Quantitative methods for understanding physical mechanisms of neutrophil adhesion

Thesis (Ph. D.)--University of Rochester. Dept. of Biomedical Engineering, 2008.During inflammation, neutrophils first roll and then firmly adhere to activated endothelium in a sequence known as the adhesion cascade. Knowing the mobility and localization of the receptors that mediate binding between endothelium and neutrophils is key to building an accurate biophysical model of the adhesion cascade. The objective of this thesis is to characterize the mobility and localization of the main neutrophil adhesion receptors involved in the rolling and firm arrest stages of neutrophil recruitment To measure the natural movements of neutrophil membrane receptors, we built a novel fluorescence recovery after photobleaching (FRAP) experimental system and developed a set of complementary analytical tools to interpret data. Experiments examined non-activated neutrophils in their normal, spherical morphology. Prior studies have been performed on artificially flattened cells to accommodate the requirements of established methods for measuring diffusion of membrane proteins. Further, our studies are the first to measure the mobility of all four neutrophil receptors involved in early adhesion: the rolling associated adhesion molecules L-selectin and PSGL-1, and the β2 integrins LFA-1 and Mac-1. On resting neutrophils we found that β2 integrins have mobilities 3-7x greater than rolling associated adhesion molecules, but that mobilities within these classes was indistinguishable. We also found that β2 integrin mobility (LFA-1, D = 1.2 x 10-10 cm2/sec; 37oC, Fab) approached the expected value for free protein diffusion and was not affected by cytoskeletal poisons. These results suggest any cytoskeletal hindrance does not limit bulk integrin diffusion in resting neutrophils over distances and times important for adhesive plaque formation. Another important physical parameter in adhesion regulation is localization on the ruffled neutrophil surface. Total internal reflection fluorescence microscopy confirms for the first time on live resting neutrophils that L-selectin is localized to the microvilli tips, while LFA-1 is restricted to the cell body, away from potential contact surfaces. All four key adhesion receptors were observed to be localized to dynamic, mobile domains that corresponded to this topological positioning. We adapted image correlation microscopy (ICM) to wide-field use with the goal of measuring lateral mobility of these domains. We find that domain lateral mobility is similar to measurements made with FRAP, suggesting domain movement is a significant component of adhesion molecule lateral mobility in the membrane. Our results help elucidate how adhesion receptors move on the neutrophil surface and how adhesive plaques can form and strengthen following rolling. Beyond adding insight and building intuition, the quantitative results will aid computer simulations of the recruitment process, helping predict behavior and test theorems based on empirical evidence