title: Alterations in membrane cholesterol cause mobilization of lipid rafts from specific granules and prime human neutrophils for enhanced adherence-dependent oxidant production.
abstract: The present study sought to examine the function of membrane lipid rafts in adherence-dependent oxidant production in human neutrophils. Rafts are membrane domains that are rich in glycosphingolipids and cholesterol and are thought to be the foci for formation of signaling complexes in a variety of cells. Disruption of lipid rafts by depletion of membrane cholesterol with the chelating agent methyl-beta-cyclodextrin (MbetaCD) has been widely used to examine the function of lipid rafts. Here, we report that treatment of human neutrophils with MbetaCD unexpectedly caused priming of these cells, manifested as enhanced adherence-dependent oxidant production. Treatment of neutrophils with MbetaCD dose-dependently increased oxidant production after adhesion to fibronectin-coated plates. This priming effect was associated with recruitment of CD11b- and CD66b-rich raft domains from the specific granules, as determined by immunoblot and flow cytometry. Confocal microscopy showed that MbetaCD caused otherwise untreated neutrophils to rapidly adhere and spread on fibronectin-coated plates. Furthermore, three-dimensional reconstruction microscopy studies showed that MbetaCD caused expansion and coalescence of raft domains that covered most of the cell surface. These large raft domains expressed CD11b primarily in the core of these regions. Our studies demonstrate that cholesterol depletion with MbetaCD results in neutrophil priming manifested as enhanced adherence-dependent oxidant production. These studies caution against assumption that any observed MbetaCD effects are a function of reduced raft formation.
authors.affiliation: The Laboratory of Trauma, Sepsis and Inflammation Research, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0558, USA.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Solomkin
authors.forename: Chad T
authors.initials: CT
authors.lastname: Robinson
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Cave
authors.forename: Birgit
authors.initials: B
authors.lastname: Ehmer
authors.forename: Alex B
authors.initials: AB
authors.lastname: Lentsch
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D058851:GPI-Linked Proteins; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007104:Immune Adherence Reaction; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D015703:Antigens, CD; D039481:CD11b Antigen; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; C509295:ITGAM protein, human; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D006861:Hydrogen Peroxide