Interplay of Ethanol and Cholesterol in the Neutrophil Membrane: Effects on Membrane and Cell Mechanics and Neutrophil Adhesion.

Previous work from our lab has demonstrated that ethanol and cholesterol each have significant effects on neutrophil tethering, rolling, and adhesion. Since ethanol and cholesterol are often present within the body simultaneously, we endeavored to characterize their combined effects on neutrophil membrane mechanics and adhesion. The effect of ethanol on membrane cholesterol loading was ascertained by incubating neutrophils with 0.3% ethanol before or after cholesterol manipulation with methyl-beta-cyclodextrin (MβCD) or MβCD/cholesterol complexes. Microcapillary flow chamber assays at a wall shear rate of 100 s-1 were used to examine the effects of ethanol on cholesterol-loaded neutrophils with respect to: (1) collision efficiency and membrane tethering to P-selectin-coated microbeads, (2) rolling on P-selectin-coated surfaces, and (3) primary and secondary interactions with neutrophils preadhered to intercellular adhesion molecule-1 (ICAM-1). We performed flow cytometry experiments to assess the effects of ethanol and cholesterol together on adhesion molecule expression. Microcapillary flow chambers presenting chamber-adherent, non-adhesive and P-selectin-coated 10 μm beads were used to assess neutrophil whole-cell deformation changes with separate ethanol and cholesterol treatment, and varied shear rate. The individual effects of ethanol and cholesterol on membrane fluidity, measured by membrane lipid diffusivity, were determined using fluorescence recovery after photobleaching (FRAP) experiments to obtain lipid diffusion coefficients. We found that membrane cholesterol was increased over control in the absence of ethanol; ethanol pre- or post- loading reduced this increase. Cholesterol enrichment did not alter CD11b expression; however, PSGL-1 and L-selectin expression were lowered by cholesterol enrichment plus or minus ethanol. Ethanol appeared to abrogate the pro-adhesive effects of cholesterol, but it did not impact cholesterol's ability to enhance tethering probability. Ethanol enhanced membrane fluidity of cholesterol-enriched neutrophils as indicated by tethering metrics. Ethanol and cholesterol enrichment both increased neutrophil deformation, while cholesterol depletion decreased neutrophil deformation. Neutrophil deformation increased in a dose-dependent manner with shear rate. Membrane lipid diffusivity was increased by ethanol administration and cholesterol enrichment, and decreased by cholesterol depletion. Our research shows that ethanol and cholesterol interact in a complex manner in the neutrophil membrane, sometimes in concert, and sometimes in conflict, to influence neutrophil adhesion via biomechanical and biochemical effects.