Assay method for myeloperoxidase in human polymorphonuclear leukocytes

A simple assay method for measuring myeloperoxidase (MPO) has been developed. MPO is found in polymorphonuclear leukocytes and is important as a bactericidal agent in the presence of H2O2 and halide ions. This improved assay method is based on work of Andrews and Krinsky using tetramethylbenzidine (TMB) a noncarcinogenic substrate. By assaying MPO under optimal conditions of TMB at 1.6 mM, H2O2 concentration of 0.3 mM, pH 5.4, and incubation temperature of 37 degrees C, sensitivity of MPO measurements increased eightfold in comparison with the original TMB method. A method has been established to determine absorbance at 655 nm of the reaction mixture by incubation for 3 min and then stopping the reaction by the addition of pH 3.0 buffer. An attempt was also made to raise the sensitivity by using 3,3'-dimethyoxybenzidine (DMB), a carcinogenic substrate. The improved TMB method was 34 times more sensitive than the DMB method.     

CO-reacting haemoproteins of neutrophils: Evidence for cytochrome b-245 and myeloperoxidase as potential oxidases during the respiratory burst

Room temperature, CO-difference spectra of intact rat polymorphonuclear leucocytes (neutrophils) revealed the presence of a number of CO-binding haemoproteins. Absorption maxima at 413, 540 and 570 nm were attributed to the CO-complex of cytochromeb-245 whereas an absorption maximum at 595 nm was assigned to the contribution from a myeloperoxidase complex, since an identical absorption maximum was observed in CO-difference spectra of purified myeloperoxidase in the presence of H₂O₂. Photochemical action spectra for the relief of CO-inhibited O₂ uptake revealed contributions from both cytochromeb-245 and myeloperoxidase. The potential of these two O₂- and CO-binding haemoproteins to function as oxidases during the respiratory burst is discussed.     

Chemiluminescence of human bloodstream monocytes and neutrophils: An unusual oxidant(s) generated by monocytes during the respiratory burst

Maximal rates of O and H₂O₂ production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCI and related compounds. These combined reductions in O generating ability and lower myeloperoxidase levels result in low luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O and H₂O₂ than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O, H₂O₂, · OH, ¹O₂ or NO, but is derived from O generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.     

Temporal adaptation of human neutrophil metabolic responsiveness to the peptide formylmethionyl-leucyl phenylalanine: a comparison between human neutrophils and granule-depleted neutrophil cytoplasts

When polymorphonuclear leukocytes (neutrophils) and soluble or particulate matter interact, the cells produce superoxide anions (O2-) and hydrogen peroxide (H2O2). The chemotactic peptide formylmethionyl-leucyl-phenylalanine (FMLP) induced a very weak response in normal neutrophils. The cellular response was changed, however, as a result of in vitro aging of the cells, i.e. the magnitude of the response was increased following storage of the cells at 22 degrees C for up to 120 min, in the absence of any stimulus, and before the addition of the peptide. When phorbol myristate acetate was used as a stimulus, there was a pronounced production of O2- and H2O2, but no change in magnitude as a result of in vitro aging. When neutrophil cytoplasts (granule-free vesicles of cytoplasm enclosed by plasmalemma) were exposed to the peptide FMLP of PMA, the vesicles produced both O2- and H2O2. There was, however, no increase in oxidative metabolite production in cytoplasts as a result of in vitro aging when either FMLP or PMA was used as a stimulus. The results thus indicate that mere incubation at room temperature primed the cells to increase their production of oxidative metabolites as a result of spontaneous exposure of hidden receptors. The fact that no such effects were observed with cytoplasts indicates that spontaneous receptor recruitment is a granule-dependent process.     

Influence of 4-hydroxynonenal on chemiluminescence production by unstimulated and opsonized zymosan-stimulated human neutrophils

The lipid peroxidation product 4-hydroxy-2, 3-trans-nonenal (HNE) has a spectrum of biological effects on different cell types depending on the concentrations tested. In particular micromolar HNE concentrations stimulate neutrophil migration and polarization whereas higher doses inhibit. In our experimental conditions, fMet-Leu-Phe (fMLP) increased CL production of both unstimulated and zymosan-stimulated neutrophils, whereas cell stimulation with low HNE concentrations as well as zymosan addition to HNE incubated cells did not enhance light emission. In contrast 10(-4) M HNE reduced CL emission by unstimulated cells nearly to background values, completely depressed CL production by zymosan-stimulated cells and reduced phagocytosis. Cysteine was found to be able to counteract the HNE effect by about 70 per cent. The possibility that this aldehyde could exert its inhibitory effect through the alkylation of NADPH-oxidase SH-groups is postulated. Moreover, our present data on differences observed between fMLP and HNE indicate a different chemotactic mechanism induced by these two classes of compounds and lead to the conclusion that the local functional features of the attracted cells may be different.     

pH-dependent regulation of myeloperoxidase activity

The balance between peroxidase and chlorinating activities of myeloperoxidase (MPO) is very important for the enhancement of antimicrobial action and prevention of damage caused by hypochlorite. In the present paper, the peroxidase and chlorinating activities have been studied at various pH values. The possibility of using neutrophil protein solution for the evaluation of MPO activity has been demonstrated. It is shown that at neutral pH MPO had higher affinity to peroxidase substrate guaiacol: at pH 7.4, chloride ions did not compete with guaiacol up to the concentration of 150 mM. At acidic pH, chlorinating activity of MPO dominates: only hypochlorite production can be detected at equal chloride and guaiacol concentrations of 15 mM. However, horseradish peroxidase does not exhibit any difference in activity in the presence of chloride ions even at acidic pH values. It was demonstrated by MALDI-TOF mass-spectrometry that the amount of hypochlorite produced is sufficient to modify phospholipids (with formation of Cl- and Br-hydrins and lyso-derivatives) only at acidic pH (5.0). Thus, in the presence of phenolic peroxidase substrate, MPO chlorinating activity can be displayed at acidic pH only. It can lead to elimination of hypochlorite production in normal tissues at neutral pH (7.4) and its enhancement in phagosomes where the pH range is 4.7-6.0.     

Surface-bound myeloperoxidase is a ligand for recognition of late apoptotic neutrophils by human lung surfactant proteins A and D

Surfactant proteins A (SP-A) and D (SP-D), both members of the collectin family, play a well established role in apoptotic cell recognition and clearance. Recent in vitro data show that SP-A and SP-D interact with apoptotic neutrophils in a distinct manner. SP-A and SP-D bind in a Ca²⁺-dependent manner to viable and early apoptotic neutrophils whereas the much greater interaction with late apoptotic neutrophils is Ca²⁺-independent. Cell surface molecules on the apoptotic target cells responsible for these interactions had not been identified and this study was done to find candidate target molecules. Myeloperoxidase (MPO), a specific intracellular defense molecule of neutrophils that becomes exposed on the outside of the cell upon apoptosis, was identified by affinity purification, mass-spectrometry and western blotting as a novel binding molecule for SP-A and SP-D. To confirm its role in recognition, it was shown that purified immobilised MPO binds SP-A and SP-D, and that MPO is surface-exposed on late apoptotic neutrophils. SP-A and SP-D inhibit binding of an anti-MPO monoclonal Ab to late apoptotic cells. Fluorescence microscopy confirmed that anti-MPO mAb and SP-A/SP-D colocalise on late apoptotic neutrophils. Desmoplakin was identified as a further potential ligand for SP-A, and neutrophil defensin as a target for both proteins.     

Leukotriene B4 is a complete secretagogue in human neutrophils: a kinetic analysis

The interactions have been studied of leukotriene B₄ (LTB₄) and 20-COOH-LTB₄ with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB₄ activates PMN with respect to aggregation, mobilization of membrane-associated Ca²⁺, $\underset{\dot{}}{?}$ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB₄ is much more potent than its dicar?ylic acid derivative (20-COOH-LTB₄) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB₄ rapidly activates PMN and indicate that LTB₄ serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.     

Effect of a new fluorinated quinolone (ofloxacin) on the chemiluminescence of phagocytosing human neutrophils

We measured the chemiluminescence (CL) of human neutrophils (PMNLs) exposed to different concentrations of ofloxacin (2, 4, and 6 μg/ml) readily achievable in therapy. CL reaction during zymosan phagocytosis by PMNLs obtained from human healthy volunteers was registered in a computer-linked LKB 1251 luminometer. Ofloxacin did not induce significant variations on the respiratory burst of PMNLs.     

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O₂ ^–) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca²⁺ was absent (0.56±0.13 nmol/min per 10⁶ cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca²⁺. Moreover, the O₂ ^– production by fMLP in the presence of extracellular Ca²⁺ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca²⁺ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O₂ ^– production of electropermeabilized neutrophils in which the intracellular Ca²⁺ concentration was fixed to about 100 nM. These observations suggest that not only Ca²⁺ influx but the inhibition of PKA is necessary for the maximum O₂ ^– production by fMLP and that the O₂ ^– production is partially suppressed by the activation of PKA induced by fMLP.     

Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 μmol/l (9-fold difference), but not by calcium ionophore A23187 (15 μmol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.     

Chemotactically active proteins of neutrophils

Polymorphonuclear leukocytes (neutrophils) are the first cells that arrive at sites of infection or injury. There, besides their microorganism-targeted effector functions, activated neutrophils secrete numerous chemoattractants that recruit other leukocyte subtypes into the inflamed tissue. First, neutrophil activation leads to the upregulation of the gene expression of several classical chemokines of the CXC and CC families. Second, neutrophil granules contain preformed intracellular storage pools of chemotactically active proteins that are rapidly released upon neutrophil degranulation. The third pathway of generation of chemotactically active proteins by activated neutrophils--shedding and concomitant proteolytic processing of a membrane protein--has recently been demonstrated in our laboratory. In this review, we summarize the essential features of chemoattractant production by neutrophils and their contribution to orchestrating the recruitment of leukocyte subtypes during inflammatory response.     

Properties, Functions, and Secretion of Human Myeloperoxidase

The heme-containing protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes at sites of inflammation. It is involved in the generation of reactive oxygen and nitrogen species and tissue damage. The general properties and functional aspects of this enzyme are reviewed. Special attention is given to luminescence methods for investigating the release of myeloperoxidase from stimulated cells.     

Ceruloplasmin decreases respiratory burst reaction during pregnancy

Testing of pregnant women reveals weakening of neutrophil-mediated effector functions, such as reactive oxygen species generation. This study provides data confirming the phenomenon, gained through application of the flow cytometry technique. Key factors influencing neutrophil functional activity in blood plasma of pregnant women have not been detected so far. At the same time, concentration of ceruloplasmin – a copper-containing glycoprotein – is known to increase in blood significantly during pregnancy. We observed the negative correlation between ceruloplasmin concentration in blood plasma of pregnant women and the intensity of respiratory burst of neutrophils. Fractionation of plasma using gel-filtration revealed that ceruloplasmin-containing fraction demonstrated suppression of the respiratory burst reaction. Partial elimination of ceruloplasmin from the blood of pregnant women, performed with the help of specific antibodies and followed by immunoprecipitation, leads to an increased respiratory burst reaction. On the contrary, addition of ceruloplasmin to blood samples of healthy donors noticeably decreases the respiratory burst reaction. The results presented prove that change in ceruloplasmin level in plasma is necessary and sufficient for modulating the ability of neutrophils to produce reactive oxygen species during pregnancy.     

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

A fast and sensitive chemiluminescence assay for the determination of H₂O₂ in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H₂O₂ concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H₂O₂ without influence of H₂O₂ decomposition by cellular enzymes or newly produced H₂O₂ due to dismutation of superoxide anion radicals. Concentrations of H₂O₂ were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H₂O₂ concentration upon stimulation could be observed at 3000 cell/mL.     

Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway

3β-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3β-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ～30% was converted to its aldehyde-oxidation product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.     

Modulatory effect of chiral nonsteroidal anti-inflammatory drugs on apoptosis of human neutrophils

Polymorphonuclear neutrophils (PMNs) are short-lived leukocytes that die by apoptosis. Although PMNs are crucial in the defense against infection, they have been implicated in the pathogenesis of tissue injury observed in inflammatory diseases. The induction or prevention of PMN apoptosis is currently discussed as a key event in the control of inflammation. Caspase-3 activation is the first step in the execution phase of apoptosis. In the study, effect of racemic mixtures and enantiomers of 2-arylpropionic acid derivatives: ketoprofen, flurbiprofen (FBP), and (+)-S-naproxen and 2-arylbutyric acid: indobufen on apoptosis activation via caspase-3 and phosphatidylserine (PS) translocation (annexin-V binding) in human neutrophils in vitro has been investigated. Caspase-3 activation was detected by Western blotting, fluorometric assay of DEVD-AMC cleavage, and flow cytometry with carboxyfluorescein (FAM) labeled caspase inhibitor. PMNs were isolated and cultured up to 24 h. The chiral nonsteroidal anti-inflammatory drugs (NSAIDs) were found to modulate human PMN apoptosis in a dose- and time-dependent manner. The greater activation of caspase was found at 75-150 microg/ml concentration of racemates as well enantiomers, especially for FBP, whereas NSAIDs at smaller quantities (15 microg/ml) were inactive. At concentration of 75 microg/ml, NSAIDs increased the rate of PS externalization in PMA-stimulated and non-stimulated neutrophils. Additionally, no cytotoxic effect of the NSAIDs was observed at concentration up to 75 microg/ml that induce apoptosis. Regulation of caspase activity by NSAIDs may represent a potent target to trigger apoptosis and resolve inflammatory disorders.     

Prevalence of inherited myeloperoxidase deficiency in Japan

The microbicidal activity of the myeloperoxidase (MPO)-hydrogen peroxide-halide system has been implicated as the most efficient, oxygen-dependent antimicrobial component of neutrophil host defense. Unexpectedly, individuals with MPO deficiency suffer few clinical consequences. In order to understand better the clinical impact of MPO deficiency, we surveyed several clinical hematology laboratories in Japan to assess the prevalence of MPO deficiency in the general population. MPO activity was determined by flow cytometry using the Technicon H series of automated systems. We identified 26 cases of complete MPO deficiency, prevalence 1 in 57,135, and 129 cases of partial deficiency, prevalence 1 in 17,501. The distribution of complete and partial deficiencies differed among the laboratories studied.     

Effects of L-citrulline oral supplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise

Seventeen volunteer male professional cyclists were randomly assigned to control or supplemented (6 g L-citrulline-malate) groups and participated in a cycling stage. Blood samples were taken in basal conditions, after the race and 3 h post-race. Citrulline supplementation significantly increased plasma concentration of both arginine and citrulline after the stage only in the supplemented group. Polymorphonuclear neutrophils (PMNs) from controls responded to exercise with a progressive decrease in ROS production. Supplemented PMNs significantly increased ROS production after exercise compared to basal values and diminished to values lower than basal at recovery. PMN nitrite concentration was significantly higher after exercise and recovery only in the supplemented group. Markers of oxidative damage—CK, LDH, malondialdehyde—and DNA damage remained unchanged in both groups. In conclusion, oral L-citrulline administration previous to a cycling stage increases plasma arginine availability for NO synthesis and PMNs priming for oxidative burst without oxidative damage.