Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.     

Contribution of CD54 to human eosinophil and neutrophil superoxide production.

We have reported that CD54 on eosinophils is involved in eosinophil degranulation. However, the role of CD54 in eosinophil and neutrophil superoxide production is still uncertain. We assessed the effect of CD54 on eosinophils and neutrophils in recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF)- or phorbol myristate acetate (PMA)-induced superoxide production through CD18. Anti-CD54 monoclonal antibody attenuated leukocyte aggregation and superoxide production of rGM-CSF- or PMA-stimulated neutrophils and PMA-stimulated eosinophils. Anti-CD18 monoclonal antibody or theophylline attenuated superoxide production of eosinophils and neutrophils stimulated by either stimuli. Flow cytometric analysis demonstrated CD54 expression on freshly isolated neutrophils but not on freshly isolated eosinophils. CD54 newly expressed on eosinophils reached its peak expression 30 min after PMA stimulation. The increase in CD18 and CD54 expression on neutrophils caused by rGM-CSF stimulation was partially inhibited by theophylline. These data demonstrated that CD54 and CD18 interaction of eosinophils or neutrophils is involved in superoxide production and that the inhibition of superoxide production by theophylline may be at least partly due to the inhibition of CD54 and CD18.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Inhibition of neutrophil superoxide production by human plasma alpha 1-antitrypsin.

We report here that human plasma alpha 1-antitrypsin (alpha 1-AT) inhibited human neutrophil O2.- release elicited by a variety of stimulants. In comparison, the inhibitory capacities of two serine protease inhibitors, L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK) and soybean trypsin inhibitor (SBTI), and the human recombinant alpha 1-AT mutant, alpha 1-AT-Arg358 were in the order: alpha 1-AT = TPCK much greater than alpha 1-AT-Arg358 greater than SBTI when cells were stimulated with concanavalin A plus cytochalasin E. These data suggest that, in human inflammatory fluids containing relatively high concentrations of alpha 1-AT (such as rheumatoid arthritis synovial fluid), (i) alpha 1-AT may down-regulate the inflammatory process by inhibiting the neutrophil respiratory burst and (ii) serpin oxidation by neutrophil-released reactive oxygen species is unlikely to occur.     

Apolipoprotein A-I decreases neutrophil degranulation and superoxide production.

Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response.     

Direct relationship between intracellular calcium mobilization and phospholipase D activation in prostaglandin E-stimulated human erythroleukemia cells.

The relationship between calcium mobilization and phospholipase D (PLD) activation in response to E-series prostaglandins (PGEs) was investigated in human erythroleukemia cells. Intracellular free Ca2+ concentration ([Ca2+]i) was increased by PGE1 and PGE2 over the same concentration range at which PLD activation was seen. Pretreatment of cells with pertussis toxin greatly inhibited the PGE-stimulated increase in [Ca2+]i, implying that a G protein participates in the PGE receptor signaling process. The peak level and also the plateau level of Ca2+ mobilization stimulated by these prostaglandins were markedly decreased in Ca(2+)-depleted medium, indicating that both extracellular and intracellular Ca2+ stores contribute to the changes in [Ca2+]i. Likewise, activation of PLD by PGE1 and PGE2 was abolished by pertussis toxin pretreatment or incubation in Ca(2+)-depleted medium. U73122, a putative phospholipase C inhibitor, blocked both Ca2+ mobilization and PLD activation in PGE-stimulated cells. Furthermore, the intracellular loading of BAPTA, a Ca2+ chelator, inhibited both Ca2+ mobilization and PLD activation by PGE1 and PGE2 in a similar dose-dependent manner. Simultaneous measurement of [Ca2+]i and PLD activity in the same cell samples indicated that PLD activity increases as a function of [Ca2+]i in a similar fashion in cells stimulated either by PGEs or by the calcium ionophore ionomycin. Taken together, these findings suggest that a rise in [Ca2+]i is necessary for PGE-stimulated PLD activity in human erythroleukemia cells.     

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and increased neutrophil production of the NF-kappaB-dependent cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-alpha or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-kappaB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-kappaB and increases expression of NF-kappaB-dependent cytokines.     

Role of different Ca2+ sources in the superoxide production of human neutrophil granulocytes.

The role of different Ca2+ sources in the activation of the NADPH oxidase was investigated in human neutrophil granulocytes. Selective depletion of the stimulus-responsive intracellular Ca2+ -pool and the consequent opening of the store-dependent Ca2+ channel of the plasma membrane was achieved with thapsigargin, an inhibitor of microsomal Ca2+ -ATPase. Low concentration (10-100 nM) of thapsigargin did not induce any O2*- -production, indicating that elevation of [Ca2+]ic to similar level and probably via similar route as following stimulation of chemotactic receptors, by itself is not sufficient to activate the NADPH oxidase. In significantly higher concentration (1-10 microM) thapsigargin did induce O2*- -generation but this effect was not the result of elevation of [Ca2+]ic. In the absence of external Ca2+ a gradual decrease of the responsive Ca2+ pool was accompanied by a gradual decrease of the rate and duration of the respiratory response stimulated by formyl-methionyl-leucyl-phenylalanin. Maximal extent of receptor-initiated O2*- -production could only be obtained when the intracellular [Ca2+] was higher than the resting level. Under this condition Ca2+ originating from intracellular or external source was equally effective in supporting the biological response.     

The temporal relationship between protein phosphatase, mitochondrial cytochrome c release, and caspase activation in apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochrome c. Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochrome c in apoptosis induced by the Fas receptor. We demonstrate that cytochrome c is released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochrome c. The protein phosphatase inhibitor calyculin A prevented cytochrome c release and apoptosis induced by both agents, suggesting that release of cytochrome c is required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochrome c. The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochrome c and apoptosis induced by both insults.     

Rac2 regulates neutrophil chemotaxis, superoxide production, and myeloid colony formation through multiple distinct effector pathways

Polymorphonuclear neutrophils (PMN) are an important component of the innate immune system. We have shown previously that migration and superoxide (O2*-) production, as well as some kinase signaling pathways are compromised in mice deficient in the Ras-related Rho GTPase Rac2. In this study, we demonstrate that Rac2 controls chemotaxis and superoxide production via distinct pathways and is critical for development of myeloid colonies in vitro. The Rac2 mutants V36A, F37A, and N39A all bind to both Pak1 and p67(phox), yet are unable to rescue superoxide production and chemotaxis when expressed in Rac2-/- PMN. In contrast, the N43A mutant, which binds to Por1 (Arfaptin 2), p67phox, and Pak1, is able to rescue superoxide production but not chemotaxis. The F37A mutant, demonstrated to have reduced binding to Por1, shows reduced rescue of fMLP-induced chemotaxis. Finally, the Rac2Y40C mutant that is defective in binding to all three potential downstream effectors (Pak1, p67phox, and Por1) is unable to rescue chemotaxis, motility, or superoxide production, but is able to rescue defective growth of myeloid colonies in vitro. These findings suggest that binding to any single effector is not sufficient to rescue the distinct cellular phenotypes of Rac2-/- PMN, implicating multiple, distinct, and potentially parallel effector pathways.     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.     

Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase

Abstract Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 min after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by luminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca²⁺ and phospholipase A₂ activity, but not the activities of phospholipase D or D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.     

Fluoride activates diradylglycerol and superoxide generation in human neutrophils via PLD/PA phosphohydrolase-dependent and -independent pathways

In contrast to the rapid, ethanol-inhibited superoxide generation by the receptor-linked agonist formyl-methionyl-leucyl-phenylalanine (fMLP), fluoride-activated superoxide generation occurs after a prolonged lag, and as shown herein is relatively ethanol-insensitive. We have investigated fluoride-activation of diradylglycerol generation and phospholipase D activity. Fluoride induces a very large increase in diradylglycerol mass (both 1,2-diacylglycerol (DAG) and 1-O-alkyl,2-acylglycerol (EAG)), with kinetics similar to superoxide generation. Unlike fMLP-activated diglyceride generation which is completely inhibited by ethanol, that produced by fluoride is only partially (30%) blocked. When the phosphatidylcholine pool is 3H-prelabeled, fluoride activates both [3H]phosphatidic acid (PA) and [3H]diglyceride generation with similar kinetics. Partial inhibition of the production of these species by ethanol was seen, coincident with the appearance of [3H]phosphatidylethanol, indicating phospholipase D-dependent transphosphatidylation had occurred. The data are consistent with the fluoride activation of PA and diglyceride generation by both phospholipase D-dependent and -independent (presumably phospholipase C) mechanisms.     

A phospholipase A2-activating protein (PLAP) stimulates human neutrophil aggregation and release of lysosomal enzymes, superoxide, and eicosanoids

We have recently isolated a human phospholipase A2-activating protein (PLAP) that shares antigenic and biochemical similarities with melittin, a well characterized bee venom phospholipase-stimulatory peptide. To explore the potential mechanisms of action of PLAP that extend beyond its effects on eicosanoid synthesis, we examined its effects on the release of human neutrophil lysosomal enzymes and superoxide, and on RBC hemolysis. These results were compared to the effects of melittin, which has been reported to induce enzyme release and hemolysis. We also examined the effects of PLAP on neutrophil aggregation and chemotaxis. PLAP induced neutrophils to release beta-glucuronidase and metalloproteinase enzyme activities as well as produce superoxide ion in both a dose- and time-dependent manner. Eicosanoid synthesis inhibitors did not abrogate these responses. PLAP induced release of arachidonic acid metabolites, but this response could be abrogated by eicosanoid synthesis inhibitors. PLAP also induced neutrophil aggregation and chemokinesis, but not chemotaxis. Concentrations of PLAP that induced these responses did not induce cellular toxicity as determined by light and electron microscopy, lactic dehydrogenase release, trypan blue dye exclusion, and RBC hemolysis. In contrast, prolonged incubation with higher concentrations of PLAP induced cell death that was similar to that observed with melittin. These findings suggest that the mechanisms of action of PLAP extend beyond the eicosanoid synthetic pathway, and that disordered regulation of PLAP may be responsible, at least in part, for chronic immune and inflammatory states.     

FMLP诱导的嗜中性白细胞呼吸爆发与凋亡的关系研究

The relationship between apoptosis of neutrophils and the change of their intracellular free Ca2+ concentration [Ca2+]i was studied. FMLP and A23187 were used to elevate the [Ca2+]i while BAPTA was used to deplete it. Fluorescence microscope, flow cytometry and gel electrophoresis were used to study the percentage of cell apoptosis and the change of f-actin during apoptosis. The results showed that the apoptosis was obviously inhibited by fMLP and A23187, while accelerated by BAPTA. The detection of f-actin showed that the f-actin depolymerized obviously during apoptosis. The elevation of [Ca2+]i inhibit the actin depolymerization while depletion of [Ca2+]i accelerated it. This result indicated that the apoptosis of neutrophil was obviously inhibited by [Ca2+]i elevation but accelerated by [Ca2+]i depletion.     

The human neutrophil lipocalin supports the allosteric activation of matrix metalloproteinases.

The human neutrophil lipocalin (HNL), a member of the large family of lipocalins that exhibit various physiological functions, is coexpressed in granulocytes with progelatinase B (MMP-9). Part of it is covalently bound to the proenzyme and therefore may play a possible role in the activation process of promatrix metalloproteinases. We now report that HNL is able to accelerate the direct activation of promatrix metalloproteinases slightly. A significant enhancement of the activity could be demonstrated for the HgCl2- and the plasma kallikrein-induced activation of all three secretory forms of proMMP-9 and of proMMP-8. The same activating effects were exerted by HNL isolated from granulocytes as well as by the recombinant forms expressed by the yeast Pichia pastoris or by Escherichia coli. This demonstrates that the carbohydrate moiety is not essential for the biological activity of HNL. Activation and activity enhancement are obviously mediated by entrapping the remaining N-terminal sequence residues of the partially truncated proenzyme into the hydrophobic binding pocket of the HNL. In conclusion these results document that HNL can exert an enzyme-activating effect in the regulation of inflammatory and pathophysiological responses of granulocytes in the physiological activation of MMPs that have been subject to limited proteolytic processing.     

Differential activation by fMet-Leu-Phe and phorbol ester of a plasma membrane phosphatidylcholine-specific phospholipase D in human neutrophil

Signal transduction involving phosphatidylcholine hydrolysis has been investigated in human neutrophils (PMN) after in situ generation of [3H]alkylacyl-sn-glycero-3-phosphocholine ([3H]alkylacyl-GPC) by cell incubation with [3H]alkylacetyl-GPC. When PMN were stimulated with the chemotactic peptide N-formyl-Met-Leu-Phe(fMLP) or phorbol myristate acetate (PMA) in the presence of cytochalasin B, both 1-O-alkyl-2-acyl-sn-glycero-3-phosphate (PA) and 1-O-alkyl-2-acyl-sn-glycerol (AAG) were generated. On addition of the agonists in the presence of ethanol, phosphatidylethanol (PEt) [corrected] was formed with a concomitant decrease in PA and AAG. These results indicate the presence of a phospholipase D (PLD) acting on phosphatidylcholine in human PMN. The kinetics of hydrolysis were quite different according to the stimulus. Whereas fMLP induced a maximum rise in PA and AAG at 30-45 s, these products began to appear only after 1 min upon cell incubation with PMA. Similar amounts of products were formed at 1 min with fMLP and only at 5 min with PMA. Although similar time courses of PA generation were obtained in the absence of cytochalasin B, AAG were no longer involved and therefore cannot account for intracellular second messenger under physiological conditions. Subcellular distribution studies demonstrated the exclusive location of PA and PEt [corrected] in the plasma membrane. The possible involvement of PA in respiratory burst activation is discussed.