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. 2011 Mar;44(3):333-40.
doi: 10.1165/rcmb.2009-0408OC. Epub 2010 Apr 23.

Hemorrhagic shock activates lung endothelial reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase via neutrophil NADPH oxidase

Meng Xiang  1 , Lianhua Yin, Yuehua Li, Guozhi Xiao, Yoram Vodovotz, Timothy R Billiar, Mark A Wilson, Jie Fan
Affiliations

Hemorrhagic shock activates lung endothelial reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase via neutrophil NADPH oxidase

Meng Xiang et al. Am J Respir Cell Mol Biol. 2011 Mar.

Abstract

The vascular endothelium plays an important role in the regulation of inflammatory responses after trauma and hemorrhage. Interactions of neutrophils with endothelial cells (ECs) contribute to the activation of specific EC responses involved in innate immunity. We have previously reported that oxidants derived from the neutrophil reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a critical regulator to EC activation. Our objective was to test the role of neutrophil NADPH oxidase-derived oxidants in mediating and enhancing hemorrhagic shock (HS)-induced activation of lung endothelial NADPH oxidase. Mice were subjected to HS and neutrophil depletion. The mice were also replenished with the neutrophil from NADPH oxidase-deficient mice. The resultant activation of lung NADPH oxidase was analyzed. The in vivo studies were also recapitulated with in vitro neutrophil-EC coculture system. HS induces NADPH oxidase activation in neutrophils and lung through high-mobility group box 1/Toll-like receptor 4-dependent signaling. In neutropenic mice, shock-induced NADPH oxidase activation in the lung was reduced significantly, but was restored upon repletion with neutrophils obtained from wild-type mice subjected to shock, but not with neutrophils from shock mice lacking the gp91(phox) subunit of NADPH oxidase. The findings were recapitulated in mouse lung vascular ECs cocultured with neutrophils. The data further demonstrate that neutrophil-derived oxidants are key factors mediating augmented High mobility group box 1 (HMGB1)-induced endothelial NADPH oxidase activation through a Rac1-dependent, but p38 mitogen-activated protein kinase-independent, pathway. Oxidant signaling by neutrophil NADPH oxidase is an important determinant of activation of endothelial NADPH oxidase after HS.

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Figures

Figure 1.
Figure 1.
Hemorrhagic shock (HS) induction of phosphorylation of p47phox in the polymorphonuclear leukocyte (PMN) and lung. (A) Wild-type (WT; C57BL/6) mice were subjected to HS or sham operation. PMN and lung tissue were obtained 1, 2, 4, and 6 hours after HS, or 6 hours after sham operation. Total and phospho-p47phox in the PMN and lung were detected using immunoprecipitation and immunoblotting, as described in Materials and Methods. The graph depicts the mean and SEM of the changes in ratio of phospho:total p47phox from five mice. *P < 0.01 compared with the groups labeled with no asterisk. (B) high mobility group box 1 (HMGB1) mediates HS-induced p47phox phosphorylation in the lung. WT mice and Toll-like receptor (TLR) 4–mutant mice received anti-HMGB1 antibody (600 μg per mouse) or nonimmune control IgG by intraperitoneal injection 10 minutes before hemorrhage or sham operation. Lung tissue was then collected from the mice at 4 hours after HS or sham operation for detection of total and phospho-p47phox using immunoprecipitation and immunoblotting. The images are representative of four independent studies. The graph depicts the mean and SEM of the changes in ratio of phospho:total p47phox from four mice. *P < 0.01 compared with all other groups.
Figure 2.
Figure 2.
PMN reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase signals HS-induced activation of endothelial cell (EC) NADPH oxidase. (A) Effect of PMN depletion and repletion on the HS-induced p47phox phosphorylation in the lung. PMN depletion was performed as described in the Materials and Methods 16 hours before hemorrhage or sham (SM) operation. PMN repletion was performed in neutropenic mice during resuscitation using tail vein injection of PMNs that were isolated from blood of WT or gp91phox−/− mice subjected to sham or HS. The lungs were harvested from the mice 4 hours after resuscitation or sham operation for the detection of p47phox phosphorylation by immunoprecipitation and immunoblotting. The graph depicts the mean and SEM of the changes in ratio of phospho:total p47phox from four mice. **P < 0.01 compared with all other groups; *P < 0.01 compared with the groups labeled with no asterisk. (B) Myeloperoxidase (MPO) concentration in the unflushed and flushed lungs after HS. MPO concentration in the lungs, which were flushed through bronchoalveolar lavage (BAL) and received a right ventricular injection of PBS to remove PMNs and other myeloid cells before the tissue harvesting, was measured by ELISA, and compared with that in the lungs that were not flushed (unflushed lung). The lungs were collected at 2, 4, and 6 hours after HS, or 6 hours after sham operation (SM). The graph depicts the mean and SEM of the changes in the lung MPO concentration from four mice. *P < 0.01 compared with the groups labeled with no asterisk. (C) PMN-derived p47phox-gp91phox binding was undetectable in the flushed lung. PMNs in gp91phox−/− mice were replaced with WT PMNs using PMN depletion and repletion techniques before the mice were subjected to HS. At 4 hours after HS, the lungs were flushed through BAL and received a right ventricular injection of PBS, and the p47phox-gp91phox binding in the lungs was then measured by immunoprecipitation and immunoblotting. The gel as such is a representative of three independent experiments.
Figure 3.
Figure 3.
Effect of oxidants derived from PMN NADPH oxidase on HMGB1-induced binding of p47phox-gp91phox and p47phox phosphorylation in mouse lung vascular ECs (MLVECs). WT and TLR4-mutant MLVECs were coincubated with PMNs that were isolated from the blood of WT or gp91phox−/− (gp91−/−) mice in the presence or absence of HMGB1 (0.5 μg/ml) for 2 hours. Membrane-permeable polyethylene glycol (PEG) catalase (1,000 U/ml) was applied to the coculture system as indicated. The MLVECs were then recovered for the detection of p47phox-gp91phox binding and p47phox phosphorylation using immunoprecipitation and immunoblotting. The graph depicts the mean and SEM of the percentage changes in the p47phox-gp91phox binding in the MLVECs isolated from WT (solid bars) and TLR4 mutant (open bars), which were normalized by the density of total p47phox, from four experiments. **P< 0.01 compared with all other groups; *P < 0.01 compared with the groups labeled with no asterisk.
Figure 4.
Figure 4.
Role of H2O2 in enhancing NADPH oxidase activation and reactive oxygen species (ROS) production in MLVECs. WT MLVECs were treated with HMGB1 (0.5 μg/ml) in the presence or absence of H2O2 (250 μM) for 1 hour. The MLVECs were then recovered for the detection of p47phox-gp91phox binding and p47phox phosphorylation using immunoprecipitation and immunoblotting. H2O2 enhanced the p47phox-gp91phox binding and p47phox phosphorylation in response to HMGB1. The graph depicts the mean and SEM of the percent changes in the p47phox-gp91phox binding, which were normalized by the density of total p47phox, from four experiments. **P < 0.01 compared with all other groups; *P < 0.01 compared with the groups labeled with no asterisk. (B) ROS production in live MLVECs. WT MLVECs that were cultured in 12-well cell culture plates were stained with the cell-permeable ROS detection reagent, H2DFFDA, in the concentration of 10 μM for 10 minutes. The cells were then washed three times with Hanks' balanced salt solution, followed by incubation in the growth medium in the presence or absence of HMGB1 (0.5 μg/ml) and/or H2O2 (250 μM) for 2 hours. The ROS production was detected by fluorescence microscopy at different time points, as indicated. To define the source of the ROS, the NADPH oxidase–specific inhibitor, diphenyleneiodonium (DPI; 100 μM), was added to the MLVECs immediately before the treatment of HMGB1 and H2O2. The images are representative of four independent studies.
Figure 5.
Figure 5.
Effect of inhibitors on HMGB1 and/or H2O2 induction of NADPH oxidase activation in MLVECs. Inhibitors of Rac1 (NSC23766, 200 μM), Akt (4 μM), and p38 mitogen-activated protein kinase (MAPK) (SB202190, 4 μM) were added to WT MLVECs, which were then treated with HMGB1 and/or H2O2 for 1 hour. Inhibitors of Rac1 and p38 MAPK, but not inhibitor of Akt, markedly attenuated the p47phox-gp91phox binding and p47phox phosphorylation in response to HMGB1 alone. In the groups treated with HMGB1 and H2O2, the inhibitor of Rac1 decreased the p47phox-gp91phox binding and p47phox phosphorylation, whereas inhibitors of p38 MAPK and Akt failed to exhibit an effect in the MLVECs. The graph depicts the mean and SEM of the percent changes in the p47phox-gp91phox binding, which were normalized by the density of total p47phox, from four experiments. **P < 0.01 compared with all other groups; *P < 0.01 compared with the groups labeled with no asterisk.
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