title: Oxidant alterations in CD16 expression are cytoskeletal induced.
abstract: Oxidative stress during reperfusion of ischemia is associated with a phenotypic change in circulating monocytes from CD14++CD16- to a proinflammatory CD14+CD16+ subpopulation resulting in altered immunity and development of organ failure. However, the mechanism responsible remains unknown. We hypothesize that this phenotypic change, modeled by hydrogen peroxide exposure in vitro, is due to oxidative-induced intracellular calcium flux and distinct cytoskeletal and lipid raft changes. Peripheral blood monocytes obtained from healthy volunteers underwent 100 mM H2O2 exposure for 0 to 24 h. Selected cells were pretreated with 2 microM cytochalasin D, 1 microM lactrunculin A, or 30 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid for 30 min. Cells underwent fluorescence-activated cell sorter for CD14, CD16, and cytokine expression. Cellular and lipid raft CD16 expression was determined by immunoblot and confocal microscopy. H2O2 exposed monocytes underwent a rapid time-dependent increase in the surface expression of CD16 from 12.81% +/- 3.53% to 37.12% +/- 7.61% at 24 h (P = 0.001). Total cellular CD16 was not changed by H2O2, but an increase in lipid raft and decrease in intracellular CD16 expression were seen after H2O2 exposure. This increase in CD16 expression was associated with a 27% increase in intracellular TNF-alpha, an alteration in actin polymerization, and the formation of raft macrodomains. These changes induced by H2O2 were inhibited by inhibition of actin polymerization (cytochalasin D and lactrunculin A) and intracellular calcium flux [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. This study provides the first evidence that phenotypic alterations induced by oxidative stress during reperfusion may occur as a result of changes in cytoskeletal architecture due to calcium flux that result in lipid raft alterations rather than solely from demargination and/or production of bone marrow-derived CD16+ monocytes.
authors.affiliation: Department of Surgery, University of Washington, Seattle, Washington, USA. jcuschie@u.washington.edu
authors.forename: Joseph
authors.initials: J
authors.lastname: Cuschieri
authors.forename: Sana
authors.initials: S
authors.lastname: Sakr
authors.forename: Eileen
authors.initials: E
authors.lastname: Bulger
authors.forename: Megan
authors.initials: M
authors.lastname: Knoll
authors.forename: Saman
authors.initials: S
authors.lastname: Arbabi
authors.forename: Ronald V
authors.initials: RV
authors.lastname: Maier