Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.     

Role of myeloperoxidase in the killing of Naegleria fowleri by lymphokine-altered human neutrophils.

Previously we have shown that human neutrophils treated with conditioned medium from phytohemagglutinin-stimulated mononuclear leukocytes (sCM) in the presence of antisera have amoebicidal properties for Naegleria fowleri, a pathogenic free-living amoeba. The data now presented show that neutrophils which lack myeloperoxidase (MPO) but have a normal oxygen-dependent respiratory burst could not be altered by sCM to express the amoebicidal activity. Catalase inhibited this amoebicidal activity of sCM-treated neutrophils. Various components and products of the neutrophils were examined for effects on naegleriae. A granule extract was found to have no effect at concentrations up to 100-fold that which killed Salmonella minnesota R595. Hydrogen peroxide appeared to have little effect even at 100 microM. However, in the presence of MPO, H2O2 was amoebicidal at 2.5 microM. The generation of amoebicidal activity required the presence of chloride ions. Azide inhibited the effects of the MPO-H2O2-Cl- system. Arginine, a scavenger of hypochlorite, significantly depressed the ability of sCM-treated neutrophils to kill amoebae and also prevented the amoebicidal properties of the MPO-H2O2-halide system. These results suggest that the MPO-H2O2-halide system is important in the killing of naegleriae by sCM-treated neutrophils and that hypochlorite may be the amoebicidal agent.     

IgE-dependent release of myeloperoxidase by neutrophils from allergic patients

BACKGROUND: The three forms of IgE receptor: the heterotrimeric high-affinity receptor for IgE (FcepsilonRI), the low-affinity receptor for IgE (FcepsilonRII/CD23) and the Mac-2/IgE-binding protein (epsilonBP), have previously been found on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present in the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether specific allergens are able to elicit MPO release by neutrophils from allergic patients. METHODS: Neutrophils were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. Also, the cells were challenged with allergens that the patients were not sensitive to. Neutrophils from normal subjects were also challenged with allergens. RESULTS: The in vitro challenge of neutrophils with allergens that the patients were sensitive to elicited a release of MPO by these cells. The in vitro activation of neutrophils was highly allergen-specific, in such a way that allergens other than those accounting for clinical symptoms did not evoke MPO release, and allergens were ineffective on neutrophils from healthy donors. CONCLUSION: An IgE-dependent mechanism might promote MPO release by neutrophils at allergic sites.     

Delayed apoptosis of circulating neutrophils in Kawasaki disease.

Circulating polymorphonuclear neutrophils (PMNs) are known to increase in number and are functionally activated in the acute phase of Kawasaki disease (KD). In the present study, we investigated whether the apoptosis of PMNs is deregulated in KD. When the isolated PMNs were cultured in vitro, the proportions of spontaneous apoptotic PMNs (annexin V+ cells and cells with fragmented DNA) were found to be significantly lower (P < 0.01) in the patients with KD (n = 25) than in the patients with a bacterial infection (n = 20) or a viral infection (n = 20), or in healthy children (n = 20). The proportion of circulating Fas-positive PMNs was also significantly lower (P < 0.01) in the acute KD patients than in the other groups. In the acute phase of KD, the proportion of spontaneous apoptotic PMNs showed a significant positive correlation (P < 0.01) with the proportions of circulating Fas-positive PMNs. Furthermore, the agonistic anti-Fas MoAb (CH-11) induced a significant increase in the proportion of apoptotic PMNs in the patients with a viral infection and healthy children, but not in either the patients with KD or the patients with a bacterial infection. In the intracellular expression of anti- and pro-apoptotic proteins, the A1/Bax ratio was significantly higher in acute KD than in the other groups. These findings indicate that PMN apoptosis is inhibited during the acute phase of KD and also suggest that both the resistance against the Fas-mediated death signal and the down-regulation of the mitochondrial apoptotic signalling pathway due to an altered balance of Bcl-2 protein expression are responsible for the delayed PMN apoptosis.     

Effects of (d-Ala2) methionine enkephalinamide in PMA-induced mouse ear edema

The synthetic opioid peptide (d-Ala²) methionine enkephalinamide (DAMEA) was reported to be an extremely potent inhibitor of the respiratory burst of polymorphonuclear neutrophils (PMNs). In the present paper the activity of DAMEA was investigated in the PMA-induced mouse ear edema since there is strong evidence that reactive oxygen species (ROS) are involved in the pathogenesis of this inflammatory reaction. DAMEA inhibited PMA-induced mouse ear edema in a dose-dependent manner between 10^–6 and 10^–9 mol/ear. The well-known anti-inflammatory activity of narcotic analgesics could be explained partly by an inhibition of ROS production.     

Differentiated HL60 promyelocytic leukaemia cells have a deficient myeloperoxidase/halide killing system.

Following induction of differentiation by incubation with 1.25% dimethylsulfoxide (DMSO), cells of the HL60 promyelocytic leukaemia cell line acquire certain characteristics of the mature polymorphonuclear leucocyte (PMN) including the ability to produce oxygen radicals and to phagocytose opsonized bacteria. However, these cells are unable to fix 125I during phagocytosis and are only able to kill phagocytosed microorganisms (C. albicans and S. aureus) to a small degree compared to mature PMN. Further, release of myeloperoxidase (MPO) from cytoplasmic granules occurs to approximately 20% of control levels after 6 days culture with DMSO, and drops to negligible levels by 7 days. These data suggest an immature or inactive MPO/peroxide/halide killing system. Insensitivity to the cyclooxygenase pathway inhibitor indomethacin suggests that there may also be a defect in this pathway.     

Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.     

Histamine synthesis in mouse polymorphonuclear neutrophils

Inflammation Research -     

Alteration of constitutive apoptosis in neutrophils by quinolones

Neutrophils constitutively undergo apoptosis at sites of infection. The process of apoptosis controls inflammatory responses of neutrophils. However, little is known about the abilities of quinolones, which are often administered to patients showing infection disease, on constitutive apoptosis of neutrophils. The aim of this study is to evaluate abilities of quinolones on constitutive apoptosis of neutrophils. Tosufloxacin delayed neutrophil death and delayed neutrophil apoptosis. In contrast, ofloxacin, lomefloxacin, fleroxacin, sparfloxacin, and levofloxacin markedly promoted neutrophil death without affecting neutrophil apoptosis. Inhibitors of phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (MAPK) attenuated the delay of neutrophil apoptosis by tosufloxacin, respectively. However, an inhibitor of extracellular-signal–related kinase did not alter the delay of neutrophil apoptosis by tosufloxacin. Moreover, tosufloxacin increases the expression of p85, p110, and Akt protein in neutrophils. These results suggest that tosufloxacin may delay neutrophil apoptosis via activation of PI3K/Akt and/or p38 MAPK, and the other quinolones may promote neutrophil death without affecting their apoptosis.     

Transgenic mouse model of early-onset DYT1 dystonia

Early-onset dystonia is an autosomal dominant movement disorder associated with deletion of a glutamic acid residue in torsinA. We generated four independent lines of transgenic mice by overexpressing human DeltaE-torsinA using a neuron specific enolase promoter. The transgenic mice developed abnormal involuntary movements with dystonic-appearing, self-clasping of limbs, as early as 3 weeks after birth. Animals also showed hyperkinesia and rapid bi-directional circling. Approximately 40% of transgenic mice from each line demonstrated these severe behavioral abnormalities. Neurochemical analyses revealed decreases in striatal dopamine in affected transgenic mice, although levels were increased in those that had no behavioral changes. Immunohistochemistry demonstrated perinuclear inclusions and aggregates that stained positively for ubiquitin, torsinA and lamin, a marker of the nuclear envelope. Inclusions were detected in neurons of the pedunculopontine nucleus and in other brain stem regions in a pattern similar to what has been described in DYT1 patients. This transgenic mouse model demonstrates behavioral and pathologic features similar to patients with early-onset dystonia and may help to better understand the pathophysiology of this disorder and to develop more effective therapies.     

Interleukin-2 prevention of apoptosis in human neutrophils

Evidence is presented that interleukin (IL)-2 maintains viability of human polymorphonuclear cells (PMN) in culture by preventing these cells from undergoing programmed cell death (PCD) and induces the synthesis of new RNA and protein. Our laboratory has recently discovered that human PMN constitutively express IL-2β receptor and more importantly, PMN are able to respond functionally to IL-2 by enhanced growth inhibitory activity against an opportunistic fungal pathogen, Candida albicans. We now report that IL-2 was able to interfere with the PCD process and reduce the number of apoptotic PMN to < 40% in 72-h culture. Freshly isolated PMN usually underwent a timedependent aging process and > 80% of PMN cultured in medium alone for 72 h showed morphologic features of PCD as depicted by hematoxylin and eosin staining as well as by electron microscopy. During the PCD process, untreated PMN not only exhibited condensed nuclear structure and decrease in cell size, but also displayed DNA fragmentation. DNA fragmentation in PMN was prevented by IL-2. Prevention of PCD by IL-2 was associated with an increase in new RNA and protein synthesis in PMN, which may reflect cytokine induction, such as tumor necrosis factor, as we have recently shown. Thus, our data expands our current understanding of PMN in that they may be an active component of the immune system, with a longer life-span when activated than expected.     

Colocalization of elastase and myeloperoxidase in human blood and bone marrow neutrophils using a monoclonal antibody and immunogold.

The authors have localized elastase in human blood and bone marrow neutrophils by immunoelectron microscopy using a monoclonal anti-human elastase antibody (NP 57) and compared its distribution with myeloperoxidase (MPO) and lactoferrin (LF), which mark primary and secondary neutrophil granule, respectively. Human bone marrow and blood polymorphonuclear leukocytes (PMN), either unstimulated or after phagocytosis of latex microbeads, were fixed in 4% paraformaldehyde. Ultrathin frozen sections were immunolabeled with NP 57, followed by an immunogold probe. In bone marrow granulocyte precursors elastase appeared simultaneously in the immature first granules of myeloblasts with MPO. As these granules became denser with maturation, labeling for both enzymes became weaker and sometimes negative (possibly due to masking of immunoreactivity). The ellipsoidal primary granules were strongly labeled by NP57. LF positive granules appeared later, at the myelocyte stage, and contained neither MPO nor elastase. In mature neutrophils, immunolabeling for elastase was found together with MPO in the large electron-dense primary granules and in a different granule population from the LF-positive secondary granules. Double labeling with two different-sized gold particles was used to compare the kinetics of degranulation of secondary and primary granules. The observation and the analysis of single phagosome content was made possible by this new technique. In conclusion, immunoelectron microscopy was used to show elastase in the primary granules of neutrophils, where it appears simultaneously with MPO. This technique has also allowed comparison of the kinetics of degranulation of both types of granules, and could be applied to different experimental and pathologic conditions.     

Effect of exercise to exhaustion on myeloperoxidase and lysozyme release from blood neutrophils

Exercise sessions (swimming in rats and treadmill running in humans) resulted in stimulation of neutrophil degranulation in the experiments with animals and in the human study. Myeloperoxidase (MPO) (+67%) and lysozyme (+51%) quantities in the plasma of rats increased significantly immediately after exercise. The blood plasma lysozyme concentration was increased by 41% at the 6th min of treadmill exercise in athletes. The blood concentrations of neutrophil proteins normalized both in humans and animals at rest. The neutrophil protein concentrations in blood increased in parallel with the decrease of their level in leukocytes. The neutrophil capacity for an oxidative burst was not changed by the exercise, but decreased for 3–6 h in the post-exercise period. Such dynamics of the oxidative burst activity suggest a lack of association between this parameter and the degranulation process. The neutrophil proteins that appear in blood during degranulation can be involved in enhancing the bactericidal potency of blood, the activation of granulopoiesis, neutrophil efflux from bone marrow, and the conditioning of blood endothelium for leukocyte extravasation. Electronic Publication     

Elevated secretion of myeloperoxidase by neutrophils from asthmatic patients: the effect of immunotherapy

BACKGROUND: There is increasing evidence of neutrophil participation in asthma and the allergic process. After activation, neutrophils release myeloperoxidase (MPO) together with other granule enzymes. OBJECTIVES: In this study we attempted to evaluate the release of MPO in vitro by neutrophils from asthmatic patients and the relationship between neutrophil degranulation and lung function, measured as FEV(1), of the patients. We also investigated the possible role of immunotherapy in the release of MPO by neutrophils. METHODS: Neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine for 45 minutes at 37 degrees C. MPO released from neutrophils was assayed by using an MPO enzyme immunoassay. RESULTS: Neutrophils released statistically significantly higher MPO levels in the asthmatic patients not receiving immunotherapy than in the healthy group. A significant inverse correlation was observed in the asthmatic group not receiving immunotherapy between MPO secretion and lung function, measured as FEV(1), of the patients. Neutrophils of the asthmatic group receiving immunotherapy released significantly less MPO than did those of the asthmatic group not receiving immunotherapy, with MPO levels equal to those from nonallergic subjects. CONCLUSIONS: We conclude that neutrophils obtained from allergic asthmatic patients have an increased propensity to release MPO. The experiments described here provide evidence that there is a significant inverse relationship between levels of MPO released by neutrophils from allergic patients and lung function, as assessed by FEV(1). Our study suggests that immunotherapy actively modifies the release of MPO in vitro by neutrophils from allergic asthmatic patients.     

Inhibition by hydroxyachillin,sesquiterpene lactone fromTanacetum microphyllum,of PMA-induced mouse ear oedema

4--phorbol 12-myristate 13-acetate (PMA), when administered topically to mouse ear, induces a pronounced inflammatory response mediated by protein kinase C (PKC). Activation of PKC is implicated in the pathogenesis of inflammation, with phospholipase A₂-dependent arachidonic acid release and eicosanoid production. We have investigated the effects of hydroxyachillin, a sesquiterpene lactone fromTanacetum microphyllum DC., on mouse ear oedema induced by PMA. The effects of this compound on swelling and other inflammatory parameters are described. Hydroxyachillin significantly (p0.01) inhibited ear swelling in a dose-dependent manner, and was as effective as the reference drugs. The PMA-induced vascular permeability was significantly (p0.05) reduced by hydroxyachillin at the highest dose (3 mg/ear). Histologically, the signs of inflammation were greatly reduced in the hydroxyachillin-treated ear lesions. These data suggest that hydroxyachillin is an effective anti-inflammatory agent in this model, and that the inhibition of PKC may be one of the mechanisms of hydroxyachillin's effect.accepted by K. Brune     

Lysophospholipase activity of Ascaris suum-induced mouse peritoneal neutrophils and eosinophils.

Lysophospholipase activity of mouse peritoneal neutrophils and eosinophils was studied to determine if neutrophils and eosinophils have lysophospholipase activity when treated with Ascaris suum whole worm extract, if zymosan activated complement can induce increased lysophospholipase activity, or if the immune status of the host has an effect on lysophospholipase activity. Neutrophils from noninfected or infected (immunized) mice were found to have increased lysophospholipase activity when treated with A. suum whole worm extract or zymosan activated complement demonstrating neutrophils as a source of lysophospholipase activity in the presence or absence of an immune response. Eosinophils from immunized mice had increased lysophospholipase activity when treated with either A. suum whole worm extract or zymosan activated complement.