Selective Inhibition of Cytosolic Phospholipase A2 in Activated Human Monocytes
REGULATION OF SUPEROXIDE ANION PRODUCTION AND LOW DENSITY LIPOPROTEIN OXIDATION*

(Received for publication, March 5, 1996, and in revised form, November 5, 1996)

Qing Li and Martha K. Cathcart Dagger

From the Department of Cell Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Our previous studies have shown that monocyte activation and release of Obardot 2 are required for monocyte-mediated low density lipoprotein (LDL) lipid oxidation. We have also found that intracellular Ca2+ levels and protein kinase C activity are requisite participants in this potentially pathogenic process. In these studies, we further investigated the mechanisms involved in the oxidation of LDL lipids by activated human monocytes, particularly the potential contributions of the cytosolic phospholipase A2 (cPLA2) signaling pathway. The most well-studied cPLA2, has a molecular mass of 85 kDa and has been reported to be regulated by both Ca2+ and phosphorylation. We found that cPLA2 protein levels and cPLA2 enzymatic activity were induced upon activation of human monocytes by opsonized zymosan. Pharmacologic inhibition of cPLA2 activity by AACOCF3, which has been reported to be a specific inhibitor of cPLA2 as compared with sPLA2, caused a dose-dependent inhibition of cPLA2 enzymatic activity and LDL lipid oxidation by activated human monocytes, whereas sPLA2 activity was not affected. To corroborate these findings, we used specific antisense oligonucleotides to inhibit cPLA2. We observed that treatment with antisense oligonucleotides caused suppression of both cPLA2 protein expression and enzymatic activity as well as monocyte-mediated LDL lipid oxidation. Furthermore, antisense oligonucleotide treatment caused a substantial inhibition of Obardot 2 production by activated human monocytes. In parallel experimental groups, cPLA2 sense oligonucleotides did not affect cPLA2 protein expression, cPLA2 enzymatic activity, Obardot 2 production, or monoctye-mediated LDL lipid oxidation. These studies support the proposal that cPLA2 activity is required for activated monocytes to oxidize LDL lipids.


INTRODUCTION

Human native low density lipoprotein (LDL)1 can be oxidized by activated human monocytes, neutrophils, and cells of the monocytoid cell line U937 (1, 2) as well as endothelial cells and smooth muscle cells (3). Once oxidized, LDL is chemotactic for monocytes (4), serves as a cytotoxin for target cells (1, 5-7), and hinders the movement of macrophages (8). It is recognized by scavenger and oxidized LDL receptors on macrophages and is taken up by these receptors in an unregulated fashion (9-12). Oxidized LDL has been detected in atherosclerotic lesions (13, 14). Macrophages trapped in the artery wall may take up oxidized LDL, thus contributing to the formation of foam cells and fatty streak lesions. Cell-mediated oxidation of LDL has therefore been suggested to be a key event in atherogenesis as well as in inflammatory tissue injury (15).

In our culture system, human monocyte oxidation of LDL is dependent on monocyte activation. Since activation of monocytes is a complex process, one that involves a series of secondary messengers that mediate signal transduction and alter cell function, we have begun to identify several key signaling pathways that are required for converting a blood monocyte to an activated monocyte that can mediate LDL lipid oxidation. We have found that superoxide anion (Obardot 2) production is required for this process and that intracellular Ca2+ levels are integrally involved in oxidation of LDL lipids by activated human monocytes. Both the influx of extracellular Ca2+ and the release of intracellular Ca2+ are involved (16). Recently, we demonstrated that a Ca2+-regulated, intracellular signaling pathway, protein kinase C (PKC), was required. Our experimental results showed that depletion of PKC activity by phorbol 12-myristate 13-acetate, inhibition of PKC activity by pharmacologic inhibitors, or suppression of PKC levels by antisense oligonucleotides caused an inhibition of LDL lipid oxidation by activated human monocytes (17). The isoenzyme of PKC, required for oxidation of LDL by activated monocytes, was shown to be a member of the cPKC group of isoenzymes.

The rise of intracellular Ca2+ levels and activation of PKC elicit a variety of cellular responses including phosphorylation of target proteins which are located throughout the cell, on the plasma membrane, in the cytosol, and in the nucleus. This can initiate a cascade of other second messengers to transmit intracellular signals that ultimately alter cell function (18). Ca2+- and PKC-dependent signaling, therefore, provide exceptionally versatile signaling mechanisms. Downstream effects of Ca2+ and PKC have been reported to be related to the induction of several other intracellular signal transduction pathways, one of these pathways involves phospholipase A2 (PLA2) which hydrolyzes the sn-2 fatty acid on phospholipids producing free fatty acid and lysophospholipid (18, 19). Both free fatty acid and lysophospholipid serve as lipid mediators to regulate cell functions. PLA2 also plays a critical role in providing substrate for the biosynthesis of prostaglandins and leukotrienes by releasing arachidonic acid (AA) from membrane phospholipids. Consequently, PLA2s have been implicated in many cellular processes and disease states, such as maintenance of cellular phospholipid pools, participation in inflammatory reactions and host defense, and involvement in myocardial ischemia. Furthermore, AA has been reported to induce Obardot 2 production in human neutrophils (20) and monocytes (21) by activation of NADPH oxidase or by its metabolism via lipoxygenase pathways (22). Our laboratory has previously shown that both Obardot 2 production and lipoxygenase are involved in monocyte-mediated LDL lipid oxidation (5, 16, 17), suggesting that PLA2 might participate in this process.

Phospholipases A2 are a diverse family of enzymes with a growing number of members. Among the mammalian enzymes, the most well-characterized are the 14-kDa secretory PLA2 (sPLA2) and the 85-kDa cytosolic PLA2 (cPLA2) (19). The sPLA2 is Ca2+-dependent and requires mM levels of Ca2+ for activity. It also has seven disulfide bonds that are required for activity and therefore is sensitive to treatment with reducing agents such as dithiothreitol (DTT). In contrast, the 85-kDa cPLA2 requires only µM levels of Ca2+, levels that can be reached intracellularly, and it does not have disulfide bonds so its activity is not susceptible to reducing agents. Although cPLA2 is 85 kDa, it migrates as an 110-kDa protein in SDS gels. Unlike the sPLA2, cPLA2 shows a preference for arachidonic acid in the sn-2 position of substrate phospholipid. The activity of this latter enzyme is induced by protein phosphorylation and Ca2+-dependent translocation to membranes from the cytosol. In addition to these two enzymes several Ca2+-independent PLA2 (iPLA2) have been described, including the canine myocardial 40-kDa iPLA2 (23), the murine macrophage-like cell line P388D1 80-kDa iPLA2 (24), bovine brain 100-kDa iPLA2 (25), and an 80-kDa iPLA2 from CHO cells (46). These iPLA2 are Ca2+-independent and activated by ATP or detergent. To date, only the latter iPLA2 has been cloned (46).

The 85-kDa cPLA2 is believed to be an important regulator of arachidonic acid availability and thereby controls the production of potent lipid mediators. We were particularly interested in this enzyme because its activity has been shown to be regulated by PKC phosphorylation and by Ca2+ levels, and both PKC and Ca2+ have been shown to be key participants in monocyte oxidation of LDL. We therefore designed a series of experiments to test the hypothesis that cPLA2 participates in regulating the activation-dependent oxidation of LDL lipids by human monocytes.


MATERIALS AND METHODS

Chemicals

DEDA (7,7-dimethyleicosadienoic acid), ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-cholorobenzoic acid), 4-BpB (4-bromophenacyl bromide), aristolochic acid, and AACOCF3 (arachidonyl trifluoromethyl ketone) were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). DEDA was dissolved in ethanol. ONO-RS-082, 4-BpB, and AACOCF3 were dissolved in dimethyl sulfoxide. Aristolochic acid was dissolved in water. Arachidonic acid (AA) and L-alpha -lysophosphatidylcholine (lyso-PC, Sigma) were dissolved in ethanol. All these reagents were made as 100-fold stock solutions and stored at -20 °C prior to use.

Zymosan, obtained from ICN Biochemicals (Cleveland, OH), was opsonized (26) and used at a concentration of 2 mg/ml to activate human monocytes and U937 cells. Opsonized zymosan (ZOP) was suspended in phosphate-buffered saline as a 20-fold stock solution and stored at -70 °C prior to use.

Lipoprotein Preparation

Low density lipoprotein (LDL) was prepared according to previously described methods which minimize oxidation and exposure to endotoxin (2). All reagents used for LDL isolation were prepared with Chelex-treated MilliQ water. Each batch of LDL was assayed for endotoxin contamination by the limulus amebocyte lysate assay (kit QCL-1000, Whittaker Bioproducts Inc., Walkersville, MD). Final endotoxin contamination was always <0.03 unit/mg LDL cholesterol. LDL was stored in 0.5 mM EDTA. Immediately before use, LDL was dialyzed at 4 °C against phosphate-buffered saline without calcium or magnesium (Life Technologies, Inc.) in the dark. 1 g/liter Chelex was added to the dialysis buffer. LDL was used at a final concentration of 0.5 mg of cholesterol/ml.

Isolation of Human Monocytes and Cell Culture

Human monocytes were isolated from heparinized whole blood by sequential centrifugation over a Ficoll-Paque density solution and adherence to serum-coated cell culture flasks (5). Nonadherent cells were then removed. The adherent cells were released with 5 mM EDTA and plated into multiwell tissue culture plates at 1.0 × 106 cells/ml. This cell population consisted of greater than 95% monocytes (5). The isolated human monocytes were then cultured overnight in Dulbecco's modified Eagle's medium with 10% serum before use in experiments. In the experiments, monocytes were washed twice with RPMI 1640 (Whittaker, Walkersville, MD) and incubated with LDL (0.5 mg of cholesterol/ml) and ZOP (2 mg/ml) in the presence or absence of different reagents. After 24 h incubation, cell supernatants were collected, and LDL lipid oxidation was determined.

U937 cells, obtained from the American Type Culture Collection (Rockville, MD), were cultured in 150-cm2 flasks (Corning, Corning, NY) in RPMI 1640 supplemented with 10% bovine calf serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a humidified atmosphere of 90% air, 10% CO2. U937 cells were maintained in log phase (cell number was kept between 0.1 and 1.0 × 106 cells/ml). For experiments, U937 cells (in log phase between 3 to 6 × 105 cells/ml) were washed twice with RPMI 1640. 5 × 105 cells/ml were plated into multiwell tissue culture plates (Costar, Cambridge, MA) and incubated with LDL (0.5 mg of cholesterol/ml) and ZOP (2 mg/ml) in the presence or absence of different reagents. After 24 h incubation, cell supernatants were collected, and lipid oxidation of LDL was determined.

Measurement of Lipid Oxidation

The oxidation of LDL lipids was measured by both the thiobarbituric acid (TBA) assay and the lipid peroxide (LPO) assay.

Thiobarbituric Acid Assay

The oxidation of LDL lipids was measured by the TBA assay, a modification of the assay described by Schuh et al. (27). The thiobarbituric acid assay detects malondialdehyde (MDA) and MDA-like compounds reacting with TBA. Compounds that react with TBA are referred to as TBA-reactive substances. Data are expressed in terms of MDA equivalents (nmol of MDA/ml).

Lipid Peroxide Assay

The lipid peroxide levels on LDL were determined by a modification of the assay described by El-Saadani et al. (28). This assay measures the oxidative capacity of lipid peroxides to convert iodide to iodine, which can be measured spectrophotometrically at 365 nm. Data are expressed in nanomoles of lipid peroxide/ml (nmol LPO/ml).

cPLA2 Activity Assay

Human monocytes (2.5 × 106 cells/ml) in RPMI without serum were incubated with ZOP (2 mg/ml) in the presence or absence of a variety of reagents as indicated. After incubation, cells were harvested and resuspended in Buffer A (50 mM Hepes, pH 7.5, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.1 mM sodium vanadate, 0.5 mM phosphoserine, and 0.5 mM phosphothreonine) at a concentration of 2.5 × 107 cells/ml. Cells in Buffer A were lysed by sonication, and then cell lysates were centrifuged at 1000 × g for 15 min. Supernatants were collected and 100 µg of total protein, as determined by the Lowry assay, were used in the PLA2 assay.

The substrate, L-alpha -1-palmitoyl-2-[14C]arachidonyl phosphatidylcholine (55 mCi/mmol, DuPont NEN), was dried under N2 and resuspended in dimethyl sulfoxide (final concentration in the reaction was less than 0.3% v/v) by vigorous mixing for 2 min, and then resuspended in 10 mM Hepes buffer, pH 7.4, with 5 mM CaCl2, 1 µg/ml leupeptin, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM sodium vanadate (7, 29).

For PLA2 assays, the reaction was initiated by adding cell lysate (100 µg of protein) to the substrate (100 nCi, 1.8 nmol) followed by incubation at 37 °C for 30 min. The total volume was 100 µl. Then, lipids were extracted by the method of Bligh and Dyer (30). Free fatty acid and phospholipids were separated by TLC using the solvent system, chloroform/acetone/methanol/acetic acid/H2O, 6:8:2:2:1 (v/v). The free fatty acid and phosphatidylcholine were scraped from the TLC plate, and the radioactivity was counted on a beta -counter. In some cases the DTT was not included. The DTT-sensitive activity (the activity without DTT minus that with DTT) is referred to as sPLA2 activity, and the DTT-resistant activity is referred to as cPLA2 activity. This latter activity may include that mediated by Ca2+-requiring cPLA2 and Ca2+-independent iPLA2, but for simplicity we refer to it as cPLA2 activity.

Western Blotting Analysis

Human monocytes (2.5 × 106 cells/ml) were incubated with ZOP (2 mg/ml) in the presence or absence of different reagents for 24 h as indicated in figure legends. After incubation, cells were harvested and resuspended in 200 µl of hypotonic lysis buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 0.5 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, and 0.5% Nonidet P-40). The cells were vortexed for 15 s, and cellular debris and nuclei were removed by centrifugation in a microcentrifuge at 1000 × g for 10 min. The supernatants were collected, and 100 µg of cell lysate protein was prepared for 7% SDS-PAGE (31). The SDS-PAGE gel was transferred to a polyvinylidene difluoride membrane by the semi-dry method (32). After blocking the nonspecific binding sites with 10% milk in Tris buffer (20 mM Tris-base, pH 7.4, 1.5 M NaCl, 1% Nonidet P-40) for 1 h at room temperature, cellular cPLA2 protein was detected with a 1:1000 dilution of rabbit anti-human recombinant cPLA2 polyclonal antibody (generously provided by Dr. J. Clark, Genetic Institute, Inc., Andover, MA), followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution). The polyvinylidene difluoride membrane was developed using enhanced chemiluminescence (Amersham Corp.).

For immunoprecipitation of cPLA2 protein, the total cell lysate from 10 × 106 cells was incubated with 30 µl of polyclonal antibody prebound to protein A beads for 1 h at 4 °C. After incubation, cell lysates were centrifuged at 1000 × g for 10 min. Pellets were collected and prepared for 7% SDS-PAGE. After transfer to a polyvinylidene difluoride membrane, cPLA2 protein was detected by Western blotting using anti-human recombinant cPLA2 monoclonal antibody (generously provided by Dr. J. Clark, Genetics Institute, Inc., Andover, MA).

Treatment of Cells with Oligonucleotides

The sense and antisense sequences of cPLA2 were selected from a unique area of the mRNA that is near the 5' end of the message. Prior to selection, the sequences were selected by screening for uniqueness using Blast© and were also tested for lack of secondary structure and oligo pairing using Mulfold© (33).

The antisense oligomer was complementary to nucleotides 219-238 of cPLA2 which code for amino acids 27-34 of the protein. The sequence was 5'CCC CCT TTG TCA CTT TGG TG3'. The sequence of the cPLA2 sense oligomer was 5'CAC CAA AGT GAC AAA GGG GG3'. Phosphorothioate-modified oligonucleotides were used for these studies to limit degradation. The oligonucleotides were synthesized and purified by HPLC prior to use (Genosys Biotechnologic, Inc., Woodlands, TX).

For these experiments, human monocytes (1 × 105 cells/0.1 ml/well), LDL (0.5 mg of cholesterol/ml), and ZOP (2 mg/ml) were cultured in RPMI 1640 without serum in the presence or absence of different concentrations of sense or antisense oligonucleotides in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) for 24 h. After incubation, cell-mediated LDL lipid oxidation was assessed by the TBA assay as described above. In some cultures, sense or antisense oligonucleotides were incubated with cells (2.5 × 106 cells/ml) for 24 h, and then cellular cPLA2 activity was measured by the method described above.

Detection of Toxicity of Test Agents

The toxicity of test agents to human monocytes and U937 cells was determined by the [14C]adenine release assay (34). Briefly, 1 × 107 cells in 20 ml of RPMI 1640 were labeled overnight by incubating them with 10 µCi of [14C]adenine (ICN Radiochemical, Irvine, CA). For experiments, the cells were washed twice with RPMI 1640, and human monocytes (1 × 106 cells/ml) or U937 cells (0.5 × 106 cells/ml), ZOP (2 mg/ml), and LDL (0.5 mg of cholesterol/ml) were added to 12-well tissue culture plates in the presence or absence of test agents. After a 24 h incubation, the amount of [14C] adenine released by the cells was detected by counting 100 µl of the supernatant fluid using a Beckman LS-3801 beta -counter. [14C]Adenine release from ZOP-activated cells in the absence of test agents was defined as 0% release. [14C]Adenine release by 0.2% SDS was defined as 100% release and interpreted as maximum toxicity. The data are expressed in Equation 1 as
% <UP>release</UP>=<FR><NU><UP>sample release</UP>−<UP>background</UP></NU><DE><UP>maximum release</UP>−<UP>background</UP></DE></FR>×100%. (Eq. 1)

Detection of Antioxidant Activity of Test Agents

The following method was used to assess the antioxidant effects of test agents (16, 35). 5 µM of CuSO4 and 0.5 mg of cholesterol/ml of LDL were incubated together with the various test agents at 37 °C for 24 h in RPMI 1640. After incubation, supernatants were collected, and LDL oxidation was measured by the TBA assay as described above. All experiments were performed in triplicate. If copper-induced LDL oxidation was inhibited in the presence of the test agents, this indicated that the test agent could serve as a general antioxidant.

Measurement of Superoxide Anion Production

Superoxide anion (Obardot 2) production was measured by the cytochrome c reduction assay (36). Cytochrome c can be reduced by Obardot 2 on a mol/mol basis, and the reduced cytochrome c has an increased absorbance at 550 nm. Human monocytes (1 × 106 cells/ml) and antisense oligonucleotides or sense oligonucleotide (5 µM) were preincubated for 24 h in Dulbecco's modified Eagle's medium with 10% bovine calf serum. After preincubation, cells and 320 µM cytochrome c (Sigma) were incubated in the presence or absence of 150 units/ml superoxide dismutase (from bovine erythrocytes, Sigma) in 96-well cell tissue culture plates (a total volume of 100 µl/well) in RPMI 1640 without phenol red and serum (Whittaker, Walkersville, MD) at 37 °C in a humidified incubator with 10% CO2 for 1 h. ZOP (2 mg/ml) and test agents were included during the incubation. After incubation, the absorbance was measured at 550 nm. Equation 2 was used to determine the nmol of Obardot 2 produced (where SOD is superoxide dismutase).
<UP>O&cjs1138;<SUB>2</SUB> nmol/ml = </UP>(A<SUB>550</SUB> (<UP>in the absence of SOD</UP>)×159)−(A<SUB>550</SUB> (<UP>in the presence of SOD</UP>)×159) (Eq. 2)

Statistical Analyses

The data from experiments were analyzed using the unpaired two-tailed Student's t test. Statistical tests were performed with GraphPAD InStat software (GraphPAD Software Inc., San Diego, CA). Data points with a p < 0.05 were considered to be significantly different.


RESULTS

We first investigated the induction of cellular cPLA2 by Western blotting analysis using different anti-human cPLA2-specific antibodies. In these experiments, human monocytes were incubated with the activator (ZOP) for 24 h. After incubation, cell lysates were prepared. In one set of experiments, cPLA2 protein was immunoprecipitated from cell lysates using anti-human cPLA2-specific polyclonal antibody. After immunoprecipitation, cPLA2 protein was detected by Western blotting using an anti-human cPLA2-specific monoclonal antibody as described under "Materials and Methods." The result is shown in Fig. 1A. In a similar experiment, cPLA2 cell lysates, without immunoprecipitation, were directly detected by Western blotting using the same polyclonal antibody. This result is shown in Fig. 1B. Our data indicate that human monocytes had very low levels of endogenous cPLA2 (as shown in lane 1 of Fig. 1, A and B). Upon activation, monocyte cPLA2 was induced as demonstrated in lane 2 of Fig. 1, A and B. The lysate from unstimulated U937 cells, constitutive producers of cPLA2, was included as a positive control as shown in lane 3 of Fig. 1A. These data demonstrate the induction of cPLA2 protein in activated human monocytes, and similar results were obtained regardless of whether cell lysates were first subjected to immunoprecipitation. In this study, therefore, cPLA2 proteins in cell lysates were analyzed using anti-human cPLA2-specific polyclonal antibody without immunoprecipitation.


Fig. 1. cPLA2 protein is induced in ZOP-activated human monocytes. Human monocytes (2.5 × 106 cells/ml) were incubated in the presence or absence of ZOP (2 mg/ml) for 24 h. After incubation, cells were lysed. Cellular debris was removed by centrifugation, and cellular proteins (100 µg of protein/lane) were assessed by Western blotting analysis with cPLA2-specific antibody as described under "Materials and Methods." Left side arrows indicate the position of cPLA2 protein. A, immunoprecipitation of cPLA2 protein. Lane 1, unactivated human monocytes; lane 2, ZOP-activated human monocytes; lane 3, unactivated U937 cells. Results from one of two similar experiments are shown. B, direct detection of cPLA2 protein in cell lysate. Lane 1, cPLA2 in unactivated human monocytes; lane 2, cPLA2 in ZOP-activated human monocytes. Results from one of three similar experiments are shown.
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The time course for induction of cPLA2 protein levels in activated human monocytes was also examined. In this experiment, human monocytes and ZOP were incubated for different periods. cPLA2 protein was detected using Western blotting as described under "Materials and Methods." Although total protein levels were not substantially changed in both unactivated (0.2756-0.3138 mg/10 × 106 cells) and activated cells (0.2242-0.3180 mg/10 × 106 cells), the cPLA2 protein levels were substantially induced as shown in Fig. 2A. The cPLA2 protein levels began to increase after 4 h of activation and reached maximal levels at 12 h of activation and then gradually decreased at 24 h.


Fig. 2. Induction of cPLA2 protein and enzymatic activity in ZOP-activated human monocyte. Human monocytes (2.5 × 106 cells/ml) were incubated in the presence or absence of ZOP (2 mg/ml) for different times as indicated. After incubation, cells were lysed. Cellular debris was removed by centrifugation. A, quantitation of cPLA2 protein levels in unactivated human monocytes (open circles) and in ZOP-activated human monocytes (closed circles). Inset, cPLA2 protein levels were detected by Western blotting as described under "Materials and Methods." Left side arrow indicates the position of cPLA2 protein. Results from one of two similar experiments are shown. B, cPLA2 enzymatic activity was assessed in lysates of ZOP-activated human monocytes in the presence of 2 mM DTT as described under "Materials and Methods." Data represent the mean ± S.E. obtained from three similar experiments. The significance of induction was determined by Student's t test (* indicates p < 0.05).
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In parallel experiments, the induction of cPLA2 enzymatic activity at various times following human monocyte activation was also investigated. In these experiments, human monocytes were incubated with ZOP for various times between 0 and 24 h as indicated. After incubation, cell lysates were assayed for cPLA2 activity. During the cPLA2 activity assay, DTT was included to effect selective inactivation of sPLA2-like activity. After incubation, 14C-labeled free arachidonic acid and phosphatidylcholine were separated by TLC using the solvent system as described under "Materials and Methods." The data shown in Fig. 2B indicate that there is a basal level of endogenous DTT-resistant, cPLA2 activity in unactivated human monocytes. The cPLA2 enzymatic activity began to increase after 4 h of activation. The maximal induction of cPLA2 enzymatic activity was 12 h after initiation of activation. Although the cPLA2 enzymatic activity gradually decreased after 12 h of activation, significant induction of cPLA2 enzymatic activity was still observed at 24 h of activation. Significant induction of cPLA2 enzymatic activity is indicated by asterisks (* indicates p < 0.05). These results indicate that human monocyte cPLA2 enzymatic activity is induced upon activation, and the induction of cPLA2 enzymatic activity is correlated with the rise in cPLA2 protein levels. It should be noted that this assay may detect both the activities of the cPLA2 and the Ca2+-independent cytosolic PLA2 (iPLA2).

The involvement of phospholipases A2 in the process of human monocyte and U937 cell oxidation of LDL was first evaluated using several, structurally unrelated, pharmacologic inhibitors of PLA2, including DEDA, ONO-RS-082, aristolochic acid, and 4-BpB. Freshly isolated human monocytes or U937 cells, LDL, and ZOP were incubated together in the presence or absence of the PLA2 inhibitors for 24 h. After incubation, the lipid oxidation of LDL was assessed by the TBA assay and the LPO assay. The TBA assay is a widely used method to detect malondialdehyde and MDA-like compounds derived from lipid oxidation products (27). The LPO assay detects lipid hydroperoxide which are produced upon lipid oxidation (28). Our experimental results demonstrated that each of these PLA2 inhibitors showed dose-dependent inhibition of LDL lipid oxidation by activated human monocytes and U937 cells (data not shown). These data, regardless of the fact that most of these pharmacologic PLA2 inhibitors are nonselective for sPLA2 versus cPLA2, provided the first suggestion that PLA2 activity was involved in LDL lipid oxidation by activated human monocytes and U937 cells.

To further investigate the requirement for cPLA2, we used another inhibitor, AACOCF3, which has been reported to be a selective inhibitor of cPLA2 (37). In these experiments, human monocytes and ZOP were incubated in the presence or absence of different concentrations of AACOCF3 for 24 h. After incubation, cell lysates were prepared. Then, PLA2 activities were assessed in the presence or absence of 2 mM DTT as described under "Materials and Methods." The experimental results are summarized in Fig. 3. AACOCF3 inhibited DTT-resistant PLA2 activity in a concentration-dependent fashion, indicating that cPLA2 activity was inhibited by AACOCF3 as shown in Fig. 3A. In contrast, AACOCF3 did not inhibit DTT-sensitive PLA2 activity, indicating that sPLA2 activity was not inhibited by AACOCF3 as shown in Fig. 3B.


Fig. 3. Human monocyte cPLA2 activity is inhibited by AACOCF3 in a concentration-dependent fashion. Human monocytes (2.5 × 106 cells/ml) and ZOP (2 mg/ml) were incubated in the presence or absence of different concentrations of AACOCF3 for 24 h. After incubation, cell lysates were made, and PLA2 activity was measured in the presence or absence of 2 mM DTT. A, DTT-resistant cPLA2 activity. B, DTT-sensitive sPLA2 activity (the activity in the absence of DTT minus that in the presence of DTT). PLA2 activity in unactivated human monocytes (open bars), ZOP-activated human monocytes (solid bars), and ZOP-activated human monocytes in the presence of different concentrations of AACOCF3 (hatched bars). Data are presented as the mean ± S.E. obtained in duplicate samples of three similar experiments. The significance of inhibition was determined by Student's t test (* indicates p < 0.05).
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In a parallel experiment, we also monitored monocyte-mediated LDL lipid oxidation in the presence or absence of AACOCF3. In these experiments, human monocytes were incubated with LDL and ZOP in the presence or absence of AACOCF3 for 24 h. After incubation, cell-mediated LDL lipid oxidation was assessed by both the TBA assay and the LPO assay. The experimental results are summarized in Fig. 4. Upon activation, cell-mediated LDL lipid oxidation was substantially increased as detected by the TBA assay (as shown in Fig. 4A) and the LPO assay (as shown in Fig. 4B). AACOCF3 caused a concentration-dependent inhibition of cell-mediated LDL lipid oxidation. Taken together, these data suggest that an AACOCF3-sensitive PLA2 activity is required for human monocyte-mediated LDL lipid oxidation.


Fig. 4. Human monocyte-mediated LDL lipid oxidation is inhibited by AACOCF3 in a concentration-dependent fashion. Human monocytes (1 × 106 cells/ml), ZOP (2 mg/ml), and LDL (0.5 mg of cholesterol/ml) were incubated in the presence or absence of different concentrations of AACOCF3 for 24 h. After incubation, LDL lipid oxidation was assessed by both the TBA assay and the LPO assay. A, monocyte-mediated LDL lipid oxidation detected by the TBA assay. B, monocyte-mediated LDL lipid oxidation detected by the LPO assay. The open bars represent LDL lipid oxidation by unactivated monocytes. The solid bars represent LDL lipid oxidation by ZOP-activated monocytes. The hatched bars represent LDL lipid oxidation in the presence of different concentrations of AACOCF3. The data are expressed as the mean ± S.D. of triplicate samples obtained in one of three similar experiments. The significance of inhibition was determined by Student's t test (* indicates p < 0.05).
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To investigate potential nonspecific effects of AACOCF3, we also examined its toxicity to human monocytes and its ability to function as a general antioxidant. For the cytotoxicity studies we used an assay measuring [14C]adenine metabolite release. We have shown that the results obtained with this assay correlate well with the chromium release assay of toxicity (16). The results, presented in Table I, demonstrate that AACOCF3, at doses used for these studies, showed less than 5% toxicity for human monocytes (shown in Table I) or U937 cells (data not shown). We also evaluated the general antioxidant activity of AACOCF3 as measured by its inhibition of copper-induced LDL lipid oxidation as described previously (16, 35). The results of these studies are presented in Table II. AACOCF3 showed no inhibition of copper-mediated LDL lipid oxidation at concentrations from 1 to 50 µM, indicating that AACOCF3 did not exhibit antioxidant activity at concentrations used in the studies.

Table I.

Cytotoxic effects of AACOCF3


Incubation additionsa [14C]Adenine releaseb Percent releasec

cpm × 10-3
Unactivated cells + LDL 11.72  ± 0.27
Activated cells + LDL + ... 
  Dimethyl sulfoxide (solvent   control) 7.10  ± 0.27 0
  0.2% SDS (total release) 103.58  ± 13.2 100
  AACOCF3
    1 µM 8.05  ± 0.03 0.98
    10 µM 8.22  ± 0.10 1.16
    25 µM 10.92  ± 0.10 3.96
    50 µM 11.72  ± 0.43 4.79

a  Human monocytes (1 × 106/ml) were labeled with [14C]adenine and then incubated together with ZOP (2 mg/ml), LDL (0.5 mg of cholesterol/ml), and different concentrations of AACOCF3 as indicated for 24 h. After incubation, the supernatants were collected.
b  Release of radioactivity was determined as described under "Materials and Methods." All experiments were performed in triplicate. The results are expressed as mean ± S.D. of data obtained in one of three similar experiments.
c  Percent release of [14C]adenine was determined by the equation described under "Materials and Methods."

Table II.

Effect of AACOCF3 on copper-mediated LDL lipid oxidation


Incubation conditionsa TBAb

nmol MDA/ml
LDL 0.40  ± 0.01
LDL + 5 µM CuSO4 + ... 
  Dimethyl sulfoxide (solvent control) 2.76  ± 0.08
  AACOCF3
    1 µM 2.69  ± 0.03
    10 µM 2.76  ± 0.04
    50 µM 2.69  ± 0.08

a  LDL (0.5 mg of cholesterol/ml) and 5 µM CuSO4 were incubated in the presence or absence of different concentrations of AACOCF3 at 37 °C for 24 h. After incubation, the supernatants were collected, and LDL lipid oxidation was determined by the TBA assay.
b  The TBA assay was performed as described under "Materials and Methods." LDL lipid oxidation is expressed as the mean of triplicate determinations ± S.D. in nmol of MDA/ml from one of three similar experiments.

To corroborate our findings with AACOCF3 and to determine whether, indeed, cPLA2 was required, we used another approach to regulate cPLA2 activity. For these studies, we used cPLA2-specific antisense oligonucleotides to inhibit cPLA2 expression. We also used a cPLA2 sense oligonucleotide as a control in parallel cultures. In these experiments, human monocytes were incubated with sense or antisense phosphorothioate-modified oligonucleotides (HPLC-purified) in the presence or absence of ZOP and LDL as described under "Materials and Methods." After incubation, both cPLA2 protein expression and cPLA2 enzymatic activity were determined. As shown in Fig. 5A, cPLA2 protein expression in activated human monocytes was inhibited by cPLA2-specific antisense oligonucleotide treatment (lane 3 of Fig. 5A) as detected by Western blotting analysis using cPLA2-specific antibody. Sense oligonucleotide treatment had no effect on human monocyte cPLA2 protein expression (lane 4 of Fig. 5A). We also examined the cPLA2 activity in lysates of monocytes that had been treated with antisense or sense oligonucleotides. These data are shown in Fig. 5B. cPLA2 activity was increased upon monocyte activation as previously observed (Fig. 2B) and antisense oligonucleotide treatment substantially inhibited cPLA2 activity. In contrast, treatment with the sense oligonucleotides did not alter cPLA2 activity. Furthermore, we also monitored whether oligonucleotide treatment had any nonspecific inhibitory effects on the assay. In this experiment, U937 cell lysates and oligonucleotides were included together during the cPLA2 activity assay. Neither antisense oligonucleotides nor sense oligonucleotides affected the cPLA2 activity assay itself (data not shown).


Fig. 5. cPLA2 antisense oligonucleotides inhibit both cPLA2 protein expression and enzymatic activity in ZOP-activated human monocytes. Human monocyte (2.5 × 106 cells/ml) and ZOP (2 mg/ml) were incubated with either 5 µM cPLA2-specific antisense or sense oligonucleotides for 24 h. After incubation, cell lysates were made. Both cPLA2 protein expression and enzymatic activity were assessed as described under "Materials and Methods." A, induction of cPLA2 proteins. Experimental results represent data obtained from one of three similar experiments. B, induction of cPLA2 enzymatic activity. Data represent the mean ± data range of the duplicate samples obtained in one of three similar experiments. The open bars represent results in unactivated human monocytes. The solid bars represent results in ZOP-activated human monocytes. The hatched bars on the left represent results in ZOP-activated human monocytes treated with 5 µM of cPLA2-specific antisense oligonucleotides. The hatched bars on the right represent results in ZOP-activated human monocytes treated with 5 µM of control sense oligonucleotides. A, inset, cPLA2 Western blot of unactivated human monocytes (lane 1), activated human monocytes (lane 2), activated human monocytes treated with 5 µM of cPLA2-specific antisense oligonucleotides (lane 3), and activated human monocytes treated with 5 µM of control sense oligonucleotides (lane 4). The significance of inhibition was determined by Student's t test (* indicates p < 0.05).
[View Larger Version of this Image (37K GIF file)]


Next, we evaluated human monocyte-mediated LDL lipid oxidation after treatment with cPLA2-specific antisense or sense oligonucleotides. Treatment with cPLA2-specific antisense oligonucleotides caused a dose-dependent decrease in monocyte-mediated LDL lipid oxidation as detected by the TBA assay (Fig. 6). In contrast, treatment with sense oligonucleotides caused no significant inhibition of LDL lipid oxidation (* indicates p < 0.05). These data suggest that cPLA2 activity is a critical regulator of the oxidation of LDL by activated human monocytes.


Fig. 6. Human monocyte-mediated LDL lipid oxidation is inhibited by cPLA2-specific antisense oligonucleotides. Human monocytes (1 × 106 cells/ml), ZOP (2 mg/ml), and LDL (0.5 mg cholesterol/ml) were incubated in the presence or absence of different concentrations of cPLA2-specific antisense or control sense oligonucleotides for 24 h. After incubation LDL lipid oxidation was assessed by the TBA assay as described under "Materials and Methods." The levels of monocyte-mediated LDL lipid oxidation in the presence of different concentrations of cPLA2-specific antisense (closed circles) or control sense (open circles) oligonucleotides are shown. Data are expressed as the mean ± S.E. of triplicate samples obtained in three similar experiments. The significance of inhibition was determined by Student's t test (* indicates p < 0.05).
[View Larger Version of this Image (18K GIF file)]


Previously, our laboratory has shown that Obardot 2 production is required for monocyte-mediated LDL lipid oxidation, and arachidonic acid has been shown to regulate the activity of the NADPH oxidase Obardot 2 generating complex. We therefore examined whether cPLA2 activity was essential for Obardot 2 production. In this experiment, human monocytes were preincubated with either cPLA2-specific antisense or sense oligonucleotides for 24 h and then Obardot 2 production was quantified in response to activation. As expected, Obardot 2 production was increased upon monocyte activation as shown in Fig. 7A. Antisense oligonucleotide treatment significantly inhibited Obardot 2 production in activated human monocytes, whereas sense oligonucleotide treatment was without significant effect. Furthermore, we found that the inhibitory effect of antisense treatment could be negated by addition of arachidonic (AA) one product of cPLA2 activity. AA alone or with ZOP did not alter Obardot 2 production except to restore levels to normal in antisense-treated, activated monocytes.


Fig. 7. Human monocyte-mediated LDL lipid oxidation and Obardot 2 production are inhibited by cPLA2-specific antisense oligonucleotides. Human monocytes (1 × 106 cells/ml), ZOP (2 mg/ml), LDL (0.5 mg cholesterol/ml), and cPLA2-specific antisense or control sense oligonucleotides (5 µM) were incubated in the presence or absence of arachidonic acid (AA, 3 µM) for 1 h or 24 h. After 1 h incubation, Obardot 2 production was assessed as described under "Materials and Methods" (results shown in A). After 24 h incubation, LDL lipid oxidation was assessed by the TBA assay as described under "Materials and Methods" (results shown in B). Determinations were made in unactivated monocytes (open bar), unactivated monocytes in the presence of AA (stippled bar), ZOP-activated monocytes (solid bar), ZOP-activated monocytes treated with antisense or sense oligonucleotides in the absence of AA (hatched bars), and ZOP-activated monocytes treated with antisense or sense oligonucleotides in the presence of AA (cross-hatched bars). Data are presented as the mean ± S.D. of triplicate samples obtained in three similar experiments. * indicates significant differences between activated cells and antisense-treated, activated cells (p < 0.05). ** indicates significant differences between antisense-treated cells in the presence or absence of AA (p < 0.05).
[View Larger Version of this Image (44K GIF file)]


We then conducted similar experiments to attempt to restore the ability of antisense-treated, activated monocytes to oxidize LDL lipids. In these experiments (see Fig. 7B), addition of AA restored LDL lipid oxidation by 50% in antisense-treated cells. Addition of lysophosphatidyl choline (lyso-PC) or lyso-PC plus AA did not fully restore LDL lipid oxidation in antisense-treated cells (data not shown). Treatment with AA and/or lyso-PC did not alter levels of LDL lipid oxidation in unactivated monocytes nor in ZOP-activated monocytes that were not treated with oligonucleotides or were treated with sense oligonucleotides.


DISCUSSION

In our previous studies, we found that human peripheral blood monocytes could oxidize LDL in an activation-dependent manner (1, 2). We also found that Obardot 2 production (2), increases in intracellular Ca2+ levels (16), and induction of PKC activity were required as well (17). These observations suggested that one or more Ca2+- and protein phosphorylation-dependent intracellular signaling pathways regulated monocyte function and participated in the process of monocyte-mediated LDL lipid oxidation. A potential candidate for one of these pathways was the high molecular weight cPLA2. We hypothesized that cPLA2 might prove to be an important regulatory pathway in the oxidation of LDL by activated monocytes.

We found that low levels of cPLA2 protein were detectable in unactivated human monocytes, and upon activation, the cPLA2 protein levels and enzymatic activity were substantially increased. The time course studies showed a correlation in the increase of cPLA2 protein and enzymatic activity, with both reaching a maximum at 12 h of activation. Our previous studies documented an increase in intracellular Ca2+ levels and induction of PKC activity occurring within 30 min of monocyte activation, demonstrating that these two events occur early in the course of human monocyte activation (16, 17). Our previous studies also demonstrated that LDL lipid oxidation begins to be detectable from 4 to 6 h after monocyte activation and then increases and begins to plateau at 12 h of activation and gradually increases to 24-h levels (5). Taking all of these observations together, our studies demonstrate that increases in intracellular Ca2+ levels and activation of PKC precede the induction of cPLA2 activity and that the induction of cellular cPLA2 activity closely correlates with that of monocyte-mediated LDL lipid oxidation (5).

In experiments using several types of functionally and structurally diverse pharmacologic inhibitors of PLA2, results indicated that cPLA2 activity was required for monocyte-mediated LDL lipid oxidation (data not shown). To confirm this observation we used another inhibitor, AACOCF3. AACOCF3 is an analog of arachidonic acid in which the COOH group is replaced with COCF3 (trifluoromethyl ketone) (37). It has been reported to be a selective inhibitor of cPLA2 in that it is 500-fold more potent as an inhibitor of cPLA2 as compared with sPLA2 (37). Since sPLA2 has seven disulfide bridges and is inactivated by DTT, we could distinguish between sPLA2 and cPLA2 activities. AACOCF3 only inhibited DTT-resistant PLA2 activity but not DTT-sensitive PLA2 activity, and inhibition was concentration-dependent, demonstrating that cPLA2 activity was selectively inhibited. Importantly, AACOCF3 also inhibited human monocyte-mediated LDL lipid oxidation in a dose-dependent fashion. In concert, these data supported our hypothesis that cPLA2 activity was required for monocyte-mediated LDL lipid oxidation; recently, however, it has been reported that AACOCF3 can inhibit cytosolic iPLA2 activity in a mouse macrophage cell line, P388D1 (38). Technically, it is difficult to distinguish cPLA2 enzymatic activity from iPLA2 activity in this assay, because both cPLA2 and iPLA2 are insensitive to DTT and both activities would be detected in our assay system (19, 39).

To more specifically address the participation of cPLA2 in this process, cPLA2-specific antisense and sense oligonucleotides were developed. The sequence was carefully chosen from a region lacking substantial homology with other sequenced human genes. The oligonucleotides were phosphorothioate-modified to limit degradation and purified by HPLC prior to use to remove all incomplete synthesis products thereby limiting nonspecific effects. We have found this latter step to be critical in rendering specificity to antisense oligonucleotide regulation in human monocytes. The finding that antisense treatment, but not treatment with sense oligonucleotides, resulted in decreased cPLA2 protein expression and decreased enzymatic activity leads us to believe that the decrease in monocyte oxidation of LDL was indeed due to inhibition of cPLA2. Recent reports have also shown that cPLA2 protein expression and enzymatic activity can be inhibited by cPLA2 antisense oligonucleotide treatment in human monocytes (40, 41). In these studies, different cPLA2 antisense oligonucleotide sequences were used including sequences directly recognizing the initiation site of transcription (40) and sequences recognizing cPLA2 mRNA downstream from the initiation site (41).

An important mechanistic finding of this study is that monocyte-mediated Obardot 2 production is inhibited by suppression of cPLA2 activity (Fig. 7A). Although numerous studies report that AA and phospholipase A2 appear to regulate Obardot 2 production, this is the first report that specific suppression of cPLA2 protein expression and enzymatic activity, using cPLA2-specific antisense oligonucleotide treatment, inhibited Obardot 2 production by activated human monocytes. The finding that cPLA2 regulates LDL lipid oxidation is also novel. Interestingly, both Obardot 2 production and LDL lipid oxidation were inhibited to the same extent, but as discussed below, neither was inhibited completely (see Fig. 7).

We have previously reported that Obardot 2 is required for monocyte oxidation of LDL, yet it is clear from data from our laboratory and others that Obardot 2 alone is not sufficient for LDL oxidation (42).2 Activated monocytes must provide Obardot 2 plus some unidentified cofactor for oxidation to proceed. In this regard, it is interesting that the addition of AA completely restored Obardot 2 production by antisense-treated, activated monocytes (Fig. 7A), whereas AA only partially restored the capacity of antisense-treated, activated monocytes to oxidize LDL (Fig. 7B). We also examined whether the addition of lyso-PC or both lyso-PC + AA could restore the LDL oxidation mediated by antisense-treated, activated monocytes, but some inhibition of LDL oxidation remained (data not shown). These data suggest that AA is the cPLA2 product required for regulating Obardot 2 production, whereas AA in addition to another product, likely a specific lysophospholipid, both participate in modulating the process of LDL lipid oxidation. The fact that lyso-PC was not restorative for this function even in the presence of AA indicates that a phospholipid other than PC is the essential substrate in regulating this process.

Our published studies have shown that inhibition of PKC decreases Obardot 2 production by activated monocytes as well as inhibiting LDL oxidation (17). cPLA2 activity is reportedly regulated by both PKC-dependent and PKC-independent pathways (29). In recent studies, we have found that inhibition of PKC activity caused a related inhibition of cPLA2 activity in activated human monocytes,3 thus suggesting that PKC-dependent phosphorylation events regulate cPLA2 activity. It would appear then that cPLA2 is an intermediary enzyme in the signal transduction pathway involving PKC regulation of Obardot 2 production and LDL oxidation.

Another observation from these studies was that antisense oligonucleotide treatment almost completely inhibited cPLA2 protein expression and enzymatic activity, as measured in monocyte lysates; however, monocyte-mediated Obardot 2 production and LDL lipid oxidation were not completely suppressed. Our data indicate the presence of a constitutive PLA2 activity that was not inhibited by antisense to cPLA2 (see Fig. 5 and Fig. 7). This activity might be due to another PLA2, which may also participate in regulating monocyte Obardot 2 production and LDL oxidation. Multiple forms of PLA2s in individual cell types have been reported, such as in the mouse macrophage-like cell line P388D1 (43), canine vascular smooth muscle cells (44), and the rat mast cell line, RBL-2H3 (45). Further investigations are needed to define the involvement of other PLA2 activities. In addition, the incomplete inhibition of monocyte-mediated LDL lipid oxidation by both AACOCF3 and cPLA2-specific antisense oligonucleotide treatment may also suggest that one or more parallel, cPLA2-independent pathways may regulate monocyte activation and be involved in LDL oxidation.

In summary, our data demonstrate that cPLA2 activity plays an important role in both Obardot 2 production and optimal LDL lipid oxidation by activated human monocytes. Our current working hypothesis regarding the events required for monocyte oxidation of LDL lipids is that after activation, intracellular Ca2+ levels are increased, by both the influx of extracellular Ca2+ and the release of intracellular Ca2+, thus causing the induction of PKC activity, which together with Ca2+ can regulate cPLA2 activity (1, 2, 16, 17). The activation of cPLA2 then, in concert with Ca2+ and PKC, causes the induction of Obardot 2 production which participates in the oxidation of LDL. These signaling pathways form a network and induce optimal LDL lipid oxidation by activated human monocytes. Knowledge acquired from studies such as these will contribute to the understanding of the mechanisms of monocyte oxidation of LDL lipids and may suggest optimal points for intervening in this process.


FOOTNOTES

*   This investigation was supported by National American Heart Association Grant 91012010 and the NHLBI, National Institutes of Health, Grant HL51068. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Cell Biology, Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-444-5222; Fax: 216-444-9404.
1    The abbreviations used are: LDL, low density lipoprotein(s); MDA, malondialdehyde; ZOP, opsonized zymosan; TBA, thiobarbituric acid; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; cPLA2, cytosolic phospholipase A2; sPLA2, secretory PLA2; iPLA2, Ca2+-independent PLA2; lyso-PC, lysophosphatidylcholine; PKC, protein kinase C; AA, arachidonic acid; DEDA, 7,7-dimethyleicosadienoic acid; 4-BpB, 4-bromophenacyl bromide; LPO, lipid peroxide; HPLC, high performance liquid chromatography; AACOCF3, arachidonyl trifluoromethyl ketone.
2    A. Zendedel-Haghighi and M. K. Cathcart, unpublished observations.
3    Q. Li and M. K. Cathcart manuscript in preparation.

Acknowledgments

We thank Dr. Meredith Bond for helpful discussions, Josette Gatewood for technical assistance, and Dr. James Clark, Genetic Institute for facilitating acquisition of the antibody to human recombinant cPLA2.


REFERENCES

  1. Cathcart, M. K., Morel, D. W., and Chisolm, G. M. (1985) J. Leukocyte Biol. 38, 341-350 [Abstract]
  2. Cathcart, M. K., Chisolm, G. M., McNally, A. K., and Morel, D. W. (1988) In Vitro Cell. & Dev. Biol. 24, 1001-1008 [Medline] [Order article via Infotrieve]
  3. Morel, D. W., DiCorleto, P. E., and Chisolm, G. M. (1984) Arteriosclerosis 4, 357-364 [Abstract]
  4. Quinn, M. T., Parthasarathy, S., and Steinberg, D. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5949-5953 [Abstract]
  5. Cathcart, M. K., McNally, A. K., Morel, D. W., and Chisolm, G. M. (1989) J. Immunol. 142, 1963-1969 [Abstract/Free Full Text]
  6. Hessler, J. R., Morel, D. W., Lewis, L. J., and Chisolm, G. M. (1983) Arteriosclerosis 3, 215-222 [Abstract]
  7. Cathcart, M. K., McNally, A. K., and Chisolm, G. M. (1991) J. Lipid Res. 32, 63-70 [Abstract]
  8. Quinn, M. T., Parthasarathy, S., Fong, L. G., and Steinberg, D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2995-2998 [Abstract]
  9. Brown, M. S., and Goldstein, J. L. (1983) Annu. Rev. Biochem. 52, 223-261 [CrossRef][Medline] [Order article via Infotrieve]
  10. Endemann, G., Stanton, L. W., Madden, K. S., Bryant, C. M., White, R. T., and Protter, A. A. (1993) J. Biol. Chem. 268, 11811-11816 [Abstract/Free Full Text]
  11. Stanton, L. W., White, R. T., Bryant, C. M., Protter, A. A., and Endemann, G. (1992) J. Biol. Chem. 267, 22446-22451 [Abstract/Free Full Text]
  12. Ottnad, E., Parthasarathy, S., Sambrano, G. R., Ramprasad, M. P., Quehenberger, O., Kondratenko, N., Green, S., and Steinberg, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1391-1395 [Abstract]
  13. Yla-Herttuala, S., Palinski, W., Rosenfeld, M. E., Parthasarathy, S., Carew, T. E., Butler, S., Witztum, J. L., and Steinberg, D. (1989) J. Clin. Invest. 84, 1086-1095 [Medline] [Order article via Infotrieve]
  14. Hoff, H. F., and O'Neil, J. (1991) Arterioscler. Thromb. 11, 1209-1222 [Abstract]
  15. Jurgens, G., Hoff, H. F., Chisolm, G. M., and Esterbauer, H. (1987) Chem. Phys. Lipids 45, 315-336 [CrossRef][Medline] [Order article via Infotrieve]
  16. Li, Q., Tallant, A., and Cathcart, M. K. (1993) J. Clin. Invest. 91, 1499-1506 [Medline] [Order article via Infotrieve]
  17. Li, Q., and Cathcart, M. K. (1994) J. Biol. Chem. 269, 17508-17515 [Abstract/Free Full Text]
  18. Nishizuka, Y. (1992) Science 258, 607-614 [Medline] [Order article via Infotrieve]
  19. Dennis, E. A. (1994) J. Biol. Chem. 269, 13057-13060 [Free Full Text]
  20. Henderson, L. M., Moule, S. K., and Chappell, J. B. (1993) Eur. J. Biochem. 211, 157-162 [Abstract]
  21. Kadri-Hassani, N., Leger, C. L., and Descomps, B. (1995) J. Biol. Chem. 270, 15111-15118 [Abstract/Free Full Text]
  22. Morel, F., Doussiere, J., and Vignais, P. V. (1991) Eur. J. Biochem. 201, 523-546 [Abstract]
  23. Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630 [Abstract/Free Full Text]
  24. Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233 [Abstract/Free Full Text]
  25. Hirashima, Y., Farooqui, A. A., Mills, J. S., and Horrocks, L. A. (1992) J. Neurochem. 59, 708-714 [Medline] [Order article via Infotrieve]
  26. Johnston, R. B. (1981) in Methods for Studying Mononuclear Phagocytes (Adams, D. O., Edelson, P. J., and Koren, H., eds), pp. 489-497, Academic Press, New York
  27. Schuh, J., Fairclough, G. F., and Haschemeyer, R. H. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 3173-3177 [Abstract]
  28. El-Saadani, M., Esterbauer, H., El-Sayed, M., Goher, M., Nassar, A. Y., and Jurgens, G. (1989) J. Lipid Res. 30, 627-630 [Abstract]
  29. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269-278 [Medline] [Order article via Infotrieve]
  30. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  31. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  32. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  33. Jaeger, J. A., Turner, D. H., and Zuker, M. (1989) Methods Enzymol. 183, 281-306
  34. Shirhatti, V., and Krishna, G. (1985) Anal. Biochem. 147, 410-418 [Medline] [Order article via Infotrieve]
  35. Folcik, V. A., and Cathcart, M. K. (1993) J. Lipid Res. 34, 69-79 [Abstract]
  36. Pick, E., and Mizel, D. (1981) J. Immunol. Methods 46, 211-226 [CrossRef][Medline] [Order article via Infotrieve]
  37. Street, I. P., Lin, H.-K., Laliberte, F., Ghomashchi, F., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940 [Medline] [Order article via Infotrieve]
  38. Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) J. Biol. Chem. 270, 445-450 [Abstract/Free Full Text]
  39. Ackermann, E. J., and Dennis, E. A. (1995) Biochim. Biophys. Acta 1259, 125-136 [Medline] [Order article via Infotrieve]
  40. Roshak, A., Sathe, G., and Marshall, L. A. (1994) J. Biol. Chem. 269, 25999-26005 [Abstract/Free Full Text]
  41. Locati, M., Lamorte, G., Luini, W., Introna, M., Bernasconi, S., Mantovani, A., and Sozzani, S. (1996) J. Biol. Chem. 271, 6010-6016 [Abstract/Free Full Text]
  42. Bedwell, S., Dean, R. T., and Jessup, W. (1989) Biochem. J. 262, 707-712 [Medline] [Order article via Infotrieve]
  43. Dennis, E. A., Ackermann, E. J., Deems, R. A., and Reynolds, L. J. (1995) Adv. Prostaglandin Thromboxane Leukotriene Res. 23, 75-80 [Medline] [Order article via Infotrieve]
  44. Miyake, R., and Gross, R. W. (1992) Biochim. Biophys. Acta 1165, 167-176 [Medline] [Order article via Infotrieve]
  45. Murakami, M., Kudo, I., Umeda, M., Matsuzawa, A., Takeda, M., Komada, M., Fujimori, Y., Takahashi, K., and Inoue, K. (1992) J. Biochem. (Tokyo) 111, 175-181 [Abstract]
  46. Jones, S. S., Tang, J., Kriz, R., Shaffer, M., Knopf, J., and Seehra, J. (1996) FASEB J. 10, L15 (abstr.)

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