To characterize the sequential cellular processes involved in mucosal repair.
Main Methods:
- In vitro modeling of epithelial wound closure using T84 intestinal epithelial cells and PMNs.
- Analysis of wound closure via confocal microscopy and barrier function assessment.
- Labeling of wounds with antibodies to beta1-integrin to track epithelial dynamics.
Main Results:
- PMN migration induced multifocal epithelial wounds that impaired barrier function, with recovery within 12-20 hours.
- Large wound closure involved cell flattening and lamellipodia extension (F-actin, vinculin, paxillin).
- Small wounds (<30 microm) were sealed by apical F-actin/myosin II rings, suggesting a contractile mechanism.
Conclusions:
- The study models mucosal repair in acute inflammatory conditions.
- Sequential early (cell extension) and late (contractile ring) mechanisms of epithelial discontinuity repair are identified.
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