Expression of Fc(gamma) receptors on cytokine activated endothelial cells: Potential roles of Fc(gamma) receptors in endothelial cell function and vasculitis
Abstract
Endothelial cell (EC) injury is a major pathologic feature of vasculitis, but the cause of EC injury is less clear. The purpose of this study was to investigate the presence of Fc$\gamma$ receptors on canine ECs, and to elucidate possible roles of Fc$\gamma$ receptors in initiating immune complexes deposition and mediating EC injury in canine vasculitis. Canine aortic ECs (CAECs) were isolated and characterized. These cells displayed several common EC characteristics including monolayer growth and "cobblestone" morphology, expression of Von Willebrand factor, and uptake of acetylated LDL molecules. Stimulation of CAECs with lipopolysaccharide induced adherence of canine PMN in a dose-dependent manner. An enzyme-containing peroxidase:anti-peroxidase immune complex (rabbit PAP) was characterized by both capillary electrophoresis and size exclusion HPLC, and selected as a ligand for Fc$\gamma$ receptor recognition. A PAP-based ELISA technique was developed and evaluated as an appropriate method for detecting Fc$\gamma$ receptors on monocytes and CAECs. Several different cytokines including IL-1$\beta$, TNF-$\alpha$, $\gamma$-INF, and GMCSF were investigated for their ability to modulate expression of Fc$\gamma$ receptors on CAECs. Only canine GMCSF induced a significant increase in Fc specific binding which reached a peak at 5 days stimulation. The binding of PAP to GMCSF activated CAECs was saturable, time and dose dependent, and could be inhibited by rabbit monomeric IgG; increasing concentrations of BSA had no impact on PAP binding. The binding of Fab-PAP or free peroxidase to GMCSF activated CAECs was minimal. In addition to the PAP assay, a modified rosette formation assay, dot blotting, and Western blotting techniques were also used to detect the presence of Fc-specific receptor expression on GMCSF activated CAECs. Functional studies were conducted to document the roles and functions of Fc-receptor like molecules in leukocyte-mediated EC injury. Cytotoxicity of PMNs and monocytes (U937) toward GMCSF activated CAECs was evaluated by tetrazollum bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays. The preliminary data suggested that increased expression of Fc$\gamma$ receptors on CAECs can enhance CAEC injury.
Degree
Ph.D.
Advisors
Kreisle, Purdue University.
Subject Area
Immunology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.