Stimulated neutrophils induce myosin light chain phosphorylation and isometric tension in endothelial cells

Abstract

The mechanism or mechanisms by which polymorphonuclear leukocytes (PMN) penetrate junctions between neighboring endothelial cells (EC) to traverse endothelial barriers remain unresolved. We report that chemoattractant-stimulated PMN induce a coordinate increase in both phosphorylation of serine 19 and threonine 18 of EC myosin regulatory light chains and isometric tension generation by EC monolayers. Unstimulated PMN had no effect on either parameter. These findings, coupled with our previous report (Huang et al., J. Cell Biol. 120: 1371-1380, 1993) that chemoattractant-stimulated PMN cause a rise in EC cytosolic free Ca2+, provide strong presumptive evidence that myosin light chain kinase is the EC enzyme responsible for initiating myosin light chain phosphorylation, EC contraction, and isometric tension generation in response to chemoattractant-stimulated PMN. We suggest that, by inducing phosphorylation of EC cytoskeletal proteins, chemoattractant-stimulated PMN induce EC to open their intercellular junctions, thereby facilitating transendothelial movement of these leukocytes.

Leukocyte transendothelial migration: A local affair

“…The first is based on research conducted on GPCR signaling in ECs, such as thrombin induced junctional opening 51 and postulates that leukocytes induce actomyosin contraction in ECs triggering junctional opening. [52][53][54] The second hypothesis anticipates that EC junctions are locally destabilized to allow migrating cells to squeeze through the transient opening in the junction. Recent evidence supporting the latter hypothesis shows that leukocytes trigger rapid dephosphorylation of Tyr731 on VE-cadherin via the tyrosine phosphatase SHP-2, which allows the adaptin AP-2 to bind and initiates endocytosis of VE-cadherin (Fig.…”

Unique Structural Features That Influence Neutrophil Emigration Into the Lung

“…Neutrophil adhesion is known to induce a coordinate increase in endothelial cytosolic free calcium, phosphorylation of serine-19 and threonine-18 on endothelial myosin regulatory light chains, and isometric tension generation by endothelial monolayers (80,101,109,222). Neutrophilmediated outside-in endothelial signaling likely involves the same molecules that mediate neutrophil adhesion to the endothelial surface.…”

“…In a study of human microvascular endothelial monolayers, disruption of endothelial microfilaments by cytochalasin B or latrunculin A inhibits monocyte transmigration (128). On HUVEC monolayers, inhibition of myosin light-chain kinase (MLCK) diminishes neutrophil transmigration and inhibits endothelial F-actin formation, myosin filament formation, and myosin light chain (MLC) phosphorylation (101,222). As mentioned in the preceding paragraph, neutrophil adhesion to human pulmonary microvascular endothelial cells (HPMVEC) treated with TNF-␣ also leads to endothelial cell stiffening and F-actin reorganization.…”

ICAM-1/LFA-1 interaction contributes to the induction of endothelial cell-cell separation: implication for enhanced leukocyte diapedesis

“…While the "contraction-respreading" cycle of ECs is mainly mediated by ICAM-1/LFA-1, the detailed mechanism was not determined in the current study and requires further exploration. Previous reports have shown that ligation of ICAM-1 triggers an increase in intracellular calcium and activation of myosin light chains, MAPK, and Rho GTPase, thereby enhancing cell contraction and promoting the opening of interendothelial contacts (Hixenbaugh et al, 1997;Greenwood et al, 2003;Muller, 2003;Ley et al, 2007;Oh et al, 2007). Further studies are necessary to examine a potential involvement of these ICAM-1-mediated signaling cascades in the "contraction-respreading" cycle of ECs.…”

“…EC height was calculated by the sum of the z-axes in an orthogonal view (height of combined Z-sections) using FLUOVIEW software. (Hixenbaugh et al, 1997;Greenwood et al, 2003;Muller, 2003;Ley et al, 2007;Oh et al, 2007). In the present study, we investigated whether ICAM-1/LFA-1-mediated EC contraction is …”