Activation of endothelial cell phospholipase D by migrating neutrophils

Background: Vascular endothelium plays a critical role in regulating the integrity of intercellular adhesive and junctional contacts in response to migrating neutrophils during the inflammatory process. Biochemical responses induced in endothelial cells by adherent, migrating neutrophils are poorly understood. This study was undertaken to explore the possibility that endothelial cell phospholipase D (PLD) is activated when neutrophils migrate through endothelial cell monolayers in response to chemotactic stimuli. Methods: Bovine pulmonary artery endothelial cells (BPAEC) were cultured on polycarbonate filters at the base of inserts in Transwell chambers (Costar, Cambridge, MA) and subsequently labeled with H-3-myristic acid. Human neutrophil migration through BPAEC monolayers was measured in response to a chemoattractant gradient produced by leukotriene B-4 (LTB4). After neutrophils were induced to migrate through endothelial monolayers in the presence of 0.1% ethanol, the filters were excised, and cellular lipids were extracted. Levels of labeled phosphatidylethanol (PEt), an index of activation of endothelial cell PLD, were measured in chromatographs of resolved phospholipids. Results: When neutrophils migrated through endothelial monolayers in response to LTB4, endothelial cells accumulated significant levels of PEt, indicating activation of endothelial cell PLD. Kinetic analysis demonstrated that PEt generation closely paralleled chemotactic migration over a 2-hour time period. Direct contact of neutrophils with the endothelium failed to induce PEt formation in the absence of neutrophil chemotaxis, and LTB4 was an ineffective stimulus of endothelial cell PLD activity in the absence of migrating neutrophils. Neutrophil migration-dependent endothelial PLD activation was observed when chemotaxis was induced by an unrelated chemoattractant, serum activated by exposure to Escherichia coli. However, neutrophil migration alone could not account for activation of endothelial cell PLD, since the peptide chemoattractant f-met-leu-phe (FMLP) induced comparable migration of neutrophils through endothelial monolayers but did not induce PEt generation. Conclusions: Our study indicates that chemotactic migration of neutrophils through endothelial monolayers results in endothelial cell PLD activation. This process may amplify both target and effector cell reactivity during the inflammatory response.