Introduction

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The xanthogenate compound tricyclodecan-9-yl-xanthate (D609; Fig. 1) has antiviral and antitumor properties (Amtmann et al., 1985; Waldeck, 1990; Villanueva et al., 1991; Walro and Rosenthal, 1997), and also inhibits events related to the activation of leukocytes during inflammation (Schutze et al., 1991; Bauldry et al., 1996; Spitsin et al., 1997; Carter et al., 1998; Tschaikowsky et al., 1998; Monick et al., 1999; Wooten et al., 1999; Zhang et al., 2001). D609 is a well characterized competitive inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC) with a K_i of 6.4 µM, but shows no inhibitory activity toward phospholipase A₂ and D (Amtmann, 1996). Thus, D609 is frequently used in signal transduction research as a specific inhibitor of PC-PLC. Phospholipase D and sphingomyelinase, which are downstream effectors of PC-PLC, are indirectly inhibited by D609.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structures of D609 and ethyl xanthate.

The respiratory burst in alveolar macrophages can be primed in vitro by pre-exposure to nontoxic concentrations of hydroperoxides (H₂O₂ or tert-butylhydroperoxide) (Murphy et al., 1995; Hoyal et al., 1998; Giron-Calle and Forman, 2000). In primary isolates the respiratory burst is enhanced by up to 40% when a population of suspended cells is exposed to about 25 µM H₂O₂ for 15 min before stimulation (Murphy et al., 1995). In the NR8383 alveolar macrophage cell line the enhancement is maximal, 100% above the basal stimulated level, when cells are exposed to 50 to 100 µM H₂O₂ before stimulation with the agonist ADP (Giron-Calle and Forman, 2000). Using primary isolates of alveolar macrophages, Hoyal et al. (1996a) discovered that this effect depends on a transient increase of the cytosolic concentration of Ca²⁺ ([Ca²⁺]_c), and correlates with increased translocation of p47^phox to the plasma membrane (Zhou et al., 1997). Higher, but still nontoxic, concentrations of H₂O₂ cause inhibition of the respiratory burst, which correlates with a more sustained increase in the [Ca²⁺]_c and decreased translocation of p47^phox to the plasma membrane. Phosphorylation of p47^phox, which increased upon stimulation of the respiratory burst, was unaffected by preincubation with H₂O₂ (Zhou et al., 1997). The possible involvement in this phenomenon of Ca²⁺-dependent phospholipases that can be activated by H₂O₂ has been investigated. Activation of the respiratory burst by receptor-mediated agonists such as ADP involves the activation of phosphatidylinositol-specific phospholipase C (PI-PLC) to produce inositol-1,4,5-trisphosphate. Nonetheless, priming of the respiratory burst is not mediated by alteration of the production of this second messenger (Robison et al., 1995). Using NR8383 macrophages, Giron-Calle and Forman (2000) later showed that this enhancement is also not mediated by phospholipase D.

Hydrolysis of phosphatidylcholine to yield diacylglycerol and phosphocholine upon activation by an agonist depends on increases in the [Ca²⁺]_c in different cell types (Exton, 1994). PC-PLC can be activated by H₂O₂ in a Ca²⁺-dependent and -independent manner (Rice et al., 1992). Consequently, PC-PLC is another potential mediator of the effect of hydroperoxides on the respiratory burst. The PC-PLC inhibitor D609 was used to test this hypothesis, but it became apparent that D609 was a reducing agent that interfered with the commonly used ferricytochrome c reduction assay for the respiratory burst. It was suspected that D609 might also have other effects on macrophage function through its reductive capacity, which would be consistent with a very recent report by Zhou et al. (2001), showing that D609 inhibits cellular damage due to ionizing radiation by acting as an antioxidant. A variety of antioxidants and analogs of D609 have therefore been used in the present work to investigate whether the effect of D609 on the enhancement of the respiratory burst by H₂O₂ is due to its reducing or antioxidant properties. The effect of the generic phospholipase C inhibitor 1-(6-((17-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) has also been investigated. A view emerges from these studies in which PC-PLC has a function very different from that of the better characterized PI-PLC in the stimulation of the respiratory burst; whereas PI-PLC is essential for receptor-mediated stimulation of the respiratory burst, PC-PLC is involved only in the priming by H₂O₂.

	Experimental Procedures

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Reagents and Materials. Chemicals and enzymes were purchased from the following suppliers: H₂O₂, nitro blue tetrazolium (NBT), superoxide dismutase, fatty acid-free bovine serum albumin, superoxide dismutase, cytochrome c, and ADP (Sigma-Aldrich, St. Louis, MO); catalase (Worthington Biochemicals, Lakewood, NJ); and D609 and U73122 (BIOMOL Research Laboratories, Plymouth Meeting, PA). D609 was kept as lyophilized aliquots and reconstituted in phosphate-buffered saline (PBS). U73122 was dissolved in chloroform, aliquoted, kept dried under nitrogen gas, and reconstituted in PBS containing 1% bovine serum albumin (BSA). Cell culture medium was purchased from Invitrogen (Carlsbad, CA).

Cell Culture and Preparation for Incubations. NR8383 rat alveolar macrophages were obtained by Helmke et al. (1989), who kindly provided the cell line to us. They were cultured in F-12K nutrient mixture containing 15% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells in suspension were collected and resuspended in fresh medium twice a week when concentration was about 2 × 10⁶ cells/ml (Helmke et al., 1989).

Spectrophotometric Analysis of Reaction of D609 with Cytochrome c or H₂O₂. The time course of the reaction of D609 with cytochrome c or H₂O₂ was followed by UV/VIS spectrophotometry. Stock solutions of the reagents were prepared in phosphate-buffered saline (D609, cytochrome c, dithiothreitol) or water (H₂O₂). Reactions were carried out in KRP pH 7.4 in a cuvette kept at 37°C, by using a Spectromax Plus spectrophotometer (Molecular Dynamics, Sunnyvale, CA).

Respiratory Burst Determination. Analysis of superoxide production by determination of cytochrome c reduction was carried out essentially as described (Babior et al., 1973) by using a 20 µM concentration of cytochrome c, and cells were suspended in KRP at a concentration of 1 × 10⁶ cells/ml. Reduced cytochrome c was measured spectrophotometrically as the difference between the absorbance at 550 nm (reduced cytochrome c) and 540 nm (background given by the isosbestic point between the absorbance peaks of oxidized and reduced cytochrome c). Superoxide production was given by the difference between samples incubated in the absence or presence of superoxide dismutase (120 U/ml).

	Results

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D609 Reacts with Cytochrome c and H₂O₂. Superoxide dismutase-inhibitable reduction of cytochrome c is the basis for the most widely used assay for determination of extracellular superoxide production by phagocytes (Babior et al., 1973). Nevertheless, this assay has been found to be inappropriate when incubations include the PC-PLC inhibitor D609, because D609 reacts with cytochrome c. As shown in Fig. 2A, addition of D609 at a concentration normally used for the purpose of PC-PLC inhibition caused increased absorption at 550 nm, characteristic of reduced cytochrome c. Concomitantly, the absorbance at 302 nm, maximum of absorbance for D609, decreased (Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Reduction of cytochrome c by D609. D609 (100 µM) was added to 20 µM cytochrome c in KRP buffer, pH 7.4, 37°C. A, spectra were taken before () and 15 () and 30 () min after addition of D609. B, time course of the same reaction followed at 302 nm.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Reaction of H2O2 with D609. H2O2 (2 mM) was added to 50 µM D609 in KRP buffer pH 7.4, 37°C. A, spectra were taken before and 1, 5, and 15 min after addition of D609. B, time course of the reaction of a lower H2O2 concentration (100 µM) with 50 µM D609, followed at 302 and 348 nm; catalase (CAT, 23 units/ml) and dithiothreitol (DTT, 1 mM) were added 10 and 20 min after addition of H2O2, respectively.

Effect of D609 on Modulation of Respiratory Burst by H₂O₂. NBT is another chemical that can be used to measure superoxide production by phagocytes (Elferink, 1984). NBT is a yellowish, water-soluble tetrazolium salt, which upon reduction by superoxide, forms a purple insoluble formazan. Incubation of NBT with D609 yielded no formazan product (data not shown). Thus, although cytochrome c is readily reduced by D609, NBT is not. Consequently, reduction of NBT has been chosen to study the effect of PC-PLC inhibition by D609 on the enhancement of the respiratory burst by H₂O₂.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effect of D609 on the priming of the respiratory burst by H2O2. Cells were allowed to attach to BSA-coated plates for 15 min. Cells were then exposed to a gradient of H2O2 formed by diffusion of peroxide added in the center of the wells (400 nmol, 10 µl of 40 mM). D609 (50 or 100 µM) was added 5 min before or 10 min after addition of H2O2. The respiratory burst was stimulated 15 min after addition of H2O2 by replacing the buffer for prewarmed KRP containing 400 µM ADP and 25 mM NBT. Superoxide production was allowed to proceed for 10 min before fixation for image recording.

D609 Inhibition of Priming of Respiratory Burst by H₂O₂ Is Not Due to Its Antioxidant Properties. As shown above, D609 acts as a reductant in its reaction with cytochrome c, and is also able to react with H₂O₂. This points to the possibility that the mechanism of action of D609 may not only involve inhibition of PC-PLC as commonly argued but also the reducing power of the dithiocarbonate group. Therefore, the potential effects on priming by H₂O₂ of several related and unrelated compounds with reductive capacity were examined for comparison. The effect of the D609 analog ethyl xanthate (Fig. 1) and the sulfhydryl agents glutathione, glutathione ester, N-acetyl cysteine, and dithiothreitol, and the lipid antioxidant butylhydroxytoluene were studied. The effect of pyrrolidinedithiocarbamate was studied as well. Pyrrolidinedithiocarbamate contains a moiety that resembles the chemical structure of the dithiocarbonate group, and is frequently used as an antioxidant, although its effect may be more related to metal chelating activity. As reported by Zhou et al. (2001), pyrrolidinedithiocarbamate reproduced the protective effect of D609 against ionizing radiation, supporting an antioxidant effect of the latter. If the antioxidant properties of D609 caused the inhibition of the enhancement of the respiratory burst by H₂O₂, at least some other antioxidants should mimic this effect; however, none of them were able to mimic the effect of D609 in a wide range of concentrations tested. Despite being able to react with H₂O₂ as well as D609, ethyl xanthate also failed to reproduce the effect of D609 (Table 1). Some of the treatments are also shown in Fig. 5.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of analog ethyl xanthate and antioxidants on the enhancement of the respiratory burst by H2O2. Cells were allowed to attach to BSA-coated plates for 15 min. Cells were then exposed to a gradient of H2O2 formed by diffusion of peroxide added in the center of the wells (400 nmol, 10 µl of 40 mM). D609 (100 µM), 100 µM ethyl xanthate, 100 µM glutathione ethyl ester, or 100 µM pyrrolidinedithiocarbamate was added 5 min earlier. The respiratory burst was stimulated 15 min after addition of H2O2 by replacing the buffer for prewarmed KRP containing 400 µM ADP and 25 mM NBT. Superoxide production was allowed to proceed for 10 min before fixation.

Inhibition of PI-PLC, but not PC-PLC, Blocks Activation of Respiratory Burst. The effect of D609 is suggestive of an involvement of PC-PLC in the priming of the respiratory burst by H₂O₂, but not in the signal transduction pathway leading to stimulation of the respiratory burst by ADP directly. Another isoform of PLC, PI-PLC, is involved in the stimulation of the respiratory burst in phagocytes. Production of inositol-1,4,5-trisphosphate by PI-PLC leads to phosphorylation of certain cytosolic components of the NADPH oxidase, most prominently p47^phox, which allows for assembly of the functional NADPH oxidase enzymatic complex (Dusi et al., 1993; Lopes et al., 1999). The effect of the generic PLC inhibitor U73122 corroborated the involvement of PLC in the respiratory burst (Fig. 6). In contrast to D609, U73122 inhibited the respiratory burst not only when added before H₂O₂ but also when added after preincubation with H₂O₂ but before addition of ADP, which is consistent with its inhibition of PI-PLC.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of U73122 on the modulation of the respiratory burst by H2O2. Cells were allowed to attach to BSA-coated plates for 15 min. Cells were then exposed to a gradient of H2O2 formed by diffusion of peroxide added in the center of the wells (400 nmol, 10 µl of 40 mM). U73122 (10 or 20 µM) was added 5 min before or 10 min after addition of H2O2. The respiratory burst was stimulated 15 min later by replacing the buffer for prewarmed KRP containing 400 µM ADP and 25 mM NBT. Superoxide production was allowed to proceed for 10 min before fixation.

Priming of Respiratory Burst by Pre-exposure to H₂O₂ Is Mimicked by Pre-exposure to Substimulatory Concentrations of Phorbol-12-myristate-13-acetate. PMA is a tumor promoter frequently used in signal transduction research as an activator of protein kinase C. PMA acts as an analog of the second messenger diacylglycerol, although activation of protein kinase C by PMA lasts longer (Castagna et al., 1981). If the priming effect of H₂O₂ depends on activation of PC-PLC, leading to release of diacylglycerol, PMA could mimic the effect of H₂O₂. We examined whether pre-exposure to PMA before activation of the respiratory burst has an effect similar to exposure to H₂O₂. PMA is able to stimulate a respiratory burst by itself (Giron-Calle and Forman, 2000), which can be enhanced by pre-exposure to H₂O₂ in a D609-inihibitable manner (Fig. 7A). Thus, to test the hypothesis that pre-exposure to PMA mimics the effect of pre-exposure to H₂O₂, PMA concentrations below the threshold for activation of the respiratory burst were used. Figure 7B shows the result of adding substimulatory concentrations of PMA to the cells instead of H₂O₂, 15 min before stimulation by ADP. A concentration of PMA that was not sufficient to activate a respiratory burst by itself was able to prime the respiratory burst subsequently activated by ADP.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Effect of pre-exposure to PMA on the ADP-stimulated respiratory burst. A, cells were preexposed to D609 and H2O2 before activation of the respiratory burst as described in Fig. 4, except that in this case the agonist added 15 min after addition of H2O2 was 100 ng/ml PMA instead of ADP. B, cells were allowed to attach to BSA-coated plates for 15 min before addition of PMA (1.6 or 6.5 pmol, 10 µl of 0.16 or 0.65 µM in PBS) in the center of the wells. Determination of superoxide production was started 15 min later by replacing the buffer for prewarmed KRP containing 25 mM NBT alone or 25 mM NBT plus 400 µM ADP. Cells were fixed 10 min later.

	Discussion

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Reactive oxygen species were considered for a long time only as toxic by-products of xenobiotics or cell metabolism with deleterious effects on cells; however, considerable evidence has accumulated for their participation in the regulation of cell growth and function (Suzuki et al., 1997). The modulation of the respiratory burst by pre-exposure to H₂O₂ in alveolar macrophages constitutes an example of a signaling role (Hoyal et al., 1998). Generation of secondary messengers by phospholipases plays a major role in the signal transduction pathways leading to activation of the respiratory burst (Bauldry et al., 1988; Burnham et al., 1989; Smith et al., 1990; Cockcroft, 1992). Stimulation of macrophages by adenine nucleotides leads to assembly of the respiratory burst oxidase in a Ca²⁺-dependent manner (Murphy et al., 1993; Hoyal et al., 1996a). Because the modulation of the respiratory burst in alveolar macrophages also depends on increases in the [Ca²⁺]_c, Ca²⁺-dependent phospholipases have been suggested as possible mediators of the modulatory effect of H₂O₂.

As a well documented inhibitor of PC-PLC, D609 was chosen to ascertain whether this enzyme might be involved in the response to H₂O₂. We found however that D609 is able to reduce cytochrome c and react with H₂O₂, although the reaction with H₂O₂ proceeds slowly at physiologically relevant concentrations. Oxidation of the dithiocarbonate group present in the D609 molecule is responsible for these chemical properties, which indicate that D609 may be considered as a reducing agent with potential antioxidant properties. Reaction with H₂O₂ affords a single product that can be reduced by dithiothreitol, probably reflecting the formation of an oxidized D609 dimer. During the course of our studies, Zhou et al. (2001) demonstrated that D609 inhibits lipid peroxidation, glutathione consumption, and protein oxidation in lymphocytes exposed in vitro to ionizing radiation. They also showed that the antioxidant effect of pyrrolidinedithiocarbamate on the oxidation of dihydrorhodamine by a Fenton reaction system (FeSO₄/H₂O₂) was mimicked by D609. Our observations are consistent with theirs, and point to the need for considering the potential antioxidant effects of D609. With this in mind, the D609 analog ethyl xanthate can be used as a control because it possesses a reactive dithiocarbonate group, but does not inhibit PC-PLC.

Ethyl xanthate and a variety of cell-permeable as well as -impermeable antioxidants did not reproduce the inhibitory effect of D609 on the priming of the respiratory burst by H₂O₂. Although D609 or ethyl xanthate react with H₂O₂, at the H₂O₂ concentrations used in this study, the reaction rate is too slow to be of any consequence. The total lack of effect of ethyl xanthate on priming is consistent with the conclusion that, in this case, the antioxidant properties of D609 are not a factor.

D609 inhibited priming of the respiratory burst by H₂O₂ if present during incubation with H₂O₂, but not if added just before stimulation, suggesting that D609 affected priming but not the respiratory burst per se. In contrast, the generic PLC inhibitor U73122 inhibited the stimulation of the respiratory burst when added before or after preincubation with H₂O₂. This is consistent with the established involvement of PI-PLC in signaling for the receptor-mediated stimulation of the respiratory burst (Burnham et al., 1989; Edwards, 1994).

It is interesting to note that although D609 is generally considered a specific inhibitor of PC-PLC, some reports claim that D609 can inhibit phospholipase D. This inhibition was observed at concentrations similar to those that inhibit PC-PLC (Kiss and Tomono, 1995) or higher (Gratas and Powis, 1993). Nevertheless, it was previously shown that activation of phospholipase D by H₂O₂ is not responsible for the enhancement of the respiratory burst by H₂O₂ in NR8383 macrophages (Giron-Calle and Forman, 2000), so that the hypothetical inhibition of phospholipase D cannot explain the effects of D609 described in this article.

An elegant regulatory mechanism involving two isoforms of PLC emerges from our results, in which activation of PC-PLC has a priming effect, whereas PI-PLC participates in the signaling for basal stimulation of the respiratory burst. There are two main differences in the signaling triggered by hydrolysis of phosphatidylcholine versus hydrolysis of phosphatidylinositol. These are production of diacylglycerol without causing changes in the [Ca²⁺]_c, and a longer duration of diacylglycerol release with prolonged activation of protein kinase C. Some data also indicate that diacylglycerol derived from phosphatidylcholine may activate different protein kinase C isoforms than those activated by phosphatidylinositol-derived diacylglycerol (Exton, 1994). The modulation of the respiratory burst by peroxides is mediated by an increase in [Ca²⁺]_c (Hoyal et al., 1996a), but the source of this Ca²⁺ and the characteristics of the increase are different from agonist (ADP)-triggered Ca²⁺ release, which depends on release from the endoplasmic reticulum due to phosphatidylinositol hydrolysis (Robison et al., 1995; Hoyal et al., 1996b). Interestingly, pretreatment with substimulating doses of PMA, a phorbol ester that mimics prolonged release of diacylglycerol without inducing changes in the [Ca²⁺]_c, was effective in enhancing the respiratory burst with a time frame similar to H₂O₂. Thus, the Ca²⁺ release that is necessary for the modulation by H₂O₂ most likely occurs upstream of PC-PLC activation. A likely downstream target of the PC-PLC-mediated diacylglycerol release is activation of protein kinase C. A relatively prolonged activation of protein kinase C may conceivably put the cell into a primed state that would allow an enhanced burst upon stimulation by a receptor-mediated agonist. Another possible downstream target of diacylglycerol production is phospholipase A₂, which has been linked with priming by diacylglycerol in neutrophils (Bauldry et al., 1988).

Using D609 as a specific inhibitor of PC-PLC, recent reports claim the involvement of this enzyme in various events related with the activation of leukocytes. Thus, D609 has been shown to inhibit mitogen-activated protein kinases activation by lipopolysaccharide (Monick et al., 1999), protein kinase C activation by tumor necrosis factor (Schutze et al., 1991), nitric-oxide synthase activation (Spitsin et al., 1997; Zhang et al., 2001), outside-in signaling for spreading of lymphocytes (Wooten et al., 1999), nuclear factor-B activation (Yamamoto et al., 1997; Carter et al., 1998; Zhou et al., 2001), and cytokine release (Carter et al., 1998). Using radiolabeled phospholipids, Grove et al. (1990) determined that a phosphatidylcholine-specific phospholipase is involved in the activation of macrophages by lipopolysaccharide. D609 also inhibited endotoxin shock in whole animals, and has been proposed as an anti-inflammatory agent (Tschaikowsky et al., 1998). In this context, D609 inhibition of priming by H₂O₂ of the respiratory burst adds another dimension to its anti-inflammatory action.

Although priming of the respiratory burst by H₂O₂ has only been demonstrated in vitro, it may constitute a mechanism of regulation of the respiratory burst in the lung. Potentially, generation of hydrogen peroxide derived from the superoxide generated by macrophages and neutrophils during their respiratory burst could prime neighboring macrophages for greater subsequent superoxide production. Considering the concentrations of H₂O₂ that are effective for priming the respiratory burst (25-100 µM, or 25-100 nmol/10⁶ cells, for maximal enhancement of the respiratory burst), this phenomenon would probably be of relevance in sites of inflammation in which vigorous production of superoxide by phagocytes is taking place. At the same time, it would be advantageous if this priming were self-limiting to avoid excessive superoxide production. Previously published studies show that concentrations of H₂O₂ higher than those causing priming can inhibit the respiratory burst without being toxic to alveolar macrophages (Murphy et al., 1995).