Endothelial-leukocyte interactions

This thesis describes early inflammatory responses involving interactions between leukocytes and the endothelial lining of the blood vessel wall. An adhesion molecule, E-selectin, which is upregulated on interleukin-1 activated endothelium, was found to mediate rolling between leukocytes and endothelial cells in vivo. Immunohistochemistry revealed that E-selectin was preferentially expressed in venules and not in arterioles. In most inflammatory conditions, leukocyte rolling and subsequent extravasation to surrounding tissues take place in the postcapillary venules which is also the site for inflammation mediated vascular permeability. We found that a gradient of the inflammatory mediator, LTB4, was essential for the induction of neutrophil-mediated increased permeability and migration. Furthermore, permeability was found to be increased by the cationic and neutrophilic granular protein heparin binding protein (HBP, CAP37, azurocidin). The effects of HBP on endothelial barrier function could be partially neutralized by anti-HBP antibodies. In addition, HBP was shown to increase intracellular Ca2+-levels and rearrange actin-filaments in endothelial cells which showed that HBP was capable of inducing the cellular changes that are required for cell contraction and intercellular gap formation. Affinity chromatography of HBP binding molecules showed that HBP bound to all known endothelial cell surface proteoglycans. Flow cytometry and microscopical investigations demonstrated that HBP was rapidly internalized into endothelial cells. Subcellular fractionation, confocal microscopy and electron microscopy studies led us to the conclusion that HBP was localized to the mitochondrial compartment. Addition of HBP to endothelial cells prior to the induction of apoptosis, by growth factor withdrawal, showed that HBP protected the endothelial cells from apoptosis. HBP was also shown to interact with a mitochondrial resident protein, p33, and to decrease incorporation of 32PO4 into endothelial proteins