J Biol Chem, Vol. 275, Issue 7, 4783-4786, February 18, 2000

Group V Phospholipase A₂-mediated Oleic Acid Mobilization in Lipopolysaccharide-stimulated P388D₁ Macrophages^*

Jesús Balsinde^§, María A. Balboa, Saul Yedgar^¶, and Edward A. Dennis

From the  Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601 and the ^¶ Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel 91120

	ABSTRACT

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P388D₁ macrophages prelabeled with [³H]arachidonic acid (AA) respond to bacterial lipopolysaccharide (LPS) by mobilizing AA in a process that takes several hours and is mediated by the concerted actions of the group IV cytosolic phospholipase A₂ and the group V secretory phospholipase A₂ (sPLA₂). Here we show that when the LPS-activated cells are prelabeled with [³H]oleic acid (OA), they also mobilize and release OA to the extracellular medium. The time and concentration dependence of the LPS effect on OA release fully resemble those of the AA release. Experiments in which both AA and OA release are measured simultaneously indicate that AA is released 3 times more efficiently than OA. Importantly, LPS-stimulated OA release is strongly inhibited by the selective sPLA₂ inhibitors 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid and carboxymethylcellulose-linked phosphatidylethanolamine. The addition of exogenous recombinant sPLA₂ to the cells also triggers OA release. These data implicate a functionally active sPLA₂ as being essential for the cells to release OA upon stimulation with LPS. OA release is also inhibited by methyl arachidonyl fluorophosphonate but not by bromoenol lactone, indicating that the group IV cytosolic phospholipase A₂ is also involved in the process. Together, these data reveal that OA release occurs during stimulation of the P388D₁ macrophages by LPS and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.

	INTRODUCTION

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Using the murine macrophage-like cell line P388D₁, we have recently shown that arachidonic acid (AA)¹ mobilization and prostaglandin production stimulated by platelet-activating factor and/or lipopolysaccharide (LPS) involves the participation of three effectors, namely group IV cytosolic PLA₂ (cPLA₂), secretory group V PLA₂ (sPLA₂), and COX-2. In this system, the cPLA₂ fundamentally plays a regulatory role, whereas the sPLA₂ plays an augmentative role by providing most of the AA metabolized by COX-2 (1-6).

The different roles for both cPLA₂ and sPLA₂ during stimulus-response coupling have now been recognized in a number of different systems (7-14). Interestingly, in some instances the sPLA₂ involved is not a group V sPLA₂ but rather a closely related group IIA enzyme (15). Nevertheless, group IIA sPLA₂ appears to serve in the same augmentative role (9, 10, 16, 17).

Recently, a surface receptor that recognizes certain sPLA₂ forms with high affinity has been cloned (18). In line with the existence of putative sPLA₂ receptors, it has been suggested that sPLA₂-mediated AA release in some systems may not involve the hydrolytic activity of the sPLA₂. Rather, the sPLA₂ would act as a ligand-like agonist that stimulates the cPLA₂ for an increased AA release response (19-21). A major argument in favor of the above scenario is the finding that no fatty acids other than AA are detected in the extracellular medium (19-21). Specific AA release would be inconsistent with the involvement of a sPLA₂, since this enzyme shows little or no fatty acid preference (22).

Since the augmentative role that group V sPLA₂ plays in LPS-activated P388D₁ macrophages appears to depend on enzyme activity (5, 6), we have now examined the hypothesis of whether these cells mobilize other fatty acids in addition to AA. Our results show that the activated cells do release measurable amounts of oleic acid (OA), that this release appears to be due to the hydrolytic action of the sPLA₂ acting on the cellular surface, and that the regulatory features of the OA release are strikingly similar to those previously found for the AA release.

	EXPERIMENTAL PROCEDURES

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Materials-- Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum was from Hyclone Laboratories (Logan, UT). Nonessential amino acids were from Irvine Scientific (Santa Ana, CA). [9,10-³H]Oleic acid (specific activity 55 Ci/mmol) and [5,6,8,9,11,12,14,15-³H]arachidonic acid (specific activity 100 Ci/mmol) were from NEN Life Science Products. [1-¹⁴C]Oleic acid (specific activity 56 mCi/mmol) was from Amersham Pharmacia Biotech. LPS (E. coli 0111:B4) was from Sigma. Methyl arachidonyl fluorophosphonate (MAFP) and (E)-6(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone (BEL)) were from Biomol (Plymouth Meeting, PA). The sPLA₂ inhibitor 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid (LY311727) was generously provided by Dr. Edward Mihelich (Lilly). Human recombinant group V sPLA₂ was produced in our laboratory utilizing the Pichia pastoris expression system (6). The sPLA₂ inhibitor CMPE (phosphatidylethanolamine covalently linked to carboxymethylcellulose) was synthesized in Dr. Yedgar's laboratory by Arie Dagan and Miron Krimsky (23).

Cell Culture and Labeling Conditions-- P388D₁ cells (MAB clone) (5, 6) were maintained at 37 °C in a humidified atmosphere at 90% air and 10% CO₂ in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids. P388D₁ cells were plated at 10⁶/well, allowed to adhere overnight, and used for experiments the following day. All experiments were conducted in serum-free Iscove's modified Dulbecco's medium. When required, radiolabeling of the P388D₁ cells was achieved by including 0.5 µCi/ml [³H]OA or 0.1 µCi/ml [¹⁴C]OA plus 0.5 µCi/ml [³H]AA during the overnight adherence period (20 h). Labeled fatty acid that had not been incorporated into cellular lipids was removed by washing the cells six times with serum-free medium containing 1 mg/ml albumin.

Measurement of Extracellular Fatty Acid Release-- The cells were placed in serum-free medium for 30 min before the addition of LPS or exogenous sPLA₂ for different periods of time in the presence of 0.5 mg/ml bovine serum albumin. The supernatants were removed, cleared of detached cells by centrifugation, and assayed for radioactivity by liquid scintillation counting. When inhibitors were used, they were added 30 min before the addition of LPS.

Data Presentation-- Except for the data in Fig. 2, which are given as percentage of release with respect to total cellular radioactivity levels, agonist-stimulated OA release is expressed by subtracting the basal rate observed in the absence of agonist and inhibitor. These background values were in the range of 1000-2000 cpm. Each set of experiments was repeated at least three times with similar results. Unless otherwise indicated, the data presented are from representative experiments.

	RESULTS

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OA Release in LPS-stimulated P388D₁ Cells-- We have previously shown that exposure of P388D₁ macrophages (MAB clone) to LPS induces a concentration-dependent release of AA to the extracellular medium that spans several hours (5). We began the current study by determining whether LPS was able to cause the extracellular release of OA as well. To this end, the cells, labeled with 0.5 µCi/ml [³H]oleic acid, were exposed to different concentrations of LPS for various periods of time. As shown in Fig. 1, LPS did induce a time- (Fig. 1A) and concentration- (Fig. 1B) dependent release of [³H]OA from the cells. As shown in Fig. 1A, the kinetics of the LPS effect on OA release was very similar to that previously found for the LPS-induced AA release (5). Thus, after a lag of about 3 h, OA release proceeded linearly up to about 10 h, after which it continued at a slower rate. The concentration dependence of the LPS-induced OA equally resembled that of the LPS-induced AA release (5).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   LPS-stimulated [3H]OA release in P388D1 macrophages. A, time course of [3H]OA release upon stimulation with 100 ng/ml LPS (closed circles) and in the absence of stimulation (open circles). B, dose response of the LPS effect (20-h incubation).

By simultaneously labeling the cells with [³H]AA and [¹⁴C]OA, it was possible to measure under identical settings the release of these two fatty acids in response to LPS. To allow for a direct comparison, the results are given as the percentage of labeled fatty acid incorporated into cells that is released. Fig. 2 shows that despite the fact that the LPS-activated cells released OA to a significant level (2-fold above basal), AA was released about 3 times more efficiently.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Simultaneous measurement of OA and AA in activated P388D1 macrophages. The cells, prelabeled with [3H]AA and [14C]OA, were stimulated with 100 ng/ml LPS for 20 h (closed bars). Afterward, the supernatants were collected and assayed for 3H radioactivity (AA release) and 14C radioactivity (OA release). Open bars denote control incubations (i.e. those that did not receive LPS).

PLA₂ Inhibition Studies-- To address the involvement of the different PLA₂ forms in LPS-induced OA release, we first utilized MAFP (2), a dual cPLA₂/iPLA₂ inhibitor that has previously been found to block the cPLA₂-dependent release of AA from LPS-stimulated P388D₁ macrophages (2, 5). As shown in Fig. 3A, MAFP strongly blocked the LPS-induced [³H]OA release.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of MAFP and BEL on LPS-induced OA release. The cells were treated with the indicated concentrations of MAFP (A) or BEL (B) for 30 min before the addition of 100 ng/ml LPS (closed symbols), and the incubations proceeded for 20 h. Open circles denote control incubations (i.e. those that did not receive LPS).

iPLA₂ involvement was studied with BEL, a compound that manifests a 1000-fold selectivity for inhibition of the iPLA₂ versus the cPLA₂ in vitro (2). As shown in Fig. 3B, BEL had no measurable inhibitory effect on LPS-induced [³H]OA release. Nevertheless, it completely inhibited iPLA₂ activity in homogenates prepared from LPS-treated cells (not shown). In turn, these data indicate that the effects of MAFP shown above are due to inhibition of the cPLA₂.

To assess the involvement of sPLA₂, we utilized two structurally unrelated inhibitors, namely LY311727 and CMPE (Fig. 4). The first compound is an indole derivative (24), and the second one is composed of N-derivatized phosphatidylethanolamine covalently linked via the head group to carboxymethyl cellulose (23). Both of these compounds strongly inhibited [³H]oleic acid release (Fig. 4). When, in the experiment shown in Fig. 4B, carboxymethyl cellulose alone was added instead of CMPE, no effect on LPS-induced [³H]OA release was observed at all (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of LY311727 and CMPE on LPS-induced OA release. The cells were treated with the indicated concentrations of LY311727 (A) or CMPE (B) for 30 min before the addition of LPS (closed symbols), and the incubations were allowed to proceed for 20 h. Open circles denote control incubations (i.e. those that did not receive LPS).

Exogenous Group V sPLA₂ Triggers OA Release-- CMPE is a cell-impermeable inhibitor that prevents the sPLA₂ from attacking the phospholipids on the outer surface (23). Thus, the data shown in Fig. 4B imply that the extracellular sPLA₂ pool is the one that participates in OA release in the LPS-treated cells. Given that group V sPLA₂ is active per se toward cell membranes (i.e. no "membrane rearrangement" is needed for this enzyme to attack the outer membrane) (6, 25), we reasoned that the addition of exogenous group V sPLA₂ to the macrophages should result in an enhanced release of OA to the extracellular medium. This is exactly what happened in the experiment shown in Fig. 5.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of exogenous group V sPLA2 on OA release. The cells were treated with the indicated concentrations of recombinant group V sPLA2 for 1 h. Afterward, supernatants were assayed for [3H]OA release.

	DISCUSSION

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Recent work by several laboratories has highlighted the importance of sPLA₂ (either group V or group IIA) in AA mobilization and attendant prostaglandin formation (26, 27). The sPLA₂ is thought to amplify the AA release signal initiated by the cPLA₂ to generate large amounts of free AA, part of which will eventually be converted into eicosanoids (26, 27).

That the sPLA₂ plays merely a hydrolytic role in the process of AA release has been argued against recently on the basis that AA mobilization in some cell types appears to be highly specific for AA (i.e. no release of other fatty acids is detected) (19-21). The latter finding would be inconsistent with the hydrolytic action of an enzyme such as the sPLA₂, which shows no fatty acid preference (22). Thus, an alternative explanation has been proposed that involves the sPLA₂ acting as a ligand-like molecule independent of enzyme activity. According to this hypothesis, the sPLA₂ acts as a a receptor-directed agonist that stimulates the selective release of AA via cPLA₂ activation. In addition to the lack of release of fatty acids other than AA (19-21), this hypothesis is also supported by data showing that sPLA₂s from different sources that have been rendered catalytically inactive by inhibitors are still able to elicit the AA release (20, 21).

In contrast, a large number of studies have shown that sPLA₂ inhibitors markedly diminish the release of AA (2, 5, 8, 10, 14, 28-31), thus supporting a hydrolytic role for the sPLA₂ in the process. Moreover, we (6) have recently found that the addition of exogenous group V sPLA₂ to the cells induces an AA release response that is not observed if chemically inactivated enzyme is used. In agreement with our data, Tada et al. (8) have found that catalytically inactive group IIA sPLA₂ mutants are incapable of promoting AA release from cytokine-primed cells.

The current results clearly show that the LPS-activated P388D₁ macrophages do release OA and that the regulatory features of the OA release are strikingly similar to those found previously for the AA release (2). Simultaneous measurement of the OA release versus AA release revealed that the activated cells appear to release AA in preference over OA, which is fully consistent with recent data of Murakami and colleagues (10, 17). However, it is important to note that OA release was not detected in these previous studies, or it was detected at a very low level (10, 17). In contrast, we show in this study that OA release in the LPS-activated cells is actually quite significant (2-fold over basal).

LPS-activated OA release can be blocked by sPLA₂ inhibitors, which implies that a catalytically active sPLA₂ is needed for the OA release to occur. Hence, in the LPS-activated cells, AA-containing phospholipids are not the only substrates for the sPLA₂. Thus, it is tempting to speculate that the fatty release observed in cells may reflect the fatty acid composition of the specific phospholipid pools that come in contact with the sPLA₂. sPLA₂ docking in a membrane domain highly enriched in AA-containing phospholipids could explain why this enzyme appears to release AA in preference to other fatty acids in vivo.

Importantly, at least one of the sPLA₂ inhibitors utilized in this study, CMPE, is cell-impermeable. CMPE anchors to the extracellular leaflet of the plasma membrane by its phospholipid moiety, thereby protecting the membrane from the hydrolytic action of the sPLA₂ (23). From the results obtained with this compound, it can be concluded that the sPLA₂ pool involved in the OA release is the one on the cellular surface, because if the sPLA₂ were acting inside the cell, CMPE would not have had any effect on the release. Similar to OA release, we have observed that CMPE also strongly blunts AA release in the LPS-activated P388D₁ cells,² which also points at the cell surface as the site for sPLA₂-dependent AA mobilization. In agreement with these observations, we show that exogenous sPLA₂ is able to induce both AA (6) and OA (this study) release from the P388D₁ cells.

Our inhibitor studies indicate that, in addition to the sPLA₂, OA release in the LPS-activated P388D₁ cells also involves the cPLA₂. Since the cPLA₂ is highly AA-specific, it appears very unlikely that this enzyme contributes to the OA release by directly cleaving OA-containing phospholipids. We (5) have previously demonstrated that a catalytically active cPLA₂ is required for the cells to show enhanced expression of sPLA₂ in response to LPS. Thus, inhibition of OA release by cPLA₂ inhibitors most likely reflects the diminished capacity of the cells to synthesize sPLA₂, in an analogous manner to what we (5) and others (13) have previously described for the AA release.

P388D₁ macrophages contain a third PLA₂ type, the group VI iPLA₂. Given that iPLA₂ is not fatty acid-selective, a possible role for this enzyme in OA mobilization could in principle be envisioned. However, in analogy again with the AA release, we have failed to detect any role for this enzyme in the LPS-induced OA release. Under conditions wherein cellular iPLA₂ is completely inhibited by BEL, no effect on LPS-stimulated OA release is observed. In agreement with our data, a recent study by Ito et al. (32) also failed to detect any effect of BEL on OA release. Thus, these data lend further support to the idea that the group VI iPLA₂ may not be involved in cellular signaling in P388D₁ macrophages (33). In support of this view, elegant studies by Murakami et al. (10) have shown that overexpression of iPLA₂ in 293 fibroblasts does not modify the AA release response triggered by interleukin-1, but overexpression of either cPLA₂, group IIA sPLA₂, or group V sPLA₂ does result in all cases in an increased AA release in response to interleukin-1 (10).

In summary, we have found that LPS-stimulated P388D₁ macrophages release OA by a mechanism that involves the hydrolytic actions of both group V sPLA₂ and cPLA₂ and appears to be strikingly similar to the one previously described for AA release. The one other PLA₂ present in these cells (i.e. the group VI iPLA₂) appears not to be required for stimulated release.

	ACKNOWLEDGEMENTS

We thank Dr. Edward Mihelich for providing LY311727 and Drs. M. Krimsky and A. Dagan from the laboratory of S. Yedgar for synthesizing the CMPE.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants HD26171 and GM2051 and University of California BioStar Project/Lilly Laboratories Grant S96-08.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence may be addressed. Tel.: 858-534-8902; Fax: 858-534-7390; E-mail: jbalsinde@ucsd.edu.

To whom correspondence may be addressed. Tel.: 858-534-3055; Fax: 858-534-7390; E-mail: edennis@ucsd.edu.

² J. Balsinde and E. A. Dennis, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: AA, arachidonic acid; OA, oleic acid; PLA₂, phospholipase A₂, cPLA₂, group IV cytosolic PLA₂; sPLA₂, secretory PLA₂; iPLA₂, group VI Ca²⁺-independent PLA₂; MAFP, methyl arachidonyl fluorophosphonate; BEL, bromoenol lactone; CMPE, phosphatidyletanolamine linked to carboxymethyl cellulose; LY311727, 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propane sulfonic acid; LPS, lipopolysaccharide.

	REFERENCES

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