The human neutrophil oxygen armory

The human neutrophil respiratory burst, initiated by the activation of the flavoenzyme NADPH-oxidase, generates the one-electron reduction product of oxygen, O₂⁻. Derivative free radicals and products of the myeloperoxidase-H₂O₂-halide system are toxic to micro-organisms and cytolytic to normal and malignant mammalian cells. Current interest has focused on the intra- and extra-cellular molecular targets of the respiratory burst products to define the complex pathways by which these metabolites participate in the inflammatory reaction.     

Expression of functional mammal flavocytochrome b558 in yeast: Comparison with improved insect cell system

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b₅₅₈ (Cytb₅₅₈) which consists of a heterodimer of the gp91^phox and p22^phox subunits. The Cytb₅₅₈ is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb₅₅₈ (rCytb₅₅₈). We expressed the gp91^phox and p22^phox subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91^phox undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb₅₅₈ displayed comparable spectral properties to neutrophil Cytb₅₅₈. In contrast to rCytb₅₅₈ produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.     

Modulation of microglial superoxide production by α-tocopherol in vitro: attenuation of p67phox translocation by a protein phosphatase-dependent pathway

As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.     

Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.     

Regulation of Nox and Duox enzymatic activity and expression

In recent years, it has become clear that reactive oxygen species (ROS, which include superoxide, hydrogen peroxide, and other metabolites) are produced in biological systems. Rather than being simply a by-product of aerobic metabolism, it is now recognized that specific enzymes--the Nox (NADPH oxidase) and Duox (Dual oxidase) enzymes--seem to have the sole function of generating ROS in a carefully regulated manner, and key roles in signal transduction, immune function, hormone biosynthesis, and other normal biological functions are being uncovered. The prototypical Nox is the respiratory burst oxidase or phagocyte oxidase, which generates large amounts of superoxide and other reactive species in the phagosomes of neutrophils and macrophages, playing a central role in innate immunity by killing microbes. This enzyme system has been extensively studied over the past two decades, and provides a basis for comparison with the more recently described Nox and Duox enzymes, which generate ROS in a variety of cells and tissues. This review first considers the structure and regulation of the respiratory burst oxidase, and then reviews recent studies relating to the regulation of the activity of the novel Nox/Duox enzymes. The regulation of Nox and Duox expression in tissues and by specific stimuli is also considered here. An accompanying review considers biological and pathological roles of the Nox family of enzymes.     

Respiratory burst in human B lymphocytes. Triggering of surface Ig receptors induces modulation of chemiluminescence signal.

B lymphocytes have been shown to proliferate and release oxygen metabolites when surface Ig is cross-linked and when stimulated with phorbol ester. Biochemical evidence has been provided for the presence of a superoxide generating system in B cells, which seems to be identical to the well-characterized NADPH-oxidase of phagocytes. In this report, we show that normal and EBV-transformed B cells produce superoxide anions after stimulation with phorbol ester and when surface Ig was cross-linked, as detected by lucigenin-dependent chemiluminescence. Anti-surface IgG antibodies induced a significant respiratory burst whereas those directed against surface IgM had no effect on B cell oxidative metabolism. Prestimulated B lymphocytes responded to further triggering by the same or another ligand. Pretreatment with Staphlococcus aureus Cowan I strain (SAC) or anti-IgM antibodies resulted in complete unresponsiveness to subsequent SAC or anti-IgG stimulation, but it did not affect PMA- and ionomycin-mediated B cell chemiluminescence. In contrast to preincubation with anti-IgM antibodies, the pretreatment of B cells with SAC induced a transient inhibitory effect on B cell signaling. In fact, SAC-pretreated B lymphocytes could be restimulated with the same ligand when blast cells were isolated. Furthermore, a 24-h incubation of the pretreated B cells in the absence of SAC completely restored the SAC-mediated respiratory burst. These results suggest that two distinct mechanisms may account for SAC- and anti-IgM-induced inhibition: a transient and reversible modulation of surface Ig, induced by SAC, and a long-lasting desensitization of the surface Ig receptors, respectively. These findings may have interesting implications for understanding the transduction of negative signals in B lymphocytes.     

Subcellular localization and dynamics of components of the respiratory burst oxidase

Membrane and cytosolic factors cooperate to generate NADPH-oxidase. The study of the syndrome of NADPH-oxidase deficiencies, chronic granulomatous disease, has enabled the identification of two membrane factors: a flavin adenine dinucleotide flavoprotein and ab cytochrome. The nature of the cytosolic components is still unknown, but a 47-kD protein, whose phosphorylation occurs in parallel with the generation of a respiratory burst in intact cells, seems to be one of the cytosolic factors. The subcellular localization of the membrane-bound NADPH-oxidase components has been studied in neutrophils: In unstimulated cells, only a minute fraction of the NADPH-oxidase components is localized in the plasma membrane, whereas 80% is localized in the membrane of the specific granules and the majority of the rest is in a newly described membrane-bound compartment, the secretory granules, identified by latent alkaline phosphatase. During stimulation, these NADPH-oxidase components are translocated to the plasma membrane as a result of fusion of granule membrane with plasma membrane. Only the NADPH-oxidase components present in the plasma membrane are incorporated in the respiratory burst oxidase generated in intact cells.     

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.     

Tyrosine phosphorylation: a signal for the activation of the phagocyte respiratory burst

The respiratory burst of the phagocytic cell is a unique function which provides the cell with a series of reactive oxygen intermediates which it can use to kill ingested microorganisms. The complex signal transduction pathways responsible for regulating the respiratory burst are yet to be fully elucidated. Protein tyrosine kinases are now recognised as being critical components in the regulatory pathways controlling many cellular functions. In this report we review the evidence implicating tyrosine phosphorylation as an important signal for the activation of the phagocyte respiratory burst.     

Versatile roles of plant NADPH oxidases and emerging concepts

NADPH oxidase (NOX) is a key player in the network of reactive oxygen species (ROS) producing enzymes. It catalyzes the production of superoxide (O₂⁻), that in turn regulates a wide range of biological functions in a broad range of organisms. Plant Noxes are known as respiratory burst oxidase homologs (Rbohs) and are homologs of catalytic subunit of mammalian phagocyte gp91^phox. They are unique among other ROS producing mechanisms in plants as they integrate different signal transduction pathways in plants. In recent years, there has been addition of knowledge on various aspects related to its structure, regulatory components and associated mechanisms, and its plethora of biological functions. This update highlights some of the recent developments in the field with particular reference to important members of the plant kingdom.     

RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes

Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1 μM fMLF and 1 μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells.     

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils

Background  

Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.     

Opening the black box: lessons from cell-free systems on the phagocyte NADPH-oxidase

The NADPH-oxidase complex of phagocytic cells plays a key role in the defense against invading pathogens, through the release of superoxide anion, precursor of other reactive oxygen species (ROS). NADPH-oxidase deficiency is called Chronic Granulomatous Disease (CGD), in which patients suffer from recurrent infections and from the formation of granulomas in various organs. Research on NADPH-oxidase has much benefited from the discovery of cell-free systems, i.e. reconstitution assays from broken resting (unstimulated) phagocytes, in which activation of the oxidase is elicited in vitro. Cell-free systems were developed in parallel to studies of molecular defects of patients with CGD, both approaches leading to the identification of the major proteins implicated in enzyme activation. Variations around the cell-free system allowed molecular dissection of the mechanism of NADPH-oxidase activation and provided insights into its relationship to phagocytosis.     

Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with beta-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O-2 dismutation to H(2)O(2), supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca(++) pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

The phagocytosis-associated respiratory burst in human monocytes is associated with increased uptake of glutathione

During the phagocytic respiratory burst, oxygen is converted to potent cytotoxic oxidants. Monocytes and macrophages are potentially long-lived, and we have hypothesized that protective mechanisms against oxidant stress are varied and fully expressed in these cells. We report here that the respiratory burst in monocytes is accompanied by an increase in the uptake of [35S]glutathione ([35S]GSH) after 20-30 min to levels up to 10-fold greater than those at baseline. By 30 min, 49% of the cell-associated radioactivity was in the cytosol, 41% was in membrane, and 10% was associated with the nuclear fraction. GSH uptake was inhibited by catalase, which removes hydrogen peroxide (H2O2), and micromolar H2O2 stimulated GSH uptake effectively in monocytes and also lymphocytes. Oxidation of GSH to glutathione disulfide with H2O2 and glutathione peroxidase prevented uptake. Acivicin, which inhibits GSH breakdown by gamma-glutamyl transpeptidase (GGT), had no effect on the enhanced uptake seen during the respiratory burst. Uptake of cysteine or cystine, possible products of GGT activity, stayed the same or decreased during the respiratory burst. These results suggest that a GGT-independent mechanism is responsible for the enhanced GSH uptake seen during the respiratory burst. We describe here a sodium-independent, methionine-inhibitable transport system with a Km (8.5 microM) for GSH approximating the plasma GSH concentration. These results suggest that monocytes have a specific GSH transporter that is triggered by the release of H2O2 during the respiratory burst and that induces the uptake of GSH into the cell. Such a mechanism has the potential to protect the phagocyte against oxidant damage.     

Studies of cytochrome b-245 translocation in the PMA stimulation of the human neutrophil NADPH-oxidase

The human neutrophil respiratory burst, activated by phorbol 12-myristate 13-acetate (PMA), results from specific receptor-ligand binding and activation of the NADPH-oxidase in the plasma membrane. The role of granule membrane constituents has been elucidated with neutrophils disrupted by nitrogen cavitation and then fractionated in Percoll gradients to resolve four postnuclear fractions: cytoplasm, light membranes or gamma fraction (site of the NADPH-oxidase), a light granule (beta) fraction containing putative constituents of the NADPH-oxidase (cytochrome b-245 and an associated flavoprotein), and a fraction of heavy granules. Cytochrome b-245 is localized to two pools of specific granules within the beta fraction as assessed by differing sedimentation in narrow Percoll gradients and translocates upon PMA-stimulation from one of these specific granule sub-pools to the plasma membrane where it exhibits no change in its midpoint redox potential. Translocation of cytochrome b-245 parallels O2-production initiated by PMA stimulation as assessed in the time course of each activity. The finding of increased amounts of the b cytochrome in cytoplast membranes relative to plasma membranes of unstimulated cells suggests that the cytoplasts, devoid of granules yet capable of O2-generation upon PMA-stimulation, are useful in assessing post-translocation events in the activation pathway of the NADPH-oxidase. These data support the hypothesis that translocation of NADPH-oxidase components from an intracellular granular pool contributes to respiratory burst expression.     

Formation of reactive oxygen species following bioactivation of gentamicin

The present study investigated the ability of gentamicin to catalyze free radical reactions and probed the underlying mechanisms by hydroethidine imaging, oxygen consumption, and reduction of cytochrome c. In Epstein-Barr virus-transformed lymphoblastoid cells, a respiratory burst was induced by phorbol ester and detected by hydroethidine, a fluorescent indicator of superoxide radical. The addition of gentamicin increased the fluorescence two-fold while gentamicin did not produce fluorescence in the absence of phorbol ester. In membrane preparations, gentamicin did not enhance NADPH consumption ruling out a direct activation of NADPH oxidase. The formation of reactive oxygen species by gentamicin was additionally supported by experiments that showed gentamicin increased oxygen consumption two-fold in intact cells and a cell-free system. In addition, generation of superoxide was indicated by the gentamicin-stimulated reduction of cytochrome c. The stimulation by gentamicin depended upon the presence of iron (Fe^II/Fe^III) and of arachidonic acid as an electron donor. These results support the hypothesis that an iron-gentamicin complex can increase reactive oxygen species in nonenzymatic and in biological systems. The requirement for a reductive activation in intact cells (e.g., by a respiratory burst) is interpreted as the conversion of an inactive Fe^III-gentamicin to a redox-active Fe^II-gentamicin complex.     

The influence of selenium and vitamin E on the enhanced respiratory burst reaction in smokers

The respiratory burst reaction (RBR) of neutrophilic granulocytes of the peripheral blood was estimated by means of the luminol reaction in 10 smokers and in 10 nonsmokers. Compared to the nonsmokers, the RBR of smokers' granulocytes showed a significantly higher rate of RBR. RBR consists of two enzymatic systems, i.e., NADPH-oxidase generating superoxide anions and myeloperoxidase, generating hypochlorous acid. Furthermore the superoxide anion may undergo dismutation to oxygen and peroxide. Thus, since the RBR may cause an oxidative stress, the smokers were supplemented for 10 d with antioxidants, i.e., 200 micrograms L-Se-methionine and 1000 mg vitamin E/d. After 10 d of supplementation with the antioxidants, the RBR of the smokers was significantly decreased by 20-75 percent. Since the oxidative stress associated with RBR may cause autodigestive reactions in the lungs of smokers, it may be beneficial for smokers to use relatively high doses of such antioxidants in order to hamper the pathological processes associated with smoking.     

Chemiluminescence response of β-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with β-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O−2 dismutation to H₂O₂, supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca⁺⁺ pump channels and calmodulin are involved in a metabolic energy-dependent pathway.