title: Real-time imaging of endothelial cell-cell junctions during neutrophil transmigration under physiological flow.
abstract: During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.
authors.affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Kroon
authors.affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Daniel
authors.affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam.
authors.forename: Mark
authors.initials: M
authors.lastname: Hoogenboezem
authors.affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, AMC at University of Amsterdam; j.vanbuul@sanquin.nl.
authors.forename: Jaap D
authors.initials: JD
authors.lastname: van Buul
mesh_terms: D015703:Antigens, CD / Q000378:metabolism*; D015820:Cadherins / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism*; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D000099152:Cadherin 5