Molecular mechanisms of monocyte adhesion to endothelial cells under flow conditions
McIntire, Larry V.
Doctor of Philosophy
Monocyte adhesion to the endothelium is one of the earliest events leading to the pathogenesis of various inflammatory disorders like atherosclerosis. Since these interactions normally occur in flowing blood, it becomes essential to elucidate receptor mediated molecular mechanisms underlying monocyte-endothelial adhesion under physiologic flow conditions. This work uses a parallel plate flow chamber to simulate dynamic flow conditions in vitro and image processing through videomicroscopy to visualize and quantitate adhesion of monocytes to endothelial cells. Monocyte interaction with P-selectin was investigated on histamine stimulated endothelial cells (HUVECs) and chinese hamster ovary (CHO) cells stably expressing P-selectin. Results indicate that monocytes bind to P-selectin through ligands containing O-linked oligosaccharides with terminal sialylated structures. On 4 hour IL-1$\beta$ stimulated human umbilical vein endothelial cells (HUVECs), monocytes appeared to use multiple pathways at both primary and secondary (firm) adhesion stages. Simultaneous blocking of VLA-4, L-selectin and sialylated moieties on the monocyte cell surface abolished the primary adhesive mechanisms of monocytes, consequently inhibiting monocyte adhesion by more than 80%. Blocking VCAM-1/VLA-4 and ICAM-1/$\beta\sb2$ pathways together eliminated firm adhesion of monocytes and shifted the pattern of interaction to that of pure rolling along the endothelium at 2 dynes/cm$\sp2.$ On 24 hour IL-4 stimulated endothelium, monocytes predominantly used VLA-4/VCAM-1 pathway for adhesion. When IL-4 (24 hr) treated HUVECs were further exposed to histamine, both P-selectin and VLA-4 pathways appeared to act in concert to support monocyte adhesion under flow conditions. Monocytes were also able to adhere on IL-1 (24 hr) stimulated HUVECs. However, when a combined stimulation of IL-1 + IL-4 (24 hr) was used, monocytes failed to attach under flow conditions whereas under static conditions large amounts of adhesion occurred. Further stimulation with histamine of IL-1 + IL-4 (24 hr) treated HUVECs was unable to restore a primary adhesion mechanism for monocytes.
Cell biology; Chemical engineering