Modulation of Leukocyte Mechanotransduction of Fluid Shear Stress by the Cell Membrane

Fluid shear stresses due to blood flow controls leukocyte activity by down-regulating pseudopod formation and surface expression of CD18 adhesion molecules, key events during acute inflammation. These shear responses prevent adhesion and microvascular entrapment of leukocytes under physiological conditions and, if impaired, may lead to chronic cell activity in the microcirculation related to cardiovascular disease (e.g., hypercholesterolemia, hypertension, etc.). Recently, I revealed an underlying shear-related mechanism involving structural changes in CD18 integrins that promotes its own cleavage by lysosomal cathepsin B (catB), a protease linked to flow-induced pseudopod retraction. An implication of this discovery is that the cell membrane influences leukocyte shear responses due to its location between the cytosol and the extracellular fluid as well as its role as a substrate for CD18 receptors and a barrier between catB and its extracellular target(s). As such, changes in the cell membrane properties (e.g., fluidity), such as due to circulatory disorders (e.g., hypercholesterolemia), may alter the ability of shear to induce changes in the CD18 structure or secrete catB and thus affect the degree to which leukocytes respond to flow (i.e., mechanosensitivity) with pathological consequences (e.g., capillary rarefaction). My hypothesis is that the fluidity of the cell membrane influences leukocyte mechanosensitivity by influencing shear-induced activation and catB-mediated cleavage of CD18 leading to a phenotype that is insensitive to flow. I propose to address this hypothesis with the following 3 specific aims: 1) to establish membrane fluidity as a physical modulator of leukocyte functional (i.e., pseudopod retraction) responses to shear stress; 2) to confirm membrane fluidity as a physical factor that influences shear-induced catB release and CD18 cleavage, an underlying cellular mechanotransducing mechanism; and 3) to correlate elevated blood cholesterol levels in hypercholesterolemic mice with altered membrane fluidity and impaired leukocyte mechanosensitivity to fluid shear stress. For this purpose, I will utilize in vitro cell mechanics methodologies and cell/molecular biology tools to elucidate quantitative relationships between the cell membrane properties and neutrophil responses to shear. The results of these studies will shed new insight into the importance of cell mechanosensitivity in the control of acute inflammatory processes.