VISCOELASTICITY OF WHITE BLOOD CELLS

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Our studies this past year have focused on basic mechanisms of adhesion at the level of individual molecular bonds. We have focused our efforts on the adhesion and detachment of the unstimulated and stimulated neutrophil from artificial surfaces and from endothelial cells and other neutrophils. We have successfully cultured both human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) on large solid and porous microcarriers and we are studying the interaction of antibody coated beads with the endothelial cells. By using antibody-coated beads rather than neutrophils, we can specify the ligands that interact with the receptors on the surface of the endothelial cells. We are using a positive pressure to gently press a latex bead, which slides freely in a pipette like a piston in a cylinder, against the endothelial cell. Using this micropipet suction technique we plan to measure the interactive forces between beads coated with an antibody to E-Selectin and endothelial cells following the up-regulation of E-selectin caused by the stimulation of endothelial cells. In our preliminary experiments, we have observed a gradual increase (2-3x control) in adhesion frequency in the first 4 hours and a marked increase (15-20x control) in the frequency of adhesion between 4 and 5 hours after stimulation of endothelial cells by the inflammation factor TNFa or IL-1a. We plan to use this technique to characterize the time course of adhesion receptor expression on the surface of HMVECs in real time. Once characterized, this technique will provide a simple but effective way to quantify the adhesion events between specific receptors and ligands on cells in vitro. We have measured, we believe for the first time, the on-rate between receptors on neutrophils and ligands (in this case antibodies) to these receptors bound to another surface. To date we have used only latex beads coated with antibodies either to L-selectin, an important receptor that mediates neutrophil rolling, or the membrane protein CD45, a ubiquitous receptor present on all white cells. The receptors, either L-selectin or CD45, are basely expressed on the surface of a resting neutrophil. The neutrophil is held in one micropipette so that it slides freely and oscillates back and forth like a piston in a cylinder because of an oscillating pressure in a reservoir. The bead, held in another pipette, is positioned at the entrance to the pipette that contains the oscillating neutrophil. The neutrophil repeatedly contacts the bead for a set time, usually about 0.1 s, and for a set contact area that is on the order of 1 m2. Occasionally, less than 20% of the time, the cell and bead adhere. This frequency of adhesion depends on the on-rate and on the number of receptors and ligands in the contact area. The number of receptors are estimated from published values of the total number on the neutrophil surface and our measurement of the contact area. The number of ligands in the contact area are determined by noting that the adhesion frequency steadily decreases as the number of adhesive events increases. We believe this decrease is caused by the extraction of receptors from the cell, which then block these particular ligands from further binding with a receptor in the cell membrane. From an analysis of the rate of decrease of adhesion events plotted as function of the number of contacts, we are able to calculate the number of ligands present in the contact area as long as we assume that the off-rate for these antigen-antibody reactions is negligible compared to the on-rate and the time of the experiment. (Antigen-antibody reactions have very high affinities, which imply that the off-rates are very slow compared to the on-rates.) Typical ligand densities calculated from our binding experiments are on the order of a few hundred antibodies per square micron. Estimated values for the receptor density are of the same order. Thus, for an adhesion frequency of about one receptor-antibody bond formed about every 0.5 s and for, say, 200 receptors and 200 ligands in the contact area, the forward rate constant is: kf = [2 receptor-ligand bonds/s]/[200 receptors 200 ligands] = 5 10-5/s. These experiments demonstrate the feasibility of actually measuring, for the first time, the rate of binding of receptors and ligands when both receptor and ligand are at (or in) a surface. A goal is to extend this new technique for characterizing surface binding to cases where the off-rate is significant and to cases involving cell-cell adhesion, especially, white cell-endothelial cell adhesion. Another goal is to model this process using Monte-Carlo simulations of the movement of receptors in a membrane.