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The Journal of Immunology, 2006, 176: 2555-2561.
Copyright © 2006 by The American Association of Immunologists

Involvement of Group VIA Calcium-Independent Phospholipase A2 in Macrophage Engulfment of Hydrogen Peroxide-Treated U937 Cells1

Rebeca Pérez, María A. Balboa and Jesús Balsinde2

Institute of Molecular Biology and Genetics, Spanish Research Council and University of Valladolid School of Medicine, Valladolid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hydrogen peroxide-induced apoptosis of U937 cells results in substantial hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A2 (iPLA2-VIA). However, abrogation of cellular iPLA2-VIA neither delays nor decreases apoptosis, suggesting that, beyond a mere destructive role, iPLA2-VIA may serve other specific roles. In this study, we report that phagocytosis of apoptosing U937 cells by macrophages is blunted if the cells are depleted of iPLA2-VIA by treatment with an inhibitor or an antisense oligonucleotide, and it is augmented by overexpression of iPLA2-VIA in the dying cells. Thus, the magnitude of macrophage phagocytosis correlates with the level of iPLA2-VIA activity of the dying cells. Eliminating by antisense oligonucleotide technology of cytosolic group IVA phospholipase A2 does not attenuate phagocytosis of U937 dying cells by macrophages. Incubation of U937 cells with different fatty acids has no effect on either the extent of hydrogen peroxide-induced apoptosis or the degree of phagocytosis of the dying cells by macrophages. However, preincubation of the macrophages with lysophosphatidylcholine before exposing them to the dying cells blocks phagocytosis of the latter. These results indicate that formation of lysophosphatidylcholine by iPLA2-VIA in hydrogen peroxide-treated U937 cells to induce apoptosis directly contributes to their efficient clearance by macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Apoptosis is a type of cell death that does not involve an inflammatory response and occurs in a tightly controlled manner. In contrast with cell death by necrosis, apoptosis involves the activation of catabolic mediators and enzymes before cytolysis. Many cellular changes that take place during apoptosis play important roles in the safe and efficient recognition and clearance of the dying cells by neighboring phagocytes. One of these changes is the scrambling of plasma membrane phospholipids, which results in loss of membrane asymmetry and the exposure of anionic phospholipids such as phosphatidylserine (PS)3 on the cell surface (1, 2). This event constitutes one of the best-described "eat-me" signals that allow professional phagocytes to identify an apoptotic cell as such and proceed to its engulfment. PS is recognized by a recently cloned receptor on the phagocyte surface whose blockade abrogates the phagocytosis of the apoptotic cell (1, 2, 3, 4).

Apoptosis induced by different agents also is associated with the hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A2 (iPLA2-VIA) (5, 6, 7, 8, 9). In addition to merely serving a destructive function, iPLA2-VIA-catalyzed phospholipid hydrolysis gives rise to the formation of lysophosphatidylcholine (lysoPC) in the apoptotic cell. A portion of this lysoPC is secreted to the extracellular milieu, where it is thought to function as an attraction signal for the phagocytes to find the apoptotic cell (7).

The involvement of iPLA2-VIA in apoptosis was first demonstrated by Atsumi et al. (5, 6) in U937 promonocytes treated with Fas ligand or TNF-{alpha} plus cycloheximide. We have shown recently that apoptosis of U937 cells in response to hydrogen peroxide also is associated with increased iPLA2-VIA activity (9). In this system, overexpression of iPLA2-VIA increases membrane phospholipid hydrolysis and augments the rate of apoptosis (9). However, importantly, inhibition of endogenous iPLA2-VIA activity by either methylarachidonyl fluorophosphonate (MAFP) or an antisense oligonucleotide does not prevent apoptosis, suggesting that, although iPLA2-VIA does participate in membrane phospholipid destruction during apoptosis, it is not absolutely required for the apoptotic process to fully develop (9). Collectively, these results suggest that, beyond a merely destructive role, iPLA2-VIA may be centrally involved in providing accessory signals that are triggered along with the destructive process itself.

In the present study, we provide evidence that iPLA2-VIA-mediated membrane phospholipid hydrolysis in apoptotic cells plays a role in enabling macrophages to engulf the dying cells. We show that the iPLA2-VIA metabolite responsible for this effect is lysoPC. Thus, iPLA2-VIA-induced lysoPC within the dying cell may function as a direct "eat me" signal that helps macrophages recognize an engulf the apoptotic cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Human U937 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. P388D1 macrophages stably expressing the enhanced GFP (EGFP) were provided by Dr. Y. Shirai (Biosignal Research Center, Kobe University, Kobe, Japan). Pyrrophenone was provided by Dr. T. Ono (Shionogi, Osaka, Japan). MAFP (methylacetate solution), free fatty acids, and the anti-iPLA2-VIA were from Cayman Chemical. Anti-cPLA2{alpha} Ab was from BD. All other reagents were obtained from either Sigma-Aldrich or BIOMOL.

Cell culture

Murine P388D1 macrophages and human U937 promonocytic cells were maintained in RPMI 1640 medium supplemented with 10% (v/v) FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Phagocytosis assay

Phagocytosis of apoptotic cells by macrophages was determined by flow cytometry. Murine P388D1 macrophages stably transfected with EGFP were used as effector cells. One million P388D1 cells were seeded in 24-well culture plates in RPMI 1640 medium containing 10% serum. As a cell target, apoptotic U937 cells were used. Apoptotic cell death was induced in these cells by treating them with 500 µM H2O2 for 20 h (9). Afterward, the U937 cells were stained with 500 µM propidium iodide for 20 min. In some experiments, the apoptotic cells were labeled with Cy3-annexin V for 5 min. After washing the cells twice with PBS, target cells were added to the P388D1 macrophage monolayers in a final volume of 1.5 ml of RPMI 1640 medium without serum. The plates were centrifuged at 300 x g for 3 min so as to bring the target cells into direct contact with the macrophages, thus avoiding the necessity for "find me" signals. The ratio of target cells to macrophages was 3:1. The phagocytosis reaction was allowed to proceed for 2 h in a humidified CO2 incubator at 37°C, after which the macrophages were trypsinized by incubating them with a solution 0.25% trypsin/EDTA (Sigma-Aldrich) for 5 min. This additional step helps to detach undigested U937 cells from the macrophages. The macrophages were then scraped from the wells by using a cell scraper, washed twice with PBS, and analyzed by flow cytometry using a Coulter Epics XL-MCL cytofluorimeter. Green fluorescence from the EGFP was analyzed in FL1 (505–545 nm), while red fluorescence from propidium iodide or annexin V-Cy3 was analyzed in FL2 (555–600 nm). To quantify phagocytosis, red fluorescence was analyzed only in the cell populations exhibiting significant green fluorescence (EGFP-positive cells, i.e., macrophages). The EGFP-containing macrophages are positive for red fluorescence only if they have ingested the propidium iodide-labeled U937 cells. Phagocytosis was confirmed by confocal microscopy using a laser scanning system (Radiance 2100; Bio-Rad) coupled to a Nikon inverted microscope with a thermostatized chamber. The objective was a x60, 1.4 numerical aperture, oil immersion. Green fluorescence was monitored at 488-nm argon excitation using a combination of HQ500 long-bandpass and a HQ560 short-bandpass blocking filters. Red fluorescence was monitored sequentially at 543-nm HeNe excitation using a HQ590/570 long-bandpass blocking filter.

Measurement of apoptosis

Apoptosis was analyzed by labeling with the Annexin-V Apoptosis Detection Kit (BD Pharmingen) that recognizes PS exposure on the outer leaflet of the plasma membrane. The cells were analyzed by flow cytometry using an Epics XL-MCL cytofluorimeter.

Antisense oligonucleotide treatments

The iPLA2-VIA antisense oligonucleotide used in this study has been described in previous studies from our laboratory (9, 10, 11). The antisense or 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 under standard cell culture conditions. The final concentrations of oligonucleotide and LipofectAMINE 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 measuring adherent cell protein.

The human cPLA2{alpha} antisense oligonucleotide used in this work was originally described by Tomassini and Cantoni (12). The oligonucleotides used were as follows: (antisense) 5'-GTA AGG ATC TAT AAA TGA CAT-3'; and (sense; random sequence of the antisense bases), 5'-GAT GAT CAG ATA TAC GAT AAT-3'). The oligonucleotides were phosphorothioate-modified (MWG Biotech). Nucleotides were introduced into the cell by the Nucleofector method (Amaxa Biosystems), following the manufacturer’s instructions (program V-001). The cells were used 48 h after nucleofection.

Production of transfectants stably expressing iPLA2-VIA

pcDNA3.1 vector containing group VIA iPLA2 (provided by Dr. S. Jackowski, St. Jude Children’s Research Hospital, Memphis, TN) (~2 µg per 106 cells) was transfected by electroporation at 270 V (975 µF) using a Gene Pulser II electroporator (Bio-Rad). To select for the transfected cells, they were incubated in medium containing 1 mg/ml geneticin. To obtain the transfectants stably expressing iPLA2-VIA, the transfected cells were cloned by limiting dilution in medium containing 300 µg/ml geneticin. After 2 wk, wells containing a single colony were chosen for further expansion, and the iPLA2-VIA expression was analyzed by immunoblot and measurement of iPLA2 activity (9). The clones were always grown in medium containing 300 µg/ml geneticin.

Data presentation

Assays were conducted in duplicate or triplicate. Each set of experiments was repeated at least three times with similar results. Paired Student’s t test was used to compare treated samples with controls. Values of p are given in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
We have demonstrated previously that iPLA2-VIA-mediated phospholipid hydrolysis occurs during oxidative stress-induced apoptosis (9, 10). However, iPLA2-VIA appears to be dispensable for the apoptotic process to occur, because inhibition of enzyme activity does not prevent apoptosis (9). These data suggested that, beyond a mere destructive role, iPLA2-VIA may be playing other roles during apoptosis.

We set up a flow cytometry assay of phagocytosis of apoptotic cells in which U937 cells exposed to H2O2 to induce apoptosis were coincubated with P388D1 macrophages, and a centrifugation was conducted to bring target and effector cells together. This procedure ensured that the extent of phagocytosis was not limited by the generation of attraction signals by the apoptotic cells, because macrophages were already put into direct contact with the dying cells. The apoptotic cells were stained with propidium iodide and/or Cy3-annexin V and exposed to P388D1 macrophages stably expressing EGFP. The number of macrophage cells positive for both red and green colors is determined by flow cytometry, and this provides a direct estimation of phagocytosis of apoptotic cells by the macrophages (Fig. 1). For most experiments described below, propidium iodide-labeled cells were used, which indicates cells in late apoptosis. However, we also used cells in early apoptosis (Cy3 annexin V-positive, propidium iodide-negative; treatment with H2O2 for periods of time up to 12 h) and obtained essentially the same results. Thus, the results described below apply to both H2O2-induced early and late apoptosis


Figure 1
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  		FIGURE 1. Phagocytosis of apoptotic U937 cells by P388D1 macrophages. EGFP-transfected P388D1 macrophages were analyzed by flow cytometry for green (A and C) and red fluorescence (B and D) before (A and B) and after (C and D) coincubating them with propidium iodide (PrI)-loaded apoptotic U937 cells for 2 h. Apoptosis was induced in these cells by treating them with 500 µM H2O2 for 20 h. Red fluorescence was analyzed only in the population of cells with intense green fluorescence (macrophages; 86% of the cells in A and 36% of the cells in C). Phagocytosis also was studied by confocal microscopy (E).

 
As a first approach to study whether iPLA2-VIA was responsible for inducing changes that lead to recognition of the apoptotic cells by the macrophages, we used the pharmacological inhibitors MAFP (a dual cPLA2{alpha}-iPLA2 inhibitor) (13) and pyrrophenone (a selective cPLA2{alpha} inhibitor) (14). The iPLA2-selective inhibitor bromoenol lactone cannot be used for these studies, because long-term incubation with this compound induces apoptosis per se (15). Apoptosis was induced by treating the U937 cells with H2O2 for 24 h in the absence or presence of the aforementioned inhibitors. In previous studies, we characterized the H2O2-induced U937 cell apoptosis by using the annexin V binding assay (9). Here, we confirmed that H2O2 does render the U937 cells apoptotic, as judged by chromatin condensation, measured by 4',6'-diamidino-2-phenylindole staining (Fig. 2). We have shown previously that neither MAFP nor pyrrophenone modifies the H2O2-induced apoptosis of U937 cells (9). After the different treatments, the cells were labeled with propidium iodide and coincubated with the EGFP-labeled P388D1 macrophages. Results in Fig. 3A show that phagocytosis of apoptotic U937 cells was markedly reduced if the cells were treated with MAFP but not with pyrrophenone. In vitro activity assays confirmed that, at the concentrations used, MAFP and pyrrophenone completely abrogated iPLA2 and cPLA2{alpha} activities, respectively (data not shown). These results suggest that inhibition of iPLA2-VIA but not of cPLA2{alpha}- impairs phagocytosis of apoptotic cells.


Figure 2
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  		FIGURE 2. Effect of H2O2 on nuclear morphology of U937 cells. The cells were treated without (A and B) or with (C and D) 500 µM H2O2 for 20 h, stained with 4',6'-diamidino-2-phenylindole, and examined by fluorescence microscopy. A and C are the Nomarski images, and B and D show the fluorescence of the same cells.

 

Figure 3
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  		FIGURE 3. iPLA2-VIA modulates phagocytosis of apoptotic cells by macrophages. A, U937 cells were rendered apoptotic by incubating them with 500 µM H2O2 for 20 h in the absence (Control) or presence of the cPLA2{alpha} inhibitor pyrrophenone (Pyrr; 2 µM) or the dual cPLA2{alpha}-iPLA2-VIA inhibitor MAFP (10 µM), as indicated. B, U937 cells were treated with an antisense oligonucleotide specific for iPLA2-VIA or a control sense oligonucleotide, as indicated. iPLA2 protein level after the treatment with the oligonucleotides was analyzed by immunoblot (see inset). C, U937 cells stably overexpressing iPLA2-VIA (referred to in the abscissa as Overexpression, Ovx) were prepared, and the level of iPLA2 immunoreactive protein was analyzed by immunoblot (see inset). Apoptosis was induced by incubating the U937 cells with 500 µM H2O2 for 20 h. The apoptotic cells were washed and coincubated with the EGFP-transfected macrophages for 2 h. Phagocytosis was determined by flow cytometry, and it is presented as a percent of the number of green cells exhibiting red fluorescence with respect to the total number of green cells. Results are given as means ± SEM of three independent determinations (*, p < 0.01).

 
To test the above suggestion more rigorously, the effect of an iPLA-2-VIA antisense oligonucleotide on phagocytosis of apoptotic cells was evaluated. The antisense oligonucleotide used is the human counterpart of the murine one that we and others have successfully used elsewhere (16, 17, 18). Using this antisense in the U937 cells, we typically detect a 70–75% decrease of both immunoreactive iPLA2-VIA protein and cellular iPLA2 activity (Fig. 3B and Refs.9, 10, 11). Phagocytosis of the iPLA2-VIA-deficient cells by P388D1 macrophages was considerably lower (Fig. 3B), thus providing again strong evidence for the involvement of iPLA2-VIA in this process. Because cells made deficient in iPLA2-VIA by this antisense procedure undergo apoptosis at levels similar to those exhibiting normal iPLA2-VIA levels (Ref.9 and data not shown), the percent apoptotic cells in the experiments depicted in Fig. 3 is the same under all conditions.

In the next set of experiments, U937 cells stably overexpressing iPLA2-VIA (9) were used. iPLA2-VIA-overexpressing cells rendered apoptotic by treatment with H2O2 were found to be phagocytosed by P388D1 macrophages to a greater extent than apoptotic cells expressing normal iPLA2-VIA levels (Fig. 3C). Collectively, the data in Fig. 3 do support the involvement of iPLA2-VIA of apoptosing U937 cells in enabling phagocytosis by the macrophages.

To study whether the role of iPLA2-VIA in H2O2-treated cells is generalizable to other cell types, we conducted studies with the lymphocyte-like cell line Jurkat. Although in these cells H2O2 did induce apoptosis, as judged by the annexin V binding assay, phagocytosis of apoptotic Jurkat cells by macrophages was not altered by inhibiting endogenous iPLA2 activity with 10 µM. In keeping with this finding, and in stark contrast with U937 cells (10), Jurkat cells did not readily release fatty acids into the incubation medium in response to H2O2. These data suggest that the role of iPLA2 during apoptosis appears to be peculiar to cells in which iPLA2-VIA can be potently activated by H2O2 (i.e., U937 cells but not Jurkat cells). In this regard, it is interesting to note that Jurkat cells appear to contain 2- to 3-fold less iPLA2 activity than U937 cells (R. Pérez, M. A. Balboa, and J. Balsinde, unpublished data).

In the next series of experiments, we used TNF-{alpha}, a well known inducer of apoptosis in the various cell types, including U937 cells (5, 6). We confirmed that U937 cells exposed to TNF-{alpha} for 24 h underwent apoptosis, as judged by the annexin V binding assay, and were readily engulfed by the P388D1 macrophages when assayed according to our phagocytosis protocol described above. However, phagocytosis of TNF-{alpha}-treated cells was not inhibited if the cells were first treated with 10 µM MAFP to block cellular iPLA2. These results indicate that the role of iPLA2-VIA during H2O2-induced apoptosis may not be generalizable to other apoptotic stimuli or cell systems.

In the H2O2-treated U937 cells, the data show a correlation between the levels of cellular iPLA2-VIA activity within the apoptotic cell and the extent of phagocytosis. Thus, it could be that a metabolite generated by iPLA2-VIA in the apoptotic cell is responsible for facilitated phagocytosis by the macrophage.

iPLA2-VIA-mediated fatty acid release occurs abundantly during H2O2-induced apoptosis of U937 cells (9, 10). Thus, we investigated whether iPLA2-VIA-mediated fatty acid deacylation reactions during apoptosis could be involved in providing signals that facilitate phagocytosis. In the first series of experiments, we prepared cells enriched in different fatty acids by culturing them for 3 days in medium containing 10 µM corresponding fatty acid. The fatty acids tested were arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and palmitic acid. If a free fatty acid was responsible for favoring macrophage phagocytosis of dying cells, one would expect these fatty acid-enriched cells to be ingested by macrophages to a greater extent than cells incubated under standard conditions (i.e., no fatty acid supply). Apoptosis of the fatty acid-enriched cells in response to H2O2 was no different from that observed in cells cultured under normal incubation conditions. Similarly, phagocytosis of the fatty acid-enriched apoptotic cells was not changed (data not shown). Next, we added different fatty acids at the same time the apoptotic cells were coincubated with the macrophages. The fatty acids tested were arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and palmitic acid, all at 10 µM. Phagocytosis was again not changed whether or not any fatty acid was present during the 2-h incubation period (data not shown). Collectively, these data suggest that fatty acids and/or phospholipid fatty acid composition do not affect phagocytosis of apoptotic U937 cells.

LysoPC is the other major by-product of iPLA2-VIA-mediated hydrolysis of cellular phospholipids (19, 20, 21). We have shown previously that U937 cells made deficient in iPLA2-VIA by treatment with the antisense oligonucleotide exhibit decreased lysoPC levels, compared with untreated cells (11), and, conversely, iPLA2-VIA-overexpressing cells exhibit larger lysoPC levels than untreated cells (9). To study the possibility that lysoPC on the surface of apoptotic cells facilitates engulfment by the macrophages, we exposed the H2O2-treated U937 cells to P388D1 macrophages that had been preincubated with different concentrations of lysoPC for 30 min. Thus, if engulfment by macrophages of apoptotic cells occurs at least in part by recognition of lysoPC on the surface of the dying cells, blockade of the putative lysoPC receptors on the macrophage surface by soluble lysoPC should inhibit phagocytosis. Fig. 4 shows that phagocytosis of apoptotic cells by the macrophages was inhibited by lysoPC in a concentration-dependent manner. Importantly, if lysoPC was added after the macrophages had been exposed to the apoptotic cells, no significant inhibition of phagocytosis was observed. For these experiments, lysoPC was added in the absence of serum or albumin in the incubation medium. The experiments also were repeated using resident peritoneal macrophages from C57BL/6 mice (University of Valladolid Animal House, Valladolid, Spain), and the results obtained were the same as those obtained with P388D1 macrophage-like cells and shown in Fig. 4.


Figure 4
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  		FIGURE 4. LysoPC blocks phagocytosis of apoptotic cells by macrophages. U937 cell apoptosis was induced by incubating the cells with 500 µM H2O2 for 20 h. The EGFP-transfected macrophages were exposed to the indicated amounts of lysoPC for 30–60 min before coincubating them with the dying cells for 2 h. Phagocytosis was determined by flow cytometry, and it is presented as a percent of the number of green cells exhibiting red fluorescence with respect to the total number of green cells. Results are given as means ± SEM of three independent determinations.

 
The concentrations of lysoPC used in these experiments (<5 µM) were not stimulatory for the macrophages, as judged by Ca2+ mobilization and p42/p44MAPK phosphorylation measurements. In addition, 5 µM lysoPC did not induce apoptosis or necrosis of P388D1 macrophages, or enhanced cell death of the targets, as judged by the annexin V binding assay or the trypan blue exclusion assay (results not shown). Concentrations of lysoPC >5 µM could not be used, because they activated the macrophages per se. This is likely due to the changes in the physical state of the phospholipid at higher concentrations, because lysoPC’s critical micellar concentration under these conditions is ~7 µM (22). As a control for these experiments, the effect of other lysophospholipids, namely lysophosphatidylethanolamine and lysophosphatidic acid, on macrophage phagocytosis of apoptotic cells also was assayed. Neither of these lysophospholipids, used at 5 µM, exerted any effect, thus reinforcing the notion that lysoPC is specifically involved in the recognition of apoptotic cells by the macrophages.

As another control, we studied the effect of lysoPC on macrophage phagocytosis of yeast-derived zymosan. Alexa Fluor 594-labeled zymosan (Molecular Probes) was used at a concentration of 5 particles per 106 macrophages. The extent of zymosan phagocytosis by lysoPC-treated macrophages was no different from the one observed with macrophages not exposed to the lysophospholipids. Thus, the inhibitory effect of exogenous lysoPC appears to be specific for the engulfment of apoptotic cells.

Although the molecular composition of the fatty acid at the sn-1 position of cellular lysoPC can be regulated by multiple CoA-dependent and -independent transacylation reactions, formation of lysoPC in cells can be conducted only by A-type PLs. Having established previously that iPLA2-VIA does regulate lysoPC levels in U937 cells (9, 11), we wanted to investigate whether other PLA2 forms present in the U937 cells also catalyzed lysoPC formation during apoptosis. Using RT-PCR, we investigated the expression of different PLA2 forms in the U937 cells. PLA2s sought for were those of groups IB, IIA, IID, IIE, IIF, III, IVA, IVB, IVC, V, VIB, and X. Of those, only cytosolic group IVA PLA2 (cPLA2{alpha}) was detected in the U937 cells (Table I). To study the role of this enzyme in lysoPC production during apoptosis, we inhibited its expression by antisense oligonucleotide technology. Fig. 5 shows that, 48 h after transfection, immunoreactive cPLA2{alpha} was barely detectable, but robust expression was observed in homogenates treated with a random oligonucleotide control. The apoptotic response of the cPLA2{alpha}-depleted cells to H2O2 was identical with those of cells expressing normal cPLA2{alpha} levels (Fig. 5). These data are consistent with the lack of effect of pyrrophenone (Fig. 3A) and, together, demonstrate that cPLA2{alpha} is not required for this type of apoptosis. Importantly, phagocytosis of the cPLA2{alpha}-deficient apoptotic cells by the macrophages was also not impaired, indicating that, unlike iPLA2-VIA, cPLA2{alpha} is not involved in providing "eat me" signals (Fig. 5).


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  		Table I. Oligonucleotide primers used for amplification of PLA2 groups from U937 cell cDNAa

 

Figure 5
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  		FIGURE 5. Antisense inhibition of cPLA2{alpha} does not affect U937 cell apoptosis or phagocytosis of the apoptotic cells by macrophages. U937 cells were treated with an antisense oligonucleotide specific for cPLA2{alpha} or a control random oligonucleotide, as indicated. cPLA2{alpha} protein level after the treatment with the oligonucleotides was analyzed by immunoblot (see inset). U937 cell apoptosis was induced by incubating the cells with 500 µM H2O2 for 20 h. U937 cell apoptosis ({blacksquare}) was analyzed by the annexin V-binding assay. Apoptotic U937 cells were washed and coincubated with the macrophages for 2 h. Phagocytosis ({cjs2108}) was determined by flow cytometry, and it is presented as a percentage of the number of green cells exhibiting red fluorescence with respect to the total number of green cells (EGFP-transfected macrophages). Results are given as means ± SEM of three independent determinations (differences are statistically not significant).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Efficient clearance of apoptotic cells, a critical process for tissue homeostasis, is conducted by phagocytic cells. This process is initiated by the display of "eat me" signals on the surface of the dying cells. Thus, "eat me" signals represent markers for phagocytes to identify and engulf dying cells. Different types of "eat me" signals have been identified, including new molecules that appear on the surface (e.g., PS or annexin I) (1, 2, 3, 4, 23), surface molecules that undergo modifications (e.g., ICAM-3 or CD31) (24, 25), and indirect signals generated by interaction of serum components with the apoptotic cell surface (26).

In the present study, we have found that a functionally active iPLA2-VIA enzyme within the H2O2-treated, apoptosing cells is required for them to be efficiently recognized and engulfed by macrophages. By either blunting or increasing the levels of iPLA2-VIA in the apoptotic cells, we have found a correlation between iPLA2-VIA activity and the extent of phagocytosis of the dying cells by the macrophages, suggesting that a metabolic product of iPLA2-VIA is involved in the process. We have identified this metabolite as lysoPC. Thus, the present studies identify lysoPC as a direct "eat me" signal for phagocytes. Importantly, it is the lysoPC that is generated specifically by iPLA2-VIA that functions as an "eat me" signal, because abrogation of the one other PLA2 present in the cells, cPLA2{alpha}, has no discernible effect. The latter result would be consistent with previous studies suggesting that cPLA2{alpha} is inactivated during U937 cell apoptosis induced by Fas ligand or TNF-{alpha} (5, 6, 27).

However, the role of lysoPC as an "eat me" signal for apoptosis may be limited to certain conditions involving sustained iPLA2-VIA activity. The lysoPC role could not be appreciated with Jurkat cells or with U937 cells when TNF-{alpha} was used as an inducer of apoptosis. In both of these cases, iPLA2-VIA-mediated phospholipid hydrolysis does not occur at the high levels observed in H2O2-treated U937 cells (9, 10). Thus, it appears that the lysoPC role as an "eat me" signal may be significant only when iPLA2-VIA substantially participates in phospholipid degradation during apoptosis.

Of considerable interest to our present results, elegant studies by Kim et al. (26) have demonstrated that apoptotic cells generate a surface epitope recognized by natural IgM Abs that results in complement activation and phagocytosis. The epitope recognized was the phosphoryl choline moiety of lysoPC (26). Interestingly, enhanced IgM binding could be suppressed by the unspecific iPLA2 inhibitor bromoenol lactone, making it likely that also in this system, lysoPC was being generated by an iPLA2, although the molecular identity of the actual iPLA2 form involved was not determined (26).

In H2O2-treated, apoptosing U937 cells, different molecular approaches, together with the fact that U937 appear not to express the group VIB iPLA2 isoform, have clearly indicated the specific involvement of the iPLA2-VIA isoform in lysoPC production. Also, our phagocytosis studies were conducted in the total absence of serum, thus ruling out the participation of any serum component in the recognition of apoptotic cells by the macrophages. Moreover, phagocytosis of dying cells by macrophages could be inhibited by preincubating the macrophages with lysoPC, clearly suggesting that macrophages can recognize lysoPC directly. The ability of lysoPC to act as a direct (this study) or indirect (26) "eat me" signal may depend on cell type or apoptotic stimuli used, but it appears likely that the two lysoPC functions may coexist during phagocytosis of apoptotic cells.

It is interesting to speculate with the mechanism by which lysoPC produced by iPLA2-VIA attack on membrane phospholipids gets exposed to the cell surface and how the lipid is recognized by phagocytes. As discussed elsewhere (28), it appears likely that phospholipid scrambling may represent the main mechanism for lysoPC exposure to occur. Phospholipid scrambling is an early event in apoptosis and results in the outer exposure of the negatively charged aminophospholipid PS. PS exposure constitutes a primary "eat me" signal for phagocytes, and these cells possess receptors for PS that mediate engulfment of the dying cell (1, 2, 3, 4), Although recent studies have questioned the view that the PS receptor primarily functions in apoptotic cell clearance (29), it is possible that macrophages possess receptors that specifically recognize lysoPC on the surface of the dying cell. This lysoPC receptor may either mediate engulfment of the dying cell or act indirectly to promote binding and recruit other signaling receptors involved in the engulfment. A G-protein-coupled receptor present in macrophages and other cells, G2A, was reported to bind lysoPC with high affinity (30). Although more recent results have failed to confirm that lysoPC directly binds to G2A (31), contrasted evidence exists that expression of G2A in Jurkat T cells confers the ability to migrate in response to lysoPC (32, 33) Thus, G2A may effect certain cellular responses to lysoPC by an indirect mechanism in which lysoPC modifies another receptor or process that in turn regulates the G2A receptor.

To conclude, our studies highlight a key role for iPLA2-VIA and its by-product lysoPC in mediating the recognition and subsequent engulfment of H2O2-treated, apoptosing cells by macrophages. It is noteworthy that the control of the steady-state level of lysoPC is one the earliest biological functions discovered for iPLA2-VIA in cells (for reviews, see Refs.19, 20, 21). In resting cells, iPLA2-VIA activity is quite significant, as judged by the measurable decrease of lysoPC levels after acute inhibition of the enzyme (19, 20, 21). Importantly, iPLA2-VIA is cleaved by caspase-3 during apoptosis, yet the specific activity of the enzyme remains unchanged (5, 6). This suggests that certain apoptotic signals may render the membrane more susceptible to iPLA2-VIA (10), or that a putative iPLA2-VIA-regulatory protein may be modified in vivo, leading to increased phospholipid hydrolysis and the accumulation of lysoPC. Studies are currently in progress to elucidate the mechanisms of regulation of iPLA2-VIA activity during apoptosis induced by H2O2.


   Acknowledgments
 
We thank Yolanda Sáez and Montserrat Duque for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Spanish Ministry of Education and Science Grants BFU2004-01886/BMC and SAF2004-04676. Back

2 Address correspondence and reprint requests to Dr. Jesús Balsinde, Instituto de Biología y Genética Molecular, Universidad de Valladolid, Calle Sanz y Forés s/n, 47003 Valladolid, Spain. E-mail address: jbalsinde{at}ibgm.uva.es Back

3 Abbreviations used in this paper: PS, phosphatidylserine; PL, phospholipase; iPLA2-VIA, calcium-independent group VIA PLA2; cPLA2{alpha}, cytosolic group IVA PLA2; MAFP, methylarachidonyl flurophosphonate; lysoPC, lysophosphatidylcholine (1-acyl-2-lyso-sn-glycero-3-phosphocholine); EGFP, enhanced GFP. Back

Received for publication July 1, 2005. Accepted for publication December 1, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Fadok, V. A., D. L. de Cathelineau, P. M. Henson Daleke, D. L. Bratton. 2001. Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J. Biol. Chem. 276: 1071-1077. [Abstract/Free Full Text]
  2. Hoffmann, P. R., A. M. de Cathelineau, C. A. Ogden, Y. Leverrier, D. L. Bratton, D. L. Daleke, A. J. Ridley, V. A. Fadok, P. M. Henson. 2001. Phosphatidylserine (PS) induces PS receptor-mediated macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 155: 649-659. [Abstract/Free Full Text]
  3. Fadok, V. A., D. L. Bratton, D. M. Rose, A. Pearson, R. A. Ezekowitz, P. M. Henson. 2000. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405: 85-90. [Medline]
  4. Savill, J., V. Fadok. 2000. Corpse clearance defines the meaning of cell death. Nature 407: 784-788. [Medline]
  5. Atsumi, G., M. Tajima, A. Hadano, Y. Nakatani, M. Murakami, I. Kudo. 1998. Fas-induced arachidonic acid release is mediated by Ca2+-independent phospholipase A2 but not by cytosolic phospholipase A2, which undergoes proteolytic degradation. J. Biol. Chem. 273: 13870-13877. [Abstract/Free Full Text]
  6. Atsumi, G., M. Murakami, K. Kojima, A. Hadano, M. Tajima, I. Kudo. 2000. Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway: proteolytic fragment of type IVA cytosolic phospholipase A2{alpha} inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release. J. Biol. Chem. 275: 18248-18258. [Abstract/Free Full Text]
  7. Lauber, K., E. Bohn, S. M. Krober, Y. J. Xiao, S. G. Blumenthal, R. K. Lindemann, P. Marini, C. Wiedig., A. Zobywalski, S. Baksh, et alApoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell 113: 717-730.
  8. Ramanadham, S., F. F. Hsu, S. Zhang, C. Jin, A. Bohrer, H. Song, S. Bao, Z. Ma, J. Turk. 2004. Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2beta) and suppressed by inhibition of iPLA2beta. Biochemistry 43: 918-930. [Medline]
  9. Pérez, R., R. Melero, M. A. Balboa, J. Balsinde. 2004. Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide. J. Biol. Chem. 279: 40385-40391. [Abstract/Free Full Text]
  10. Balboa, M. A., J. Balsinde. 2002. Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells. J. Biol. Chem. 277: 40384-40389. [Abstract/Free Full Text]
  11. Balboa, M. A., Y. Sáez, J. Balsinde. 2003. Calcium-independent phospholipase A2 is required for lysozyme secretion in U937 promonocytes. J. Immunol. 170: 5276-5280. [Abstract/Free Full Text]
  12. Tommasini, I., O. Cantoni. 2004. Dexamethasone promotes toxicity in U937 cells exposed to otherwise nontoxic concentrations of peroxynitrite: pivotal role for lipocortin 1-mediated inhibition of cytosolic phospholipase A2. Mol. Pharmacol. 65: 964-972. [Abstract/Free Full Text]
  13. Balsinde, J., E. A. Dennis. 1996. Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. J. Biol. Chem. 271: 6758-6765. [Abstract/Free Full Text]
  14. Ono, T., K. Yamada, Y. Chikazawa, M. Ueno, S. Nakamoto, T. Okuno, K. Seno. 2002. Characterization of a novel inhibitor of cytosolic phospholipase A2{alpha}, pyrrophenone. Biochem. J. 363: 727-735. [Medline]
  15. Fuentes, L., R. Pérez, M. L. Nieto, J. Balsinde, M. A. Balboa. 2003. Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2. J. Biol. Chem. 278: 44683-44690. [Abstract/Free Full Text]
  16. Balsinde, J., M. A. Balboa, E. A. Dennis. 1997. Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages. J. Biol. Chem. 272: 29317-29321. [Abstract/Free Full Text]
  17. Carnevale, K. A., M. K. Cathcart. 2001. Calcium-independent phospholipase A2 is required for human monocyte chemotaxis to monocyte chemoattractant protein 1. J. Immunol. 167: 3414-3421. [Abstract/Free Full Text]
  18. Smani, T., S. I. Zakharov, E. Leno, P. Csutora, E. S. Trepakova, V. M. Bolotina. 2003. Ca2+-independent phospholipase A2 is a novel determinant of store-operated Ca2+ entry. J. Biol. Chem. 278: 11909-11915. [Abstract/Free Full Text]
  19. Balsinde, J., E. A. Dennis. 1997. Function and inhibition of intracellular calcium-independent phospholipase A2. J. Biol. Chem. 272: 16069-16072. [Free Full Text]
  20. Winstead, M. V., J. Balsinde, E. A. Dennis. 2000. Calcium-independent phospholipase A2: structure and function. Biochim. Biophys. Acta 1488: 28-39. [Medline]
  21. Balsinde, J., M. A. Balboa. 2005. Cellular regulation and proposed biological functions of group VIA calcium-independent phospholipase A2 in activated cells. Cell. Signal. 17: 1052-1062. [Medline]
  22. Wang, A., H. C. Yang, P. Friedman, C. A. Johnson, E. A. Dennis. 1999. A specific human lysophospholipase: cDNa cloning, tissue distribution and kinetic characterization. Biochim. Biophys. Acta 1437: 157-169. [Medline]
  23. Arur, S., U. E. Uche, K. Rezaul, M. Fong, V. Scranton, A. E. Cowan, W. Mohler, D. K. Han. 2003. Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev. Cell 4: 587-598. [Medline]
  24. Brown, S., I. Heinisch, E. Ross, K. Shaw, C. D. Buckley, J. Savill. 2002. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 418: 200-203. [Medline]
  25. Moffatt, O. D., A. Devitt, E. D. Bell, D. L. Simmons, C. D. Gregory. 1999. Macrophage recognition of ICAM-3 on apoptotic leukocytes. J. Immunol. 162: 6800-6810. [Abstract/Free Full Text]
  26. Kim, S. J., D. Gershov, X. Ma, N. Brot, K. B. Elkon. 2002. I-PLA2 activation during apoptosis promotes the exposure of membrane lysophosphatidylcholine leading to binding by natural immunoglobulin M antibodies and complement activation. J. Exp. Med. 196: 655-665. [Abstract/Free Full Text]
  27. Fadok, V. A.. 2003. The sirens’ call. Nat. Cell Biol. 5: 697-699. [Medline]
  28. Krönke, M., S. Adam-Klages. 2002. Role of caspases in TNF-mediated regulation of cPLA2. FEBS Lett. 531: 18-22. [Medline]
  29. Böse, J., A. D. Gruber, L. Helming, S. Schiebe, I. Wegener, M. Hafner, M. Beales, F. Kontgen, A. Lengeling. 2004. The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal. J. Biol. 3: 15[Medline]
  30. Kabarowski, J. H. S., K. Zhu, L. Q. Le, O. N. Witte, Y. Xu. 2001. Lysophosphatidylcholine as a ligand for the immunoregulatory receptor G2A. Science 293: 702-705. [Abstract/Free Full Text]
  31. Witte, O. N., J. H. S. Kabarowski, Y. Xu, L. Q. Le, K. Zhu. 2005. Science 307: 206
  32. Yang, L. V., C. G. Radu, L. Wang, M. Riedinger, O. N. Witte. 2005. Gi-independent macrophage chemotaxis to lysophosphatidylcholine via the immunoregulatory GPCR G2A. Blood 105: 1127-1134. [Abstract/Free Full Text]
  33. Wang, L., C. G. Radu, L. V. Yang, L. A. Bentolila, M. Riedinger, O. N. Witte. 2005. Lysophosphatidylcholine-induced surface redistribution regulates signaling of the murine G protein-coupled receptor G2A. Mol. Biol. Cell 16: 2234-2247. [Abstract/Free Full Text]



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