Vascular endothelial cells (ECs) play important roles in inflammation, thrombosis and immunity. Efforts to study the properties of these cells have been hampered by their intimate intermixing with other cells and connective tissue components in the various organs of the body. This has been especially true of the barrier functions of the endothelium. While it is possible to make sophisticated physiologic measurements using single capillaries, it is difficult to study biochemical processes in vivo. Biochemical studies also are hampered by the heterogeneity of ECs. Even with a single capillary bed there are structural and functional differences between arteriolar, capillary and veinular endothelium. To overcome these difficulties we have developed a system for maintaining human ECs in monolayer cultures on a substrate of human amnion. ECs maintained in this way form confluent monolayers. The cells exhibit many of the ultrastructural and functional properties of endothelium in vivo. Their intercellular junctions stain with silver; they exhibit electron dense plaques characteristic of tight junctions at zones of intercellular contact; they exhibit numerous cell surface caveolae, they restrict the transendothelial passage of ions and macromolecules; they support an oncotic pressure gradient; their surfaces are not thrombogenic; and they support the adhesion and transendothelial migration of leucocytes only when the leucocytes are stimulated by a chemoattractant gradient or the ECs are treated with cytokines. Thus they monolayers exhibit many of the barrier functions of endothelium in vivo. We have used these monolayers to examine the mechanisms by which polymorphonuclear leucocytes (PMNs) traverse the endothelium. Our studies show that the interaction of chemoattractant stimulated, but not of unstimulated, PMNs with EC monolayers stimulates a pronounced but transient rise in EC intracellular calcium ([Ca++]i). We believe that this rise in [Ca++]i reflects the active participation of ECs in reshaping their cytoskeleton and opening their intercellular junctions to allow transendothelial passage of PMN. The studies proposed here are designed to investigate communication between these two cell types, to characterize the processes by which chemoattractants activate the adhesive properties of PMN receptors; and to determine the mechanisms by which cytokines stimulate transendothelial migration of PMNs in the absence of a gradient of chemoattractants.
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