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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:1290.)
© 2009 American Heart Association, Inc.

National Cholesterol Awareness Month

Membrane Cholesterol Is a Biomechanical Regulator of Neutrophil Adhesion

Hana Oh; Emile R. Mohler, III; Aiwei Tian; Tobias Baumgart; Scott L. Diamond

From the Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering (H.O., S.L.D.), the Department of Medicine (E.R.M.), and the Department of Chemistry (A.T., T.B.), University of Pennsylvania, Philadelphia.

Correspondence to Scott L. Diamond, 1024 Vagelos Research Laboratory, 3340 Smith Walk, Philadelphia, PA 19104. E-mail sld{at}seas.upenn.edu

Abstract

Objective— The purpose of this study was 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, whereas 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.

Neutrophils and HL-60 cells were perfused into flow chambers at 100 s^–1 after cholesterol enrichment or depletion. Cholesterol enrichment increased P-selectin–mediated adhesion, membrane tethering fraction, membrane tether length and growth rate. Cells with higher cholesterol content were more deformable and rolled more slowly and uniformly on P-selectin or activated endothelium.

Key Words: neutrophils • cholesterol • cell adhesion • endothelium • membrane • inflammation