Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages

Biochimica et Biophysica Acta 1530 (2001) 67^76 www.elsevier.com/locate/bba

Group X secretory phospholipase A2 induces potent productions of various lipid mediators in mouse peritoneal macrophages Akihiko Saiga, Yasuhide Morioka, Takashi Ono, Kazumi Nakano, Yoshikazu Ishimoto, Hitoshi Arita, Kohji Hanasaki * Shionogi Research Laboratories, ShionogipCo., Ltd., 12-4 Sagisu, 5-Chome, Fukushima-ku, Osaka 553-0002, Japan Received 20 July 2000; received in revised form 12 October 2000; accepted 19 October 2000

Abstract We have previously shown the expression of group X secretory phospholipase A2 (sPLA2 -X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA2 -X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA2 -X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA2 s. In resting macrophages, sPLA2 -X elicited a modest production of prostaglandin E2 and thromboxane A2 . After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA2 -X-treated macrophages, which was completely blocked by the addition of either the specific sPLA2 inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA2 -X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA2 -X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Phospholipase A2 ; Cyclooxygenase; Arachidonic acid; Eicosanoid; Lysophosphatidylcholine; Lipid mediator

1. Introduction

Abbreviations: PLA2 , phospholipase A2 ; cPLA2 , group IV cytosolic PLA2 ; sPLA2 , secretory PLA2 ; PG, prostaglandin ; TXA2 , thromboxane A2 ; TXB2 , thromboxane B2 ; COX, cyclooxygenase; sPLA2 -IB, IIA, IID, IIE, V and X, group IB, IIA, IID, IIE, V and X sPLA2 , respectively; PC, phosphatidylcholine; LysoPC, lysophosphatidylcholine; LPS, lipopolysaccharide; PLA2 R, PLA2 receptor; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; TBS, Tris-bu¡ered saline; PE, phosphatidylethanolamine; 13-HODE, 13-hydroxyoctadecadienoic acid * Corresponding author. Fax: +81-6-6458-0987; E-mail: [email protected]

In chronic in£ammatory diseases, macrophages are recruited to the sites of in£ammation and play a critical role as a source and target of numerous in£ammatory mediators, such as cytokines, growth factors and lipid mediators [1^3]. Upon macrophage activation, the expression levels of several enzymes involved in the lipid metabolisms are up-regulated. Cyclooxygenase-2 (COX-2), which catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2 leading to the production of various bioactive PGs in concert with the respective terminal synthases, is one example of this up-regulation [4^6]. The release of

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arachidonic acid from activated macrophages is mainly catalyzed by phospholipase A2 (PLA2 ). To date, numerous types of PLA2 s have been characterized and classi¢ed into di¡erent families according to their biochemical features [7^9]. Among them, cytosolic PLA2 K (cPLA2 K) has been identi¢ed as an essential enzyme for the arachidonic acid release linked to the production of various lipid mediators during activation of mouse peritoneal macrophages with the Ca2‡ ionophore A23187 and lipopolysaccharide (LPS) [10,11]. Although cPLA2 K-de¢cient mice have been reported to exhibit reduced reactions in allergy, postischemic brain injury and parturition [10,11], various phenotypic changes observed in mice de¢cient in COX and PG receptors, such as nephropathy, have not been reported [4]. These ¢ndings suggest a potential involvement of other types of PLA2 s in the production of lipid mediators during the progression of some pathological states. Secretory phospholipase A2 s (sPLA2 s) have several characteristics which di¡er from cPLA2 , including a relatively low molecular mass (13^18 kDa), the presence of 6^8 disul¢de bridges, an absolute catalytic requirement for millimolar concentrations of Ca2‡ and a broad speci¢city for phospholipids [7^9]. Depending on the primary structure characterized by the number and positions of cysteine residues, mammalian sPLA2 s have been now classi¢ed into nine di¡erent groups [7^9], including IID and IIE that we have recently cloned [12,13]. Among them, the group II subfamily (IIA, IID, IIE and V) is thought to play a role in the production of several lipid mediators, especially in the delayed phase of the cell activation process, because their expression levels are up-regulated under various in£ammatory conditions [14,15]. In particular, the expression of group IIA sPLA2 (sPLA2 -IIA) is greatly elevated in various macrophage-like cell lines after stimulation with pro-in£ammatory cytokines or LPS [16,17]. In contrast, group IB sPLA2 (sPLA2 -IB) acts as an endogenous ligand for the PLA2 receptor (PLA2 R) to induce various biological responses, including eicosanoid production and regulation of pro-in£ammatory cytokine synthesis during murine endotoxic shock [18,19]. The recently cloned group X sPLA2 (sPLA2 -X) possesses several characteristics distinct from other sPLA2 s [20]. It is the most acidic sPLA2 (pI 5.3) of

the seven types of human sPLA2 s. It has 16 cysteine residues located at positions characteristic of both sPLA2 -IB and sPLA2 -IIA and also possesses an amino acid C-terminal extension that is typical of sPLA2 -IIA. The pro-form of sPLA2 -X possesses a propeptide sequence attached at the NH2 -terminus of the mature protein, and the proteolytic removal of the propeptide is essential for optimal activity, similar to the case of sPLA2 -IB [21,22]. We have recently shown that, among the human sPLA2 species, sPLA2 -X has the most potent hydrolyzing activity toward phosphatidylcholine (PC) and elicits a marked release of arachidonic acid from various intact cell membranes [21]. Moreover, mouse sPLA2 -X was identi¢ed as one of the high-a¤nity ligands for murine PLA2 R [22]. Recent immunohistochemical analysis revealed the expression of sPLA2 -X in mouse splenic macrophages [22], suggesting its potential role in the production of lipid mediators from macrophages during the progression of in£ammatory conditions. In the present study, we examined and compared the potencies of sPLA2 -IB, IIA and X in the fatty acid release and eicosanoid production in isolated mouse peritoneal macrophages. We found that sPLA2 -X induced the most potent release of arachidonic acid leading to COX-2-dependent production of PGE2 and thromboxane A2 (TXA2 ). In accordance with its preferred hydrolyzing activity for PC, sPLA2 -X also induced a prominent production of lysophosphatidylcholine (lysoPC), one of the proin£ammatory lipid mediators [23^27]. 2. Materials and methods 2.1. Materials Puri¢ed recombinant mouse pro and mature forms of sPLA2 -X, human sPLA2 -IB and sPLA2 -X were prepared as described previously [21,22]. Recombinant human sPLA2 -IIA was a generous gift from Dr. Ruth Kramer (Eli Lilly, Indianapolis, IN, USA). Bovine serum albumin (BSA), indomethacin and A23187 were obtained from Sigma. Indoxam was synthesized at Shionogi Research Laboratories [28]. LPS (Salmonella typhosa 0901) was purchased from Difco Laboratories.

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2.2. Assay for fatty acid release and eicosanoid production by sPLA2 -stimulated mouse peritoneal macrophages Peritoneal macrophages were isolated from the peritoneal lavage £uids of male C57BL/6J mice (8 weeks) 4 days after injection of thioglycollate (Difco Laboratories), as described previously [29^31]. Brie£y, the cells recovered from the peritoneal £uids were plated in 6-well plates at 2.5U106 cells/well. After incubation for 1 h at 37³C, non-adherent cells were removed by thorough washing and adherent macrophages were incubated either with or without LPS (1 Wg/ml) for 6 h at 37³C [29]. After extensive washing, macrophages were preincubated with or without several inhibitors in Hanks' bu¡ered saline (pH 7.6), containing 0.1% BSA for 10 min at 37³C, and then stimulated with various concentrations of sPLA2 s or 10 WM A23187 in a ¢nal volume of 1 ml. To analyze the released fatty acids, the reaction was stopped by the addition of 2 ml Dole's reagent. The released fatty acids were extracted as described previously [21], labeled with 9-anthryldiazomethane (Funakoshi Co.), and then analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a LiChroCART 125-4 Superspher 100 RP-18 column (Merck), as described by Tojo et al. [32]. To measure the released PGE2 and TXB2 contents, the reaction was stopped by rapidly cooling the cells on ice. The supernatant was collected after centrifugation, and the PGE2 and TXB2 levels were quanti¢ed using speci¢c enzyme immunoassay kits (Cayman Chemicals Co.). 2.3. Western blot analysis of COX-2 expression Macrophages were pretreated either with or without LPS for 6 h, and then solubilized with Laemmli sample bu¡er containing 5% 2-mercaptoethanol. Equal volumes of the samples were separated by SDS^PAGE using a 4^20% gradient gel (Daiichi Chemicals Co., Ltd.). Proteins were transferred to Immobilon-P membrane (Millipore Co., Ltd.), blocked in BlockAce solution (Dainippon Pharma) in Tris-bu¡ered saline (TBS; 250 mM NaCl, 20 mM Tris^HCl, pH 7.4) and washed with TBS with 0.5% Tween 20. The membrane was incubated for 2 h with anti-COX-1 or anti-COX-2 antibody (0.6 Wg/

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ml; Cayman Chemicals Co.) in TBS with 1% BSA, and washed three times with TBS with 0.5% Tween 20. The blot was then incubated with peroxidaseconjugated goat anti-rabbit IgG for 2 h, and washed ¢ve times with TBS with 0.5% Tween 20. The blot was incubated with chemiluminescent detection reagent (ECL Western blotting detection reagents, Amersham Pharmacia Biotech) according to the manufacturer's instructions, and the signals were analyzed with an image analyzer (Fluor-S MultiImager, Bio-Rad). 2.4. Assay for lysoPC production in sPLA2 -X-treated mouse macrophages Peritoneal macrophages were stimulated with 100 nM mouse sPLA2 -X for the appropriate times in the presence or absence of 20 WM indoxam or 10 WM indomethacin. After the reaction, the supernatant was removed, and the phospholipid fractions in the medium and the resultant cells were extracted as described previously [33]. The extracted phospholipids were then separated by normal-phase HPLC on an Ultrasphere silica 4.6U250 mm column (Beckman, connected with a guard column of 4.6U45 mm), using acetonitrile/methanol/sulfuric acid (100:7:0.05, v/v) with a £ow rate of 1 ml/min at room temperature. Fractions corresponding to authentic L-K-lysoPC (Sigma), detected at the wavelength of 202 nm, were pooled and subjected to quantitative phosphorus analysis [34]. 3. Results 3.1. Potent release of fatty acids from sPLA2 -Xstimulated mouse peritoneal macrophages We ¢rst examined the potency of three types of sPLA2 s for the release of fatty acids from adherent peritoneal macrophages. Fig. 1 summarizes the amount of fatty acids released by the action of 100 nM sPLA2 s and 10 WM A23187 within 30 min. Among the human sPLA2 species examined, sPLA2 -X induced a marked release of all types of unsaturated fatty acids measured, whereas sPLA2 IB and IIA caused little release response. The cPLA2 activator A23187 induced a slight release of linoleic

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acid and oleic acid from the macrophages. Mouse sPLA2 -X also elicited a potent fatty acid release with an increased preference for linoleic acid and oleic acid, as compared to the human enzyme. Notably, a marked release of arachidonic acid was detected in the stimulation with both mouse and human sPLA2 -X. The amount of arachidonic acid released by sPLA2 -X was calculated to be about 5.6 nmol/106 cells, which was 30- and 60-fold higher than that released by sPLA2 -IB and IIA, respectively. The sPLA2 -X-induced fatty acid release was detected within 2 min and the reaction reached a steady-state level within 30 min (data not shown). Fig. 2 shows the dose dependence of mouse sPLA2 -X on the release of arachidonic acid. A signi¢cant release of arachidonic acid was detected at 0.1 nM sPLA2 -X. Mouse pro-sPLA2 -X induced much weaker release responses compared to the mature enzyme, and there was no release of lactate dehydrogenase activity from macrophages during the incubation of these sPLA2 s (data not shown). The sPLA2 -X-induced arachidonic acid release was blocked by the addition of the sPLA2 inhibitor indoxam [28] at 20 WM. Conversely, the arachidonic acid release was not suppressed by a known cPLA2

Fig. 2. Dose-dependent release of arachidonic acid by mouse sPLA2 -X. Mouse peritoneal macrophages were incubated with various concentrations of mouse sPLA2 -X for 30 min at 37³C, and the released arachidonic acid was quanti¢ed. The results are expressed as the increase in arachidonic acid levels above that obtained from the incubation in the absence of sPLA2 -X. Each point represents the mean þ S.D. of triplicate measurements. The data are representative of three experiments.

inhibitor, AACOCF3 [35], as previously reported for human monocytic THP-1 cells and mouse spleen cells [21,22] (data not shown). Taken together, these results demonstrate that sPLA2 -X induces a prompt and marked release of arachidonic acid from mouse peritoneal macrophages and that sPLA2 -X is more e¡ective than sPLA2 -IB, sPLA2 -IIA or A23187. 3.2. COX-2-dependent eicosanoid production in sPLA2 -X-stimulated mouse peritoneal macrophages

Fig. 1. Potent fatty acid release from mouse peritoneal macrophages by sPLA2 -X. Mouse peritoneal macrophages were incubated with 100 nM mouse sPLA2 -X (m-X), human sPLA2 -X (h-X), sPLA2 -IB (h-IB), sPLA2 -IIA (h-IIA) or 10 WM A23187 for 30 min at 37³C, and the released fatty acids were quanti¢ed as described in Section 2. The results are expressed as the increase in fatty acid levels above that obtained from the incubation in the absence of sPLA2 s. Each point represents the mean þ S.D. of triplicate measurements. The data are representative of three experiments.

Upon stimulation with pro-in£ammatory cytokines and LPS, the eicosanoid biosynthesis was dramatically enhanced in accordance with up-regulation of several enzymes involved in the conversion of arachidonic acid to eicosanoids in various cell types, including vascular smooth muscle cells, ¢broblasts, synovial cells and monocytes/macrophages [4^6]. In mouse peritoneal macrophages, COX-1 protein was constitutively expressed and was not altered by LPS treatment, as previously reported (data not shown) [30,31]. In contrast, COX-2 protein was slightly expressed in the resting state, and its level was markedly increased after 6 h of LPS treatment (Fig. 3A). Accordingly, the conversion of exogenously added arachidonic acid to PGE2 and TXA2 was markedly

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of PGE2 (Fig. 3A) and TXA2 (Fig. 3B); in contrast, little, if any, production by sPLA2 -IB and IIA was observed (P 6 0.01 for mouse and human sPLA2 -Xstimulated cells compared to non-treated cells in both eicosanoid productions). Upon LPS activation, the sPLA2 -X-induced eicosanoid formation was markedly enhanced. In particular, the PGE2 level produced by human sPLA2 -X was increased up to 18-fold by pretreatment with LPS (Fig. 3A) and the amounts of PGE2 and TXA2 produced by human sPLA2 -X were about 11% of the maximum levels induced by 10 WM A23187. The potency of mouse pro-sPLA2 -X in the eicosanoid production was much lower as compared to the mature form. Human sPLA2 -IIA also induced a signi¢cant eicosanoid formation in LPS-treated macrophages, although the elevated eicosanoid levels were less than 12% of those elicited by human sPLA2 -X. As shown in Fig. 4, the sPLA2 -X-induced PGE2 production was blocked by the addition of the sPLA2 inhibitor indoxam, as well as by the COX inhibitor indomethacin. These ¢ndings demonstrate that sPLA2 -X-induced arachidonic acid release was e¤ciently coupled with COX-2, leading to potent eicosanoid production in LPS-activated macrophages. Fig. 3. Eicosanoid formation in resting and LPS-pretreated peritoneal macrophages. Mouse peritoneal macrophages were pretreated with (closed column) or without (open column) LPS (1 Wg/ml) for 6 h. After washing, the cells were stimulated with 100 nM mouse sPLA2 -X (m-X), human sPLA2 -X (h-X), sPLA2 -IB (h-IB), sPLA2 -IIA (h-IIA) or the mouse pro form of sPLA2 -X (pro m-X) for 30 min at 37³C. The amounts of PGE2 (A) or TXA2 (B) were quanti¢ed as described in Section 2. The results are expressed as the increase in eicosanoid levels above that obtained from the incubation in the absence of sPLA2 s. Each point represents the mean þ S.D. of triplicate measurements. Statistical signi¢cance was determined by Student's t-test (*P 6 0.05, **P 6 0.01 compared to non-stimulated cells) in the resting or LPS-pretreatment states, respectively. The data are representative of three experiments. (Inset) Increased expression of COX-2 protein in LPS-pretreated macrophages, assessed by Western blot analysis.

augmented in the LPS-pretreated macrophages, compared to quiescent cells (data not shown). Therefore, the potency of sPLA2 s in eicosanoid formation was examined in the resting and LPS-stimulated macrophages. In the resting state, mouse and human sPLA2 -X (100 nM) induced a slight but signi¢cant production

Fig. 4. E¡ects of various inhibitors on sPLA2 -X-induced PGE2 production in LPS-pretreated macrophages. LPS-pretreated macrophages were incubated with 20 WM indoxam or 10 WM indomethacin for 10 min at 37³C, and then stimulated with 100 nM mouse sPLA2 -X for 30 min at 37³C. The results are expressed as the percent of PGE2 produced by sPLA2 -X in the absence of inhibitors. Each point represents the mean þ S.D. of triplicate measurements. The data are representative of three experiments.

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3.3. LysoPC production in sPLA2 -X-stimulated peritoneal macrophages Previous analysis of the substrate speci¢city of

mouse sPLA2 -X in the mixed phospholipid vesicles revealed its substrate preferences: PC s phosphatidylethanolamine (PE) = phosphatidylglycerol among the 1-palmitoyl-2-oleoyl phospholipids. This is in contrast with the substrate speci¢city of human sPLA2 -IB and IIA (phosphatidylglycerolEPE s PC) [22]. Since the outer lea£et of the plasma membrane of mammalian cells is largely composed of zwitterionic PC and sphingomyelin [36], and sPLA2 -X induces a potent release of unsaturated fatty acids from mouse peritoneal macrophages (Fig. 1), we next examined the potency of sPLA2 -X in the production of lysoPC. After treatment of resting macrophages with mouse sPLA2 -X, lysoPC was puri¢ed by reverse-phase HPLC and then quanti¢ed by phosphorus analysis. As shown in Fig. 5A, mouse sPLA2 -X elicited a signi¢cant production of lysoPC. The maximum production of lysoPC (250 pmol/106 cells) was observed with 10 min stimulation. We could not detect any signi¢cant elevation of lysoPC in the reaction medium during sPLA2 -X stimulation and found that the lysoPC produced was retained in the cell fractions. The sPLA2 -X-induced lysoPC production was completely suppressed by the addition of 20 WM indoxam, but not blocked by indomethacin (Fig. 5B). These results demonstrate that sPLA2 -X also induces the production of lysoPC, one of the bioactive lysophospholipids in the macrophages. 4. Discussion

Fig. 5. LysoPC production in sPLA2 -X-stimulated macrophages. (A) Time-dependent lysoPC production by sPLA2 -X. Peritoneal macrophages were stimulated with 100 nM mouse sPLA2 -X for the indicated times at 37³C, and the amount of lysoPC was quanti¢ed as described in Section 4. The results are expressed as the increase in lysoPC levels above that obtained in the incubation in the absence of sPLA2 -X. Each point represents the mean þ S.D. of triplicate measurements. The data are representative of three experiments. (B) E¡ects of indoxam and indomethacin on sPLA2 -X-induced lysoPC production. Peritoneal macrophages were incubated with 20 WM indoxam or 10 WM indomethacin for 10 min at 37³C, and then stimulated with 100 nM mouse sPLA2 -X for 30 min at 37³C. The results are expressed as the percent of lysoPC produced by sPLA2 -X in the absence of inhibitors. Each point represents the mean þ S.D. of triplicate measurements. The data are representative of three experiments.

The present study demonstrates that sPLA2 -X induces a prompt and marked release of arachidonic acid leading to COX-2-dependent eicosanoid formation in mouse peritoneal macrophages. Among the sPLA2 enzymes examined, potent fatty acid release responses were observed only with the treatment of sPLA2 -X, which is conceivably due to its higher hydrolyzing activity toward PC [21,22], one of the major phospholipid species in the outer lea£et of the plasma membrane [36]. The amount of arachidonic acid released from sPLA2 -X-stimulated macrophages (5600 and 5900 pmol/106 cells at 100 nM mouse and human sPLA2 -X, respectively) was much higher than the levels in human monocytic THP-1 cells (ca. 220 pmol/106 cells stimulated with 350 nM human sPLA2 -X) [21] and mouse spleen cells (ca. 145

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pmol/106 cells stimulated with 500 nM mouse sPLA2 -X) [22]. The great releasing activity of sPLA2 -X in mouse peritoneal macrophages may be due to the presence of larger amounts of arachidonic acid in the total fatty acids (25%) in this cell type compared to a few percent in most other cell types [37]. Alternatively, the accessibility of sPLA2 -X to the cell membrane phospholipids might be di¡erent between the adhesive and suspended cell conditions, as suggested in a previous paper [38]. Recently, we identi¢ed mouse sPLA2 -X as one of the high-a¤nity ligands for PLA2 R [22]. The PLA2 R is known to play a role in the arachidonic acid release and eicosanoid production in various murine cells [18]. In the present study, human sPLA2 -IB showed little activity in both responses despite its high-a¤nity binding ability for PLA2 R (Fig. 1). In addition, reverse transcription-polymerase chain reaction analysis revealed no signi¢cant expression of PLA2 R mRNA in isolated peritoneal macrophages, and we could not detect any high-a¤nity and speci¢c binding sites of 125 I-labeled mouse sPLA2 -X (K. Hanasaki and K. Nakano, unpublished data). These ¢ndings suggest that sPLA2 -X-induced acute release responses depend on its enzymatic activity toward cell membrane phospholipids. In resting macrophages, sPLA2 -X elicited a modest but signi¢cant production of PGE2 and TXA2 (Fig. 3). After the induction of COX-2 by LPS treatment, the eicosanoid formation was dramatically enhanced, demonstrating that the arachidonic acid released by sPLA2 -X is e¤ciently converted into these eicosanoids by the action of endogenous COX-2. This is consistent with previous results in transfection experiments with HEK293 cells, in which human sPLA2 -X could induce potent PGE2 production in COX-2-overexpressing cells compared to mocktransfected cells [38,39]. In LPS-treated cells, sPLA2 -IB and sPLA2 -IIA could not induce any signi¢cant eicosanoid production (Fig. 3). Since some inbred mouse strains have a natural frameshift mutation in the sPLA2 -IIA gene [40] and the mast cells derived from these mice exhibit normal PG production to ligand activation [41], its contribution to the eicosanoid production could be much lower, especially in mice. Recently, we have detected the expression of sPLA2 -X in mouse splenic macrophages and human alveolar type II epithelial cells [21,22], where

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high levels of COX-2 can be induced by several in£ammatory cytokines [5,6,42]. Although the enzymatic activity of sPLA2 -X is regulated by the conversion of its pro form to the mature enzyme via proteolytic removal of the propeptide [21,22], the expression of several proteinases is up-regulated in cytokine-activated macrophages [43,44]. Furthermore, the protease activities in the tissues and/or circulation are elevated in acute pancreatitis and sepsis [45,46], suggesting that the conversion of pro-sPLA2 -X into the active enzyme could be accelerated in some pathological states. Taken together, these ¢ndings strongly suggest that sPLA2 -X plays a critical role in eicosanoid production during the development of in£ammatory conditions. It is well known that cPLA2 K is involved in the arachidonic acid release coupled with eicosanoid production during various cell activation processes [47]. In mouse peritoneal macrophages, the A23187-induced PGE2 production is completely abrogated by the de¢ciency in cPLA2 K [10,11]. However, sPLA2 -X can induce a fraction (11%) of the eicosanoid production evoked by A23187 in LPS-activated macrophages. Furthermore, the sPLA2 -X-induced arachidonic acid release was suppressed by a sPLA2 inhibitor, but not blocked by a cPLA2 inhibitor. These results suggest the existence of a cPLA2 -independent mechanism for mobilization of arachidonic acid from cell membrane phospholipids. Mice de¢cient in cPLA2 K exhibited reduced reactions in various in£ammatory disease models, such as allergy and acute lung injury [10,48]. However, several phenotypic changes observed in mice de¢cient in COX and PG receptors, such as severe nephropathy [4], impaired febrile response [49], and reduced colon carcinogenesis [50,51], have not been reported in cPLA2 K mutant mice. In particular, recent studies have shown that cPLA2 K is not involved in the colonic polyp formation in a mouse model for human familial adenomatous polyposis in spite of its important role in the expansion of polyps in the small intestine [52]. Further studies are required to clarify the potential involvement of sPLA2 -X in the progression of these pathological conditions. Previous studies have revealed the potency of sPLA2 -X in the production of various types of lysophospholipids in mixed phospholipid vesicle [38]. In the present study, we have presented evidence that

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sPLA2 -X can induce the production of a large amount of lysoPC in the macrophages (Fig. 5). The sPLA2 -X-elicited lysoPC production reached its maximum at 10 min followed by a decline to the sustained level, which may be due to di¡erences in the kinetics of the release and uptake of fatty acids in PC, as macrophages can actively incorporate exogenous fatty acids into cell membrane phospholipids [53]. In addition, the degradation of lysoPC might be accelerated by the actions of lysophospholipases or phospholipase D (PLD) because the released unsaturated fatty acids, such as oleic acid and arachidonic acid, can stimulate the PLD activity in various cell types [54]. Since lysoPC has been shown to induce diverse biological responses, including the promotion of cell growth and chemotaxis in monocytes/ macrophages [25] and the activation of protein kinase C in T cells [55], sPLA2 -X may play a role in the progression of in£ammatory diseases via lysoPC production. In vascular endothelial cells, lysoPC can induce the expression of various cell adhesion molecules and growth factors involved in the migration and di¡erentiation of monocytes [56]. sPLA2 -X also induced a potent release of linoleic acid (Fig. 1), which can be metabolized by 12/15-lipoxygenase into its oxidized metabolites such as 13-hydroxyoctadecadienoic acid (13-HODE). Since 13-HODE can act as one of the endogenous ligands of peroxisome proliferator-activated receptor Q that is a key regulator of gene expression involved in the scavenger function of foam cells [57], sPLA2 -X also might play a role in the pathogenesis of atherosclerosis. In conclusion, we have demonstrated here that sPLA2 -X induces the production of various lipid mediators in mouse peritoneal macrophages. In contrast to selective inhibition of COX inhibitor for sPLA2 X-induced eicosanoid production, the sPLA2 inhibitor indoxam can suppress the formation of all types of lipid mediators, including unsaturated fatty acids, eicosanoids, and lysoPC. Further studies on the physiological and pathological functions of sPLA2 X should provide more information regarding the therapeutic potential of sPLA2 inhibitors for various human diseases including chronic in£ammation and atherosclerosis.

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