Spermine suppresses the activation of human neutrophil NADPH oxidase in cell-free and semi-recombinant systems.
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ABSTRACT:
SUBMITTER: Ogata K
PROVIDER: S-EPMC1216942 | biostudies-other | 1996 Jan
REPOSITORIES: biostudies-other
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Project description:The phagocyte reduced NAD phosphate (NADPH) oxidase generates superoxide, the precursor to reactive oxygen species (ROS) that has both antimicrobial and immunoregulatory functions. Inactivating mutations in NADPH oxidase alleles cause chronic granulomatous disease (CGD), characterized by enhanced susceptibility to life-threatening microbial infections and inflammatory disorders; hypomorphic NADPH oxidase alleles are associated with autoimmunity. Impaired apoptotic cell (AC) clearance is implicated as an important contributing factor in chronic inflammation and autoimmunity, but the role of NADPH oxidase-derived ROS in this process is incompletely understood. Here, we demonstrate that phagocytosis of AC (efferocytosis) potently activated NADPH oxidase in mouse peritoneal exudate macrophages (PEMs). ROS generation was dependent on macrophage CD11b, Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response 88 (MyD88), and was also regulated by phosphatidylinositol 3-phosphate binding to the p40 phox oxidase subunit. Maturation of efferosomes containing apoptotic neutrophils was significantly delayed in CGD PEMs, including acidification and acquisition of proteolytic activity, and was associated with slower digestion of apoptotic neutrophil proteins. Treatment of wild-type macrophages with the vacuolar-type H+ ATPase inhibitor bafilomycin also delayed proteolysis within efferosomes, showing that luminal acidification was essential for efficient digestion of efferosome proteins. Finally, cross-presentation of AC-associated antigens by CGD PEMs to CD8 T cells was increased. These studies unravel a key role for the NADPH oxidase in the disposal of ACs by inflammatory macrophages. The oxidants generated promote efferosome maturation and acidification that facilitate the degradation of ingested ACs.
| S-EPMC5969376 | biostudies-literature
Project description:The p47(phox) cytosolic factor from neutrophilic NADPH oxidase has always been resistant to crystallogenesis trials due to its modular organization leading to relative flexibility. Hydrogen/deuterium exchange coupled to mass spectrometry was used to obtain structural information on the conformational mechanism that underlies p47(phox) activation. We confirmed a relative opening of the protein with exposure of the SH3 Src loops that are known to bind p22(phox) upon activation. A new surface was shown to be unmasked after activation, representing a potential autoinhibitory surface that may block the interaction of the PX domain with the membrane in the resting state. Within this surface, we identified 2 residues involved in the interaction with the PX domain. The double mutant R162A/D166A showed a higher affinity for specific phospholipids but none for the C-terminal part of p22(phox), reflecting an intermediate conformation between the autoinhibited and activated forms.
| S-EPMC2937925 | biostudies-literature
Project description:Tonicity of saline (NaCl) is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET) formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA), Gram-negative bacterial cell wall component lipopolysaccharide (LPS), calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus), and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus). We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2)-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12), and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol). This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic solutions do not suppress NOX2-independent NETosis. Although hypertonic saline partially suppresses ionomycin-induced NETosis, it enhances A23187-induced NETosis, and it does not alter S. aureus-induced NETosis. Overall, this study determined that hypertonic saline suppresses NOX2-dependent NETosis induced by several agonists; in contrast, it has variable effects on neutrophil death induced by NOX2-independent NETosis agonists. These findings are important in understanding the regulation of NETosis and apoptosis in neutrophils.
| S-EPMC5859219 | biostudies-literature
Project description:Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5'-[gamma-thio]triphosphate and guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.
| S-EPMC1132836 | biostudies-other
Project description:The effects of neomycin, fluoride and the non-hydrolysable guanine nucleotide analogue GTP gamma S on the kinetics of cell-free activation of NADPH oxidase in membranes of resting human neutrophils were investigated. Arachidonate-mediated activation of the oxidase followed a first-order reaction course (kobs. = 0.39 min-1 at 26 degrees C). In the presence of NaF during the activation process, activity was enhanced while the activation rate was slightly reduced (kobs. = 0.25 min-1 at 26 degrees C). Neomycin blocked activation (half-maximal effect at 25 microM) without affecting rates of superoxide release by preactivated enzyme in vitro or in vivo. In spite of reduced specific activity neither the first-order rate constant of the activation nor the Km of the oxidase were altered by neomycin. Oxidase activated in the presence of GTP gamma S exhibited increased specific activity and unchanged Km; the course of the reaction deviated from first-order kinetics. Kinetic evidence is presented for two separate activation reactions: a GTP gamma S-independent, basal, first-order process and a GTP gamma S-dependent sigmoid activation process. The results are compatible with the existence in neutrophil membranes of two separate pools of dormant oxidase. An alternative scheme of the formation of two active forms of NADPH oxidase is also presented.
| S-EPMC1138850 | biostudies-other
Project description:The assembly of cytosolic subunits p47(phox), p67(phox), and p40(phox) with flavocytochrome b(558) at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47(phox) subunit plays a critical role in oxidase assembly. Recent studies showed that the p47(phox) Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47(phox) PX domain in neutrophils is unclear, we investigated its role using p47(phox) knock-out (KO) mouse neutrophils to express human p47(phox) and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47(phox) proteins were expressed at levels similar to endogenous murine p47(phox), with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47(phox) rescued p47(phox) KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47(phox) with Arg(90) substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47(phox) Arg(90) mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47(phox) PX domain for neutrophil NADPH oxidase activation.
| S-EPMC2966130 | biostudies-literature
Project description: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.
| S-EPMC3095934 | biostudies-literature
Project description:In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40(phox), p47(phox), and p67(phox), and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40(phox) remains less well defined than those of p47(phox) and p67(phox). We investigated the biological role of p40(phox) in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40(phox) using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67(phox) levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40(phox) reduces both the maximal rate and total amount of ROS produced without altering the K(M) value of the oxidase for NADPH. Using a series of mutants, p47PX-p40(phox) chimeras, and deletion constructs, we found that the p40(phox) PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67(phox) requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40(phox), however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40(phox) can increase oxidase activity approximately 2.5-fold above that of wild-type p40(phox) but maintain the requirement for PX and SH3 domain function. We present a model where p40(phox) translocates p67(phox) to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.
| S-EPMC2755574 | biostudies-literature
Project description:Recent evidence suggests that oxidative stress contributes to the pathogenesis of prostate cancer. The present study focused on the effect of apocynin, an inhibitor of NADPH oxidase, on prostate carcinogenesis using the transgenic rat for adenocarcinoma of prostate (TRAP) model. There were no toxic effects with apocynin treatment. The percentages and numbers of carcinomas in both the ventral and lateral prostate were significantly reduced by apocynin treatment, with dose dependence. Reduction of reactive oxygen species by apocynin was confirmed by immunohistochemistry of 8-OHdG and dihydroethidium staining. Positivity of Ki67 was significantly reduced by apocynin treatment, and downregulation of clusterin expression, as well as inactivation of the MEK-ERK1/2 pathway, was a feature of the apocynin treated groups. In human prostate cancer cell line LNCaP, apocynin also inhibited reactive oxygen species production and blocked cell growth by inducing G0/G1 arrest with downregulation of clusterin and cyclin D1. These data suggest that apocynin possesses chemopreventive potential against prostate cancer.
| S-EPMC7654245 | biostudies-literature
Project description:The cell-free activation of human neutrophil NADPH oxidase (O(2)(-)-generating enzyme) is enhanced by actin [Morimatsu, Kawagoshi, Yoshida and Tamura (1997) Biochem. Biophys. Res. Commun. 230, 206--210]. In an attempt to elucidate the mechanism, we examined the effect of actin-depolymerizing agents on the duration of NADPH oxidase in a cell-free system. The addition of DNase I, an F-actin-depolymerizing protein, caused an accelerated deactivation of the oxidase. The deactivation was also facilitated by latrunculin A, a sponge toxin that depolymerizes F-actin. Exogenously added actin prevented the deactivation by DNase I or latrunculin A, whereas EDTA accelerated a dilution-induced deactivation of the oxidase and Mg(2+) ions retarded it. The stability in dilution was found to correlate well with free Mg(2+) concentration. Estimation of F-actin in the system showed that F-actin increased during the oxidase activation and that DNase I or EDTA decreased F-actin content in parallel with the activity. Treatment of the cell-free mixture with a chemical cross-linker prevented the deactivation and F-actin decrease by EDTA. Taken together, these results suggest that actin filaments which grow during the activation of NADPH oxidase prolong the lifetime of the oxidase.
| S-EPMC1221158 | biostudies-other