(649a) Membrane Cholesterol Regulates Neutrophil Membrane Tether Mechanics and Rolling Dynamics

Oh, H., University of Pennsylvania
Diamond, S. L., University of Pennsylvania

We evaluated the role of membrane cholesterol on human neutrophil and HL-60 biomechanics, capture, rolling, and arrest to P-selectin or IL-1-activated endothelium. Cholesterol content was modulated using Methyl-β-cyclodextrin (MβCD), which removed up to 73% and 45% of membrane cholesterol from HL-60 cells and neutrophils, while MβCD/cholesterol complexes resulted in maximum enrichment of 65% and 40%, respectively, above control levels. Using parallel-plate flow chambers, cells were perfused at a venous wall shear rate of 100 s-1 over adherent P-selectin-coated 1-μm diameter beads, uncoated 10-μm diameter beads, P-selectin-coated surfaces, or activated endothelium. Elevated cholesterol enhanced capture efficiency to 1-μm beads and increased membrane tether growth rate by 1.5- to 2-fold, whereas cholesterol depletion greatly reduced tether formation. Increased membrane cholesterol levels enhanced tether lifetime in neutrophils and adhesion lifetime in HL-60 cells. Cholesterol-enriched neutrophils displayed significantly higher deformability during collisions with 10-μm-diameter beads, which increased the contact time by 32% and the contact area by 7-fold. Neutrophils with higher cholesterol content rolled more slowly, more stably, and were more likely to firmly arrest on both P-selectin surfaces and endothelial-cell monolayers, Cholesterol depletion resulted in opposite effects. Our results show that enhanced membrane cholesterol increased membrane tether formation and whole cell deformability, contributing to slower, more stable rolling on P-selectin and increased firm arrest on activated endothelium.