Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

Activation of cytosolic phospholipase A2 in permeabilized human neutrophils

Neutrophils (PMN) contain two types of phospholipase A₂ (PLA₂), a 14 kDa ‘secretory’ Type II PLA₂ (sPLA₂) and an 85 kDa ‘cytosolic’ PLA₂ (cPLA₂), that differ in a number of key characteristics: (1) cPLA₂ prefers arachidonate (AA) as a substrate but hydrolyzes all phospholipids; sPLA₂ is not AA specific but prefers ethanolamine containing phosphoacylglycerols. (2) cPLA₂ is active at nM calcium (Ca²⁺) concentrations; sPLA₂ requires μM Ca²⁺ levels. (3) cPLA₂ activity is regulated by phosphorylation; sPLA₂ lacks phosphorylation sites. (4) cPLA₂ is insensitive to reduction; sPLA₂ is inactivated by agents that reduce disulfide bonds. We utilized PMN permeabilized with Staphylococcus aureus α-toxin to determine whether one or both forms of PLA₂ were activated in porated cells under conditions designed to differentiate between the two enzymes. PMN were labeled with [³H]AA to measure release from phosphatidylcholine and phosphatidylinositol; gas chromatography-mass spectrometry was utilized to determine total AA release (mainly from phosphatidylethanolamine) and to asses oleate and linoleate mass. A combination of 500 nM Ca²⁺, a guanine nucleotide, and stimulation with n-formyl-met-leu-phe (FMLP) were necessary to induce maximal AA release in permeabilized PMN measured by either method; AA was preferentially released. [³H]AA and AA mass release occurred in parallel over time. A hydrolyzable form of ATP was necessary for maximum AA release and staurosporin inhibited PLA₂ activation. Dithiothreitol treatment had little affect on [³H]AA release and metabolism but inhibited AA mass release. Assay of cell supernatants after cofactor addition did not detect sPLA₂ activity and the cytosolic buffer utilized did not support activity of recombinant sPLA₂. These results strongly suggested that cPLA₂ was the enzyme activated in the permeabilized cell model and this is the first report which unambiguously demonstrates AA release in response to activation of a specific type of PLA₂ in PMN.     

Lysophosphatidic acid increases phosphatidic acid formation, phospholipase D activity and degranulation by human neutrophils

I-oleoyl-sn-glycero-3-phosphate, a lysophosphatidic acid (LPA), in serum is a biologically active lipid and has multiple functions depending on the cell types. Several studies have shown that LPA stimulates phospholipase D (PLD) activity in fibroblasts and prostate cancer cells in culture. PLD plays a central role in regulating neutrophil functions. One of the functions of the lipid product, phosphatidic acid (PA), of PLD action in neutrophils is to promote degranulation. In the present study, we examined the effect of LPA on PLD activity and degranulation by human neutrophils. The results show that exogenous LPA increased PA formation, PLD activity and degranulation by human neutrophils in a time and concentration dependent manner. These findings suggest that LPA released from activated platelets during blood clotting may participate in bacterial killing and wound healing process. On the other hand, augmented LPA production might be involved in inflammation, causing damage of the host tissues.     

Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of the NADPH oxidase by FMLP in human neutrophils

It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.     

Inositol lipids and phosphatidic acid inhibit cell-free activation of neutrophil NADPH oxidase

The effect of inositol lipids on the SDS-initiated cell-free activation of NADPH oxidase in membranes of human neutrophils was investigated. In a system consisting of low density membranes, cytosol and SDS, low doses of phosphatidylinositol, phosphatidylinositol mono- and biphosphates and phosphatidic acid interfered with activation of the oxidase. The inhibition was relieved by increasing concentrations of the cytosol. Conversely, preincubation of multilamellar phosphoinositide vesicles with cytosol reduced its ability to support activation of the oxidase.     

Activation of NADPH oxidase in human neutrophils permeabilized with Staphylococcus aureus alpha-toxin. A lower Km when the enzyme is activated in situ.

The NADPH oxidase is a multicomponent enzyme system that produces the reduced oxygen species essential for bacterial killing by polymorphonuclear leukocytes (PMN). Study of the oxidase has typically been carried out in cell-free systems in which Km values of 20-150 microM NADPH have been reported. However, when compared with affinities reported for other flavoprotein dehydrogenases and when considering the cellular concentration of NADPH/NADP+ of approximately 35 microM, the reported affinity of the oxidase for NADPH appears low. To investigate this apparent discrepancy we have studied the kinetics of NADPH oxidase activation in situ in human PMN permeabilized with Staphylococcus aureus alpha-toxin. alpha-Toxin permeabilization of human PMN did not initiate NADPH oxidase activation at physiologic concentrations of NADPH. If permeabilized cells were stimulated with 1 microM formyl-methionyl-leucyl-phenylalanine, 10 microM guanosine 5'-O-(3-thiotriphosphate), 0.5 mM Ca2+, 5 micrograms/ml cytochalasin B in the presence of varying concentrations of NADPH, we were able to demonstrate activation of the oxidase complex as shown by superoxide dismutase-inhibitable reduction of cytochrome c. In this system we determined that the Km for oxidase activation was 4-7 microM NADPH, a 4-10-fold decrease from reported values. The oxidase was the enzyme being studied as shown by the absence of enzymatic activity in patients with chronic granulomatous disease. In addition, if the enzyme was initially activated in permeabilized cells, the cells homogenized, and the Km for the oxidase determined in a cell-free system, the observed Km reverted to previously reported values (36 microM). These results indicate that NADPH oxidase, studied in situ, has a significantly higher substrate affinity than that observed in isolated membranes and, moreover, indicate that substrate affinity is optimal for catalysis at reported concentrations of cytosolic NADPH.     

Activation of human neutrophil NADPH oxidase by phosphatidic acid or diacylglycerol in a cell-free system. Activity of diacylglycerol is dependent on its conversion to phosphatidic acid.

The superoxide-generating neutrophil NADPH oxidase can be activated in cell-free reconstitution systems by several agonists, most notably arachidonic acid and the detergent sodium dodecyl sulfate. In this study, we show that both phosphatidic acids and diacylglycerols can serve separately as potent, physiologic activators of NADPH oxidase in a cell-free system. Stimulation of superoxide generation by these lipids was dependent upon both Mg(2+) and agonist concentration. Activation of NADPH oxidase by phosphatidic acids did not appear to require their conversion to corresponding diacylglycerols by phosphatidate phosphohydrolase, since diacylglycerols were much slower than phosphatidic acids to activate the system and required the presence of ATP. Stimulation of the oxidase by dioctanoylglycerol proved to be by a means other than the activation of protein kinase C. Instead, dioctanoylglycerol was converted to dioctanoylphosphatidic acid by an endogenous diacylglycerol kinase present in the cell-free reaction system. This conversion was sensitive to the diacylglycerol kinase inhibitor R59949 and explains the markedly slower kinetics of activation and the novel ATP requirement seen with dioctanoylglycerol. The level of dioctanoylphosphatidic acid formed was suboptimal for NADPH oxidase activation but could synergize with the unmetabolized dioctanoylglycerol to activate superoxide generation.     

Mitogenic effects of phospholipase D and phosphatidic acid in transiently permeabilized astrocytes: effects of ethanol

Investigations of lipid-mediated signalling pathways are often limited by a lack of methods for the intracellular delivery of lipid messengers. We established a procedure for the transient permeabilization of astrocytes by an oxygen-insensitive mutant of streptolysin-O (SLO) to investigate the participation of the phospholipase D (PLD) signalling pathway in astroglial cell proliferation. Exogenous PLD, when incubated in the presence of SLO, caused an increase in DNA synthesis (measured by thymidine incorporation) which was completely suppressed by ethanol (0.3%, v/v). In parallel experiments, phosphatidic acid also induced a dose-dependent mitogenic response which, however, was not affected by the presence of ethanol. Phosphatidic acid was more effective in this assay than diacylglycerol but its effect was sensitive to the protein kinase inhibitor Ro 31-8220. Our findings provide direct evidence that disruption of the PLD signalling pathway by ethanol is sufficient to suppress astroglial proliferation, an effect that might contribute to the inhibition of brain growth in alcoholic embryopathy.     

Lectin-induced activation of plasma membrane NADPH oxidase in cholesterol-depleted human neutrophils

The gp91phox subunit of flavocytochrome b₅₅₈ is the catalytic core of the phagocyte plasma membrane NADPH oxidase. Its activation occurs within lipid rafts and requires translocation of four subunits to flavocytochrome b₅₅₈. gp91phox is the only glycosylated subunit of NADPH oxidase and no data exist about the structure or function of its glycans. Glycans, however, bind to lectins and this can stimulate NADPH oxidase activity. Given this information, we hypothesized that lectin–gp91phox interactions would facilitate the assembly of a functionally active NADPH oxidase in the absence of lipid rafts. To test this, we used lectins with different carbohydrate-binding specificity to examine the effects on H₂O₂ generation by human neutrophils treated with the lipid raft disrupting agent methyl-β-cyclodextrin (MβCD). MβCD treatment removed membrane cholesterol, caused changes in cell morphology, inhibited lectin-induced cell aggregation, and delayed lectin-induced assembly of the NADPH oxidase complex. More importantly, MβCD treatment either stimulated or inhibited H₂O₂ production in a lectin-dependent manner. Together, these results show selectivity in lectin binding to gp91phox, and provide evidence for the biochemical structures of the gp91phox glycans. Furthermore, the data also indicate that in the absence of lipid rafts, neutrophil NADPH oxidase activity can be altered by these select lectins.     

Activation of NADPH oxidase in human neutrophils. Synergism between fMLP and the neutrophil products PAF and LTB4

The induction of the respiratory burst in human neutrophils by combinations of fMLP and either PAF or LTB4 was studied. Pretreatment with PAF (0.0001 to 10 uM), which by itself did not elicit the burst, greatly enhanced the rate and extent of fMLP-induced superoxide production. A synergism of a different kind was observed with the reversed stimulus sequence: Pretreatment with fMLP made the neutrophils capable to respond to PAF with superoxide production. A moderate enhancement of the fMLP response was also obtained following pretreatment with LTB4. The response of the cells to LTB4, however, was not influenced by fMLP, and no synergism was observed between the two neutrophil products PAF and LTB4. The results of this study demonstrate a marked synergism between fMLP and PAF and suggest that PAF may function as an amplifier of the respiratory burst response of stimulated neutrophils.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Activation of NADPH oxidase of human neutrophils. Potentiation of chemotactic peptide by a diacylglycerol

Formyl-methionyl-leucyl-phenylalanine (fMLP) and 1-oleoyl-2-acetyl-glycerol (OAG) are synergistic stimuli of the respiratory burst of neutrophils. Simultaneous exposure to both agents greatly enhanced superoxide production, both in rate and extent. OAG potentiated the response to fMLP also in Ca++ -free medium. Pretreatment of the neutrophils with fMLP drastically shortened the lag of superoxide production in response to OAG. Our findings lead to the following conclusions: (i) Protein kinase C is likely to be involved in the activation of the NADPH oxidase by fMLP; (ii) OAG appears to be utilized as an intermediate in the activation process; (iii) prestimulation of the cells with fMLP facilitates the response to OAG.     

Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils

The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production.     

Phospholipid signalling through phospholipase D and phosphatidic acid

Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, phosphatidylcholine producing phosphatidic acid and free choline. This activity can participate in signal transduction pathways and impact on vesicle trafficking for secretion and endocytosis, as well as receptor signalling. Phospholipids can regulate PLD activity directly, through specific intermolecular interactions, or indirectly, through their effect on the localization or activity of PLD's protein effectors. This short review highlights these various phospholipid inputs into the regulation of PLD activity and also reviews potential roles for PLD-generated phosphatidic acid, particularly a mechanism by which the phospholipid may participate in the process of vesicular trafficking.     

Heat shock inhibits NADPH oxidase in human neutrophils

The heat shock response is a conserved, physiological, transient cellular response to injury. Several studies have suggested a link between the heat shock response and oxidative injury. We have investigated the effects of heat shock on superoxide anion generation by human neutrophils stimulated with opsonized zymosan or phorbol myristate acetate. Human neutrophils exposed to elevated temperatures or to the heavy metal cadmium synthetized a variety of heat shock proteins. In parallel to this protein synthesis, we observed a selective, reversible and temperature-dependent inhibition of NADPH oxidase activation, which was independent from variations of cytosolic pH or thiol group oxidation. Inhibition of NADPH oxidase by heat shock appeared related to the synthesis of heat shock proteins and may represent an intrinsic cellular mechanism to down regulate superoxide production.     

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.     

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid

Phosphatidic acid (PA), a molecule that is rapidly produced by the stimulated turnover of phospholipids in a variety of cells including blood neutrophils, elicited NADPH-dependent superoxide anion (O2-) production in detergent extracts from membranes of resting pig neutrophils. The stimulatory effect of PA was independent of cytosolic factors, differing from arachidonic acid and sodium dodecyl sulfate which, on the contrary, absolutely required the presence of cytosol to elicit the same result. The O2(-)-forming activity of the detergent extract activable by PA, as that by sodium dodecyl sulfate and arachidonic acid plus cytosol, was found in the chromatographic fractions containing cytochrome b558 and presented a chromatographic profile identical to that of the activated NADPH oxidase, which was obtained from neutrophils prestimulated with phorbol 12-myristate 13-acetate. The PA-induced NADPH-dependent O2(-)-forming activity showed kinetic properties and sensitivity to the inhibitors similar to the classical ones of the activated neutrophil NADPH oxidase. The data suggest that, in this cell-free system, PA may stimulate O2- formation by direct interaction with latent NADPH oxidase of neutrophils or with some of its regulatory components.     

Roles of phosphatidylinositol 3-kinase and phospholipase D in temporal activation of superoxide production in FMLP-stimulated human neutrophils

To determine the temporal roles of phosphatidylinositol 3-kinase (PI3-kinase) and phospholipase D (PLD) during human neutrophil activation stimulated by a chemotactic peptide, we examined the kinetics of these enzymes and related them to a neutrophil function (superoxide production). Both wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), potent and specific inhibitors of PI3-kinase, inhibit PI3-kinase activity in human neutrophils and significantly inhibit superoxide production from the early phase. Ethanol has no effect on PI3-kinase and markedly inhibits superoxide production at the late phase. Although these agents are inhibitory to different degrees, when neutrophils are simultaneously treated with ethanol and PI3-kinase inhibitors, superoxide is not produced. These results suggest that PI3-kinase and PLD play a pivotal role in the signal transduction pathway of the chemo-attractant-receptor involved neutrophil activation. These enzymes produce second messengers which are required for subsequent superoxide production in human neutrophils. NADPH oxidase is activated in a PI3-kinase-dependent manner at the early phase, and PLD activity follows it and is related to superoxide production at the late phase in human neutrophils by stimulation with FMLP.     

Activation of neutrophils NADPH oxidase by PMA: Cytosol activity is translocated in phorbol-primed neutrophils

• 1.1. Translocation of cytosol activity in phorbol-primed neutrophils was studied.
• 2.2. Prior exposure of PMA or FMLP could potentiate the oxidative response by subsequent heterogeneous stimulus, FMLP or PMA.
• 3.3. In FMLP-primed neutrophils, the cytosol had almost the same activity as resting one and cytosol activity was not eluted from the membrane.
• 4.4. In PMA-primed neutrophils, however, the cytosol had less activity and cytosol activity was correspondingly eluted from the membrane.
• 5.5. These observations suggested that cytosol activity was translocated in PMA-primed cells.