Superoxide-producing Activity Research Articles

Long COVID syndrome in children: neutrophilic granulocyte dysfunction and its correlation with disease severity.

Many children suffer from lingering symptoms after COVID-19, known as long COVID syndrome (LCS), otherwise called Post COVID-19 Condition (PCC). Despite extensive research, the prevalence of symptoms, its impact on quality of life, and underlying mechanisms still need to be fully understood. As neutrophilic granulocytes play an essential role in COVID-19, and their prolonged disruption was found to cause immunological diseases, we hypothesized their ongoing disturbed functionality in LCS. We studied 129 children with LCS, 32 convalescent children (CG+), and 8 uninfected children (CG-). Online questionnaires and in-person examinations assessed symptoms, quality of life, and functioning (QoL-F). Effector functions of neutrophilic granulocytes obtained from the venous blood of 29 LCS and 17 CG+ children were also investigated. Persistent fatigue was the most common symptom in children with LCS, while both control groups complained about anxiety most frequently. LCS children experienced significantly more symptoms, impairing their QoL-F compared to CG+. Neutrophilic granulocyte dysfunction was found in LCS children, with decreased superoxide-producing activity and phagocytosis compared to CG+. The number of complaints of children with LCS correlated significantly with altered neutrophil effector functions. Neutrophil dysfunction in children with LCS may be part of the disease pathogenesis or a predisposing factor. Using online questionnaires validated during in-person medical examinations and including two different control groups, our study compellingly supports and adds to previous clinical observations in the field. Our study provides valuable insights into the prevalence and characteristics of pediatric LCS, highlighting the significant quality of life and functioning impairment compared to control groups. By detecting neutrophilic granulocyte dysfunction in children with LCS, we shed light on a previously overlooked pathophysiological component of the condition. We demonstrate a significant correlation between clinical symptoms and superoxide production, further enhancing our understanding of the underlying mechanisms of pediatric LCS.

Functional status of blood neutrophils in congenital defects of antibody production

We evaluated some functional indices of neutrophils in 100 adult patients with a verified diagnosis of primary antibody production deficiency (44 patients with common variable immunodeficiency – CVID, and 56 patients with selective IgA deficiency – SigAD). The diagnosis was made according to the criteria of the Pan-American Group for Immunodeficiency and European Society for Immunodeficiency. The criteria for diagnosis were based on clinical data, medical history and results of laboratory tests. All patients with CVID received regular immunoglobulin replacement therapy, and the SIgAD patients received symptomatic treatment on demand. The examination was performed beyond the infectious episodes, inflammatory events or exacerbation of chronic disorders upon admission for planned hospitalisation, before any therapeutic and diagnostic procedures. In addition to standard clinical and laboratory examination appointed for the mentioned diseases, we assessed fnctional status of neutrophils: superoxide-anion radical production, NETosis, phagocytosis and apoptosis. Taking into account clinical manifestations, the patients with CVID were divided into 6 clinical subgroups: without CVID-related complications (13.6%); with infectious complications, i.e., bronchiectatic disease (15.9%), autoimmune syndromes (22.7%), polyclonal lymphoid infiltration (13.6%), enteropathic syndrome (34.1%), and malignant neoplasms (9.1%). Four phenotypes were identified in the patients with SIgAD: absence of SIgAD-related complications (28.6%); autoimmune syndrome (16.1%), non-malignant lymphoproliferation (3.6%) and enteropathy (28.6%). Higher incidence of non-infectious complications (autoimmune syndrome, non-malignant lymphoproliferation, enteropathies) was found in СVID compared to SIgAD patients (χ2 = 10.27; p = 0.001). Patients from the both groups showed changes in neutrophil reactivity compared to control values expressing higher basal generation of superoxide radicals (p < 0.001), NETosis activity (p = 0.005) and apoptosis (p < 0.001) with decreased phagocytic function (p < 0.001) and lower reserve for reactive oxygen species formation (p < 0.001). The maximal degree of changes in phagocytosis and superoxide-producing activity was observed in СVID; altered NETosis was revealed in SIgAD. The development of non-infectious complications was accompanied by a significant increase in stimulated NETosis indexes, thus suggesting a promising index in order to assess stability of clinical course and to predict development of complications in congenital defects of antibody production.

Identification and activity of superoxide-producing protein complexes of the plasma membrane of etiolated maize seedlings subjected to low positive temperature

Kinetics of superoxide anion generation by the isolated plasma membrane was determined by the rate of formazan formation from XTT in the presence of NADPH or NADH. The plasma membrane was prepared from (control) etiolated maize seedlings grown at 25°C and from (cooled) seedlings incubated at 6°C for the last day. Membrane vesicles from the control plants possessed superoxide-producing activity, and the rate of NADH oxidation was markedly higher than that of NADPH. The low-temperature incubation of the seedlings suppressed the NADPH-dependent activity, whereas the NADH-dependent one slightly increased. The solubilized by dodecyl maltoside (DDM) plasma membranes were separated into multiprotein complexes by high-resolution clear native electrophoresis (hrCN-PAGE). The aim was to find complexes exhibiting the superoxide-producing activity sensitive to inhibition by diphenylene iodonium. Several protein complexes from the plasma membrane capable of superoxide producion in the presence of NADPH or NADH were found. The maximum diphenylene iodonium-sensitive activity was found in the high-molecular weight complex, in which proteins reacting with antibodies against C-terminal peptide of phagocytic oxidase (gp91phox) were detectable. The activity of this complex was lower in the cooled than in the control seedlings and displayed higher affinity to NADPH than to NADH. To search for the cooling-induced changes in the polypeptide content of protein complexes, the two-dimensional difference gel electrophoresis (hrCN/SDS-PAGE) was used. Control and cooled samples, whose lysine had been labeled with fluorescent dyes Cy2 and Cy3, respectively, were separated by this method in one gel. Decrease in a temperature of plant growing affected the protein content of the complex so that some new proteins appeared and several polypeptides disappeared as compared with the control. There were no significant differences between the cooled and control counterparts in the content of proteins detectable with gp91phox antibodies. Therefore, the high-molecular complex containing NADPH oxidase looses proteins under low temperature that may decrease its superoxide-producing activity.

36 - Quantitative Relationship between NADPH Depletion and Inhibition of NOX2-Induced Superoxide Radical Anion

Nicotinamide adenine dinucleotide (NAD + ) is an essential cofactor controlling cell metabolism and survival. NAD + is recycled in cells at rates controlled by nicotinamide phosphoribosyl transferase (NAMPRT). The pharmacological inhibition of NAMPRT by FK866 diminishes intracellular concentration of redox couples NAD + /NADPH and NADP + /NADPH. Here we examined the quantitative relationship between inhibition of NAMPRT and superoxide-producing activity of NOX2 from PMA activated macrophage RAW 264.7 cells. Cells were treated with 10nM FK866 for 24 h showed PI staining negative and survival >80%. Superoxide (O 2 ●– ) in PMA activated cells was maximally stimulated with 100-400 ng/ml. 2-Hydroxyethidium, 2-OHE + in both supernatant and cytosolic fractions was decreased in cells pre-treated with 10nM FK866 for 24 h. This inhibition was rescued with10µM NMN co-treatment. The NOX inhibitor, VAS2870 (10-100µM) decreased O 2 ●– release although toxicity was evident. FK866 did not alter the protein expression of NOX subunits, p91phox and p47phox. HPLC analysis of FK866 treated cells showed diminished intracellular levels of NAD + and NADPH and to a lesser extent NADH, NADP + . A dose-dependent effects of FK866 (1-20nM) indicated that after 24h incubation FK866 at concentrations of 10nM led to a decrease of ~50% of NADPH (from 2.2±0.2 to 1.2±0.2 nmol/mg protein) eliciting a reduction of 2-OHE + >70%. Increasing FK to 20nM showed small effects compared to 10nM treatment. An inhibition of 50% of 2-OHE+ formation was attained with 5nM FK866 treatments indicating that NADPH EC50 for NOX2 ~1.6nmol/mg protein. These results indicate that NOX2 activity is inhibited by depletion of physiological levels of NADPH which could be highly significant in the regulation of peroxynitrite and other oxidant production.

Dexamethasone, tetrahydrobiopterin and uncoupling of endothelial nitric oxide synthase.

ObjectiveTo find out whether dexamethasone induces an uncoupling of the endothelial nitric oxide synthase (eNOS).Methods & ResultsA major cause of eNOS uncoupling is a deficiency of its cofactor tetrahydrobiopterin (BH4). Treatment of human EA.hy 926 endothelial cells with dexamethasone decreased mRNA and protein expression of both BH4-synthesizing enzymes: GTP cyclohydrolase I and dihydrofolate reductase. Consistently, a concentration- and time-dependent reduction of BH4, dihydrobiopterin (BH2) as well as BH4: BH2 ratio was observed in dexamethasone-treated cells. Surprisingly, no evidence for eNOS uncoupling was found. We then analyzed the expression and phosphorylation of the eNOS enzyme. Dexamethasone treatment led to a down-regulation of eNOS protein and a reduction of eNOS phosphorylation at serine 1177. A reduction of eNOS expression may lead to a relatively normal BH4: eNOS molar ratio in dexamethasone-treated cells. Because the BH4-eNOS stoichiometry rather than the absolute BH4 amount is the key determinant of eNOS functionality (i.e., coupled or uncoupled), the down-regulation of eNOS may represent an explanation for the absence of eNOS uncoupling. Phosphorylation of eNOS at serine 1177 is needed for both the NO-producing activity of the coupled eNOS and the superoxide-producing activity of the uncoupled eNOS. Thus, a reduction of serine 1177 phosphorylation may render a potentially uncoupled eNOS hardly detectable.ConclusionsAlthough dexamethasone reduces BH4 levels in endothelial cells, eNOS uncoupling is not evident. The reduction of NO production in dexamethasone-treated endothelial cells is mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.

The protective role of a hypothalamic proline-rich peptide during acute intoxication of rats by carbon dioxide in combination with oxygen starvation

We measured the contents of NADPH oxidases (Nox) in the membranes of erythrocytes, spleen cells, bone marrow cells, nuclei from spleen cells, and blood serum of rats that were subjected to action of carbon dioxide under conditions of oxygen starvation. We found that some increase in Nox content during intoxication with CO2 correlates with an increase in the superoxide-producing activity of Nox. Intraperitoneal administration of the synthetic analogue of the proline-rich peptide, which was previously extracted from neurosecretory granules of the hypothalamus and is referred to as galarmin, 1 hour before the treatment with CO2 resulted in a further increase in the content and superoxide-producing activity of Nox. The methemoglobin-reducing activity of Nox, which was decreased due to CO2 intoxication, increased in the presence of galarmin. We also found that considerable changes in the activities of superoxide dismutase and catalase in the cytosol of erythrocytes and the cells of rat spleen, which were induced by CO2, were reverted by galarmin, which returned these activities almost to their norm. The data we obtained suggest that galarmin may have a protective effect during CO2 intoxication under conditions of oxygen starvation.

Abstract 5471: NAD(P)H Oxidase Nox4 in Vascular Endothelial and Smooth Muscle Cells May Be Involved in the Regulation of Basal Blood Pressure

Reactive oxygen species (ROS) play an important role in mediating changes in blood pressure, vascular remodeling, and arteriosclerosis. All vascular cell types including vascular endothelial cells (ECs) and smooth muscle cells (SMCs) produce ROS, primarily through NAD(P)H oxidase (Nox) family enzymes, including Nox1, Nox2 and Nox4. Nox4 is predominantly expressed in ECs and SMCs and its expression in the vasculature is upregulated by aging. In order to elucidate the function of Nox4 in blood vessels, we established transgenic mice with EC- and SMC-specific overexpression of wild-type Nox4 (EC-Nox4, SMC-Nox4) and dominant negative Nox4 (EC-DN-Nox4, SMC-DN-Nox4). EC- and SMC-specific overexpression was achieved with SM22 α and Tie2 promoters, respectively. Enhanced expression of Nox4 or DN-Nox4 in ECs or SMCs was confirmed by immunostaining of blood vessels with a specific anti-Nox4 antibody. Superoxide producing activity in blood vessels was enhanced in EC-Nox4 (129.5±15.2%) and SMC-Nox4 (142.2±12.9%), and attenuated in EC-DN-Nox4 (30.6±4.9%) and SMC-DN-Nox4 (49.4±10.2%) compared to non-transgenic controls, as evaluated by dihydroethidium staining. At 3 months of age, EC-Nox4 exhibited significantly lower systolic blood pressure than WT (96.3±5.5 vs. 110.3±5.0 mmHg, p < 0.05) as determined by the tail-cuff method. Conversely, SMC-Nox4 exhibited significantly higher systolic blood pressure than WT (122.7±3.2 vs. 107.3±3.9 mmHg, p < 0.05). Blood pressure in EC-DN-Nox4 or SMC-DN-Nox4 was not statistically different from WT. At 3 months of age, none of the transgenic mouse lines exhibited obvious histopathological changes in vasculature, including aortas and arterioles in hearts and kidneys. Elevation of blood pressure induced by continuous infusion of angiotensin II (Ang II) (0.6 ng/g/min) in EC-Nox4 or SMC-Nox4 was not significantly different from WT at day 3, 7 and 14. Taken together, these results suggest that upregulation of Nox4 in SMCs and ECs increases superoxide production, thereby increasing or decreasing, respectively, blood pressure at baseline, without affecting Ang II-induced elevation in blood pressure.

Interleukin 10 inhibits interferon γ- and tumor necrosis factor α-stimulated activation of NADPH oxidase 1 in human colonic epithelial cells and the mouse colon

NADPH oxidase 1 (Nox1) is preferentially expressed in the colon, but its functional role is not fully understood. This study was designed to elucidate a potential role of Nox1 in inflammation of the colon. Superoxide production by T84 cells was measured by the cytochrome c method. Protein and mRNA levels of Nox1 and Nox organizer 1 (NOXO1) in the cells were measured by real-time reverse transcriptase PCR and Western blotting, respectively. Expression of Nox1, Nox2, dual oxidase 2 (Duox2), NOXO1, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha mRNAs was measured in proximal, middle, and distal portions of colonic mucosas from male wild-type C57BL/6J and interleukin (IL)-10 knockout mice at 6, 10, and 16 weeks of age. Grading of inflammation was done by scoring histological changes. IL-10 significantly inhibited IFN-gamma- or TNF-alpha-induced up-regulation of superoxide-producing activity in T84 cells by suppressing expression of Nox1 mRNA and protein. IL-10 also inhibited TNF-alpha-stimulated induction of NOXO1 and p38 MAPK phosphorylation. Levels of Nox1, but not Nox2 or Duox2 mRNA, was age-dependently increased following a gradient with low levels in the proximal colon and high levels in the distal colon of the wild-type mice. The absence of IL-10 significantly facilitated Nox1 expression in association with increased IFN-gamma mRNA expression before the development of spontaneous colitis and age-dependently accelerated their mRNA expression. IL-10 may be a possible down-regulator of the Nox1-based oxidase in the colon, suggesting a potential role of reactive oxygen species (ROS) derived from Nox1-based oxidase in inflammation of the colon.

Tumor necrosis factor α activates transcription of the NADPH oxidase organizer 1 (NOXO1) gene and upregulates superoxide production in colon epithelial cells

NADPH oxidase 1 (Nox1) is a multicomponent enzyme consisting of p22 phox , Nox organizer 1 (NOXO1), Nox1 activator 1, and Rac1. Interleukin-1β, flagellin, interferon-γ, and tumor necrosis factor α (TNF-α) similarly induced Nox1 in a colon cancer cell line (T84), whereas only TNF-α fully induced NOXO1 and upregulated superoxide-producing activity by ninefold. This upregulation was canceled by knockdown of NOXO1 with small interfering RNAs. TNF-α rapidly phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase 1/2, followed by phosphorylation of c-Jun and c-Fos and appearance of an AP-1 binding activity within 30 min. We cloned the 5′ flank of the human NOXO1 gene (– 3888 to + 263 bp), and found that the region between – 585 and – 452 bp, which contains consensus elements of YY-1, AP-1, and Ets, and the GC-rich region encoding three putative binding sites for SP-1, was crucial for TNF-α-dependent promoter activity. Serial mutation analysis of the elements identified an AP-1 binding site (from – 561 to – 551 bp, agtAAGtcatg) as a crucial element for TNF-α-stimulated transcription of the human NOXO1 gene, which was also confirmed by the AP-1 decoy experiments. Thus, TNF-α acts as a potent activator of Nox1-based oxidase in colon epithelial cells, suggesting a potential role of this oxidase in inflammation of the colon.

NAD(P)H oxidase p22phoxC242T polymorphism and ischemic stroke in Japan: the Fukuoka Stroke Registry and the Hisayama study

The C242T polymorphism of p22phox, a component of NAD(P)H oxidase, may have an impact on cardiovascular diseases; however, the association between this polymorphism and brain infarction is not fully understood. Here, we investigate the relationship between the C242T polymorphism and brain infarction in Japan. We recruited 1055 patients with brain infarction and 1055 control subjects. A chi-squared test revealed that the T-allele frequency was lower in patients with cardioembolic infarction (5.6%) than in control subjects (11.0%, P < 0.001); however, allele frequencies in patients with lacunar and atherothrombotic infarction (11.2%) were not significantly different from those in control subjects (11.0%). A multivariate-adjusted conditional logistic regression analysis also revealed no association between CT + TT genotype, and lacunar and atherothrombotic infarction (odds ratio = 0.97, 95% confidence interval: 0.72-1.32). To investigate the functional effects of the C242T polymorphism, we examined superoxide production in COS-7 cells cotransfected with Nox4 and p22phox of each genotype. The superoxide-producing activity in those cells expressing p22phox with the T allele was not significantly different from that in cells expressing p22phox with the C allele. The present results suggest that the p22phox C242T polymorphism may have a protective effect against cardioembolic infarction, but is not related to lacunar and atherothrombotic infarction in Japan.

The effect of proline-rich polypeptide on reduction of methemoglobin and formation of superoxide by the b558III cytochrome and suprol metalloproteins in vitro

A synthetic analogue of the proline-rich polypeptide (PRP-1) from neurosecretory granules of bovine neurohypophysis (1.5–7 μg/ml) was shown to stimulate reduction of methemoglobin (metHb) and NADPH-dependent formation of superoxide by the b558III cytochrome (cyt) from human erythrocyte membranes in the course of incubation under aerobic conditions at 4°C in vitro. However, increase in the PRP-1 concentration resulted in a decrease in the superoxide-producing activity of the b558III cyt. It was also demonstrated that PRP-1 inhibited superoxide formation in response to the action of the suprol lipoprotein of blood serum.

Expression and function of Noxo1γ, an alternative splicing form of the NADPH oxidase organizer 1

Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis and fetal brain. The Noxo1gamma protein contains an additional five amino acids in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1gamma shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1beta. Consistent with this, in resting cells, Noxo1gamma is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1beta, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support the superoxide-producing activity of Nox3 to the same extent in a manner independent of cell stimulation.

Activation of the superoxide-producing phagocyte NADPH oxidase requires co-operation between the tandem SH3 domains of p47phox in recognition of a polyproline type II helix and an adjacent α-helix of p22phox

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial for host defence, requires an SH3 (Src homology 3)-domain-mediated interaction of the regulatory protein p47phox with p22phox, a subunit of the oxidase catalytic core flavocytochrome b558. Although previous analysis of a crystal structure has demonstrated that the tandem SH3 domains of p47phox sandwich a short PRR (proline-rich region) of p22phox (amino acids 151-160), containing a polyproline II helix, it has remained unknown whether this model is indeed functional in activation of the oxidase. In the present paper we show that the co-operativity between the two SH3 domains of p47phox, as expected from the model, is required for oxidase activation. Deletion of the linker between the p47phox SH3 domains results not only in a defective binding to p22phox but also in a loss of the activity to support superoxide production. The present analysis using alanine-scanning mutagenesis identifies Pro152, Pro156 and Arg158 in the p22phox PRR as residues indispensable for the interaction with p47phox. Pro152 and Pro156 are recognized by the N-terminal SH3 domain, whereas Arg158 contacts with the C-terminal SH3 domain. Amino acid substitution for any of the three residues in the p22phox PRR abrogates the superoxide-producing activity of the oxidase reconstituted in intact cells. The bis-SH3-mediated interaction of p47phox with p22phox thus functions to activate the phagocyte oxidase. Furthermore, we provide evidence that a region C-terminal to the PRR of p22phox (amino acids 161-164), adopting an a-helical conformation, participates in full activation of the phagocyte oxidase by fortifying the association with the p47phox SH3 domains.

The superoxide‐producing NAD(P)H oxidase Nox4 in the nucleus of human vascular endothelial cells

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase.

Recent evidence has suggested that reactive oxygen species are important signaling molecules in vascular cells and play a pivotal role in the development of vascular diseases. The activity of NAD(P)H oxidase has been identified as the major source of reactive oxygen species in vascular endothelial cells. However, the precise molecular structure and the mechanism of activation of the oxidase have remained poorly understood. Here, we investigated the molecular identities and the superoxide-producing activity of endothelial NAD(P)H oxidase. We found that Nox4, a homologue of gp91phox/Nox2, was abundantly expressed in endothelial cells. The expression of Nox4 in endothelial cells markedly exceeded that of other Nox proteins, including gp91phox/Nox2, and was affected by cell growth. Using electron spin resonance and chemiluminescence, we measured the superoxide production and found that the endothelial membranes had an NAD(P)H-dependent superoxide-producing activity comparable to that of the neutrophil membranes, whereas the activity was not enhanced by the 2 recombinant proteins p47phox and p67phox, in contrast to that of the neutrophil membranes. Downregulation of Nox4 by an antisense oligonucleotide reduced superoxide production in endothelial cells in vivo and in vitro. These findings suggest that Nox4 may function as the major catalytic component of an endothelial NAD(P)H oxidase.

Effect of Shiga toxins on granulocyte function

We already showed that injection of Shiga toxin (Stx) 2 into mice caused severe granulocytosis in the peripheral blood. In this study we further clarified changes of granulocyte function by Stx 2. The activity of medullasin, a neutral serine protease in granulocytes that injures endothelial cells in vessels, significantly increased when Stx 2 was injected into mice intraperitoneally. Since granulocyte count in the peripheral blood of mice was markedly increased after intraperitoneal injection of Stx 2, medullasin activity in the peripheral blood was remarkably elevated. In contrast to Stx 2, injection of Stx 1 into mice caused no elevation of medullasin activity in granulocytes nor increase in granulocyte count in the peripheral blood. Cathepsin G levels in granulocytes increased only slightly after Stx 2 injection. Granulocytes obtained from mice injected with Stx 2 showed reduced superoxide-producing activity compared with those from controls. Addition of Stx 2 or Stx 1 to human mature granulocytes in vitro decreased their superoxide-producing activity when stimulated with agonists. Therefore, these toxins produced from Escherichia coli augment toxic effect of the bacteria by reducing bactericidal activity of granulocytes. Tissue injury in organisms infected with Shiga toxin-producing E. coli is mainly derived from elevated neutral proteases, such as medullasin, in granulocytes rather than direct toxic effect of superoxide from granulocytes. Hemolytic uremic syndrome caused by Shiga toxin-producing E. coli infection is due, at least in part, to the elevation of medullasin levels produced by granulocytes.

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 clone 15 cells to eosinophilic lineage.

Superoxide-generating NADPH oxidase consists of the membrane-bound cytochrome b558 (gp91phox and p22Phox) and the cytosolic components (p67phox, p47phox, p40phox and rac). In this study, we evaluated the superoxide-generating activity and the expression of NADPH oxidase components during eosinophilic maturation using HL-60 clone 15 cell line. HL-60 clone 15 cells were matured to eosinophils by incubation with 0.5 mM butyrate for 7 days, and NADPH oxidase components were detected by Northern blot, Western blot analyses and immunocytochemical staining. Moreover, superoxide-generating activity was examined by nitro blue tetrazolium (NBT) assay. Northern blot and Western blot analyses revealed that mRNAs and proteins for gp91phox, p67phox and p47phox were expressed after eosinophilic myelocyte stages, whereas mRNAs and proteins for p40phox and rac-2 were expressed from the promyelocyte stage. Interestingly, p22phox mRNA was expressed from the promyelocyte stage, but its protein was expressed after eosinophilic myelocyte stages. Consistent with the results of Western blotting, immunocytochemical staining of butyrate-induced HL-60 clone 15 cells indicated that gp91phox, p22phox, p67phox and p47phox were detected after eosinophilic myelocyte stages (eosinophilic myelocytes, eosinophilic metamyelocytes, eosinophilic band cells and eosinophilic-segmented cells), whereas p40phox and rac-2 were expressed from the promyelocyte stage. Moreover, almost the same results as those with butyrate-treated HL-60 clone 15 cells were obtained using human bone marrow cells by immunocytochemical staining. Furthermore, nitro blue tetrazolium (NBT) assay indicated that superoxide could be produced after eosinophilic myelocyte stages but not produced before the promyelocyte stage. Together these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during eosinophilic maturation.

Impairment of Microbial Killing and Superoxide-Producing Activities of Alveolar Macrophages by a Low Level of Ozone.

Male Wistar rats were exposed to 0.2 ppm ozone for up to 14 days, during which alveolar macrophages were collected by pulmonary lavage to assess the effect of ozone on their microbial killing and superoxide-producing activities. For rapid assessment of microbial killing activity, we measured the release of 3H-radioactivity into the supernatant by deoxycholate-lysis of the macrophages that had phagosytosed and killed 3H-uridine-labeled microbes. The killing activity against Escherichia coli and Candida albicans was reduced to 70-80% of control levels on day 3. However, phagocytosis by and the activity of lysosomal enzymes of the macrophages were not impaired. On day 14 the killing activity against E. coli had returned to control levels, whereas that against C. albicans was still reduced. Because active oxygen species plays an important role in microbial killing activity of macrophages, the effects of ozone on respiratory burst and superoxide production were examined. Aliquots of alveolar macrophages were stimulated with phorbol myristate acetate (PMA), opsonized zymosan, or lipopolysaccharide (LPS) plus cytochalasin E (Cyt.E). The respiratory burst, oxygen consumption for rapid superoxide production, was decreased to 60-80% of control levels on day 3. On day 14, the respiratory burst by opsonized zymosan was still 80% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In addition, the superoxide-producing activity of ozone-exposed macrophages was 10-60% decreased on day 3. On day 14, the superoxide production by stimulation with opsonized zymosan was still 60% reduced, whereas that by PMA or LPS plus Cyt. E had returned to control levels. In conclusion, because of their decreased production of superoxide, the host defense activity of alveolar macrophages was impaired by in vivo exposure to 0.2 ppm ozone. In particular, the C. albicans-associated defect lasted throughout the exposure period.

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.

Ureides and enzymes of ureide synthesis in wheat seeds and leaves and effect of allopurinol on Puccinia recondita f. sp. tritici infection

Xanthine oxidoreductase exhibiting a superoxide-producing activity (xanthine: O 2 oxidoreductase, EC 1.2.3.2), uricase (urate: O 2 oxidoreductase, EC 1.7.3.3) and allantoinase (EC 3.5.2.5) as well as ureides (allantoin and allamtoic acid) were detected in leaves, roots and seeds of wheat. Allopurinol [4-hydroxypyrazolo(3,4- d)pyrimidine], an analogue of purine and a tightly binding inhibitor of xanthine oxidizing enzyme, applied as soil drench at 40–60 μM starting 2–3 days after planting reduced the concentration of ureides in leaves and strongly inhibits the biotrophic growth of Puccinia recondita f. sp. tritici. Both phenomena are considered consequence of the in vivo inhibition of xanthine oxidoreductase in wheat plants. These findings indicate that purine catabolic pathway is involved during host-parasite relationship in rust-affected wheat leaves.