Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.     

Zymosan but not phorbol myristate acetate induces an oxidative burst in rat bone marrow-derived macrophages

We found that rat bone marrow-derived macrophages responded to opsonized zymosan by releasing superoxide anion. However, these cells were defective in the response to the potent oxidative burst activator phorbol myristate acetate (PMA). This result was observed whatever the concentration of agonist used and with different concentrations of cells. Since it is strongly suspected that protein kinase C (PKC) is involved in the transductional pathway induced by PMA in numerous cell types, and particularly in phagocytes, we studied PKC and we observed that it was functional in rat bone marrow-derived macrophages, but only present at a low level. Thus, we suggest that our results are consistent with the possibility that zymosan-induced respiratory burst may be independent of PKC and that these cells may not possess the minimal level of PKC required for responding to PMA.     

Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.     

Tumor necrosis factor alpha stimulates mycobactericidal/mycobacteriostatic activity in human macrophages by a protein kinase C-independent pathway.

Tumor necrosis factor (TNF) is a 17-kDa protein produced by endotoxin-stimulated macrophages. We have demonstrated that recombinant human TNF activates human macrophages to kill intracellular bacteria of the Mycobacterium avium complex (MAC) in a dose-related manner. TNF also primed macrophages to produce superoxide anion (O2-) following treatment with phorbol esther PMA (0.1 micrograms/ml). To investigate the intracellular pathway involved in the TNF-mediated activation of mycobacteriostatic/mycobactericidal activity in macrophages, we used two different protein kinase C (PKC) inhibitors: H7 (10(-5)-10(7) M) and staurosporine (10(-7)-10(-9) M). Mellitin (1 and 100 mM) was used as a calmodulin inhibitor. Human peripheral blood-derived macrophages cultured for 7 days were treated with H7, mellitin, or staurosporine for 1 hr prior to incubation with TNF (10(3) U/ml). Twenty-four hours after treatment with TNF the O2- release was measured spectrophotometrically following exposure to PMA. Macrophages were infected with MAC and the viable intracellular bacilli were quantitated following 4 days of treatment with TNF. All PKC inhibitors suppressed O2- production after incubation with PMA. However, treatment with either PKC or calmodulin inhibitors did not influence the intracellular killing of M. avium by TNF-stimulated macrophages. Exposure of the macrophages to cGMP inhibitor but not to cAMP inhibitor significantly impaired the response to the stimulation with TNF. In contrast, incubation of macrophages with protein kinase A (PKA) had no effect on TNF-mediated mycobacteriostatic/mycobactericidal activity. These results suggest that the TNF-mediated mycobactericidal activity in cultured macrophages probably occurs by a PKC-independent mechanism.     

Priming of the respiratory burst of bone marrow-derived macrophages is associated with an increase in protein kinase C content.

The biochemical mechanism(s) underlying the priming of the macrophage for an enhanced PMA-induced respiratory burst is not understood. Because the cellular receptor for PMA is thought to be protein kinase C (PKC), we have investigated the effects of priming agents on cellular PKC levels. Sonicates from unprimed bone marrow-derived macrophages (BMM) were found to contain PKC activity (309 +/- 51 pmol 32P-incorporated/mg/min; mean +/- SE, n = 17) as measured by the phospholipid-, diacylglycerol-, and calcium-dependent phosphorylation of histone. Exposure of BMM to priming agents such as TNF-alpha, LPS, and granulocyte/macrophage-CSF resulted in a significant increase in both histone-phosphorylating activity and levels of immunoreactive PKC protein in these cells. A minimum of 6-h exposure, with an increasing effect up to 48 h, was required for a detectable increase in PKC level. The activity from primed BMM, like that of the untreated cells, was predominantly cytosolic. The kinetics and concentration dependence of the priming agent-induced increase in the PKC content of BMM closely paralleled the enhancing effects of these agents on the PMA-stimulated respiratory burst. Furthermore, CSF-1, a cytokine that does not prime BMM, failed to increase PKC activity. We propose that the exposure of BMM to priming agents leads to an increase in the expression of a stimulatory isozyme(s) of PKC, resulting in an enhanced ability to mount a respiratory burst in response to stimulation with PMA.     

Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages.

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.     

Colony stimulating factor-1 stimulates diacylglycerol generation in murine bone marrow-derived macrophages, but not in resident peritoneal macrophages

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in murine bone marrow-derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol-4,5-bisphosphate hydrolysis is not associated with CSF-1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF-1 transiently elevated both [3H]myristoyl- and [3H]arachidonyl-diacylglycerol (DAG) in BMM in a dose-dependent fashion. CSF-1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF-1 in macrophages. Other mitogenic agents, 12-0-tetradecanoyl phorbol 13-acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl- and [3H]arachidonyl-DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF-alpha increased only [3H]arachidonyl-DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.     

Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.     

Protein tyrosine kinase but not protein kinase C inhibition blocks receptor induced alveolar macrophage activation

The selective enzyme inhibitors genistein and Ro 31-8220 were used to assess the importance of protein tyrosine kinase (PTK) and protein kinase C (PKC), respectively, in N-formyl-methionyl-leucyl-phenylalanine (FMLP) induced generation of superoxide anion and thromboxane B(2) (TXB(2)) in guinea-pig alveolar macrophages (AM). Genistein (3-100 muM) dose dependently inhibited FMLP (3 nM) induced superoxide generation in non-primed AM and TXB(2) release in non-primed or in lipopolysaccharide (LPS) (10 ng/ml) primed AM to a level > 80% but had litle effect up to 100 muM on phorbol myristate acetate (PMA) (10 nM) induced superoxide release. Ro 31-8220 inhibited PMA induced superoxide generation (IC(50) 0.21 +/- 0.10 muM) but had no effect on or potentiated (at 3 and 10 muM) FMLP responses in non-primed AM. In contrast, when present during LPS priming as well as during FMLP challenge Ro 31-8220 (10 muM) inhibited primed TXB(2) release by > 80%. The results indicate that PTK activation is required for the generation of these inflammatory mediators by FMLP in AM. PKC activation appears to be required for LPS priming but not for transducing the FMLP signal; rather, PKC activation may modulate the signal by a negative feedback mechanism.     

Phorbol myristate acetate (PMA) augments chemoattractant-induced diglyceride generation in human neutrophils but inhibits phosphoinositide hydrolysis. Implications for the mechanism of PMA priming of the respiratory burst

Pretreatment ("priming") of neutrophils with a non-activating concentration (2 nM) of phorbol myristate acetate (PMA) augments superoxide (O2-) production in response to the chemoattractant formylmethionylleucylphenylalanine (fMLP). We initially examined the effect of sphinganine, an inhibitor of protein kinase C (Ca2+/phospholipid-dependent enzyme), on activation of primed neutrophils. In both primed and unprimed cells activation by fMLP was blocked, and inhibition occurred at identical concentrations, supporting a common inhibited site. PMA also augmented (about 2-fold) fMLP-induced generation of sn-1,2-diglyceride (DG), the level of which correlated with O2- generation. In contrast to its effects on DG, PMA diminished by about 50% the magnitude of the fMLP-stimulated rise in cytosolic Ca2+. Thus, PMA priming dissociates the fMLP-stimulated Ca2+ increase from DG and O2- generation. The effect of PMA on Ca2+ levels appeared to be due in part to lowered levels of inositol trisphosphate. Lowering of inositol phosphate levels correlated with inhibition of fMLP-induced hydrolysis of inositol-containing phospholipids, particularly phosphatidylinositol 4,5-bisphosphate. PMA did not inhibit (and in fact augmented at early time points) formation of [32P] phosphatidic acid in response to fMLP, indicating that the increase in DG was not due to inhibition of cellular diglyceride kinase. Thus, the data suggest that PMA enhances fMLP-stimulated DG generation concomitant with switching the source of DG from phosphatidylinositol 4,5-bisphosphate to an alternative lipid(s). Increased DG and inhibition of activation by sphinganine are consistent with a role for protein kinase C in activation of the respiratory burst in PMA-primed neutrophils.     

Regulation of (1-3)-beta-glucan-stimulated Ca(2+) influx by protein kinase C in NR8383 alveolar macrophages

Stimulation of (1-3)-beta-glucan receptors results in Ca(2+) influx through receptor-operated channels in alveolar macrophages (AMs), but the mechanism(s) regulating Ca(2+) influx is still undefined. In this study we investigated the role of protein kinase C (PKC) regulation of Ca(2+) influx in the NR8383 AM cell line using the particulate (1-3)-beta-glucan receptor agonist zymosan. PKC inhibition with calphostin C (CC) or bisindolymaleimide I (BSM) significantly reduced zymosan-induced Ca(2+) influx, whereas activation of PKC with phorbol-12-myristate 13-acetate (PMA) or 1, 2-dioctanoyl-sn-glycerol (DOG) mimicked zymosan, inducing a concentration-dependent Ca(2+) influx. This influx was dependent on extracellular Ca(2+) and inhibited by the receptor-operated Ca(2+) channel blocker SK&F96365, indicating that zymosan and PKC activate Ca(2+) influx through a similar pathway. NR8383 AMs expressed one new PKC isoform (delta) and two atypical PKC isoforms (iota and lambda), but conventional PKC isoforms were not present. Stimulation with zymosan resulted in a translocation of PKC-delta from the cytosol to the membrane fraction. Furthermore, inhibition of protein tyrosine kinases (PTKs) with genistein prevented zymosan-stimulated Ca(2+) influx and PKC-delta translocation. These results suggest that PKC-delta plays a critical role in regulating (1-3)-beta-glucan receptor activated Ca(2+) influx in NR8383 AMs and PKC-delta translocation is possibly dependent on PTK activity.     

Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.     

Effect of tumor necrosis factor-alpha on the stimulus-coupled responses of neutrophils and their modulation by various inhibitors.

Preincubation of human peripheral blood polymorphonuclear leukocytes (HPPMN) with recombinant human tumor necrosis factor-alpha (rHuTNF-alpha) enhanced the formylmethionyl-leucylphenylalanine (FMLP)-induced superoxide (O2-.) generation in a concentration- and preincubation time-dependent manner. The enhancement was very high for the FMLP- or opsonized zymosan (OZ)-induced O2-. generation, but was low for arachidonic acid (AA)- and phorbol myristate acetate (PMA)-induced O.2- generation. The rHuTNF-alpha has no effect on the steady state of intracellular calcium ion concentration ([Ca2+]i) nor on the membrane potential of neutrophils. The rHuTNF-alpha-primed FMLP-induced O2-. generation was inhibited by nicotineamide (NA), pertussis toxin (PT), and by the tyrosine kinase (TK) inhibitor, genistein, but was enhanced by the protein kinase C (PKC) inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-3-methyl-piperazine). The inhibitory actions of NA and PT were also observed in in vivo primed guinea pig peritoneal neutrophils (GPtPMN). However, FMLP-induced O2-. generation of GPtPMN was enhanced by genistein, but was inhibited by H-7. These data indicate that TNF-alpha does not induce changes in [Ca2+]i nor in membrane potential of HPPMN, and that TNF-alpha-primed FMLP-induced O.2- generation of HPPMN is coupled with ADP-ribosylation and activation of G-proteins, and that protein kinases, especially TK, seem to exert an important role in the priming action of TNF.     

Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages.

Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium-free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester- and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.     

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.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.     

RO 31-8220 and RO 31-7549 show improved selectivity for protein kinase C over staurosporine in macrophages

Two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, and staurosporine were found to inhibit dose-dependently the phorbol ester-induced formation of prostaglandin E2 and superoxide in cultured liver macrophages. Prostaglandin E2 formation from exogenously added arachidonate was not affected by these compounds. The zymosan-induced formation of inositol phosphates was decreased by simultaneous addition of phorbol ester and was enhanced by prior desensitization of protein kinase C indicating that protein kinase C negatively modulates phospholipase C activation in these cells. While staurosporine suppressed almost totally the zymosan-induced formation of inositol RO 31-8220 and RO 31-7549 inhibited the protein kinase C-mediated effect on inositol phosphate formation, only. Phagocytosis of zymosan was not affected by RO 31-8220 and RO 31-7549 but was decreased by staurosporine. These results demonstrate that two new potent protein kinase C inhibitors, RO 31-8220 and RO 31-7549, are more selective in their actions as staurosporine and are useful tools to determine an involvement of protein kinase C in cellular systems.