Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction

When polymorphonuclear leukocytes and soluble or particulate matter interact, the cells produce chemiluminescence, which is linked to activation of the oxidative metabolism of the cells. A luminol chemiluminescence assay in which the reaction mixture contains a relatively large amount of horseradish peroxidase combined with sodium azide has been proposed to quantitate H2O2 produced by human neutrophils during the respiratory burst (M.P. Wymann, V. von Tscharner, D. A. Deranleau, and M. Baggiolini (1987) Anal. Biochem. 165, 371-378). We found, when comparing the response to concanavalin A and a formylated peptide (formylmethionyl-leucyl-phenylalanine), that neutrophils produce H2O2 that is not detected as chemiluminescence by the horseradish peroxidase-azide-luminol system. Furthermore, the horseradish peroxidase-amplified chemiluminescence response obtained from granule-depleted neutrophil cytoplasts is inhibited by superoxide dismutase, an O2- scavanger. Based on these results, we question the specificity of the described technique for H2O2. The usefulness of the technique in the determining the extracellular and intracellular production of oxidative metabolites is discussed.     

Leukotriene production and inactivation by normal, chronic granulomatous disease and myeloperoxidase-deficient neutrophils

Appropriately stimulated neutrophils release peroxidase and undergo a respiratory burst to form hydrogen peroxide (H2O2) and hydroxyl radicals (OH). We report here that both the myeloperoxidase-H2O2-halide system and OH released in this way can degrade the leukotrienes (LT) formed by neutrophils. More LTB4 and LTC4 were recovered from the supernatants of chronic granulomatous disease neutrophils (which are unable to respond to stimulation with a respiratory burst) than from normal or myeloperoxidase-deficient neutrophils when stimulated with the calcium ionophore A23187. When radiolabeled LTC4 was added, 72% of the LTC4 was recovered from the chronic granulomatous disease cells in contrast to 0% from the myeloperoxidase-deficient and normal cells. Inhibitor studies using catalase, superoxide dismutase, azide, mannitol, or ethanol suggested that LTC4 degradation was mediated primarily by the myeloperoxidase system in normal cells and by OH in myeloperoxidase-deficient cells. LTC4 degradation by the cell-free myeloperoxidase-H2O2-halide system and the OH -generating acetaldehyde-xanthine oxidase-Fe2+ system had inhibitor profiles comparable to normal and myeloperoxidase-deficient neutrophils, respectively. LTC4 degradation products formed by the stimulated neutrophils and model systems included the 5-(S), 12-(R)- and 5-(S), 12-(S)-6-trans-isomers of LTB4. Thus phagocytes may modulate LT activity in inflammatory sites by the inactivation of these potent biologic mediators by at least two oxidative mechanisms.     

Inhibition of human neutrophil IL-8 production by hydrogen peroxide and dysregulation in chronic granulomatous disease

The innate immune response to bacterial infections includes neutrophil chemotaxis and activation, but regulation of inflammation is less well understood. Formyl peptides, byproducts of bacterial metabolism as well as mitochondrial protein biosynthesis, induce neutrophil chemotaxis, the generation of reactive oxygen intermediates (ROI), and the production of the neutrophil chemoattractant, IL-8. Patients with chronic granulomatous disease (CGD) exhibit deficient generation of ROI and hydrogen peroxide and susceptibility to bacterial and fungal pathogens, with associated dysregulated inflammation and widespread granuloma formation. We show in this study that in CGD cells, fMLF induces a 2- to 4-fold increase in IL-8 production and a sustained IL-8 mRNA response compared with normal neutrophils. Moreover, normal neutrophils treated with catalase (H(2)O(2) scavenger) or diphenyleneiodonium chloride (NADPH oxidase inhibitor) exhibit IL-8 responses comparable to those of CGD neutrophils. Addition of hydrogen peroxide or an H(2)O(2)-generating system suppresses the sustained IL-8 mRNA and increased protein production observed in CGD neutrophils. These results indicate that effectors downstream of the activation of NADPH oxidase negatively regulate IL-8 mRNA in normal neutrophils, and their absence in CGD cells results in prolonged IL-8 mRNA elevation and enhanced IL-8 levels. ROI may play a critical role in regulating inflammation through this mechanism.     

Early elicitor-induced events in chickpea cells: functional links between oxidative burst, sequential occurrence of extracellular alkalinisation and acidification, K+/H+ exchange and defence-related gene activation

Elicitation of cultured chickpea (Cicer arietinum L.) cells stimulates a signal transduction pathway leading to several rapid responses: (1) oxidative burst, (2) extracellular alkalinisation, (3) extracellular acidification, (4) transient K+ efflux, and (5) activation of defence related genes all within 2 hours. Induced genes are encoding acidic and basic chitinases, a thaumatin-like protein and isoflavone reductase. All these elicitor-induced responses are inhibited by the Ser/Thr protein kinase inhibitor staurosporine and the anion channel blocker anthracene-9-carboxylic acid but stimulated by the Ser/Thr protein phosphatase 2A inhibitor cantharidin. The oxidative burst leads to a transient extracellular H2O2 accumulation which seems to be preceded by O2- production, indicating dismutation of O2- to H2O2. The oxidative burst is accompanied by transient alkalinisation of the culture medium which is followed by long-lasting extracellular acidification. An 80 percent inhibition of the alkalinisation after complete inhibition of the H2O2 burst with diphenylene iodonium indicates that the elicitor induced increase of extracellular pH is mainly based on a proton consumption for O2-dismutation. A simultaneous deactivation of the plasma membrane H+-ATPase during oxidative burst and extracellular alkalinisation is also suggested. The elicitor-stimulated extracellular acidification is inhibited by the plasma membrane H+-ATPase inhibitor N, N'-dicyclohexylcarbodiimide assuming a reactivation of the H+-ATPase 25 min after elicitation. Extracellular acidification seems not to be necessary for elicitor-induced activation of defence related genes. Opposite modulation of K+ and proton fluxes after elicitation and/or treatment with the H+-ATPase effectors fusicoccin or N, N'-dicyclohexylcarbodiimide indicate that the elicitor induced transient K+ efflux is regulated by a K+/H+ exchange reaction.     

Immunosuppression by activated human neutrophils. Dependence on the myeloperoxidase system

An in vitro model system was used to define the mechanism of interaction between human neutrophils and lymphocytes. Blood mononuclear leukocytes were exposed to purified neutrophils in the presence of a neutrophil-activating agent (phorbol ester, lectin, or opsonized particle). The treated mononuclear cells displayed a marked decrease in both natural killer activity and mitogen-dependent DNA synthesis, but no change in viability. This functional suppression was dependent on neutrophil number, stimulus concentration, and duration of exposure. Lymphocytes were protected by addition of catalase, but not superoxide dismutase. Neutrophils defective in oxidative metabolism (chronic granulomatous disease) failed to suppress lymphocyte function unless an H2O2-generating system, glucose oxidase plus glucose, was added. The patients' neutrophils provided a factor, possibly myeloperoxidase, which interacted with the glucose oxidase system. The immunosuppressive effect of normal neutrophils was diminished when chloride was omitted from the cultures and was enhanced when chloride was replaced by iodide. Myeloperoxidase-deficient neutrophils were partially defective in suppressing lymphocytes and this was corrected by addition of purified myeloperoxidase. Paradoxically, azide caused enhancement of suppression that depended on the neutrophil oxidative burst, but not on myeloperoxidase and was mediated at least in part by an effect of azide on the target mononuclear leukocytes. These data indicate that suppression of lymphocyte function by activated neutrophils is mediated by the secretion of myeloperoxidase and H2O2 that react with halides to form immunosuppressive products. Moreover, the mononuclear leukocytes contain an azide-sensitive factor, probably catalase, which provides partial protection against injury by neutrophil products. These dynamic interactions may be important local determinants of the immune response.     

Interleukin-15 increases Paracoccidioides brasiliensis killing by human neutrophils

Interleukin-15 is a cytokine produced by a wide range of different cell types, including macrophages, in response to lipopolysaccharide or microbial infection. This cytokine may play a crucial role in the activation of phagocytic cells against pathogens, especially during innate immune response. The effects of IL-15 on human polymorphonuclear leukocyte fungicidal activity against a highly virulent Paracoccidioides brasiliensis strain were investigated. Pretreatment of human neutrophils from healthy individuals with IL-15 for 18 hours increased cell fungicidal activity in a dose-dependent manner. In addition, the exposure to IL-15 induced an increase in neutrophil oxidative burst as evaluated by superoxide anion and H(2)O(2) release. Catalase inhibited fungicidal activity supporting a role for H(2)O(2) in fungus killing. In contrast, IL-8 and TNF-alpha levels were not affected by IL-15 suggesting that its effects were not mediated by these cytokines. Together, these results show that IL-15 is a potent stimulant of antifungal activities in human neutrophils, at least in part by a mechanism dependent on oxidative metabolism.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Vav proteins in neutrophils are required for FcgammaR-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox)

Phagocytes generate reactive oxygen species, the regulation of which is important in eliminating ingested microbes while limiting tissue damage. Clustering of FcgammaRs results in the activation of Vav proteins, Rho/Rac guanine nucleotide exchange factors, and results in robust superoxide generation through the NADPH oxidase. In this study, studies in neutrophils isolated from mice deficient in Vav or Rac isoforms demonstrate a critical role for Vav3 in Rac2-dependent activation of the NADPH oxidase following FcgammaR clustering. However, studies in cytokine-primed cells revealed a strict requirement for Vav1 and Vav3 and Rac1 and Rac2 in the FcgammaR-mediated oxidative burst. In comparison, Vav was not essential for PMA or G protein-coupled receptor-mediated superoxide generation. The FcgammaR-mediated oxidative burst defect in Vav-deficient cells was linked to aberrant Rac activation as well as Rac- and actin-polymerization-independent, but PI3K-dependent, phosphorylation of the NADPH oxidase component p40(phox). In macrophages, Vav regulation of Rac GTPases was required specifically in FcgammaR-mediated activation of the oxidative burst, but not in phagocytosis. Thus, Vav proteins specifically couple FcgammaR signaling to NADPH oxidase function through a Rac-dependent as well as an unexpected Rac-independent signal that is proximal to NADPH oxidase activation and does not require actin polymerization.     

DNA damage induced by phorbol ester-stimulated neutrophils is augmented by extracellular cofactors. Role of histidine and metals

Activated neutrophils cause extensive DNA damage in neighboring nonphagocytic cells. To determine whether compounds in the extracellular milieu participate in the DNA damage process, murine neutrophils were cocultivated with target tumor cells in media of varying composition. Using the alkaline elution assay, it was found that the level of strand breaks induced was significantly higher (2.8-fold) in complex cell culture media than in minimal phosphate-buffered saline. Addition of amino acids in general and of histidine in particular increased the level of damage nearly to that observed in complete media (2.7- and 2.1-fold, respectively). The histidine stimulation was concentration-dependent and reached a maximum at 100-400 microM. The mechanism whereby this occurred is not proven but probably derived from chelation of metals and participation in a site-specific Fenton reaction. Addition of the cell-impermeable chelator EDTA dramatically inhibited induction of strand breaks by neutrophils in complete media and prevented the enhancement of damage induced by histidine in phosphate-buffered saline. None of the effects on neutrophil-induced damage could be attributed to modulation of the oxidative burst activity of the cells (O2- and H2O2 production). Histidine also enhanced induction of strand breaks by reagent H2O2. However, EDTA had no effect or actually increased the level of damage induced by both a bolus of H2O2 and a flux of H2O2 generated by glucose oxidase. The cell-permeable chelator o-phenanthroline inhibited both neutrophil- and H2O2-induced damage. The results indicate that secondary reactions involving extracellular amino acids and metals contribute significantly to neutrophil-induced DNA damage to neighboring cells. Moreover, the data show that the mechanism whereby neutrophils induce this damage cannot be attributed solely to secretion of H2O2.     

Co-oxidation of luminol by hypochlorite and hydrogen peroxide implications for neutrophil chemiluminescence

Stimulated neutrophils produce several potent oxidants including H2O2, O2- and HOCl. Previous studies have revealed all of these compounds to be capable of oxidizing luminol, a reagent often used to indicate, by its chemiluminescence, the oxidative burst of neutrophils. Data presented in this paper indicate that H2O2 and HOCl spontaneously react at physiologic pH to produce luminol-dependent chemiluminescence 100 times the sum of the chemiluminescence of either reagent alone. This enhancement is due to a co-oxidation by HOCl and H2O2, or to a novel oxidant generated by the interaction of HOCl and H2O2. The HOCl scavenger, taurine, inhibits the chemiluminescence. Evidence is presented against the participation of hydroxyl radical, O2- or singlet oxygen in the oxidation of luminol by HOCl and H2O2. These findings have implications for potential anti-inflammatory compounds.     

Generation of signals activating neutrophil functions by leukocyte integrins: LFA-1 and gp150/95, but not CR3, are able to stimulate the respiratory burst of human neutrophils

To address the question whether leukocyte integrins are able to generate signals activating neutrophil functions, we investigated the capability of mAbs against the common beta chain (CD18), or the distinct alpha chains of CR3, LFA-1, or gp150/95, to activate neutrophil respiratory burst. These investigations were performed with mAbs bound to protein A immobilized to tissue culture polystyrene. Neutrophils plated in wells coated with the anti-CD18 mAbs IB4 and 60.3 released H2O2; H2O2 release did not occur when neutrophils were plated in wells coated with an irrelevant, isotype-matched mAb (OKDR), or with mAbs against other molecules (CD16, beta 2-microglobulin) expressed on the neutrophil surface at the same density of CD18. Four different mAbs, OKM1, OKM9, OKM10, 60.1, which recognize distinct epitopes of CR3 were unable to trigger H2O2 or O2- release from neutrophils. However, mAbs against LFA-1 or gp150/95 triggered both H2O2 and O2- release from neutrophils. Stimulation of neutrophils respiratory burst by both anti-CD18, and anti-LFA-1 or gp150/95 mAbs was totally inhibited by the microfilaments disrupting agent, cytochalasin B, and by a permeable cAMP analogue. While the capability to activate neutrophil respiratory burst was restricted to anti-LFA-1 and gp150/95 mAbs, we observed that mAbs against all members of leukocyte integrins, including CR3, were able to trigger neutrophil spreading. These findings indicate that, in neutrophils, all three leukocyte integrins can generate signals activating spreading, but only LFA-1 and gp150/95 can generate signals involved in activation of the respiratory burst. This observation can be relevant to understand the mechanisms responsible for the activation of neutrophil respiratory burst by tumor necrosis factor-alpha, which has been shown to be strictly dependent on expression of leukocyte integrins (Nathan, C., S. Srimal, C. Farber, E. Sanchez, L. Kabbash, A. Asch, J. Gailit, and S. Wright. 1989. J. Cell Biol. 109:13411349.     

Oxygen metabolism and the microbicidal activity of macrophages.

Like neutrophils, phagocytizing macrophages undergo a "respiratory burst" in which significant quantities of oxygen are drawn into the cell. The consumed oxygen is not used in oxidative phosphorylation but, rather, in the formation of superoxide anion (O2) and H2O2. These oxygen metabolites and the products of their interaction, in particular hydroxyl radical (OH), have been implicated in the killing of ingested bacteria by neutrophils. Their role in macrophage microbicidal activity has not been fully defined. However, activated macrophages, which mediate increased resistance to infection in vivo, have a markedly increased capacity to generate O2 and H2O2 in vitro when stimulated by phagocytosis or surface perturbation. The enhanced capacity of activated macrophages to generate highly reactive oxygen metabolites during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of these cells.     

Effect of anti-inflammatory drugs on leukocyte superoxide production by soluble and particulate stimuli

When polymorphonuclear leukocytes are treated with soluble and particulate substances the cells produce superoxide anion O2-. which plays a role in the activation of the oxidative metabolism. The time-course of O2-. release and the extent to which the anion is produced are related to the nature of the stimulating agents. Anti-inflammatory non steroid drugs, such as indomethacin and oxamethacin, exert a variable depression of the oxidative burst, which does not seem correlated to the inhibition of prostaglandin synthesis.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.     

Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcepsilonRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H(2)O(2)) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and beta-hexosaminidase, and the secretion of newly synthesized leukotriene C(4), whereas selective scavenging H(2)O(2) by ebselen impaired leukotriene C(4) secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H(2)O(2) caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cgamma and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.