Membrane cholesterol is a biomechanical regulator of neutrophil adhesion

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-beta-cyclodextrin (MbetaCD) removed up to 73% and 45% of membrane cholesterol from HL-60 cells and neutrophils, whereas MbetaCD/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-microm diameter beads, uncoated 10-mum diameter beads, P-selectin-coated surfaces, or activated endothelium. Elevated cholesterol enhanced capture efficiency to 1-microm 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-mum 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.

Figure 1. Cholesterol depletion and enrichment of neutrophils and HL-60 cells and the effect on surface integrin expression

Cells were cholesterol-depleted by incubation with MβCD (A) or cholesterol-enriched with MβCD-cholesterol complexes (B). Cholesterol content after each treatment was normalized to control levels. N=4 donors. Mean Fluorescence Intensity (MFI) of FITC-conjugated CD11b and CD18 were normalized to control levels for each (C). N=3 donors.

Figure 2. Instantaneous tether growth of cholesterol-depleted, control, and cholesterol-enriched neutrophils

Tether length was measured frame-by-frame during neutrophil-bead adhesion for 10 representative tethers under each condition. The averages and the linear fit are compared. Mean ± standard deviation. N=3 donors.

Figure 3. Neutrophil whole-cell deformation by centroid tracking

Image sequences were obtained during neutrophil collision with 10-µm-diameter beads at 240 fps videomicroscopy for untreated neutrophils (A) and cholesterol-enriched neutrophils (B).The distance between the centroids of the cell and the bead, R, was tracked (C). Mean ± standard deviation of 10 cells under each condition.