A Heparin-sensitive Phospholipase A 2 and Prostaglandin Endoperoxide Synthase-2 Are Functionally Linked in the Delayed Phase of Prostaglandin D 2 Generation in Mouse Bone Marrow-derived Mast Cells*

BALB/cJ mouse bone marrow-derived mast cells (BMMC) developed with interleukin (IL)-3 can be stimulated by c- kit ligand (KL) in the presence of IL-10 and IL-1 (cid:98) for sequential immediate and delayed generation of prostaglandin (PG) D 2 through utilization of consti- tutive prostaglandin endoperoxide synthase (PGHS) -1 and induced PGHS-2, respectively (Murakami, M., Mat- sumoto, R., Austen, K. F., and Arm, J. P. (1994) J. Biol. Chem. 269, 22269–22275). We now report that BALB/cJ BMMC stimulated with KL (cid:49) IL-10 (cid:49) IL-1 (cid:98) also exhibit the biphasic release of [ 3 H]arachidonic acid with an immediate phase over the first 10 min followed by a delayed phase from 2 to 7 h. The delayed phase of arachidonic acid release and of PGD 2 generation was inhibited by heparin, which concomitantly released a phospholipase (PL) A 2 from the cells into the supernatant. Both dexamethasone and a type II PLA 2 inhibitor, 12-epi-sca-laradial, suppressed delayed-phase PGD 2 generation at concentrations that did not affect immediate eicosanoid generation. Transcripts for type IIA PLA 2 , as assessed by reverse transcription-polymerase chain reaction, as described under “Experimental Procedures.” RT-PCR products were resolved in 1.5% agarose gels. The RT-PCR product for type II PLA 2 is visualized by DNA blotting and probing with a cDNA for mouse type II PLA 2 that was labeled by random priming with [ 32 P]dCTP; the RT-PCR product for (cid:98) -actin was visualized by ethidium bromide staining. Representative results from three to five independent experiments BMMC were pretreated with 1 (cid:109) g/ml aspirin during the 2-h period of priming with KL (cid:49) IL-10 and sensitization with IgE anti-TNP and then washed and activated with TNP-BSA for 8 h; results are mean (cid:54) S.E. of 8, 5, and 5 independent experiments for 129/J, C57BL/ 6J, and BALB/cJ BMMC, respectively. Expression of PGHS-2 was ex- amined 5 h after activation by SDS-PAGE immunoblotting. D, alterna-tively, BMMC were pretreated with aspirin for 2 h, washed, and activated with KL (cid:49) IL-10 (cid:49) IL-1 (cid:98) for 8 h; results are mean (cid:54) S.E. of 13, 7, and 8 independent experiments for 129/J, C57BL/6J, and BALB/cJ BMMC, respectively. Expression of PGHS-2 was examined 5 h after activation by SDS-PAGE immunoblotting.

We have previously reported that the activation of mouse bone marrow-derived mast cells (BMMC) 1 for the immediate phase of prostaglandin (PG) D 2 generation over a period of minutes and the subsequent delayed phase of PGD 2 generation over a period of hours involves the presentation of substrate to glutathione-dependent PGD 2 synthase by different prostaglandin endoperoxide synthase (PGHS) isozymes (1). This biphasic response was elicited directly by the cytokine combination of c-kit ligand (KL), interleukin (IL) -10, and IL-1␤ (1) or by priming the BMMC with KL and IL-10 and then activating them with IgE and antigen (2). Immediate PGD 2 generation was associated with transient phosphorylation of cytosolic phospholipase A 2 (cPLA 2 ) (3)(4)(5), which was maximal at 2-5 min. The fact that immediate PGD 2 generation was abolished by pretreatment of the cells with indomethacin, but not with NS-398, indicated the participation of PGHS-1, which was the only PGHS isoform detectable by analysis for steady-state transcript and protein (1,6). The delayed phase of PGD 2 generation was associated with the de novo induction of the PGHS-2 transcript and protein; it was suppressed by the selective PGHS-2 inhibitor, NS-398, or by dexamethasone, which prevented PGHS-2 expression, and not by the PGHS-1 inhibitor, valeryl salicylate (1)(2)(3). The segregated processing of arachidonic acid, even when both isoforms were present, implied the existence of a distinct supply or different intracellular conditions for the metabolism of arachidonic acid by the alternative PGHS isoforms. The finding that the utilization of arachidonic acid by PGHS-2 is favored over its utilization by PGHS-1 at low peroxide concentrations (7) is compatible with the involvement of PGHS-1 in the burst of events that follow immediate activation and of PGHS-2 in the downstream events that last for hours. Furthermore, the linear, delayed generation of PGD 2 by BMMC 2-10 h after direct activation by the cyto-kine triad would seem to require a continuous supply of substrate, whereas the phosphorylation of cPLA 2 after activation of BMMC was largely reversed by 10 min (3,4).
Both cPLA 2 and type IIA (secretory) PLA 2 , which are products of substantially unrelated genes and which exhibit different structural and functional characteristics, have been described in mouse mast cells (3, 6, 8 -11). cPLA 2 depends on micromolar concentrations of Ca 2ϩ for translocation from the cytosol to the perinuclear membrane with concomitant function (12)(13)(14)(15)(16); this function is augmented by phosphorylation via the MEK-1 dual specificity pathway (17). cPLA 2 preferentially cleaves arachidonic acid from the sn-2 position of phospholipids. Type IIA PLA 2 is part of a family of low molecular weight PLA 2 enzymes, of which four have been described in mammals (18 -20). Type IIA PLA 2 requires millimolar concentrations of Ca 2ϩ for function and exhibits no preference for arachidonic acid in the sn-2 position (18). The capacity of type IIA PLA 2 to bind to heparin has been used to distinguish it from other PLA 2 enzymes and to investigate its role in eicosanoid biosynthesis (21)(22)(23)(24). Exogenous type IIA PLA 2 has been reported to stimulate immediate PGD 2 generation from rat peritoneal mast cells sensitized with IgE and activated with sub-threshold doses of antigen (25) and to directly elicit the immediate generation of PGD 2 from mouse BMMC (10). We now provide evidence that the delayed phase of PGD 2 synthesis in BMMC, elicited by cytokines alone or in response to IgE and antigen after cytokine priming, depends on an inducible, heparin-sensitive PLA 2 to supply arachidonic acid for metabolism by PGHS-2. That the delayed phase of PGD 2 generation occurred in BMMC derived from C57BL/6J mice, which have a natural disruption of the gene for type IIA PLA 2 (26,27), indicates the participation of a heparin-sensitive PLA 2 that is distinct from the previously characterized type IIA PLA 2 .

Immediate and Delayed Generation of PGD 2 from BMMC
Cytokine-initiated Activation-To assess the immediate phase of eicosanoid generation, BMMC were stimulated with 100 ng/ml KL in enriched medium at a cell density of 1 ϫ 10 7 cells/ml for 10 min at 37°C. The reaction was stopped by centrifugation of the cells at 120 ϫ g for 5 min at 4°C, and the supernatants were retained for assay of mediator release. The cell pellets were suspended in enriched medium and disrupted by freeze-thawing. ␤-Hexosaminidase, a marker of mast cell degranulation, was quantitated in the supernatants and pellets by spectrophotometric analysis of the hydrolysis of p-nitrophenyl-␤-D-2acetamido-2-deoxyglucopyranoside (33). The percent release of ␤-hexosaminidase was calculated by the formula (S/(S ϩ P)) ϫ 100, where S and P are the ␤-hexosaminidase contents of equal portions of each supernatant and cell pellet, respectively. PGD 2 and leukotriene (LT) C 4 were assayed by radioimmunoassay (Amersham Corp.).
For the delayed phase of PGD 2 generation, BMMC were cultured in enriched medium supplemented with 100 ng/ml KL, 10 units/ml IL-10, and 5 ng/ml IL-1␤ at a cell density of 1 ϫ 10 6 cells/ml for various periods. The cells were sedimented and analyzed for steady-state mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and for protein expression by SDS-polyacrylamide gel electrophoresis (PAGE)/immunoblotting. Replicate BMMC were pretreated for 2 h with 1 g/ml aspirin to abolish the immediate phase of PGD 2 generation by irreversibly inactivating pre-existing PGHS-1 (34,35) and then were washed twice, stimulated with cytokines, and sedimented. The supernatants were collected and assayed for PGD 2 .
IgE-dependent Activation-To assess IgE-dependent immediate mediator release, BMMC were suspended at a concentration of 1 ϫ 10 7 cells/ml in WEHI-3 cell-conditioned medium and sensitized with 10 g/ml mouse monoclonal IgE anti-trinitrophenyl (TNP) for 30 min. After being washed twice with enriched medium, the cells were resuspended in enriched medium at a concentration of 5 ϫ 10 6 cells/ml and were incubated at 37°C for 10 min with 50 ng/ml TNP-conjugated bovine serum albumin (BSA).
To assess the delayed phase of cytokine-primed, IgE-dependent PGD 2 generation, BMMC were resuspended in enriched medium supplemented with 100 ng/ml KL and 10 units/ml IL-10 at a cell density of 1 ϫ 10 7 cells/ml and sensitized with 10 g/ml monoclonal IgE anti-TNP in the presence of 1 g/ml aspirin for 2 h at 37°C. After being washed twice with enriched medium, the cytokine-primed, IgE-sensitized cells were resuspended at a concentration of 1 ϫ 10 6 cells/ml in enriched medium containing the same cytokines, were stimulated at 37°C with 50 ng/ml TNP-BSA, and were cultured for various periods in the continued presence of cytokines and TNP-BSA. The cells were analyzed for steady-state mRNA by RT-PCR and for protein expression by SDS-PAGE/immunoblotting.

Assessment of Activation
Arachidonic Acid Release-BMMC, suspended in 50% WEHI-3 cellconditioned medium at a density of 1 ϫ 10 6 cells/ml, were incubated with 1 Ci/ml [ 3 H]arachidonic acid (100 Ci/mmol) (DuPont NEN) at 37°C for 12 h. The cells were washed three times with enriched medium, stimulated for 10 min at a density of 1 ϫ 10 7 cells/ml with KL as described above, and divided into two portions. One portion was sedimented and processed for determination of immediate arachidonic acid release, whereas the other portion was sedimented, resuspended in enriched medium at a density of 1 ϫ 10 7 cells/ml in the continued presence of various cytokines, and cultured for various periods. The radioactivity in the cell pellets and supernatants was measured by liquid ␤-scintillation counting, and the release was expressed as a percentage of the total counts recovered from cells and supernatant.
In certain experiments the lipids in the cell pellets and supernatants were extracted from BMMC at various times after activation according to the method of Bligh and Dyer (36) and were developed by thin-layer chromatography (TLC) on silica gel plates (Sigma) with a solvent system of diethyl ether/methanol/acetic acid (90/2/0.2, v/v) at Ϫ20°C. The zones on the silica gel corresponding to free arachidonic acid, LTC 4 , PGD 2 , and phospholipids were determined by comparison with the mobility of authentic standards (Cayman Chemical, Ann Arbor, MI). Each zone was scraped into a separate vial, and the radioactivity was counted in a liquid ␤-scintillation counter (Beckman Instruments, Palo Alto, CA). Arachidonic acid release was expressed as a percentage of the total radioactivity recovered.
PLA 2 Release-BMMC were cultured with KL ϩ IL-10 ϩ IL-1␤ or were primed for 2 h with KL ϩ IL-10, sensitized with hapten-specific IgE, and activated with antigen for various periods at a density of 1 ϫ 10 7 cells/ml as described above, in the presence or absence of heparin. The PLA 2 activity released into the supernatant was assessed by the hydrolysis of 1-acyl-2-[ 14 C]arachidonylphosphatidylethanolamine (30 Ci/mol) (DuPont NEN) to liberate [ 14 C]arachidonic acid as described previously (37). Briefly, an 85-l sample of the BMMC supernatant was adjusted to a final volume of 125 l containing 4 mM CaCl 2 , 100 mM Tris-HCl, pH 9.0, and 10 M 1-acyl-2-[ 14 C]arachidonylphosphatidylethanolamine and was incubated for 2 h at 37°C. The reaction was stopped by the addition of 625 l of isopropyl alcohol/n-heptane/1 N H 2 SO 4 (78/20/2, v/v/v), and 370 l of n-heptane and 250 l of distilled water were added to the reaction mixture with vigorous stirring. Free [ 14 C]arachidonic acid was extracted in n-heptane and was counted in a liquid ␤-scintillation counter.
SDS-PAGE/Immunoblot Analysis-cPLA 2 and PGHS-2 were analyzed by SDS-PAGE/immunoblot with the antiserum to each at a dilution of 1:2000. Proteins were visualized with an enhanced chemiluminescence Western blot analysis system (Amersham Corp.) as described (1,6). Expression of Steady-state Transcripts for Type IIA PLA 2 by RT-PCR-The cDNA for mouse type IIA PLA 2 was originally described as a cDNA encoding mouse Paneth cell-enhancing factor (39). Poly(A) ϩ RNA, extracted from 5 ϫ 10 6 BMMC (Micro FastTrack, mRNA Isolation Kit, Invitrogen, San Diego, CA), was incubated with a 20-mer oligonucleotide primer, 5Ј-TCAGCATTTGGGCTTCTTCC-3Ј, corresponding to the C terminus of mouse intestinal enhancing factor (39), and with an oligo(dT) primer for 10 min at 65°C and then for 2 min at room temperature. Reverse transcription was carried out by incubation with avian myeloblastosis virus reverse transcriptase at 42°C for 1 h. A 1-l portion of the resulting reaction mixture was combined with a 21-mer sense primer 5Ј-AGTTTGGGGAAATGATTCGGC-3Ј and a 21-mer antisense primer 5Ј-GCTTTATCGCACTGACACAGC-3Ј, corresponding to nucleotides 11-31 and 261-281, respectively, of the reported cDNA for mouse intestinal enhancing factor (39) or with mouse ␤-actin primers (Clontech, Palo Alto, CA). PCR was performed with Taq polymerase (Invitrogen) with a cycle of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min in 15 mM (NH 4 ) 2 SO 4 , 60 mM Tris-HCl, pH 8.5, containing 2.5 mM Mg 2ϩ for ␤-actin or 3.5 mM Mg 2ϩ for type IIA PLA 2 . 30 cycles of PCR were performed for ␤-actin and 35 cycles for type IIA PLA 2 . The PCR products were resolved in 1.5% agarose gels containing ethidium bromide, transferred to Immobilon-N (Millipore, Bedford, MA), and analyzed with cDNA probes for mouse type IIA PLA 2 and ␤-actin that were labeled by random priming (Megaprime, Amersham Corp.) with [ 32 P]dCTP (3000 Ci/mmol, DuPont NEN). The relative amount of each transcript was estimated by quantitating the associated radioactivity with a Betascope 603 Blot Analyzer (Betagen, Waltham, MA). The fold increase in steady-state mRNA was calculated as the ratio of radioactivity associated with a specific transcript in treated cells to that in control cells (BMMC maintained in IL-3) and was corrected for changes in steady-state levels of ␤-actin transcript.

Cloning and Expression of Mouse Type IIA PLA 2
Cloning of a cDNA That Encodes Mouse Type IIA PLA 2 -Total RNA was extracted from BALB/cJ BMMC cultured for 5 h with KL ϩ IL-10 ϩ IL-1␤ or from BALB/cJ mouse intestine in guanidinium thiocyanate using TRI-Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions (38) and was quantitated by measurement of its optical density at 260 nm. RNA from each source was mixed with the 20-mer primer corresponding to the C terminus of the mouse intestinal enhancing factor (39) and with avian myeloblastosis virus reverse transcriptase and was incubated for 1 h at 42°C. At the time that these experiments were performed, the sequence of the signal peptide and 5Ј-untranslated region of the mouse type IIA PLA 2 had not been described. Therefore, PCR of the resulting cDNA was carried out with a 21-mer sense primer, 5Ј CCATCCAAGAGAGCTGA-CAGC 3Ј, that corresponds to the 5Ј-untranslated sequence adjacent to the signal sequence of rat type IIA PLA 2 and an antisense primer corresponding to the C terminus of mouse intestinal enhancing factor with a cycle of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min in 15 mM (NH 4 ) 2 SO 4 , 3.5 mM Mg 2ϩ , 60 mM Tris-HCl, pH 8.5. After 35 cycles of amplification of BMMC cDNA and 30 cycles of amplification of mouse intestinal cDNA, a major product with an estimated size of ϳ500 base pairs (bp) was obtained. The 500-bp cDNA was resolved in 1.5% agarose, purified with silica gel (Qiagen, Chatsworth, CA), subcloned into the pCR II cloning vector (Invitrogen), and sequenced in both strands with the dideoxynucleotide termination reaction (40).
Expression of Mouse Type IIA PLA 2 in COS-7 Cells-The mouse type IIA PLA 2 cDNA was subcloned into pcDNA3 (Clontech) at the EcoRI site and transfected into COS-7 cells with either liposomes or electroporation. COS-7 cells (American Type Culture Collection) maintained in enriched medium were seeded into 6-well plates (Corning, Corning, NY) 1-2 days before transfection so as to be 40 -60% confluent at the time of transfection. One g of plasmid DNA was mixed with 20 g/ml Lipofectin (Life Technologies, Inc.) in 200 l of RPMI 1640 medium without fetal calf serum, and the mixture was incubated for 10 min at room temperature. The mixture of plasmid and Lipofectin was added to COS-7 cells in wells containing 1 ml of RPMI 1640 medium without fetal calf serum. After 6 h of incubation at 37°C, 1 ml of enriched medium was added, and the cells were cultured for 1 day. Then, the medium was aspirated and replaced with 2 ml of fresh enriched medium, the cells were cultured for another 3 days, and the supernatants were collected and assayed for PLA 2 activity. Alternatively, COS-7 cells were transfected by electroporation as described previously (1, 6). After harvest of the medium containing type IIA PLA 2 , the cells were incubated for 1 h with 2 ml of 1 M NaCl in RPMI 1640 without fetal calf serum or with 20 mg/ml heparin at room temperature. The supernatants were collected and assayed for type II PLA 2 activity.
A 10-l portion of the supernatant was applied to 16% SDS-PAGE under nonreducing conditions, transferred to a nitrocellulose membrane, immunoblotted with 1 g/ml of anti-rat type II PLA 2 antibody, and visualized by enhanced chemiluminescence.
PCR Amplification of Genomic DNA-Genomic DNA was isolated from BMMC derived from BALB/cJ, 129/J, and C57BL/6J mice according to published protocols (41). The gene for type IIA PLA 2 in 129/J and C57BL/6J mice is disrupted by the insertion of a thymidine residue at a BamHI site in exon 3 (26,27). A 467-bp portion of the type IIA PLA 2 gene, encompassing the BamHI site in exon 3, was amplified by PCR with primers corresponding to nucleotides 1567-1585 and 2013-2033 of the gene (27). The PCR products were purified on a silica matrix (GeneClean, Bio 101, Inc., La Jolla, CA), were digested with BamHI, and were resolved in 2% agarose gels.

Cytokine-initiated Arachidonic Acid Release-After a 12-h incubation of BALB/cJ BMMC with [ 3 H]arachidonic acid,
ϳ99% of the cell-associated radioactivity was incorporated into phospholipid pools (3). Labeled BALB/cJ BMMC, stimulated for 10 min with 100 ng/ml KL, released 7.3 Ϯ 0.9% (mean Ϯ S.E., n ϭ 3) of their radioactivity into the supernatant. TLC revealed that 78% of the radioactivity released into the supernatant during this immediate phase of activation was associated with free arachidonic acid, 3.8% with eicosanoids, and 18% with phospholipids. To assess the delayed phase of cytokineinitiated arachidonic acid release, BALB/cJ BMMC were treated with KL for 10 min, washed, and exposed to various cytokines. By 6.5 h, IL-3, KL, or a combination of KL ϩ IL-10 ϩ IL-1␤, released 1.98 Ϯ 0.25, 1.95 Ϯ 0.46, and 3.33 Ϯ 0.65%, respectively, of the incorporated radioactivity. The combination of KL ϩ IL-10 ϩ IL-1␤ was distinguished by a statistically significant greater release of radioactivity at the 5-and the 6.5-h time points, respectively (p Ͻ 0.05; n ϭ 4). The delayed phase of arachidonic acid release began after 2 h of cell stimulation with KL ϩ IL-10 ϩ IL-1␤ (Fig. 1). The combination of cytokines was required because the release of radioactive counts from BALB/cJ BMMC cultured with KL alone or KL in combination with IL-1␤, IL-3, IL-4, IL-9, or IL-10 was no greater than that from BALB/cJ BMMC cultured with IL-3 alone for 5 h (p Ͼ 0.05; n ϭ 4). TLC revealed that 7.1 Ϯ 1.8% of the radioactivity was released into the supernatant at 5 h as free arachidonic acid when cells were treated with KL alone, as compared with 25.2 Ϯ 2.8% when cells were stimulated with KL ϩ IL-10 ϩ IL-1␤ (p ϭ 0.001, n ϭ 4). Whereas none of the counts migrated with PGD 2 on TLC 5 h after cell stimulation with KL alone, 3.7 Ϯ 0.6% of the counts released from cells activated with KL ϩ IL-10 ϩ IL-1␤ were associated with PGD 2 , representing the metabolism of 16.6 Ϯ 2.4% of the released arachidonic acid to delayed-phase PGD 2 generation (n ϭ 4). The remaining counts migrated with phospholipids.
When 20 mg/ml heparin or 10 Ϫ6 M dexamethasone was added to BALB/cJ BMMC during the 5-h culture with KL ϩ IL-10 ϩ IL-1␤, the delayed-phase release of incorporated radioactivity was reduced from 1.65 Ϯ 0.26% for cells cultured without drug to 0.97 Ϯ 0.07% in the presence of heparin and 0.88 Ϯ 0.17% in the presence of dexamethasone (n ϭ 3, p Ͻ 0.01). The latter values were not significantly different from the release of incorporated radioactivity from replicate cells treated with KL alone (0.89 Ϯ 0.04%, n ϭ 3, p Ͼ 0.05), indicating virtually complete inhibition of delayed-phase PGD 2 generation by either heparin or dexamethasone.
To limit any effect of heparin to the delayed phase of cytokineinitiated PGD 2 generation, the immediate phase was eliminated by preincubation of BALB/cJ BMMC with 1 g/ml aspirin (1) for 2 h to irreversibly inactivate pre-existing PGHS-1. BALB/cJ BMMC were then washed in enriched medium and stimulated with KL ϩ IL-10 ϩ IL-1␤ in the presence or absence of 20 mg/ml heparin. BALB/cJ BMMC pretreated with aspirin and stimulated with KL ϩ IL-10 ϩ IL-1␤ generated 2.0 Ϯ 0.2 ng of PGD 2 per 10 6 cells over 10 h (n ϭ 3) in the absence of heparin (Fig. 2B, open circles) compared with 0.6 Ϯ 0.2 ng PGD 2 per 10 6 cells when heparin was added 10 min after activation (Fig. 2B, closed circles; n ϭ 3, p Ͻ 0.01). When BALB/cJ BMMC were cultured for 5 h with KL ϩ IL-10 ϩ IL-1␤ in the absence of heparin and were then exposed to heparin for the next 5 h, the ongoing phase of delayed PGD 2 generation was terminated with no further increase at 7 or at 10 h (Fig. 2B, closed squares). The interruption of PGD 2 generation by heparin coincided with the rapid 4-fold increase in activity of PLA 2 in cells stimulated and processed for PLA 2 release ( Fig. 2A, closed squares). The induction of PGHS-2 protein by KL ϩ IL-10 ϩ IL-1␤, which was detectable at 2 h and reached a maximum at 5 to 10 h (1), was not inhibited by heparin (Fig. 2C).
The dose-related ability of heparin to increase the release of PLA 2 and suppress the delayed phase of PGD 2 generation was assessed in BALB/cJ BMMC stimulated for 10 h with KL ϩ IL-10 ϩ IL-1␤ (Fig. 3). Heparin augmented PLA 2 release and inhibited delayed-phase PGD 2 generation in a concentrationdependent manner, with EC 50 and IC 50 values, respectively, of ϳ10 mg/ml. The maximum release of PLA 2 with almost complete suppression of PGD 2 generation occurred in response to 20 mg/ml heparin. The 2-3-fold increase from 30 min to 10 h of PLA 2 released by heparin after cytokine stimulation of the cells suggested that the PLA 2 was inducible. To further evaluate the possible contribution of newly synthesized, heparin-sensitive PLA 2 in the delayed phase of PGD 2 generation, BALB/cJ BMMC were stimulated with KL ϩ IL-10 ϩ IL-1␤ with or without the addition of heparin 10 min after activation to release constitutively expressed or exocytosis-dependent, membrane-associated, heparin-sensitive PLA 2 during 2 h of culture. The cells were then washed, and replicates were cultured in the same cytokines for an additional 8 h in the presence or absence of heparin. The delayed phase of PGD 2 generation was not significantly impaired by removal of the constitutive or exocytosisdependent heparin-sensitive PLA 2 before the wash step but was inhibited if heparin was included in the medium during the subsequent delayed response to cytokine activation (Table I).  (Fig. 4A, closed squares; n ϭ 4, p Ͻ 0.05). By 10 h the amount of PLA 2 released into the medium by heparin was the same whether heparin was added 10 min or 5 h after activation, implying that heparin did not modulate the induction of the enzyme.
To limit the effect of heparin to the IgE-dependent delayed phase of PGD 2 generation, BALB/cJ BMMC were treated with 1 g/ml aspirin during the 2-h period of IgE sensitization and cytokine priming that preceded activation with antigen for 5 h in the presence or absence of 20 mg/ml heparin (Fig. 4B). BALB/cJ BMMC generated 1.55 Ϯ 0.32 ng of PGD 2 per 10 6 cells over 5 h in the absence of heparin (n ϭ 3; Fig. 4B, open circles) compared with 0.50 Ϯ 0.10 ng of PGD 2 per 10 6 cells when heparin was added 10 min after activation (Fig. 4B, closed circles; n ϭ 3, p Ͻ 0.05). The IgE-dependent induction of PGHS-2 expression was not inhibited by heparin (Fig. 4C).
Effect of Heparin and 12-epi-Scalaradial on Mediator Release-To examine the effect of heparin on the immediate phase of mediator release, BALB/cJ BMMC were activated   with KL or were sensitized with IgE anti-TNP and activated with TNP-BSA for 10 min in the presence or absence of 20 mg/ml heparin. In contrast to its effect on delayed phase PGD 2 generation, heparin did not significantly inhibit the immediate phase of PGD 2 and LTC 4 generation elicited either by KL or by IgE-dependent activation (Table II). However, heparin did significantly inhibit the immediate release of ␤-hexosaminidase ϳ30%.
When BALB/cJ BMMC, pretreated with aspirin, were activated with KL ϩ IL-10 ϩ IL-1␤ or were primed with KL ϩ IL-10, sensitized with IgE anti-TNP, and activated with TNP-BSA, the delayed phase of PGD 2 generation at 5 h was suppressed in a dose-dependent manner by 12-epi-scalaradial, a type II PLA 2 inhibitor (42) with an IC 50 of ϳ0.2 M in both protocols. In contrast, the immediate phase of LTC 4 and PGD 2 generation elicited by KL or by IgE and antigen was resistant to 12-epi-scalaradial even at a concentration of 1 M, the IC 50 for inhibition of exocytosis. 12-epi-Scalaradial, at a concentration of 1 M, did not inhibit the induction of PGHS-2 (data not shown).
Characterization of the Heparin-sensitive PLA 2 Involved in the Delayed-phase Generation of PGD 2 -The capacity of heparin and 12-epi-scalaradial to inhibit the delayed phase of PGD 2 generation from BALB/cJ BMMC suggested that the inducible PLA 2 providing arachidonic acid to PGHS-2 had the properties of a low molecular weight type II PLA 2 . Furthermore, as assessed by SDS-PAGE/immunoblotting, the expression of cPLA 2 protein did not change appreciably during the initial 7 h of cell culture with KL alone or with KL ϩ IL-10 ϩ IL-1␤ (data not shown), and the initial phosphorylation was substantially reversed by 10 min (3, 4).
A mouse cDNA encoding Paneth cell enhancing factor was reported to be homologous to rat type IIA PLA 2 (39). A cDNA encoding the open reading frame of mouse type IIA PLA 2 was amplified from the RNA of BALB/cJ BMMC stimulated for 5 h with KL ϩ IL-10 ϩ IL-1␤ and from mouse intestinal RNA by RT-PCR using oligonucleotides corresponding to the 5Ј-untranslated region adjacent to the signal sequence of the rat type IIA PLA 2 cDNA and the 3Ј end of the coding region of the enhancing factor cDNA, respectively. A major product of 460 bp was obtained and subcloned into pCRII. Sequencing, with the dideoxynucleotide termination reaction of Sanger, revealed that each cDNA was identical to the subsequently reported mouse type IIA PLA 2 cDNA (26) with the exception that a cytosine at position 15 of the coding sequence was replaced by guanidine. This substitution is silent and is found in the nucleotide sequence of mouse type IIA PLA 2 reported by others (27).
The cDNA from BALB/cJ mouse intestinal RNA was subcloned into a mammalian cell expression vector, pcDNA3, and transfected into COS-7 cells. 2.45 Ϯ 0.87 nmol/h/ml PLA 2 activity (n ϭ 7) was released into the supernatant of COS-7 cells transfected with the mouse cDNA; none was obtained from COS-7 cells transfected with vector alone. When transfected COS-7 cells were washed with serum-free RPMI 1640 medium and then exposed for 1 h to serum-free RPMI medium containing 20 mg/ml heparin or 1 M NaCl, a further 8.23 Ϯ 2.5 (n ϭ 4) and 5.5 Ϯ 0.79 (n ϭ 2), respectively, nmol/h of PLA 2 activity per ml of original culture buffer was solubilized into the supernatant. Thus, 75 Ϯ 7% of the PLA 2 activity of transfected COS-7 cells was cell-associated and could be released into the supernatant by heparin. The PLA 2 activity of transfected cells migrated as a 14-kDa protein by SDS-PAGE/immunoblotting with an antibody raised against rat type II PLA 2 (data not shown).
RT-PCR was used to examine the induction of type IIA PLA 2 in BALB/cJ BMMC activated with cytokines alone or primed with cytokines and activated with IgE and antigen. Stimulation of the cells with KL ϩ IL-10 ϩ IL-1␤ resulted in the marked increase of transcripts for type IIA PLA 2 by 2 h, reaching a maximum ϳ25-fold increase (n ϭ 4, p Ͻ 0.01 versus IL-3 alone) at 5 h (Fig. 5A). Type IIA PLA 2 mRNA increased ϳ17fold (n ϭ 4, p Ͻ 0.01 versus IL-3 alone) in BALB/cJ BMMC primed for 2 h with KL ϩ IL-10 and activated for 2 h with IgE and antigen (Fig. 5B). In contrast, in cells stimulated with KL alone type IIA PLA 2 transcripts increased only ϳ2-fold (Fig.  5B). Dexamethasone fully suppressed the ability of KL ϩ IL-10 ϩ IL-1␤ to increase the expression of type IIA PLA 2 mRNA (Fig. 5C).
Mediator Release from BMMC Derived from 129/J and C57BL/6J Mice-The role of type IIA PLA 2 in the immediate and delayed phases of eicosanoid generation was assessed in BMMC derived from 129/J and C57BL/6J mice (Fig. 6), which have a natural disruption of the type IIA PLA 2 gene (26,27). We first confirmed that the type IIA gene was disrupted in genomic DNA derived from 129/J and C57BL/6J BMMC. The 467-bp product amplified from genomic DNA of BALB/cJ BMMC was digested to two fragments of 345 and 122 bp, whereas the PCR product amplified from 129/J BMMC and C57BL/6J BMMC resisted digestion with BamHI (data not shown). Sequencing of the PCR product of 129/J BMMC and C57BL/6J BMMC confirmed the disruption of the BamHI site by the insertion of a thymidine residue in exon 3, as described previously (26,27).
There was no significant difference in the immediate generation of PGD 2 by 129/J BMMC, C57BL/6J BMMC, and  5. Induction of transcripts for type II PLA 2 in activated BALB/cJ BMMC, assessed by RT-PCR. A, BALB/cJ BMMC were activated with KL ϩ IL-10 ϩ IL-1␤ for various periods. B, BALB/cJ BMMC were cultured for 5 h in the presence of IL-3 alone, KL alone, or KL ϩ IL-10 ϩ IL-1␤; alternatively, BALB/cJ BMMC were primed with KL ϩ IL-10 and sensitized with IgE anti-TNP for 2 h and then stimulated with TNP-BSA for 5 h. C, BALB/cJ BMMC were cultured in IL-3 alone or in KL ϩ IL-10 ϩ IL-1␤ for 5 h in the presence or absence of 10 Ϫ6 M dexamethasone (Dex.). Poly(A) ϩ RNA was extracted from 5 ϫ 10 6 BMMC, and the expression of transcripts for type II PLA 2 and ␤-actin was assessed by RT-PCR as described under "Experimental Procedures." RT-PCR products were resolved in 1.5% agarose gels. The RT-PCR product for type II PLA 2 is visualized by DNA blotting and probing with a cDNA for mouse type II PLA 2 that was labeled by random priming with [ 32 P]dCTP; the RT-PCR product for ␤-actin was visualized by ethidium bromide staining. Representative results from three to five independent experiments are shown.
BALB/cJ BMMC in response to IgE and antigen or to KL (Fig.  6, A and B). Cytokine-primed, IgE-dependent, delayed-phase PGD 2 generation and cytokine-initiated, delayed-phase PGD 2 generation were both attenuated 80 -90% in 129/J BMMC compared with BALB/cJ BMMC. However, the delayed-phase induction of PGHS-2 in response to either stimulus was severely attenuated in 129/J BMMC (Fig. 6, C and D). In contrast, both direct, cytokine-initiated and cytokine-primed, IgE-dependent delayed-phase PGD 2 generation occurred in C57BL/6J BMMC, in which PGHS-2 was induced (Fig. 6, C and D). Whereas heparin treatment of C57BL/6J BMMC from 10 min to 2 h after activation by KL ϩ IL-10 ϩ IL-1␤, followed by a wash, had no effect on subsequent delayed-phase PGD 2 generation, the inclusion of heparin after the wash step inhibited the delayedphase PGD 2 generation efficiently (Table III). Thus, in the C57BL/6J BMMC an inducible, heparin-sensitive PLA 2 , distinct from the type IIA PLA 2 that is deficient in these mice, supplies arachidonic acid to PGHS-2 in the delayed phase of PGD 2 generation. DISCUSSION In earlier studies we identified two combinations of agonists that recruited constitutive and then inducible steps for the immediate and delayed generation of PGD 2 , respectively, namely the cytokine triad of KL ϩ IL-10 ϩ IL-1␤ (1) and priming with KL ϩ IL-10 during sensitization with IgE fol-lowed by activation with antigen (2). The separation of PGD 2 production into an immediate phase within 10 min and a delayed phase beginning after 2 h of activation and continuing to 10 h argued for a biochemical source of arachidonic acid distinct from that provided by the transient translocation and phosphorylation of cytosolic PLA 2 . Kinetic, pharmacologic, and genetic evidence now suggests that arachidonic acid is supplied to PGHS-2 in the delayed phase of PGD 2 generation in BMMC by an inducible, heparin-sensitive PLA 2 distinct from the type IIA PLA 2 that has previously been characterized.
When BALB/cJ BMMC were stimulated with KL ϩ IL-10 ϩ IL-1␤, there were two phases of arachidonic acid release. The first phase, which required only KL (3), was complete within 10 min, whereas a second, delayed phase of release of free arachidonic acid from 2 to 7 h of culture required the entire cytokine combination of KL ϩ IL-10 ϩ IL-1␤ (Fig. 1). The kinetics and cytokine requirements for the delayed phase of arachidonic acid release were similar to those observed for the induction of PGHS-2 and the delayed-phase generation of PGD 2 (1). The delayed phase was not accompanied by any change in expression of cPLA 2 in BALB/cJ BMMC, which increased only after 24 h in response to KL alone with no augmentation by the accessory cytokines (6). The suppression of the delayed phase of arachidonic acid release by dexamethasone suggested the involvement of a steroid-sensitive, inducible PLA 2 in the delayedphase response.
The inhibition of the delayed phase of PGD 2 generation (Fig.  2) and of arachidonic acid release by heparin revealed the possibility that an enzyme with the properties of type II PLA 2 , rather than cPLA 2 , was functionally associated with the delayed response. The inhibition of the delayed phase of PGD 2 generation by heparin demonstrated a close correspondence in both kinetics (Fig. 2) and dose dependence (Fig. 3) with the appearance of bioactive PLA 2 in the supernatant of activated BALB/cJ BMMC. PLA 2 activity was detectable within 30 min of stimulation of BALB/cJ BMMC with KL ϩ IL-10 ϩ IL-1␤ (Fig. 2) and progressively increased over 10 h of culture. When added at 10 min or 5 h after cytokine stimulation, heparin acutely augmented the release of PLA 2 with concomitant inhibition of the delayed phase of PGD 2 generation. The heparin dose-response indicated an inverse relationship between augmented PLA 2 release and inhibition of PGD 2 generation (Fig.  3). When BMMC were treated with heparin 10 min after activation by KL ϩ IL-10 ϩ IL-1␤ to remove constitutive and/or exocytosis-dependent PLA 2 , cultured for 2 h, and washed, the subsequent delayed phase of PGD 2 generation was not inhibited (Table I). However, delayed-phase PGD 2 generation was inhibited when heparin was added after a wash step 2 h after activation so as to be present for the subsequent 8 h of delayed phase PGD 2 generation. These observations suggest that, as is  the case with endothelial cells (21) and rat BRL-3A liver cells (24), a PLA 2 attached to the surface of BALB/cJ BMMC via heparan sulfate or other proteoglycans participates in PGD 2 biosynthesis and is displaced by heparin. Importantly, heparin did not inhibit the induction of PGHS-2 (Fig. 2C), and the total release of PLA 2 was no different whether heparin was added at 10 min or at 5 h ( Fig. 2A), indicating that heparin did not inhibit the induction of the heparin-sensitive PLA 2 . Delayed-phase PGD 2 generation occurs not only after activation of BALB/cJ BMMC with KL ϩ IL-10 ϩ IL-1␤ but also after priming of BALB/cJ BMMC with KL ϩ IL-10 and activation through the high affinity Fc receptor for IgE (Fig. 4). Once again, heparin treatment resulted in substantial inhibition of delayed-phase PGD 2 generation and the augmentation of release of PLA 2 activity into the supernatant. There was an immediate release of PLA 2 activity during the first 30 -60 min after the addition of heparin, followed by a more gradual accumulation of PLA 2 activity in the supernatant. Not only heparin but also 12-epi-scalaradial, an inhibitor of type II PLA 2 (42), inhibited both the cytokine-induced and the cytokine-primed, IgE-dependent delayed-phase generation of PGD 2 from BALB/cJ BMMC. These agents did not inhibit the immediate generation of PGD 2 and LTC 4 in response to either stimuli (Table II). These data therefore suggested a role for a type II PLA 2 in the delayed phase of PGD 2 generation.
A mouse cDNA with 89 and 75% sequence identity, respectively, to rat and human type IIA PLA 2 was originally characterized as a cDNA encoding Paneth cell enhancing factor (39).
To confirm that Paneth cell enhancing factor encodes a heparin-sensitive type II PLA 2 activity, its cDNA was amplified by RT-PCR both from BALB/cJ mouse intestine and from BALB/cJ BMMC that had been activated with KL ϩ IL-10 ϩ IL-1␤ for 10 h and was expressed in COS-7 cells. ϳ75% of the PLA 2 activity was cell-associated and was released into the culture medium by heparin. The enzyme migrated as a 14-kDa protein recognized by an antibody to rat type II PLA 2 on SDS-PAGE immunoblotting. Primers based on the mouse sequence were therefore used in RT-PCR to demonstrate that the transcript for type IIA PLA 2 increased markedly from 2 to 7 h (Fig.  5A) in response to a cytokine stimulus that elicited delayedphase PGD 2 generation. Such transcripts were not increased in response to IL-3 or KL (Fig. 5), cytokines that alone do not elicit delayed arachidonic acid release (Fig. 1) or PGD 2 production (1). Furthermore, the induction of transcripts encoding type IIA PLA 2 was inhibited by dexamethasone (Fig. 5C). The nucleotide sequence of the PCR product from BALB/cJ BMMC was identical to that amplified from BALB/cJ mouse intestine.
That the kinetics, cytokine requirements, and sensitivity to dexamethasone of the induction of type IIA PLA 2 transcripts were in agreement with those of delayed-phase arachidonic acid release (Fig. 1) and delayed-phase PGD 2 generation (1) did not prove conclusively that these transcripts encoded the functional species of PLA 2 . To definitively test the role of type IIA PLA 2 in arachidonic acid mobilization in BMMC, we examined the immediate and delayed phases of mediator generation in BMMC derived from mice in which there is a natural disruption of the type IIA PLA 2 gene. In these strains of mice (129/J, C57BL/6J, P/J, A/J, and C58) an extra thymidine residue is incorporated into exon 3, leading to disruption of a BamHI restriction site and a shift in reading frame (26,27). The mutated full-length transcript has a premature stop codon and encodes a severely truncated protein. Alternative transcripts lacking either exon 3 or exon 4 are also generated, leading to protein products that lack the critical Ca 2ϩ -binding domain and/or residues that form the catalytic site. No PLA 2 activity was detected in the supernatant of COS-7 cells transfected with these mutated cDNAs, whereas COS-7 cells transfected with the intact cDNA released significant PLA 2 activity into the culture supernatant (27). It was considered unlikely that IL-3-dependent BMMC could be cultured from A/J and C58 mice because they are deficient in the expression of the ␣ chain of the IL-3 receptor (43), and therefore BMMC were derived from 129/J and C57BL/6J mice. Delayed-phase PGD 2 generation in response to KL ϩ IL-10 ϩ IL-1␤ and in response to IgE and antigen after priming with KL ϩ IL-10 was markedly attenuated in the 129/J BMMC. However, we were unable to demonstrate the induction of PGHS-2 protein in 129/J BMMC in response to cytokines alone, and its induction with KL ϩ IL-10 priming and IgE-dependent activation was also greatly attenuated. These findings preclude any comment on the possible role of the disruption of the type IIA PLA 2 gene in the lack of delayed-phase PGD 2 generation of the 129/J strain.
In contrast, delayed-phase PGD 2 generation occurred in the C57BL/6J BMMC (Fig. 6, Table III). PCR analysis of genomic DNA from the C57BL/6J BMMC with sequencing of the PCR product confirmed the disruption of the BamHI site in the type IIA PLA 2 gene. Therefore, in this strain of mouse arachidonic acid must be supplied to PGHS-2 by a heparin-sensitive, inducible PLA 2 distinct from the type IIA PLA 2 that has been characterized (39) and that is disrupted in this strain of mouse (26,27). Two low molecular weight PLA 2 enzymes, distinct from type I and type IIA PLA 2 , have been described (44). Type IIC PLA 2 is a 14.7-kDa protein with 16 cysteine residues that lacks the "elapid loop" characteristic of type I PLA 2 enzymes and contains the C-terminal extension characteristic of type II PLA 2 (19); it is preferentially expressed in testis. Type V PLA 2 is a 13.6-kDa protein containing 12 cysteine residues, no elapid loop, and no C-terminal extension (20); it is expressed in heart, placenta, lung, and liver. Neither the specific cellular expression of these enzymes nor their sensitivity to heparin and to pharmacologic inhibitors of PLA 2 is known. Murakami and colleagues (8) described a heparin-sensitive PLA 2 in rat RBL-2H3 cells and in a mouse mast cell line derived from BALB/c mice, distinguished from conventional type II PLA 2 by its mobility in high performance liquid chromatography and by a preference for phosphatidylserine as substrate. The enzyme had poor catalytic activity toward phosphatidylethanolamine, the substrate used in our current studies.
Previous studies have used antibodies to type II PLA 2 , pharmacologic inhibitors, heparin sensitivity, and/or antisense oligonucleotides to implicate endogenous type II PLA 2 in prostanoid biosynthesis. In human endothelial cells stimulated by tumor necrosis factor ␣ (21), antibodies that neutralize type II PLA 2 effectively suppressed prostanoid biosynthesis induced by proinflammatory cytokines. In both tumor necrosis factor ␣-stimulated human endothelial cells and rat BRL-3A liver cells prostaglandin synthesis was inhibited by heparin (21,24). In mouse P388D 1 cells activated with lipopolysaccharide and platelet-activating factor, an antisense oligonucleotide was used to demonstrate the role of type II PLA 2 in the delayed phase of PGE 2 generation (22). Our results indicate that heparin sensitivity is not confined to a specific type II PLA 2 . The heparin-sensitive PLA 2 operative in C57BL/6J BMMC may share significant cross-reactive epitopes or significant DNA sequence homology with the cloned type IIA PLA 2 initially described for mouse intestine (39).
A heparin-sensitive PLA 2 with the characteristics of type II PLA 2 is not implicated in the immediate phase of eicosanoid generation since neither heparin nor 12-epi-scalaradial was inhibitory (Table II), and the response was intact in 129/J BMMC and C57BL/6J BMMC (Fig. 6). Fonteh et al. (10) concluded that type II PLA 2 was involved in the immediate phase of IgE-dependent prostanoid generation in BMMC derived from CBA/J mice. They demonstrated release of a PLA 2 activity into culture supernatants of BMMC that was inhibitable by dithiothreitol and by an antibody raised against human type II PLA 2 . Furthermore, when type I or type II PLA 2 was added to BMMC at a concentration of 1 to 2 g/ml, a relatively selective release of arachidonic acid was observed within 10 min, with attendant prostanoid formation. However, the participation of type II PLA 2 in IgE-dependent prostanoid formation was not definitively demonstrated by pharmacologic or antibody inhibition of PGD 2 biosynthesis, and as defined by PLA 2 activity, endogenous type II PLA 2 contained in BMMC is likely less than 1 ng/10 6 cells (8). An alternative role for mast cell-derived type II PLA 2 is illustrated by the transcellular, PGHS-1-dependent PGE 2 generation by fibroblasts (11). That degranulation was partially suppressed by 12-epi-scalaradial and by heparin without concomitant inhibition of PGD 2 generation (Table I) is consistent with previous reports with rat peritoneal mast cells that the inhibition of type II PLA 2 suppressed histamine release without affecting the immediate phase of PGD 2 generation (23). These observations therefore support the possible function of a constitutively expressed PLA 2 with type II characteristics in the regulation of degranulation but not eicosanoid synthesis in the immediate phase of mast cell mediator release.
The role of cPLA 2 in supplying free arachidonic acid in the immediate phase of eicosanoid generation is suggested by its activation and translocation in response to stimuli that elicit acute Ca 2ϩ flux (12)(13)(14). There is no increased expression of cPLA 2 and no detectable activation of 5-lipoxygenase, a Ca 2ϩdependent cytosolic enzyme, in the delayed phase of eicosanoid release in BALB/cJ BMMC stimulated via either c-kit or the high affinity Fc receptor for IgE (3,45). Increased expression or phosphorylation of cPLA 2 has been reported in cytokine-stimulated fibroblasts (46 -48) and in monocytes/macrophages (49) in association with the induction of PGHS-2. However, others have shown that increased expression or phosphorylation of cPLA 2 per se was insufficient to elicit prostaglandin biosynthesis unless Ca 2ϩ mobilization was elicited by a second agonist (50 -52). That antisense inhibition of cPLA 2 expression in human monocytes resulted in marked reduction of lipopolysaccharide-stimulated PGE 2 generation over 5-18 h (53) suggests activation of cPLA 2 at a time remote from the immediate stimulus in this circumstance.
We have previously established that the immediate generation of PGD 2 from BALB/cJ BMMC is dependent on PGHS-1, whereas the delayed phase of PGD 2 generation from BALB/cJ BMMC in response to direct cytokine stimulation or in response to cytokine priming for activation by IgE and antigen is dependent on PGHS-2. We now find that the segregation of arachidonic acid metabolism between the two isoforms of PGHS in the immediate and delayed phases of PGD 2 generation in BALB/cJ BMMC is linked to different PLA 2 enzymes. Specifically, cPLA 2 is phosphorylated and translocates to a cell membrane in response to Ca 2ϩ flux to supply arachidonic acid in the immediate phase of PGD 2 generation, whereas an induced, heparin-sensitive enzyme supplies arachidonic acid to PGHS-2 in the delayed phase. Analyses of cells from a mouse strain genetically deficient in the previously characterized type IIA PLA 2 indicate that the heparin-sensitive, inducible PLA 2 involved in delayed-phase PGD 2 generation is distinct and could be either a novel enzyme or a new function for a low molecular weight PLA 2 .