LEUKOCYTE TRANSENDOTHELIAL MIGRATION PATHWAYS

Brigham And Women'S Hospital, Boston MA

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Abstract

Transendothelial migration (TEM) of leukocytes is an essential component of the inflammatory response. The overall goal of this project is to characterize the endothelial junctional dependent and independent (non-junctional) pathways of leukocyte transmigration using in vivo and in vitro models of inflammation. Our laboratory has observed that the VE-cadherin complex is rapidly displaced and subsequently reorganized in minutes during neutrophil, monocyte and T cell subset transmigration (2). The mechanisms that control VE-cadherin mobility and the dynamics of its assembly with cytosolic alpha- and beta-catenin in live endothelial cells at baseline and during leukocyte transmigration are not well understood. Our hypothesis is that the mobility of VE-cadherin is increased at sites of TEM. In Specific Aim # 1 the role of the cytoplasmic tail of VE-cadherin and its association with cytosolic catenins in controlling VE-cadherin junctional stability will be assessed by multiple strategies using quantitative live cell fluorescence analytical techniques. Specifically, the studies will determine the mobility (diffusion coefficient) of VE-cadherin in endothelial junctions using Green Fluorescent Protein-tagged VE-cadherin (VEcadGFP) molecules by Fluorescence Recovery After Photobleaching (FRAP), and two color live cell fluorescence imaging of VEcadGFP and catenin molecules tagged with fluorescent GFP mutants (CFP, YFP) under a variety of experimental conditions. In Specific Aim #2 evidence for both junctional and non-junctional leukocyte transendothelial pathways will be sought in vivo, using unique reagents including transgenic animals with GFP-tagged junctional molecules (eg, VE-cadherin) in appropriate vascular beds by fast-scanning confocal intravital microscopy. Experiments from our laboratory using live cell fluorescence imaging of endothelium showed that in contrast to VE-cadherin gap formation, JAM-1 formed "rings" at sites of leukocyte transmigration. JAM1 is an endothelial junctional protein also expressed on leukocytes, and has been implicated in leukocyte TEM in vivo and in vitro. This ring structure is rich in cytoskeletal actin and contains endothelial JAM1, but not VE-cadherin, and leukocyte JAM1 and LFA-1. We have termed these rings TEM "tunnels". We hypothesize that the endothelial TEM tunnels are rapidly assembled actin scaffolds that provide temporal and spatial oraanization of the endothelial and leukocyte TEM machinery (a sleeve) for leukocytes to pass through. Specific Aim # 3 will characterize the endothelial cell components that constitute this tunnel (cytoskeletal components and adhesion molecules) during T cell, monocyte and neutrophil TEM, in endothelial cell monolayers under flow in vitro using quantitative fluorescence microscopy of fluorescent-tagged mutant and wild-type molecules. The overall results of the proposed studies will provide new information on the integrative role of endothelial cytoskeleton, surface adhesion molecules and lateral junction molecules in TEM and hence increase our understanding of leukocyte egress at sites of inflammation.