The interactions of leukocytes, in particular polymorphonuclear leukocytes (PMN), with the endothelial lining of blood vessels are important events in the inflammatory response. After the initial attachment, the PMNs migrate through the endothelial lining and migrate to the site of inflammation. At present most of the available information concerns the initial interactions and what affects adhesion, from the standpoint of the PMN and, more recently, the endothelial cell. There is very little data about what occurs in each cell once they have made contact with each another. It is, therefore, the overall aim of this proposal to investigate the effects on endothelial cells, in particular, subsequent to PMN attachment. Specifically, we propose to look at changes in the levels of expression of TGF-beta, PDGF and metalloproteinases by measuring release into conditioned media and by Northern analyses. To determine the contribution made by each cell type, in situ hybridization techniques on PMN attached to monolayer of cultured endothelial cells will be carried out. We also propose to characterize the arachidonic acid metabolites, particularly those of the cytochrome P-450 dependent monooxygenase pathway, that are synthesized and released by the endothelial cells, in particular, as a result of these interactions. Finally, we will investigate intracellular events that occur subsequent to PMN contact. Specifically, we will monitor changes in intracellular pH, Ca++ mobilization and phosophoinositol turnover. All of these measurements will be made on an in vitro model of the acute inflammatory response composed of cultured endothelial cells and isolated PMN. In all of these experiments, analyses will be carried out on stimulated and unstimulated endothelial cells in contact with PMN, both activated and non-activated. Endothelial cells isolated from large and small vessels of several different species, including human, ovine, bovine and rat will be used. It is hoped that the results of these experiments will provide new and useful information leading to a better understanding at the molecular level of some of the mechanisms which regulate the acute inflammatory response.
