Biosynthesis in Rat Peritoneal Macrophages Synthases in Different ...

Segregated Coupling of Phospholipases A2, Cyclooxygenases, and Terminal Prostanoid Synthases in Different Phases of Prostanoid Biosynthesis in Rat Peritoneal Macrophages1 Hiroaki Naraba,2* Makoto Murakami,† Hideki Matsumoto,* Satoko Shimbara,† Akinori Ueno,* Ichiro Kudo,† and Sachiko Oh-ishi* We examined herein the functional linkage of enzymes regulating the initial, intermediate, and terminal steps of PG biosynthesis to provide PGs in rat peritoneal macrophages stimulated with LPS and/or A23187. Quiescent cells stimulated with A23187 produced thromboxane B2 (TXB2) in marked preference to PGE2 within 30 to 60 min (constitutive immediate response), which was mediated by preexisting cytosolic phospholipase A2 (cPLA2), cyclooxygenase-1 (COX-1), and TX synthase. Cells treated with LPS predominantly produced PGE2 during culture for 3 to 24 h (delayed response), where cPLA2 and secretory PLA2 functioned cooperatively with inducible COX-2, which was, in turn, coupled with inducible PGE2 synthase. Cells primed for 12 h with LPS and stimulated for 30 min with A23187 produced PGE2 in marked preference to TXB2 (induced immediate response), in which three inducible enzymes, cPLA2, COX-2, and PGE2 synthase, were functionally linked. Preferred coupling of the two inducible enzymes, COX-2 and PGE2 synthase, was further confirmed by the ability of LPS-treated cells to convert exogenous arachidonic acid to PGE2 optimally at a time when both enzymes were simultaneously induced. These results suggest that distinct PG biosynthetic enzymes display segregated functional coupling following different transmembrane stimulation events even when enzymes that catalyze similar reactions in vitro coexist in the same cells. The Journal of Immunology, 1998, 160: 2974 –2982.

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egulation of prostanoid generation by mammalian cells occurs at three sequential biosynthetic steps. Arachidonic acid, which is stored in membrane glycerophospholipids, is liberated by phospholipase A2 (PLA2)3 enzymes and is sequentially converted to PGH2 by PGH2 synthases (cyclooxygenase (COXs)) and then to various bioactive PGs, such as PGE2, PGD2, PGF2a, PGI2, and thromboxane A2 (TXA2), by the respective terminal synthases. PLA2 comprises a large superfamily of distinct enzymes that exhibit different substrate specificity, cofactor requirement, and subcellular localization (for review, see Ref. 1). Cytosolic (cPLA2) and secretory (sPLA2) subfamilies of PLA2 represent a class of Ca21-dependent enzymes implicated in eicosanoid biosynthesis. During stimulus-initiated arachidonic acid release accompanied by an increase in the intracellular Ca21 level, cPLA2 is phosphorylated and activated by mitogen-activated protein kinases (2) and is translocated from the cytosol to the perinuclear and endoplasmic reticular membranes (3), where several enzymes involved in the

*Department of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku; and †Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan Received for publication July 21, 1997. Accepted for publication November 20, 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. 2 Address correspondence and reprint requests to Dr. Hiroaki Naraba, Department of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108, Japan. E-mail address: [email protected]. kitasato-u.ac.jp 3

Abbreviations used in this paper: PLA2, phospholipase A2; COX, cyclooxygenase; TX, thromboxane; cPLA2, cytosolic phospholipase A2; sPLA2, secretory phospholipase A2; TBS, 10 mM Tris-HCl containing 150 mM NaCl, pH 7.4; TBS-T, TBS containing 0.1% Tween-20; RT-PCR, reverse transcription-polymerase chain reaction. Copyright © 1998 by The American Association of Immunologists

downstream COX and 5-lipoxygenase pathways are reported to colocalize (4, 5). Type IIA sPLA2, one of the most characterized of the sPLA2 isoforms, is secreted and becomes associated with cell surfaces, where it may contribute to augmentation of certain phases of eicosanoid biosynthesis (6 – 8). Inflammatory stimuli often induce the expression of type IIA sPLA2 (7, 9, 10) and cPLA2 (11), whereas anti-inflammatory agents down-regulate their expression and function (9, 11, 12). More recently, type V sPLA2 has been shown to be an alternative effector of stimulus-initiated arachidonic acid release (13, 14). COX-1 is a ubiquitously and constitutively expressed isoform that is postulated to have housekeeping functions, and COX-2 is an inducible isoform implicated in inflammatory responses and cell growth/differentiation regulation (for review, see Ref. 15). Several lines of evidence have shown that the two COX isoforms regulate different phases of prostanoid biosynthesis, rather than exerting overlapping functions, in activated cells (16 –18). For instance, COX-1, but not COX-2, functions in TX generation by activated platelets (16) and immediate PGD2 generation by IgE/Ag-activated mast cells (17, 18), whereas de novo induced COX-2 is an absolute requirement for delayed prostanoid generation extending over several hours elicited by proinflammatory stimuli, even though COX-1 coexists in the same cell (17–21). In addition to their different subcellular localizations (4) and their different substrate concentration requirements (22), their selective coupling with distinct PLA2 enzymes (19, 23, 24), which defines the specific intracellular arachidonate-presenting route, has been recently proposed to account for how each COX isoform is selectively used in a particular phase of cell activation, although only a limited amount of evidence has yet become available. Terminal prostanoid synthases catalyze the conversion of PGH2 to each bioactive prostanoid. TX synthase, abundantly expressed in platelets, is a microsomal 60-kDa protein with the highest identity with cytochrome P450 isozymes (25). Little is unknown about 0022-1767/98/$02.00

The Journal of Immunology the properties of PGE2 synthase, whose activity reported to date is glutathione dependent and is detected in both soluble and membrane-bound fractions (26). Since the production of PGE2 is often induced by several stimuli in a variety of cells and is coupled with the induction of COX-2 (8, 18, 20), one might speculate that the COX-2-dependent pathway is more selectively linked to PGE2 synthase. Indeed, Harada et al. (27) reported that PGE2, but not TX or PGI2, accumulation was suppressed by COX-2 inhibitors in rat carrageenan-induced pleurisy. Macrophages are known to produce PGE2 via the COX-2-dependent pathway in response to proinflammatory cytokines or bacterial LPS in long term cultures (20). Furthermore, we recently found that rat peritoneal macrophages have the capacity to metabolize exogenous arachidonic acid to TX via COX-1 and to PGE2 via COX-2 before and after culture with LPS, respectively (28), thereby formulating the hypothesis that there is a preferential, phase-specific correlation between the two COX isoforms and the downstream respective terminal PG synthases. We now provide evidence that rat peritoneal macrophages exhibit three different PG biosynthetic responses from endogenous arachidonic acid, namely, constitutive immediate, inducible immediate (priming), and delayed responses, in which the initial (cPLA2 and sPLA2), immediate (COX-1 and COX-2), and terminal (TX and PGE2 synthases) enzymes display differential functional coupling. Thus, the A23187-induced constitutive immediate response is mediated by cPLA2/COX-1/TX synthase; the LPS-primed, A23187-induced immediate response is mediated by cPLA2/COX-2/PGE2 synthase; and the LPS-initiated delayed response is mediated by cPLA2 and sPLA2/COX-2/PGE2 synthase.

Materials and Methods Materials The type IIA sPLA2 inhibitor thielocin A1 (29) was donated from Shionogi Pharmaceutical Co. (Osaka, Japan). The COX-2 inhibitor NS-398 (30) was a gift from Taisho Pharmaceutical Co. (Tokyo, Japan). Rabbit antisera to COX-1 and COX-2 were provided by Dr. W. L. Smith (Michigan State University, Lansing, MI). Mouse cDNA probe for COX-2 was a gift from J. Trzaskos (Merck-DuPont, Wilmington, DE), rabbit antiserum to human cPLA2 was obtained from J. D. Clark (Genetics Institute, Cambridge, MA), and rat type V sPLA2 cDNA was obtained from J. A. Tischfield (Indiana University School of Medicine, Indianapolis, IN). Rabbit antiserum for rat type IIA sPLA2 (31), rat type IIA sPLA2 cDNA (32), and rat cPLA2 cDNA (8) were described previously. Recombinant rat type IIA sPLA2 and mouse cPLA2 were purified from Sf9 insect cells (Invitrogen, San Diego, CA) using the baculovirus system (PharMingen, San Diego, CA). Bacterial LPS (Escherichia coli O111: B4) and calcium ionophore A23187 were purchased from Sigma Chemical Co. (St. Louis, MO). Arachidonic acid, PGH2, AACOCF3, and enzyme immunoassay kits for PGE2 and TXB2 were obtained from Cayman Chemical Co. (Ann Arbor, MI).

Preparation and activation of rat peritoneal macrophages Adherent macrophages were prepared and activated as previously reported (28). Briefly, 5% (w/v) bacto-peptone (Life Technologies, Grand Island, NY) in saline (5 ml/100 g body weight) was injected i.p. into SpragueDawley rats (Japan SLC, Hamamatsu, Japan), and peritoneal exudate cells were collected on day 4 by washing the cavity with 20 ml of ice-cold Ca21and Mg21-free HBSS. The cells were washed twice and plated onto 24well plastic plates or 60-mm diameter plastic dishes (Corning, Corning, NY) at a density of 4.8 3 105 cells/cm2 in RPMI 1640 medium (Life Technologies) containing 10% (v/v) FBS (Atlanta Biologics, Atlanta, GA). After 2 h of incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% air, nonadherent cells were removed by rinsing. Then RPMI 1640 medium containing 10% FBS was added to the adherent cells, and they were used as macrophages. The viability of macrophages was .95% as assessed by trypan blue dye exclusion. Macrophages were incubated in the medium with or without 10 mg/ml LPS up to 24 h. After incubation, supernatants were collected, and PGE2 and TXB2 levels were measured by enzyme immunoassay kits. In the experiments for A23187 stimulation, cells cultured for various periods with LPS were washed and then challenged with 1 mM A23187 in RPMI 1640

2975 medium containing 1% FBS for up to 60 min; PGE2 and TXB2 levels in the supernatants were then measured. The ability of macrophages to yield COX metabolites from exogenous arachidonic acid was evaluated as previously reported (28). Briefly, macrophages cultured in 35-mm diameter dishes at density of 4.5 3 106 cells/dish with or without LPS were washed and incubated in HBSS containing 1% FBS and 30 mM arachidonic acid for 40 min; PGE2 and TXB2 accumulated in the supernatants were subsequently measured by enzyme immunoassay kits.

Assay of sPLA2 and cPLA2 activities Macrophages incubated with or without LPS were washed with ice-cold PBS and then scraped off the dishes. The cells were disrupted by sonication (10 s, three times, 1-min interval) in 1 ml of 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl (TBS), 1 mM EDTA, 50 mg/ml leupeptin (Peptide Institute, Osaka, Japan), 1.5 mM pepstatin A (Sigma Chemical Co.), 1 mM PMSF (Sigma Chemical Co.), and 1 mM okadaic acid (Wako Chemicals, Osaka, Japan) with a Branson Sonifer model 250 (Branson Co., Danbury, CT) and centrifuged at 1700 3 g for 10 min at 4°C. The resultant supernatants were used as the enzyme source. For the measurement of sPLA2, 5 ml of 1 N H2SO4 was added to the lysates, and incubation was conducted for 3 h at 4°C, under which conditions sPLA2 was efficiently extracted, while cPLA2 was inactivated (33). For the measurement of cPLA2 activity, 0.5 mM DTT (Sigma Chemical Co.) was added to the PLA2 assay mixture, by which treatment only sPLA2 was inactivated (34). After incubation for 20 min with 2 mM 1-acyl-2-[14C]arachidonoyl-snglycero-3-phosphatidylethanolamine (DuPont-New England Nuclear, Boston, MA) in 0.25 ml of 0.1 M Tris (pH 9.0) in the presence of 4 mM CaCl2, liberated [14C]arachidonic acid was extracted by Dole’s extraction procedure (35).

SDS-PAGE/immunoblotting Macrophages were lysed in PBS containing 0.1% SDS at 4 3 107 cells/ml, applied to SDS-polyacrylamide gels, and electrophoresed as previously reported (36). Then proteins were electroblotted onto nitrocellulose membranes with a semidry blotter (MilliBlot-SDE system, Millipore Corp., Bedford, MA). The membranes were blocked for 1 h in 10 mM TBS containing 0.1% Tween-20 (TBS-T) and 3% skim milk. After washing the membranes with TBS-T, Ab against cPLA2, type IIA sPLA2, COX-1, or COX-2 was added at a 1/4500, 1/3500, 1/3500, or 1/7,000 dilution, respectively, in TBS-T and incubated for 2 h. After washing the membranes with TBS-T, horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed, San Francisco, CA) was added at a 1/7000 dilution in TBS-T and incubated for 1 h. After a final wash with TBS-T, protein bands were visualized with an ECL Western blot analysis system (Amersham, Arlington Heights, IL).

RNA blotting Total RNA extracted with TRIzol reagent (Life Technologies) from macrophages were electrophoresed in formaldehyde/agarose gels, transferred to Immobilon-N (Millipore), and hybridized with cDNA probes for type IIA sPLA2, cPLA2, COX-2, and b-actin labeled with [32P]dCTP (DuPontNew England Nuclear) by a random priming labeling system (Takara Biomedical, Kyoto, Japan). After overnight hybridization, the membranes were washed under high stringency conditions as described previously (36), and RNA bands were visualized by autoradiography with Kodak X-AR films (Eastman Kodak, Rochester, NY).

Reverse transcription-PCR The type IIA sPLA2 primers used were 59-ATCCCATCCAA GAGAGCTGA-39 and 59-CCTGCTTCTAGGGTTGGAGA-39, the type V sPLA2 primers used were 59-ATCCATCCTTCCTGTGTTGC-39 and 59TCAGGCAGTAGACCAGCTTC-39, and the glyceraldehyde-3-phosphate dehydrogenase primers used were 59-AGACAGCCGCATCTTCTTGT-39 and 59-CCACAGTCTTCTGAGTGGCA-39. RT-PCR was conducted using an RNA PCR kit (AMV, version 2, Takara Biomedical), according to the manufacturer’s instructions, using 1 mg of total RNA from macrophages as a template. Equal amounts of each RT product were amplified by PCR with Taq polymerase (Takara Biomedical) for 30 cycles consisting of 30 s each at 94, 55, and 72°C. The amplified cDNA fragments were resolved electrophoretically on 1.5% (w/v) agarose gels and visualized by ethidium bromide.

Recombinant expression of type V sPLA2 Approximately 1 mg of rat type V sPLA2 cDNA (37) subcloned into pCR3.1 (Invitrogen) was mixed with 5 ml of CellFection (Life Technologies) in 200 ml of Opti-MEM medium (Life Technologies) for 15 min and

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REGULATION OF PROSTANOID GENERATION IN RAT MACROPHAGES

FIGURE 1. LPS-stimulated delayed prostanoid synthesis in rat peritoneal macrophages. Peritoneal macrophages were incubated in 24-well plates at a density of 9 3 105 cells/well in the presence (closed symbols) or the absence (open symbols) of 10 mg/ml of LPS for the indicated time periods, after which time the amounts of PGE2 (A) and TXB2 (B) secreted into the supernatants were determined as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. ** Indicates a statistically significant difference between stimulated and nonstimulated cells at P , 0.01.

then added to human embryonic kidney 293 cells (Japanese Cancer Research Resources Bank, Tokyo, Japan) that had attained 60 to 80% confluence in six-well plates and had been supplemented with 800 ml of OptiMEM. After incubation for 6 h, the medium was replaced with 2 ml of RPMI 1640 medium containing 10% FBS, the cells were cultured overnight, and the medium was replaced again with 2 ml of fresh medium and cultured for an additional 2 days. The supernatant was used as a source of type V sPLA2.

Assay of terminal prostanoid synthase activities Terminal prostanoid synthase activities in cell lysates were measured by assessment of conversion of PGH2 to PGE2 and TXB2 as previously reported (28). Macrophages, seeded into 150-mm diameter plastic plates (Corning) at a density of 4.8 3 105 cells/cm2 in RPMI 1640 medium containing 10% FBS, were incubated with or without 10 mg/ml LPS for up to 24 h. After incubation, the cells were scraped off the dish and disrupted by sonication (10 s, three times, 1-min interval) in 400 ml of 2 M Tris-HCl (pH 8.0). After centrifugation of the sonicates at 1700 3 g for 10 min at 4°C, the supernatants were used as the enzyme source. For assessment of PGE2 synthase activity, an aliquot of each lysate (100 mg protein equivalents) was incubated with 2 mg of PGH2 for 30 s at 24°C in 0.1 ml of 1 M Tris-HCl (pH 8.0) containing 2 mM glutathione (Sigma Chemical Co.). The reaction was terminated by the addition of 100 mM FeCl2. The reaction mixtures were left at room temperature for 15 min, mixed with 13,14dihydro-15-dehydro-PGF2a as an internal standard, adjusted to pH 3.0, further mixed vigorously with 3 ml of ether and 0.5 g of sodium sulfate, and centrifuged for 10 min at 430 3 g at 4°C. Extraction was repeated twice, and the pooled ether phase was condensed by evaporation. The resulting residue was reacted with 9-anthryldiazo methane and was analyzed by HPLC as described previously (38). TX synthase activity was measured by incubating an aliquot of lysate (100 mg protein equivalents) with 2 mg of PGH2 for 30 s at 24°C in a 0.1 ml of 1 M Tris-HCl (pH 7.4). After the reaction had been stopped by the addition of FeCl2, TXB2 was quantified using an enzyme immunoassay kit.

Statistical analysis Data were expressed as the mean 6 SEM of more than three independent experiments. Statistical analysis was performed using Student’s t test.

Results Delayed prostanoid production by LPS-stimulated macrophages When rat peritoneal macrophages were cultured for up to 24 h with LPS in medium containing 10% FBS, a gradual accumulation of PGE2 in the supernatants occurred after a lag phase of 3 h, reaching 9.8 6 1.7 ng/well (fivefold increase over that in control cells) at 12 h and then a plateau by 24 h (Fig. 1A). On the other hand, accumulation of TXB2 was less obvious than that of PGE2 and did not depend on LPS stimulation (Fig. 1B). In addition, PGD2 was produced only minimally (,1.5 ng/well at 12 h) and did not change appreciably following LPS stimulation (data not shown). These results confirm that rat peritoneal

macrophages produce PGE2 predominantly in response to LPS. This contrasts with our previous observation that they had the ability to produce TXB2 when excess arachidonic acid was supplied exogenously (28). Immediate prostanoid production by A23187-stimulated macrophages with or without LPS priming When quiescent macrophages were stimulated with A23187, they produced TXB2 predominantly, reaching 10.6 6 1.1 ng/well, and only a small amount of PGE2, reaching 1.1 6 0.2 ng/well, by 60 min (Fig. 2A). When replicate cells were cultured with LPS for 12 h, washed, and then stimulated with A23187 for up to 60 min, PGE2 production increased sevenfold to reach 4.9 6 0.2 and 7.1 6 1.9 ng/well at 10 and 60 min, respectively, whereas TXB2 generation was about one-third of the amount observed in unprimed cells (Fig. 2B). Replicate cells cultured for 24 h with LPS and then activated with A23187 produced smaller amounts of PGE2 and TXB2 than those cultured for 12 h with LPS (Fig. 2C). Thus, priming with LPS changed the cells’ capacity to produce terminal products in response to a secondary Ca21-mobilizing stimulus in terms of PG species and amounts, raising the question of what mechanisms are involved in these events. Expression of PLA2 and COX enzymes Expression of proteins and transcripts for type IIA sPLA2, cPLA2, and two COX isozymes were assessed by immunoblotting (Fig. 3A) and RNA blotting (Fig. 3B). Type IIA sPLA2 protein and mRNA were abundantly expressed in cells before culture and declined gradually over the culture period regardless of whether the cells were treated with LPS (Fig. 3, A and B). The level of cPLA2 protein increased significantly 6 to 12 h after treatment with LPS (Fig. 3A), without being accompanied by a concomitant change in its mRNA expression (Fig. 3B), reflecting post-transcriptional regulation of cPLA2 expression as reported previously (35, 37). The changes in expression of type IIA sPLA2 and cPLA2 proteins, visualized by immunoblotting (Fig. 3A), correlated to the changes in their enzyme activities, in which sPLA2 activity decreased over the culture period (Fig. 4A) and cPLA2 activity significantly increased at 12 h in LPS-stimulated cells (Fig. 4B). COX-1 protein was constitutively expressed and was not altered by treatment with LPS (Fig. 3A). COX-2 protein and mRNA were minimally expressed before culture, but their levels increased dramatically 6 to 24 h after culture with LPS (Fig. 3, A and B). RT-PCR analysis was conducted to assess the expression of type V sPLA2, which has been reported to be involved in arachidonate metabolism in the mouse macrophage-like cell line P388D1 (13)

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FIGURE 3. Changes in expression of cPLA2, type IIA sPLA2, COX-1, and COX-2. Lysates and total RNA were prepared from macrophages incubated in 60-mm dishes at a density of 1.35 3 107 cells/dish with or without 10 mg/ml of LPS for the indicated time periods and were processed for immunoblot (A) and RNA blot (B) analyses, respectively, as described in Materials and Methods. Representative results of three independent experiments are shown. FIGURE 2. A23187-stimulated immediate prostanoid synthesis in LPSprimed peritoneal macrophages. Peritoneal macrophages were incubated in 24-well plates at a density of 9 3 105 cells/well in the absence (open symbols) or the presence (closed symbols) of 10 mg/ml of LPS for 0 (A), 12 (B), and 24 h (C). Then the cells were washed with PBS and incubated in medium for the indicated time periods with (triangles) or without (circles) 1 mM A23187 as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. * and **, Indicate statistically significant differences from the control value at P , 0.05 and P , 0.01, respectively.

and certain cultured mast cells (14). Whereas type V sPLA2 transcript was abundantly expressed in rat heart, used as a positive control (37), its expression level in rat macrophages was low in unstimulated cells and was barely detectable in cells incubated for 12 h with LPS (Fig. 5). By comparison, type IIA sPLA2 was readily detected under the same conditions, with higher level of expression before LPS treatment (Fig. 5), consistent with the data shown in Figure 3. Type V sPLA2 mRNA was undetectable by RNA blotting (data not shown). PLA2 and COX enzymes involved in delayed prostanoid generation To assess which PLA2 enzymes are involved in delayed PGE2 production by LPS, we examined the effects of inhibitors and Ab against these PLA2s. First, we confirmed the specificity of the cPLA2 inhibitor, AACOCF3, and that of the type IIA sPLA2 inhibitor, thielocin A1, toward these PLA2s by assaying their enzymatic activities in the presence of these inhibitors (Table I). AA

COCF3 suppressed cPLA2, but not sPLA2, activity in cell lysates. Conversely, thielocin A1 suppressed sPLA2, but not cPLA2, activity in the lysates. Experiments using recombinant PLA2 enzymes revealed that AACOCF3 inhibited cPLA2, but not sPLA2, enzymes, whereas thielocin A1 inhibited type IIA sPLA2 specifically (Fig. 6). These results suggest that AACOCF3 and thielocin A1, at the concentrations used, are selective to cPLA2 and type IIA sPLA2, respectively, and that type IIA sPLA2 is the major sPLA2 isozyme present in rat peritoneal macrophages. We then examined the effects of AACOCF3 and thielocin A1 on LPS-stimulated delayed PGE2 generation. As shown in Figure 7A, each inhibitor markedly suppressed the PGE2 generation assessed at 12 h in a dose-dependent manner. Conversion of exogenous arachidonic acid to PGE2 by these cells was not suppressed by these inhibitors (data not shown), indicating that they indeed acted on the respective PLA2s and not on the downstream COXs and PGE2 synthase. Furthermore, the addition of an Ab raised against type IIA sPLA2, which neutralized type IIA sPLA2 but not type V sPLA2 and cPLA2 (Fig. 6), attenuated delayed PGE2 generation to an extent comparable to that achieved with thielocin A1 (Fig. 7A). LPS-induced PGE2 generation was suppressed markedly by the COX-2 inhibitor NS-398 (Fig. 7A), but was not suppressed by pretreatment of the cells with aspirin, which inactivated the preexisting COX-1 (data not shown), confirming its dependence on the inducible COX-2. These results suggest that delayed PGE2 generation induced by LPS requires both cPLA2 and type IIA sPLA2, which are functionally coupled with COX-2.

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FIGURE 4. Changes in the activities of cPLA2 and sPLA2. Cells incubated with (closed symbols) or without (open symbols) LPS for the indicated time periods were lysed, and cPLA2 (A) and sPLA2 (B) activities were assayed as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. * Indicates a statistically significant difference between stimulated and nonstimulated cells at p , 0.05.

PLA2 and COX enzymes involved in immediate prostanoid generation Next, we examined the effect of AACOCF3, thielocin A1, antitype IIA sPLA2 Ab, and NS-398 on A23187-induced TXB2 generation by quiescent cells (0 h) and PGE2 generation by LPSprimed cells (12 and 24 h; Table II). Each inhibitor was added 10 min before stimulation with A23187. AACOCF3 markedly reduced both TXB2 and PGE2 production, whereas both thielocin A1 and anti-type IIA sPLA2 Ab failed to do so under any condition. Thielocin A1 and anti-type IIA sPLA2 Ab did not reduce A23187induced PGE2 generation appreciably even after 12-h pretreatment of the cells (data not shown). These results imply that cPLA2 is the dominant enzyme involved in A23187-induced immediate prostanoid production, even when type IIA sPLA2 coexists and is enzymatically active (Figs. 3A and 4A). PGE2 production by LPSprimed cells was suppressed markedly by NS-398, whereas TXB2 generation by quiescent cells was insensitive to it (Table II). Collectively, arachidonic acid released by cPLA2 after A23187 stimulation was mainly converted to TXB2 by COX-1 in quiescent cells and to PGE2 by COX-2 in LPS-primed cells. Assessment of terminal synthases Delayed PGE2 generation after LPS stimulation proceeded for 6 to 12 h and tended to terminate thereafter (Fig. 1). Furthermore, the priming effect of LPS on A23187-induced PGE2 generation reached a peak at 12 h and declined at 24 h (Fig. 2). Despite these findings, COX-2 protein expression continued to increase over 24 h (Fig. 3A), suggesting that the PGE2-producing capacity was determined not only by the COX-2 level but also by some other regulating steps. The expression of cPLA2, which increased at 12 h

and returned to nearly the basal level at 24 h after LPS stimulation (Figs. 3A and 4B), might be responsible for this event. To address a regulating step other than one involving cPLA2, we measured the conversion of exogenous arachidonic acid to prostanoids by intact cells, thereby allowing bypassing of the PLA2 reaction. When macrophages cultured with LPS for 12 h were incubated for an additional 40 min with exogenous arachidonic acid, they produced large amounts of PGE2, reaching 105.8 6 15.9 ng/dish, whereas cells cultured for 24 h with LPS produced only 23.5 6 4.9 ng/dish of PGE2 from exogenous arachidonic acid, corresponding to only 25% of that produced by cells primed for 12 h with LPS (Fig. 8A). Since this reaction involves two sequential biosynthetic steps, COX and terminal PGE2 synthase, we next sought to determine whether the PGE2 synthase level could account for the temporal change in the PGE2-producing capability of the cells. The activity of terminal PGE2 synthase in lysates increased approximately 20-fold in cells primed for 12 h with LPS compared with that of lysates obtained before LPS priming (Fig. 8B). This increase in PGE2 synthase activity at 12 h was sensitive to cycloheximide and actinomycin D (data not shown), implying its de novo synthesis (28). Notably, PGE2 synthase activity in lysates of cells primed for 24 h with LPS was decreased by 60% compared with that in cells primed for 12 h. Thus, changes in PGE2 synthase activity after LPS treatment correlated with those in A23187-induced PGE2 generation (Fig. 2) and the conversion of exogenous arachidonic acid to PGE2 (Fig. 8A). These results Table I. Effects of AACOCF3 and thielocin A1 on PLA2 activities in cell lysatesa PLA2 Activity in Cell Lysates (pmol/min/mg protein) Inhibitors

Control AACOCF3 0.1 mM 1 mM 10 mM Thielocin A1 0.3 mM 3 mM 30 mM FIGURE 5. Expression of type V sPLA2 as assessed by RT-PCR. Total RNA, prepared from cells before (A) and 12 h after (B) stimulation with LPS, were subjected to RT-PCR analysis for type V sPLA2, type IIA sPLA2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression as described in Materials and Methods.

sPLA2

cPLA2

60.1 6 8.2

25.1 6 8.4

64.6 6 10.1 58.3 6 5.6 49.2 6 6.9

27.2 6 1.9 10.3 6 1.1** 9.1 6 2.2**

43.2 6 3.9 12.3 6 2.1** 4.8 6 0.39**

26.8 6 10.1 25.5 6 5.6 23.4 6 4.9

a For measurement of sPLA2 and cPLA2 activities, cell lysates were prepared from quiescent and LPS-stimulated (12 h) cells, respectively. Each PLA2 activity was assayed under different conditions as described in Materials and Methods. AACOCF3 and thielocin A1 were added to the reaction mixture 10 min before measurement of the enzymatic activity. Values are expressed as the mean 6 SE of three independent experiments. **Statistically significant difference between stimulated and nonstimulated cells at p , 0.01.

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FIGURE 6. Effects of AACOCF3, thielocin A1, and anti-type IIA sPLA2 Ab on enzymatic activities of recombinant cPLA2 (open circles), type IIA sPLA2 (closed circles), and type V sPLA2 (closed triangles). Equivalent units of each PLA2 were preincubated for 10 min with the indicated concentrations of inhibitors or Ab and then taken for PLA2 enzyme assay in their continued presence. Values are expressed as the residual activity, which is calculated by the formula (PLA2 activity in the presence of inhibitors/PLA2 activity in the absence of inhibitors) 3 100%.

strongly suggest that both inducible COX-2 and inducible terminal PGE2 synthase are required for optimal PGE2 generation. TX synthase activity tended to decrease over 12 to 24 h of culture (Fig. 8B) in parallel with changes in the TX-producing ability of cells after stimulation with A23187 (Fig. 2) and exposure to exogenous arachidonic acid (Fig. 8A).

Discussion We have shown that rat peritoneal macrophages exhibit three different PG biosynthetic responses from endogenous arachidonic acid. Quiescent cells stimulated for 10 to 60 min with A23187 produced TXB2 in preference to PGE2 (constitutive immediate response), cells cultured with LPS for 3 to 24 h produced PGE2 gradually (delayed response), and cells cultured for 12 h with LPS and then stimulated for 10 to 60 min with A23187 produced PGE2 in preference to TXB2 (induced immediate response). These results raise the possibility that different PG biosynthetic enzymes are preferentially coupled to provide a phase-specific prostanoid synthesis. Indeed, we demonstrated that the constitutive COX-1 and the inducible COX-2 are preferentially coupled to distinct terminal prostanoid synthases and that a Ca21-mobilizing stimulus activates cPLA2, which can couple to the constitutive COX-1/TX synthase and to the inducible COX-2/PGE2 synthase in quiescent and LPS-primed cells, respectively. Furthermore, in the LPS-ini-

FIGURE 7. Effects of AACOCF3, thielocin A1, anti-type IIA sPLA2 Ab, and NS-398 on delayed prostanoid synthesis in LPS-stimulated macrophages. Peritoneal macrophages were incubated in 24-well plates at a density of 9 3 105 cells/well with or without (control) 10 mg/ml of LPS in the presence of AACOCF3, thielocin A1, Ab to type II sPLA2, NS-398, control IgG, or vehicle for 12 h. The amount of PGE2 secreted into the supernatants was determined as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. * and **, Indicate statistically significant differences from the control value at P , 0.05 and P , 0.01, respectively.

tiated delayed response, sPLA2, in concert with cPLA2, serves as an arachidonic acid-providing enzyme to the inducible COX-2/ PGE2 synthase. These three segregated biosynthetic pathways are reminiscent of mouse bone marrow-derived mast cells stimulated with a cytokine triad, in which cPLA2 and COX-1 couple to provide immediate PGD2 generation, inducible sPLA2 and COX-2 are essential for delayed PGD2 generation, and priming for increased IgE-dependent immediate PGD2 generation is accompanied by a concordant increase in the expression of cPLA2, COX-1, and PGD2 synthase, with COX-1 and COX-2 showing no overlapping function in each phase (17, 36, 39, 40). Constitutive immediate response When rat peritoneal macrophages were stimulated with A23187, rapid production of TXB2 occurred, whereas there was minimal generation of PGE2. This finding is compatible with the observation that replicate cells exposed to exogenous arachidonic acid produced a large amount of TXB2 with minimal PGE2 (28) and that the activity of TX synthase in cell lysates was higher than that of PGE2 synthase. Pharmacologic studies revealed the involvement of cPLA2 and COX-1 in A23187-induced TXB2 production, in line with the fact that COX-1 was the dominant COX isoform expressed in these cells. A23187 rapidly increases intracellular Ca21 concentrations, which is likely to promote the translocation of cPLA2 to the perinuclear or endoplasmic reticular membranes where it liberates arachidonic acid (3). We also observed that cPLA2 activity was increased about threefold in A23187-stimulated macrophages (data not shown), most likely reflecting its phosphorylation by mitogen-activated protein kinases (2). In contrast, the involvement of type IIA sPLA2 in the immediate arachidonic acid release was negligible, even though it is abundantly expressed. The association of cPLA2, but not type IIA sPLA2, with the immediate PG biosynthesis has been observed in a number of cell systems, such as thrombin-activated human platelets (41), norepinephrine-stimulated rabbit aortic smooth muscle cells (42), A23187-stimulated human monocytes (43), and acetylcholinestimulated rabbit coronary endothelial cells (44). Our recent finding that cytokine-stimulated delayed, but not A23187-induced immediate, arachidonic acid release was augmented in a CHO-K1 cell transformant overexpressing type IIA sPLA2 (8) is also consistent with the present results. Thus, although type IIA sPLA2 could augment the immediate PG biosynthesis when added exogenously in excess amounts (7, 45– 47), it might not mediate it at endogenously expressed levels. Recently, a novel type V sPLA2 has been shown to be involved in immediate prostanoid generation in several cells (13, 14). Dennis and colleagues (13, 23) demonstrated that in the mouse macrophage cell line P388D1, which expresses type V, but not IIA,

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Table II. Effect of AACOCF3, thielocin A1, Ab to type IIA sPLA2, and NS-398 on A23187-induced prostanoid synthesisa Prostanoids (ng) LPS-Priming Period (h)

Metabolite

Control

AACOCF3

Thielocin A1

Anti-Type IIA sPLA2 Ab

NS-398

0 12 24

TXB2 PGE2 PGE2

15.9 6 3.9 12.3 6 2.1 3.8 6 0.5

3.4 6 0.7** 1.2 6 0.1** 1.2 6 0.2**

16.7 6 2.8 11.5 6 3.1 3.9 6 0.4

17.2 6 2.8 11.5 6 2.1 3.9 6 0.4

12.1 6 0.2 2.9 6 0.3** 1.3 6 0.2**

a Peritoneal macrophages, incubated with LPS (10 mg/ml) for 0, 12, or 24 h, were washed, pretreated for 10 min with AACOCF3 (10 mM), thielocin A1 (30 mM), anti-sPLA2 Ab (10 mg/ml), or NS-398 (1 mM), and incubated for an additional 30 min with A23187 (1 mM). TXB2 (0 h) and PGE2 (12 and 24 h) released into the supernatants were quantified as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. **Statistically significant difference from the value for the control at p , 0.01.

sPLA2, PGE2 generation elicited by platelet-activating factor following LPS priming (therefore probably corresponding to the induced immediate response) is attenuated by the type V sPLA2directed antisense nucleotide and that prior activation of cPLA2 is necessary for type V sPLA2 to act. Similarly, Herschman and colleagues (14, 19) showed that both cPLA2 and type V sPLA2 are required for IgE-dependent immediate PGD2 generation by several mast cells. Therefore, implication of type V sPLA2 as the sPLA2 isoform in immediate prostanoid biosynthesis is plausible, and several sPLA2 inhibitors originally developed on the basis of type IIA sPLA2 inhibition might have exhibited their inhibitory actions on immediate eicosanoid biosynthesis via inhibition of type V sPLA2. Nonetheless, reagents directed to sPLA2 that we used here, thie-

FIGURE 8. Changes in expression of COX and terminal synthases in LPS-stimulated macrophages. A, Peritoneal macrophages, incubated in the presence or the absence of 10 mg/ml of LPS for 0, 12, and 24 h, were washed and then incubated with exogenous arachidonic acid (30 mM) for 40 min, and the amounts of PGE2 (open columns) and TXB2 (hatched columns) produced and secreted into the supernatants were determined. B, PGE2 (open columns) and TX (hatched columns) synthase activities at each time point were determined as described in Materials and Methods. Values are expressed as the mean 6 SE of three independent experiments. * and **, Indicate statistically significant differences from the corresponding 0 h value at P , 0.05 and P , 0.01, respectively.

locin A1 and anti-type IIA sPLA2 Ab, neither inhibited the activity of recombinant type V sPLA2 in vitro nor suppressed the immediate response in vivo. A conclusion concerning whether type V sPLA2 is involved in immediate prostanoid biosynthesis in rat peritoneal macrophages, even though its expression level is low, must await the future development of the type V sPLA2-selective inhibitor. Delayed response Macrophages cultured with LPS for up to 24 h produced PGE2 gradually, accompanied by the induction of COX-2 expression over the culture period. The cPLA2 expression also increased and peaked at 12 h after treatment with LPS, whereas sPLA2 expression declined gradually, prompting us to speculate that induced cPLA2 could provide arachidonic acid to inducible COX-2. Indeed, the cPLA2 inhibitor AACOCF3 suppressed delayed PGE2 production considerably. In addition, the type IIA sPLA2 inhibitor thielocin A1 as well as the Ab to type IIA sPLA2 suppressed this PGE2 production markedly, suggesting that type IIA sPLA2 also plays a crucial role in LPS-induced delayed PGE2 production despite the decreasing type IIA sPLA2 expression over the culture. Recent studies have shown that each PLA2 isozyme could regulate delayed PG generation by use of inhibitors, overexpression, and antisense oligonucleotides. The participation of cPLA2 in the delayed response has been demonstrated in cells such as LPSstimulated human monocytes (48), IgE-activated mouse mast cells (19), TNF-stimulated mouse fibroblasts L929 (49), and IL-1- and TNF-stimulated mouse osteoblastic cells (50); that of sPLA2 has been implicated in cells such as IL-1-stimulated rat mesangial cells (7), TNF-stimulated human endothelial cells (46), and TNF-stimulated rat hepatocytes BRL-3A (51), in all of which type IIA sPLA2 expression is dramatically induced, as well as in IL-1-stimulated CHO-K1 cells overexpressing type IIA sPLA2 (8). Our present study provides evidence that both cPLA2 and sPLA2 are required for the delayed PG generation. In rat mesangial cells, type IIA sPLA2 is reported to activate mitogen-activated protein kinases (52); therefore, a possible pathway is that sPLA2 leads to activation of cPLA2, which, in turn, links to COX-2-dependent PGE2 generation, probably through an as yet unidentified Ca21independent cPLA2 activation pathway. Alternatively, as has been recently suggested (19, 23), functional cPLA2 may be essential for sPLA2 to function. In this case, cPLA2 activation might modify the structure of the plasma membrane, rendering it susceptible to sPLA2. The sPLA2 then releases arachidonic acid, which is, in turn, incorporated and delivered to intracellular COX-2. Nonetheless, this kind of cross-talk between the two Ca21-dependent PLA2s may occur in various, if not all, cell types and could account for the implication of both enzymes in the delayed response. The fact that PGE2 is the only prostanoid produced in the delayed response suggests that the profile of PG biosynthesis is also

The Journal of Immunology determined at the level of terminal synthases. The measurement of terminal PGE2 synthase activity revealed that this enzyme was inducible, which increased 20-fold after 12 h of culture with LPS. In contrast, no increase in TX synthase or PGD2 synthase (data not shown) activity was found, implying the selective up-regulation of the PGE2 biosynthetic pathway. In our preliminary study, the induction of PGE2 synthase was also observed in several other cells stimulated with proinflammatory stimuli (unpublished observations). Thus, the cooperative induction of COX-2 and PGE2 synthase appears to determine the capacity of cells to produce PGE2 optimally and may explain a mechanism by which COX-2 is selectively used in PGE2 generation in the delayed response. Induced immediate response In contrast to quiescent cells, cells primed with LPS and stimulated with A23187 produced PGE2 in marked preference to TXB2. Pharmacologic studies revealed the involvement of cPLA2 and COX-2, rather than COX-1, in A23187-induced PGE2 production by LPSprimed cells. Since LPS increased the expression of cPLA2, COX-2, and PGE2 synthase at 12 h, functional coupling of these coinduced enzymes appears to contribute to rapid and selective production of PGE2 over TXB2 in response to a Ca21-mobilizing stimulus. More importantly, after the induced immediate PGE2 generation reached a peak 12 h after priming with LPS, it declined to nearly the basal level by 24 h regardless of the sustained high level of COX-2 expression. This again implies the importance of the expression level of PGE2 synthase, which correlated with the cells’ capacity to produce PGE2 in the induced immediate response to A23187 and to metabolize exogenous arachidonic acid to PGE2. Specific inhibition of inducible PGE2 synthase may, therefore, be an important pharmacologic target for several modes of PGE2 generation in chronic and acute inflammatory responses. Furthermore, since PGE2 is often produced by several cells without priming via the COX-1 pathway (18), we assume that, as in the case of PLA2 and COX isozymes, there might be constitutive and inducible PGE2 synthase isoforms that exhibit differential coupling with the upstream enzymes and play distinct roles in the PGE2 biosynthetic pathway.

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9.

10.

11.

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13.

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15. 16.

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23.

Acknowledgments We thank Drs. J. D. Clark (Genetics Institute), W. L. Smith (Michigan State University), J. Trzaskos (Merck-DuPont), and J. A. Tischfield (Indiana University School of Medicine) for providing Abs and/or cDNA for COX-1, COX-2, sPLA2s, and cPLA2. Thielocin A1 and NS-398 were generously provided by Shionogi Pharmaceutical Co. and Taisho Pharmaceutical Co., respectively.

24.

25.

26.

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