The perturbed membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A2 to liberate arachidonic acid

Biochimica et Biophysica Acta 1349 Ž1997. 43–54

The perturbed membrane of cells undergoing apoptosis is susceptible to type II secretory phospholipase A 2 to liberate arachidonic acid Gen-ichi Atsumi, Makoto Murakami, Masae Tajima, Satoko Shimbara, Noriko Hara, Ichiro Kudo ) Department of Health Chemistry, School of Pharmaceutical Sciences, Showa UniÕersity, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan Received 1 April 1997; revised 12 May 1997; accepted 20 May 1997

Abstract Several lines of evidence have suggested that the plasma membranes of cells elicited by proinflammatory stimuli or microvesicles shed from activated cells are sensitive to extracellular type II secretory phospholipase A 2 ŽsPLA 2 . that liberates fatty acids and lysophospholipids. Here we report that the membranes of cells undergoing apoptosis are highly susceptible to type II sPLA 2 . When neuronally differentiated rat pheochromocytoma PC12 cells deprived of nerve growth factor and serum, mouse mast cells deprived of hematopoietic cytokines or human monocytic U937 cells stimulated via Fas antigen Ža receptor for the death factor Fas ligand., were exposed to type II sPLA 2 at concentrations comparable to those detected at inflamed sites, the release of arachidonic acid was significantly accelerated in association with the process of programmed cell death. Arachidonic acid release by sPLA 2 was dependent on the extracellular Ca2q and was accompanied by preferential hydrolysis of phosphatidylethanolamine and phosphatidylserine in the membrane phospholipids. Association of sPLA 2 with cell surface proteoglycan, which has been shown to be a prerequisite for endogenous sPLA 2-dependent arachidonic acid release from the plasma membranes of live cells, was not essential for sPLA 2-mediated hydrolysis of apoptotic cell membranes. Taking these results together, the apoptotic cell membrane is a potential target for extracellular type II sPLA 2 . The present findings may be relevant to events occurring at inflammatory or ischemic disease sites where apoptotic cells accumulate. q 1997 Elsevier Science B.V. Keywords: Type II sPLA 2 ; Apoptosis; Arachidonic acid; Phospholipid

1. Introduction Abbreviations: BMMC, bone marrow-derived mast cells; cPLA 2 , cytosolic PLA 2 ; DAPI, 4X ,6X-diamino-2-phenylindole; IL, interleukin; JNK, c-jun N-terminal kinase; KL, c-kit ligand; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PLA 2 , phospholipase A 2 ; PS, phosphatidylserine; sPLA 2 , secretory PLA 2 ; TNF, tumor necrosis factor ) Corresponding author. Fax: q81 3 37848245.

Arachidonic acid is liberated from membrane phospholipids by the action of phospholipase A 2 ŽPLA 2 . w1,2x. The crucial role of 85 kDa cytosolic PLA 2 ŽcPLA 2 . in the rapid release of arachidonic acid after stimulus-coupled cell activation is well established. cPLA 2 preferentially liberates arachidonic acid following translocation from the cytosol to the nuclear envelope in response to stimulus-initiated

0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 0 8 2 - 9

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G.-i. Atsumi et al.r Biochimica et Biophysica Acta 1349 (1997) 43–54

transient increases in cytoplasmic Ca2q concentrations w3,4x and phosphorylation by mitogen-activated protein kinase ŽMAPK. w5x. Type II secretory PLA 2 ŽsPLA 2 ., one of the low-molecular-weight PLA 2 isozymes w6x, is often detected at sites of inflammation, is exocytosed from activated inflammatory cells, is induced by proinflammatory stimuli and downregulated by anti-inflammatory glucocorticoids, and exacerbates inflammation when injected in vivo Žreviewed in w1,2,7x.. Several lines of evidence suggest that type II sPLA 2 also participates in the generation of eicosanoids by various cell types under appropriate conditions w7–15x. Whereas quiescent cells usually show low sensitivity to exogenous type II sPLA 2 , the plasma membranes of cells activated by certain stimuli w7,8x or microvesicles shed from activated cells w9x become susceptible to it. Thus, it has been postulated that transcellular activation of eicosanoid biosynthesis by type II sPLA 2 w10x is important in certain tissue disorders, in which it acts as an extracellular signal transducer to promote expansion of local cellular responses to surrounding tissue microenvironments. In support of this hypothesis, recent in vivo studies using type II sPLA 2 inhibitors w11,16x and transgenic mice w17x have provided further evidence that this enzyme contributes to the exacerbation, rather than the initiation, of inflammatory responses. Apoptosis, also known as programmed cell death, involves cell shrinkage, nuclear condensation, and endonucleolytic cleavage of DNA into oligonucleosomal fragments, as well as plasma membrane blebbing w18x. Apoptosis is observed in a variety of physiologic and non-physiologic events, including negative selection of thymocytes in the thymus, CD8q T lymphocyte-mediated cytotoxicity, neuronal cell death after deprivation of neurotrophins, or hematopoietic cell death after cytokine deprivation w18x. Apoptosis is stimulated through receptors for death-inducing cytokines, such as Fas ligand and tumor necrosis factor Ž TNF. w19,20x. Identification of the signaling pathway from the Fas antigen or TNF receptor I on the plasma membranes to the intracellular ICErCED3 family of proteases resulting in the proteolysis of particular proteins has provided new insights into the molecular mechanisms leading to apoptotic events in the cytoplasm and nucleus w21x. The fact that apoptosis is accompanied by plasma membrane blebbing suggests that there are also significant changes in the

phospholipids that form a major component of the plasma membrane. Although sphingomyelin metabolism, leading to the formation of ceramide, has been extensively studied w22x, there is now a limited amount of information on changes in glycerophospholipid metabolism during apoptosis. In the present study we examined changes in glycerophospholipid metabolism induced in cells undergoing apoptosis by a particular PLA 2 isozyme, type II sPLA 2 . We show that cells undergoing apoptosis are highly sensitive to exogenous type II sPLA 2 , which hydrolyzes membrane phospholipids to liberate free arachidonic acid. This study not only identifies the PLA 2 isozyme that affects phospholipid metabolism during apoptosis at the molecular level, but also describes an alternative route for the supply of arachidonic acid by this particular extracellular enzyme, which is associated with inflammatory diseases.

2. Materials and methods 2.1. Materials Rat type II sPLA 2 was purified to near homogeneity from rat platelets by immuno-affinity chromatography, as described previously w23x. Recombinant mouse type II sPLA 2 and its mutant KE4, which has reduced affinity for heparin w24x, were expressed in baculovirus-transfected Sf9 cells and purified by sequential heparin-Sepharose ŽPharmacia, Uppsala, Sweden. and anti-mouse type II sPLA 2 antibodyconjugated column chromatography, as described previously w25x. Mouse nerve growth factor Ž NGF. 2.5S was purchased from Chemicon International ŽTemecula, CA, USA. . Recombinant mouse interleukin Ž IL.-3, IL-4, IL-10 and c-kit ligand ŽKL. , cytokines known to promote the proliferation of mast cells, were expressed in baculovirus-transfected Sf9 cells, as described previously w26x. Anti-Fas antibody ŽCH-11. w27x was purchased from MBL Ž Nagoya, Japan. . 2.2. Cell culture Rat pheochromocytoma PC12 cells Ž Health Science Research Resources Bank. were maintained in

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RPMI 1640 medium ŽNissui Pharmaceutical, Tokyo, Japan. supplemented with 10% horse serum and 5% fetal calf serum. For neuronal differentiation, 2 = 10 4 PC12 cellsrcm2 were cultured with 5 ngrml NGF in collagen-coated 60 mm culture dishes ŽIwaki, Funabashi, Chiba, Japan. for 6 days. A mouse IL-3-dependent BMMC line, MC-MKM w28x, was maintained in 50% enriched medium ŽRPMI 1640 containing 10% fetal calf serum, 2 mM glutamine, 100 Urml penicillin, 100 m grml streptomycin, 50 m grml gentamicin and 0.1 mM non-essential amino acids. with 50% WEHI-3B ŽJapanese Cancer Research Resources Bank. -conditioned medium as a source of IL-3, as described previously w26x. Human monocytic U937 cells ŽRIKEN Cell Bank. were maintained in enriched medium. 2.3. Induction of apoptosis in PC12 cells After culture for 1 week with 5 ngrml NGF in collagen-coated dishes w29x, neuronally differentiated PC12 cells were incubated for 24 h with 0.1 m Cirml w 3 Hxarachidonic acid ŽDuPont NEN, Boston, MA, USA. or 0.6 m Cirml w 3 Hxoleic acid ŽDuPont NEN.. After three washes with serum-free RPMI 1640, the cells were dispersed in serum-free RPMI 1640 using a cell scraper, washed five times with serum-free RPMI 1640, seeded into 96-well plates at 1 = 10 7 cellsrml and cultured for various periods Žup to 24 h. in serum-free RPMI 1640 in the presence or absence of NGF andror rat type II sPLA 2 . In some experiments, mouse type II sPLA 2 , its mutant KE4 w24x, or 5 mM EGTA were also added. The process of cell death was monitored by morphological changes and trypan blue dye exclusion. Apoptosis was quantified using 4X ,6X-diamino-2-phenylindole Ž DAPI; Sigma, St. Louis, MO, USA. as described previously w30x. The free 3 H-labeled fatty acids released were extracted by the method of Dole and Meinertz w31x, and resultant radioactivity was counted in a liquid b-scintillation counter. 2.4. Induction of apoptosis in BMMC BMMC, maintained in 50% WEHI-CM, were adjusted to 1 = 10 6 cellsrml and preincubated with 1 m Cirml w 3 Hxarachidonic acid or w 3 Hxoleic acid for 24 h at 378C. The cells were then washed three times

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with enriched medium, resuspended in enriched medium with or without recombinant cytokines at 2.5 = 10 6 cellsrml, and incubated for various periods Žup to 24 h. in the presence or absence of rat type II sPLA 2 . The concentrations of cytokines used were 100 Urml, 6 ngrml, 100 Urml and 10 ngrml of IL-3, IL-4, IL-10 and KL, respectively. The viability of BMMC was assessed by trypan blue dye exclusion. To investigate DNA fragmentation, cells cultured under various conditions were washed and resuspended in lysis buffer composed of 10 mM TrisHCl ŽpH 7.4. containing 0.1 M NaCl, 1 mM EDTA and 0.3% SDS. The lysate was incubated with 100 m grml proteinase K Ž Boehringer Mannheim, Mannheim, Germany. and incubated overnight at 558C. DNA was extracted with phenolrchloroform, precipitated with ethanol and reconstituted in 10 mM Tris-HCl ŽpH 8.0. and 1 mM EDTA. A portion of this DNA was separated on 1% agarose gel containing ethidium bromide. Electrophoresis was conducted for 1 h at 100 V, and the resulting gel was viewed and photographed under UV light. The free 3 H-labeled fatty acids released were assessed by Dole’s extraction w31x as above. 2.5. Induction of apoptosis in U937 cells U937 cells were preincubated with 0.1 m Cirml w 3 Hxarachidonic acid for 24 h, washed three times, and resuspended in enriched medium with or without anti-Fas antibody at 1 = 10 7 cellsrml. After 12 h of culture, rat type II sPLA 2 was added and culture continued for up to 12 h more. Apoptosis was monitored by trypan blue dye exclusion and DNA fragmentation and the release of free w 3 Hxarachidonic acid was examined by Dole’s extraction w31x as above. 2.6. Two-dimensional thin layer chromatography Total lipids from w 3 Hxarachidonic acid-labeled cells and their supernatants were extracted by the method of Bligh and Dyer w32x and developed by two-dimensional thin layer chromatography on silica 60 gel plates ŽMerck, Darmstadt, Germany. according to a previously described method w28x with a slight modification. The first solvent system consisted of chloroformrmethanolracetic acidrwater Ž 65:25:4:2, vrvrv., followed by a second solvent system of

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chloroformrmethanolrformic acid Ž 65:25:8.8, vrvrv.. The zones on the silica gel corresponding to arachidonic acid and phospholipids were identified by comparison with the mobility of authentic standards, visualized with iodine vapor. Each zone was scraped into a vial, and radioactivity was counted in a liquid b-scintillation counter.

3. Results 3.1. Type II sPLA 2 releases arachidonic acid from NGF- and serum-depriÕed PC12 neuronal cells To investigate the function of type II sPLA 2 in arachidonate metabolism in neuronal cells, rat pheochromocytoma PC12 cells that had been stimulated to differentiate into sympathetic neuron-like cells by culture for 1 week with NGF were prelabeled with w 3 Hxarachidonic acid and exposed to 10 m grml rat type II sPLA 2 for an additional 24 h to assess any changes in free w 3 Hxarachidonic acid levels. Whereas no appreciable change in arachidonic acid release was found in cells maintained in the continued presence of NGF with or without sPLA 2 , replicate cells deprived of NGF and serum for 24 h exhibited a significant increase in arachidonic acid release. These increases reached 48.3 " 5.8% and 12.0 " 2.3% Žmean " S.E. Ž n s 7., P - 0.05 vs. 0 h. with and without sPLA 2 , respectively Ž Fig. 1. . Thus, arachidonic acid release from NGF-deprived cells after sPLA 2 addition was about 13-fold greater than in cells before sPLA 2 addition Ž0 h. and 4-fold greater than in cells cultured for 24 h in the absence of both NGF and sPLA 2 ŽFig. 1. . Withdrawal of the trophic support provided by NGF from neuronally differentiated PC12 cells and primary sympathetic neurons triggers apoptotic death w29,33,34x. PC12 cells cultured with NGF remained viable and extended neurites over the collagen-coated plates ŽFig. 2A, left., whereas those cultured in the absence of NGF exhibited a rounded shape Ž Fig. 2A, right.. The number of trypan blue-positive cells increased Ž approx. 70% at 24 h.. When apoptosis was quantified by a fluorometric method using DAPI, DNA fragmentation was undetectable during the initial 6 h, then increased progressively over 24 h. The increase reached 16% at 12 h and 62% at 24 h ŽFig.

Fig. 1. The effect of sPLA 2 on arachidonic acid release from neuronally differentiated PC12 cells. w 3 HxArachidonic acid-prelabeled PC12 cells, which had been cultured for 1 week with NGF, were cultured for an additional 24 h under various conditions Žcolumns B–H. and w 3 Hxarachidonic acid release was assessed as described in Section 2. A, before incubation Ž ns 7.; B, medium alone Ž ns 7.; C, 10 m grml rat sPLA 2 Ž ns 7.; D, 10 m grml rat sPLA 2 plus 5 mM EGTA Ž ns 4.; E, 5 ngrml NGF Ž ns 3.; F, 5 ngrml NGF plus 10 m grml rat sPLA 2 Ž ns 3.; G, 10 m grml mouse sPLA 2 Ž ns6.; H, 10 m grml KE4 Ž ns6.. Values are expressed as means"S.E.

2B.. Release of arachidonic acid from NGF-deprived PC12 cells increased only modestly over 24 h of culture. Further addition of 10 m grml rat type II sPLA 2 to replicate cells dramatically increased the level of arachidonic acid released, increasing from 5% after 6 h to 50% after 24 h of culture ŽFig. 2C. and plateauing thereafter Ždata not shown. . These kinetic studies revealed the close correlation between the ability of sPLA 2 to liberate arachidonic acid and the process of apoptosis in PC12 cells following NGF depletion. sPLA 2 addition did not alter the number of trypan blue-positive cells appreciably as assessed at 24 h Ždata not shown.. If the effect of sPLA 2 is a direct one on apoptotic cell membranes, it should be possible to increase the arachidonate release by adding the sPLA 2 into the cells only for the last hours of the culture. Thus, PC12 cells were precultured for 12 h in the absence of NGF to induce apoptosis and then incubated for an additional 12 h with sPLA 2 . In this setting, arachidonic acid release reached approx. 55% after addition of sPLA 2 . The concentration of rat type II sPLA 2 used in the kinetic studies Ž Fig. 2C. was based on the dose-response experiments depicted in Fig. 3. When as-

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tration of 1 m grml and reached a maximum at 5–10 m grml ŽFig. 3.. When similar experiments were performed with PC12 cells prelabeled with w 3 Hxoleic acid, net oleate release 24 h after withdrawal of NGF was about 20% by the addition of 10 m grml sPLA 2 , revealing that sPLA 2-induced fatty acid release from apoptotic PC12 cells was not selective for arachidonic acid. Addition of 5 mM EGTA to the medium to remove extracellular Ca2q, which is essential for the catalytic action of sPLA 2 w1,2,6x, totally abolished the effect of rat type II sPLA 2 on NGF-deprived PC12 cells Ž 6.4 " 1.9% Ž n s 4., P - 0.05 vs. NGFdeprived cells cultured in the presence of sPLA 2 without EGTA. ŽFig. 1.. Recombinant mouse type II sPLA 2 released arachidonic acid from NGF-deprived cells as effectively as the rat enzyme Ž50.8 " 9.3%, n s 6. ŽFig. 1., revealing a lack of species specificity. Mouse type II sPLA 2 mutant KE4, which does not bind to cell surface proteoglycan owing to the replacement of four Cys residues with Glu in the C-terminal domain w24x, released arachidonic acid from NGF-deprived PC12 cells to a similar extent as the native enzyme Ž 55.8 " 6.0%, n s 6. Ž Fig. 1.. After culture of NGF-deprived PC12 cells with or without type II sPLA 2 , total lipid was extracted from the cells, and phospholipids were separated by twodimensional thin layer chromatography and quanti-

Fig. 2. Time dependence of the effect of sPLA 2 on arachidonic acid release from neuronally differentiated PC12 cells undergoing apoptosis. ŽA. Morphology of neuronally differentiated PC12 cells before Žleft. and 24 h after Žright. removal of NGF and serum Ž=100.. ŽB. Quantification of DNA fragmentation using DAPI. ŽC. Release of w 3 Hxarachidonic acid from NGF-deprived PC12 cells in the presence Žclosed circles. or absence Žopen circles. of 10 m grml rat type II sPLA 2 . Values are expressed as the mean"S.D. of seven independent experiments.

sessed after 24 h of culture, rat type II sPLA 2-dependent liberation of arachidonic acid from NGF-deprived PC12 cells consistently increased at a concen-

Fig. 3. Dose dependence of the effect of sPLA 2 on arachidonic acid release from neuronally differentiated PC12 cells undergoing apoptosis. Neuronally differentiated PC12 cells were deprived of NGF and serum and cultured for 24 h in the presence of the indicated concentrations of rat type II sPLA 2 . Release of w 3 Hxarachidonic acid was then assessed. Values are expressed as the mean"S.E. of four independent experiments.

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Table 1 Changes in the phospholipid composition after incubation with sPLA 2 in PC12 cells undergoing apoptosis Lipids

sPLA 2 Žy.

sPLA 2 Žq.

SM PC PI PS PE PA AA Others

0.43"0.13 17.71"2.93 11.50"2.06 1.43"0.37 21.31"3.09 0.76"0.07 22.81"4.85 24.04"4.32

0.20"0.04 10.77"1.94 8.53"2.02 0.49"0.07 ) 7.84"2.00 ) 0.71"0.14 54.50"3.39 ) 16.96"4.68

Neuronally differentiated PC12 cells prelabeled with w 3 Hxarachidonic acid for 24 h were cultured with or without 10 m grml type II sPLA 2 in the absence of NGF for an additional 24 h. After separation of lipids by two-dimensional thin layer chromatography, the radioactivity associated with each spot was visualized using iodine vapor and measured. SM, sphingomyelin; PC, phosphatidylcholine; PI, phosphatidylinositol; PS, phosphatidylserine; PE, phosphatidylethanolamine; PA, phosphatidic acid; AA, arachidonic acid; others, sum of other lipids including neutral lipids and lysophospholipids. Results Ž% radioactivity associated with each lipid relative to the radioactivity incorporated into total lipids. are expressed as the mean"S.E. of six Žwithout sPLA 2 . and eight Žwith sPLA 2 . independent experiments. ) P - 0.05 vs. without sPLA 2 .

fied by measuring the associated radioactivity. As shown in Table 1, the increase in free arachidonic acid produced by sPLA 2 was accompanied by preferential decreases in phosphatidylethanolamine Ž PE. and phosphatidylserine ŽPS., each of which exhibited

Fig. 4. Effect of sPLA 2 on BMMC undergoing apoptosis following deprivation of IL-3. ŽA. Fragmentation of chromosomal DNA 6 and 24 h after withdrawal of IL-3. Each experiment was carried out in duplicate. MW, molecular weight marker. ŽB,C. Time course of changes in arachidonic acid release and cell viability of BMMC treated with type II sPLA 2 , with or without IL-3. BMMC prelabeled with w 3 Hxarachidonic acid were cultured for the periods indicated with 100 Urml IL-3 Žopen squares., 100 Urml IL-3 plus 10 m grml type II sPLA 2 Žclosed squares., 10 m grml type II sPLA 2 Žclosed circles., or in their absence Žopen circles.. Cell viability ŽB. and arachidonic acid release ŽC. were assessed as described in Section 2. ŽD. Dose dependence on sPLA 2 . BMMC prelabeled with w 3 Hxarachidonic acid were cultured for 12 h either with or without IL-3, in the presence of the concentrations of type II sPLA 2 indicated and arachidonic acid release was assessed. Values are expressed as the mean"S.E. of four ŽB,C. or three ŽD. independent experiments.

a reduction of about 65% relative to cells cultured in the absence of sPLA 2 . Phosphatidylcholine Ž PC., phosphatidylinositol ŽPI. and phosphatidic acid Ž PA.

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also decreased consistently, although these changes were not significant. 3.2. Type II sPLA 2 releases arachidonic acid from cytokine-depriÕed mast cells BMMC, grown in WEHI-CM as a source of IL-3, are dependent on several cytokines for their growth and survival in culture w35x. BMMC cultured with recombinant IL-3 maintained their viability, whereas those cultured in the absence of IL-3 became progressively less viable over 24 h, with viabilities of 46 " 5% and 25 " 3% Žmean " S.E. Ž n s 4., P - 0.05 vs. cells cultured with IL-3 at each time point. at 12 and 24 h, respectively ŽFig. 4B. . The characteristic changes of apoptosis were demonstrated by DNA fragmentation into oligonucleosomal fragments after deprivation of IL-3 ŽFig. 4A. . Addition of 10 m grml type II sPLA 2 to BMMC cultured with or without IL-3 did not appreciably affect their viability under each culture condition ŽFig. 4B. . After incubation with type II sPLA 2 in culture without IL-3, levels of free arachidonic acid increased from 1.0 " 0.1% to 8.0 " 4.5% at 12 h and to 21.5 " 6.8% at 24 h Žmean " S.E. Ž n s 4., P - 0.05 vs. cells cultured with IL-3 at each time point. Ž Fig. 4C. . In the absence of type II sPLA 2 , these levels did not increase appreciably up to 12 h, and then increased non-significantly to 7% by 24 h. In contrast, release of arachidonic acid from BMMC increased only minimally when type II sPLA 2 was added in the continued presence of IL-3 ŽFig. 4C. . Liberation of arachidonic acid from IL-3-deprived BMMC was dependent on the concentration of type II sPLA 2 ; when assessed after 12 h of culture, at which time there was no appreciable increase in the release of arachidonic acid from BMMC in the absence of type II sPLA 2 ŽFig. 4C. , a significant effect became evident at concentrations greater than 1 m grml and reached a maximum at 10 m grml ŽFig. 4D.. Similar results were obtained when w 3 Hxoleate-labeled cells were used Ždata not shown. . The three mast cell-poietic cytokines tested w36–38x each prevented apoptosis of BMMC following WEHI-CM deprivation. The viability of BMMC 12 h after WEHI-CM deprivation was around 90% when cultured with recombinant mouse IL-3, IL-4 or KL and 42% in their absence ŽFig. 5A.. Type II sPLA 2

Fig. 5. Prevention of sPLA 2-mediated arachidonic acid release from apoptotic BMMC by mast cell-poietic cytokines. BMMC prelabeled with w 3 Hxarachidonic acid were cultured for 12 h with recombinant mouse IL-3 Ž100 Urml., IL-4 Ž6 ngrml., KL Ž10 ngrml. or IL-10 Ž100 Urml., or in their absence, either with Žclosed bars. or without Žopen bars. 10 m grml type II sPLA 2 . Cell viability ŽA. and arachidonic acid release ŽB. were assessed as described in Section 2. Values are expressed as the mean"S.E. of four independent experiments. ) P - 0.05 vs. BMMC cultured in the presence of IL-3 alone.

had no significant effect on the viability of BMMC following each treatment. Although type II sPLA 2 increased arachidonate release only minimally from BMMC maintained by these cytokines, it increased release markedly when BMMC were cultured without cytokines Žup to approx. 8% of total release. ŽFig. 5B.. In the presence of recombinant mouse IL-10 w37x, which partly suppressed apoptosis in BMMC Ž64% viability after 12 h of culture., type II sPLA 2 increased arachidonic acid release to some extent Ž up to approx. 5%. . Two-dimensional thin layer chromatography revealed that when PLA 2 was added to the culture

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without IL-3, arachidonic acid levels increased significantly from 3.9 to 8.8%, and there was a concurrent significant decrease in the percentage of total w 3 Hxarachidonic acid contained in PE and to a lesser extent in PS, without any appreciable changes in other phospholipids ŽTable 2.. When BMMC were cultured in the continued presence of IL-3, type II sPLA 2 caused no change in lipid composition Ždata not shown.. 3.3. Type II sPLA 2 augments arachidonic acid release by U937 cells undergoing Fas-mediated apoptosis Whereas ‘passive’ apoptosis of neuronal and hematopoietic cells after removal of supporting cytokines provided a useful tool for demonstrating this novel aspect of type II sPLA 2 function, whether the cells undergoing ‘active’ apoptosis, which is induced by death factors and is more relevant to physiological andror pathological events, would become susceptible to type II sPLA 2 remains to be elucidated. Therefore our last experiment investigated the effect of type II sPLA 2 on arachidonic acid release from cells undergoing active apoptosis after stimulation of Fas antigen. Crosslinking of Fas by the agonistic anti-Fas antibody w27x induced dose-dependent apoptosis in huTable 2 Changes in the phospholipid composition after incubation with sPLA 2 in BMMC undergoing apoptosis Lipids

sPLA 2 Žy.

sPLA 2 Žq.

SM PC PI PS PE PA AA

0.57"0.18 20.95"6.86 12.88"9.68 5.94"1.53 54.08"2.01 1.65"0.33 3.89"1.03

0.42" 0.10 21.83" 7.68 14.29"10.01 4.60" 0.79 ) 48.53" 1.06 ) 1.52" 0.23 8.78" 3.59 )

BMMC prelabeled with w 3 Hxarachidonic acid for 24 h were cultured with or without 10 m grml type II sPLA 2 in the absence of IL-3 for an additional 24 h. After separation of lipids by two-dimensional thin layer chromatography, the radioactivity associated with each spot was visualized using iodine vapor and measured. Results Ž% radioactivity associated with each lipid relative to the radioactivity incorporated into total lipids. are expressed as the mean"S.E. of four independent experiments. ) P - 0.05 vs. without sPLA 2 . Abbreviations as for Table 1.

Fig. 6. Effect of sPLA 2 on U937 cells undergoing apoptosis following treatment with anti-Fas antibody. w 3 HxArachidonic acid-prelabeled U937 cells were cultured for 12 h with 0, 5 and 50 ngrml anti-Fas antibody. The cells were then incubated with Žclosed bars. or without Žopen bars. 10 m grml rat type II sPLA 2 in the continued presence of the same amounts of anti-Fas antibody up to 24 h to assess release of w 3 Hxarachidonic acid. Values are expressed as the mean"S.E. of three independent experiments.

man monocytic leukemia U937 cells as evidenced by DNA ladder formation Ž data not shown. . Arachidonic acid release increased significantly after culture with 50 ngrml anti-Fas antibody alone, reaching 15.4 " 2.3% at 24 h Ž Fig. 6.. When type II sPLA 2 was added to cells that had been cultured for 12 h with 50 ngrml anti-Fas antibody, at which time about 45% of the cells were trypan blue-positive Ždata not shown., release of arachidonic acid was further augmented by 26.8 " 6.6% at 24 h, a 1.7-fold augmentation relative to cells treated with 50 ngrml anti-Fas antibody alone and a 6-fold increase over those cultured without anti-Fas antibody ŽFig. 6.. A minimal increase in arachidonic acid release was produced by as little as 5 ngrml anti-Fas antibody, at which concentration little DNA fragmentation occurred Ž Fig. 6. . Thus, the effect of type II sPLA 2 on arachidonic acid release was dependent on Fas-induced apoptosis. It should be noted that although arachidonic acid release increased significantly in cells treated with the anti-Fas antibody alone, this was not due to the autocrine function of endogenous type II sPLA 2 , since its mRNA was barely detectable in U937 cells and the PLA 2 inhibitor p-bromophenacyl bromide did not affect

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arachidonic acid release from anti-Fas antibodytreated cells Ždata not shown..

4. Discussion Whereas apoptosis has been shown to be accompanied by substantial changes in cytosolic and nuclear proteins and DNA w18x, little is known as to how glycerophospholipid metabolism is affected. Whilst some investigators have shown that PLA 2 inhibitors such as p-bromophenacyl bromide and quinacrine suppress apoptosis to some extent w39–41x, the utilization of these non-specific inhibitors will preclude investigation of the role of PLA 2 isoenzymes in the apoptotic process. Others have demonstrated that the uneven arachidonic acid redistribution between glycerophospholipid subclasses, which is caused by the inhibition of CoA-independent transacylase, is correlated with apoptosis w42x. One report of a TNF-resistant subline of L929 cells, which showed reduced expression of cPLA 2 , regaining a sensitive phenotype after overexpression of transfected cPLA 2 w43x is the convincing example of the correlation between arachidonic acid release and apoptosis at the molecular level. Type II sPLA 2 , a member of the lowmolecular-weight PLA 2 family, is released from cells activated by various stimuli and its accumulation often correlates with disease pathology w2,6x. Its proinflammatory action has been reported to be at least in part due to the promotion of eicosanoid generation by target cells exposed to inflammatory stimuli w7–15x. We have now shown that PC12 neuron-like cells deprived of NGF, mast cells deprived of hematopoietic cytokines, and anti-Fas antibodytreated U937 monocytic leukemia cells, all of which display the classical changes of apoptosis, become sensitive to type II sPLA 2-mediated membrane glycerophospholipid hydrolysis and liberate arachidonic acid. The fact that three different systems have yielded similar results strongly suggests that apoptotic cells are potential pathophysiological targets for extracellular type II sPLA 2 . When these apoptotic cells were cultured in the presence of type II sPLA 2 , release of arachidonic acid increased markedly in a time- and dose-dependent manner. Since oleic acid is released to a similar extent to arachidonic acid, it seems unlikely that

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phospholipid hydrolysis is mediated indirectly by the intracellular activation of cPLA 2 , which exhibits a marked preference for arachidonic acid w3x. The concentrations of type II sPLA 2 required for fatty acid release from apoptotic cells were 1–10 m grml. These levels are comparable to those required for immediate activation of various cell types when combined with a co-stimulator w7,8x and to those detected at inflamed sites w2,6x. These results suggest that although type II sPLA 2 has little effect on the plasma membranes of intact cells, it recognizes and degrades the ‘perturbed’ structure of apoptotic cell membranes. Since incorporation of arachidonic acid into cells decreased by half during apoptosis, increase in free arachidonic acid level may be a reflection of both increased release and decreased incorporation into cells. Recent studies have demonstrated that the apoptotic death process in NGF-deprived PC12 cells requires activation of the c-jun N-terminal kinase Ž JNK. pathway, which presumably leads to the transcription of particular genes involved in apoptosis w33,34x, and of the caspase ŽICErCED-3. family of proteases, which appears to be regulated independently of the JNK pathway w34x. The MAPK pathway is crucial for the continued survival of hematopoietic cells and its interruption following cytokine withdrawal leads to apoptosis of these cells w44x. Fas-induced active apoptosis occurs through interaction of the death domaincontaining proteins with caspase family proteases and does not require new protein synthesis w45–47x. Nonetheless, since type II sPLA 2 could liberate fatty acids in these three different situations, changes in the plasma membrane structure which lead to the acquisition of sensitivity to type II sPLA 2 may lie downstream from the common events following various apoptotic stimuli. Phospholipid analysis revealed that, among the three major glycerophospholipid components in apoptotic cells, hydrolysis of PE and PS occurred in preference to that of PC or PI Ž Tables 1 and 2. . This agrees with the substrate specificity demonstrated by type II sPLA 2 in in vitro enzyme assays using dispersed phospholipid substrates w2,6x. The preferential hydrolysis of these acidic phospholipids, which are normally distributed in the inner leaflet of the plasma membrane bilayer w48x, by extracellular type II sPLA 2 in apoptotic cells suggests that these acidic phospholipids translocate to the outer leaflet during apoptosis

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so as to be accessible to type II sPLA 2 . This hypothesis is supported by the fact that macrophage recognition of apoptotic cells for phagocytosis depends on the availability of external PS w49x and that externalization of PS is preceded by activation of the caspase family of proteases w50x, a general event occurring in the process of apoptosis w21x. Of note, a type II sPLA 2 mutant, which lacks the C-terminal cell surface proteoglycan-binding domain but retains intact catalytic activity toward dispersed phospholipid substrates w24x, released arachidonic acid from apoptotic PC12 cells as effectively as the native enzyme Ž Fig. 1.. This observation is in marked contrast to our recent finding that this proteoglycan-binding domain is essential for the action of endogenous type II sPLA 2 on live cells, which augments cytokine-induced delayed eicosanoid generation w24x. Thus, type II sPLA 2 may recognize the plasma membrane phospholipids of apoptotic cells in a similar way to dispersed phospholipids, the hydrolysis of which preferentially occurs in acidic phospholipids and does not require proteoglycan anchoring. Fourcade et al. w9x reported that type II sPLA 2 is stimulated by certain sphingomyelin degradation products in membrane microvesicles shed from activated platelets or erythrocytes. The observations that sphingomyelin degradation products induce apoptosis w22x, and that death factors such as TNF w51x or Fas ligand w52x stimulate sphingomyelin hydrolysis may provide an alternative explanation as to why cells undergoing apoptosis become susceptible to the action of type II sPLA 2 . Indeed, TNF does render some cells more sensitive to type II sPLA 2 w8,12x, suggesting the presence of a common signaling pathway for ‘membrane rearrangement’ involving sphingomyelin turnover, which could be responsible for type II sPLA 2 sensitivity. It has been reported that cPLA 2 may play a crucial role in TNF-induced apoptosis w41,43x. In contrast to these studies, more recent evidence suggests that the TNF signaling pathway leading to apoptosis is dissociated from pathways linked to the activation of NFk B and JNK w53x. This would imply that cPLA 2 is not involved in apoptosis, since cPLA 2 activation is generally believed to be coupled with the MAPKrJNK pathway w5,54x. Indeed, the facts that IL-1, a potent activator of NFk B and JNK and therefore cPLA 2 , does not induce apoptosis w55x and

that Fas ligand, which shares the apoptosis pathway with TNF w56x but unlike TNF minimally activates the NFk B and JNK pathways, fails to activate cPLA 2 w57x provide support for the dissociation of cPLA 2 activation from apoptosis. Consistent with this, pbromophenacyl bromide, which inactivates both sPLA 2 and cPLA 2 w2x, failed to suppress arachidonic acid release from U937 cells induced by the anti-Fas antibody alone ŽAtsumi, G. et al., unpublished data. . However, the possibility still remains that cPLA 2 could participate in certain stages of apoptosis in some way, for example through the ceramide-dependent pathway, which appears to represent an alternative route for TNF-induced apoptosis w51x and to be associated with the MAPK pathway through activation of ceramide-activated protein kinaseŽ s. which links to Raf-1 w58x. cPLA 2 has also been implicated in H 2 O 2-induced cell death, although how it participates in cytotoxicity in this situation remains uncertain w59x. This study has identified type II sPLA 2 as an enzyme that can modulate glycerophospholipid metabolism in cells undergoing apoptosis. It is possible that the arachidonic acid released from apoptotic cells is delivered to neighboring cells and is then converted into prostanoids. Indeed, arachidonic acid released from apoptotic BMMC by type II sPLA 2 could be supplied to live BMMC, which metabolize it to prostaglandin D 2 via cyclooxygenase-1 Ž data not shown.. This provides a new insight into the target cells used by type II sPLA 2 to provide arachidonic acid at sites of inflammation or ischemic tissues, where both type II sPLA 2 and apoptotic cells accumulate in high concentrations. Our present finding appears to be compatible with the recent report by Kolko et al. w60x, who showed that several sPLA 2 s from bee and snake venom elicited arachidonate release in association with glutamate-induced death of primary cortical neurons.

Acknowledgements We thank K. Yamakawa, S. Yanagimoto and N. Gotoh for their technical assistance. This work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.

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