HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Endothelial [Ca]i signaling during transmigration of polymorphonuclear leukocytes

Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, the intracellular calcium ion ([Ca]i) signaling of endothelial cells (ECs) during PMN transmigration was examined at the single-cell level. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2, immersed in formyl-Met-LeuPhe, and subsequently perfused with fresh buffer to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. The data showed the following: (1) At high concentration (approximately 3 3 106/mL), both PMN suspension and its supernatant stimulated frequent EC [Ca]i elevations across the monolayer; (2) when used at lower concentration (approximately 5 3 105/mL) to avoid the interference of soluble factors, PMN transmigration, but not rolling or adhesion, was accompanied by EC [Ca]i elevation; (3) the latter EC [Ca]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those that were not in direct contact with the transmigrating PMNs; (4) this EC [Ca]i elevation was an initial and required event for PMN transmigration; and (5) PMNs pretreated with 5,5*-dimethyl-1,2-bis(2-aminophenoxy)ethane-N, N, N*, N*-tetraacetic acid transmigrated with the accompanying EC [Ca]i elevation, but they became elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca]i signaling, which apparently mediates the “gating” step for their subsequent transmigration. (Blood. 2000;96:3816-3822)