Changes in the biomechanical properties of neutrophils and endothelial cells during adhesion

Abstract

This study examined changes in the biomechanical properties of cultured pulmonary microvascular endothelial cells (ECs) and neutrophils induced by adhesion of neutrophils to these ECs. The biomechanical properties of cells were evaluated using magnetic twisting cytometry, which measures the angular rotation of ferromagnetic beads bound to cells through antibody ligation on application of a specified magnetic torque. Adhesion of neutrophils to 24-hour tumor necrosis factor-␣ (TNF-␣)-treated ECs, but not to untreated ECs, induced an increase in EC stiffness within 2 minutes, which was accompanied by an increase and a reorganization of F-actin in ECs. A cell-permeant, phosphoinositide-binding peptide attenuated the EC stiffening response, suggesting that intracellular phosphoinositides are required. The stiffening response was not inhibited by ML-7, a myosin light-chain kinase inhibitor, or BAPTA, an intracellular Ca 2؉ chelator. Moreover, the phosphorylation pattern of the regulatory myosin light chains was unaltered within 15 minutes of neutrophil adherence. These data suggested that the EC stiffening response appeared not to be mediated by myosin light-chain-dependent mechanisms. Concomitantly, neutrophil adhesion to 24-hour TNF-␣-treated ECs also induced changes in the biomechanical properties of neutrophils compared to neutrophils bound to untreated ECs. Taken together, these results demonstrated that neutrophil adhesion to TNF-␣-treated ECs induces changes in the biomechanical properties of both cell types through actin cytoskeletal remodeling. These changes may modulate neutrophil transmigration across the endothelium during inflammation. IntroductionNeutrophil transmigration across the vascular endothelium is a highly regulated process that requires the up-regulation of neutrophil and endothelial cell (EC) adhesion molecules. 1 During neutrophil adhesion to ECs, binding of the neutrophil or EC adhesion molecules to their ligands may induce intracellular signaling pathways and downstream events, which may in turn modulate neutrophil transmigration. Indeed, the ligation of various adhesion molecules can initiate signal transduction pathways and induce subsequent cellular changes. Cross-linking of neutrophil CD11/CD18 induces intracellular Ca 2ϩ increases, CD11/ CD18 up-regulation, and F-actin polymerization. 2 Ligation of EC E-selectin, P-selectin, ICAM-1, and VCAM-1 by antibodies induces transient increases in cytosolic Ca 2ϩ . [3][4][5] Similarly, neutrophil adherence and emigration also induce increases in intracellular Ca 2ϩ levels in ECs. 3,6 One of the downstream targets of these signaling events in ECs after neutrophil adherence is the actin cytoskeleton. Neutrophil adherence induces stress fiber formation in ECs, 3,7 and inhibition of the F-actin changes and actin polymerization in ECs reduces neutrophil transmigration as observed both in vivo and in vitro. [7][8][9] However, the mechanisms involved in mediating the EC actin cytoskeletal changes on neutrophil adhesion are not understood.In the system...

Lipopolysaccharide-Induced Leukocyte-Endothelial Cell Interactions: A Role for CD14 Versus Toll-Like Receptor 4 Within Microvessels

“…Wang et al (30) have reported that the endothelium increases in rigidity and perhaps contributes to the physical trapping of the general circulating pool of leukocytes. There may also be a direct increase in leukocyte rigidity in response to LPS and thereby increased sequestration within the pulmonary microvascular bed (30). Clearly, it is not surprising that some studies have reported only partial or no decrease in LPS-induced leukocyte sequestration with anti-adhesion therapy (13,31).…”

Unique Structural Features That Influence Neutrophil Emigration Into the Lung

“…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. Interestingly, unlike HUVEC monolayers, endothelial stiffening does not appear to rely on MLCK or MLC phosphorylation (280). This may reflect phenotypic differences between HUVEC and HPMVEC.…”

Activation of SRC Tyrosine Kinases in Response to ICAM-1 Ligation in Pulmonary Microvascular Endothelial Cells

“…ECs were grown to confluence and used 2-3 days post-confluence. These cells can be induced to up-regulate ICAM-1 expression upon TNF-␣ stimulation (17,18).…”

“…Cultivation of Human Pulmonary Microvascular ECs-Human pulmonary microvascular ECs were obtained from Clonetics (Walkersville, MD) and plated onto fibronectin-coated culture dishes, as described previously (17,18). ECs were used between passage 7 and 15.…”

“…Signaling pathways initiated by ICAM-1 ligation with neutrophils or cross-linking antibodies include ICAM-1 redistribution on EC surface, activation of xanthine oxidase, production of reactive oxygen species (ROS), and activation of p38 MAPK (4,17). Activation of p38 MAPK results in phosphorylation of heat shock protein 27, an actinbinding protein that may induce actin polymerization when phosphorylated, and p38 activation is required for the cytoskeletal rearrangement induced by ICAM-1 ligation, as well as for neutrophil migration toward EC borders (4,17,18). The signaling pathways induced by ROS production that lead to p38 MAPK activation are not understood.…”