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J Biol Chem, Vol. 274, Issue 44, 31476-31484, October 29, 1999

Low Molecular Weight Group IIA and Group V Phospholipase A₂ Enzymes Have Different Intracellular Locations in Mouse Bone Marrow-derived Mast Cells^*

Clifton O. Bingham III^§¶, Remond J. A. Fijneman^§, Daniel S. Friend^§**, Richard P. Goddeau^§, Rick A. Rogers, K. Frank Austen^§§§, and Jonathan P. Arm^§§§¶¶

From the Departments of  Medicine and ^** Pathology, Harvard Medical School, the ^§ Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, the ^§§ Partners Asthma Center, Brigham and Women's Hospital, and the  Biomedical Imaging Laboratory, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The subcellular location of the enzymes of eicosanoid biosynthesis is critical for their co-ordinate action in the generation of leukotrienes and prostaglandins. This activity is thought to occur predominantly at a perinuclear location. Whereas the subcellular locations of cytosolic phospholipase (PL) A₂ and each of the pathway enzymes of eicosanoid generation have been defined, the distribution of the low molecular weight species of PLA₂ has remained elusive because of the lack of antibodies that distinguish among homologous family members. We have prepared affinity-purified rabbit antipeptide IgG antibodies that distinguish mouse group IIA PLA₂ and group V PLA₂. Immunofluorescence staining and immunogold electron microscopy reveal different subcellular locations for the enzymes. Group IIA₂ PLA₂ is present in the secretory granules of mouse bone marrow-derived mast cells, consistent with its putative role in facilitating secretory granule exocytosis and its consequent extracellular action. In contrast, group V PLA₂ is associated with various membranous organelles including the Golgi apparatus, nuclear envelope, and plasma membrane. The perinuclear location of group V PLA₂ is consistent with a putative interaction with translocated cytosolic PLA₂ in supplying arachidonic acid for generation of eicosanoid products, while the location in Golgi cisternae may also reflect its action as a secreted enzyme. The spatial segregation of group IIA PLA₂ and group V PLA₂ implies that these enzymes are not functionally redundant.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Arachidonic acid, released from membrane phospholipids by phospholipase (PL)¹ A₂, undergoes oxidative metabolism to generate eicosanoid lipid mediators such as leukotrienes and prostanoids. These eicosanoids have been implicated in diverse physiologic and pathologic processes including maintenance of normal renal function, protection of the gastric mucosa, hemostasis, parturition, pain, various forms of inflammation, asthma, and cancer (1, 2). Mammalian PLA₂ enzymes, which are classified according to structure (3-5), include the cysteine-rich low molecular weight groups IB, IIA, IIC, V, and X enzymes (5, 6); the 85-kDa group IV cytosolic PLA₂ (cPLA₂) (7); and the group VI calcium-independent PLA₂ (8).

Of the PLA₂ enzymes that have been described to date, cPLA₂, group IIA PLA₂, and group V PLA₂ have been implicated in eicosanoid generation. The participation of cPLA₂ in leukotriene (LT) and prostaglandin (PG) generation is established by studies with cells from mice in which the gene for cPLA₂ has been disrupted (9-11). In peritoneal macrophages from cPLA₂-deficient mice the immediate phase of LTB₄, LTC₄, and PGE₂ generation that occurs in minutes in response to A23187 and the delayed phase of PGE₂ generation that occurs over several hours in response to lipopolysaccharide (LPS) were both markedly attenuated (9, 10). Similarly, both immediate-phase leukotriene and PGD₂ generation in response to dimerization of c-kit by stem cell factor or to cross-linking of the high affinity Fc receptor for IgE (FcRI) by hapten-specific IgE and antigen, and cytokine-initiated, prostaglandin endoperoxide synthase (PGHS) 2-dependent, delayed phase PGD₂ generation were completely absent in bone marrow-derived mast cells (BMMC) from cPLA₂-deficient mice (11).

Much of the early data implicating group IIA PLA₂ in eicosanoid generation were based on reagents that are now known to cross-react with group V PLA₂ (5). Nevertheless, that group IIA PLA₂ may participate in the delayed phase of PGE₂ generation by LPS-stimulated rat peritoneal macrophages is indicated by the inhibitory effects of the low molecular weight PLA₂ inhibitor, thielocin A1, and an antibody to group IIA PLA₂ that did not recognize group V PLA₂ (12). Furthermore, group V PLA₂ was minimally expressed in the LPS-treated peritoneal macrophages (12). By contrast, pharmacologic and antisense inhibition experiments in LPS-primed P388D1 macrophages have implicated group V PLA₂ in supplying arachidonic acid to PGHS-2 for a rapid phase of PGE₂ generation in response to platelet-activating factor (13-15). Pharmacologic inhibition experiments in BMMC from mice lacking group IIA PLA₂ have implicated group V PLA₂ in immediate PGD₂ generation via PGHS-1 (16, 17) and alternatively in delayed PGD₂ generation via PGHS-2 (18). In transfection studies group IIA PLA₂ and group V PLA₂ have been essentially interchangeable (19), even though their catalytic functions appear to differ substantially for phospholipid substrates (20).

Eicosanoid generation depends on the subcellular localization and/or translocation of individual enzymes of leukotriene and prostaglandin biosynthesis. Group IV cPLA₂ translocates from the cytosol to a perinuclear location after cell activation (21-23). 5-Lipoxygenase (5-LO) is present in the cytosol and/or nucleosol of unstimulated cells but translocates to the nuclear envelope with cell activation (23-27). The integral perinuclear membrane protein 5-LO activating protein (FLAP) is essential for the cellular processing of released arachidonic acid by 5-LO to 5-hydroperoxyeicosatetraenoic acid and then to LTA₄ (25, 28, 29). LTA₄ is either conjugated by integral perinuclear LTC₄ synthase with glutathione to form LTC₄ (30) or is converted by cytosolic LTA₄ epoxide hydrolase to LTB₄. The intermediate enzymes of prostaglandin biosynthesis, constitutive PGHS-1 and induced PGHS-2, are localized to the nuclear envelope and contiguous endoplasmic reticulum (31). Thus, cPLA₂, 5-LO, FLAP, PGHS-1, PGHS-2, and LTC₄ synthase either are integral or translocate to a perinuclear site. In contrast, the locations of the group IIA and group V PLA₂ enzymes have not been established. The studies of the group IIA enzyme were carried out before the size of the family of low molecular weight enzymes was recognized, and used antibodies of poorly defined specificity or antibodies that are now known to cross-react with other low molecular weight enzymes (5). There have been no studies of the subcellular localization of the group V enzyme.

We sought to compare the subcellular localization of group IIA PLA₂ and group V PLA₂ in primary cultures of mouse BMMC, which have transcripts for both enzymes (17, 18, 32). Using affinity-purified specific rabbit antipeptide IgG with specificity to the recombinant proteins, we demonstrate that these two PLA₂ enzymes exist in different subcellular compartments in these cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

Chemicals were purchased from Sigma unless otherwise noted. Restriction endonucleases were purchased from Roche Molecular Biochemicals. The pCEP expression vector was provided by Jay A. Tischfield (Rutgers University, Piscataway, NJ), and the mouse monoclonal IgG anti-rat group IIA PLA₂ was a gift from Makato Murakami (Showa University, Tokyo, Japan). Oligonucleotides were synthesized by Oligos, Etc. (Wilsonville, OR).

Culture of BMMC

Mouse BMMC from BALB/c and C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were cultured for 4-9 weeks as described (18). Mast cells were at least 97% pure, as assessed by staining with toluidine blue or with alcian blue and safranin. The 293S human embryonal kidney cell line (ATCC) was cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 4.5 g/liter glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 10 µg/ml gentamycin, 1.0 mM sodium pyruvate, and 2 mM L-glutamine. Spodoptera frugiperda (Sf9) and HighFive^TM insect cells (Invitrogen, Carlsbad, CA) were cultured in Grace's insect medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin-B (Life Technologies, Inc.).

Cloning, Expression, and Protein Purification of Mouse PLA₂ Enzymes

Cloning of Mouse Group V PLA₂ cDNA-- At the time these experiments were conducted, the cDNA for human and rat, but not mouse, group V PLA₂ had been reported (33, 34). To clone the cDNA encoding the mouse group V PLA₂ enzyme, total RNA was isolated from the hearts of BALB/c mice in guanidine thiocyanate with TriReagent^TM (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. One µg of total RNA was mixed with 20 pmol of oligo(dT) for 2 min at 70 °C and then incubated with Moloney murine leukemia virus (MMLV) reverse transcriptase (RT) (Advantage^TM RT-for-PCR, CLONTECH, Palo Alto, CA) for 1 h at 42 °C to generate a cDNA template for polymerase chain reaction (PCR). Homologous regions of the rat and human group V PLA₂ cDNAs (33) (GenBank accession nos. U03763 and U03090, respectively) were used to design PCR primers. The sense primer, PV-1 5'-ACGCTGGCTTGGTTCCTGGC-3', and the antisense primer, PV-2 5'-GACATTAGCAGAGGAAGTTGGG-3', correspond to nucleotides 231-249 and 612-633, respectively, of the rat cDNA. PCR was carried out for 30 cycles of 94 °C for 1 min, 62 °C for 1 min, and 72 °C for 2 min in 15 mM (NH₄)₂SO₄, 2.0 mM Mg²⁺, 60 mM Tris-HCl, pH 9.5, with 0.3 units of Taq polymerase (Perkin-Elmer, Foster City, CA). A major PCR product of ~400 bp was purified with silica gel (Qiagen, Valencia, CA), subcloned into pCRII (Invitrogen), and sequenced in both strands with an Applied Biosystems model 377 cycle sequencer. A BALB/c mouse heart cDNA library in Lambda Zap II (Stratagene, La Jolla, CA) was screened with the 400-bp mouse group V cDNA labeled by random priming as described previously (35). A 1.9-kb mouse group V PLA₂ cDNA was isolated and sequenced in both strands.

The full-length 1.9-kb mouse group V PLA₂ cDNA encoded a 222-bp 5'-untranslated region, a 411-bp open reading frame, and a 1279-bp 3'-untranslated region including a classical AATAAA polyadenylylation signal 14 bp upstream from a poly(A) tail. The open reading frame of mouse group V PLA₂ encodes 137 amino acids. A putative 20-amino acid signal peptide is identified (36), resulting in a mature protein of 117 amino acids with an estimated molecular mass of 13.8 kDa and an estimated pI of 8.25.

Expression of Mouse Group V PLA₂-- The mouse group V PLA₂ cDNA in Lambda Zap II was used as a template for a 20-cycle PCR reaction with primers flanking the open reading frame of mouse group V PLA₂ (PV-3 5'-TCGGATCCAGGCTACAAAGAACCCAA-3', and PV-4 5'-ATCTCGAGATTAGCAGAGGAAGTTGGGG-3') with cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min. The PCR product was subcloned into pCRBac (Invitrogen) and sequenced to ensure the absence of PCR artifacts. The 455-bp PCR product was excised with NheI and XhoI, subcloned into the pCEP mammalian expression vector (34), and stably expressed in 293S cells with calcium phosphate transfection (CalPhos^TM Maximizer, CLONTECH) with hygromycin B (Calbiochem, La Jolla, CA) selection. Supernatants from transfected cells and from untransfected cells were collected and analyzed for PLA₂ activity as described (18). Briefly, an 85-µl portion of supernatant was incubated at 37 °C for 1 h with 10 µM 1-palmitoyl-2-[¹⁴C]arachidonyl-phosphatidylethanolamine (NEN Life Science Products) in 100 mM Tris-HCl, at variable pH and calcium concentrations in a final volume of 125 µl. PLA₂ activity was measured by the release of [¹⁴C]- or [³H]arachidonic acid. A 25-ml portion of transfected 293S cell supernatant was loaded sequentially over a Protein A-Sepharose column (Amersham Pharmacia Biotech) followed by a Q-Sepharose anion exchange column (Amersham Pharmacia Biotech). After being washed extensively with 20 mM Tris-HCl, pH 7.4, the Q-Sepharose column was eluted with a NaCl gradient from 200 mM to 1 M. Then, 1-ml fractions were collected and assayed for PLA₂ activity with phosphatidylethanolamine as substrate at pH 9, with 4 mM Ca²⁺; these conditions were determined to be optimal for the assessment of PLA₂ enzymatic activity. Fractions were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) immunoblotting with the rabbit anti-group V PLA₂ (described below).

Cloning of Mouse Group IB PLA₂ cDNA-- Total RNA was isolated from BALB/c mouse lung tissue and cDNA was generated with MMLV reverse transcriptase as described above. PCR primers were designed based on the homologous rat and human group IB PLA₂ cDNAs (GenBank accession nos. D00036 and M21054, respectively). The primers, PIB-1 (5'-GGCTGTGTGGCAGTTCCGC-3') and PIB-2 (5'-GTGTTGGTGTAGGGGTTGTC-3'), corresponding to nucleotides 77-95 and 273-292, respectively, of the rat sequence, were used in a 25-cycle PCR reaction with the mouse lung cDNA as a template at an annealing temperature of 55 °C. A 500-fold dilution of the PCR product thus generated was used in a second 30-cycle PCR reaction with a set of nested primers, PIB-3 (5'-GTTCCGCAATATGATCAAGTGC-3') and PIB-4 (5'-CTTTCCAGCTTCTTGGCCTG-3'), corresponding to nucleotides 89-110 and 237-256, respectively, of the rat sequence.

A BALB/c mouse lung 5'-STRETCH^TM cDNA library in gt11 (CLONTECH) was screened with the 168-bp mouse group IB PLA₂ nested PCR product as a probe. A 551-bp mouse group IB PLA₂ cDNA was isolated, amplified by PCR with phage DNA as template and gt11 primers, sequenced in both strands, and subcloned into pCR2.1. Cycle sequencing revealed 90% nucleotide identity to the rat group IB PLA₂ cDNA. Amplification by RT-PCR and sequencing of the entire group IB PLA₂ coding region from C57BL/6 lung revealed complete identity with the BALB/c nucleotide sequences.

The full-length 551-bp mouse group IB PLA₂ cDNA encoded a 21-bp 5'-untranslated region, a 438-bp open reading frame, and an 89-bp 3'-untranslated region including a classical AATAAA polyadenylylation signal 14 bp upstream from a poly(A) tail. The open reading frame of mouse group IB PLA₂ encodes 146 amino acids. A putative 15-amino acid signal peptide (36) and a 7-amino acid activation peptide (37) are identified (Fig. 1). The mature protein of 124 amino acids has an estimated molecular mass of 14.1 kDa and an estimated pI of 6.11.

Expression of Group IIA PLA₂-- The cDNA encoding the open reading frame of mouse group IIA PLA₂ (18) was subcloned into pVL1393 (PharMingen, San Diego, CA) and co-transfected with Baculo-Gold^TM linearized baculovirus DNA (PharMingen) into Sf9 cells with calcium phosphate according to the manufacturer's instructions. After plaque purification and amplification of recombinants, HighFive insect cells were infected with high titer viral stocks. Supernatants were assayed for PLA₂ activity with phosphatidylethanolamine as substrate at pH 9.0 and 4 mM Ca²⁺, as described above. A 50-ml portion of insect cell culture supernatant was loaded sequentially over a Protein A-Sepharose column and then a heparin-Sepharose column (Amersham Pharmacia Biotech). After being washed extensively with phosphate-buffered saline (PBS) pH 7.4, the heparin-Sepharose column was eluted with a NaCl gradient from 250 mM to 1 M. One-ml fractions were assayed for PLA₂ activity. Fractions with PLA₂ activity were pooled and loaded over an affinity column of mouse IgG anti-rat group IIA PLA₂ coupled to formyl Cellulofine. After being washed extensively, the column was eluted with 0.2 M glycine, pH 2.5; fractions were collected and assayed for PLA₂ activity, and protein content was measured by determination of the optical density at 280 nm. Fractions containing PLA₂ activity were resolved in 12% SDS-PAGE (Novex) under reducing conditions and silver-stained according to manufacturer's instructions (Sigma) to reveal a single protein band of ~17 kDa.

RT-PCR Analysis of Transcripts for Group IB and Group V PLA₂

Total RNA was extracted from mouse BMMC, heart, and lung, and cDNA was synthesized with MMLV reverse transcriptase as described above. cDNA encoding group IB PLA₂ was amplified with primers PIB-5 (5'-CCTCACTCCTTCTGAAGATG-3') and PIB-6 (5'-CTGACAGCAGGTACTTTATTAG-3'), corresponding to nucleotides 5-24 and 530-551, respectively, of the mouse group IBPLA₂ cDNA. cDNA encoding group V PLA₂ was amplified with primers PV-1 and PV-2. The amplified products were resolved in 2% agarose gels and visualized with ethidium bromide.

Generation and Characterization of Anti-peptide Antibodies for Group IIA and Group V PLA₂

The predicted amino acid sequences for the mouse group IB, IIA, IIC, and V PLA₂ enzymes were aligned (Fig. 1). Peptide sequences unique for group IIA and group V PLA₂ were identified that did not show significant homology to other proteins in the Swiss-Protein and translated GenBank data bases and that were considered immunogenic based on amino acid composition. Synthetic peptides that had been purified with high pressure liquid chromatography were synthesized and coupled to keyhole limpet hemocyanin (KLH) (Quality Controlled Biochemicals, Hopkinton, MA). New Zealand White rabbits were immunized subcutaneously with 500 µg of peptide-KLH conjugate in complete Freund's adjuvant. At 3, 6, and 9 weeks, the rabbits received booster doses subcutaneously of 250 µg of the peptide-KLH conjugate in incomplete Freund's adjuvant. Antisera to group IIA and group V PLA₂ were positive in enzyme-linked immunosorbent assay against immunizing peptides conjugated to bovine serum albumin (BSA) at titers of >1:12,000 and >1:100,000, respectively. High titer sera were affinity-purified over a column of the immunizing peptide linked to thiol coupling gel (Quality Controlled Biochemicals).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Alignment of the predicted amino acid sequences of mouse low molecular weight PLA2 enzymes. The signal peptide, start of the mature protein, and the Ca2+-binding domain are indicated. The activation peptide of mouse group IB PLA2 is boxed. The peptides of group V PLA2 and group IIA PLA2 that were used as immunogens are underlined. *, catalytic residues; , gap introduced into the alignment.

SDS-PAGE Immunoblotting

BALB/c BMMC and C57BL/6 BMMC, minced mouse hearts, and distal small intestines were lysed by sequential freezing and thawing in a buffer containing 50 mM Tris-HCl, pH 7.4, 0.1% SDS, 0.5% Nonidet P-40 (Roche Molecular Biochemicals), 5 mM Na₃VO₄, 50 µg/ml leupeptin, 1.5 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Lysates from 2 × 10⁵ BMMC, 2 mg of tissues, 150 ng of purified mouse group IIA PLA₂, and 10 µl of partially purified mouse group V PLA₂ were applied to 16% polyacrylamide gels (Novex) under reducing conditions. Gels were transferred to nitrocellulose membranes (Bio-Rad), and blocked with 5% nonfat dry milk, 0.5% normal goat serum (Jackson Immunoresearch, West Grove, PA) in 150 mM NaCl buffered with 10 mM Tris-HCl, pH 7.4, 0.1% Tween 20. Immunoblotting was performed for 2 h at room temperature with rabbit IgG anti-group IIA PLA₂ at 0.5 µg/ml, and for 1 h at room temperature with rabbit IgG anti-group V PLA₂ at 0.58 µg/ml, or with non-immune rabbit IgG (Jackson Immunoresearch). The blots were washed with Tris-buffered saline with Tween 20, incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) for 30 min to 1 h, and washed extensively. The protein bands were visualized with enhanced chemiluminescence (SuperSignal^®, Pierce) and Biomax^TM XR film (Eastman Kodak Co.) for 30 s to 5 min.

Analytical Procedures for Subcellular Localization

Immunohistochemistry-- BMMC from BALB/c and C57BL/6 mice were cytocentrifuged onto slides, fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, and washed with PBS containing 0.1% BSA and 0.05% Tween 20 (wash buffer). After treatment with 0.025% saponin for 10 min, the slides were blocked with 3% normal goat serum (Vector Laboratories, Burlingame, CA) in wash buffer for 30 min (blocking buffer). Primary antibodies, rabbit anti-group IIA PLA₂, rabbit anti-group V PLA₂, and non-immune rabbit IgG were prepared in blocking buffer at 1 µg/ml and were applied for 2 h at room temperature in a humidified environment. The slides were washed three times, incubated with biotinylated goat anti-rabbit IgG (Vector) for 40 min, washed again, and incubated with Vectastain ABC reagent (Vector) for 40 min. After further washing, Vectastain alkaline phosphatase substrate was added, and the slides were incubated for 15 min in the dark. The slides were washed with water, counterstained with Gill's hematoxylin no. 2 (Polysciences, Worthington, PA) for 20 s, and mounted with Immun-mount (Shandon, Atlanta, GA). The slides were evaluated with a Leica microscope (model Dialux 20) equipped with a 50× objective, and the results were photographically recorded with Kodak Royal Gold film, ASA 25.

Immunofluorescence-- BMMC in suspension were fixed with 2% paraformaldehyde in PBS for 10 min at room temperature, washed once with Hanks' balanced salt solution without Mg²⁺ or Ca²⁺ containing 0.1% BSA (HBA), permeabilized with 0.025% saponin in PBS for 10 min at 4 °C, and washed once with HBA. Primary antibodies prepared in HBA were applied for 1 h at 4 °C. The rabbit anti-mouse group IIA PLA₂ was used at 10 µg/ml; the rabbit anti-mouse group V PLA₂ was used at 5 µg/ml, and purified non-immune rabbit IgG was used at 10 or 5 µg/ml, respectively, as a control. Cells were washed in HBA and then treated for 1 h at 4 °C with fluorescein isothiocyanate (FITC)-conjugated goat F(ab') ₂ anti-rabbit IgG F(ab') ₂ (Cappel, West Chester, PA) diluted 1:20 for epifluorescence and confocal laser scanning microscopy or 1:50 for cytofluorographic analysis. For epifluorescence, BMMC were resuspended in Vectashield^TM mounting medium (Vector). For confocal laser scanning microscopy, BMMC were suspended in ProLong^TM antifade mounting medium (Molecular Probes, Eugene, OR).

Alternatively, BMMC were washed in cold HBA, fixed in 2% paraformaldehyde for 10 min at 4 °C, washed again, and resuspended in cold HBA. 4 × 10⁴ BMMC were pelleted onto 12-mm circular glass coverslips (Fisher Scientific) with a cytocentrifuge and were placed in wells of a 24-well tissue culture dish. BMMC were permeabilized in 100% methanol at 20 °C for 20 min, washed in HBA, and blocked in HBA containing 2.5% normal donkey serum (Sigma) for 30 min at room temperature. BMMC were then incubated with 5 µg/ml rabbit anti-group V PLA₂ in blocking buffer for 1 h at room temperature, washed with HBA, and incubated with Texas Red-conjugated donkey anti-rabbit IgG (heavy and light chains) (Jackson Immunoresearch) for 1 h at room temperature. Cells were washed in HBA and mounted in 15% w/v Vinol 205 (Air Products and Chemicals, Allentown, PA), 33% v/v glycerol, 0.1% azide in PBS, pH 8.5.

Cytofluorographic analysis of cells was performed on a FACSort^TM machine (Becton Dickinson, Oxnard, CA). The results are presented as overlaid histograms. For epifluorescence, cells were visualized at 40× or 60× with oil with a Nikon Optishot-2 microscope, or at 100× with a Nikon Eclipse 800 microscope. For confocal laser scanning microscopy, BMMC were examined with a Leica TCSNT confocal laser scanning microscope (Leica Inc, Exton, PA) fitted with air-cooled argon and krypton lasers. Fields of view were selected and brought into view under bright-field imaging conditions. Confocal micrographs of emission spectra (>510 nm) were recorded under fluorescent imaging mode with an excitation wavelength of 488 nm. Images were collected from a 100× oil objective lens with a 0.02-µm pixel size. Micrographs were examined with ImageSpace software (Molecular Dynamics, Sunnyvale, CA) with a pseudocolor thermal gradient map applied.

Immunogold/Electron Microscopy-- BMMC were resuspended in 2% paraformaldehyde in 200 mM Hepes, pH 7.4. After 1 h of fixation at room temperature, the samples were pelleted at 14,000 rpm in a tabletop centrifuge for 5 min and incubated overnight at 4 °C. The pellets were infiltrated with 2.1 M sucrose in PBS containing 0.2 M glycine for 15 min and then frozen in liquid nitrogen. Ultrathin (60 nm) sections were cut at 120 °C with a cryo diamond knife, picked up with a loop dipped in 2.3 M sucrose, and transferred to a Formvar-carbon-coated copper grid (Electron Microscopy Sciences, Fort Washington, PA). The grids were placed on a drop of PBS and processed for immunogold labeling. The grids were wet with 0.1% BSA in PBS for 15 min. Stock solutions containing the primary antibodies and the protein A-gold conjugate (Janslot, Utrecht, Netherlands) were diluted in 1% BSA in PBS with 0.5% fish skin gelatin and centrifuged for 1 min at 14,000 rpm before use. Primary antibodies were used at the following concentrations: rabbit anti-group IIA PLA_2, 25 µg/ml; rabbit anti-group V PLA₂, 18 µg/ml; and non-immune rabbit IgG at the same concentrations. The grids were incubated with the primary antibodies for 30 min and rinsed with 0.1% BSA in PBS for 15 min. The protein A-gold conjugate was applied for 20 min. The grids were washed for 15 min with 0.1% BSA in PBS, floated for 10 min in a mixture of 0.3% uranyl acetate dissolved in 2% methyl cellulose (Electron Microscopy Sciences), and examined in a Jeol 1200 EX transmission electron microscope. Images were recorded at a primary magnification of between 10,000× and 30,000×.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Transcripts for Group V PLA₂ but Not Group IB PLA₂ in BMMC-- We previously identified group IIA PLA₂ transcripts by RT-PCR in BMMC from BALB/c mice (18) and confirmed the work of others (38, 39) that BMMC from the C57BL/6 strain are deficient in group IIA PLA₂. We extended these data to examine the presence of transcripts for the group V and group IB PLA₂ in BALB/c and C57BL/6 BMMC using RT-PCR with specific primers (PV-1 and PV-2, or PIB-5 and PIB-6, respectively). We identified an appropriately sized 404-bp RT-PCR product for group V PLA₂ in BMMC from both BALB/c and C57BL/6 mice (Fig. 2A). A 547-bp RT-PCR product for group IB PLA₂ was detected in mouse lung but not in BMMC (Fig. 2B). Further PCR amplification with nested primers also failed to reveal group IB PLA₂ transcripts in BMMC (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   RT-PCR analysis of transcripts for group V PLA2 (A) and group IB PLA2 (B). RNA was extracted from the tissues and cells of BALB/c and C57BL/6 mice as indicated, cDNA was synthesized, and transcripts for group IB PLA2 and group V PLA2 were amplified by RT-PCR as described under "Experimental Procedures." The products were resolved in 2% agarose gels and visualized with ethidium bromide. The size of the RT-PCR products in base pairs (bp) is shown.

Specificity of Group IIA and Group V PLA₂ Antibodies-- Sequence alignment of the mouse low molecular weight PLA₂ enzymes (Fig. 1) allowed the identification of unique peptide sequences in the group IIA and group V PLA₂ proteins that were used as immunogens to generate affinity-purified polyclonal antiserum specific for each enzyme. The specificity of each antibody was initially assessed in SDS-PAGE immunoblotting of intestine (group IIA) (40), heart (group V) (34), BMMC from BALB/c and C57BL/6 mice, partially purified recombinant mouse group V PLA₂, and purified recombinant mouse group IIA PLA₂. C57BL/6 mice lack group IIA PLA₂ due to a natural disruption of the group IIA PLA₂ gene (38, 39). The rabbit anti-mouse group IIA PLA₂, but not rabbit non-immune IgG, recognized an ~17-kDa protein in intestines from BALB/c but not C57BL/6 mice that co-migrated with purified recombinant mouse group IIA PLA₂ on SDS-PAGE immunoblotting (Fig. 3A). The rabbit anti-mouse group IIA PLA₂ did not recognize recombinant group V PLA₂ (Fig. 3C). The rabbit anti-mouse group V PLA₂ recognized a ~17-kDa protein on SDS/PAGE immunoblotting of mouse heart, an organ with abundant group V PLA₂ transcripts (34), and of BMMC from both BALB/c and C57BL/6 mice (Fig. 3B). The rabbit anti-mouse group V PLA₂ recognized a protein of ~17 kDa in fractions of partially purified recombinant group V PLA₂ but did not recognize recombinant group IIA PLA₂ (Fig. 3D). The immunoreactivity of each antibody was absorbed in a dose-dependent fashion by the corresponding immunizing peptide, but not by an irrelevant peptide (data not shown).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   SDS-PAGE immunoblotting of mouse group IIA PLA2 (A and C) and group V PLA2 (B and D). Purified recombinant group IIA PLA2 (rIIA), partially purified recombinant group V PLA2 (rV), and lysates of heart, intestine, and BMMC from BALB/c and C57BL/6 mice were resolved in 16% SDS-PAGE gels, transferred to Immobilon-N, and stained with rabbit IgG anti-group IIA PLA2 (A and C) or with rabbit IgG anti-group V PLA2 (B and D) as described under "Experimental Procedures."

BMMC from BALB/c mice were further evaluated for expression of group IIA and group V PLA₂ by flow cytometry before and after permeabilization with 0.025% saponin. Non-immune rabbit IgG served as a negative control for each antibody. Group IIA and group V PLA₂ were detected in only a small proportion of nonpermeabilized cells by flow cytometry (data not shown). Permeabilization of cells with 0.025% saponin revealed appreciable intracellular staining for both group IIA PLA₂ and group V PLA₂. Rabbit anti-mouse group IIA PLA₂ recognized epitopes in BMMC from BALB/c but not C57BL/6 mice (Fig. 4, A and B). Rabbit anti-group V PLA₂ recognized epitopes in BMMC from both BALB/c and C57BL/6 mice (Fig. 4, C and D).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Flow cytometric analysis of the expression of group IIA PLA2 and group V PLA2 in mouse BMMC. BMMC from BALB/c and C57BL/6 mice were fixed in 2% paraformaldehyde, permeabilized with 0.025% saponin, and stained with affinity-purified rabbit IgG anti-mouse group IIA PLA2, affinity-purified rabbit IgG anti-mouse group V PLA2, or non-immune rabbit IgG followed by FITC-conjugated goat anti-rabbit IgG as described under "Experimental Procedures." Cells were analyzed by flow cytometry. Staining with non-immune IgG is indicated by the thin line; staining with specific antibodies is indicated by the thick line.

BMMC were evaluated by alkaline phosphatase immunohistochemistry after fixation with paraformaldehyde and permeabilization with saponin. Rabbit anti-group IIA PLA₂ recognized a cytoplasmic epitope in BMMC from BALB/c but not C57BL/6 mice (Fig. 5A). Rabbit anti-group V PLA₂ recognized epitopes in both BALB/c and C57BL/6 BMMC with accentuation of staining at the plasma membrane and also in a juxtanuclear location suggestive of Golgi staining in cells from both strains of mice (Fig. 5B). The non-immune rabbit IgG control antibody did not stain BMMC from BALB/c or C57BL/6 mice (Fig. 5C). Toluidine blue staining yielded typical images of BMMC from both strains (Fig. 5D).

View larger version (55K): [in this window] [in a new window]   Fig. 5.   Immunoalkaline phosphatase staining of group IIA PLA2 and group V PLA2 in mouse BMMC. BMMC from BALB/c and C57BL/6 mice were spun onto glass slides, fixed in 4% paraformaldehyde, permeabilized with 0.025% saponin, and stained with affinity-purified rabbit IgG anti-mouse group IIA PLA2 (A), affinity-purified rabbit IgG anti-mouse group V PLA2 (B), or non-immune rabbit IgG (C), followed by biotinylated goat anti-rabbit IgG. Bound antibody was visualized with the Vectastain ABC system for alkaline phosphatase. Replicate cells were stained with toluidine blue (D).

Subcellular Distribution of Group IIA PLA₂ and Group V PLA₂-- To define more accurately the intracellular localization of group IIA and group V PLA₂, BMMC from BALB/c mice were examined by immunofluorescence microscopy after saponin permeabilization to expose intracellular epitopes. Approximately 30% of cells were stained with antibodies to group IIA and group V PLA₂ after permeabilization with 0.025% saponin. Increasing the concentration or duration of saponin permeabilization led to significant loss of cells, and conditions were chosen that gave optimal staining. Epifluorescence with rabbit IgG anti-group IIA PLA₂ revealed a coarse granular cytoplasmic pattern (Fig. 6). In contrast, the rabbit IgG anti-group V PLA₂ detected a reticular pattern of cytoplasmic staining with plasma membrane and perinuclear accentuation (Fig. 6), the predominant site of staining varying somewhat from cell to cell. Confocal laser scanning microscopy confirmed the localization of the group IIA and group V PLA₂ enzymes in BMMC. Density gradient maps confirmed the coarse granular staining for group IIA PLA₂ (Fig. 7A), whereas group V PLA₂ was distributed in the cytoplasm and perinuclear area with a preferential localization on one side of the nucleus consistent with a Golgi pattern of staining (Fig. 7B).

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Immunofluorescence staining for group IIA PLA2 and group V PLA2 in saponin-permeabilized BALB/c mouse BMMC. BMMC were fixed in 2% paraformaldehyde, permeabilized with 0.025% saponin, and stained with affinity-purified rabbit IgG anti-mouse group IIA PLA2 (left), or affinity-purified rabbit IgG anti-mouse group V PLA2 (right), followed by FITC-conjugated goat anti-rabbit IgG as described under "Experimental Procedures." Cells were spun onto glass slides and visualized with a Nikon Optishot-2 microscope.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Confocal laser scanning microscopy of group IIA PLA2 and group V PLA2 in saponin-permeabilized BALB/c mouse BMMC. BMMC were fixed in 2% paraformaldehyde, permeabilized with 0.025% saponin, and stained with affinity-purified rabbit IgG anti-mouse group IIA PLA2 (a), affinity-purified rabbit IgG anti-mouse group V PLA2 (b), or non-immune rabbit IgG (c) followed by FITC-conjugated goat anti-rabbit IgG as described under "Experimental Procedures." Cells were suspended in ProLong antifade mounting medium and visualized by confocal laser scanning microscopy. False color images are presented. N, the location of the nucleus determined from bright field images. Bar = 2 µm.

Because different methods of cell permeabilization allow access to different subcellular compartments (41) and 0.025% saponin did not provide access to the endoplasmic reticulum or nucleus of BMMC as judged by failure to stain with antibodies to Grp78 and 5-LO, respectively (data not shown), we also evaluated methanol-permeabilized BMMC. Rabbit anti-group IIA PLA₂ failed to stain methanol-permeabilized BMMC (data not shown). The fact that the antibody had access to secretory granules was indicated by granule staining with an antibody to mouse mast cell secretory granule carboxypeptidase A (data not shown). This suggests either that the group IIA PLA₂ was solubilized by methanol or that the epitopes recognized by the rabbit anti-group IIA PLA₂ were denatured by methanol. After methanol permeabilization, 100% of cells were stained with rabbit anti-group V PLA₂. As after saponin permeabilization, rabbit anti-group V PLA₂ revealed a reticular pattern of cytoplasmic staining. Staining at the plasma membrane was more marked with methanol permeabilization than with saponin permeabilization. Staining after methanol permeabilization did not include the Golgi and sometimes extended to the nucleus (Fig. 8).

View larger version (56K): [in this window] [in a new window]   Fig. 8.   Immunofluorescence microscopy for group IIA PLA2 and group V PLA2 in methanol-permeabilized BALB/c mouse BMMC. BMMC were fixed in 2% paraformaldehyde, cytospun onto glass coverslips, and permeabilized with methanol. Cells were incubated with affinity-purified rabbit IgG anti-mouse group V PLA2 followed by Texas Red-conjugated donkey anti-rabbit IgG. Cells were visualized with a Nikon Eclipse 800 microscope. The faint intranuclear staining that is apparent in some cells is indicated by arrows.

Immunohistochemistry and immunofluorescence analyses based on saponin or methanol permeabilization to expose intracellular epitopes may imply an apparent localization of proteins that is perturbated by these procedures (31, 53). We therefore sought to confirm our observations with ultrathin cryosectioning and immunogold staining to expose epitopes in all intracellular compartments without detergent solubilization. The secretory granules of mouse BMMC are spherical bodies of varying density, 0.5-1 µm in diameter, composed of vesicles and amorphous material encased together in a limiting membrane. Gold-labeled antibodies to group IIA PLA₂ predominantly labeled these vesicular structures, preferentially marking the denser granules (Fig. 9A). There was a lesser degree of labeling of mitochondria and an even sparser distribution of gold particles over some Golgi elements. Otherwise, background labeling was comparable to the labeling of cells exposed to control IgG. The predominant structures labeled with rabbit IgG anti-group V PLA₂ were vesicular and cisternal elements of the Golgi apparatus (Fig. 9C) and in some cells the plasma membrane (Fig. 9D) and to a lesser extent the nuclear envelope (Fig. 9B). As with the antibody to group IIA PLA₂, a few gold grains were also found overlying mitochondria. The secretory granules did not label.

View larger version (97K): [in this window] [in a new window]   Fig. 9.   Immunoelectron microscopy for group IIA PLA2 and group V PLA2 in BALB/c mouse BMMC. BMMC were prepared for electron microscopy and processed for immunogold staining with affinity-purified rabbit IgG anti-mouse group IIA PLA2 (a) or affinity-purified rabbit IgG anti-mouse group V PLA2 (b-d) as described under "Experimental Procedures." Sections were visualized with a Jeol 1200 EX transmission electron microscope.

	DISCUSSION

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Evidence is accumulating that the cellular functions of the enzymes involved in leukotriene and prostaglandin biosynthesis are regulated in part by their induced expression (PGHS-2), subcellular localization (FLAP, LTC₄ synthase, PGHS-1, PGHS-2), and translocation (5-LO and cPLA₂). The nuclear envelope appears to be a major site of eicosanoid biosynthesis. In contrast, the low molecular weight PLA₂ enzymes have been described as secreted proteins that may release arachidonic acid from plasma membrane phospholipids (42, 43) or act at a PLA₂ receptor, (44-46) likely the M-type PLA₂ receptor, in a paracrine or autocrine manner. Group IIA PLA₂ was the initial low molecular weight PLA₂ to be implicated in eicosanoid generation in mast cells, as indicated by the ability of group IIA PLA₂ to stimulate arachidonic acid release from mouse BMMC (43) and from antigen-primed rat serosal mast cells (47). Although transcripts for group IIA PLA₂ are present in BMMC from BALB/c mice (18, 32), BMMC from mice genetically deficient in group IIA PLA₂ are not impaired in their ability to generate eicosanoids even though secretory granule exocytosis is somewhat attenuated (17, 18). Furthermore, BMMC from mice deficient in group IIA PLA₂ exhibited low molecular weight PLA₂ activity, indicating that an alternative PLA₂ was active and was a candidate for the role attributed to this class of PLA₂ (17, 18). Among this class, transcripts for group IIC PLA₂ have been described in BMMC (32); however, group IIC PLA₂ failed to couple to eicosanoid generation in transfected 293 cells (19), and the group IIC PLA₂ gene is likely a pseudogene in humans (48). Transcripts for group IB PLA₂ were not detected in BMMC (Fig. 2). Transcripts for the group V enzyme are present in BMMC (17) (Fig. 2), and antisense inhibition of group V PLA₂ abrogated immediate PGD₂ generation in the MMC-34 mast cell line (17). Therefore, the low molecular weight PLA₂ likely participating in eicosanoid generation in BMMC is the group V enzyme. Although transfection studies indicated that group IIA PLA₂ and group V PLA₂ were interchangeable in that experimental setting (19), it seemed to us that these actions in cells not of mast cell lineage may be the result of a shared catalytic capability that does not reflect functional differences in their appropriate physiologic subcellular locations.

Several extracellular mechanisms of action have been proposed for the low molecular weight PLA₂ enzymes. As secreted enzymes they may directly hydrolyze cell membrane phospholipids, an action that would be potentiated by binding of the enzyme to cell surface proteoglycan (18, 49). Arachidonic acid thus released at the plasma membrane would have to be metabolized by downstream enzymes at the plasma membrane or would need to travel to the nuclear envelope and endoplasmic reticulum, either by passive diffusion or by active transport mechanisms, for metabolism by 5-LO or isoforms of PGHS. Alternatively, the secreted PLA₂ enzymes may act as ligands for cell surface receptors to elicit activation of cPLA₂ (45, 46, 50, 51), an action that is independent of the catalytic activity of the enzymes. Two types of mammalian PLA₂ receptor have been described, the neuronal (N-type) receptor and the muscle (M-type) receptor; of these, the M-type receptor is the better characterized (52). The capacity of low molecular weight PLA₂ enzymes to signal through each receptor is species-dependent. In the mouse, the M-type receptor recognizes group IB PLA₂ from several species, group IIA PLA₂ of mouse but not human, and the snake venom PLA₂, OS₁, but not bee venom PLA₂. Recent studies have indicated specific and saturable binding of porcine group IB PLA₂ to mouse BMMC with a K_d of 0.56 nM; subnanomolar concentrations of porcine group IB PLA₂ selectively released arachidonic acid from BMMC (44). The observation that bee venom PLA₂ also elicited release of arachidonic acid from BMMC suggests a separate mode of action or that the low molecular weight PLA₂ enzymes may act at a receptor distinct from the M-type receptor. The capacity of exogenous group V PLA₂ to bind to BMMC and to release arachidonic acid has not been evaluated. Furthermore, the capacity of the low molecular weight enzymes to act at intracellular locations, rather than as secreted enzymes, requires consideration.

To set some physiologic constraints, our studies focused on the subcellular location of the group IIA and group V enzymes. The antibodies previously used to implicate group IIA PLA₂ in eicosanoid biosynthesis either are of unknown specificity or are now known to recognize both the group IIA and group V PLA₂ enzymes (5). Alignment of the mouse low molecular weight enzymes, including group IB PLA₂ and group IIC PLA₂, allowed the identification of immunogenic peptides with which to raise specific anti-peptide antibodies to the group IIA and group V enzymes (Fig. 1). These antibodies showed specificity for the enzymes to which they were raised and clearly distinguish between the group V PLA₂ and the group IIA PLA₂ (Figs. 3-6), allowing a comparison of the subcellular distribution of these two proteins.

The localization of group IIA PLA₂ in the secretory granules of BMMC (Figs. 6, 7, and 9) is consistent with the results of other studies demonstrating the localization of a low molecular weight PLA₂ enzyme in the secretory granule matrix of the rat peritoneal mast cell and its release with exocytosis (54). The latter study, however, relied on an antibody generated against the calcium-binding domain of group IB PLA₂ that is conserved among all low molecular weight PLA₂ enzymes (55). Other investigators have detected an immunoreactive low molecular weight PLA₂ in the granules of resting human neutrophils (56) and rat platelets (57) with the use of antibodies raised against a PLA₂ enzyme purified from rat liver mitochondria, the nature of which was not determined (58). Our present studies provide definitive localization for the group IIA enzyme in secretory granules of mouse mast cells. This location is consistent with the putative role for the group IIA PLA₂ in facilitating secretory granule exocytosis by generation of lysophospholipids to promote fusion of the perigranular and cell membranes (59, 60). The low molecular weight PLA₂ inhibitor, 12-epi-scalaradial, and heparin each inhibited c-kit ligand- and FcRI-dependent -hexosaminidase release from BMMC (18). Similarly, the degranulation of rat peritoneal mast cells was inhibited by the low molecular weight PLA₂ inhibitors mepacrine and thielocin (60). After its release from BMMC during secretory granule exocytosis, group IIA PLA₂ may act directly to release arachidonic acid from plasma membrane phospholipids or may serve in an autocrine or paracrine manner by binding to cell surface receptors to elicit intracellular signaling (44-46, 61). Either or both of these mechanisms may contribute to the prostanoid generation observed in response to exogenous group IIA PLA₂ in primed rat or mouse mast cells (44, 47).

The demonstration of group V PLA₂ in association with membrane compartments, including the Golgi and plasma membrane (Figs. 6-9), is the first definitive intracellular localization of this enzyme. Although low molecular weight PLA₂ enzymes have been identified in the perinuclear region and Golgi in other cells, these analyses have relied on antibodies with unproved specificity in the family of low molecular weight enzymes and generally were believed to represent group IIA PLA₂ (58, 62). A PLA₂ was localized by immunofluorescence in the Golgi apparatus and in punctate cytoplasmic structures of rat mesangial cells with a monoclonal antibody raised against a purified gel filtration fraction with PLA₂ activity isolated from rat liver mitochondria, the particular identification of which was unknown (58). A perinuclear and cytoplasmic pattern of staining was seen in immunofluorescence studies of human megakaryocytes with a monoclonal antibody raised against recombinant human group IIA PLA₂ (62). In contrast, Balsinde and colleagues (15) inferred the expression of group V PLA₂ on the surface of the P388D₁ mouse macrophage-like cell line by fluorescence with a cross-reacting antibody directed to mouse group IIA PLA₂ and recognized an increase in fluorescence with cell activation. In the current study, the prominent localization of group V PLA₂ in the perinuclear region of BMMC with saponin permeabilization (Figs. 6 and 7) is noteworthy, because the perinuclear region is the site of translocation of group IV cPLA₂, 5-LO, and other intermediate enzymes of eicosanoid biosynthesis (63). This localization might spatially permit the participation of group V PLA₂ in the supply of arachidonic acid for eicosanoid generation. Localization of the group V enzyme at the Golgi (Figs. 6, 7, and 9) may represent an intermediate compartment in the trafficking of group V PLA₂ and its function as a putative secreted enzyme acting at the plasma membrane (61).

A dependence of group V PLA₂ function on a cooperative interaction with cPLA₂ has been suggested for the generation of PGE₂ from the mouse P388D₁ macrophage cell line in response to platelet-activating factor after priming with LPS (13-15). Dissection of events with various pharmacologic and antisense inhibitors suggests that the intracellular release of arachidonic acid within 2 min of platelet-activating factor activation is due to cPLA₂ and that this intracellular arachidonic acid is required for the subsequent action of the group V PLA₂ in releasing additional arachidonic acid for PGHS-2-dependent PGE₂ generation that is maximal within 10 min. In mouse BMMC, pharmacologic studies have also implicated both cPLA₂ and group V PLA₂ in the immediate and delayed phases of prostanoid generation (16-18). Our recent studies with BMMC from mice in which the gene for cPLA₂ has been disrupted (11) have proved that cPLA₂ is absolutely required for both phases of eicosanoid generation in BMMC. Hence, any role for group V PLA₂ would depend on cPLA₂, as suggested for p388D₁ macrophages. Because the immediate and delayed phases of PGD₂ generation occur in mice deficient in group IIA PLA₂, it is conceivable that the perinuclear group V PLA₂ can enhance the immediate response under certain experimental conditions and that the secreted, plasma membrane-associated group V PLA₂ is essential to the delayed phase of PGD₂ generation, which requires the induction of PGHS-2.

Our studies demonstrate for the first time that the subcellular locations of different types of low molecular weight PLA₂ enzymes are distinct, and this finding must be considered in any functional interpretation of transfected cells that lack the key compartments involved. The different subcellular locations of group IIA PLA₂ and group V PLA₂ in single cells suggest that these enzymes serve different functions and are not functionally redundant.

	ACKNOWLEDGEMENTS

We thank Chioma Nwankwo (Brigham and Women's Hospital) for expert technical assistance, Maria Ericsson (Harvard Medical School) for assistance with electron microscopy, Jean Lai (Harvard School of Public Health) for assistance with Confocal Laser Scanning Microscopy, and Michelle Winstead and Jay A. Tischfield for making available the sequence of the mouse cDNA for group V PLA₂ before its release to GenBank.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HL36110, AI22531, and AI31599; American Cancer Society Grant RPG-97-001-01-BE; and a grant from the Hyde and Watson Foundation.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF162712 and AF162713.

^¶ Supported by the Arthritis Foundation and by a President's grant-in-aid award from the American Academy of Allergy, Asthma, and Immunology.

Supported by the Dutch Cancer Society.

^¶¶ Supported by a Burroughs Wellcome Developing Investigator Award. To whom correspondence should be addressed: Div. of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Smith Research Bldg., Rm. 638B, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1305; Fax: 617-525-1310; E-mail: jarm@rics.bwh.harvard.edu.

	ABBREVIATIONS

The abbreviations used are: PL, phospholipase; BMMC, bone marrow-derived mast cells; bp, base pair(s); BSA, bovine serum albumin; cPLA₂, cytosolic PLA₂; FcRI, high affinity Fc receptor for IgE; 5-LO, 5-lipoxygenase; FITC, fluorescein isothiocyanate; FLAP, 5-lipoxygenase activating protein; HBA, Hanks' balanced salt solution without Mg²⁺ or Ca²⁺ and containing 0.1% BSA; kb, kilobase pair(s); KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; LT, leukotriene; MMLV, Moloney murine leukemia virus; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PG, prostaglandin; PGHS, prostaglandin endoperoxide synthase; RT, reverse transcriptase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES