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The Journal of Immunology, 2001, 167: 3414-3421.
Copyright © 2001 by The American Association of Immunologists

Calcium-Independent Phospholipase A2 Is Required for Human Monocyte Chemotaxis to Monocyte Chemoattractant Protein 11

Kevin A. Carnevale and Martha K. Cathcart

Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A2 (cPLA2) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA2, calcium-independent PLA2 (iPLA2) in comparison to cPLA2. Pharmacological inhibitors of PLA2 were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA2 expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA2 or cPLA2 expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA2-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA2-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA2-deficient monocytes while having no effect on the iPLA2-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA2 and cPLA2 represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocyte migration into the intima of an arterial wall is thought to be one of the key initial steps in atherogenesis. Recruitment of monocytes from the peripheral blood is a multistep process in which locally produced chemokines are believed to play a crucial role (1).

Monocyte chemoattractant protein 1 (MCP-1)3 is a member of the CC or {beta} chemokine subfamily and exhibits its most potent chemotactic activity toward monocytes. MCP-1 binds to the CCR2 and recently identified CCR11 receptors (2). It plays an important role in inflammation and the inflammatory response. MCP-1 has also been associated with a series of human inflammatory diseases including rheumatoid arthritis, viral meningitis, psoriasis, and inflammatory bowel disease (3).

The importance of MCP-1 in inflammation has been determined by numerous knockout and transgenic studies focusing on the inflammatory response and infection in mice (4, 5, 6, 7, 8, 9, 10). CCR2-deficient mice have a severe reduction in leukocyte adhesion and extravasation (4, 5, 6). Conversely, MCP-1-transgenic mice with tissue-specific promoters developed considerable increases in monocyte infiltration into tissues in which MCP-1 expression was detected (7, 8). MCP-1-deficient mice or mice overexpressing MCP-1 have also been shown to have altered Th1 and/or Th2 responses (11, 12, 13). These in vivo studies demonstrate how MCP-1 and its receptor are important in the inflammatory response involving monocytes and T lymphocytes.

Furthermore, there is growing evidence that MCP-1 plays an important role in atherogenesis (9, 10, 14, 15). Two well-documented atherogenic stimuli such as modified low-density lipoprotein (16) and fluid shear stress (17) induce MCP-1 expression. The cellular components of the arterial wall, including endothelial cells and smooth muscle cells, secrete MCP-1 (18, 19, 20), and MCP-1 is up-regulated in human atherosclerotic plaques (21). These studies indirectly support a role for MCP-1 in atherogenesis, however, direct evidence was recently obtained with double-knockout mice fed Western diets. Low density lipoprotein receptor/MCP-1-deficient mice fed a Western diet had 83% less plaque formation throughout the aorta as compared with controls (9). Mice lacking apolipoprotein E and the CCR2 receptor also showed significant reduction in the size of atherosclerotic lesions covering the aorta after 13 wk on a Western diet (10). Finally, apolipoprotein E-deficient mice with macrophages overexpressing the MCP-1 transgene developed increased atherosclerotic lesions in their aorta and aortic valve regions (22). These in vivo studies provide strong direct evidence that MCP-1 is important in atherogenesis.

CCR2 is one of numerous serpentine receptors characterized by seven-transmembrane domains and coupled to a GTP binding protein (23). When MCP-1 binds to CCR2 it induces a rapid (15 s) and transient (15 min) release of [3H]arachidonic acid (AA) from radiolabeled human monocytes (24, 25). This effect was inhibited by Bordetella pertussis toxin treatment, was dependent on the influx of extracellular calcium, and was increased in a synergistic fashion by platelet-activating factor. Also, general pharmacological inhibitors of PLA2 such as mepacrine, p-bromophenacyl bromide, and manoalide inhibited monocyte chemotaxis to MCP-1 (24). In addition, human monocyte chemotaxis to MCP-1 and AA release were inhibited by an antisense oligonucleotide specific for cytosolic phospholipase A2 (cPLA2) (26). These results suggest a role for PLA2 products, AA, and/or lysolipids, as second messengers in monocyte migration to MCP-1.

PLA2 enzymes hydrolyze the fatty acyl group from the sn-2 position of phospholipid with the concomitant production of lysophospholipid. Cells of monocyte lineage have three different PLA2: 1) secretory, referred to as sPLA2; 2) cPLA2; and 3) calcium independent (iPLA2). sPLA2 is a low-molecular-mass (14 kDa) secreted enzyme that requires millimolar concentrations of calcium for its catalytic activity and does not show a selectivity for fatty acid esterification at the sn-2 position (27, 28). cPLA2 is an 85-kDa protein that requires nanomolar to micromolar concentrations of calcium, is the only PLA2 identified that has selectivity for sn-2 AA, and plays an important role in agonist-induced AA release (29, 30). iPLA2 is an 85-kDa protein that requires no calcium for its catalytic activity. iPLA2 has no acyl specificity and has been suggested to function in the steady-state remodeling of phospholipid fatty acyl groups (31).

In this paper we report that selective pharmacologic inhibition of iPLA2 substantially and significantly decreased the chemotactic response of human monocytes to MCP-1. With the use of specific antisense oligodeoxyribonucleotides (ODN), we were able to specifically inhibit expression of iPLA2 and confirmed its importance in this monocyte chemotactic response. Our results indicate requisite roles for both iPLA2 and cPLA2 enzymes. To evaluate whether these phospholipases contribute redundant or unique functions, we examined the ability of PLA2 products to restore migration when these enzymes were selectively inhibited. Our findings from these studies suggest that iPLA2 and cPLA2 perform distinct functional roles in regulating the monocyte chemotactic response to MCP-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Aristolochic acid, ONO-RS-082 (2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid), AACOCF3 (arachidonyl trifluoromethyl ketone), and BEL (E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2h-pyran-2-one) were purchased from Biomol (Plymouth Meeting, Pa). Each of these reagents was dissolved in DMSO as 100-fold stock solutions and stored at -20°C before use. Human MCP-1 obtained from BD PharMingen (San Diego, CA) was diluted to 50 µg/ml with Dulbecco’s PBS containing 1 mg/ml BSA as a 1000-fold stock solution and stored at -80°C. MCP-1 was used at 50 ng/ml to attract human monocytes. Lysophosphatidic acid (LPA), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC), phosphatidylcholine, and phosphatidic acid (PA) were purchased from Avanti Polar Lipids (Alabaster, AL), whereas AA, linoleic acid, and palmitic acid were purchased from Sigma (St. Louis, MO). LPA was dissolved in distilled deionized H2O, and all other lipids were dissolved in ethanol at 1 M concentrations and stored under N2 gas.

Isolation of human monocytes and cell culture

Human monocytes were isolated from heparinized whole blood by sequential centrifugation over a Ficoll-Paque density solution, removal of platelets, and adherence to tissue culture flasks precoated with bovine calf serum (BCS) (32, 33). Nonadherent cells were removed. The adherent cells were released with 5 mM EDTA, washed twice with PBS, and added to polypropylene tubes at 2 x 106 cells/ml. This cell population consisted of greater than 95% monocytes (33). The isolated monocytes were usually rested for 1 h in DMEM with 10% BCS at 37°C in 10% CO2 before use in experiments.

Treatment of cells with pharmacologic inhibitors

Monocytes were washed once in PBS and resuspended in DMEM without serum. The cells were treated with the pharmacologic inhibitor and incubated for 1 h at 37°C with 10% CO2 before performing the chemotaxis assay, enzyme activity assay, or flow cytometry.

Treatment of cells with ODN

The antisense ODNs used in our studies were derived from prior publications reporting their efficacy. The cPLA2 sequence was complementary to nucleotides 219–238 of cPLA2, coding for amino acids 27–34 of the protein. The sequence was 5'-CCC CCT TTG TCA CTT TGG TG-3'. The sequence of the cPLA2 sense ODN was 5'-CAC CAA AGT GAC AAA GGG GG-3' (34). The iPLA2 antisense ODN corresponded to nucleotides 59–78 in the murine group VI iPLA2 sequence (35). We confirmed that this region was conserved in human group VI iPLA2. The sequence was 5'-CTC CTT CAC CCG GAA TGG GT-3'. As a control, the iPLA2 sense ODN sequence was 5'-ACC CAT TCC GGG TGA AGG AG-3'. Phosphorothioate-modified ODNs were used for these studies to limit degradation. Importantly, the oligonucleotides were purified by HPLC before use (Sigma-Genosys, The Woodlands, TX).

For these experiments, human monocytes (0.4 ml of 2 x 106 cells/ml in each tube) were cultured in DMEM with 10% BCS in the presence or absence of different concentrations of sense or antisense ODNs in polypropylene tubes (Falcon; BD Labware, Lincoln Park, NJ). Cells were incubated for 24 h at 37°C with 10% CO2 before performing the chemotaxis assay, enzyme activity assay, or flow cytometry.

Western blotting analysis

Human monocytes (8 x 105 cells/0.4 ml/tube, in seven tubes; total cells = 5.6 x 106) were incubated in polypropylene tubes with sense or antisense ODN for either cPLA2 or iPLA2 for 24 h in DMEM with 10% BCS. Subsequently, the cells were washed three times with PBS to remove traces of DMEM and 10% BCS. The tubes were placed on ice, and the cells were lysed using 200 µl of lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCL, pH 7.4, 1 mM PMSF, and 10 µl of protease inhibitor mix (Sigma) per milliliter of lysis buffer). After 30 min, the lysate was centrifuged for 15 min at 9300 x g. The supernatant was collected, and the protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA) and loaded on a 7.5% SDS-PAGE gel (150 µg of lysate/well). The proteins were transferred to a polyvinylidene difluoride membrane (0.2 µm; Bio-Rad) using a TRANS-BLOT SD electrophoretic transfer cell (Bio-Rad). The membrane was blocked in 5% nonfat milk in PBS and 1% Tween 20 overnight at 4°C and then probed with primary Ab. cPLA2 protein was detected with a 1/1000 dilution of anti-human recombinant cPLA2 mAb (generously provided by the Bioanalytical Science Department of Genetics Institute, Andover, MA), followed by incubation with anti-mouse IgG HRP (1/1000 dilution; Transduction Laboratories, Lexington, KY). This Ab recognizes a distinct cPLA2 protein band running at ~110 kDa and comigrates with recombinant cPLA2 (34).

iPLA2 protein was detected with a 1/2000 dilution of rabbit polyclonal Ab to Escherichia coli inclusion body-denatured iPLA2 (also generously provided by Bioanalytical Science Department of Genetics Institute, and purchased from Upstate Biotechnology, Lake Placid, NY) or by a rabbit polyclonal Ab to a synthetic peptide of Chinese hamster ovary iPLA2 (1/1000; Cayman Chemical, Ann Arbor, MI) followed by incubation with goat anti-rabbit IgG Fc HRP (1/1000; Pierce, Rockford, IL). The hybridization signals were detected using ECL detection reagents (Pierce) according to the manufacturer’s guide, and followed by autoradiography. These Abs recognized a band at the predicted migration of 85 kDa. This band was the predominant band and was not detected when an irrelevant primary Ab was used.

Treatment of cells with fatty acids and phospholipids

After a 24-h treatment with ODN, various concentrations of fatty acids or lysolipids were added to cells and incubated for 1 h at 37°C with 10% CO2 before performing the chemotaxis assay. The fatty acids and phospholipids were added as 250x stocks made up in ethanol (for arachidonic, palmitic, linoleic, lysophosphatidylcholine, PA) or water (for LPA).

iPLA2 activity assay

After treatment of monocytes, 1 x 107 cells were washed three times in PBS and sonicated (3 x 15 s cycles) in 500 µl of buffer containing 10 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM DTT, and 0.34 M sucrose. The Bradford assay (Bio-Rad) was performed to determine protein concentration. The substrate was prepared by previously described methods (35). Dipalmitoyl phosphatidylcholine (100 µM) and 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphocholine (300,000 cpm/assay) were evaporated to dryness under a N2 stream and then 250 µl of buffer containing 800 µM Triton X-100, 10 mM EDTA, and 200 mM HEPES (pH 7.5) was added. Micelles were then formed by a combination of heating above 40°C, vortexing, and water bath sonicating until the solution was clarified.

The reaction was conducted as previously described (36). Cell lysates (200 µg) were mixed with substrate and 0.8 mM ATP and brought up to a final volume of 500 µl. The samples were then incubated at 40°C for 1 h. After incubation, the assay was quenched by adding 1 ml of chloroform/methanol/acetic acid (2:4:1, v/v/v), followed by the addition of 0.5 ml of chloroform and 0.5 ml of water. The chloroform layer was dried under a stream of N2, resuspended in chloroform/methanol (2:1, v/v), and spotted onto a Silica Gel G TLC plate. The lipids were separated by a solvent composed of chloroform/methanol/acetic acid/water (25:15:4:2, v/v/v/v). The lipids were visualized with iodine vapor, and the zones corresponding to fatty acid and dipalmitoyl phosphatidylcholine (as determined by the migration of purified standards) were scraped and counted. Normalized activity represents cpm of free fatty acid divided by the cpm of free fatty acid plus the cpm of the dipalmitoyl phosphatidylcholine times the total added counts (300,000 cpm). This calculation was performed to correct for any variability in the efficiency of lipid extraction between samples.

cPLA2 activity assay

This assay was performed according to published protocols with the exception that the sonicated lysate was not centrifuged before assay performance (34).

Chemotaxis assay

Monocyte migration was evaluated using a microchamber technique (37). Human recombinant MCP-1 (50 ng/ml) in DMEM with 0.1% BSA was added to the lower compartment of the disposable 96-well chemotaxis chamber (NeuroProbe, Gaithersburg, MD) in a volume totaling 29 µl. The cell suspension (50 µl of 2 x 106 cells/ml; 1 x 105 cells/well) was added to the upper compartment of the chamber that had been precoated with BCS for 2 h. The two compartments were separated by a 5-µm pore size, polycarbonate, polyvinylpyrrolidone-free filter. The chamber was incubated at 37°C in air with 10% CO2 for 90 min. At the end of the incubation, the filter facing the upper compartment was scraped with a sponge and rinsed gently with PBS to remove all nonmigrated cells. The side of the filter with the migrated cells was fixed and stained with Hema 3 Stain Set (Biochemical Science, distributed by Fisher Scientific, Pittsburgh, PA). Migrated monocytes were counted in five high-power fields (x400) using a light microscope. All samples were tested in triplicate, and the data are expressed as the mean ± SD.

Flow cytometry

After isolation, 2 x 106 suspended human monocytes were washed in 1 ml of PBS with 3% BSA. The PBS was removed and the cells were resuspended in 10 µl of PE-labeled monoclonal mouse anti-human CCR2 IgG2b Ab (R&D Systems, Minneapolis, MN) and incubated for 30 min at room temperature. PE-labeled anti-human CD20 and CD3 IgG2b Abs (BD PharMingen) were used as a negative control. After incubation, the cells were washed three times in PBS and centrifuged at 200 x g. Labeled cells were detected using FACScan (BD Biosciences, San Jose, CA) in which 10,000 cells were analyzed in each gated event using CellQuest software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aristolochic acid and ONO-RS-082, general PLA2 inhibitors, both inhibited migration of human monocytes to MCP-1 (Fig. 1Go). Aristolochic acid showed significant inhibition at all three doses (Fig. 1GoA) with the greatest being 80.7% at 100 µM (Fig. 1GoA). ONO-RS-082 inhibition was significant and dose-dependent with 88.9% inhibition at 10 µM (Fig. 1GoB). We next tested more selective inhibitors that have not previously been examined for their effects on monocyte chemotaxis to MCP-1. AACOCF3 has been reported to inhibit both cPLA2 and iPLA2, but not sPLA2 (38, 39). AACOCF3 caused significant, dose-dependent inhibition of monocyte migration to MCP-1 with complete inhibition of migration at 50 µM (Fig. 1GoC).



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  		FIGURE 1. Pharmacological inhibitors of PLA2 suppress human monocyte chemotaxis. Monocytes were cultured in DMEM without serum and in the presence or absence of various concentrations of aristolochic acid (A), ONO-RS-082 (B), or AACOCF3 (C) for 1 h. Monocyte chemotaxis across a polycarbonate filter in response to MCP-1 (50 ng/ml) was then measured. All samples were performed in triplicate, and experiments were repeated three times. The results are expressed as the mean number of migrated monocytes in five high-power fields ± SD (x400 light microscope) and are from a representative experiment. Percent inhibition is shown in parenthesis. *, p < 0.05; **, p < 0.005 from unpaired t tests. {blacksquare}, Number of monocytes migrating in response to MCP-1; {blacktriangleup}, basal migration of monocytes in the absence of MCP-1.

 
BEL is a selective inhibitor of iPLA2 that does not affect cPLA2 or sPLA2 activities (38, 39, 40). BEL significantly inhibited iPLA2 activity by 66 and 94.8% at concentrations of 0.1 and 1 µM, respectively (Fig. 2GoA). At these concentrations, BEL also significantly suppressed MCP-1-stimulated migration by 63.5 and 87.1%, respectively (Fig. 2GoB). It should be noted that the level of activity in the no lysate control is due to contaminating free fatty acid in the substrate preparation and does not represent phospholipase activity.



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  		FIGURE 2. BEL inhibits iPLA2 activity and MCP-1-stimulated chemotaxis of human monocytes. A, Monocytes were incubated with 0.1 and 1 µM of BEL in serum-free DMEM for 1 h, and then iPLA2 activity was assayed in duplicate samples of cell lysates. Substrate incubated without lysate is labeled NL, and lysates from untreated cells represent positive controls (Con). Percent inhibition is shown in parenthesis. *, p < 0.05; **, p < .005 from paired t tests. B, Monocytes were cultured in serum-free DMEM and different concentrations of BEL for 1 h, and then monocyte chemotaxis in response to MCP-1 (50 ng/ml) was evaluated. All samples were examined in triplicate, and experiments were repeated three times. The results are expressed as the mean number of migrated monocytes in five high-power fields ± SD (x400 light microscope) and are from a representative experiment. {blacksquare}, Number of monocytes migrating in response to MCP-1; {blacktriangleup}, basal migration of monocytes in the absence of MCP-1. Percent inhibition is shown in parenthesis. *, p < 0.05; **, p < 0.005 from unpaired t tests.

 
Because BEL caused inhibition of chemotaxis, this strongly suggested that iPLA2 was playing a role in monocyte migration to MCP-1. To test this more rigorously, antisense ODN to iPLA2 was evaluated for effects on MCP-1-stimulated monocyte migration. iPLA2 antisense ODN reduced iPLA2 protein by ~90% (Fig. 3GoA, top). Compiled data from five experiments indicated a range of inhibition between 75 and 100% and a mean percent inhibition of 91% as determined by densitometry (data not shown). This level of protein inhibition is greater than that observed in the original report by Balsinde et al. (35) using this conserved antisense ODN in murine cells. The blots were stripped and reprobed with cPLA2 Ab. Antisense ODN to iPLA2 did not inhibit the expression of cPLA2 protein (Fig. 3GoA, bottom). Thus the antisense ODN inhibition was selective for iPLA2.



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  		FIGURE 3. iPLA2 antisense ODN inhibits iPLA2 protein expression, iPLA2 activity, and MCP-1-stimulated chemotaxis of human monocytes. A, Monocytes were incubated with no ODN, 5 µM iPLA2 sense ODN (S), or 5 µM antisense (AS) ODN at 37°C for 24 h. Total cytoplasmic protein (100 µg) was evaluated by Western blot for iPLA2 protein (top). The same blot was stripped and reprobed with Ab to cPLA2 (bottom). B, Monocytes were incubated with iPLA2 sense (S, hatched bars) or antisense (AS, cross-hatched bars) ODN at 37°C for 24 h in DMEM with 10% BCS, and then iPLA2 activity was assayed in duplicate samples of cell lysates. The data are expressed as the mean ± SEM from three experiments. Substrate incubated without lysate is labeled NL, and lysates from untreated cells represent positive controls (Con). Percent inhibition is shown in parenthesis. **, p < 0.005 from paired t tests. C, Monocytes were cultured in DMEM with 10% BCS and two different concentrations (1 and 5 µM) of iPLA2 sense (hatched bars) or antisense (cross-hatched bars) ODN. Monocyte chemotaxis to MCP-1 (50 ng/ml) was then measured. All samples were run in triplicate. Results are expressed as the mean number of migrated monocytes ± SD in five high-power fields (x400 light microscope) from a representative experiment of three that were performed. Percent inhibition is shown in parenthesis. *, p < 0.05; **, p < 0.005 from paired t tests.

 
The effect of inhibition of iPLA2 expression on iPLA2 activity assays was also assessed. iPLA2 antisense ODN significantly reduced iPLA2 activity by greater than 95% at 5 µM (Fig. 3GoB). Again, it should be noted that the level of activity in the no lysate control is due to contaminating free fatty acid in the substrate preparation. iPLA2 activity, which was significant in monocytes in the absence of MCP-1 (see "Con" group in Fig. 3GoB), was induced 2-fold by exposure to MCP-1 (Fig. 4GoA). Treatment with iPLA2 antisense inhibited both basal and induced iPLA2 activity while having no inhibitory effect on the activity of cPLA2 (Fig. 4GoA). Thus the decrease in protein expression correlates with decreased enzymatic activity.



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  		FIGURE 4. iPLA2 and cPLA2 activities are inhibited by their respective antisense ODN. Monocytes were incubated at 37°C with 10% CO2 for 24 h in DMEM with 10% BCS in the presence of 5 µM iPLA2 or 10 µM cPLA2 sense ODN (hatched bars) or antisense ODN (cross-hatched bars). Monocytes were either treated with 50 ng/ml MCP1 (+) or untreated (-) for each group. iPLA2 activity (A) and cPLA2 activity (B) were assayed in duplicate samples of cell lysates. Substrate incubated without lysate is labeled NL.

 
iPLA2 antisense ODN was next evaluated for its effect on monocyte chemotaxis to MCP-1. Doses of antisense ODN that were effective in inhibiting protein expression and enzymatic activity were also found to totally inhibit MCP-1-stimulated monocyte chemotaxis, whereas iPLA2 sense ODN caused no inhibition (Fig. 3GoC).

We next evaluated whether certain products of PLA2 activity could restore monocyte migration when iPLA2 activity was inhibited by antisense ODN treatment. Numerous fatty acids and lysophospholipids were tested. Addition of AA did not influence the chemotactic response of iPLA2-deficient monocytes (Fig. 5GoA), however, LPA restored the MCP-1-stimulated chemotactic response of iPLA2-deficient monocytes at 50 µM and above (Fig. 5GoB). Importantly, LPA did not induce migration on its own. Addition of other free fatty acids, linoleic and palmitic, or addition of LPC or PA did not restore the chemotactic response of iPLA2-deficient monocytes (data not shown). LPA was the only lipid tested that restored monocyte migration to MCP-1 in monocytes with diminished iPLA2 expression.



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  		FIGURE 5. LPA restores monocyte chemotaxis in iPLA2-deficient monocytes, whereas AA was without effect. Monocytes were cultured for 24 h with 5 µM iPLA2 sense ODN (S) or antisense ODN (AS). Different concentrations of AA (A) or LPA (B) were added 1 h before the chemotaxis assay. Monocyte chemotaxis across a polycarbonate filter in response to no MCP-1 (open bars) or 50 ng/ml MCP-1 (filled bars) was then assessed. Filled bars were not treated with ODN. Cross-hatched bars represent the chemotactic response of monocytes that were treated with antisense ODN. Hatched bars represent the chemotactic response of monocytes that were treated with sense ODN. All samples were tested in triplicate. The results are expressed as the mean number of migrated monocytes in five high-power fields ± SD (x400 light microscope) and are from a representative study of three performed.

 
Because cPLA2 had been previously reported to participate in monocyte chemotaxis to MCP-1, we designed an experiment to confirm this finding and then to extend the prior work by examining the ability of PLA2 products to restore migration in the cPLA2-deficient monocytes. First we confirmed that we could create cPLA2-deficient cells using antisense ODN. Antisense ODN treatment resulted in decreasing cPLA2 protein expression by greater than 90% at 10 µM (Fig. 6GoA, top). The blot was stripped and reprobed with iPLA2 Ab to demonstrate the specificity of the inhibition. iPLA2 protein was not reduced when monocytes were treated with 10 µM of cPLA2 antisense ODN (Fig. 6GoA, bottom).



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  		FIGURE 6. cPLA2 antisense ODN inhibits cPLA2 protein expression and MCP-1-stimulated chemotaxis of human monocytes. A, Monocytes were incubated with no ODN, 10 µM cPLA2 sense ODN, or 10 µM antisense ODN at 37°C for 24 h. Total cytoplasmic protein (100 µg) was evaluated by Western blot for cPLA2 protein (top). The same blot was stripped and reprobed with iPLA2 Ab (bottom). B, Monocytes were cultured in DMEM with 10% BCS and 5 and 10 µM of cPLA2 sense (hatched bars) or antisense (cross-hatched bars) ODN for 24 h. Monocyte chemotaxis across a polycarbonate filter in response to MCP-1 (50 ng/ml) was then measured. All samples were run in triplicate. The results are expressed as the mean number of migrated monocytes ± SEM in five high-power fields (x400 light microscope) and are compiled results from three similar experiments. Percent inhibition is shown in parenthesis. *, p < 0.05; **, p < 0.005 from paired t tests.

 
cPLA2 antisense ODN was also evaluated for its effect on PLA2 activities (Fig. 4Go). We have previously demonstrated that cPLA2 antisense ODN effectively inhibits cPLA2 activity (34). Basal levels of cPLA2 activity are extremely low; however, activity was stimulated 25-fold by exposure of the monocytes to MCP-1. Our results confirm that cPLA2 antisense ODN treatment caused substantial inhibition of cPLA2 enzyme activity (Fig. 4GoB). We also examined the effect on iPLA2 activity and found that cPLA2 antisense ODN treatment had little or no effect on iPLA2 activity (Fig. 4GoA).

cPLA2 antisense ODN also inhibited monocyte chemotaxis to MCP-1 at 5 and 10 µM showing 75.3 and 100% inhibition, respectively (Fig. 6GoB), whereas cPLA2 sense ODN had little to no inhibitory effect at these concentrations (Fig. 6GoB). In contrast to results obtained with iPLA2-deficient monocytes, when AA was added back at various concentrations (0.3, 1.5, and 3 µM) to see whether it would restore the chemotaxis of cPLA2-deficient monocytes; it fully restored chemotaxis at 3 µM (Fig. 7GoA). Furthermore, LPA had no effect, even at 100 µM (Fig. 7GoB). Adding back other lipids such as LPC, PA, linoleic acid, and palmitic acid had no effect on restoring the chemotactic response of cPLA2-deficient monocytes (data not shown).



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  		FIGURE 7. AA restores monocyte chemotaxis in cPLA2-deficient monocytes, whereas LPA was without effect. Monocytes were cultured for 24 h with 10 µM cPLA2 sense ODN (S) or antisense ODN (AS). Different concentrations of AA (A) or LPA (B) were added 1 h before the chemotaxis assay. Monocyte chemotaxis in response to no MCP-1 (open bars) or 50 ng/ml MCP-1 (filled bars) was then assessed. Filled bars were not treated with ODN. Cross-hatched bars represent the chemotactic response of monocytes that were treated with antisense ODN. Hatched bars represent the chemotactic response of monocytes that were treated with sense ODN. All samples were run in triplicate. The results are expressed as the number of migrated monocytes in five high-power fields ± SD (x400 light microscope) and are from a representative study of three performed.

 
One possible mechanism for inhibiting chemotaxis to MCP-1 might be through the inhibition of CCR2 expression. We measured CCR2 expression on monocytes exposed to each of the antisense and sense ODN reagents, AACOCF3 and BEL. CCR2 expression was detected in human monocytes, and expression was not substantially modulated by any of these treatments (Fig. 8Go).



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  		FIGURE 8. CCR2 levels are not modulated by the different ODNs or by BEL or AACOCF3. Flow cytometry was performed on human monocytes labeled with PE-tagged Abs to CD20 (A) and CCR2 (B–H). Monocytes were treated for 24 h with 5 µM iPLA2 sense ODN (C), 5 µM iPLA2 antisense ODN (D), 10 µM cPLA2 sense ODN (E), or 10 µM cPLA2 antisense ODN (F) at 37°C with 10% CO2 for 24 h in DMEM with 10% BCS before labeling. Monocytes were also treated with 1 µM BEL (G) and 50 µM AACOCF3 (H) at 37°C with 10% CO2 for 1 h in DMEM before labeling.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Relatively little is known about the regulation of monocyte chemotaxis to MCP-1. The majority of studies have used pharmacologic inhibitors to understand possible pathways that may be involved. Some pathways that have been implicated as regulators of monocyte chemotaxis to MCP-1 include phosphatidylinositol 3 kinase, mitogen-activated protein kinases, protein kinase C, and PLA2 (23, 41, 42, 43). Although studies from knockout mice indicate the importance of phosphatidylinositol 3 kinase in macrophage chemotaxis to a variety of stimuli, chemotaxis to MCP-1 was not evaluated (44). One protein that has been specifically inhibited using antisense oligonucleotides and evaluated for its effect on MCP-1-induced monocyte chemotaxis is cPLA2 (26). The results from our experiments are the first report that iPLA2 is a key regulator of monocyte chemotaxis to MCP-1. Therefore, based on prior studies and our results presented here, two PLA2 enzymes are required for this important inflammatory event (Figs. 3GoC and 6B). The expression of CCR2 does not appear to be regulated by either of these enzymes, thus alternative regulatory pathways should be considered (Fig. 8Go).

It has been suggested that iPLA2 plays an important role in phospholipid remodeling of membranes (31). In the remodeling pathway, preformed diacylglycero-phosphatides (phospholipids) are acted on by iPLA2 to produce their respective monoacylglycerol-phosphatides (lysophospholipids) (45). Inhibition of iPLA2 expression by antisense ODN has been reported to decrease both steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate AA into membrane phospholipids, whereas iPLA2 antisense had no effect on platelet-activating factor-stimulated AA release in P388D1 macrophages (35). In contrast, induction of AA release in zymosan-stimulated P388D1 macrophages was in part due to iPLA2 (46). Therefore, iPLA2 is thought to be involved in phospholipid remodeling in membranes and also AA release in certain situations.

LPA but not AA fully restored the MCP-1-stimulated chemotaxis of iPLA2-deficient monocytes (Fig. 5Go). We hypothesize that phospholipid remodeling of membranes may be important for chemotaxis in which iPLA2 generation of LPA is playing a key role. LPA has been reported to enhance migration of a variety of cell types (47, 48); however, under the conditions of our experiments, LPA alone did not induce monocyte migration nor modify the MCP-1-induced chemotaxis of untreated monocytes. LPA has also been reported to be a chemotactic agent and to mediate a haptotactic response (49, 50, 51). We do not believe that this activity is contributing to our results because the LPA was present on the side of the filter with the monocytes and not on the side with the MCP-1. Therefore, if LPA was serving as a potent chemotactic agent it might have been expected to inhibit migration by keeping monocytes on the side of the filter where they were seeded. Additional evidence that the LPA effect is not due to its haptotactic activity is derived from the observation that PA was shown to cause equal haptotaxis (51), whereas PA was without effect in any of our chemotaxis experiments. Further evidence for specificity is that concentrations of LPA, severalfold higher than those used in our experiments to restore chemotaxis, have been shown to have no untoward morphologic changes on leukocytes as determined by transmission electron microscopy (50). We examined our cells by trypan blue exclusion and found no toxicity by these concentrations of any of the reagents used in these experiments. The exact monocyte function requiring iPLA2 that is essential for monocyte chemotaxis to MCP-1 deserves further investigation. Interestingly, iPLA2 has recently been implicated in regulating the actin cytoskeleton in preadipocytes. In these studies, BEL totally inhibited {alpha}2-adrenergic induced spreading of preadipocytes, and this inhibition was completely restored by LPA but not AA or other fatty acids (52).

It has previously been demonstrated that AA release and chemotaxis of human monocytes to MCP-1 are dependent on cPLA2 (26). We confirmed that cPLA2 is required for human monocyte chemotaxis to MCP-1. In our system cPLA2 antisense ODN inhibited cPLA2 protein by greater than 90% (Fig. 6GoA) and showed total inhibition of chemotaxis at this dose (Fig. 6GoB). This level of inhibition is consistent with our prior observations using this antisense ODN (34). Our novel finding presented in this manuscript is that addition of AA could completely circumvent the inhibitory effects of cPLA2 antisense ODN (Fig. 7GoA) thus suggesting that cPLA2 production of AA is essential in human monocyte migration to MCP-1. Remarkably, LPA had no effect on restoring the chemotactic response of cPLA2-deficient monocytes (Fig. 7GoB). Other fatty acids or phospholipids that were tested also had no effect on restoring this chemotactic response. Because eicosanoids are potent modulators of chemotaxis (53, 54, 55, 56), we hypothesize that cPLA2 may mobilize AA for eicosanoid production and thereby regulate chemotaxis; however, the role of eicosanoids in the process of chemotaxis of human monocytes to MCP-1 remains unknown.

The complementary yet distinct roles of these two phospholipases in regulating monocyte chemotaxis suggest that iPLA2 and cPLA2 may be located in different cellular compartments and controlling unique aspects of the monocyte response to MCP-1. Localization studies have been difficult to conduct because the location of the active enzymes, not inactive enzymes, is the critical issue. Alternatively, these two enzymes may have distinct substrate preferences allowing for discrete product formation. The activity assays reported in Fig. 4Go suggest that these two enzymes work in parallel and not in sequence, and thereby appear to represent key enzymes in a bifurcated signal transduction pathway. Future studies will focus on the unique contributions of the two lipid products that we have shown to provide essential regulatory functions in monocyte chemotaxis.

In summary, our studies are the first to find a critical role for iPLA2 in the chemotactic response of monocytes to MCP-1. Because LPA restored chemotaxis to iPLA2-deficient monocytes and AA restored chemotaxis to cPLA2-deficient monocytes, the requisite roles for these two enzymes appear to be substantially different. Our results indicate that iPLA2 and cPLA2 are separately and uniquely required for human monocyte chemotaxis to MCP-1. These findings suggest that iPLA2, as well as cPLA2, may serve as novel therapeutic targets in certain disease conditions where MCP-1 and monocyte migration are involved in the pathogenesis.


   Acknowledgments
 
We thank Dr. Bela Anand-Apte for advice on the chemotaxis assay, and Portia Payton and Hongmin Xu for help in counting cells and compiling data from the chemotaxis assays.


   Footnotes
 
1 Funding for these studies was derived from National Heart Blood Lung Institute, National Institutes of Health Grants HL51068 and HL61971 (to M.K.C.). Back

2 Address correspondence and reprint requests to Dr. Martha K. Cathcart, Department of Cell Biology/NC10, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: cathcam{at}ccf.org Back

3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein 1; cPLA2, cytosolic phospholipase A2; iPLA2, calcium-independent PLA2; ODN, oligodeoxyribonucleotide; LPA, lysophosphatidic acid; sPLA2, secretory PLA2; PA, phosphatidic acid; BCS, bovine calf serum; AA, arachidonic acid. Back

Received for publication September 21, 2000. Accepted for publication July 10, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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