Analysis of the Secretory Phospholipase A2 That Mediates Prostaglandin Production in Mast Cells*

(Received for publication, December 3, 1996, and in revised form, March 26, 1997)

Srinivasa T. Reddy , Michelle V. Winstead Dagger , Jay A. Tischfield Dagger and Harvey R. Herschman §

From the Departments of Biological Chemistry and Molecular and Medical Pharmacology and the Molecular Biology Institute, UCLA Center for the Health Sciences, Los Angeles, California 90095-1570 and the Dagger  Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202-5251

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Prostaglandin D2 (PGD2) synthesis in activated mast cells occurs in two phases, an early phase that is dependent on prostaglandin synthase 1 and a delayed phase that is dependent on activation-induced prostaglandin synthase 2 gene expression. Early phase PGD2 synthesis in activated mast cells also requires the activity of a secretory phospholipase A2 (PLA2). It has been thought that the secretory PLA2 expressed in mast cells is group IIa PLA2, encoded by the Pla2 g2a gene. However, activated bone marrow-derived mast cells prepared from Pla2 g2a+/+ mice and mast cells prepared from mice with a mutation in the Pla2 g2a gene both demonstrate early phase PGD2 synthesis. Moreover, mast cells from both murine strains secrete PLA2 activity following activation. Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that mast cells from Pla2 g2a+/+ and Pla2 g2a-/- mice do not express group IIa PLA2 message. Instead, Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that both Pla2 g2a+/+ and Pla2 g2a-/- mast cells express mRNA for group V PLA2, encoded by the Pla2gV gene. An antisense oligonucleotide directed against group V PLA2 is also able to inhibit both the early phase of PGD2 production and the secretion of PLA2 activity by activated mast cells. Our data suggest that (i) group IIa PLA2 does not play a significant role in murine mast cell prostaglandin synthesis, (ii) group V PLA2 mediates early mast cell PGD2 production and transcellular PGE2 production in murine mast cells, and (iii) much of the data, based on studies with chemical inhibitors and antibodies, suggesting that group IIa PLA2 is responsible for arachidonic acid mobilization needs to be reevaluated.


INTRODUCTION

Four low molecular weight, "secretory" phospholipase A2 (PLA2)1 enzymes are present in mammals. These enzymes cleave glycerophospholipids, in the presence of millimolar levels of calcium, to release arachidonic acid. Arachidonate is the substrate for lipoxygenase-mediated leukotriene synthesis and prostaglandin synthase-mediated prostanoid synthesis (1, 2). The most well studied of these low molecular weight phospholipases are the group I PLA2, present in pancreas, and the "group II PLA2" (now termed group IIa PLA2) present in synovial fluid, intestine, and a wide range of other organs. One of our laboratories recently identified genes for two additional proteins with related sequences and similar enzymatic properties (3-5). Murine group IIc PLA2 is found in testis, while the group V PLA2 mRNA is most actively expressed in the heart.

Group IIa PLA2 has been proposed to play a role in many pathological conditions, such as rheumatoid arthritis (6-8), septic shock (9, 10), pancreatitis (11), and psoriasis (12, 13), that are mediated by arachidonic acid release. The Pla2 g2a gene, encoding group IIa PLA2, has also been implicated in colon cancer. The "murine intestinal neoplasia" or min1 gene is the murine orthologue of the APC gene, mutated in human familial adenomatosis polyposis, a hereditary form of colon cancer. The number of intestinal tumors and the longevity of mice with the min1- mutation is greatly influenced by their genetic background. Recently, mice carrying the "modifier of min1" or mom1 mutation have been shown to have a frameshift mutation in the Pla2ga gene. Mice with this mutation express marginally detectable group IIa PLA2 mRNA and no group IIa PLA2 protein (14, 15). Identification of a naturally occurring null mutation in the gene encoding group IIa PLA2 presents an opportunity to examine the role of this gene and its product.

Mast cells play a critical role in immune responses and in allergic disease. When activated, either by antigen aggregation of cell surface high affinity IgE receptors or by other effectors, mast cells degranulate and release a variety of agents, including histamine and serotonin, that modulate inflammatory responses. In addition, activation of mast cells also induces the synthesis and release of leukotrienes and of prostaglandin D2 (PGD2).

PGD2 production in activated mast cells occurs in two distinct stages, an immediate release that is completed within 10-15 min and a delayed phase of PGD2 synthesis and secretion that peaks at 4-6 h. The early phase of PGD2 synthesis in mast cells following activation is due to the conversion of arachidonic acid to prostaglandin by preexisting prostaglandin synthase 1. In contrast, the late phase of PGD2 synthesis and secretion following mast cell activation requires activation-induced synthesis of prostaglandin synthase 2 mRNA and functional protein (16, 17).

We recently used both a chemical inhibitor of the group IIa PLA2 and a monoclonal antibody produced against human group IIa PLA2 to demonstrate that the early, prostaglandin synthase 1-dependent phase of PGD2 production requires the release of PLA2 from the activated mast cell (18). We also showed that both recombinant group IIa PLA2 and mast cell supernatants can elicit "transcellular" prostaglandin synthesis in fibroblasts, releasing from the fibroblast membranes arachidonic acid that can serve as substrate for the fibroblast prostaglandin synthase 1 (19). The ability of mast cell supernatants to elicit transcellular prostaglandin synthesis was also blocked by both the monoclonal antibody to human group IIa PLA2 and a chemical inhibitor of group IIa PLA2. Like others, we attributed the mast cell PLA2 activity essential for prostanoid synthesis to group IIa PLA2. However, we now report that mast cells derived from animals homozygous for the mom1-/- incapacitating mutation in the Pla2 g2a gene, which encodes group IIa PLA2, demonstrate normal prostaglandin production. Moreover, mast cells derived from mice wild type for the Pla2 g2a gene do not produce detectable message for group IIa PLA2 or for group IIc PLA2. However, group V PLA2 message is present in mast cells derived from both mom1+ and mom1- mice. Moreover, preincubation with an antisense oligonucleotide directed against group V PLA2 message can block both the early phase of PGD2 production and the secretion of PLA2 activity by activated mast cells.


EXPERIMENTAL PROCEDURES

Cell Culture

AKR/J and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Bone marrow mast cell lines were derived from these strains according to published protocols (20). Briefly, bone marrow from the femurs was flushed out with RPMI, 5% fetal bovine serum. The cells were centrifuged, resuspended, and plated in RPMI 1640 medium containing 10% fetal bovine serum and 50% WeHi-conditioned medium. The conditioned medium serves as a source of IL-3. Culture media were changed twice each week. The cells were transferred to new culture flasks at each medium change, to remove adherent cells. By 3-4 weeks in culture, this procedure yielded >95% pure mast cell cultures. Murine MMC-34 mast cells (16), AKJ mast cells, and B6 mast cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Swiss 3T3 cells were cultured as described previously (19).

Reagents

Murine IgE and monoclonal anti-IgE were purchased from Pharmingen (San Diego, CA). PGD2 assay kits, Hybond-N membranes, and Hybond-N+ membranes were purchased from Amersham Corp. (United Kingdom). Aminopropyl solid-phase columns number 9070 (100 mg/ml) were from Burdick and Jackson (Muskeogon, MI). Arachidonate 3H-labeled Escherichia coli suspension was from DuPont NEN. AmpliTaq DNA polymerase was obtained from Perkin-Elmer (Norwalk, CT). BamHI was from New England Biolabs. The SuperScript cDNA synthesis kit and the Lipofectamine reagent were purchased from Life Technologies, Inc. Oligonucleotide primers were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Phosphorothioate oligonucleotides for the antisense experiments were purchased from Oligos Etc, Inc. (Wilsonville, OR). RPMI 1640 medium was purchased from ICN (Cleveland, OH). Fetal calf serum was from Gemini Bioproducts Inc. (Calabasas, CA). Monoclonal antibody F10 (mAb F10), directed against recombinant human group IIa PLA2, and SB 203347, an inhibitor of PLA2 (21), were gifts of Dr. Lisa Marshall (SmithKline Beecham Pharmaceuticals, King of Prussia, PA). Murine group IIa PLA2 cDNA was a gift from Dr. Brian Kennedy (Merck Frosst, Canada). NS-398 was the gift of Taisho Chemical Co. (Japan).

RNA Isolation

Heart and testis tissues removed from Swiss Webster mice and intestines removed from AKR/J and C57BL/6J mice were frozen immediately in liquid nitrogen. One gram of each tissue was ground to a fine powder, using a precooled (dry ice) mortar and pestle and homogenized in guanidine isothiocyanate solution (16). Total RNA from tissue powders and from mast cell cultures was prepared as described previously (16).

Northern Blot Analysis

Ten micrograms of total RNA from each sample was subjected to electrophoresis in a 1% agarose gel, transferred to Hybond-N membranes, and hybridized with a cDNA probes for group IIa PLA2 and GAPDH. Group V PLA2 probe was generated by subjecting total RNA from heart tissue (Swiss Webster mice) for RT-PCR, using Group V PLA2-specific primers: primer 1, 5'-CAG GGG GCT TGC TAG AAC TCA A-3'; primer 2, 5'-AAG AGG GTT GTA AGT CCA GAG G-3'. Electrophoresis and hybridization procedures have been described previously (16).

Genomic DNA Isolation and Southern Analysis

Washed intestines (1 g) and mast cells (5 × 107) were incubated in 1 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5% SDS, 100 mM EDTA, and 100 µg of proteinase K/ml) at 55 °C overnight. DNA was isolated by phenol/chloroform extraction (22). DNAs (20 µg) were digested with BamHI, subjected to electrophoresis on a 1% agarose gel, and transferred to a Hybond N+ membrane. The membrane was hybridized to a group IIa PLA2 cDNA probe according to standard protocols.

Reverse Transcriptase-Polymerase Chain Reaction Measurement of PLA2 Messages

Single strand cDNA was synthesized in a 20-µl reverse transcription reaction that contained 4 µg of total RNA, oligo(dT) primers, and SuperScript reverse transcriptase (Life Technologies, Inc.). The resulting cDNAs were directly subjected to polymerase chain reaction (PCR). All amplifications were performed with 30 cycles, in a SingleBlock thermal cycler (Ericomp). Each cycle consisted of denaturation for 1 min at 94 °C, annealing for 1 min at 58 °C, and extension at 72 °C for 1 min and 20 s. The 30 cycles were followed by a final extension for 5 min at 72 °C. The PCR reactions contained 30 pmol of each primer, 4 units of AmpliTaq DNA polymerase, and 4 µl of cDNA from the reverse transcription reactions. The following PCR primers were used: group IIa PLA2, 5'-CAG TTT GGG GAA ATG ATT CGG C-3' and 5'-GAA ACA TTC AGC GGC GGC TTT A-3'; group IIc PLA2, 5'-GGC ATT GCC ATC TTC CTT GTC T-3' and 5'-TAA GCT TGT GGT AGC AGC AGT C-3'; group V PLA2, 5'-CAG GGG GCT TGC TAG AAC TCA A-3' and 5'-AAG AGG GTT GTA AGT CCA GAG G-3'; and GAPDH, 5'-TCC AGT ATG ACT CCA CTC-3' and 5'-ATT TCT CGT GGT TCA CAC-3'. Fifteen microliters of each PCR reaction product was subjected to electrophoresis on a 1.7% agarose gel.

Prostaglandin Determinations

Medium was collected by centrifugation and analyzed for PGD2 or PGE2, using kits purchased from Amersham.

Assay of PLA2 Enzyme Activity

Phospholipase activity was determined as described previously (19). Briefly, cells were treated as described in the figure legends and under "Results." The supernatants were collected by centrifugation and incubated in a reaction mixture (150 µl) containing 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2 and 0.1 microcurie of [3H]arachidonate-labeled E. coli membranes for 1 h at 37 °C. Free arachidonic acid was separated by elution of the sample over aminopropyl solid phase columns and quantitated by scintillation counting. Data are expressed as pmol of phospholipid hydrolyzed per min per ml and are calculated from the radioactivity and phospholipid content of the E. coli membranes, as supplied by the manufacturer.

Antisense Inhibition of Early Phase PGD2 Production and PLA2 Secretion in Activated Mast Cells

MMC-34 mast cells (2 × 106 cells/ml/well) were plated into six-well culture dishes in serum-free RPMI medium. Antisense murine group V PLA2 and sense murine group V PLA2 oligonucleotides, as described by Balboa et. al. (23) were used at concentrations indicated in the figure legends. Oligonucleotides and Lipofectamine reagent solutions were prepared separately, each in 100 µl of serum-free medium. The oligonucleotides and Lipofectamine reagent were mixed and allowed to incubate at room temperature for 45 min before addition of the mixtures to the MMC-34 mast cell cultures. The final concentration of the Lipofectamine reagent was 5 µg/ml. Four hours after oligonucleotide transfection, 1 ml of RPMI medium containing 10% heat-inactivated fetal bovine serum was added to each well. The oligonucleotide transfection procedure was repeated 12 h after the first transfection. Twelve hours after the second transfection, the MMC-34 mast cells were activated by aggregation of their IgE receptors. One hour after activation, the cell supernatants were analyzed for PGD2 accumulation and phospholipase activity.


RESULTS

Activated Mast Cells Derived from Pla2 g2a+/+ and Pla2 g2a-/- Mice Have Identical PGD2 Induction Profiles

To investigate the role of the group IIa PLA2 enzyme in PGD2 synthesis following mast cell aggregation, we established bone marrow-derived mast cell lines from AKJ mice (wild type for the Pla2 g2a gene encoding group IIa PLA2 protein) and from B6 mice (mutant for the Pla2 g2a gene). When IgE receptors on either AKJ or B6 mast cells were aggregated with IgE and anti-IgE, PGD2 synthesis occurred in two phases, an early phase complete within the first 10-20 min after receptor aggregation and a late phase of PGD2 synthesis that continued throughout the 6-h period of this experiment (Fig. 1).


Fig. 1. Activated mast cells derived from Pla2 g2a+/+ and Pla2 g2a-/- mice have identical PGD2 induction profiles. AKJ and B6 mast cell cultures were plated in 12-well culture dishes (106 cells/ml). Mast cells were activated by aggregating their IgE receptors, using sequential treatment with IgE followed by anti-IgE. For activation, IgE (1 µg/ml) was first added to all wells. Two hours later, cells in each well were washed and replated, and anti-IgE (1 µg/ml) was added. Media were collected at the times indicated after the addition of anti-IgE and used for PGD2 assays. All PGD2 assays were performed on triplicate wells. Data are expressed as averages ± S.D.
[View Larger Version of this Image (15K GIF file)]

To be certain that the genotypes of our AKJ and B6 bone marrow-derived mast cell cultures were correct, we performed Southern blots on DNA taken from the intestines of the parental strains and on DNA prepared from the two mast cell cultures (Fig. 2). Strains with a wild-type Pla2 g2a gene, encoding functional group IIa PLA2 enzyme, have a BamHI restriction site within the coding region. A single base pair insertion into the Pla2 g2a gene eliminates this BamHI restriction site in Pla2 g2a-/- (mom1-/-) mice and renders the gene product nonfunctional (14, 15). As expected, DNA from the intestines of AKJ mice, wild type for the Pla2 g2a gene encoding group IIa PLA2, have the internal BamHI site, while DNA from the intestines of B6 mice, mutant for the Pla2 g2a gene, do not demonstrate this restriction site. Digestion with BamHI of DNA isolated from mast cells derived from AKJ and B6 mice demonstrated that the genotypes of the two murine mast cell lines are correct; PGD2 production in activated mast cells derived from B6 mice occurs normally (Fig. 1), despite the presence of the incapacitating mutation in the Pla2 g2a gene (Fig. 2).


Fig. 2. Southern blot analysis of the Pla2 g2a genotype of mast cells derived from AKJ and B6 mice. DNA was prepared from intestine and mast cell lines of B6 and AKJ mice, as described under "Experimental Procedures." After digestion with BamHI, the DNA samples were subjected to electrophoresis, and the restriction fragments were transferred to a Hybond N+ membrane. The blot was hybridized with a Pla2 g2a cDNA probe spanning the internal BamHI site present in the wild-type gene.
[View Larger Version of this Image (48K GIF file)]

Mast Cells from Mice That Contain a Functional Pla2 g2a Gene Encoding Group IIa PLA2 Do Not Express mRNA for This Enzyme

The group IIa PLA2 enzyme has been thought to be the PLA2 isoform expressed in mast cells. We examined mast cells prepared from B6 and AKJ mice for group IIa PLA2 mRNA by Northern blot analysis. As expected, mast cells from B6 mice, mutant for the Pla2 g2a gene, did not contain detectable group IIa PLA2 mRNA (Fig. 3). However, we were also unable to demonstrate mRNA for group IIa PLA2 in mast cells prepared from wild-type AKJ mice.


Fig. 3. Mast cells from Pla2 g2a+/+ mice do not express group IIa PLA2 (sPLA2 II) mRNA. Total RNA isolated from AKJ intestine, AKJ mast cells, B6 intestine, and B6 mast cells was subjected to electrophoresis, transferred to hybond membranes, and probed with cDNAs for the group IIa PLA2 message and the GAPDH message. Each lane contains 10 µg of total RNA.
[View Larger Version of this Image (39K GIF file)]

The RNA preparation from the B6 mast cell cultures was not degraded in this experiment, and RNA loading was equivalent for B6 and AKJ mast cell preparations, since hybridization to a GAPDH probe demonstrated equivalent signal for GAPDH mRNA in the B6 and AKJ mast cell RNA samples (Fig. 3). Failure to detect group IIa PLA2 message in AKJ mast cells was not due to a problem with the group IIa PLA2 cDNA probe, since mRNA for this gene can be detected in RNA prepared from intestine of AKJ mice. As expected, intestine from B6 mice, mutant for the Pla2 g2a gene, does not contain mRNA for group IIa PLA2.

Mast Cells from Mice That Do Not Contain a Functional Pla2 g2a Gene Encoding Group IIa PLA2 Secrete Phospholipase A2 Activity into the Medium following Activation

We have previously demonstrated that activated MMC-34 mast cells rapidly secrete PLA2 activity into the culture medium when their IgE receptors are aggregated (19). When mast cells derived from AKJ and B6 mice are activated by aggregating their IgE receptors, similar levels of PLA2 activity are secreted into the medium (Fig. 4). Thus, mast cells from animals that are mutant in the Pla2 g2a gene encoding group IIa PLA2 gene still secrete active PLA2 enzyme when their IgE receptors are aggregated.


Fig. 4. Activated mast cells from Pla2 g2a+/+ and Pla2 g2a-/- mice secrete PLA2 (SPLA2) activity. Mast cells were cultured and activated by aggregation of their IgE receptors, as described in the legend to Fig. 1. Supernatants were collected by centrifugation 1 h after the addition of anti-IgE and were assayed for phospholipase activity. All analyses were performed on triplicate wells. Data are expressed as averages ± S.D.
[View Larger Version of this Image (20K GIF file)]

Mast Cells Express mRNA for the Group V PLA2 Protein

Although mast cells from both AKJ (Pla2 g2a+/+) and B6 (Pla2 g2a-/-) mice do not express message for the group IIa PLA2 protein (Fig. 3), both AKJ and B6 mast cell lines secrete PLA2 activity following activation (Fig. 4). We have recently identified genes for two additional low molecular weight, calcium-dependent phospholipases, group IIc PLA2 and group V PLA2 (2-5). Group IIc PLA2 mRNA is observed predominantly in testis, while group V PLA2 message has been observed predominantly in heart. We examined mast cells prepared from B6 and AKJ mice for group V PLA2 mRNA by Northern blot analysis, using RNA from heart as a positive control. Both mast cell populations, one with a wild-type Pla2 g2a gene and one with a mutated Pla2 g2a gene (Fig. 2) express mRNA for the group V PLA2 (Fig. 5). In contrast, intestine from either strain of mice does not contain mRNA for the group V PLA2 gene.


Fig. 5. Mast cells from Pla2 g2a +/+ and Pla2 g2a -/- mice express group V PLA2 mRNA. Total RNA isolated from Swiss Webster mouse heart, AKJ intestine, AKJ mast cells, B6 intestine, and B6 mast cells was subjected to electrophoresis, transferred to hybond membranes, and probed with cDNAs for the group V PLA2 message and the GAPDH message. Each lane contains 10 µg of total RNA.
[View Larger Version of this Image (40K GIF file)]

We also used reverse transcription of mRNA followed by polymerase chain reaction (RT-PCR) with primers for the messages from the group IIa PLA2, the group IIc PLA2, and the group V PLA2 to examine control tissues and mast cells from Pla2 g2a+/+ and Pla2 g2a-/- mice for expression of the three PLA2 isoforms. Heart tissue of Pla2 g2a+/+ Swiss Webster (SW) mice contains mRNA for all three PLA2 isoforms (Fig. 6). Although mRNA for group IIc PLA2 is present in Swiss Webster heart RNA, the signal is not strong. In contrast, message for RNA prepared from testis of the Swiss Webster mice demonstrated a clear signal for group IIc PLA2 mRNA, as expected (5), but did not contain detectable message for the other two PLA2 isoforms. When RNAs from intestines of AKJ (Pla2 g2a+/+) and B6 (Pla2 g2a-/-) mice were used as templates for RT-PCR, message for group IIa PLA2 was present in the AKJ preparation but not in the B6 RNA (Fig. 6), in agreement with our Northern blot analysis (Fig. 3) and data from the literature (14, 15). When RNAs from the AKJ and B6 mast cells were used as templates, no mRNA encoding group IIa PLA2 was observed in either RNA preparation. This result is also in agreement with the Northern blot analysis shown in Fig. 3. In contrast, RNA preparations from both AKJ mast cells and B6 mast cells did express message for the group V PLA2 protein. The RT-PCR data for the expression of group IIa PLA2 and group V PLA2 in mast cells and intestine are in complete agreement with the Northern analysis data presented in Figs. 3 and 5. In addition, mast cell RNA did not contain message for the group IIc PLA2 (Fig. 6). Group V PLA2 appears to be the major, if not the only, secretory PLA2 expressed in activated murine mast cells.


Fig. 6. Reverse transcriptase-polymerse chain reaction analysis of the PLA2 isoforms present in murine heart, intestine, and mast cells. Total RNA was prepared from tissues and from cell cultures. RT-PCR was performed as described under "Experimental Procedures," using primers for GAPDH, group IIa PLA2, group IIc PLA2, and group V PLA2. The sizes of the expected amplified fragments are as follows: GAPDH, 278 nucleotides; group IIa PLA2, 288 nucleotides; group IIc PLA2, 217 nucleotides, and group V PLA2, 329 nucleotides. The presence of the GAPDH product in each tissue set provides an internal normalization for each of the three different PLA2 bands and provides a reference to determine that the appropriate sized band is amplified in each primer reaction.
[View Larger Version of this Image (39K GIF file)]

The PLA2 Secreted by Activated AKJ Mast Cells and B6 Mast Cells Mediates both Their Early Phase of PGD2 Synthesis and Transcellular PGE2 Synthesis in Fibroblasts

We previously demonstrated that the early phase of PGD2 synthesis following aggregation of IgE receptors on MMC-34 murine mast cells can be prevented by co-incubation of the cells either with SB203347, an inhibitor of group IIa PLA2 (21), or with a monoclonal antibody (mAb F10) prepared against recombinant human group IIa PLA2 (24). We have also shown that the ability of medium from activated MMC-34 mast cells to induce transcellular, prostaglandin synthase 1-dependent PGE2 synthesis in 3T3 fibroblast cells can be inhibited by both SB203347 and by mAb F10 (19). Despite the fact that these two PLA2 inhibitors were initially characterized by their ability to inhibit human group IIa PLA2, SB203347 and mAb F10 block the early phase of PGD2 accumulation in activated mast cells derived from both Pla2 g2a+/+ and Pla2 g2a-/- mice (Fig. 7) and the ability of supernatants from mast cells of either strain to induce transcellular prostaglandin synthesis in Swiss 3T3 fibroblasts (Fig. 8).


Fig. 7. PLA2 secreted by activated AKJ mast cells and activated B6 mast cells mediates the early phase of PGD2 synthesis. Mast cells were cultured and activated by aggregration of their IgE receptors, as described in the legend to Fig. 1. SB203347 (1 µM) was added, as indicated, at the time of addition of anti-IgE. NS-398, a prostaglandin synthase 2-specific inhibitor (27) that does not interfere with the early prostaglandin synthase 1-dependent phase of PGD2 synthesis in activated mast cells (16), was added to inhibit the activity of any induced prostaglandin synthase 2. Supernatants were collected by centrifugation from all cultures 1 h after the addition of anti-IgE and were assayed for PGD2. All analyses were performed on triplicate culture wells. Data are expressed as averages ± S.D.
[View Larger Version of this Image (18K GIF file)]


Fig. 8. PLA2 secreted by activated AKJ mast cells and activated B6 mast cells mediates transcellular PGE2 synthesis in Swiss 3T3 fibroblasts. Supernatants were prepared from control mast cells and from cells treated with IgE plus anti-IgE. Where indicated, SB203347 was added at the same time as anti-IgE. Media were collected after 1 h, and 0.9-ml samples were placed on confluent cultures of Swiss 3T3 cells. Culture media were collected 1 h later and assayed for PGE2. All analyses were performed on triplicate culture wells. Data are expressed as averages ± S.D.
[View Larger Version of this Image (16K GIF file)]

An Antisense Oligonucleotide for Group V PLA2 Inhibits both Early Phase PGD2 Production and Secretion of PLA2 in Activated Mast Cells

Balboa et al. (23) demonstrated that P388D1 macrophage-like cells also produce easily detectable levels of group V PLA2. They were unable to detect group IIa PLA2 mRNA by either Northern or RT-PCR analysis. They used antisense oligonucleotides to demonstrate that arachidonic acid mobilization by endotoxin and platelet-activating factor was dependent on the presence of group V PLA2. We have used the same antisense and sense oligonucleotides for murine group V PLA2 (23) to investigate the role of this enzyme in the early phase of PGD2 production in activated mast cells. Preliminary titration experiments suggested that pretreatment of MMC-34 mast cells with 1 µM antisense oligonucleotide for group V PLA2 (AS5) would block activation-induced early phase PGD2 production. Pretreatment of MMC-34 mast cells with the AS5 antisense oligonucleotide was as effective as the PLA2 inhibitor SB203347 in preventing the activation-induced early phase of PGD2 production (Fig. 9). In contrast, the S5 sense oligonucleotide had no effect on activation-induced PGD2 production. Pretreatment with the AS5 antisense oligonucleotide also blocked the release of the bulk of PLA2 enzyme activity associated with mast cell activation.


Fig. 9. An antisense oligonucleotide for group V PLA2 inhibits both early phase PGD2 production and secretion of PLA2 (sPLA2) in activated mast cells. MMC-34 mast cells were cultured and activated by aggregation of their IgE receptors, as described in the legend to Fig. 1. Cells were pretreated with the antisense oligonucleotide AS5 or the sense oligonucleotide S5 as described under "Experimental Procedures." SB203347 (1 µM) was added, as indicated, at the time of the addition of anti-IgE. Supernatants were collected by centrifugation 1 h after the addition of anti-IgE and assayed for PGD2 levels (left panel) and for phospholipase activity (right panel). All analyses were performed on triplicate wells. Data are expressed as averages ± S.D.
[View Larger Version of this Image (21K GIF file)]


DISCUSSION

We initially expected that mast cells derived from animals with a defective Pla2 g2a gene, unable to produce functional group IIa PLA2 (14, 15), would be incapable of the early phase of PGD2 production and would not engage in transcellular prostaglandin synthesis. This assumption was based on (i) previous conclusions that group IIa PLA2 is the low molecular weight phospholipase active in mast cells and (ii) our previous demonstration that both the early phase of PGD2 production in activated mast cells and mast cell/fibroblast transcellular prostaglandin production are blocked by an inhibitor (SB203347) characterized as a group IIa PLA2 inhibitor and by a monoclonal antibody to human group IIa PLA2 (18, 19). However, it is clear that mast cells derived from Pla2 g2a-/- mice (i) cannot, as expected, express group IIa PLA2, (ii) do secrete a low molecular weight PLA2 in response to aggregation of their IgE receptors, (iii) are able to participate in the early phase of PGD2 production following activation, and (iv) secrete the active PLA2 responsible for mast cell/fibroblast transcellular prostaglandin synthesis. These data suggest that another PLA2 enzyme, and not group IIa PLA2, must be responsible for the early phase of PGD2 production following mast cell activation.

Northern and RT-PCR analyses demonstrated that mast cells derived from both AKJ (Pla2 g2a+/+) mice and B6 (Pla2 g2a-/-) mice express message for group V PLA2. In contrast, group IIc PLA2 message could not be detected in either mast cell preparation. These data suggested that the group V PLA2 enzyme mediates the early phase of PGD2 production in activated mast cells. The ability of an antisense oligonucleotide, directed against the group V PLA2 message, to inhibit the secretion of PLA2 and the early phase of PGD2 production in activated mast cells confirmed this suggestion. Northern analyses of the levels of group V PLA2 message in mast cells derived from AKJ and B6 mice demonstrated that the production of group V PLA2 in mice with a mutation in the Pla2 g2a gene (encoding group IIa PLA2) is not due to a developmentally regulated compensating response to the Pla2 g2a mutation. We conclude that, in murine mast cells, PLA2-mediated arachidonic acid release is not mediated by group IIa PLA2 (the product of the Pla2 g2a gene) and is mediated by the group V PLA2 (the product of the Pla2gV gene).

Balboa et al. (23) provided compelling evidence that P388D1 macrophage-like cells do not produce detectable levels of message for the group IIa PLA2 enzyme. Nevertheless, activation of these cells by endotoxin priming followed by platelet activating factor stimulation resulted in secretion of substantial PLA2 activity. P388D1 cells are wild type for the Pla2 g2a gene. Northern and RT-PCR analysis of message levels for the products of the Pla2 g2a, Pla2 g2c, and Pla2gV genes demonstrated the presence of mRNA for group V PLA2, but not for the other two low molecular weight phospholipases. Balboa et al. (23) conclude that group V PLA2, not group IIa PLA2, mediates arachidonic acid release in activated P388D1 macrophages. Their results for LPS/platelet activating factor stimulated P388D1 cells are, therefore, very similar to our results with activated mast cells.

While our studies were in progress, Bingham et al. (25) also reported that activated mast cells derived from C57BL/6J (Pla2 g2a-/-) mice demonstrated normal early and delayed phase PGD2 production. However, they suggest that a secretory phospholipase activity is required for the late phase of PGD2 synthesis in mast cells. Bingham et al. (25) report that mast cells derived from Pla2 g2a+/+ mice could produce some mRNA for group II PLA2. They did not, however, compare the levels of phospholipase enzyme activity secreted from activated mast cells derived from Pla2 g2a-/- and Pla2 g2a+/+ mice, nor did they determine whether group IIc PLA2 or group V PLA2, the products of the Pla2 g2c or Pla2gV genes, are expressed in mast cells. Our data showing that mast cells from Pla2 g2a+/+ and Pla2 g2a-/- (group IIa PLA2-deficient) mice (i) secrete similar levels of phospholipase activity (Fig. 4), (ii) produce equivalent amounts of PGD2 (Fig. 1), and (iii) both stimulate transcellular prostaglandin synthesis in fibroblasts (Fig. 8) all suggest that group IIa PLA2, the product of the Pla2 g2a gene, does not play a major role in prostaglandin production. While additional experiments in which mast cell preparations and inhibitors will need to be compared to understand the differences in the results of our two laboratories, it seems clear from both the results of Bingham et al. (25) and our data that the Pla2 g2a gene plays little or no role in mast cell prostaglandin production.

High levels of PLA2 activity have been described in serum and/or tissue exudates of patients with a variety of inflammatory illnesses, including arthritis, pancreatitis, adult respiratory distress syndrome, and septic shock (6-13). The identification of the PLA2 activity present in normal immune responses and inflammatory conditions has often been based on enzymatic assays, enzyme inhibitor studies, and/or immunologic detection methods that probably cannot discriminate between the enzymatic activities of the group IIa PLA2, group IIc PLA2, and group V PLA2 isoforms. Our demonstration that murine mast cells express message from the Pla2gV gene encoding message for the group V PLA2 and do not express messages for the other low molecular weight phospholipases, along with the observation that group V PLA2, and not group IIa PLA2, mediates arachidonic acid release and prostaglandin synthesis in P388D1 macrophages (23), suggests that the recently discovered group V PLA2 may play a much more prominent role in mediating normal immune responses and pathologic inflammatory responses than previously thought.

Development of phospholipase A2 inhibitors has been an important topic of pharmacologic research because of the potential of such molecules for use as anti-inflammatory agents (26). Although a variety of compounds has been described that can discriminate between secretory PLA2 enzymes and the type IV cytoplasmic PLA2, these compounds have not been analyzed for their ability to discriminate between group IIa PLA2, group IIc PLA2, and group V PLA2 enzyme activity. The demonstration that group V PLA2 mediates both mast cell and macrophage prostaglandin production following ligand activation should rekindle interest in this area of drug design and screening.


FOOTNOTES

*   These studies were supported by the UCLA Asthma, Allergic and Immunologic Diseases Center (AI34567) funded by the NIAID and by the NIEHS (National Institutes of Health) (to H. R. H.), and by National Institutes of Health Grant DK38185 (to J. A. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: 341A Molecular Biology Institute, UCLA, Los Angeles, CA 90095. Tel.: 310-825-8735; Fax: 310-825-1447; E-mail: harvey{at}lbes.medsch.ucla.edu.
1   The abbreviations used are: PLA2, secretory phospholipase A2; PGD2, prostaglandin D2; PGE2, prostaglandin E2; mAb, monoclonal antibody; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

ACKNOWLEDGEMENTS

We thank Raymond Basconcillo and Arthur Catapang for technical assistance. We also thank Lisa Marshall for the gifts of mAb F10, recombinant PLA2, and SB203347.


REFERENCES

  1. Glaser, K. B. (1995) Adv. Pharmacol. 32, 31-66 [Medline] [Order article via Infotrieve]
  2. Dennis, E. A. (1994) J. Biol. Chem. 269, 13057-13060 [Free Full Text]
  3. Tischfield, J. A., Xia, Y. R., Shih, D. M., Klisak, I., Chen, J., Engle, S. J., Siakotos, A. N., Winstead, M. V., Seilhamer, J. J., Allamand, V., Gyapay, G., and Lusis, A. (1996) Genomics 32, 328-333 [CrossRef][Medline] [Order article via Infotrieve]
  4. Chen, J., Engle, S. J., Seilhamer, J. J., and Tischfield, J. A. (1994) J. Biol. Chem. 269, 2365-2368 [Abstract/Free Full Text]
  5. Chen, J., Engle, S. J., Seilhamer, J. J., and Tischfield, J. A. (1994) J. Biol. Chem. 269, 23018-23024 [Abstract/Free Full Text]
  6. Hara, S., Hudo, I., Chang, H. W., Matsusta, K., Miyamoto, T., and Inoue, K. (1989) J. Biochem. (Tokyo) 105, 395-399 [Abstract]
  7. Vadas, P., Stefanski, E., and Pruzanski, W. (1985) Life Sci. 36, 579-587 [Medline] [Order article via Infotrieve]
  8. Seilhamer, J. J., Pruzanski, W., Vadas, P., Plant, S., Miller, J. A., Kloss, J., and Johnson, L. K. (1989) J. Biol. Chem. 264, 5335-5338 [Abstract/Free Full Text]
  9. Vadas, P., Pruzanski, W., Stefanski, E., Sternby, B., Mustard, R., Bohnen, J., Fraser, I., Farewell, V., and Bombardier, C. (1988) Crit. Care Med. 16, 1-7 [Medline] [Order article via Infotrieve]
  10. Vadas, P. (1984) J. Lab. Clin. Med. 104, 873-881 [Medline] [Order article via Infotrieve]
  11. Schroder, T., Kivilaakso, E., Kinnunen, P. K. J., and Lempinen, M. (1980) Scand. J. Gastroenterol. 15, 633-636 [Medline] [Order article via Infotrieve]
  12. Forster, S., Ilderton, E., Norris, J. F. B., Summerly, R., and Yardley, H. J. (1985) Br. J. Dermatol. 112, 135-147 [Medline] [Order article via Infotrieve]
  13. Forster, S., Ilderton, E., Summerly, R., and Yardley, H. J. (1983) Br. J. Dermatol. 108, 103-105 [Medline] [Order article via Infotrieve]
  14. Kennedy, B. P., Payette, P., Mudgett, J., Vadas, P., Pruzanski, W., Kwan, M., Tang, C., Rancourt, D. E., and Cromlish, W. A. (1995) J. Biol. Chem. 270, 22375-22385
  15. MacPhee, M., Chepenik, K. P., Liddell, R. A., Nelson, K. K., Siracusa, L. D., and Buchberg, A. M. (1995) Cell 81, 957-966 [Medline] [Order article via Infotrieve]
  16. Kawata, R., Reddy, S. T., Wolner, B., and Herschman, H. R. (1995) J. Immunol. 155, 818-825 [Abstract]
  17. Murakami, M., Bingham, C. O., III, Matsumoto, R., Austen, K. F., and Arm, J. P. (1995) J. Immunol. 155, 4445-4453 [Abstract]
  18. Reddy, S. T., and Herschman, H. R. (1997) J. Biol. Chem. 272, 3231-3237 [Abstract/Free Full Text]
  19. Reddy, S. T., and Herschman, H. R. (1996) J. Biol. Chem. 271, 186-191 [Abstract/Free Full Text]
  20. Razin, E., Mencia-Huerta, J-M., Stevens, R. L., Lewis, R. A., Liu, F-T., Corey, E. J., and Austen, K. F. (1983) J. Exp. Med. 157, 189-201 [Abstract]
  21. Marshall, L. A., Hall, R. H., Winkler, J. D., Badger, A., Bolognese, B., Roshak, A., Louis-Flamberg, P., Sung, C-M., Chabot-Fletcher, M., Adams, J. L., and Mayer, R. J. (1995) J. Pharm. Exp. Ther. 274, 1254-1262 [Abstract]
  22. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., pp. 14-26, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Balboa, M. A., Balsinde, J., Winstead, M. V., Tischfield, J. A., and Dennis, E. A. (1996) J. Biol. Chem. 271, 32381-32384 [Abstract/Free Full Text]
  24. Fonteh, A. N., Bass, D. A., Marshall, L. A., Seeds, M., Samet, J. M., and Chilton, F. H. (1994) J. Immunol. 152, 5438-5446 [Abstract/Free Full Text]
  25. Bingham, C. O., III, Murakami, M, Fujishima, H., Hunt, J. E., Austen, K. F., and Arm, J. P. (1996) J. Biol. Chem. 271, 25936-25944 [Abstract/Free Full Text]
  26. Gelb, M. H., Jain, M. K., and Berg, O. G. (1994) FASEB J. 8, 916-924 [Abstract/Free Full Text]
  27. Futaki, N., Takahashi, S., Yokoyama, M., Arai, I., Higuchi, S., and Otomo, S. (1994) Prostaglandins 47, 55-59 [CrossRef][Medline] [Order article via Infotrieve]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.