©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Lysophospholipase Activity of the 85-kDa Phospholipase A and Activation in Mouse Peritoneal Macrophages (*)

(Received for publication, November 14, 1994; and in revised form, June 9, 1995)

Marianne G. S. de Carvalho Joanna Garritano Christina C. Leslie (§)

From the Division of Basic Science, Department of Pediatrics, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206 and the Department of Pathology, University of Colorado School of Medicine, Denver, Colorado 80262

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The regulation of the lysophospholipase activity of the 85-kDa cytosolic phospholipase A(2) (PLA(2)) was studied in vitro and in stimulated macrophages. Bovine serum albumin was found to inhibit lysophospholipase activity of the recombinant 85-kDa PLA(2) when assayed at a relatively low substrate concentration. Inhibition could be reversed if the substrate concentration was increased or if Ca was present in the assay. Incubation of recombinant enzyme with macrophage membranes and lipid extracts from macrophage membranes resulted in the release of arachidonic acid, as well as, stearic acid, which is enriched at the sn-1 position of macrophage phospholipids. This suggests that with a bilayer substrate the PLA(2) can sequentially deacylate the sn-2 then sn-1 acyl groups. This was verified by demonstrating that the phospholipids, phosphatidylcholine and phosphatidylinositol, were hydrolyzed to glycerophosphocholine and glycerophosphoinositol by incubation with recombinant 85-kDa PLA(2). The 85-kDa enzyme was identified as the main lysophospholipase activity in mouse peritoneal macrophage cytosols. Addition of Ca to the assay enhanced activity, but this effect decreased as the substrate concentration was increased. Incubation of macrophages with zymosan increased the lysophospholipase activity of the 85-kDa PLA(2) in cytosols. Phosphorylation of recombinant PLA(2) with mitogen-activated protein kinase resulted in an increase in lysophospholipase, as well as, PLA(2) activity. In macrophages stimulated with zymosan release of stearic acid (18:0) and palmitic acid (16:0) was observed in addition to arachidonic acid (20:4). These results are consistent with a role of the 85-kDa PLA(2) in regulating lysophospholipid levels in macrophages during zymosan stimulation.


INTRODUCTION

Lysophospholipids and arachidonic acid are produced by the action of phospholipase A(2) (PLA(2)) (^1)on membrane phospholipids during an inflammatory response. Arachidonic acid can be metabolized to a number of potent proinflammatory eicosanoids. In addition, arachidonic acid itself can act as a second messenger and modulate a number of cellular functions(1) . Lysophospholipids occur naturally at low levels (0.5-6%) in membranes of unstimulated cells and are produced transiently during phospholipid remodeling reactions (2, 3) . In stimulated cells some of the 1-alkyl-linked lysophosphatidylcholine produced is further metabolized into the inflammatory mediator platelet activating factor(4) . Recent studies suggest that lysophospholipids themselves can cause a variety of cellular responses such as changes in intracellular Ca and modulation of chemotaxis(5, 6, 7) . In addition, at higher concentrations lysophospholipids act as natural detergents and can disrupt membrane structure(3) . Accumulation of lysophospholipid can be controlled by reacylation to phospholipid or by metabolism to water soluble glycerophosphocholine by lysophospholipases. Multiple forms of lysophospholipases have been identified and frequently coexist in cells, suggesting there may be specific functional roles for individual lysophospholipases(8) . However, little is known about the mechanisms involved in lysophospholipase regulation.

The 85-kDa PLA(2) is a highly regulated enzyme that is now thought to play an important role in mediating arachidonic acid release in stimulated cells(9) . Interestingly, this enzyme also exhibits a relatively high lysophospholipase activity and a low level of transacylase activity(10, 11) . The lysophospholipase and PLA(2) activities share one catalytic domain(12) . Whether the 85-kDa PLA(2) acts as a lysophospholipase in vivo and whether its lysophospholipase activity is regulated during cell activation is not currently known. Simultaneous activation of both PLA(2) and lysophospholipase activities of the 85-kDa enzyme would provide an efficient mechanism for regulation of levels of arachidonic acid and lysophospholipid. There is now substantial evidence that both phosphorylation of the 85-kDa PLA(2) and Ca-dependent translocation to the membrane are necessary for activation of the PLA(2) resulting in arachidonic acid release(13) . In this study we have examined whether the lysophospholipase activity of the 85-kDa PLA(2) is regulated in zymosan-stimulated macrophages and whether it functions as a lysophospholipase under physiological conditions.


EXPERIMENTAL PROCEDURES

Materials

Pathogen-free female ICR mice (8 weeks old) were obtained from Harlan Sprague-Dawley. Zymosan, aprotinin, leupeptin, potato acid phosphatase, fatty acid-free bovine serum albumin, phenylmethylsulfonyl fluoride, glycerophosphocholine, glycerophosphoinositol, bovine liver phosphatidylinositol, and ATP were obtained from Sigma. 1-Palmitoyl-2-hydroxy-PC was obtained from Avanti Polar Lipids. 1-[^14C]Palmitoyl-2-hydroxy-PC (57 mCi/mmol), [methyl-^3H]choline chloride (81 Ci/mmol), L-alpha-[myo-inositol-2-^3H]phosphatidylinositol (11 Ci/mmol) and [-P]ATP (3000 Ci/mmol) were from DuPont NEN. Dulbecco's modified Eagle's medium and Hank's balanced salts solution were from BioWhittaker, Inc. (Walkersville, MD). Zymosan was prepared for use as described previously(14) . One mg of zymosan is equivalent to approximately 1.3 10^7 particles. Recombinant MAP kinase (ERK 2), which had been activated with MAP kinase kinase, was generously provided by Dr. Lee Graves, University of Washington, Seattle, WA. 1-O-Hexadecyl-2-[^3H]arachidonoyl-PC was prepared as described in detail elsewhere(15) . Polyclonal antibody was generated by immunizing rabbits with recombinant baculovirus-derived PLA(2) as described previously(16) .

Cell Culture

Sf9 cells were infected with recombinant virus containing the PLA(2) gene as described previously(16) . Cells were harvested 65-70 h post-infection by scraping into 10 mM HEPES, 10% glycerol, 0.34 M sucrose, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin (Buffer A) containing the phosphatase inhibitors 100 mM sodium fluoride, 3 µMp-nitrophenyl phosphate, 200 µM sodium orthovanadate, and 10 mM tetrasodium pyrophosphate. Cell suspensions were then sonicated for 50 s and centrifuged at 100,000 g for 60 min. The cytosolic fraction was removed, stored at -20 °C and thawed once before assay. Resident mouse peritoneal cells were isolated as described previously and plated at a density of 20-25 10^6 cells/60-mm tissue culture dish(17) . Cells were stimulated with zymosan (30 particles/cell) for 30 min, scraped into Buffer A, and then lysed by sonication for 10 s at 0 °C. The cell homogenate was centrifuged at 2000 g, for 5 min at 4 °C to remove whole cells and then the supernatant was centrifuged at 100,000 g for 1 h at 4 °C to obtain the cytosolic and membrane fractions.

Phospholipase Assays

Lysophospholipase activity was assayed using sonicated dispersions of 1-[^14C]palmitoyl-lyso-PC (100,000-200,000 dpm/assay) with a final substrate concentration of 50-200 µM. For assays using macrophage cytosols, 10 µg of cytosolic protein was incubated for 2 min in 50 µl of 50 mM Tris, pH 7.4, with or without 5 mM CaCl(2). For assays using PLA(2) in Sf9 cytosols 0.4 µg of cytosolic protein was incubated for 30 s, which was within the linear range of the reaction, in a total volume of 50 µl of 50 mM Tris, pH 7.6. For assays with added BSA and/or 5 mM Ca these were incubated with the substrate 10 min at 37 °C prior to the addition of cytosol. The assay was stopped by the addition of 1.25 ml of Dole reagent(18) . Fatty acid (20 µg) was added as carrier lipid and then 100 µg of silica was added. The tubes were vortexed followed by the addition of 0.75 ml of heptane and 0.75 ml of water. The tubes were vortexed, the upper heptane phase removed, and the radiolabeled fatty acids released measured by liquid scintillation spectrometry. For measuring PLA(2) activity, assays contained 1-hexadecyl-2-[^3H]arachidonoyl-PC (30 µM) (100,000 dpm/assay) cosonicated with 9 µM dioleoylglycerol, 1 mg/ml fatty acid-free BSA, 150 mM NaCl, and 50 mM Tris, pH 7.4, for macrophage cytosols or in 50 mM Tris, pH 7.6, for recombinant PLA(2). For PLA(2) assays using macrophage cytosol, 25 µg of cytosolic protein was added, and the reaction was incubated 10 min at 37 °C. PLA(2) assays with purified recombinant PLA(2) used 40 ng of enzyme purified as described previously and a reaction time of 30 s, which was within the linear range of the reaction(16) . Reactions were terminated by the addition of 2.5 ml of Dole reagent(18) . Fatty acid (20 µg) was added as carrier lipid, and the radiolabeled fatty acids released were extracted and separated by silicic acid chromatography as described previously(10) .

Analysis of Fatty Acid Release from Macrophage Membranes and Membrane Lipid Extracts

The 100,000 g membrane pellet, prepared from macrophages as described above, was resuspended in Buffer A containing 100 µg/ml of fatty acid-free BSA and then centrifuged at 100,000 g for 1 h at 4 °C to remove any free fatty acids. The pellet was resuspended in 100 mM Tris, pH 7.6, centrifuged at 100,000 g for 30 min at 4 °C, and then resuspended in 100 mM Tris, pH 7.6. Macrophage membranes were also extracted and lipid phosphorus determined as described previously(19, 20) . Membranes (20 µg of protein) or sonicated lipids extracted from 10 µg of membrane protein (0.7 µg of lipid phosphorus) were incubated with purified PLA(2) (50 ng) in 50 mM Tris, pH 7.6, containing 1 mM CaCl(2), 100 µg/ml of fatty acid-free BSA, 150 mM NaCl at 37 °C with shaking in a total volume of 50 µl. The reaction was stopped by the addition of 100 µl of ice-cold methanol, to which was added 50 ng of [C(4)]palmitic acid, 50 ng [^2H(3)]stearic acid, 10 ng of [^2H(2)]oleic acid, and 10 ng of [^2H(8)]arachidonic acid. The solution was acidified by the addition of 50 µl of 1 N HCl. The fatty acids were extracted into isooctane, then derivatized to their pentafluorobenzyl esters as described previously(21) . Fatty acid pentafluorobenzyl esters were resuspended in decane and analyzed by temperature-programmed gas chromatography-mass spectrometry (Finnigan SSQ 70 quadrupole mass spectrometer, San Jose, CA). The gas chromatograph utilized a DB-1 column (10 m, 250-µm inner diameter, and 0.25-µm film) (J & W Scientific, Folsom, CA) and helium as a carrier gas. The mass spectrometer was operated in the negative ion chemical ionization mode with methane as the reagent gas. Amounts of fatty acids were determined by stable isotope dilution calibration curves. Carboxylate anion signals were obtained by selected ion recording of ions of target fatty acids and ratioed to the carboxylate anion from the corresponding stable isotope labeled fatty acids (internal standards).

Analysis of Hydrolysis Products from [^3H]Choline-labeled PC and myo-[H]Inositol-labeled PI

[^3H]Choline-labeled macrophage lipids were prepared by labeling macrophages with [methyl-^3H]choline (20 µCi/20 10^6 cells) in 60-mm dishes for 17 h. Macrophage lipids were extracted, and the labeled PC was purified by HPLC using a Hewlett-Packard series 1050 system with a variable wavelength detector set at 205 nm(20) . The lipid extract was applied to a 250 4.6-mm Phenomenex column packed with Lichrosorb 5 Sil 60A which was equilibrated in 47% solvent B (hexane/isopropanol/water, 300:400:70) in solvent A (hexane/isopropanol, 300:400). Using a flow rate of 1 ml/min, the solvent was held at 47% solvent B for 6 min followed by a 20-min linear gradient from 47% solvent B to 100% solvent B. The solvent was then held at 100% solvent B until 60 min. The [^3H]choline-labeled PC eluted at approximately 35 min. The [^3H]PC (250,000 dpm/assay) or [^3H]PI (85,000 dpm/assay) (obtained from DuPont NEN) were sonicated and used at a final concentration of 70 µM. They were also tested as substrates after cosonication with extracted macrophage membrane lipid resulting in a final concentration of phospholipid of 530 µM for the experiments with PC and 490 µM for the experiments with PI. The substrates were incubated with purified PLA(2) (100 ng) in 50 mM Tris, pH 7.6, containing 1 mM CaCl(2), 100 µg/ml fatty acid-free BSA, and 150 mM NaCl at 37 °C in a total volume of 50 µl. The reaction was stopped by the addition of 100 µl of methanol. Lipids were extracted, and the organic and aqueous phases analyzed separately by TLC by comparison with appropriate standards(20) . For analysis of lyso-PC and PC the organic phase was separated on Analtech Silica Gel G plates using the solvent system CHCl(3)/CH(3)OH/CH(3)CO(2)H/H(2)O (100:50:16:8). For analysis of lyso-PI and PI, the organic phase was separated on Analtech Silica gel H plates using the solvent system CHCl(3)/CH(3)OH/NH(4)OH (28%) (45:45:2.5). For analysis of GPC and GPI the aqueous phase was separated on Silica Gel G plates using the solvent system CHCl(3)/CH(3)OH/NH(4)OH (28%) (30:50:25). The aqueous phase was also analyzed for any lyso-PC and lyso-PI. Lyso-PC carryover in the aqueous phase was negligible. Lyso-PI was found in the aqueous phase (approximately 40% of total), but was separated from GPI using the above TLC conditions for analysis of the aqueous phase (R lyso-PI = 0.75, R GPI = 0.34). Silica was scraped into 1 ml of H(2)0, and radiolabeled lipids were detected by liquid scintillation spectrometry.

Analysis of Fatty Acid Release from Intact Cells

Macrophages were plated at 1 10^6 cells/2 cm^2 (24-well plate) and stimulated with zymosan (30 particles/cell) in 1 ml of Dulbecco's modified Eagle's medium in the absence of serum for indicated times as described previously(14) . Cells were scraped into ice-cold methanol (0.5 ml) and the free fatty acids released analyzed as described above.

Phosphatase Treatment and Mono Q Chromatography

For preparation of phosphorylated PLA(2), cytosol from Sf9 cells infected with recombinant virus was prepared in Buffer A containing the phosphatase inhibitors 100 mM sodium fluoride, 3 µMp-nitrophenyl phosphate, 200 µM sodium orthovanadate, and 10 mM tetrasodium pyrophosphate. For preparation of dephosphorylated PLA(2), cytosol from Sf9 cells infected with recombinant virus was prepared in Buffer A without phosphatase inhibitors and treated with acid phosphatase. Potato acid phosphatase, which is supplied at 1 mg/ml in 3.2 M (NH(4))(2)SO pH 6.0, containing 10 mg/ml BSA, was dialyzed against 20 mM Tris, pH 7.0. Cytosols were treated with dialyzed phosphatase (60 units/mg of cytosolic protein) for 1 h at 30 °C in 100 mM HEPES, pH 6.0, containing 1 mM dithiothreitol, 2 mM MgCl(2), 5% glycerol, 1 mM EGTA, 20 µg/ml leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride or treated with a similar solution containing an equivalent amount of BSA instead of phosphatase. Reactions were adjusted to 12.5% ethylene glycol in 100 mM Tris, pH 7.6, and then loaded onto a Mono Q (HR 5/5) column which had been equilibrated with 10 mM Tris, pH 8.0, containing 10% glycerol and 230 mM NaCl. Fractions (0.5 ml) were eluted with a 40-ml gradient from 230 to 400 mM NaCl at a flow rate of 0.5 ml/min. Dithiothreitol (1 mM) was added to the fractions for long term storage at -20 °C.

PLA(2)Phosphorylation with MAP Kinase

Dephosphorylated PLA(2) (84 ng), which had been separated by Mono Q chromatography, was incubated with MAP kinase (175 ng) in 20 mM Tris, pH 7.6, 10 mM MgCl(2), 0.5 mM dithiothreitol, 50 µM ATP in a 200-µl reaction volume for 1 h at 30 °C. Parallel samples were run containing 10 µCi of [-P]ATP/200 µl of assay. PLA(2) and lysophospholipase activity was then assayed using 25 µl of the reaction mixture as described above for the purified enzyme. To detect the gel shift on Western blots, a portion of the sample was boiled in Laemmli buffer, and proteins were resolved using a 10% SDS-polyacrylamide separating gel made using a pH 8.3 Tris buffer and a concentration of bisacrylamide that was decreased to 1% of the total acrylamide(22) . The gel was run an additional 1 h and 40 min after the dye front had run off. Proteins were then transferred to nitrocellulose and incubated for 2 h with polyclonal antibody against recombinant 85-kDa PLA(2). Amersham's ECL detection system was used to detect bound antibody. For analysis of the stoichiometry of phosphorylation the P-labeled PLA(2) was separated by SDS-polyacrylamide gel electrophoresis, the band excised and counted by Cerenkov counting to determine the moles phosphate incorporated per mol of PLA(2)(16) .


RESULTS

Inhibition of Lysophospholipase Activity by Albumin

Prior to our studies to investigate lysophospholipase activity in stimulated macrophages, experiments were carried out to optimize the lysophospholipase assay of the 85-kDa PLA(2). This PLA(2) exhibits unusual kinetics characterized by a burst of activity that quickly levels off and ceases after only 5-10% substrate hydrolysis(10, 23) . This phenomenon is observed for both the Ca-dependent PLA(2) activity against PC liposomes and the Ca-independent lysophospholipase activity against lysophospholipid micelles(10) . This is thought to be due to trapping of the enzyme on substrate vesicles as product accumulates(24) . The presence of BSA in the PLA(2) assay can partially reverse this phenomenon and helps linearize the reaction curve(24) . In contrast, BSA was found to inhibit the lysophospholipase activity of the 85-kDa enzyme in a concentration-dependent manner (Fig. 1). BSA at 0.1 and 1 mg/ml inhibited lysophospholipase activity of the recombinant PLA(2) by 50 and 90%, respectively, when assayed in the absence of Ca using a substrate concentration of 50 µM. When 5 mM Ca was included in the assay, inhibition by BSA could be prevented and the degree of reversal was dependent on the BSA concentration. Ca could completely reverse inhibition at concentrations of 0.1 mg/ml BSA and lower, but only partially reversed inhibition at concentrations of 0.5 and 1 mg/ml BSA. Ca itself had no significant effect on lysophospholipase activity when assays were run in the absence of BSA, as reported previously(10, 11, 16) . Inhibition by BSA was not unique to the lysophospholipase activity in the cytosolic fraction of Sf9 cells, as BSA at 0.5 and 1 mg/ml also inhibited lysophospholipase activity of purified recombinant PLA(2) by 57 and 87%, respectively (data not shown). Although the BSA used was fatty acid-free, experiments were carried out to determine if other lipids bound to the BSA were interfering with the assay. BSA was delipidated using di-isopropyl ether and butanol which removed 75% of the contaminating lipid phosphorus(25) . The extracted BSA inhibited lysophospholipase activity similarly when compared with equivalent amounts of the unextracted BSA, demonstrating that inhibition was not due to the presence of contaminating lipids.


Figure 1: BSA inhibition of lysophospholipase activity. Lysophospholipase activity was assayed in cytosols from Sf9 cells expressing recombinant PLA(2). Activity was assayed with increasing concentrations of BSA (0-1.0 mg/ml) in the absence (1 mM EGTA) (open bars) or presence (striped bars) of 5 mM Ca. The data represent the mean ± S.E. of three experiments.



It is well established that albumin has the capacity to bind lysophospholipid and acts as a carrier of this lipid in the blood(26) . To test the possibility that BSA was binding to the lysophospholipid substrate and interfering with the lysophospholipase assay, the enzyme was assayed in the presence of increasing amounts of lysophospholipid at a fixed concentration of BSA (0.5 mg/ml) in the absence of Ca (Fig. 2). As the substrate concentration was increased from 50 to 200 µM, the degree of inhibition by BSA decreased from 68 to 24%, indicating that inhibition by BSA was dependent on the substrate concentration and suggested that BSA was interacting with the substrate.


Figure 2: Effect of increasing lysophospholipid concentration on inhibition of lysophospholipase activity by BSA. Lysophospholipase activity was assayed in the absence (open bars) or presence (striped bars) of 0.5 mg/ml BSA using cytosol (0.4 µg) from Sf9 cells infected with recombinant virus. Control activity (100%) at each substrate concentration equals the activity in the absence of BSA. The percent hydrolysis was less than 6% at each substrate concentration. The data represent the mean ± S.E. of three experiments.



Lysophospholipase Activity in Macrophage Cytosols

During stimulation of macrophages with zymosan, phosphorylation of the 85-kDa PLA(2) occurs, resulting in a stable increase in PLA(2) activity in macrophage cytosols(14) . Experiments were carried out to determine if zymosan stimulation resulted in an increase in lysophospholipase activity in the cytosol, which could be attributed to the 85-kDa PLA(2). When activity was assayed using 200 µM lysophospholipid, significant Ca-independent lysophospholipase activity was observed which was increased by 2-fold in cytosols from zymosan-stimulated cells (Fig. 3). At assay times of 1-2 min, lysophospholipase activity was increased by approximately 50% by the addition of Ca. However, as shown in Table 1, lysophospholipase activity could not be detected in macrophage cytosols when assayed using a lower substrate concentration (50 µM) unless Ca was present in the assay. The ability to detect Ca-independent lysophospholipase activity increased with increasing substrate concentration, and the stimulatory effects of Ca on activity decreased with increasing substrate concentration. These results were similar to the inhibitory effects of BSA we observed on lysophospholipase activity of recombinant PLA(2), suggesting that proteins in the macrophage cytosols might be similarly interfering with the lysophospholipase assay at low substrate concentrations, and Ca was reversing the inhibition.


Figure 3: Lysophospholipase activity in cytosols from unstimulated and zymosan-stimulated macrophages. Macrophages were incubated with (, black square) or without zymosan (, bullet) for 30 min (30 particles/cell) and the cytosolic fraction prepared as described under ``Experimental Procedures.'' Lysophospholipase activity was assayed at the times indicated in the presence (bullet, black square) or absence (, ) of Ca. Activity was assayed using 10 µg of cytosolic protein and 200 µM 1-palmitoyl-2-lyso-PC. Data from a representative experiment run in triplicate is shown and expressed as mean ± S.D.





Since mammalian cells contain multiple lysophospholipases, it was necessary to determine that the zymosan-stimulated lysophospholipase activity in macrophage cytosols was due to the 85-kDa enzyme. When a neutralizing polyclonal antibody against the recombinant baculovirus-expressed PLA(2) was incubated with purified recombinant PLA(2), both PLA(2) and lysophospholipase activities of the enzyme were inhibited, although a higher concentration of antiserum was required to inhibit the lysophospholipase activity of the enzyme (Fig. 4). PLA(2) activity was completely inhibited at a serum dilution of 1:50, whereas a 1:25 dilution of antiserum was required to inhibit lysophospholipase activity by 82%. The two enzymatic activities share one catalytic domain(12) , but the purified substrates for the two different enzymatic activities assays differ structurally and presumably in how they bind to the enzyme. Thus the differential potency of the antiserum against the two enzymatic activities could possibly be attributed to differences in the inhibition of the binding of the substrates to the enzyme. When tested on macrophage cytosols, preimmune serum did not inhibit activity, whereas immune serum inhibited PLA(2) and lysophospholipase activity in both unstimulated and zymosan-stimulated cytosols, verifying that these activities were due to the 85-kDa PLA(2) (Fig. 5). Lysophospholipase activity was similarly inhibited whether assayed in the presence or absence of Ca (data not shown).


Figure 4: Inhibition of PLA(2) and lysophospholipase activities of the 85-kDa PLA(2) by polyclonal antiserum. Recombinant PLA(2) was incubated with different dilutions of serum from rabbits, before immunization (open bars) or serum from rabbits after immunization with recombinant PLA(2) (striped bars), in 50 mM Tris, pH 7.6, containing 10% glycerol. Samples were then diluted 10-fold in the same buffer, and 25 µl (40 ng of enzyme) was assayed for lysophospholipase (A) or PLA(2) activity (B) in a total volume of 50 µl. Data from a representative experiment run in triplicate are shown and expressed as mean ± S.D.




Figure 5: Inhibition of PLA(2) and lysophospholipase activities in macrophage cytosols by polyclonal antiserum to the 85-kDa PLA(2). Macrophage cytosol from unstimulated (open bars) and zymosan-stimulated (striped bars) cells was incubated with a 1:50 dilution of preimmune (PI) or immune (I) serum from rabbits immunized with recombinant PLA(2). Samples were then assayed for lysophospholipase (10 µg of cytosolic protein) (A) or PLA(2) activity (20 µg of cytosolic protein) (B). Data are expressed as a percent of the maximum response, which is the activity in zymosan-stimulated cytosol incubated with preimmune serum (100%). The data are from a representative experiment run in triplicate and expressed as mean ± S.D.



Phosphorylation with MAP Kinase

The enhanced lysophospholipase activity in cytosols of macrophages stimulated with zymosan is consistent with our previous observation that zymosan stimulation also enhances PLA(2) activity in the cytosol. Zymosan also induces a decrease in electrophoretic mobility of the 85-kDa PLA(2), which is consistent with the phosphorylation by MAP kinase(13) . Since phosphorylation of the 85-kDa PLA(2) by MAP kinase enhances the PLA(2) activity of this enzyme in in vitro studies(13) , the effect of phosphorylation with MAP kinase on lysophospholipase activity was investigated. We have shown that the 85-kDa PLA(2) is expressed as an activated phosphoprotein in Sf9 cells, and phosphorylation of a tryptic peptide of the recombinant enzyme containing the MAP kinase site has been verified(16, 27) . Due to this evidence we treated the enzyme with phosphatase before attempting to rephosphorylate with MAP kinase. Cytosols from Sf9 cells expressing the 85-kDa enzyme were treated with phosphatase and then partially purified over an anion exchange column. Under these conditions the phosphatase completely removed P-labeled phosphate from an equivalent amount of P-labeled PLA(2) (data not shown). As shown in Fig. 6the dephosphorylated form of the enzyme eluted at a lower salt concentration than the phosphorylated form similar to the results of the PLA(2) in platelets seen by Kramer et al.(28) . Rephosphorylation of the dephosphorylated enzyme with MAP kinase resulted in a 1.8-fold increase in lysophospholipase activity and a 3-fold increase in PLA(2) activity (Fig. 7A). Rephosphorylation also resulted in a decrease in electrophoretic mobility on SDS-polyacrylamide gels, which is characteristic of phosphorylation of the 85-kDa PLA(2) by MAP kinase, and resulted in the shift of 100% of the PLA(2) molecules (13) (Fig. 7B). The stoichiometry of phosphorylation was 1.0 mol of phosphate incorporated per mol of PLA(2).


Figure 6: Separation of phosphorylated and dephosphorylated forms of recombinant PLA(2) by Mono Q chromatography. Cytosols from Sf9 cells expressing recombinant PLA(2) were prepared and treated with mock buffer (, ) or phosphatase (black square, up triangle, filled) as detailed under ``Experimental Procedures'' and loaded onto a Pharmacia Mono Q HR 5/5 column equilibrated with buffer containing 230 mM NaCl. Fractions were eluted with a gradient from 230 to 400 mM NaCl at a flow rate of 0.5 ml/min and assayed for PLA(2) (, black square) or lysophospholipase activity (, up triangle, filled) as described under ``Experimental Procedures.''




Figure 7: Rephosphorylation of PLA(2) with MAP kinase. Dephosphorylated recombinant PLA(2) purified by Mono Q chromatography was incubated with purified MAP kinase as described under ``Experimental Procedures.'' A, samples were assayed for PLA(2) (open bars) and lysophospholipase activity (shaded bars), and the data are expressed as percent of control activity in the absence of MAP kinase and represents mean ± S.E. of three experiments. B, samples were also immunoblotted using a polyclonal antibody against the recombinant PLA(2).



Fatty Acid Release with Recombinant PLA(2)

To determine whether the 85-kDa PLA(2) could act as a lysophospholipase and hydrolyze fatty acid from the sn-1 position when supplied with its native substrate (cell membranes), we incubated purified PLA(2) with macrophage membranes and monitored the release of free fatty acids by GCMS over time (Fig. 8). As would be predicted, arachidonic acid (20:4) was the predominant fatty acid released from macrophage membranes. The release of 20:4 increased steadily resulting in 20-fold greater amounts of 20:4 than oleic acid (18:1) and linoleic acid (18:2), which are also present at the sn-2 position of macrophage phospholipids. Of interest, there was also release of the saturated fatty acids, stearic acid (18:0), as well as palmitic acid (16:0), that occurred at a greater rate between 1 and 3 h after a lag. In mouse peritoneal macrophages 18:0 is enriched in the sn-1 position of cellular phospholipids and can be used as a marker for the release of fatty acids from the sn-1 position(29, 30) . These results suggest that the lysophospholipase activity of the PLA(2) released 18:0 after production of lysophospholipid in the membrane.


Figure 8: Release of fatty acids from macrophage membranes incubated with recombinant PLA(2). Macrophage membranes (20 µg of membrane protein) were incubated in the absence (open symbols) or presence (closed symbols) of purified recombinant PLA(2) (50 ng) in 50 mM Tris, pH 7.6, with 5 mM CaCl(2), 150 mM NaCl, and 100 µg/ml of fatty acid free BSA for the indicated times. The free fatty acids released were analyzed by GCMS as described under ``Experimental Procedures.'' Data from a representative experiment run in triplicate are expressed as mean ± S.D.



In order to rule out a contaminating endogenous lysophospholipase activity in the membrane, which hydrolyzed the lysophospholipid supplied by the 85-kDa PLA(2) activity, we incubated membranes under the same conditions without the PLA(2), but with 1-[^14C]palmitoyl-2-lyso-PC. Under these conditions there was detectable lysophospholipase activity in the membranes (data not shown), which might explain the slight increase in the release of 16:0 in membranes in the absence of PLA(2). By Western blotting we were able to detect low level contamination of the 85-kDa PLA(2) in macrophage membranes washed with EDTA and EGTA (data not shown), suggesting that the contaminating lysophospholipase activity could be either the 85-kDa PLA(2) or another lysophospholipase. When we heated these membranes at 65 °C for 90 min, we were able to completely abolish the endogenous lysophospholipase activity in the membranes. Similar levels of fatty acids were released from heat-treated membranes after incubation with the 85-kDa PLA(2) as was released with untreated membranes verifying that the release of 18:0 was due to the added 85-kDa enzyme (data not shown). Additionally, we measured fatty acid release from lipid extracts of macrophage membrane after incubation with PLA(2) (Fig. 9). As observed with the membrane substrate the release of fatty acid was selective for 20:4 versus the other fatty acids found at the sn-2 position. The release of 18:0 and 16:0 paralleled 20:4 release, plateauing at 1 h.


Figure 9: Release of fatty acids from lipid extracts of macrophage membranes incubated with PLA(2). Lipid (0.7 µg of lipid phosphorus) extracted from macrophage membranes was incubated in the absence (open symbols) or presence (closed symbols) of purified recombinant PLA(2) (50 ng) in 50 mM Tris, pH 7.6, with 5 mM CaCl(2), 150 mM NaCl, and 100 µg/ml of fatty acid free BSA for the indicated times. The free fatty acids released were analyzed by GCMS as described under ``Experimental Procedures.'' Data from a representative experiment run in triplicate are expressed as mean ± S.D.



Complete Phospholipid Deacylation by the 85-kDa PLA(2)

Experiments were carried out using choline- or inositol-labeled phospholipid substrates to provide additional evidence that the 85-kDa PLA(2) could completely deacylate phospholipid in a bilayer by sequential action of its PLA(2) and lysophospholipase activities. The production of lysophospholipid and the water-soluble products GPC or GPI were measured (Fig. 10). For measuring hydrolysis of PC macrophages were labeled with [^3H]choline, the lipids extracted and the labeled PC purified by HPLC. PC liposomes were prepared by sonication either in the presence or absence of a total lipid extract of macrophage membranes and incubated with the PLA(2). With PC alone as substrate and no added macrophage lipids, greater levels of lyso-PC than GPC were produced at early time points (Fig. 10A). The production of lyso-PC rapidly plateaued levelling off at 4% hydrolysis (at a concentration of 3 µM lysophospholipid) which was accompanied by a concomitant linear increase in the production of GPC. At 2 and 30 min, respectively, 3.5 and 11% of the total PC substrate was converted to lyso-PC and GPC combined. A similar pattern was observed when PC was codispersed with macrophage membrane lipids, but less of the lyso-PC produced was hydrolyzed to GPC (Fig. 10B) (49% of the lyso-PC was converted to GPC at 30 min compared with 65% conversion in the absence of added macrophage lipid). When PI liposomes, which consisted of the diacyl species sn-1-18:0, sn-2-20:4, were used as a substrate, a greater percent conversion of lyso-PI to GPI occurred than with the PC substrate (Fig. 10, C and D). This is not surprising since the defined PI substrate consisted of only the diacyl species, whereas the labeled PC substrate purified from macrophage lipids would contain alkyl-acyl and alkenyl-acyl species which would not be hydrolyzed by the lysophospholipase activity of the enzyme. The production of GPI was slower in the presence of added macrophage lipid as was seen with the PC substrate.


Figure 10: Products released from [^3H]choline-labeled PC and myo-[^3H]inositol-labeled PI incubated with PLA(2). Recombinant PLA(2) (100 ng) was incubated with liposomes of [^3H]choline-labeled PC (A, B) or myo-[^3H]inositol-labeled PI (C, D) in the absence (A, C) or the presence (B, D) of macrophage membrane lipid for the indicated times. Lipid products (, GPC; , GPI; bullet, lyso-PC; black square, lyso-PI) were extracted and separated by TLC as described under ``Experimental Procedures.'' Data from a representative experiment are expressed as the mean of triplicate values.



Release of Fatty Acids from Macrophages

Experiments were carried out to determine if the types of fatty acids released (i.e. 18:0 and 16:0) from zymosan-stimulated macrophages were consistent with activation of lysophospholipases during cell activation (Fig. 11). After zymosan stimulation there was release of the unsaturated fatty acids 20:4 > 18:1 as well as, the release of the saturated fatty acids 18:0 and 16:0. In these experiments the production of eicosanoids was not inhibited so only relatively low levels of free 20:4 were detected since most is converted to leukotriene C(4) and prostaglandin E(2). The level of 18:0 released was greater than the amount of 18:1 or 16:0 released. These results show that 18:0, which occurs at the sn-1 position in macrophage phospholipids, is released during zymosan stimulation of macrophages.


Figure 11: Release of fatty acids from zymosan-stimulated macrophages. Macrophages were incubated without (open symbols) or with zymosan (30 particles/cell, closed symbols) for the indicated times in 1 ml of Dulbecco's modified Eagle's medium. The reaction was stopped by the addition of 0.5 ml of methanol. The samples were then analyzed for free fatty acids by GCMS as described under ``Experimental Procedures.'' The data are expressed as mean ± S.E. of three experiments.




DISCUSSION

Lysophospholipid levels must be tightly controlled due to their deleterious effects on cellular membranes, and they increase only transiently after cell activation. When macrophages are incubated with lysophosphatidylcholine the majority is converted via the lysophospholipase pathway to glycerophosphocholine rather than being reacylated to phosphatidylcholine(7) . In the macrophage three Ca-independent lysophospholipases have been identified, the 85-kDa cytosolic PLA(2) and two others with molecular masses of 27 and 28 kDa(10, 31, 32) . The functional role and regulation of specific lysophospholipases in the cell is as yet unknown. In neutrophils there appears to be an unidentified lysophospholipase activity that can be stimulated during cell activation(33) . In addition, during differentiation of HL-60 cells to neutrophil-like cells, there is an up-regulation of a 20-kDa lysophospholipase(34) . In this study we show that zymosan stimulation increases lysophospholipase activity in macrophage cytosol. The 85-kDa PLA(2) is the predominant lysophospholipase and PLA(2) activity in the cytosol, since both activities can be completely abolished with specific antiserum. During zymosan stimulation of macrophages, MAP kinase is activated and the 85-kDa PLA(2) is phosphorylated, resulting in increased cytosolic PLA(2) activity(14, 17) . Our data show that the lysophospholipase activity can also be increased by phosphorylation with MAP kinase.

While exploring assay conditions for assaying the lysophospholipase activity of the 85-kDa enzyme, we observed that BSA had an inhibitory effect on activity which could be reversed as the substrate concentration was increased. In contrast, BSA increases PLA(2) activity by preventing enzyme trapping which occurs when product accumulates in liposomes(24) . Albumin can bind lysophospholipid as a monomer or can interact with lysophospholipid micelles(35, 36) . In addition, it can remove lysophospholipid bound to membranes and interacts with lysophospholipid in liposomes to cause a disruption of membrane integrity(37, 38) . One possibility for the inhibitory effects of BSA is that it binds lysophospholipid, and at low substrate concentrations there are predominately BSA-lysophospholipid complexes. Only as the substrate concentration is increased is there lysophospholipid substrate available for the enzyme.

We also observed that Ca could abolish the inhibition of lysophospholipase activity by BSA. The PLA(2) activity of the 85-kDa PLA(2) enzyme is Ca-dependent. Ca does not play a role in catalysis, but is required for binding of the enzyme to the phospholipid substrate, which is mediated by a Cadependent phospholipid binding region (39, 40) . In contrast, the lysophospholipase activity has been reported to be Ca-independent(10, 11, 16) . This lack of Ca dependence appears to only be unique to the hydrolysis of lysophospholipid micelles. Hydrolysis of lysophospholipid, when it was incorporated into a liposome substrate, was Ca-dependent, suggesting that the enzyme requires Ca to bind to a liposome, but not to a lysophospholipid micelle(41) . Consequently, another possibility is that BSA changes the structure of the lysophospholipid substrate so that the enzyme now requires Ca for binding. It has also been observed that when assays are run in the presence of Triton X-100 or glycerol that Ca has a stimulatory effect on activity (11) . More recently, Nalefski et al.(42) reported that the lysophospholipase activity of the 85-kDa enzyme against lysophospholipid micelles was Ca dependent and the Ca dependence was more pronounced at lower substrate concentrations; however, in this study the assays included BSA. Based on our observations, the stimulatory effect of Ca may have been due to the reversal of the inhibition by BSA. It is of interest that we observed that the lysophospholipase activity in macrophage cytosols was stimulated by Ca when assayed at low substrate concentration, but was less affected by Ca at higher substrate concentrations. Possibly, the presence of cytosolic protein interferes with the substrate, as was observed with the BSA, since cytosolic proteins can also bind lysophosphatidylcholine(43) . This effect was not observed in Sf9 cytosols, but the ratio of PLA(2) to extraneous protein is much greater and the total protein in the assay was 25-fold less than in assays run with macrophage cytosols.

Hydrolysis of sn-2 fatty acids from diacyl-linked cellular phospholipids by the 85-kDa PLA(2) would generate substrate for the lysophospholipase activity of this enzyme. Although 20:4 is enriched in ether-linked PC and PE pools, in macrophages significant amount of acyl-20:4 pools exist that would be a substrate for the arachidonate-preferring PLA(2)(29, 30) . Lysophospholipids generated in membranes can be reacylated or hydrolyzed by lysophospholipases, resulting in the release of sn-1 fatty acid. Of the total 18:0 present in the PE and PC pools (both the sn-1 and sn-2 positions) of macrophages, 95 and 86% is found in the sn-1 position, respectively(30) . When fatty acid release was measured from macrophages after stimulation with zymosan we saw an increase in the release of 18:0 consistent with a role of lysophospholipase activation during zymosan stimulation, although we cannot rule out direct release of fatty acid by a phospholipase A(1) or other complex pathways. Also, we found that the 85-kDa enzyme could catalyze release of 18:0 from macrophage membranes, which occurred when apparent lysophospholipid levels accumulated to only a small percentage of the total phospholipid present in the membrane. The amount of lysophospholipid produced after incubation of membranes with recombinant PLA(2) can be estimated based on the moles of 20:4 released assuming no reacylation of lysophospholipid occurs. At 1 h when the release of 18:0 started to increase, the amount of lysophospholipid was 0.7%, or 7 µM, of the amount of phospholipid present (960 µM) in the membrane.

These data suggest that the cPLA(2) releases 18:0 from the sn-1 position through its lysophospholipase activity, although we cannot rule out some release of 18:0 from the sn-2 position. However, this is unlikely since sn-2 saturated fatty acids and even monoenoic fatty acids are poor substrates for the 85-kDa PLA(2)(44, 45) . In addition the lysophospholipase activity of the 85-kDa PLA(2) can hydrolyze 1-stearoyl-lyso-PC, although 1-palmitoyl-lyso-PC is a better substrate(46) . Experiments measuring the production of lysophospholipid and glycerophosphocholine or glycerophosphoinositol, after incubation of the 85-kDa enzyme with PC and PI, confirmed that the 85-kDa enzyme can completely deacylate diacyl phospholipid in a bilayer. Particularly, the results with the diacyl substrate stearoyl-arachidonoyl-PI show that levels of lysophospholipid are kept low in the bilayer due to efficient conversion of the lysophospholipid to GPI. In summary, the results suggest that the 85-kDa enzyme can hydrolyze lysophospholipid as it accumulates in membranes, suggesting a role for the lysophospholipase activity of the 85-kDa PLA(2) in regulating cellular lysophospholipid levels.


FOOTNOTES

*
This work was conducted in the F. L. Bryant Research Laboratory for the Mechanisms of Lung Disease and supported by National Institutes of Health Grant HL 34303. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: National Jewish Center for Immunology and Respiratory Medicine, Dept. of Pediatrics, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1214; Fax: 303-398-1851.

(^1)
The abbreviations used are: PLA(2), phospholipase A(2); PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; GPI, glycerophosphoinositol; GPC, glycerophosphocholine; MAP kinase, mitogen-activated protein kinase; BSA, bovine serum albumin; 20:4, arachidonic acid; 18:0, stearic acid; 16:0, palmitic acid; 18:1, oleic acid; 18:2, linoleic acid; GCMS, gas chromatography mass spectrometry; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We are grateful to Dr. Robert Murphy and Dr. Keith Clay for fatty acid analysis and to Chris Johnson for the excellent technical assistance.


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