Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Abstract

Objective-To evaluate the role of membrane cholesterol on human neutrophil and HL-60 biomechanics, capture, rolling, and arrest to P-selectin or IL-1-activated endothelium.Methods and Results-Methyl-β-cyclodextrin (MβCD) 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. 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. Elevated cholesterol levels increased tether lifetime by 17% in neutrophils and adhesion lifetime by 63% in HL-60 cells. Deformation of cholesterol-enriched neutrophils increased the contact time with 10-µm beads by 32% and the contact area by 7-fold. On both P-selectin surfaces and endothelial-cell monolayers, cholesterol-enriched neutrophils rolled more slowly, more stably, and were more likely to firmly arrest. Cholesterol depletion resulted in opposite effects.Conclusions-Increasing membrane cholesterol enhanced membrane tether formation and whole cell deformability, contributing to slower, more stable rolling on P-selectin and increased firm arrest on activated endothelium.

Shear-Sensitive Regulation of Neutrophil Flow Behavior and Its Potential Impact on Microvascular Blood Flow Dysregulation in Hypercholesterolemia

“…10,18 Notably, cholesterol influences the regulation of CD18-related neutrophil adhesivity. 41,42 Its enrichment in the cell membrane raises CD18 surface levels and neutrophil adhesion. 42,43 Thus, the major impact of hypercholesterolemia on RH may, in fact, be attributable to its effects on shear-sensitive CD18 proteolysis.…”

“…41,42 Its enrichment in the cell membrane raises CD18 surface levels and neutrophil adhesion. 42,43 Thus, the major impact of hypercholesterolemia on RH may, in fact, be attributable to its effects on shear-sensitive CD18 proteolysis. Despite this, our data are still the first to implicate neutrophil shear sensitivity in RH.…”

“…10,18 Notably, cholesterol influences the regulation of CD18-related neutrophil adhesivity. 41,42 Its enrichment in the cell membrane raises CD18 surface levels and neutrophil adhesion.…”

Linking the Pathobiology of Hypercholesterolemia with the Neutrophil Mechanotransduction

“…In addition to de novo biosynthesis, mammalian cells can take up cholesterol from the extracellular milieu. Exposure to elevated cholesterol levels both in vivo and in vitro enhances cholesterol abundance within the plasma membrane of neutrophils and other blood cells [39][40][41][42]. These findings, in conjunction with the wealth of evidence demonstrating the influence of the extracellular cholesterol levels on neutrophil activity, point to membrane cholesterol enrichment as a potential link between hypercholesterolemia and chronic neutrophil activity.…”

“…For example, neutrophils with increased membrane cholesterol exhibit enhanced tethering and firm arrest on activated endothelial cell monolayers [41,71,72]. Moreover, neutrophils exposed to hypercholesterolemia display increased heterotypic adhesion to platelets [73] as well as increased homotypic aggregation in response to 10 M N-formyl-Met-Leu-Phe (fMLP) [74].…”