Adherent neutrophils activate endothelial myosin light chain kinase: Role in transendothelial migration

Joe G.N. Garcia, Alexander D. Verin, Maria Herenyiova, Denis English

Research output: Contribution to journalArticle

127 Scopus citations

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 μM) 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.

Original languageEnglish (US)
Pages (from-to)1817-1821
Number of pages5
JournalJournal of Applied Physiology
Volume84
Issue number5
Publication statusPublished - May 1 1998
Externally publishedYes

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Keywords

  • Cytoskeleton
  • Inflammation
  • Neutrophil diapedesis
  • Paracellular gap formation

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)

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