Experimental and computational studies of selectin -mediated leukocyte adhesion

Selectins are cell adhesion molecules that initiate recruitment of white blood cells to targeted sites on the blood vessel wall. This process is necessary for numerous immune functions, including inflammation, lymphocyte homing, and bone marrow replenishment after transplantation. Cell recruitment proceeds via a multi-step process, with selectin-mediated rolling followed by integrin-mediated firm adhesion. This thesis extends current knowledge of the mechanisms governing selectin-mediated leukocyte adhesion by examining the kinematics of selectin-mediated rolling, structure-function relationships in selectins and their ligands, and the roles of selectins and integrins in promoting cell adhesion. We characterized a yeast display system that may be used for directed evolution and structure-function studies of selectins. E-selectin-expressing yeast were perfused over sialyl-Lewisx-bearing surfaces in a parallel plate flow chamber. We find that E-selectin-expressing yeast are functional, interacting specifically with sLex ligands. Measurement of yeast adhesion under flow provides a functional assay for the activity of selectin-ligand interactions. Kinematic analysis of budding yeast demonstrates that selectin-expressing cells roll rather than slip across ligand-coated surfaces. Structure-function relationships in selectin ligands were studied using the method of cell-free rolling. The influence of sLex sulfation, sLex density, and L-selectin density on the shear threshold effect was examined in cell-free systems, in which carbohydrate-coated microspheres are perfused over L-selectin substrate. We find that a shear threshold is required for L-selectin-mediated rolling only when low numbers of receptor-ligand bonds can be formed. Sulfation of sLex does not affect the appearance or location of the shear threshold. The roles of selectin-sLex interactions and integrin-ICAM-1 interactions in mediating cell adhesion were investigated using two-receptor adhesive dynamic (AD) simulations. AD simulations predict synergistic functions of the two receptors in mediating adhesion. We present this relationship in a two-receptor state diagram, a map that relates the densities and properties of adhesion molecules to various adhesive behaviors that they code, such as rolling or firm adhesion. We also present a state diagram for neutrophil activation, which relates β2 integrin density and integrin-ICAM-1 kinetic on-rate to neutrophil adhesive behavior. Simulation predictions are validated by the ability of the model to reproduce in vivo neutrophil rolling velocities from animal experiments