Neutrophil Adhesion Mechanics Under Hydrodynamic Shear

DESCRIPTION (Verbatim from Applicant's Abstract): Appropriate neutrophil adhesion is essential for normal human immune system filnction. Abnormal cell adhesion results in either a lack of immune response (e.g. in leukocyte adhesion deficiency (LAD) patients) or in excessive immune response (e.g. in inflammatory disease and dunng certain types of cardiovascular disorders). Elimination of these pathophysiological reactions lies in our ability to control the rate at which neutrophils bind to vascular tissue. Neutrophil adhesion in the vasculature is mediated via the selectin and integrin fam aboutlies of adhesion molecules, and this occurs in the milieu of hydrodynamic shear and cellular activating factors. In the current project we propose to better understand some of the key features of these adhesion proteins and their ligands which contribute to cell adhesion. We anticipate that this quantitative investigation of the cellular, molecular and hydrodynamic factors that control cell binding will in the long run provide the framework for the development of therapeutic strategies. The methodologies we employed involve in vitro measurement of cell adhesion kinetics under shear, flow cytometry, video- and electron- microscopy, and detailed mathematical analysis. The specific aims are: 1) To determine the cutical biophysical and chem aboutcal parameters that control L-, E- and P-selectin mediated adhesion to their natural ligands and to synthetic carbohydrate ligand analogues. These features will be determined by systematically altering the structure of both the cherrucally synthesized selectin-ligand analogue and the amino acid residues in the lectin domain of the selectin molecule, and examining how these structural changes influence the binding function of the selectins. An understanding of these features will aid to more effectively develop selectin-ligand analogues that can be used for anti-adhesion therapy. 2) To deterrnine the factors that contnbute to the differential roles of the p2-integnn subunits (LFA-1 & Mac-1) during neutrophil adhesion, with time following stimulation. Here, we examine how changes in the ,32-integnn receptor number, affinity, topography and cytoskeletal interactions contribute to the time-dependent changes in neutrophil adhesivity following cell stimulation. This fundamental understanding is necessar for the development of anti-adhesive therapies under low shear conditions in the vasculature where adhesion may be purely , beta2-integrin dependent.