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. The increased cell deformability and faster membrane tether growth was consistent with the slower rolling and increased probability of firm arrest we observed for cholesterol-enriched cells (). 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.
More than 90% of cellular cholesterol is found in the plasma membrane (30
). Thus, we examined tether extrusion from cells under flow which provides insight to the lipid flow from the membrane as well as the adhesion strength between the bilayer and the underlying cytoskeleton, with important implications on bond loading and neutrophil rolling dynamics as studied previously(18
). We have found that with increasing cholesterol content, a higher fraction of both neutrophils and HL-60 pulled tethers (). Also, the 1.5- to 2-fold increase in average tether length suggests that the cholesterol-enriched membrane is more likely to dissociate from the cytoskeleton to form longer tethers, while the increase in the growth velocity of the tethers indicate increased fluidity.
A mechanical mechanism is well supported by the experimental data. As previously shown(18
), longer tethers reduce the force loading on the bonds by changing the angle of the mechanical lever arm(17
), thus resulting in a longer lifetime of bonds. To help verify the mechanical-level mechanism underpinning the observation of enhanced tether growth with elevated cholesterol, we have taken FRAP measurements of lipid diffusivity in a pulled tether. We found that cholesterol loading increased lipid diffusivity in the tether by 1.38-fold (p
= 0.04). By lowering the viscosity, the elevated cholesterol makes it easier to pull a tether at a given mechanical loading, which leads to faster growing and longer tethers () that reduced the lever arm and consequently shielded the bond from force loading, thereby lengthening the bond lifetimes () and stabilizing rolling ().
Cholesterol-enriched membrane fluidity has been previously measured using fluorescence polarization (FP) to probe the molecular rotational diffusivity of fluorescent lipids. Typically, cholesterol will reduce “membrane fluidity”(28
), but molecular membrane fluidity is not predictive of translational diffusivity measured by FRAP. In contrast, others have shown an increase in fluidity with cholesterol loading(8
). The FP probe localizes in the membrane whereas FRAP samples larger areas of membrane that may have heterogeneity. Cholesterol effects on the membrane-cytoskeletal linkage, which would also alter mechanically-driven lipid flow into tethers, would not be predicted by FP. Changes in cell shape () indicate that cholesterol effects on the cytoskeleton result in more compliant cells and faster tether growth. Clearly, the effect of membrane cholesterol varies greatly from one cell type to another and depend on experimental technique which measures lipid mobility on length scales of nanometer (FP), micron (FRAP) or several microns (tether pulling and deformation).
We also examined the effect of varying cholesterol levels on whole cell deformation. Previous studies have suggested that the mechanism of actin polymerization may be separate from the mechanism governing pseudopod formation and viscoelastic recovery (35
). During collision with 10-µm diameter beads, cells with elevated cholesterol deformed to an extent that the distance between the centroids of the cell and the bead decreased by 1.8 µm compared to two hard spheres during collision. The magnitude of the effect of cholesterol on cell rigidity becomes more apparent when compared to the effect of ethanol (0.3% by vol.), which also influences tether mechanics and rolling behavior (20
Deformability of neutrophils at the whole-cell level has several physiological implications. In vivo, leukocytes flatten against the endothelial wall to reach aspect ratios of up to 1.4 (36
). Mechanosensing abilities of compliant neutrophils have been suggested from studies of mechanical deformation into narrow channels (19
), while large-scale deformation of neutrophils has been shown to lead to activation (37
). According to computational studies, cellular deformation decreases the drag on the cell and may help account for a plateau of rolling velocity with increasing shear rate (38
). We conclude that cholesterol-induced changes in membrane tether growth and cell deformability are likely to contribute toward enhanced adhesion and firm arrest that have been observed in vivo in high-cholesterol environment (4
The increased deformability after cholesterol enrichment led to a 7-fold increase in contact area between cells and 10-micron beads, as well as a 32% increase in contact time (), both of which may favor increased bonding. In fact, cholesterol enrichment stabilized rolling behavior, decreased rolling velocity, and increased firm arrest ().
Cholesterol enrichment and depletion are expected to have a number of differing effects on neutrophils and neutrophil signaling beyond the direct effects of changing membrane mechanics, which in itself is a complex effect that impacts bonding dynamics with P-selectin. The prominent effect of cholesterol on both tether mechanics and cellular rigidity suggests a link between cholesterol and the actin cytoskeleton (8
). Since cholesterol removal by MβCD has been shown to inhibit polymerization (13
) and disruption of actin cytoskeleton has been shown to interfere with intrinsic selectin adhesiveness (16
), it is also possible that cholesterol effects on the actin network alter the mobility of adhesion molecules in the membrane which affects their binding ability. Also, disruption of the interaction between PSGL-1 and the actin cytoskeleton has been previously shown to reduce adhesion and rolling on P-selectin (39
We observed faster P-selectin-mediated rolling and reduced adhesion for cholesterol-depleted cells, in contrast to cholesterol-enriched cells, which confirms previous observations (10
) and computational modeling studies (40
) of faster rolling of stiffer cells. It is also consistent with prior observation that faster rolling attenuates activation of integrin-mediated arrest (41
), and that cholesterol extraction disrupts integrin-mediated adhesive process (16
The effect of membrane tethers on rolling stability (i.e. reduced variance, ) has been well established (42
). Since rolling has been shown to be unaffected by the intracellular domain of PSGL-1 (43
), we conclude that the reduction in variance of rolling velocity by cholesterol loading or with neutrophils from HC patients was due to enhanced membrane tether growth () and increased deformability with a consequent increase in contact area during rolling (). A functional consequence of altered rolling dynamics, namely increased firm arrest, was detected with neutrophils from HC patients.
Inspection of indicates that by all measures the neutrophils from HC patients are strikingly similar to cholesterol-loaded neutrophils (from healthy donors) and strikingly dissimilar from cholesterol-depleted neutrophils. However, cholesterol depletion has complex biochemical effects on neutrophils, distinct from mechanical changes, that include ablation of caveolae and changes in calcium entry (44
). Still, the rolling flux and percent arrested on P-selectin surfaces were not statistically different for cholesterol depleted cells and control neutrophils (), however both these attributes were reduced (but not ablated) on IL-1 stimulated HAEC. These observations are consistent with cholesterol depletion causing a biomechanical change in rolling dynamics, but a biochemical change in signaling dynamics related to firm arrest. Clearly, PSGL-1 dependent signaling dynamics such as Syk-dependent activation of LFA-1 (43
) and subsequent conversion to firm arrest of neutrophils from HC patients or cholesterol-enriched neutrophils from healthy donors remains an important subject of future study. Furthermore, the cell softening in cholesterol-loaded cells () was clearly a result of changes in whole cell mechanics that include cytoskeletal function.
Overall, our results show that elevated cholesterol levels yield a slower, more stable rolling behavior, likely due to longer tethers with increased lifetime and increased whole cell deformability with increased contact area during rolling. Distinct from purely biochemical effects, membrane cholesterol is a biomechanical regulator of tether growth and whole-cell mechanics of neutrophil which, in turn, modulates their neutrophil rolling and adhesion.