Clamping Human Umbilical Vein Endothelial Cell Cytosolic Free Calcium at Resting Levels Reduces Polymorphonuclear Leukocyte Induced Increases in Human Umbilical Vein Endothelial Cell Monolayer

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]t) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387)and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell BioL 42:617-672). Substances such as leukotriene (24 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca~]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca§ and that this increase in [Ca++]~ causes an increase in EC monolayer permeability. Such [Ca§ increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]t regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes. ritical and precisely regulated step in acute inflammation is the movement of polymorphonuclear leukocytes (PMN) t across the vascular endothelium. This process can be initiated by PMN in response to soluble chemoattractants or by endothelial cells (EC) in response to cytokines such as interleukin 1 OL-1) and tumor necrosis factor (8, 29). Cell surface molecules including the CD11/CD18 complex of proteins on the PMN surface (12) and intercelluAddress correspondence to Dr. Huang, College of P & S, Columbia University, Department of Physiology, 630 West 168th Street, New York, NY 10032. 1. Abbreviations used in this paper: [Ca++]i, cytosolie free calcium concentration; EC, endothelial cell; ELAM 1, endothelial leukocyte adhesion molecule 1; fMLP, formyl-methionyl-leucyl-phenylaianine; HBS, Hepes buffered saline; HIFBS, heat inactivated fetal bovine serum; HUVEC, human umbilical vein endothelial cells; ICAM 1, intercellular adhesion molecule 1; IL-1, interleukin 1; LTC4, leukotriene C,; M199/FBS, M199 medium containing 20% FBS, 100 U/ml penicillin, 100 ttg/ml streptomycin, and 2 ttg/ml amphotericin B; PMN, polymorphonuclear leukoeytes. lar adhesion molecule 1 (ICAM 1) (4), endothelial leukocyte adhesion molecule 1 (ELAM 1) (2), and platelet activating factor (35) on the EC surface have been shown to mediate the adhesion of PMN to EC, but the mechanism(s) by which junctions between EC open during PMN emigration from the blood remain unresolved. Monolayers of cultured EC respond to soluble mediators such as histamine by increasing their permeability to water and macromolecules. Such an EC response requires the soluble mediator to bind to a corresponding receptor on the EC surface. Binding of histamine to histamine receptors on EC results in an increase in EC cytosolic free calcium concentration ([Ca++]i), assembly of actin filaments, and an increase in transendothelial albumin flux in vitro (30). Morphologic studies in vivo demonstrate that changes in EC shape and opening of the interendothelial cell junctions accompany this increase in vascular permeability to fluid and macromolecules (26). Similar changes in EC shape accompany PMN adhesion to and migration across an endothelium 9 The Rockefeller University Press, 0021-9525/93/03/1371/10 $2.00 The Journal of Cell Biology, Volume 120, Number 6, March 1993 1371-138