Identification of Proteases Involved in the Proteolysis of Vascular Endothelium Cadherin during Neutrophil Transmigration

Abstract

Transmigration of neutrophils across the endothelium occurs at the cell-cell junctions where the vascular endothelium cadherin (VE cadherin) is expressed. This adhesive receptor was previously demonstrated to be involved in the maintenance of endothelium integrity. We propose that neutrophil transmigration across the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This hypothesis is supported by the following lines of evidence. 1) Proteolytic fragments of VE cadherin are released into the culture medium upon adhesion of neutrophils to endothelial cell monolayers; 2) conditioned culture medium, obtained after neutrophil adhesion to endothelial monolayers, cleaves the recombinantly expressed VE cadherin extracellular domain; 3) these cleavages are inhibited by inhibitors of elastase; 4) VE cadherin fragments produced by conditioned culture medium or by exogenously added elastase are identical as shown by N-terminal sequencing and mass spectrometry analysis; 5) both elastase-and cathepsin G-specific VE cadherin cleavage patterns are produced upon incubation with tumor necrosis factor ␣-stimulated and fixed neutrophils; 6) transendothelial permeability increases in vitro upon addition of either elastase or cathepsin G; and 7) neutrophil transmigration is reduced in vitro in the presence of elastase and cathepsin G inhibitors. Our results suggest that cleavage of VE cadherin by neutrophil surface-bound proteases induces formation of gaps through which neutrophils transmigrate.

A new insight into phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of polymorphonuclear leukocytes by macrophages

“…Data are the mean7S.D. from two independent experiments, each performed in triplicate: *Po0.05; **Po0.01; ***Po0.001 significance as compared to untreated controls receptor involved in clearance by CG associated with the PMN surface, 35,36 CG-treated PMNs were incubated with Suc-Ala-Pro-Phe-cmk, a potent CG inhibitor, before being cocultured with macrophages. Such treatment did not improve engulfment of CG-treated PMNs indicating that the enzyme apparently proteolytically inactivates an 'eat-me' signal(s) on the cell surface of PMNs (data not shown).…”

Blood cells and endothelial barrier function

“…85 Neutrophils are also able to enhance transendothelial protein exchange by releasing proteases, like elastase and matrix metalloproteinases (MMP), which appear to alter barrier function by disrupting junctional complexes and inducing endothelial cell retraction. [86][87][88] Elastase has also been shown to promote the adhesion and transendothelial migration of leukocytes in the microcirculation, 89 suggesting that the permeability enhancing effect of the protease may also be related to an enhancement of neutrophilendothelial cell adhesion. This possibility is supported by reports describing diminished endothelial barrier function, resulting from junctional disassembly and cytoskeletal reorganization, following the ligation of neutrophil adhesion molecules with their counterreceptors on endothelial cells, such as the binding of b-2 integrins with either ICAM-1 or VCAM-1.…”

Modeling the Molecular Network Controlling Adhesion Between Human Endothelial Cells: Inference and Simulation Using Constraint Logic Programming

“…VE-cadherin is directly involved in the maintenance of endothelium permeability [9,12] and in the control of the traffic of leukocytes from blood toward inflamed tissues [10]. Thus, following close contact between leukocytes and endothelial cells, proteases previously stored within leukocytes are transported at the cell surface and locally cleave VE-cadherin.…”