title: Analysis of tight junctions during neutrophil transendothelial migration.
abstract: Intercellular junctions have long been considered the main sites through which adherent neutrophils (PMNs) penetrate the endothelium. Tight junctions (TJs; zonula occludens) are the most apical component of the intercellular cleft and they form circumferential belt-like regions of intimate contact between adjacent endothelial cells. Whether PMN transmigration involves disruption of the TJ complex is unknown. We report here that endothelial TJs appear to remain intact during PMN adhesion and transmigration. Human umbilical vein endothelial cell (HUVEC) monolayers, a commonly used model for studying leukocyte trafficking, were cultured in astrocyte-conditioned medium to enhance TJ expression. Immunofluorescence microscopy and immunoblot analysis showed that activated PMN adhesion to resting monolayers or PMN migration across interleukin-1-treated monolayers does not result in widespread proteolytic loss of TJ proteins (ZO-1, ZO-2, and occludin) from endothelial borders. Ultrastructurally, TJs appear intact during and immediately following PMN transendothelial migration. Similarly, transendothelial electrical resistance is unaffected by PMN adhesion and migration. Previously, we showed that TJs are inherently discontinuous at tricellular corners where the borders of three endothelial cells meet and PMNs migrate preferentially at tricellular corners. Collectively, these results suggest that PMN migration at tricellular corners preserves the barrier properties of the endothelium and does not involve widespread disruption of endothelial TJs.
authors.affiliation: Cardiovascular Sciences, Department of Medicine, Baylor College of Medicine, Houston, TX, USA. aburns@bcm.tmc.edu.
authors.forename: A R
authors.initials: AR
authors.lastname: Burns
authors.forename: R A
authors.initials: RA
authors.lastname: Bowden
authors.forename: S D
authors.initials: SD
authors.lastname: MacDonell
authors.forename: D C
authors.initials: DC
authors.lastname: Walker
authors.forename: T O
authors.initials: TO
authors.lastname: Odebunmi
authors.forename: E M
authors.initials: EM
authors.lastname: Donnachie
authors.forename: S I
authors.initials: SI
authors.lastname: Simon
authors.forename: M L
authors.initials: ML
authors.lastname: Entman
authors.forename: C W
authors.initials: CW
authors.lastname: Smith
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D004553:Electric Conductivity; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D005614:Freeze Fracturing; D006358:Hot Temperature; D006801:Humans; D007378:Interleukins / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein
chemical_list: D017077:Culture Media, Conditioned; D007378:Interleukins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D010972:Platelet Activating Factor; C569042:TJP1 protein, human; C569046:TJP2 protein, human; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D010450:Endopeptidases