title: Adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration.
abstract: Increased vascular endothelial cell (EC) permeability and neutrophilic leukocyte (PMN) diapedesis through paracellular gaps are cardinal features of acute inflammation. Activation of the EC contractile apparatus is necessary and sufficient to increase vascular permeability in specific models of EC barrier dysfunction. However, it is unknown whether EC contraction with subsequent paracellular gap formation is required for PMN transendothelial migration in response to chemotactic factors. To test this possibility, we assessed migration of human PMNs across confluent bovine pulmonary arterial EC monolayers. Transendothelial PMN migration in the absence of a chemotactic gradient was minimal, whereas abluminal addition of leukotriene B4 (LTB4; 5 microM) resulted in significantly increased PMN migration. Reductions in EC myosin light chain kinase (MLCK) activity by EC monolayer pretreatment with specific MLCK inhibitors (KT-5926 or ML-7) or by increases in cAMP-protein kinase A activity (cholera toxin) significantly reduced PMN transmigration (30-70% inhibition). In contrast, pretreatment with the myosin-associated phosphatase inhibitor calyculin resulted in the accumulation of phosphorylated myosin light chains, EC contraction, and significantly enhanced PMN migration. Finally, the interaction of PMNs with 32P-labeled EC monolayers was shown to directly increase EC myosin phosphorylation in a time-dependent fashion. Taken together, these results are consistent with the hypothesis that the phosphorylation status of EC myosin regulates PMN migration and further indicate that EC MLCK is activated by chemoattractant-stimulated PMNs. Neutrophil-dependent activation of the EC contractile apparatus with subsequent paracellular gap formation may be a key determinant of transendothelial PMN migration responses to chemotactic agents.
authors.affiliation: Departments of Medicine, Physiology, and Biophysics, Indiana University School of Medicine, Richard Roudebush Veterans Affairs Center, and the Methodist Research Institute, Indianapolis, Indiana 46202, USA.
authors.forename: J G
authors.initials: JG
authors.lastname: Garcia
authors.forename: A D
authors.initials: AD
authors.lastname: Verin
authors.forename: M
authors.initials: M
authors.lastname: Herenyiova
authors.forename: D
authors.initials: D
authors.lastname: English
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D002199:Capillary Permeability / Q000502:physiology; D002227:Carbazoles; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles; D007249:Inflammation / Q000503:physiopathology; D007975:Leukotriene B4 / Q000494:pharmacology; D008387:Marine Toxins; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology*; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D011651:Pulmonary Artery / Q000502:physiology
chemical_list: D000470:Alkaloids; D001381:Azepines; D002227:Carbazoles; D002630:Chemotactic Factors; D004791:Enzyme Inhibitors; D007211:Indoles; D008387:Marine Toxins; D009281:Naphthalenes; D010080:Oxazoles; C070571:ML 7; C063769:KT 5926; D007975:Leukotriene B4; C059041:calyculin A; D017868:Cyclic AMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase