©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Transcellular Prostaglandin Production following Mast Cell Activation Is Mediated by Proximal Secretory Phospholipase A and Distal Prostaglandin Synthase 1 (*)

(Received for publication, June 5, 1995; and in revised form, October 25, 1995)

Srinivasa T. Reddy Harvey R. Herschman (§)

From the Departments of Biological Chemistry and Pharmacology, Laboratory of Structural Biology and Molecular Medicine, and the Molecular Biology Institute, UCLA Center for the Health Sciences, Los Angeles, California 90095

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Prostaglandins mediate many biological processes. Arachidonic acid, the common precursor for all prostaglandins, is released from membrane phospholipids by both secretory and cytoplasmic forms of phospholipase A(2). Free arachidonate is converted to prostaglandin H(2), the common precursor to all prostanoids, by prostaglandin synthase. Both mitogen-induced prostaglandin synthesis in fibroblasts and endotoxin-induced prostaglandin synthesis in macrophages require expression of the inducible prostaglandin synthase-2; arachidonate released in these contexts is unavailable to prostaglandin synthase-1 constitutively present in fibroblasts or macrophages.

In contrast to the results for fibroblasts and macrophages, prostaglandin synthesis by activated mast cells is mediated by prostaglandin synthase-1. Mast cell activation also provokes release of secretory phospholipase A(2) (sPLA(2)). We now demonstrate that sPLA(2) released from activated mast cells can mobilize arachidonate from distal Swiss 3T3 cells. This arachidonate is then used by prostaglandin synthase-1 present in 3T3 cells for prostaglandin synthesis. We thus distinguish two pathways for prostaglandin synthesis: (i) an intracellular pathway by which arachidonate released following ligand stimulation is made available only to prostaglandin synthase-2, and (ii) a transcellular pathway by which sPLA(2) of proximal cells mobilizes, in distal cells, arachidonate available to prostaglandin synthase-1. Molecular and pharmacologic approaches to modulating prostaglandin-mediated events will differ for these two pathways.


INTRODUCTION

The prostanoids (prostaglandins, thromboxanes, and prostacyclins) mediate a wide variety of physiological processes, including ovulation, hemostasis, platelet aggregation, kidney water balance, and immune responses(1) . Prostanoid production is regulated both by release of arachidonic acid from membrane lipid stores by phospholipase A(2) and by conversion of arachidonic acid to prostaglandin H(2) (PGH(2)), (^1)the common precursor for all prostanoids, by cyclooxygenase/prostaglandin synthase (PGS)(2) .

Arachidonic acid can be released from cellular membranes either by the 14 kilodalton secretory phospholipase A(2) (sPLA(2)), or by the 85 kilodalton intracellular isoform of the enzyme, cytoplasmic phospholipase A(2) (cPLA(2))(3) . Two forms of prostaglandin synthase, the rate-limiting enzyme in the conversion of free arachidonic acid to prostanoids, also exist. Most cells contain a constitutively-expressed prostaglandin synthase, PGS1, but can also express an inducible form of the enzyme, PGS2, in response to stimulation by appropriate ligands. Ligand-induced PGS2 gene expression has been observed in fibroblasts, macrophages, endothelial cells, epithelial cells, ovarian granulosa cells, neurons, and smooth muscle cells(4) . Aspirin and other nonsteroidal anti-inflammatory drugs exert their antipyretic, analgesic, and anti-inflammatory actions by inhibiting the activity of the prostaglandin synthases(5, 6) .

Experiments utilizing antisense oligonucleotide inhibition demonstrate that both mitogen-stimulated PGE(2) production in fibroblasts and endotoxin-stimulated PGE(2) production in macrophages require the induced expression and activity of the PGS2 gene and protein, despite the presence of constitutive, enzymatically active PGS1(7) . In contrast to the requirement that PGS2 enzyme be synthesized for prostaglandin production following activation by many ligands in fibroblasts and macrophages, PGD(2) synthesis occurs rapidly in activated mast cells. Moreover, the rapid PGD(2) production in mast cells activated by aggregation of IgE receptors is dependent only on the presence and activity of constitutive PGS1 (8) .

Following activation, mast cells degranulate to release preformed ligands such as histamine and serotonin. In addition, activation releases stored sPLA(2) from mast cells(9) . Activated mast cells can use arachidonic acid released by sPLA(2) for prostaglandin synthesis(9) . In this report we demonstrate that sPLA(2) released from activated MMC-34 mast cells can stimulate PGS1-dependent synthesis of PGE(2) in co-cultured Swiss 3T3 cells. These results provide evidence for distinct pathways for sPLA(2) mediated, PGS1-dependent transcellular prostaglandin production and for mitogen-stimulated, PGS2-dependent intracellular prostaglandin production.


EXPERIMENTAL PROCEDURES

Cell Culture

Mouse MMC-34 mastocytoma cells (10) were grown in RPMI 1640 medium (ICN, Cleveland, OH) supplemented with 10% fetal calf serum (Gemini Bioproducts, Inc., Calabasas, CA). Swiss 3T3 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) in 10% fetal calf serum. Co-culture conditions are described in the text or figure legends.

Reagents

Naja naja sPLA(2) and aspirin were purchased from Sigma, tetradecanoyl phorbol acetate (TPA) from Chemsyn Science Laboratories (Lenexa, KN), murine IgE and rat monoclonal anti-IgE from Pharmingen (San Diego, CA), PGD(2) and PGE(2) assay kits from Amersham Corp., tritiated arachidonic acid (76 Ci/mmol) from DuPont NEN. Aminopropyl (NH(2)) solid-phase silica columns no. 9070 (100 mg/ml) were obtained from Burdick and Jackson (Muskeogon, MI). Arachidonate ^3H-labeled Escherichia coli suspension (8 mCi/mmol) was from DuPont NEN. NS-398 was a gift from Taisho Corp (Japan). Recombinant human sPLA(2) (rPLA(2)), monoclonal antibody F10 (mAb F10) directed against rPLA(2), and SB 203347, a specific inhibitor of sPLA(2), were the gifts of Dr. Lisa Marshall (SmithKline Beecham Pharmaceticals, King of Prussia, PA).

Prostaglandin Determinations

Cell cultures were treated with IgE + Anti-IgE, MMC-34 mast cell supernatants, or sPLA(2) as described in the figure legends or text. Medium was collected by centrifugation and analyzed for PGD(2) and PGE(2) levels, using Amersham kits, as described by the manufacturer. In some cases, PGE(2) determinations were preformed by enzyme-linked immunosorbent assay(11) .

Assay of sPLA(2) Enzymatic Activity

Phospholipase activity was determined by a modification of the method of Marshall et al.,(12) , which utilizes the acylhydrolysis of [^3H] E. coli membranes by PLA(2). The reaction mixture (150 µl total volume) contained 25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl(2) and 0.1 µCi of [^3H]arachidonate-labeled E. coli membranes. The assays were incubated at 37 °C for 1 h. The reactions were stopped by the addition of 1 ml of tetrahydrofuran. Free arachidonic acid was separated by elution of the sample over aminopropyl solid-phase silica columns with tetrahydrofuran/acetic acid (49:1) and quantitated by liquid scintillation counting(12) .

Arachidonic Acid Labeling of Cells and Measurement of Radioactive PGE(2)

Swiss 3T3 cells or MMC-34 mast cells were plated on 12-well culture dishes. Twenty-four hours later, [^3H]arachidonic acid (1.5 µCi/ml) was added and cells were cultured for 48 h. Cells were then washed and cultured overnight in medium containing 0.5% serum. Cells were then washed and either treated directly with IgE + anti-IgE, or co-cultured and activated as described in the text. All treatments and co-cultures were in medium containing 0.5% serum. One hour after addition of anti-IgE, medium samples were collected. Lipids were isolated from the medium and analyzed by HPLC and scintillation counting (13) for accumulation of radioactive PGE(2).


RESULTS

sPLA(2) Stimulates PGE(2) Synthesis in Swiss 3T3 Cells

PGE(2) production by Swiss 3T3 cells following stimulation with serum, platelet-derived growth factor, or TPA requires the ligand-induced synthesis of PGS2(7) , and follows kinetics consistent with induced PGS2 expression(14) . In contrast, mast cell PGD(2) production following activation by aggregation of IgE receptors is rapid(8) . Moreover, PGD(2) production in activated mast cells is derived, at least in part, from arachidonic acid released in response to sPLA(2)(9) . To determine whether other cells show rapid production of prostaglandins in response to sPLA(2), we compared the ability of TPA and the sPLA(2) from N. naja venom to induce PGE(2) production in Swiss 3T3 cells. Although both agents cause substantial PGE(2) production, the kinetics differ. sPLA(2) treatment causes rapid PGE(2) production, which reaches a plateau by the first (10 min) time point measured (Fig. 1). At this time no PGS2 enzyme is detectable in 3T3 cells stimulated with TPA. In contrast, TPA-induced PGE(2) accumulation does not significantly increase until 2 h after TPA administration, and is substantially elevated only at later times (Fig. 1).


Figure 1: N. naja sPLA(2) stimulates PGE(2) synthesis in Swiss 3T3 cells. Confluent Swiss 3T3 cells in 12-well culture dishes were treated either with N. naja sPLA(2) (2 units/ml; closed circles) or with TPA (50 ng/ml; open circles). At the times shown, media were collected and assayed for PGE(2). Data are the averages of triplicate determinations, ±S.D.



PGE(2) Production in Swiss 3T3 Cells following sPLA(2) and TPA Treatment Is Mediated by Distinct Prostaglandin Synthase Isoforms

Swiss 3T3 cells express PGS1 constitutively(7, 14) . Preincubation with aspirin and washing irreversibly inhibits PGS1 activity of 3T3 cells, without inhibiting subsequent TPA-induced, PGS2-dependent PGE(2) production(7) . Aspirin preincubation and washing prevents all PGE(2) production in 3T3 cells in response to sPLA(2) (Fig. 2). In contrast, NS-398, a recently described nonsteroidal anti-inflammatory drug (NSAID) that inhibits PGS2 enzyme activity much more effectively than PGS1 activity(15) , has no effect on sPLA(2)-induced PGE(2) production in Swiss 3T3 cells. These data demonstrate that sPLA(2) induces PGE(2) production through the action of preexisting, constitutive PGS1 enzyme.


Figure 2: PGE(2) production in Swiss 3T3 cells following sPLA(2) and TPA treatment occurs via distinct prostaglandin synthase isoforms. 3T3 cells in 12-well culture dishes were either untreated (lane 1), treated with N. naja sPLA(2) for 1 h (lane 2), pretreated for 30 min with 200 µM aspirin, washed with fresh medium, and then treated with N. naja sPLA(2) for 1 h (lane 3), treated with sPLA(2) and 1 µM NS-398 for 1 h (lane 4), treated with 50 ng/ml TPA for 6 h (lane 5), pretreated for 30 min with aspirin, washed with fresh medium, and then treated with TPA for 6 h (lane 6), or treated with TPA + NS-398 for 6 h (lane 7). Media were collected and assayed for PGE(2). Data are averages of triplicate determinations, ±S.D.



Production of PGE(2) by TPA-treated cells is abolished by the PGS2-specific inhibitor NS-398 (Fig. 2). In contrast, inactivaton of PGS(1) by aspirin pretreatment and washing has no effect on subsequent TPA-induced PGE(2) induction in 3T3 cells. We conclude that sPLA(2)-induced PGE(2) synthesis in 3T3 cells is mediated by preexisting PGS1 enzyme, while TPA-induced PGE(2) synthesis in 3T3 cells requires synthesis and activity of PGS2 enzyme.

Mast Cell Activation Induces PGE(2) Synthesis in Mast Cell/Fibroblast Co-cultures

Because (i) activated mast cells secrete sPLA(2)(9) , (ii) mast cell prostaglandin synthesis following activation is, at least in part, dependent on sPLA(2) production(9) , and (iii) sPLA(2) can stimulate prostaglandin synthesis in 3T3 cells ( Fig. 1and Fig. 2), we thought it possible that activated mast cells might be able to induce transcellular prostaglandin synthesis by release of sPLA(2) following activation. Since mast cell-produced PGD(2) and fibroblast-produced PGE(2) can be distinguished, we tested this proposal by analyzing PGE(2) production in mast cell/fibroblast co-cultures.

If MMC-34 mast cells are simply co-cultured with 3T3 cells, little PGE(2) is present in the medium (Fig. 3, lane 1). If IgE receptors are aggregated on mast cells by treatment with IgE followed by anti-IgE, PGD(2) is produced as expected (data not shown), but no production of PGE(2) is observed (Fig. 3, lane 2). When Swiss 3T3 cells are treated with IgE followed by anti-IgE, there is also no significant increase in PGE(2) levels observed in the medium, since 3T3 cells do not have IgE receptors (Fig. 3, lane 3). However, substantial production of PGE(2) is observed in MMC-34 mast cell/Swiss 3T3 co-cultures following addition of IgE + anti-IgE (Fig. 3, lane 4). The PGE(2) present in these co-cultures after stimulation with IgE + anti-IgE must come from the 3T3 cells, since activation of mast cells alone by IgE + anti-IgE does not produce PGE(2). Activation-induced PGE(2) production in the co-cultures must, therefore, be due to a mast cell-mediated effect on the 3T3 cells. Medium from activated mast cells can also stimulate PGE(2) production when applied to 3T3 cells (Fig. 3, lane 5). We conclude that a secreted intermediate from activated mast cells mediates PGE(2) production in co-cultured Swiss 3T3 cells.


Figure 3: Activation of mast cells induces PGE(2) synthesis in MMC-34 mast cell/Swiss 3T3 cell co-cultures. MMC-34 mast cells and Swiss 3T3 cells were grown to confluence (2 times 10^6 cells per well, for both cell types). Although the MMC-34 cells are not attached tightly to the plastic, they settle to the bottom of the wells during culture. For co-culture experiments, the medium from the Swiss 3T3 cells was replaced with the medium and cells from the wells containing the MMC-34 cells. First lane; Swiss 3T3 cells and MMC-34 mast cells were co-cultured for 1 h with no additions. Media were collected and assayed for prostaglandins. Second lane; MMC-34 cells were incubated with IgE (1 µg/ml) for 2 h, then treated with anti-IgE (1 µg/ml). One hour later prostaglandins in the medium were assayed. Lane 3, 3T3 cells were incubated with IgE for 2 h, then incubated with anti-IgE for 1 h, and prostaglandins were assayed. Lane 4, MMC-34 cells were treated with IgE for 2 h. The media and MMC-34 cells were then transferred to confluent wells of 3T3 cells. Anti-IgE was added to the co-cultures, and 1 h later the supernatants were assayed for prostaglandins. Lane 5, MMC-34 mast cells were incubated with IgE for 2 h, then treated with anti-IgE for 1 h. The medium from activated MMC-34 cells (indicated by the asterisk) was collected by centrifugation and added to Swiss 3T3 cells. One hour later medium was again collected, and prostaglandins were assayed. All experiments were performed in triplicate. Data are the averages, ±S.D. PGD(2) present in the supernatants from activated mast cells did not interfere with the PGE(2) determinations (data not shown).



sPLA2 Released by Activated Mast Cells Stimulates PGE(2) Production by Swiss 3T3 Cells

As expected(9) , supernatants from MMC-34 mast cells activated by exposure to IgE + Anti-IgE contain substantially elevated levels of PLA(2) enzyme activity, when compared to supernatants from unactivated cells (Fig. 4, left panel, lanes 1 and 2). Once again, the supernatant from activated mast cells is able to induce PGE(2) production from 3T3 cells (Fig. 4, right panel, lanes 1 and 2).


Figure 4: Both phospholipase A(2) activity and fibroblast PGE(2) inducing activity of activated mast cell supernatants are inhibited by a monoclonal antibody to recombinant PLA(2) and by SB 203347. Supernatants were prepared from control MMC-34 cells and from cells treated with IgE (2 h) + anti-IgE (1 h). Activated supernatants (indicated by the asterisk) were treated with mAb F10 (10 µg/ml), a control mAb preparation, or SB 203347 (1 µM) as indicated in the figure. Samples (0.1 ml) of each supernatant were assayed for phospholipase activity as described under ``Experimental Procedures.'' Phospholipase activity is expressed as arachidonic acid (a.a.) released. Samples (0.9 ml) of the same supernatants were placed on confluent cultures of Swiss 3T3 cells. Media were collected after 1 h and assayed for PGE(2). Data are the averages of triplicate determinations, ±S.D.



It seems likely that the sPLA(2) released following activation of mast cells is responsible for the production of PGE(2) in the mast cell/fibroblast co-cultures. We used mAb F10, a monoclonal antibody directed against recombinant sPLA(2)(9) , and SB 203347, a recently described inhibitor of sPLA(2) enzyme activity(16) , to determine whether the sPLA(2) released by activated MMC-34 mast cells is responsible for PGE(2) production by 3T3 cells in the mast cell/fibroblast co-cultures. Recombinant sPLA(2) (rPLA(2)), like the N. naja sPLA(2) and the supernatants from activated mast cells, can induce PGE(2) production in 3T3 cells (Fig. 5). Both mAb F10 and SB 203347 inhibit PGE(2) induction in 3T3 cells in response to rPLA(2) treatment (Fig. 5). mAb F10 and SB 203347 block 72% and 74% of the phospholipase activity of the rPLA(2) protein used in the experiment illustrated in Fig. 5(data not shown). The amount of rPLA(2) activity (150 pmol of [^3H]arachidonic acid released/min/ml) used in this experiment was chosen to resemble that found in the supernatants of activated MMC-34 mast cells (Fig. 4).


Figure 5: The ability of recombinant PLA(2) to induce PGE(2) production in fibroblasts is inhibited by mAb F10 and by SB 203347. The enzymatic activity of rPLA(2) was determined as described under ``Experimental Procedures.'' Samples of rPLA(2) with an activity of 150 pmol of arachidonic acid released/min were treated with mAb F10 (10 µg), control mAb, or SB 203347 (1 µM). The treated and untreated rPLA(2) samples were incubated with confluent cultures of Swiss 3T3 cells, in fresh medium. Media were collected after 1 h and assayed for PGE(2). Data are the averages of triplicate determinations, ±S.D.



The PLA(2) enzymatic activity released into MMC 34 mast cell supernatants following IgE + Anti-IgE activation can be neutralized by mAb F10, and inhibited by SB 203347 (Fig. 4, left panel). In contrast, a control mAb has no effect on PLA(2) activity in activated mast cell supernatants. Similarly, addition of either the anti-rPLA(2) mAb F10 or the sPLA(2) inhibitor SB 203347 block the ability of supernatants from activated MMC 34 mast cells to induce PGE(2) production by 3T3 cells (Fig. 4, right panel). These data demonstrate that the sPLA(2) released by mast cells following activation is essential for transcellular prostaglandin synthesis in 3T3 cells.

Arachidonic Acid from 3T3 Cells Is Used for PGE(2) Production in Activated Mast Cell/Fibroblast Co-cultures

Activation of mast cells leads to release of arachidonic acid(9) , as well as sPLA(2). Since exogenous arachidonic acid can be used by PGS1 to produce PGE(2) in Swiss 3T3 cells (7) , it is possible that the PGE(2) produced in MMC-34/3T3 co-cultures following activation by IgE + anti-IgE could, at least in part, be synthesized by PGS1 in 3T3 cells from arachidonic acid released by the activated mast cells. However, supernatants from mast cells treated with either mAb F10 or SB 203347, which neuteralize and/or inhibit PLA(2) activity after mast cell activation, should contain arachidonic acid released from the mast cells prior to the addition of the inhibitors. These neuteralized/inhibited supernatants from activated mast cells do not elicit PGE(2) production in 3T3 cells (Fig. 4), suggesting that arachidonic acid from activated mast cells does not serve as substrate for transcellular prostaglandin synthesis by fibroblasts. In the co-cultures, and in in vivo contexts where transcellular prostaglandin synthesis may be occurring as a result of release of PLA(2) from proximal cells, it is likely that arachidonic acid released from the proximal cell is sequestered by plasma proteins such as albumin, and is not available as substrate for PGS in the distal cell.

To investigate more extensively the source of arachidonic acid substrate in the distal 3T3 cells engaged in transcellular prostaglandin production, we labeled the membranes of either proximal MMC-34 mast cells or distal Swiss 3T3 cells with [^3H]arachidonic acid, and examined radioactive PGE(2) synthesis in [IgE + anti-IgE]-treated cultures of each cell type individually, and in co-cultures. No radioactive PGE(2) is produced following stimulation of [^3H]arachidonic acid-labeled 3T3 cells (Fig. 6). Although radioactive arachidonate is released by the medium change that accompanied this treatment (eluting from the chromatogram at a later time; data not shown), this arachidonate cannot be converted to PGE(2), because no PGS2 is present(7) . No radioactive PGE(2) is produced following activation of [^3H]arachidonic acid-labeled MMC-34 mast cells, since mast cells make PGD(2), but not PGE(2). When co-cultures of MMC-34 mast cells and [^3H]arachidonic acid-labeled Swiss 3T3 cells are stimulated with IgE + anti-IgE, synthesis of radioactive PGE(2) is observed. In contrast, when co-cultures containing [^3H]arachidonate-labeled MMC-34 cells and unlabeled Swiss 3T3 cells are treated with IgE + anti-IgE, no radioactive PGE(2) is produced. We conclude (i) that arachidonic acid released from activated mast cells in co-culture is not available as substrate for 3T3 cells (presumably because of sequestration by plasma proteins) and (ii) that arachidonic acid present in membranes of 3T3 cells is released by mast cell sPLA(2) following activation, and is utilized by PGS1 present in the 3T3 cells for PGE(2) production.


Figure 6: Arachidonic acid from 3T3 cells is used for PGE(2) production in activated co-cultures. Swiss 3T3 cells and MMC-34 mast cells were labeled with [^3H]arachidonic acid as described in ``Experimental Procedures.'' Labeled 3T3 cells (top panel) or MMC-34 cells (second panel) were treated with IgE, followed by anti-IgE for 1 h before media were collected. For co-culture experiments, unlabeled MMC-34 cells (third panel) or labeled MMC-34 (bottom panel) cells were treated with IgE, washed, and added to 3T3 cultures. All co-culture experiments were in medium containing 0.5% serum. The asterisk (*) denotes the cell population labeled with [^3H]arachidonate. Anti-IgE was added, and media were collected at 1 h. All samples were subjected to HPLC, and radioactivity was determined by scintillation counting. Each experimental condition was performed in duplicate. Elution profiles indicating times of elution for radioactive compounds are shown for two cultures for all conditions. The initial peak at 3-10 min contains unidentified polar compounds. Arachidonic acid elutes at 30-38 min (not shown).




DISCUSSION

We suggest that there are two distinct routes of prostanoid biosynthesis in 3T3 fibroblasts (Fig. 7). For the first route, ligand-activated intracellular phospholipase(s) releases arachidonic acid that is not available to PGS1; this arachidonate must be converted to PGH(2) by PGS2 enzyme, following ligand-activated PGS2 gene expression(7) . Activation of an intracellular, cytoplasmic PLA(2) is likely to be required for this pathway. The second, transcellular route of prostanoid synthesis occurs via release of sPLA(2) by proximal cells. This sPLA(2) enzyme can then act as a paracrine mediator of arachidonate release in distal 3T3 cells, leading to transcellular prostaglandin production by constitutive PGS1.


Figure 7: Two routes of PGE(2) production in Swiss 3T3 cells. L, ligand; R, receptor; AA, arachidonic acid; PGS, prostaglandin synthase; PLA, phospholipase A(2).



The proposed alternative pathways of prostaglandin synthesis are illustrated in Fig. 7. The 14-kilodalton form of PLA(2) remains in the cytoplasm and/or is secreted following cellular activation(3) . In contrast, following ligand stimulation, the 85-kilodalton isoform of PLA(2) becomes phosphorylated, and then associates with cell membranes(3) . Recent studies have suggested that, following ligand stimulation, the 85-kilodalton PLA(2) molecule becomes associated with the nuclear membrane(17) . Until recently, both PGS1 and PGS2 enzymes were thought to be associated with the endoplasmic reticulum (18, 19) . However, confocal immunofluorescent microscopy studies suggest that PGS2, unlike PGS1, associates with the nuclear membrane (20) . The recent demonstrations that one isoform of PLA(2) (85-kilodalton cPLA(2)) and one isoform of PGS (PGS2) can preferentially associate with the nuclear membrane suggests a potential spatial explanation for the distinct routes of prostaglandin production observed in fibroblasts.

Inhibition of prostaglandin synthesis, either by interfering with phospholipase-mediated release of membrane-bound arachidonic acid or by interfering with prostaglandin synthase-mediated conversion of free arachidonate to PGH(2), is a major pharmacologic goal. With the discovery of alternative PLA(2) and prostaglandin synthase isoforms, new targets for specific pharmacologic intervention of prostaglandin synthesis have emerged. Demonstration that PGS2 is induced in inflammatory responses (reviewed in (4) ) and that PGS1 and PGS2 have restricted pools of precursor arachidonate (7, 8) has increased interest in identifying isoform-specific inhibitors of prostaglandin synthase and phospholipase A(2) as therapeutic agents. The goal of identifying PGS2-specific inhibitors is to produce NSAIDs without ulcerogenic or nephrotoxic side effects. A number of lead NSAIDs that are substantially more effective in inhibiting PGS2 activity relative to PGS1 have now been described(21) . Our data demonstrating that both a transcellular route of prostaglandin synthesis and an intracellular route of prostaglandin synthesis exist, and utilize alternative PGS and PLA(2) isoforms, suggests that combinatorial use of specific phospholipase and prostaglandin synthase inhibitors may be necessary to achieve suppression of prostaglandin production in cases where substantial sPLA(2) production occurs.

How general might transcellular prostanoid production by the proximal sPLA(2)/distal PGS1 pathway be? The first considerations are (i) the range of cells that produce and secrete sPLA(2), and (ii) the range of cells that respond to sPLA(2) administration by PGS1-mediated prostanoid production. sPLA(2) is present in inflammatory fluids, e.g. synovial fluid, and in inflammatory tissue exudates(22) . Activated mast cells(9) , platelets (23) , and neutrophils (24) rapidly release sPLA(2). Cytokine-induced sPLA(2) synthesis and release occurs in astrocytes (25) and mesangial cells(26) . sPLA(2) induction is also part of the septic shock and liver acute phase responses(22) . With regard to distal cell responsiveness to sPLA(2), antisense sPLA(2) oligonucleotides reduce both endotoxin-stimulated arachidonic acid release and prostaglandin synthesis in P388D(1) macrophage-like cells (27) . Extracellular sPLA(2) has also been implicated in prostaglandin production in mesangial cells (28) and endothelial cells(29) .

Co-cultivation of alveolar epithelial cells and macrophages labeled respectively with [^14C]arachidonate and [^3H]arachidonate has demonstrated that proximal macrophages can serve as a source of substrate arachidonic acid for prostaglandin synthesis by distal epithelial cells(30) . In contrast, we find that the arachidonic acid produced by activated mast cells, the proximal cells in our co-cultures that engage in transcellular prostaglandin synthesis, is not available to the distal 3T3 cell prostaglandin synthase. Although sPLA(2) is postulated to at least in part mediate antigen-induced prostaglandin synthesis in mast cells (8) and endotoxin-induced prostaglandin synthesis in P388D(1) macrophage-like cells (27) in an autocrine fashion, evidence for sPLA(2) as a transcellular mediator of prostaglandin synthesis has not previously been described.

Although previous reports of transcellular prostanoid synthesis are rare, there is a substantial literature demonstrating transcellular production of the other class of eicosanoids, the leukotrienes. LTA(4), the product of 5`-lipoxygenase, can be transformed to LTB(4) by LTA(4) hydrolase or conjugated to glutathione by LTC(4) synthase. 5`-Lipoxygenase is restricted to myeloid cells(31) . One might, therefore, expect that these are the only cells that would produce leukotrienes. However, many cell types unable to express 5`-lipoxygenase nevertheless produce LTB(4). This occurs because (i) such cells express LTA(4) hydrolase and (ii) LTA(4) can be released from proximal myeloid cells and used as substrate by LTA(4) hydrolase present in distal cells. Erythrocytes(32) , endothelial cells(33) , and epithelial cells (34) engage in transcellular LTB(4) production. LTC(4) synthase has been demonstrated in platelets, endothelial cells, and epidermal cells, in addition to myeloid cells. Transcellular synthesis of LTC(4) from LTA(4) has been documented for platelets(35) , endothelial cells(33, 34) , epithelial cells(36) , and keratinocytes (37) . It should be emphasized that substrate is passed between cells for transcellular leukotriene synthesis. Phospholipases have not been reported to act as transcellular mediators of leukotriene sythesis.


FOOTNOTES

*
These studies were supported by the UCLA Asthma, Allergic and Immunologic Diseases Center (AI34567) funded by NIAID and NIEHS, National Institutes of Health, and by Contract FC03 87ER60615 from the Department of Energy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 341A Molecular Biology Institute, UCLA, Los Angeles, CA 90095. Tel.: 310-825-8735; Fax: 310-825-1447; harvey@lbes.medsch.ucla.edu.

(^1)
The abbreviations used are: PG, prostaglandin; PGS1, prostaglandin synthase 1; PGS2, prostaglandin synthase 2; sPLA(2), secretory phospholipase A(2); cPLA(2), cytoplasmic phospholipase A(2); rPLA(2), recombinant phospholipase A(2); TPA, tetradecanoyl phorbol acetate; Ig, immunoglobulin; LT, leukotriene; NSAID, nonsteroidal anti-inflammatory drug; mAb, monoclonal antibody; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We thank Raymond Basconcillo and Arthur Catapang for technical assistance, and the members of the Herschman laboratory for helpful discussions. We also thank Nicholas Bazan, Elena Rodriguez de Turco, and Fannie Richardson (New Orleans) for performing the HPLC analyses, Jaime Masferrer (Searle) for PGE(2) ELISA assays, and Lisa Marshall (SmithKline Beecham) for the gifts of recombinant PLA(2), mAb F10, and SB 203347.


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