Neutrophils generate microparticles during exposure to inert gases due to cytoskeletal oxidative stress

Abstract

This investigation was to elucidate the mechanism for microparticle (MP) formation triggered by exposures to high pressure inert gases. Human neutrophils generate MPs at a threshold of ∼186 kilopascals with exposures of 30 min or more. Murine cells are similar, but MP production occurs at a slower rate and continues for ∼4 h, whether or not cells remain under pressure. Neutrophils exposed to elevated gas but not hydrostatic pressure produce MPs according to the potency series: argon ≃ nitrogen > helium. Following a similar pattern, gases activate type-2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). MP production does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds) or a NOS-2 inhibitor (1400W) or with cells from mice lacking NOS-2. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and immunoprecipitation studies indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, the H(+)/K(+) ATPase β (flippase), the hematopoietic cell multidrug resistance protein ABC transporter (floppase), and protein-disulfide isomerase in proximity to short actin filaments. Using chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates NOS-2 activation, reactive species generation, actin polymerization, and MP production. These effects were also inhibited in cells exposed to UV light, which photoreverses S-nitrosylated cysteine residues and by co-incubations with the antioxidant ebselen or cytochalasin D. The autocatalytic cycle of protein activation is initiated by inert gas-mediated singlet O2 production.

Keywords: Actin; Decompression; Focal Adhesion Kinase; NADPH Oxidase; Nitrosative Stress; Reactive Nitrogen Species (RNS); Reactive Oxygen Species (ROS); S-Nitrosylation; Singlet Oxygen.

FIGURE 1.

MP production by human neutrophils exposed to air (0 additional kPa of N₂) or air plus 186, 345, or 690 kPa of N₂ for 30 min. Data show MPs/neutrophil in suspensions immediately (open circles) after 1.8 × 10⁵ cells had been exposed to air plus various pressures of N₂ for 30 min or after suspensions were left in air at ambient pressure for 4 h before counting (closed circles). Samples exposed to only ambient air were fixed immediately after suspensions had been prepared (open circle) or after a 4-h incubation (closed circle). Data are mean ± S.E. (error bars), n = 4–9 for each value. All values with elevated pressures of N₂ are significantly different from the 0 value (air only with no added pressure). There are no significant differences between samples at each pressure that were processed immediately (open circles) versus those left for 4 h before MPs were counted (closed circles).

FIGURE 2.

MP production by murine neutrophils exposed to only air or those first exposed to air plus 690 kPa of N₂ for 30 min. Data show MPs/neutrophil in suspensions assayed at the times indicated. All post-N₂ values other than time 0 are significantly different (p < 0.05) from air-exposed control samples (open circles). Data are mean ± S.E. (error bars) (n = 4–12).

FIGURE 3.

Activity of iNOS while murine neutrophils were exposed to air (control) or air plus 345 or 690 kPa of N₂. Data show [³H]citrulline production as mean ± S.E. (error bars) (n = 3 for each 15, 30, and 120 min measurement, n = 5 for 60 min values) when [³H]arginine was added to 1.8 × 10⁵ neutrophils that were then pressurized or left exposed to ambient air. At the indicated times, cells were decompressed, TCA was added, and measurements were taken. At each time, N₂ values are statistically significantly different from air/control, and the 60 and 120 min 345 kPa N₂ values are statistically significantly different from the 690-kPa N₂ values (p < 0.05, ANOVA).

FIGURE 4.

O₂ consumption by murine neutrophils after 30-min exposures to air (control) or air plus 690 kPa of helium, N₂, or argon. Data show changes in O₂ partial pressure over 20 min in buffer containing 1.8 × 10⁵ cells studied immediately after cells had been exposed to the various inert gases as mean ± S.E. (error bars), with individual samples sizes as indicated (n). Also shown is the effect if cells were co-incubated with 10 μm Nox2ds while exposed to air plus 690 kPa of N₂. Not shown, 10 μm Nox2ds had a similar effect of eliminating virtually all O₂ consumption whether cells had been exposed to just air or air plus 690 kPa of helium or argon.

FIGURE 5.

DCF fluorescence in murine neutrophils exposed to 690 kPa of N₂. 10 μm DCF-DA was added to suspensions containing 1.8 × 10⁵ cells that were then pressurized to 690 kPa of N₂ for the indicated times on the abscissa. Fluorescence was measured immediately after cells had been decompressed. Data are mean ± S.E. (error bars), n = 3–12 for each value.

FIGURE 6.

DCF fluorescence in murine neutrophils previously exposed for 30 min to air or air plus 690 kPa of helium, N₂, or argon. Suspensions containing 1.8 × 10⁵ cells plus 10 μm DCF-DA were exposed to the indicated gas for 30 min and decompressed, and fluorescence was monitored for 10 min while cells were exposed to air at ambient pressure. Values show fluorescence subtracted from the value found when samples were first decompressed (e.g. as shown in the figure, each N₂ value was subtracted by ∼12,126). Data are mean ± S.E. (error bars); individual sample sizes were as indicated (n).

FIGURE 7.

Western blot showing biotinylated proteins. Lysates of murine neutrophils exposed only to air or to air plus 690 kPa of N₂ for 30 min were prepared according to the biotin switch assay. The entire gel is shown.

FIGURE 8.

Protein associations in the Triton-soluble short F-actin fraction. Murine neutrophils were exposed to air (control) or air plus 690 kPa of N₂ for 30 min. Where indicated, samples were then exposed to UV light for 5 min prior to the addition of DTSP to cross-link proteins and then fractioned based on Triton solubility (see “Experimental Procedures”) and subjected to Western blotting. This is a representative blot among five replicate experiments. Data based on the blots are shown in Table 4.

FIGURE 9.

Schematic of proposed mechanism for gas-mediated MP generation by neutrophils. See “Discussion” for a detailed explanation. O₂, O₂ in air-saturated solutions; NO_x, higher order reactive nitrogen species, such as nitrogen dioxide or peroxynitrite; Rac, Rac1 and -2 GTPases. FBEs, indicates FBEs as reflecting actin turnover. Manipulations used to verify a role for each protein include incubation of neutrophils with siRNA to decrease intracellular content of the protein proximal to each arrow. KO mice (iNOS knock-out mice) and the agents in boxes are inhibitors used to impede the indicated step in the cycle. Cyto D, cytochalasin D.