Changes in ascorbate levels on stimulation of human neutrophils

Changes in ascorbate levels have been measured in human neutrophils stimulated with opsonized zymosan, phorbol myristate acetate and formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), in the presence and absence of cytochalasin B. After stimulation with opsonized zymosan or phorbol myristate acetate, there was no loss of total ascorbate, but 30-40% of the reduced ascorbate was oxidized to dehydroascorbate. Superoxide dismutase and catalase added to the cell suspension did not inhibit this oxidation. fMet-Leu-Phe, however, gave no net oxidation but about 20% of the total ascorbate was lost during 2 h incubation. These results imply that there is not a simple relationship between superoxide and hydrogen peroxide production and ascorbate oxidation, and that release of ascorbate into the phagolysosomes does not occur.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.     

Characterization of the interaction of human eosinophils and neutrophils with opsonized particles

The interaction of human eosinophils with opsonized particles was compared with that of human neutrophils. When eosinophils are stimulated with serum-opsonized zymosan particles, the lag time in H2O2 production is twice as long as found with neutrophils. Moreover, the concentration of these IgG + C3-coated particles required for optimal stimulation is about four times as high for eosinophils as for neutrophils. Under these conditions, the two cell types generate similar amounts of H2O2. However, eosinophils produce twice as much H2O2 as do neutrophils when stimulated with the soluble agent phorbol myristate acetate. Thus, although the oxidase capacity of eosinophils is larger than that of neutrophils, opsonized zymosan is a weak trigger for this activity in eosinophils. This phenomenon may be due to differences between the two cell types in the plasma membrane receptors or in the receptor oxidase transducing signal. The following are indications for the first possibility. i) IgG interacts poorly with the Fc gamma receptors on the eosinophil surface compared with those on neutrophils. This was shown by the inability of IgG-coated zymosan or IgG-coated latex to trigger any substantial H2O2 production by eosinophils unless brought into close contact with these cells by centrifugation. In contrast, neutrophils are stimulated by these particles both in suspension and in a pellet. The dissimilarity of the Fc gamma receptors on eosinophils and neutrophils was also shown with respect to antigenicity, determined by the monoclonal antibodies 3G8 and CLB-FcR-1. ii) Eosinophils contain about half as many receptors for C3b and C3bi on their surface as do neutrophils, also detected with monoclonal antibodies. The interaction of IgG subclasses with functional Fc gamma receptors on eosinophils and neutrophils showed that eosinophils release twice as much H2O2 as do neutrophils upon interaction with IgG1-, IgG2-, or IgG3-coated Sepharose beads, but this difference becomes fivefold with IgG4-coated Sepharose. This might be of relevance to the situation of chronic antigenic stimulation, e.g., in chronic schistosomiasis, in which eosinophil numbers and IgG4 antibody levels are elevated.     

Ampicillin serves as an electron donor

1. The effect of ampicillin on cytochrome c reduction and on the superoxide production of human neutrophils stimulated by phorbol myristate acetate (PMA) was investigated. 2. Ampicillin did not stimulate the superoxide production of intact (resting) neutrophils and not amplify the superoxide production of neutrophils stimulated by phorbol myristate acetate (PMA). 3. However, ampicillin dose-dependently increased the reduction of cytochrome c. 4. In addition, 50 mM ampicillin stimulated a superoxide dismutase-inhibitable reduction of cytochrome c by 0.70 +/- 0.02 (mean +/- SD) nmol/min and a superoxide dismutase-noninhibitable reduction of cytochrome c by 2.08 +/- 0.03 (mean +/- SD) nmol/min. 5. These results suggest that ampicillin serves as an electron donor and/or a superoxide generator.     

Exclusive leukotriene C4 synthesis by purified human eosinophils induced by opsonized zymosan

Purified human eosinophils were challenged with N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, platelet-activating-factor, valyl-glycyl-seryl-glutamic acid, phorbol myristate acetate, zymosan, opsonized zymosan and the calcium ionophore A23187 to induce leukotriene synthesis. Reversed-phase high performance liquid chromatography analysis demonstrated the almost exclusive synthesis of leukotriene C4 by eosinophils of 11 healthy donors after challenge with opsonized zymosan [(22 +/- 4) X 10(6) molecules LTC4/cell, mean +/- SE] or the calcium ionophore A23187 [(54 +/- 7) X 10(6) molecules LTC4/cell, mean +/- SE]. The other agents were not capable of inducing leukotriene formation. When in addition to opsonized zymosan N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor were added a significant increase of the leukotriene C4 synthesis by eosinophils was observed. These results suggest that eosinophils might be triggered to produce considerable amounts of the spasmogenic leukotriene C4 in vivo by C3b- and/or IgG-mediated mechanisms e.g. phagocytosis.     

Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids

The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.     

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Inhibitory Effect of Bifemelane on Superoxide Generation by Activated Neutrophils Measured Using a Simple Chemiluminescence Method

We evaluated the effect of 4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane hydrochloride) on superoxide production by human neutrophils using an MCLA-dependent chemiluminescence assay. Bifemelane hydrochloride dose-dependently inhibited superoxide production by neutrophils stimulated with phorbol myristate acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-pheny-lalanine, while it had no effect on superoxide production by a hypoxanthine-xanthine oxidase system. These results indicate that bifemelane hydrochloride does not have a scavenging effect, but has an inhibitory effect on superoxide generation by neutrophils. Although this drug is commonly used for treating chronic cerebral infarction, it may also have a protective effect on acute ischemia/reperfusion injury.     

Fusion of human neutrophil phagosomes with lysosomes in vitro: involvement of tyrosine kinases of the Src family and inhibition by mycobacteria.

The intracellular killing of microorganisms in phagocytes involves the fusion of lysosomes containing bactericidal factors with phagosomes, and several intracellular pathogens are able to inhibit this fusion event. In this study, we report the reconstitution of phagosome-lysosome fusion in vitro, using an assay based on resonance energy transfer between fluorescent phospholipid analogues that were inserted into whole human NB4-neutrophil membranes from liposomes containing positively charged lipids. Cytosol was required for fusion, and fusion was stimulated 3-fold if this cytosol had been prepared from neutrophils activated by using opsonized zymosan or a combination of the calcium ionophore (A23187) and phorbol myristate acetate (PMA). Fusion was inhibited by the addition of PP1, an inhibitor of Src family protein kinases, or GTPgammaS. We have previously reported that the biogenesis of phagolysosomes in human neutrophils is inhibited by mycobacteria. Here we show that cytosol from cells having internalized live (not heat-killed) Mycobacterium smegmatis or cytosol simply incubated with mycobacteria inhibited fusion, indicating that soluble factors are involved in mycobacterial inhibition of phagosome-lysosome fusion.     

Oscillation of Reactive Oxygen Species Released by Activated Neutrophils

Phagocytic leukocytes, such as neutrophils and macrophages release reactive oxygen species (ROS) to the surrounding medium upon appropriate stimulation as part of their cytocidal activity. The release of ROS by inflammatory neutrophils, obtained by peritoneal injection of 12% caseinate-PBS was measured by the reduction of ferricytochrome c and luminol chemiluminescence (LCL). Neutrophils were harvested every 4 hours with cold PBS and stimulated with phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). On a regimen providing light between 6:00 to 18:00, PMA-stimulated neutrophils (1.0 x 10 7 neutrophil/ml) were found to release twice as much superoxide anion at night as they did during the day (clock time; 2:00 = 1.43 nmol/min vs. clock time 14:00 = 0.65 nmol/min). Neither FMLP- nor OZ-stimulated neutrophils displayed similar fluctuations. Thus, the qualitative and quantitative aspects of ROS generation by activated neutrophils differ significantly from one ligand to another. These results suggest that the effect of neutrophils on microorganisms and the surrounding tissues may differ with the nature of the stimulus and the time the stimulus is given.     

Stoichiometric conversion of oxygen to superoxide anion during the respiratory burst in neutrophils. Direct evidence by a new method for measurement of superoxide anion with diacetyldeuteroheme-substituted horseradish peroxidase

Extracellular release of superoxide anion (O-2) and hydrogen peroxide (H2O2) during the respiratory burst of porcine and human neutrophils was studied by using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for these oxygen derivatives. The method permitted simultaneous measurement of oxygen consumption and formation of both O-2 and H2O2 in a single reaction mixture. When neutrophils were stimulated with phorbol myristate acetate in the presence of the heme-substituted peroxidase, a rapid accumulation of compound III, a complex of the enzyme with O-2, was observed accompanying an increase in oxygen consumption. During the process, amounts of compound III formed and oxygen consumed were stoichiometric, and no compound II, an indicator of H2O2 formation, was observed. These results establish that neutrophils stimulated with the phorbol ester produce exclusively O-2 as the primary oxygen metabolite and release it into the extracellular medium. When a limited amount of opsonized zymosan was used as the stimulus, compound III formation was also observed but it ceased at an early stage of oxygen consumption. When a sufficient amount of azide was included in the system, however, formation of compound II was noted in the later stage of oxygen consumption. The findings suggest that O-2, formed during phagocytosis, is converted to H2O2 within phagosomes and then diffuses out into the extracellular medium when its decomposition by catalase and/or peroxidases is blocked by azide.     

Stimulus-specific enhancement of luminol chemiluminescence in neutrophils by phosphatidylserine liposomes.

When stimulated with different stimuli, neutrophils generate various active oxygen species. These active oxygen molecules can be analyzed by luminol chemiluminescence (LCL). Phosphatidylserine (PS)-liposomes increased the formylmethionyl-leucyl-phenylalanine-induced LCL of guinea pig peritoneal neutrophils without affecting their oxygen consumption and superoxide (O2.-) generation. Similar effects of PS-liposomes were also observed in LCL of neutrophils stimulated by phorbol myristate acetate or arachidonic acid but not by opsonized zymosan. Kinetic analysis revealed that the PS-liposome-induced increase in LCL depended on extracellulary generated O2.-. Moreover, the stimulatory effect of PS could be seen only when it formed liposomal membranes. The effect of PS-liposomes was also inhibited by superoxide dismutase, catalase, and deferoxamine, an iron chelator, but not by azide, an inhibitor of myeloperoxidase. Similar enhancement of stimulation-dependent LCL response was also observed with Fe3+ and ADP-Fe3+, but the degree of enhancement was much greater with PS-liposomes than with iron and its complex. The increase in hydroxyl radical generation by PS-liposome-treated neutrophils was confirmed by experiments with EPR spectrometry using spin-trapping agents. These results suggested that the interaction of neutrophils with PS-containing membrane surface might generate reactive oxygen species that enhance the stimulus-dependent LCL response of neutrophils.     

Serum of Behqet's Disease Enhances Super oxide Production of Normal Neutrophils

Using an MCLA-dependent chemiluminescence technique, we evaluated superoxide production by neutrophils isolated from 7 patients with Behget's disease. After stimulation by phorbol myristate acetate, N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan, neutrophils from the patients produced significantly more superoxide than those from 20 controls. Pretreatment of control neutrophils with serum prominently enhanced superoxide production, and serum from Behcet's disease patients had a significantly greater effect than that from controls. These findings suggest that serum from patients with Behget disease contains the priming factor(s) that can enhance enhanced superoxide production by neutrophils in response to stimulation.     

Factors affecting the chemiluminescent response of fish phagocytes

The effects of several factors on the phagocytic activity of cells isolated from the pronephros of striped bass, Morone saxatilis , were measured using a chemiluminescence (CL) assay. The CL responses of phagocytes to varying concentrations of bacteria, phorbol myristate acetate (PMA), and zymosan were shown to be dose-dependent. Incubation of phagocytes with PMA resulted in a decrease in cell numbers related to the concentration of PMA used in the assay. Opsonization of Aeromonas hydrophila with normal pooled bass serum decreased the number of colony forming units present in suspension while enhancing the CL response by striped bass phagocytes. Opsonization of zymosan also resulted in an enhanced CL response. Aeromonas hydrophila and Aerococcus viridans killed by heat treatment, incubation with formalin, or exposure to UV radiation elicited little or no CL when incubated with phagocytes.     

The respiratory burst response to Histoplasma capsulatum by human neutrophils. Evidence for intracellular trapping of superoxide anion

Human neutrophils (PMN) have received little attention as to the role they play in host defense against Histoplasma capsulatum (Hc). We have characterized the binding and phagocytosis of Hc yeasts by human PMN and quantified the PMN respiratory burst in response to this organism. mAb specific for CD11a, CD11b, and CD11c all partially blocked the attachment of unopsonized yeasts to PMN; a mAb to CD18 inhibited attachment by greater than 90%. Thus, human PMN recognize and bind Hc yeasts via CD18 adhesion receptors as has been found for human cultured macrophages and alveolar macrophages. Unopsonized yeasts were phagocytosed by PMN, but phagocytosis was increased markedly by heat-labile and heat-stable serum opsonins. These opsonins promoted enhanced phagocytosis of yeasts by increasing the attachment of Hc yeasts to the PMN membrane. Phagocytosis of viable or heat-killed Hc yeasts by PMN did not induce the secretion of superoxide anion (O2-) as quantified by the reduction of cytochrome c. O2- was not detected when yeasts were opsonized in normal serum or immune serum, or at a ratio of yeasts to PMN of up to a 100:1. However, phagocytosis of opsonized yeasts by PMN did not prevent them from subsequently releasing O2- after further incubation with opsonized zymosan or PMA. Opsonized Hc yeasts clearly stimulated the PMN respiratory burst as quantified by intracellular reduction of nitroblue tetrazolium, reduction of cytochrome c in the presence of cytochalasin D, oxygen consumption, luminol-enhanced and nonenhanced chemiluminescence, and H2O2 production. These data suggest that phagocytosis of Hc yeasts by PMN is associated with intracellular entrapment of O2- that is not detectable by reduction of extracellular cytochrome c.     

Ferrous iron release from transferrin by human neutrophil-derived superoxide anion: effect of pH and iron saturation.

The ability of superoxide anion (O2-) from stimulated human neutrophils (PMNs) to release ferrous iron (Fe2+) from transferrin was assessed. At pH 7.4, unstimulated PMNs released minimal amounts of O2- and failed to facilitate the release of Fe2+ from holosaturated transferrin. In contrast, incubation of phorbol myristate acetate (PMA)-stimulated PMNs with holosaturated transferrin at pH 7.4 enhanced the release of Fe2+ from transferrin eightfold in association with marked generation of O2-. The release of Fe2+ was inhibited by addition of superoxide dismutase (SOD), indicating that the release of Fe2+ was dependent on PMN-derived extracellular O2-. In contrast, at physiologic pH (7.4), incubation of transferrin at physiological levels of iron saturation (e.g. 32%) with unstimulated or PMA stimulated PMNs failed to facilitate the release of Fe2+. The effect of decreasing the pH on the release of Fe2+ from transferrin by PMN-derived O2- was determined. Decreasing the pH greatly facilitated the release of Fe2+ from both holosaturated transferrin and from transferrin at physiological levels of iron saturation by PMN-derived O2-. Release of Fe2+ occurred despite a decrease in the amount of extracellular O2- generated by PMNs in an acidic environment. These results suggest that transferrin at physiologic levels of iron saturation may serve as a source of Fe2+ for biological reactions in disease states where activated phagocytes are present and there is a decrease in tissue pH. The unbound iron could participate in biological reactions including promoting propagation of lipid peroxidation reactions or hydroxyl radical formation following reaction with phagocytic cell-derived hydrogen peroxide.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Fc-gamma- and complement receptor mediated elevation in the cytosolic calcium level in human neutrophils.

The effects of differently opsonized zymosan particles, acting solely at Fc-gamma or at complement receptors or at both, on the level of intracellular calcium ([Ca2+]i) in human neutrophils were studied. A biphasic, long-lasting increase in [Ca2+]i was seen in response to IgG-, C3- and fresh serum-opsonized zymosan particles in the presence of extracellular Ca2+. Unopsonized zymosan, acting mainly at CR3 failed to elevate [Ca2+]i. Addition of 1.4 mM EGTA reduced but did not abolish the rise in [Ca2+]i triggered by opsonized zymosan, indicating that Ca2+ is released from intracellular stores. EGTA changed also the kinetic patterns of Ca(2+)-responses possibly by indirectly affecting the extrusion of Ca2+ in neutrophils.     

Transmembrane signaling: an ion-flux-independent model for signal transduction by complexed Fc receptors

Fluxes of Na+/K+ that precede effector functions in stimulated phagocytes are thought to play a role in signal transduction. To examine this hypothesis, phagocytosis, phagosomal acidification, and superoxide anion generation (O2-) were stimulated in media in which the Na+ was replaced with K+ or choline+. Counts of particles internalized and assessment of acidification of the phagosomes by acridine orange staining indicated that Na+/K+ fluxes were not necessary for phagocytosis or phagosomal acidification in J774.2 macrophages. Phagocytosis mediated by the ionophoretic Fc receptor gamma 2b/gamma 1 of J774.2 macrophages was equally independent of a Na+ gradient. Na+/K+ fluxes did not dictate the rate of O2- generation in human monocytes. Therefore, in at least these three effector functions, Na+/K+ fluxes stimulated by Fc- and non-specific receptor binding play neither a signaling nor an enhancing role. An ion-flux-independent model for transmembrane signaling by the Fc receptor is proposed. Others have shown that there is an apparent dependence on the external Na+ concentration for O2- generation and lysosomal secretion by neutrophils. These neutrophils had been pre-treated with NH4+ during a routine purification step. O2- generation stimulated by opsonized zymosan or phorbol myristate acetate, by monocytes or monocyte-derived macrophages, and phagocytosis of opsonized zymosan by J774.2 macrophages, showed dependence on external Na+ only if these cells had been pre-treated with NH4+. Brief NH4+ pre-treatment would be expected to acidify the cytoplasm of the cells. The reversal of this acidification is known to require Na+ for H+ extrusion through the Na+/H+ antiport, thus explaining the apparent Na+ dependence.