This project will focus on the chemical and physical mechanisms that control the accumulation of neutrophils at a site of inflammation. The previous focus of this project was on the role of the physical chemistry of selectins and their carbohydrate ligands in mediating neutrophil rolling. We developed a """"""""cell-free"""""""" system to recreate leukocyte rolling with carbohydrate ligand-coated hard spheres adhering to selectin-coated surfaces in a flow chamber. Selectin-mediated leukocyte rolling is inherently noisy, both in cellular and cell-free systems. Our hypothesis is that the period of pauses during rolling facilitates firm neutrophils adhesion via b/2-integrins, and ultimately extravasation during inflammation. Furthermore, we hypothesize that both receptor chemistry (density, identify of ligands) and cell-cell interactions contribute to the dynamics of rolling, and thus to firm adhesion, but the quantitative contribution of each of these factors is poorly understood. To address these hypothesis, we will perform flow chamber adhesion experiments, micropipette aspiration experiments, and computer simulations. We will extend measurements of leukocyte adhesion under flow through experiments at higher cell or particle densities, examining the role of particle concentration (using hard spheres), hematocrit (using erythrocytes), and neutrophil-neutrophils interactions (using different concentrations of neutrophils), on the dynamics of rolling and firm arrest on substrates coated with selectin and integrin ligands. To understand better the mechanism of cell-cell adhesion under flow, we will use the same chemistries to measure the kinetics of activation and the strength of adhesion between cell-cell pairs using a novel micropipette aspiration technique. In addition, we will develop a novel calculational method, combining cell-cell and cell-surface interactions to calculate, predict, and confirm the relative contribution of chemical (receptor-mediated) and hydrodynamic interactions in the control of the dynamics of neutrophil rolling and firm adhesion.
Showing the most recent 10 out of 249 publications
