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. 2009 Dec;32(6):572-7.
doi: 10.1097/SHK.0b013e3181a72530.

Oxidant alterations in CD16 expression are cytoskeletal induced

Joseph Cuschieri  1 , Sana Sakr, Eileen Bulger, Megan Knoll, Saman Arbabi, Ronald V Maier
Affiliations

Oxidant alterations in CD16 expression are cytoskeletal induced

Joseph Cuschieri et al. Shock. 2009 Dec.

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.

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Figures

Figure 1
Figure 1. Oxidant exposure results in a time dependent surface expression of CD16 that requires maintenance of cytoskeletal integrity
Human monocytes were stimulated with 100 mM H2O2 for 24 hours. Selected cells were pretreated with either 2 μM CD or 1 μM LA. (A) Surface expression of both CD16 was then examined under all conditions with appropriate isotype control antibody (demonstrated in grey) by FACscan.
Figure 2
Figure 2
Oxidant induced CD16 surface expression is exposure dependent. To determine the rate of CD16 surface expression changes, human monocytes were stimulated with 100 mM 100 mM H2O2 for up to 24 hours. Selected cells were pretreated with either 2 μM CD or 1 μM LA. Time dependent surface expression of CD16 was determined under the various conditions by FACscan. * P <0.05 vs. time-matched control values based on 5 separately performed experiments.
Figure 3
Figure 3. Oxidant induced CD16 surface expression originates from non-raft intracellular CD16
Human monocytes were stimulated with 100 mM H2O2 for 24 hours. Selected cells were pretreated with either 2 μM CD or 1 μM LA. Lipid raft, non-raft and total protein were harvested, and analyzed by Western blot for CD16 . Constitutively found lipid raft SRC, non-raft cytoskeletal actin and cytoplasmic ERK 1 under the various extractions served as controls to access equal loading (data not shown). Representative blots from one of three separately performed experiments are shown.
Figure 4
Figure 4. Lipid raft mobilization of CD16
Human monocyte colocalization of CD16 and lipid rafts was determined by immunohistochemistry. Both control and H2O2 exposed cellular expression of lipid rafts (green) and CD16 (red) were determined. Colocalization was determined by overlaying images resulting in yellow fluorescence. Data based on 5 separately performed experiments
Figure 5
Figure 5. Oxidant exposure results in enhanced intracellular expression of TNF-α within monocyte subpopulations
Human monocytes were stimulated with 100 mM H2O2 for up to 24 hours. Selected cells were pretreated with either 2 μM CD or 1 μM LA. Intracellular TNF-a expression was then examined with appropriate isotype control antibody by FACscan. Data are expressed as percentage change in mean fluorescence intensity (ΔMFI) compared to baseline conditions (set at 100%). Significant differences: * P <0.05 vs. time-matched control values based on 6 separately performed experiments.
Figure 6
Figure 6. Oxidant induced cytoskeletal changes are calcium dependent
Human monocytes were stimulated with 100 mM H2O2 for 30 minutes. Selected cells were pretreated with 30 μM BAPTA. Cells were fixed and stained for actin, and then examined by confocal microscopy. Representative images of three separately performed experiments are shown.
Figure 7
Figure 7. Oxidant induced surface expression of CD16 requires intracellular calcium flux
Human monocytes were stimulated with 100 mM H2O2 for 24 hours. Selected cells were pretreated with 30 μM BAPTA. Surface expression of both CD14 and CD16 was then examined under all conditions with appropriate isotype control antibody by FACscan. * P <0.05 vs. time-matched control values based on 5 separately performed experiments.
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