(Received for publication, March 5, 1996, and in revised form, November 5, 1996)
From the Department of Cell Biology, Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Our previous studies have shown that monocyte
activation and release of O2 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
O
2 production by activated human monocytes. In parallel
experimental groups, cPLA2 sense oligonucleotides did not
affect cPLA2 protein expression, cPLA2
enzymatic activity, O
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.
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
(O2) 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 O2 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 O
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.
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--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.
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 CultureHuman 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 OxidationThe oxidation of LDL lipids was measured by both the thiobarbituric acid (TBA) assay and the lipid peroxide (LPO) assay.
Thiobarbituric Acid AssayThe 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 AssayThe 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 AssayHuman 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--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 -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.
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 OligonucleotidesThe 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 5CCC 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 AgentsThe 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 -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
![]() |
(Eq. 1) |
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 ProductionSuperoxide
anion (O2) production was measured by the cytochrome
c reduction assay (36). Cytochrome c can be
reduced by O
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 O
2 produced (where SOD is
superoxide dismutase).
![]() |
(Eq. 2) |
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.
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.
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.
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.
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.
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.
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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).
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.
Previously, our laboratory has shown that O2 production
is required for monocyte-mediated LDL lipid oxidation, and arachidonic acid has been shown to regulate the activity of the NADPH oxidase O
2 generating complex. We therefore examined whether
cPLA2 activity was essential for O
2
production. In this experiment, human monocytes were preincubated with
either cPLA2-specific antisense or sense oligonucleotides
for 24 h and then O
2 production was quantified in
response to activation. As expected, O
2 production was
increased upon monocyte activation as shown in Fig.
7A. Antisense oligonucleotide treatment
significantly inhibited O
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 O
2 production except to restore levels to normal in
antisense-treated, activated monocytes.
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.
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 O2 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 O2 production is inhibited by
suppression of cPLA2 activity (Fig. 7A).
Although numerous studies report that AA and phospholipase
A2 appear to regulate O
2 production, this is
the first report that specific suppression of cPLA2 protein
expression and enzymatic activity, using cPLA2-specific
antisense oligonucleotide treatment, inhibited O
2
production by activated human monocytes. The finding that
cPLA2 regulates LDL lipid oxidation is also novel. Interestingly, both O
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 O2 is required for
monocyte oxidation of LDL, yet it is clear from data from our
laboratory and others that O
2 alone is not sufficient for
LDL oxidation (42).2 Activated monocytes
must provide O
2 plus some unidentified cofactor for
oxidation to proceed. In this regard, it is interesting that the
addition of AA completely restored O
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 O
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
O2 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 O
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 O2 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 O
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 O2 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 O
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.
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.