Membrane Cholesterol Regulates Neutrophil Membrane Tether Mechanics and Rolling Dynamics

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.