Affiliations: Department of Bioengineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA | Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA, USA
Note: [] Address for correspondence: Dr. Scott L. Diamond, Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, 1024 Vagelos Research Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA. Tel.: +1 215 573 5704; Fax: +1 215 573 6815; E-mail: [email protected].
Abstract: Using microfluidic assays at a 100 s−1 wall shear rate, we examined 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 pre-adhered to intercellular adhesion molecule-1 (ICAM-1). Using methyl-β-cyclodextrin:cholesterol complexes, membrane cholesterol was increased over control by 4.6-fold (no ethanol), 3.6-fold (0.3% ethanol pre-loading), and 1.6-fold (0.3% ethanol post-loading). These treatments did not alter CD11b expression; however, PSGL-1 and L-selectin were lowered by cholesterol enrichment (±ethanol). Cholesterol enrichment enhanced microbead collision efficiency, which was abrogated by ethanol. Ethanol had no effect on elevation of tethering fraction by cholesterol enrichment. Incubation of cholesterol-loaded neutrophils with ethanol resulted in significantly longer membrane tethers, due to tether lifetime enhancement. On P-selectin-coated surfaces, cholesterol-enriched neutrophils exposed to ethanol rolled faster and with more variability than cholesterol-enriched neutrophils. Ethanol reduced homotypic collision efficiency of cholesterol-loaded neutrophils without effect on tethering fraction or secondary collision efficiency. Tether length during cholesterol-loaded neutrophil homotypic collisions was enhanced by ethanol, in part due to increased L-selectin/PSGL-1 bond tether lifetime. Overall, ethanol attenuated cholesterol-induced adhesion increases while increasing membrane fluidity as indicated by tether length.
