Effects of antibiotics on reactive oxygen species generation by neutrophils

Antibiotic therapy of patients with exacerbation of chronic obstructive bronchitis exposed and not exposed to ozone did not improve oxidative metabolism in neutrophils. Cefazolin, ceftizoxime, and gentamicin normalized functional biocidal reserves of neutrophils, which correlated with pronounced therapeutic effects.     

Effects of procainamide hydroxylamine on generation of reactive oxygen species by macrophages and production of cytokines

A series of experiments was conducted to examine the effects of the N-oxidized metabolite of procainamide, procainamide hydroxylamine (PAHA), on reactive oxygen species (ROS) production by macrophages in vitro, as well as on the release of the cytokine interleukin-1 (IL-1). Results with PAHA were compared with those from the parent compound, procainamide, and in some cases with other procainamide metabolites such as N-acetylprocainamide or nitrosoprocainamide. The effects of PAHA on ROS production by mouse and rat macrophages were complex, resulting in both stimulatory and inhibitory activity depending upon the PAHA concentration and whether macrophages were resting or elicited. The primary effect of PAHA appeared to be a stimulation of ROS production. Monocytes pretreated with PAHA (20 microM) depressed the responsiveness of lymphocytes in co-culture to a T-cell mitogen (conconavalin A) but not a B-cell mitogen (lipopolysaccharide). This effect was inhibited when monocyte pretreatment with PAHA was accompanied by the antioxidants, catalase or superoxide dismutase. IL-1 production by rat adherent splenocytes was unaffected by PAHA in concentrations that were not cytotoxic. These observations suggest that the oxidative metabolism of procainamide to PAHA may result in enhanced production of ROS by macrophages contributing its toxicity to lymphocytes.     

Walker carcinosarcoma cells damage endothelial cells by the generation of reactive oxygen species.

The passage of circulating tumor cells across vessel walls is an important step in cancer metastasis and is promoted by endothelial injury. Because Walker carcinosarcoma 256 (W256) cells generate oxygen-derived free radicals after cellular activation, the authors tested the hypothesis that these cancer cells can damage endothelial monolayers by producing such reactive oxygen species. To confirm that oxygen-derived radicals can damage endothelial cells, 3H-2-deoxyglucose-labeled human endothelial cell monolayers were exposed to xanthine oxidase in the presence of 0.2 mmol/l xanthine. 3H-2-deoxyglucose release was observed after the addition of xanthine oxidase in concentrations ranging from 6.5 x 10(-3) to 52 x 10(-3) units/ml. The extent of damage correlated with xanthine oxidase-dependent chemiluminescence (r = 0.91). Chemiluminescence assays in the presence of 5 x 10(-5) M luminol confirmed activation of the W256 cells by 1 x 10(-6) M chemotactic peptide fMLP. When fMLP-activated activated W256 cells were incubated with endothelial monolayers, concentrations of 2 x 10(6) to 6 x 10(6) W256 cells/ml were found to cause a 27% increase in the specific release of 2-deoxyglucose after a 90-minute incubation. A small but significant increase in 3H-2-deoxyglucose release also was observed in the absence of fMLP. Detection of 3H-2-deoxyglucose release in the presence of activated or unactivated tumor cells was dependent on preincubating the endothelial cell monolayer with 1 mM buthionine sulfoximine, an inhibitor of glutathione synthesis. Under these conditions, the specific release of 3H-2-deoxyglucose was increased from nondetectable levels to 21%, in the presence of 6.5 x 10(-3) units of the oxidase. Cultured W256 cells promoted isotope release from endothelial cell monolayers when activated with phorbol myristate acetate. Catalase (1000 units/ml) inhibited the tumor cell-induced release of 3H-2-deoxyglucose by 84% whereas superoxide dismutase, even at concentrations of 1 mg/ml, had no effect. A requirement for cell contact was shown because addition of cell-free supernatants from fMLP activated tumor cells did not cause 3H-2-deoxyglucose release and because pretreatment of W256 cells with 1 microM cytochalasin B inhibited their ability to promote isotope release even while increasing tumor cell-generated chemiluminescence threefold. Electron microscopy revealed that fewer cytochalasin B-treated W256 cells were attached to the endothelial cell monolayer than in untreated controls. It is concluded that the W256 tumor cells can damage endothelial cells directly via a mechanism involving production of reactive oxygen species.     

Enhanced generation of reactive oxygen species in monocytes from patients with common variable immunodeficiency.

Monocyte and macrophage dysfunction may be important for both immunopathogenesis and clinical manifestations in subgroups of patients with primary hypogammaglobulinaemia. In the present study we examined the ability to generate reactive oxygen species (ROS) in isolated monocytes from these patients by two different methods: superoxide dismutase (SOD) inhibitable cytochrome c reduction by O2- and nitroblue tetrazolium (NBT) reduction. Monocyte from patients with common variable immunodeficiency (CVI) demonstrated significantly enhanced ROS generation both unstimulated and stimulated (unopsonized zymosan and phorbol myristate acetate (PMA)). The enhanced oxidative burst response in CVI patients was found both with and without serum containing medium. Furthermore, serum from CVI patients did significantly enhance the oxidative burst response in monocytes from healthy blood donors compared with the effect of control serum. The enhanced ROS generation in CVI patients was significantly correlated with elevated serum levels of neopterin, reduced numbers of CD4+ lymphocytes in peripheral blood and occurrence of splenomegaly. In contrast to the CVI group, monocytes from patients with X-linked agammaglobulinaemia (XLA) did not show enhanced ROS generation. The increased oxidative burst response in monocytes from CVI patients most probably reflects in vivo activation of these cells. Our observations indicate the presence of a subgroup of CVI patients characterized by chronic immune activation particularly of monocytes. The enhanced ROS generation might be involved in immunopathogenesis (e.g. T cell dysfunction) and in the pathogenesis of clinical manifestations (e.g. malignancies and autoimmune disorders) in these patients.     

iNOS-mediated generation of reactive oxygen and nitrogen species by biomaterial-adherent neutrophils

Infection due to implanted cardiovascular biomaterials is a serious complication initiated by bacterial adhesion to the surface of the implant. The release of reactive oxygen species by neutrophils, particularly superoxide anion, is a well-known bactericidal mechanism. Additionally, nitric oxide (NO) has also been identified as an important cytotoxic mediator in acute and chronic inflammatory responses with enhanced NO production by upregulation of inducible nitric oxide synthase (iNOS). The interaction of NO and superoxide anion will result in the formation of peroxynitrite (OONO-), a potent cytotoxic oxidant. In this study, we have shown that biomaterial-induced neutrophil activation does not cause upregulation of iNOS and activation of iNOS-mediated pathways. However, NO and O2- production does occur over time upon adhesion to a biomaterial and is modulated by biomaterial surface chemistry. With no stimulus, the polyethylene oxide-modified polyurethane induced greater neutrophil activation than did the control as indicated by the increased production of NO and O2- over time. Adherent-stimulated neutrophils generally produced lower amounts of NO over time in comparison with unstimulated cells. Furthermore, there is no evidence of peroxynitrite activity in unstimulated neutrophils adherent to the Elasthane 80A. However, upon stimulation with adherent Staphylococcus epidermidis, peroxynitrite formation did occur. Our results suggest that bactericidal mechanisms in neutrophils involving NO generation (NOS pathway) are further compromised than O2- producing pathways (NADPH oxidase) upon exposure to biomaterials, resulting in a diminished microbial killing capacity, which can increase the probability of device-centered infections.     

胰淀素对离体大鼠胰岛细胞内活性氧生成的影响

目的：观察高浓度胰淀素对胰岛细胞内活性氧（ROS）生成的影响。方法：以体外培养胰岛单层细胞为模型,应用ROS特异性荧光染料2′,7′-二氯二氢荧光素双醋酸盐（H₂DCF）进行标记，以粘附细胞仪570型测定10 μmol/L胰淀素作用2 h后胰岛细胞内DCF荧光强度变化。结果：10 μmol/L胰淀素作用2 h，胰岛细胞内DCF荧光强度在16.7 mmol/L葡萄糖刺激后，迅速急骤升高进入平台期，升高速度为28×10^-3/s，而正常细胞DCF荧光强度无明显变化。光镜下可见部分细胞胞体缩小，胞质发生空泡变性，少数细胞核固缩、裂解，胞膜突起。结论：10 μmol/L胰淀素作用后的胰岛细胞内ROS生成增多，细胞形态受损，这些活性氧基的生成可以反映胰淀素对胰岛细胞损害程度，推测活性氧可作为胰岛细胞毒性损伤指标之一。     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H₂O₂ and O ₂ ^– in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF) and lymphotoxin (TNF ) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF and TNF favoring H₂O₂ production, C5a eliciting more O ₂ ^– than H₂O₂ and the other active stimulants being in between. Leukotriene B₄ and a novel monocytederived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the priming phenomenon since many PMN-priming mediators increase the cells' adherence.     

Stimulation of reactive oxygen species generation by disease-causing mutations of lipoamide dehydrogenase

We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.     

Sodium dependence of lysophosphatidylcholine-induced caspase-1 activity and reactive oxygen species generation

The proinflammatory cytokines interleukin (IL)-1β and IL-18 play pivotal roles in neuroinflammatory diseases. Caspase-1-mediated proteolytic cleavage is required to convert the premature, biologically inactive cytokines to their biologically active forms capable of promoting tissue inflammation. Although caspases have been recognized as potential therapeutic targets in inflammatory diseases, mechanisms regulating caspase-1 activation are not fully understood. Here we demonstrate that the proinflammatory lipid lysophosphatidylcholine (LPC) initiates microglial caspase-1 activation in a Na⁺-dependent manner. LPC-induced caspase-1 activity was almost completely inhibited upon omission of extracellular Na⁺, but was unaffected by inhibition of Na⁺/K⁺-ATPase with ouabain or by inhibition of Na⁺/H⁺ antiport with amiloride. Inhibition of caspase-1-mediated IL-1β processing by Na⁺-free medium led to reduced amounts of mature IL-1β released from LPC-stimulated microglia. Furthermore, LPC-induced production of reactive oxygen species (ROS) was abolished by Na⁺-free medium, indicating Na⁺ dependence of NADPH oxidase activity in LPC-stimulated microglia. Since ROS production was found to be crucial to caspase-1 activation in LPC-stimulated microglia, the Na⁺ dependence of caspase-1 can be related to the Na⁺ dependence of NADPH oxidase. In summary, it is suggested that in LPC-activated microglia, Na⁺ influx is required for the production of NADPH oxidase-mediated ROS, which subsequently stimulate caspase-1 activity.     

Bacteriophage preparation inhibition of reactive oxygen species generation by endotoxin-stimulated polymorphonuclear leukocytes

It has been known that administration of antibiotics may lead to excessive release of bacterial endotoxins and complicate clinical course of patients with Gram-negative infections. This concern may also apply to phages. Endotoxin may in turn activate neutrophils to produce reactive oxygen species (ROS) that are believed to play an important role in the pathogenesis of multiple organ dysfunction in the course of sepsis. We showed that a purified T4 phage preparation with low-endotoxin content could significantly diminish the luminol-dependent chemiluminescence (CL) of peripheral blood polymorphonuclear leukocytes (PMNs) both stimulated by lipopolysaccharides (LPSs) isolated from different Escherichia coli strains. This effect was also observed for live bacteria used for PMNs stimulation and was independent of bacterial susceptibility for T4-mediated lysis. Our data suggest, that phage-mediated inhibition of LPS- or bacteria-stimulated ROS production by PMNs may be attributed not only to phage-PMNs interactions, but also to phage-LPS interactions and bacterial lysis (in case of homologous phage). Interestingly, the T4 preparation did not influence ROS formation by PMNs stimulated with PMA. This suggests that the observed phenomena are also dependent upon the nature of activator. Bacteriophage-mediated inhibition of ROS formation by cells exposed to endotoxin provides new evidence for possible interactions between phages and mammalian cells. It helps in understanding the role of phages in our environment and may also be of important clinical significance.     

2-氯腺苷和异戊烯焦磷酸的细胞内抗氧化作用

目的:分析腺苷受体的兴奋剂2-氯腺苷(2-CA)和非肽类磷酸抗原异戊烯焦磷酸(IPP)在信号传导水平上对细胞内活性氧类(ROS)的调节作用。方法: SV40转化的人着色性干皮病成纤维细胞经2-CA和IPP处理30 min后, 用流式细胞仪和荧光计测定细胞内ROS水平。 结果:在适当的控制条件下, IPP可降低细胞内20%-30%左右的ROS, 2-CA对细胞内ROS调节类似IPP。结论:2-CA和IPP具有抑制细胞内ROS形成的作用, 提示其在体内有抗氧化作用。     

NF-κB "decoy"寡核苷酸对LPS诱导巨噬细胞产生NO及活性氧的影响

目的 :探讨NF -κB“decoy”寡核苷酸 (ODNs)对脂多糖 (LPS)诱导小鼠巨噬细胞株J774 1产生一氧化氮 (NO)及活性氧的影响。方法 :通过体外细胞培养技术 ,观察转染NF -κB“decoy”ODNs对LPS刺激巨噬细胞产生NO和活性氧 (ROS)的影响 ,以及诱导型一氧化氮合酶 (iNOS)活力的变化。结果 :在LPS刺激的巨噬细胞中转染NF -κB“decoy”ODNs减少NO和ROS的产生 ,降低iNOS的活力。结论 :NF -κB“decoy”ODNs可以对抗LPS诱导巨噬细胞产生的ROS和NO ,可能与其特异性竞争抑制活化NF -κB结合位点有关。     

精卵孵育时间与活性氧的关系以及对体外受精结局的影响

目的检测长、短时受精受精液中活性氧的不同,分析精卵孵育不同时间对体外受精-胚胎移植结局的影响。方法155例接受常规IVF受精的患者,每人取出的OCCC随机分为两组：A组为短时受精,B组为长时受精。检测受精液中H202、CTA的含量。移植的119名患者随机分为两组,一组60人移植短时受精的胚胎,一组59人移植长时受精的胚胎。结果B组的受精液中H202水平明显高于A组（P〈0．05）,CTA水平明显低于A组（P〈0．05）。B组的多精受精率明显高于A组（P〈0．05）,A组的优胚率明显高于B组（P〈0．05）,两组间的受精率、卵裂率、植入率、临床妊娠率无统计学差异。结论长时受精时,过量的活性氧对卵子及胚胎有不利影响。短时受精在不影响受精率的前提下,可以降低多精受精率,提高优胚率。     

The pivotal role of reactive oxygen species generation in the hypoxia-induced stimulation of adipose-derived stem cells

Adipose-derived stem cells (ASCs) offer a potential alternative for tissue repair and regeneration. We have recently shown that hypoxia stimulates ASCs and enhances the regenerative potential of ASCs, which is beneficial for ASC therapy. In the present study, we further investigated a key mediator and a signal pathway involved in the stimulation of ASC during hypoxia. Culturing ASC in a hypoxic incubator (2% oxygen tension) increased the proliferation and migration, and this was mediated by Akt and ERK pathways. To determine the generation of reactive oxygen species (ROS), 2',7'-dichlorofluorescin diacetate intensity was detected by fluorescence-activated cell sorting. Hypoxia significantly increased the dichlorofluorescin diacetate intensity, which was greatly reduced by N-acetyl-cysteine and diphenyleneiodonium treatment. Likewise, the hypoxia-induced proliferation and migration of ASCs were reversed by N-acetyl-cysteine and diphenyleneiodonium treatment, suggesting the involvement of ROS generation in ASC stimulation. Further, we examined the activation of receptor tyrosine kinases and observed that hypoxia stimulated the phosphorylation of platelet-derived growth factor receptor-β. In summary, the ROS produced by ASCs in response to hypoxia was mostly likely due to NADPH oxidase activity. The increased cellular ROS was accompanied by the phosphorylation of platelet-derived growth factor receptor-β as well as by the activation of ERK and Akt signal pathways. Our results suggest a pivotal role for ROS generation in the stimulation of ASCs by hypoxia.     

Epicatechin inhibits radiation-induced auditory cell death by suppression of reactive oxygen species generation

Radiation-induced toxicity limits the delivery of high-dose radiation to head and neck lesions. The aim of this study was to investigate the effectiveness of epicatechin (EC), a minor component of green tea extract, on radiation-induced ototoxicity in vitro and in vivo. The effect of EC on radiation-induced cytotoxicity was analyzed in the organ of Corti-derived cell lines, HEI-OC1 and UB-OC1. The cell viability, apoptosis, reactive oxygen species generation, and mitochondrial membrane potential as well as changes in the signal pathway related to apoptosis were investigated. Then, the therapeutic effects of hearing protection and drug toxicity of EC were explored in a zebrafish and rat model. Radiation-induced apoptosis and altered mitochondrial membrane potential in HEI-OC1 and UB-OC1 were observed. EC inhibited radiation-induced apoptosis and intracellular reactive oxygen species generation. EC markedly attenuated the radiation-induced embryotoxicity and protected against radiation-induced loss and changes of auditory neuromast in the zebrafish. In addition, intratympanic administration of EC was protective against radiation-induced hearing loss in the rat model, as determined by click-evoked auditory brainstem (P<0.01). EC significantly reduced the expression of p-JNK, p-ERK cleaved caspase-3, and cleaved PARP compared to their significant increase after radiation treatment. The results of this study suggest that EC significantly inhibited radiation-induced apoptosis in auditory hair cells and may be a safe and effective candidate treatment for the prevention of radiation-induced ototoxicity.     

Norepinephrine-induced myeloid-derived suppressor cells block T-cell responses via generation of reactive oxygen species

Abstract

Increased numbers of myeloid-derived suppressor cells (MDSCs) are often observed in various pathological and physiological conditions. However, the interactions between neurotransmitters and MDSCs have not been elucidated. In this study, we studied whether norepinephrine (NE), a neurotransmitter, could affect the differentiation of human MDSCs in vitro. Flow cytometric analysis showed that treatment with 20?μM NE significantly enhanced the expansion of MDSCs. The NE-generated MDSCs suppressed the T-cells proliferation, depending on the production of reactive oxygen species (ROS). Moreover, the expansion of MDSCs induced by NE resulted in a dramatic induction of nicotinamide adenine dinucleotide phosphate oxidase subunit P47^phox. Addition of the ROS inhibitor catalase into the MDSCs/T-cell co-culture system partly abrogated the suppressive effects of MDSCs on T-cell proliferation. In summary, our data have shown that NE enhanced the expansion of human MDSCs in vitro, providing important insights into the novel roles of neurotransmitters in the regulation of myeloid cell differentiation and function.     

Targeting reactive oxygen species in hypertension

Oxidative stress plays an important role in the pathogenesis of hypertension. A number of sources of reactive oxygen species have been identified including NADPH oxidase, endothelial NO synthase, and xanthine oxidase. Inhibitors of these systems reduce blood pressure in experimental models. Targeted overexpression of antioxidant systems and interference with expression of oxidant systems has also been successfully used in animal models of hypertension. It is expected that these strategies will eventually be translated to human disease, but currently, the specificity and toxicity of such measures are not yet fulfilling quality criteria for treatment of humans. In the meantime, presumably nontoxic measures, such as administration of antioxidant vitamins, are the only available treatments for oxidative stress in humans. In this review, we discuss strategies to target oxidative stress both in experimental models and in humans. We also discuss how patients could be selected who particularly benefit from antioxidant treatment. In clinical practice, diagnostic procedures beyond measurement of blood pressure will be necessary to predict the response to antioxidants; these procedures will include measurement of antioxidant status and detailed assessment of vascular structure and function.     

CO2 is a natural inhibitor of the generation of reactive oxygen species by phagocytes

The generation of reactive oxygen species in leukocytes from 73 healthy donors is studied using the luminol- and lucigenin-dependent chemiluminiscent methods and the nitroblue tetrazolium test. The results suggest CO₂ is a potent inhibitor of generation of reactive oxygen species in leukocytes under pressure close to that in the blood (37.5 mm Hg) and under high pressure (146 mm Hg). Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 10, pp. 395–398, October, 1994 Presented by F. I. Komarov, Member of the Russian Academy of Medical Sciences