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• W2029317617 abstract "Previous studies have demonstrated that P388D1 macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A2 (cPLA2), the secretory group V phospholipase A2, and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D1 macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A2 nor requires the cells to be primed by LPS. The group VI Ca2+-independent phospholipase A2 is also not involved. Zymosan appears to signal exclusively through activation of the cPLA2, which is coupled to the cyclooxygenase-2. These results define a secretory PLA2-independent pathway for AA mobilization in the P388D1 macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA2 is sufficient to generate a prostaglandin biosynthetic response in the P388D1 macrophages. Previous studies have demonstrated that P388D1 macrophages are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in two temporally distinct phases. The first phase is triggered by platelet-activating factor within minutes, but needs the cells to be previously exposed to bacterial lipopolysaccharide (LPS) for periods up to 1 h. It is thus a primed immediate phase. The second, delayed phase occurs in response to LPS alone over long incubation periods spanning several hours. Strikingly, the effector enzymes involved in both of these phases are the same, namely the cytosolic group IV phospholipase A2 (cPLA2), the secretory group V phospholipase A2, and cyclooxygenase-2, although the regulatory mechanisms differ. Here we report that P388D1 macrophages mobilize AA and produce prostaglandins in response to zymosan particles in a manner that is clearly different from the two described above. Zymosan triggers an immediate AA mobilization response from the macrophages that neither involves the group v phospholipase A2 nor requires the cells to be primed by LPS. The group VI Ca2+-independent phospholipase A2 is also not involved. Zymosan appears to signal exclusively through activation of the cPLA2, which is coupled to the cyclooxygenase-2. These results define a secretory PLA2-independent pathway for AA mobilization in the P388D1 macrophages, and demonstrate that, under certain experimental settings, stimulation of the cPLA2 is sufficient to generate a prostaglandin biosynthetic response in the P388D1 macrophages. phospholipase A2 arachidonic acid platelet-activating factor cyclooxygenase prostaglandin E2 bacterial lipopolysaccharide methyl arachidonyl fluorophosphonate bromoenol lactone diacylglycerol group IV cytosolic PLA2 secretory PLA2 group VI Ca2+-independent PLA2 Phospholipase A2(PLA2)1 enzymes play key roles in a variety of cellular processes by generating a number of bioactive mediators. PLA2-mediated hydrolysis of phospholipids results in the release of arachidonic (AA) and lysophospholipids, both of which may possess biological activity or serve as substrates for the generation of other bioactive lipid mediators such as the eicosanoids or platelet-activating factor (PAF) (1.Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar, 2.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). In major eicosanoid-producing immunoinflammatory cells such as macrophages and mast cells, prostaglandin production usually occurs in two phases (1.Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar, 2.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). The first phase takes place in minutes and is strikingly characterized by its dependence on Ca2+mobilization from internal stores; whereas, the second, delayed phase, spanning several hours, takes place in the absence of Ca2+elevations (2.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). Substantial evidence suggests that specific coupling between certain PLA2 and cyclooxygenase (COX) forms accounts for the differential regulation of the immediate and delayed responses (3–14.Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-833Crossref PubMed Scopus (84) Google Scholar). Thus, depending on whether group IV cPLA2 or sPLA2 (group IIA or group V) is the provider of free AA, either COX-1 or COX-2 would be responsible for prostaglandin production. Which PLA2 form couples to which COX isoform appears also to depend critically on cell type. We have shown that the rapid, PAF receptor-mediated phase of PGE2 production in lipopolysaccharide (LPS)-primed P388D1 macrophages involves group V sPLA2coupling to COX-2 (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). It is important to emphasize here that this response to PAF will not occur if the cells have not been first exposed to bacterial LPS for 1 h (15.Glaser K.B. Asmis R. Dennis E.A. J. Biol. Chem. 1990; 265: 8658-8664Abstract Full Text PDF PubMed Google Scholar, 16.Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Biochem. J. 1997; 321: 805-809Crossref PubMed Scopus (58) Google Scholar). Thus the P388D1 cell response to PAF is not, strictly speaking, an immediate response but rather a primed immediate one. Interestingly, we have recently discovered that group V sPLA2 also couples to COX-2 for the delayed PGE2 biosynthetic response of P388D1macrophages exposed to LPS alone (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Under those conditions, expression of both group V sPLA2 and COX-2 is markedly induced and correlates with ongoing AA release and prostaglandin biosynthesis, respectively (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), indicating that the AA produced by group V sPLA2 is used by COX-2 to produce PGE2. Importantly, expression of both group V sPLA2 and COX-2 can be abolished by pretreating the cells with the cPLA2inhibitor methyl arachidonyl fluorophosphonate, implying that a functionally active cPLA2 is essential for the delayed PGE2 response to occur (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In the current study we have uncovered a third pathway for AA mobilization and PGE2 production in activated P388D1 macrophages that appears not to require the group V sPLA2. We show here that exposure of the cells to zymosan particles triggers the immediate release of both AA and PGE2 in a process that depends only on cPLA2and COX-2. Thus in P388D1 macrophages there are at least three different phases for AA release: an immediate phase (zymosan), which does not require the participation of the sPLA2, a primed immediate phase (LPS/PAF), which does require the sPLA2, and a delayed phase (LPS), which also requires the sPLA2. All of the three phases, however, require the cPLA2. Iscove's modified Dulbecco's medium (endotoxin <0.05 ng/ml) was from Whittaker Bioproducts (Walkersville, MD). Fetal bovine serum was from Hyclone Labs. (Logan, UT). Nonessential amino acids were from Irvine Scientific (Santa Ana, CA). [5,6,8,9,11,12,14,15-3H]Arachidonic acid (specific activity 100 Ci/mmol) was from New England Nuclear (Boston, MA). PAF, LPS (Escherichia coli 0111:B4), and yeast-derived zymosan were from Sigma. Methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL), and NS-398 were from Biomol (Plymouth Meeting, PA). The sPLA2 inhibitor LY311727 was generously provided by Dr. Edward Mihelich (Lilly Research Labs, Indianapolis, IN). iPLA2 antiserum was generously provided by Dr. Simon Jones (Genetics Institute, Cambridge, MA). Human recombinant group V sPLA2 was produced in our laboratory utilizing thePichia pastoris expression system (17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). P388D1cells (MAB clone) (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) 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 with [3H]AA was achieved by including 0.5 μCi/ml [3H]AA during the overnight adherence period (20 h). Labeled AA that had not been incorporated into cellular lipids was removed by washing the cells four times with serum-free medium containing 1 mg/ml albumin. The cells were placed in serum-free medium for 30 min before the addition of zymosan for different periods of time. Afterward, the supernatants were removed and cleared of detached cells by centrifugation, and PGE2 was quantitated using a specific radioimmunoassay (PersPective Biosystems, Framingham, MA). For [3H]AA release experiments, cells labeled with [3H]AA were used, and the incubations were performed 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. Zymosan was prepared exactly as described (18.Balsinde J. Fernández B. Solı́s-Herruzo J.A. Diez E. Biochim. Biophys. Acta. 1992; 1136: 75-82Crossref PubMed Scopus (59) Google Scholar). Briefly, zymosan particles were suspended in phosphate-buffered saline, boiled for 60 min, and washed three times. The final pellet was resuspended in phosphate-buffered saline at 20 mg/ml and stored frozen. Zymosan aliquots were diluted in serum-free medium and sonicated before addition to the cells. No PLA2activity was detected in the zymosan batches used in this study, as assessed by in vitro activity assays (19.Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar). A 20-base-long antisense corresponding to nucleotides 59–78 in the murine group VI iPLA2 sequence (20.Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) was utilized (ASGVI-18, 5′-CTC CTT CAC CCG GAA TGG GT). As a control, the sense complement of ASGVI-18 was used (SGV-18, 5′-ACC CAT TCC GGG TGA AGG AG). Both ASGVI-18 and SGVI-18 contained phosphorothioate linkages to limit degradation. We have previously described a procedure of transfection that involves long incubation periods of the cells with the oligonucleotides (21.Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In the current study, we have employed the procedure recently described by Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) in which the oligonucleotides are presented to the cells in a complex with a lipophilic carrier. The antisense and sense oligonucleotides were mixed with LipofectAMINE, and complexes were allowed to form at room temperature for 10–15 min. The complexes were then added to the cells, and the incubations were allowed to proceed for 24 h under standard cell culture conditions. The final concentrations of oligonucleotide and LipofectAMINE in the incubation medium were 1 μm and 10 μg/ml, respectively. Oligonucleotide treatment and culture conditions were not toxic for the cells as assessed by the Trypan blue dye exclusion assay and by quantitating adherent cell protein. The cells were lysed in a buffer consisting of 150 mm NaCl, 20 mm Tris-HCl, 0.5% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 20 μm leupeptin, 20 μm aprotinin, 100 mm sodium vanadate, pH 7.5. The homogenates were centrifuged at 500 × g for 5 min at 4 °C to separate nuclei. Samples (50 μg) were separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide gel) and transferred to Immobilon-P (Millipore). Nonspecific binding was blocked by incubating the membranes with a buffer consisting of 5% nonfat milk, 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 150 mm NaCl, and 0.1% Triton X-100 for 60 min. Membranes were then incubated with anti-iPLA2 antiserum at a 1:1000 dilution for 30 min and then treated with horseradish peroxidase-conjugated protein A (Amersham Pharmacia Biotech). Bands were detected by enhanced chemiluminiscence (ECL, Amersham Pharmacia Biotech). After the stimulations, the cell supernatants were taken off, and the cell monolayers were scraped with 0.5% Triton X-100. Total lipids were extracted according to the method of Bligh and Dyer (23.Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). Lipids were separated by thin-layer chromatography with n-hexane/diethyl ether/acetic acid (70:30:1, by vol). This system allows a good resolution among phospholipids, monoacylglycerol, DAG, free fatty acids, and triacylglycerol (24.Balsinde J. Dennis E.A. Eur. J. Biochem. 1996; 235: 480-485Crossref PubMed Scopus (29) Google Scholar). The plates were revealed by exposing them to iodine vapors, and the spot corresponding to DAG was cut out and assayed for radioactivity by liquid scintillation counting. Except for the data in Fig. 4, zymosan-stimulated AA release is expressed by subtracting the basal rate in the absence of agonist and inhibitor. These background values were in the range 2000–3000 cpm. Assays were carried out in duplicate or triplicate. Each set of experiments was repeated at least three times with similar results. Unless otherwise indicated, the data presented are from representative experiments. We have recently reported on the use of a P388D1 cell subclone, termed MAB, which manifests considerably higher [3H]AA release responses to LPS and PAF than the ATCC cell batch from which the MAB clone was derived (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). We have repeatedly been unable to detect AA release in response to yeast-derived zymosan from P388D1 cell batches directly obtained from the ATCC (15.Glaser K.B. Asmis R. Dennis E.A. J. Biol. Chem. 1990; 265: 8658-8664Abstract Full Text PDF PubMed Google Scholar, 25.Lister M.D. Glaser K.B. Ulevitch R.J. Dennis E.A. J. Biol. Chem. 1989; 264: 8520-8528Abstract Full Text PDF PubMed Google Scholar). Highlighting another striking difference between the MAB cells and their parent ATCC cells, Fig.1 A shows that the MAB cells do respond to yeast-derived zymosan by rapidly releasing [3H]AA to the extracellular medium. The concentration dependence of the effect of zymosan on [3H]AA release is shown in Fig. 1 B. Maximal effects were observed at zymosan concentrations between 0.25–0.5 mg/ml. From these data, a zymosan concentration of 0.5 mg/ml was chosen to be employed in all subsequent experiments. Fig.2 shows that the zymosan-induced AA release was strongly inhibited by MAFP a dual cPLA2/iPLA2 inhibitor. Complete inhibition of the response was observed at a MAFP concentration of 25 μm. To distinguish whether the inhibition of MAFP of AA release was because of either cPLA2 or iPLA2, we conducted studies with BEL, a compound that manifests a marked selectivity for inhibition of the iPLA2 versusthe cPLA2 (3.Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). BEL, at concentrations that we have previously shown to block P388D1 cell iPLA2 in vitro (26.Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8527-8531Crossref PubMed Scopus (257) Google Scholar), had no significant effect on zymosan-stimulated AA release (Fig.3 A). Failure of BEL to inhibit the AA release was not because of the inhibitor not being able to cross the plasma membrane, because under identical experimental conditions BEL did inhibit zymosan-induced DAG production in a concentration-dependent manner (Fig. 3 B). The latter effect most likely reflects the inhibition by BEL of the Mg2+-dependent phosphatidate phosphohydrolase (27.Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 31937-31941Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). In turn, the absence of an effect of BEL on zymosan-induced AA release indicates that the MAFP effects shown in Fig. 2 are because of inhibition of the cPLA2, not the iPLA2.Figure 3Effect of BEL on zymosan-induced [3H] AA release (A) and [3H] DAG accumulation (B). The cells were treated with the indicated concentrations of BEL for 30 min before the addition of zymosan (closed symbols), and the incubations proceeded for 1 h. Open circles denote control incubations, i.e. those that did not receive LPS. Afterward, AA release (A) was determined in the supernatants and DAG accumulation (B) was determined in the cell monolayers as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further substantiate the lack of a role for the iPLA2 in zymosan-stimulated AA release, we also examined the effects of an iPLA2 antisense oligonucleotide, which we have previously used to attenuate the levels of immunoreactive iPLA2 in P388D1 cells (21.Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Using this antisense, we detected a decrease in the immunoreactive iPLA2 protein of about 60%, as judged by Western blot (Fig.4 A). iPLA2-deficient cells, however, did not show any significant reduction of their capacity to release AA to the extracellular medium, either spontaneously or in response to zymosan (Fig. 4 B). Collectively, the data of Figs. 3 and 4 strongly suggest that the iPLA2 does not play a significant role in mediating agonist-induced AA mobilization in P388D1cells. To investigate the involvement of the sPLA2 in zymosan-stimulated AA release, we employed LY311727, a well known sPLA2 inhibitor (1.Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Crossref PubMed Scopus (531) Google Scholar). Treatment of the cells with 25 μm LY311727 had no appreciable effect on the zymosan-stimulated AA release (Fig.5). We have previously shown that this treatment leads to a marked reduction of the AA release response of the cells to LPS/PAF (3.Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Identical results were obtained with the use of CMPE (N-derivatized phosphatidylethanolamine covalently linked via the headgroup to carboxymethyl cellulose), another sPLA2 inhibitor that is structurally unrelated to LY311727 (28.Dan P. Dagan A. Krimsky M. Pruzanski W. Vadas P. Yedgar S. Biochemistry. 1998; 37: 6199-6204Crossref PubMed Scopus (22) Google Scholar), and that we have previously shown to strongly inhibit fatty acid release in LPS-activated P388D1 macrophages (29.Balsinde J. Balboa M.A. Yedgar S. Dennis E.A. J. Biol. Chem. 2000; 275: 4783-4786Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In keeping with these previous data, CMPE strongly inhibited the LPS/PAF-induced AA release but had no measurable effect on the response to zymosan (data not shown). These results, together with the finding that zymosan did not increase cellular group V sPLA2mRNA levels for periods of time up to 1 h, strongly suggest that sPLA2 has no role in zymosan-stimulated AA release. The priming effect of LPS on PAF-induced AA mobilization in P388D1 cells is thought to involve the increased synthesis of group V sPLA2 (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Interestingly LPS also primed the AA release in response to zymosan (Fig. 5). This effect was found to be inhibited by LY311727, implying the involvement of group V sPLA2 in the priming effect (Fig. 5). Likewise, the addition of exogenous group V sPLA2 to the zymosan-treated cells increased the AA release in a concentration-dependent manner (Fig. 6). Thus, when cellular sPLA2 levels are increased because of the LPS treatment or to the addition of exogenous enzyme, the zymosan response is accordingly increased. Collectively the data indicate that the lack of involvement of sPLA2 in the response triggered by zymosan alone is not because of zymosan suppression of endogenous sPLA2 activity. We have previously observed that in addition to COX-1, P388D1 macrophages constitutively express low levels of COX-2 (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Therefore it was of interest to examine which of these COX isoforms participates in the prostaglandin response of the cells to zymosan. Neither COX-1 nor COX-2 levels were changed during incubation of the cells for 1 h with zymosan, as assessed by immunoblot. We studied the effect of NS-398, a compound that inhibits COX-2 with an IC50 <5 μm, whereas COX-1 remains unaffected at concentrations higher than 100 μm (31.Futaki N. Takahashi S. Yokoyama M. Arai I. Higuchi S. Otomo S. Prostaglandins. 1994; 47: 55-59Crossref PubMed Scopus (801) Google Scholar). Fig.7 shows complete inhibition of zymosan-induced PGE2 production by NS-398, which indicates that COX-2 is responsible for the response. We have recently identified in P388D1 macrophages (MAB clone) the existence of two distinct pathways for AA mobilization and prostaglandin production. The first one, herein referred to as the “primed immediate phase” takes place in minutes and is elicited by the Ca2+-mobilizing agonist PAF but requires the cells to be exposed first to LPS for 1 h (3.Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 32.Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar). The second pathway, or “delayed phase,” is elicited by LPS for periods of time spanning several hours (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 33.Balboa M.A. Balsinde J. Johnson C.A. Dennis E.A. J. Biol. Chem. 1999; 274: 36764-36768Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Interestingly, both pathways utilize the same effectors, namely cPLA2, sPLA2, and COX-2, although the molecular mechanisms involved dramatically differ (3.Balsinde J. Dennis E.A. J. Biol. Chem. 1996; 271: 6758-6765Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar, 4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 32.Balsinde J. Barbour S.E. Bianco I.D. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11060-11064Crossref PubMed Scopus (127) Google Scholar, 33.Balboa M.A. Balsinde J. Johnson C.A. Dennis E.A. J. Biol. Chem. 1999; 274: 36764-36768Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In both of these routes the cPLA2appears to behave primarily as an initiator of the response, whereas the sPLA2 plays an augmentative role by providing most of the AA to be converted to prostaglandins via COX-2. In the current study we show that zymosan triggers AA mobilization and prostaglandin synthesis in the P388D1 macrophages by a pathway that is clearly different from the ones identified for LPS and PAF. Thus, zymosan elicits an “immediate” AA response whose unique features are that (i) it does not cease after a few minutes but goes on for longer times, (ii) takes place in the absence of LPS priming, and (iii) utilizes only the cPLA2 to effect the AA release. In common with the response to PAF, however, prostaglandin production in the zymosan-stimulated cells is also mediated by COX-2. Thus, in zymosan-stimulated cells cPLA2 couples directly to COX-2 for prostaglandin production. This is strikingly different from the situation in the PAF-stimulated cells, where the bulk of the prostaglandins is produced by a sPLA2/COX-2 coupling mechanism (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). Exposure of the P388D1 macrophages to LPS increases group V sPLA2 levels in a concentration-dependent manner and this appears to constitute a key event of LPS priming (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar,30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). We have found here that zymosan does not trigger the increased synthesis of new sPLA2. However, resting cells still contain appreciable amounts of sPLA2 (30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Thus it is not easy to envision the reasons for the zymosan not to signal to AA release via recruitment of the endogenous sPLA2 already present in the cell. We have previously observed that the sPLA2 pool located on the cell surface appears to be involved in the LPS/PAF-induced AA mobilization and prostaglandin production (19.Barbour S.E. Dennis E.A. J. Biol. Chem. 1993; 268: 21875-21882Abstract Full Text PDF PubMed Google Scholar, 30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). sPLA2 levels on the surface of the LPS/PAF-activated cells are higher than in resting unstimulated cells (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 30.Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). This increased sPLA2 expression can be blocked by actinomycin D (4.Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar), indicating the involvement of de novoprotein synthesis. We have failed to detect an increase in group V sPLA2 mRNA levels in cells stimulated by zymosan for periods of time up to 1 h. Thus, a tempting but yet speculative idea to explain the lack of sPLA2 involvement in the zymosan response would be that the stimulus fails to recruit the constitutive sPLA2 to the appropriate cell compartment,i.e. the cell surface. Interestingly, a recent report has shown that a major portion of the group V sPLA2 that murine mast cells constitutively express is found intracellularly located during the resting state (34.Bingham C.O. Fijneman R.J.A. Friend D.S. Goddeau R.P. Rogers R.A. Austen K.F. Arm J.P. J. Biol. Chem. 1999; 274: 31476-31484Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). P388D1 macrophages contain a third PLA2 type, the group VI iPLA2. By using chemical inhibitors and antisense approaches, we have shown that this enzyme does not seem to participate in the stimulation of AA release by zymosan. In agreement with our data, Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) recently reported that MAFP strongly inhibits the zymosan-stimulated AA release in P388D1 cells. Interestingly, Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) also showed that low BEL concentrations (2 μm) partially decreased the AA release (up to 40%). This finding led the authors to suggest that, in addition to the cPLA2, the iPLA2 is also involved in the response. At 2 μm, no effect of BEL on DAG levels was detected but an inhibitory effect on the response became evident at 5 μm, which is in agreement with the results of our study. The discrepancy between the data by Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and ours regarding BEL effects on AA release probably arises from the fact that Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) have used a heterogeneous P388D1 cell population for their studies, whereas we have employed a clone of these cells, termed MAB (5.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17.Balsinde J. Shinohara H. Lefkowitz L.J. Johnson C.A. Balboa M.A. Dennis E.A. J. Biol. Chem. 1999; 274: 25967-25970Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Closer examination of the results by Akibaet al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) reveals that the concentration-response curves for the inhibitory effects of BEL on zymosan-stimulated AA release and endogenous iPLA2 activity do not correspond (cf.Fig. 2, A and B, in Ref. 22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Maximal effects of BEL on AA release are found at 2 μm, but at this concentration, endogenous iPLA2 activity is only inhibited by 40%; BEL concentrations higher than 10 μm were found to be required for full iPLA2 inhibition (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, the BEL effects on zymosan-induced AA release reported by Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) are likely not because of inhibition of the iPLA2 but of another unidentified effector. Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) also utilized antisense technology to study the role of iPLA2 in their system. Surprisingly however, the functional consequences of iPLA2 antisense depletion were investigated on prostaglandin D2 production, not on the more direct analysis of AA release (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). A potential problem with measuring prostaglandin as a marker of phospholipase activation (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) is that any effect at step(s) distal from the phospholipolytic step cannot be distinguished from effects at the phospholipolytic step itself. We have confirmed in this study that the experimental conditions employed by Akiba et al. (22.Akiba S. Mizunaga S. Kume K. Hayama M. Sato T. J. Biol. Chem. 1999; 274: 19906-19912Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) result in a decrease in cellular iPLA2, as assessed by immunoblot. We have also found that iPLA2 depletion by antisense does not result in a decreased capacity of the cells to release AA to the incubation medium in response to zymosan. We previously observed that iPLA2 depletion by the same antisense oligonucleotide also has no effect on the AA release response induced by LPS/PAF (21.Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Our previous studies have suggested that the iPLA2 serves in a phospholipid remodeling role that involves the generation of lysophospholipid precursors for incorporation of AA into phospholipids (35.Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Such a housekeeping function for the iPLA2 was deduced from experiments involving inhibition of iPLA2activity with BEL (26.Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8527-8531Crossref PubMed Scopus (257) Google Scholar, 36.Balsinde J. Dennis E.A. Adv. Exp. Med. Biol. 1997; 407: 99-103Crossref PubMed Google Scholar) or with an antisense oligonucleotide (21.Balsinde J. Balboa M.A. Dennis E.A. J. Biol. Chem. 1997; 272: 29317-29321Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Importantly, our data have now been confirmed and extended by other laboratories (37.Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 9400-9408Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 38.Alzola E. Pérez-Etxebarria A. Kabré E. Fogarty D.J. Métioui M. Chaib N. Macarulla J.M. Matute C. Dehaye J.P. Marino A. J. Biol. Chem. 1998; 273: 30208-30217Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 39.Daniele J.J. Fidelio G.D. Bianco I.D. Prostaglandins. 1999; 57: 341-350Crossref PubMed Scopus (13) Google Scholar, 40.Barbour S.E. Kapur A. Deal C.L. Biochim. Biophys. Acta. 1999; 1439: 77-88Crossref PubMed Scopus (77) Google Scholar). Thus the role of iPLA2 in regulating basal phospholipid deacylation/reacylation reactions appears not to be restricted to the P388D1 cells but rather may represent a general homeostatic mechanism for the regulation of phospholipid levels. However, a recent study in pancreatic islets failed to detect a role for the iPLA2 in basal AA incorporation into the phospholipids of these cells (41.Ramanadham S. Hsu F.F. Bohrer A. Ma Z. Turk J. J. Biol. Chem. 1999; 274: 13915-13927Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is important to note that, as the authors themselves acknowledge (41.Ramanadham S. Hsu F.F. Bohrer A. Ma Z. Turk J. J. Biol. Chem. 1999; 274: 13915-13927Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), pancreatic islets exhibit some atypical features of AA incorporation in that the basal levels of both esterified AA and lysophospholipid in these cells are substantially higher than in other tissues (41.Ramanadham S. Hsu F.F. Bohrer A. Ma Z. Turk J. J. Biol. Chem. 1999; 274: 13915-13927Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Given the high levels and apparently slow turnover of lysophosphatidylcholine in these cells (41.Ramanadham S. Hsu F.F. Bohrer A. Ma Z. Turk J. J. Biol. Chem. 1999; 274: 13915-13927Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), the finding that lysophosphatidylcholine levels do not limit AA incorporation into islet phospholipids does not come as a surprise. Whether the observations with pancreatic islets represent another mechanism for incorporation of AA into phospholipids or merely reflect atypical features of a particular cell type is not yet certain. In summary, we have described in this work the existence of a third, immediate pathway for AA mobilization and prostaglandin production that operates via activation of the cPLA2 coupled to COX-2. Neither group V sPLA2 nor group VI iPLA2 appear to be involved. While this manuscript was under review, a report appeared (Gijón, M. A., Spencer, D. M., Siddiqi, A. R., Bonventre, J. V., and Leslie, C. C. (2000) J. Biol. Chem. 275, 20146–20156) showing that in macrophages obtained from cPLA2 knock-out mice, zymosan was unable to induce AA release. These results are fully consistent with the data presented in this manuscript and confirm the usefulness of the P388D1cells as a model for macrophage activation studies. We thank Dr. Satoshi Akiba (Kyoto Pharmaceutical University, Japan) for helpful discussions during the course of this work and Dr. Michelle Winstead for reading the manuscript." @default.
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• W2029317617 title "Identification of a Third Pathway for Arachidonic Acid Mobilization and Prostaglandin Production in Activated P388D1 Macrophage-like Cells" @default.
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