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Essential Role of Phosphatidylserine Externalization in Apoptosing Cell Phagocytosis by Macrophages
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
246, 549–555 (1998)
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Essential Role of Phosphatidylserine Externalization in Apoptosing Cell Phagocytosis by Macrophages Akiko Shiratsuchi,* Sachie Osada,* Susumu Kanazawa,† and Yoshinobu Nakanishi*,‡,1 *Graduate School of Natural Science and Technology, and ‡Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan; and †Kaken Pharmaceutical Co., Ltd., Fujieda, Japan
Received April 14, 1998
In many apoptotic cells, phosphatidylserine (PS), that is normally restricted to the inner membrane layer, is externalized and subsequently recognized by phagocytes. However, it has been unclear whether PS externalization is sufficient for phagocytosis induction. In a cultured cell line undergoing Fas-mediated apoptosis, PS externalization preceded other apoptotic events. When transbilayer movement of membrane phospholipids was analyzed, a decrease of the uptake of PS and phosphatidylethanolamine and an increase of phosphatidylcholine incorporation were observed upon apoptosis induction. Apoptotic cultured cells were phagocytosed by macrophages in a manner dependent on externalized PS before plasma membrane permeability increased. Moreover, a N-ethylmaleimide treatment caused PS externalization independent of apoptosis, and such cells underwent PS-mediated phagocytosis. These results suggested that PS is externalized as a result of membrane phospholipid redistribution and externalized PS by itself induces apoptosing cell phagocytosis. q 1998 Academic Press
Apoptotic cells are engulfed by phagocytes, and this event has been presumed to prevent inflammation and damage to surrounding tissues caused by the noxious contents of dead cells (1,2). Apoptosing cells most likely expose a marker molecule(s), such as proteins, sugars, and phospholipids, which phagocytic cells recognize to capture target cells for phagocytosis (3,4). Among them, a membrane phospholipid PS has been the most extensively characterized in terms of both how it becomes 1 To whom correspondence should be addressed at Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. Fax: (81) 76-234-4480. E-mail: [email protected]. Abbreviations used: FITC, fluorescein isothiocyanate; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBD, 4-nitrobenzo-2-oxa-1,3-diazole; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.
expressed on the surface of apoptosing cells and how it is involved in phagocytosis (3-7). A variety of apoptosis triggers presumably cause redistribution of the plasma membrane phospholipid at early apoptotic stages; for example, PS and PE, which are normally confined to the inner leaflet, are transferred to the outer leaflet. With several different apoptotic cells such as lymphocytes (8), erythrocytes (9), vascular smooth muscle cells (10), and spermatogenic cells (11), externalized PS has been shown to be recognized by phagocytic cells leading to subsequent phagocytosis. However, it has been unclear whether apoptotic cells are phagocytosed before plasma membrane permeability increases and PS externalization by itself is sufficient for inducing phagocytosis. We here assessed such issues. MATERIALS AND METHODS Cell culture and apoptosis analysis. HeLa cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum at 37 7C in a humidified atmosphere with 5% CO2 in air. The cells were transfected with an expression vector pSRa (12) containing human Fas cDNA (13) using lipofectin (Gibco BRL, MD, U. S. A.). Cell clones resistant to G418 (1 mg/ml) were examined for the expression of cell surface Fas by staining them with an anti-human Fas monoclonal antibody, CH-11 (14) (MBL, Nagoya, Japan). One of the clones that gave a strong signal was selected and named HF1. The presence of cell surface Fas was determined by flow cytometry using an anti-human Fas monoclonal antibody, UB2 (MBL) and fluorescein-conjugated anti-mouse IgG (Immunotech, Marseilles, France). Normal mouse IgG (Zymed Laboratories, CA, U. S. A.) was used as a negative control. HF1 cells were induced to undergo apoptosis by cultivating them in the presence of CH-11 (0.5 mg/ml). Plasma membrane permeability, activity of mitochondrial enzyme, chromatin condensation, and DNA fragmentation were determined by standard procedures; a dye exclusion test with trypan blue, an enzyme assay with MTT (15, 16) using a commercial kit (Chemicon, CA, U. S. A.), staining with Hoechst33342, and agarose gel electrophoresis, respectively. PS externalization was determined in a flow cytometer (EPICS XL; Coulter, FL, U. S. A.) as described previously (17, 18). Briefly, FITC-labeled annexin V was added to test cells simultaneously with a plasma membrane-non-permeable dye, propidium iodide, and cells less intensely stained with propidium iodide were analyzed for the binding of FITC-annexin V. Phospholipid movement assay. Transbilayer movement of phospholipids was monitored with NBD-labeled phospholipid analogues
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(Avanti Polar Lipids, AL, U. S. A.); (palmitoyl, C6-NBD)-PS, (palmitoyl, C6-NBD)-PE, and (palmitoyl, C6-NBD)-PC, essentially as described previously (19). Briefly, NBD-phospholipids were dried down and dissolved with small amounts of ethanol. HF1 cells were suspended (1 1 106 cells/ml) in phosphate-buffered saline containing 1 mM MgCl2 and 2 mM CaCl2 , and supplemented with NBD-phospholipids (1 mM). After a 1-min incubation on ice, the cells were spun down and resuspended in the same buffer. NBD-phospholipids loaded on the outer leaflet of the plasma membrane were allowed to be transferred to the inner leaflet by incubating the cells at 25 7C for 5 min unless otherwise stated. The cells were then briefly treated with fatty acid-free bovine serum albumin (Sigma, MO, U. S. A.) (5 mg/ml) to remove the phospholipid analogues remaining on the outer leaflet; that is, ‘back exchange’ (20). These cells were then supplemented with propidium iodide and subjected to a flow cytometric analysis. The amounts of NBD-phospholipids transferred to the inner membrane leaflet were determined with cells less intensely stained with propidium iodide. Macrophage preparation and phagocytosis assay. BDF1 (female, 8-week old) mice were injected with thioglycollate-containing medium (fluid thioglycollate medium; Difco, MI, U. S. A.) (2 ml or 1.5 mg sodium thioglycollate per animal), and peritoneal fluids were obtained after 4 days. The cells in the fluid were collected by centrifugation, washed with Hank’s balanced salt solution (Nissui Pharmaceutical, Tokyo, Japan) and cultivated on a coverslip (øÅ15 mm) with RPMI1640 medium containing 10 mM Hepes, pH 7. Cells unattached to the coverslips were washed out and the remaining cells were analyzed for the identity of macrophage; the cells were mostly mononuclear and more than 90% of them showed incorporation of fluorescence-labeled latex beads (Polybead Microparticles, øÅ0.75 mm, Polysciences, PA, U. S. A.), indicating that they were at least 90% pure as macrophages. A phagocytosis assay was performed essentially as described (11). Briefly, target cells were labeled with biotin, mixed with macrophages (at a ratio of ten target cells against one macrophage), and incubated at 37 7C for 2-3 h. The reacted cells were washed with phosphate-buffered saline and subsequently treated with trypsin (0.5 mg/ml). They were then fixed and supplemented with FITC-conjugated avidin, and the extent of phagocytosis was determined under a fluorescence/phase contrast microscope (BX50; Olympus, Tokyo, Japan). The number of macrophages containing engulfed apoptotic cells was determined and shown relative to the total number of macrophages, taken as 100; that is, the phagocytic index. The mean and standard deviations of a typical example from at least three independent experiments were presented. Liposomes were prepared according to standard procedures. In brief, phospholipids (Avanti Polar Lipids) were dried as films, suspended in phosphate-buffered saline, and sonicated. The resulting liposomes were used in experiments without further fractionation at a total phospholipid concentration of 1 mM. They were composed of either PC only (PC-liposomes) or a combination of PC and PS at a molar ratio of 7:3 (PS-liposomes).
RESULTS AND DISCUSSION Apoptosis of HF1 cells. The cell surface expression of Fas in HF1 cells was evident by flow cytometric analysis while control cells containing an empty vector showed minimal fluorescence intensities (Fig. 1A). The over expression of Fas did not cause significant changes in cell growth but a slight change in morphology was noticeable; HF1 cells looked more spherical than parent HeLa cells (not shown). HF1 cells were induced to undergo apoptosis by the addition of an apoptosis-agonistic antibody, CH-11, and biochemical changes during an apoptosis course were determined (Fig. 1B). The extent
of chromatin condensation was determined by staining DNA with a fluorescence dye, Hoechst33342. Condensation was evident soon after the antibody addition and reached a maximal level at about 8 h. The integrity of mitochondria was examined by a calorimetric MTT assay, and the activity to cleave MTT began to decrease at 6 h reaching a maximal level at 12 h. Membrane permeability, assessed by a dye exclusion test with trypan blue, continued to increase up to 36 h. Internucleosomal DNA fragmentation became detectable after a 12-h incubation with CH-11 (not shown). The presence of PS on the surface of apoptotic HF1 cells was analyzed by staining the cells with FITClabeled annexin V and propidium iodide. The cells that were not stained with propidium iodide were analyzed for the amount of cell surface PS in a flow cytometer. Mean fluorescence of a main peak shifted at 3 h after apoptosis induction and did not significantly change for the next 5 h (right panels in Fig. 1B), indicating that PS externalization was maximized by 3 h. The above results showed that apoptotic events examined so far took place with different time courses and the externalization of PS reached maximal level earlier than other biochemical changes. Change of transbilayer movement of phospholipid analogues during apoptosis. Verhoven et al. reported that the efficiency by which fluorescence-labeled phospholipid analogues are transferred from the outer to the inner leaflet of the plasma membrane bilayer changes in apoptotic cells; that is, the incorporation of NBD-PS decreased and that of NBD-PC increased in a T-cell line upon apoptosis caused by an anti-CD3 antibody treatment (19). We adopted a similar method to determine the change of phospholipid analogue movement in cells undergoing Fas-dependent apoptosis. The mode of NBD-phospholipid translocation in normal HF1 cells was first examined. The cells were incubated with fluorescence-labeled phospholipid analogues, NBD-PS, -PE and -PC, for 1 min on ice; this should result in the phospholipid analogues being incorporated into the outer leaflet of the plasma membrane bilayer. In fact, more than 80% of the fluorescence signals disappeared from the labeled cells after extraction with bovine serum albumin (not shown). NBD-phospholipids loaded on the cell surface were then allowed to be transferred to the inside by raising the incubation temperature to 25 7C. The amounts of NBD-phospholipids remaining in the cells after ‘back exchange’ were determined in a flow cytometer. The incorporation of all three phospholipid analogues occurred time dependently, and NBD-PS and -PE were more efficiently transferred than NBD-PC (Fig. 2A). We then examined changes in phospholipid analogue movement in HF1 cells upon apoptosis induction with CH-11. The cells were recovered at various time points after the antibody addition and subjected to a phospho-
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FIG. 1. Apoptosis of HF1 cells by anti-Fas antibody. (A) Cell surface expression of Fas. HF1 cells and control HeLa/SRa cells (containing a vector alone) were treated with UB2 (solid lines) or control IgG (broken lines) and analyzed in a flow cytometer. (B) Time course of apoptosis. Left panels; HF1 cells were treated with CH-11 (open circles) or left untreated (closed circles), and the progress of apoptosis, in terms of chromatin condensation (top), inactivation of a mitochondrial enzyme (middle), and plasma membrane permeabilization (bottom) was analyzed. Right panels; PS externalization in apoptotic HF1 cells. The cells at each time point after apoptosis induction were analyzed for the binding of propidium iodide and FITC-annexin V in a flow cytometer. Shown on the far right are FITC profiles of cells less intensely stained with propidium iodide.
lipid movement assay. The amounts of NBD-phospholipids initially loaded on the cells needed to be determined with each cell preparation since they decreased with the progress of apoptosis (not shown). The efficiency of incorporation of NBD-PS and -PE started to decrease soon after apoptosis induction and reached 60-80% that of control cells at 3.5 h, while that of NBDPC increased up to 150% (Fig. 2B). This was explained by the appearance of cells with less NBD-PS or -PE and of cells with more NBD-PC incorporated (Fig. 2C). These results indicated that the movement of NBD-PS and -PE into the inner leaflet of the membrane bilayer decreased and that of NBD-PC contrarily increased in HF1 cells undergoing Fas-dependent apoptosis. All these changes became evident synchronously at 2-3 h after apoptosis induction (bottom panels in Fig. 2C), and this is the time point when PS externalization was observed (Fig. 1B). The above results were consistent with those obtained by Verhoven et al. (19).
PS and PE are restricted to the cytoplasmic side presumably through the action of an enzyme called aminophospholipid translocase (5-7), which was recently identified as a P-type adenosine triphosphatase (21). Verhoven et al. (19) interpreted their results as that aminophospholipid translocase is inactivated and scramblase, an enzyme responsible for the two-way movement of phospholipids between the plasma membrane leaflets (5-7), is activated in cells undergoing apoptosis, and that these changes lead to the cell surface exposure of PS. We here showed that similar changes in transbilayer movement of membrane phospholipids occurred in cells undergoing Fas-mediated apoptosis. We observed a decrease of NBD-PE incorporation in apoptotic cells, which was not investigated by Verhoven et al. This finding further supported the above speculation that aminophospholipid translocase is influenced upon apoptosis induction. All such changes may disrupt asymmetric localization of the
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FIG. 2. Transbilayer movement of fluorescence-labeled phospholipid analogues in apoptotic HF1 cells. (A) Time course with normal HF1 cells. The mean fluorescence of NBD in cells less intensely stained with propidium iodide (see MATERIALS AND METHODS) was determined at each time point in a flow cytometer and is shown relative to that in cells before ’back exchange’, which is taken as 100%. (B) Change of phospholipid analogue movement upon apoptosis induction. Cells at each time point after apoptosis induction were subjected to a NBD-phospholipid movement assay. The extent of NBD-phospholipid transfer was determined as in (A) and is shown relative to that in untreated cells (time 0), which is taken as 100%. (C) Appearance of cells with altered levels of phospholipid analogue movement upon apoptosis induction. Top nine panels; staining profiles with propidium iodide and NBD of cells after apoptosis induction. Cells with altered NBD levels are circled. Bottom panels; the number of the circled cells relative to that of total cells is shown with the mean and standard deviations from three independent experiments.
plasma membrane phospholipid and bring about its redistribution on the two membrane leaflets. Another recent study with a similar approach suggested that an increase in scramblase activity was more important than the inactivation of the other enzyme for the occurrence of PS externalization (22). Although the mechanism for asymmetric localization of the plasma membrane phospholipid has not yet been fully elucidated, the involvement of membrane-integrated enzymes with ATP-hydrolyzing activity is presumed. Tang et al. have cloned a gene coding for a Ptype adenosine triphosphatase and shown that its yeast homologue is responsible for the transport of NBD-PS from the outer to the inner leaflet of the yeast plasma membrane (21), and multidrug resistance proteins have been suggested to function as lipid flippases (23). It is naive but important to ask whether the activ-
ity and/or the concentration of such enzymes is altered upon apoptosis induction. A possible regulation is that aminophospholipid translocase is proteolytically inactivated by a caspase since PS externalization is influenced in the presence of caspase inhibitors (24). Phagocytosis of apoptotic HF1 cells by mouse peritoneal macrophages. We next asked if the apoptosing HF1 cells are susceptible to PS-mediated phagocytosis. As seen in Fig. 1B, a population of cells more intensely stained with propidium iodide appeared as apoptosis proceeded, and these were considered to be cells at late apoptotic stages with increased plasma membrane permeability. Cells became detached from the culture containers at about 4 h after apoptosis induction, and we found that such cells were mostly propidium iodide positive while cells remaining attached to dishes were negative (Fig. 3A). The attached cells were thus considered
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FIG. 3. Phagocytosis of HF1 cells undergoing Fas-mediated apoptosis by mouse peritoneal macrophages. (A) Separation of propidium iodide-positive (top square) and -negative (bottom square) cells. HF1 cells were treated with CH-11 for 10 h, and cells detached from (floating) and those remaining attached to (attaching) culture dishes were separated and analyzed for the binding of propidium iodide and FITC-annexin V in a flow cytometer. (B) Phagocytosis of the attached HF1 cells at 0 and 26 h after apoptosis induction. (C) Effect of liposomes on phagocytosis. Phagocytosis reactions were conducted in the presence of PS- and PC-liposomes, and extents of phagocytosis are shown relative to those in the reaction with no added liposomes, which is taken as 100%.
to be at early apoptotic stages with intact plasma membrane permeability. We collected the attached cells at 0 (control) and 26 h after a CH-11 treatment and analyzed them in a phagocytosis assay with peritoneal macrophages prepared from thioglycholate-treated mice. The antibody-treated HF1 cells were significantly more phagocytosed than control cells by macrophages (Fig. 3B), and all the phagocytosis reactions were inhibited in the presence of PS-liposomes while PC-liposomes showed a minimal effect (Fig. 3C). These results indicated that cells at early stages of apoptosis were phagocytosed by macrophages, at least in part, in a PS-mediated manner. Cells undergoing Fas-mediated apoptosis show PS externalization (24-27), but as to whether these cells
FIG. 4. PS externalization and phagocytosis of N-ethylmaleimide-treated HF1 cells. (A) PS externalization. HF1 cells were treated with various concentrations of N-ethylmaleimide for 6 min at 37 7C and PS externalization was analyzed by flow cytometry. Numbers are percentages of propidium iodide-negative cells. (B) Phagocytosis. The drug-treated HF1 cells were subjected to a phagocytosis assay with mouse peritoneal macrophages. (C) The effect of liposomes on phagocytosis was determined as described in the legend for Fig. 3C.
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are engulfed by phagocytes dependent on the externalized PS remained to be investigated. We here provide evidence that they are. It is thus presumed that other cells in which apoptosis is induced through the Fas system, such as T lymphocytes under activation-induced death (28-30), acute lymphocytic leukemia cell lines treated with an anti-cancer drug, doxorubicin (31), and influenza virus-infected cultured cells (27), undergo heterophagic elimination depending on the presence of PS on the cell surface. All these apoptotic cells could be cleared before their lysis, thus causing no damage to the organism. Phagocytosis of N-ethylmaleimide-treated HF1 cells. The above results suggested that PS externalization is solely responsible for the phagocytosis of apoptotic cells by macrophages. We then asked if PS externalization that occurs independent of apoptosis can be a trigger of phagocytosis. Since the activity to define the translocation of aminophospholipids is inhibited by SH-blockers such as N-ethylmaleimide (32), we determined PS externalization in HF1 cells treated with the drug and found that it induced PS externalization in a dose-responsive manner (Fig. 4A). The drug-treated cells did not seem to apoptose since most were not intensely stained with Hoechst33342 (not shown) or propidium iodide. When such cells were subjected to a phagocytosis assay with macrophages, they were phagocytosed almost in parallel with the appearance of PS on the cell surface (Fig. 4B). The extent of phagocytosis was not as large as that with apoptosing HF1 cells (see Fig. 3B), but the reaction was inhibitable with PS-liposomes (Fig. 3C). These results indicated that non-apoptotic cells were phagocytosed by macrophages in a PS-mediated manner. All the above results suggested that the externalization of PS on the surface of apoptotic cells has an essential role in their subsequent elimination by phagocytosis. It is likely that macrophages, and other phagocytic cells as well, recognize target cells through their receptors specific to PS and rapidly phagocytose them. This may prevent the surrounding tissues from being exposed to the noxious contents of lysed cells. Although members of the scavenger receptor family have been strong candidates for the PS receptor (33-37), its identity is still a matter of debate. Elucidation of the molecular basis for recognition of apoptosing cells by phagocytes is necessary for a better understanding of the fate of apoptotic cells. Such efforts should lead to the development of new therapeutics to overcome diseases for which effective medical treatment is not yet available. ACKNOWLEDGMENTS We thank R. Schlegel and K. Hanada for their important suggestions on experimental procedures and critical comments on the manuscript. We also thank M. Beppu, H. Kizaki, and F. Amano for their valuable suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science and Culture of Japan, by the Sasakawa Scientific Research Grant from the Japan Science Society and by OFUKAI (The Alumnae Association of Japan Women’s University).
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27. Fujimoto, I., Takizawa, T., Ohba, Y., and Nakanishi, Y. (1998) Cell Death Differ. in press. 28. Dhein, J., Walczak, H., Ba¨umler, C., Debatin, K.-M., and Krammer, P. H. (1995) Nature 373, 438–441. 29. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., and Green, D. R. (1995) Nature 373, 441–444. 30. Ju, S.-T., Panka, D. J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D. H., Stanger, B. Z., and Marshak-Rothstein, A. (1995) Nature 373, 444–448. 31. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K.-M. (1996) Nature Med. 2, 574–577.
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Essential Role of Phosphatidylserine Externalization in Apoptosing Cell Phagocytosis by Macrophages Akiko Shiratsuchi,* Sachie Osada,* Susumu Kanazawa,† and Yoshinobu Nakanishi*,‡,1 *Graduate School of Natural Science and Technology, and ‡Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan; and †Kaken Pharmaceutical Co., Ltd., Fujieda, Japan
Received April 14, 1998
In many apoptotic cells, phosphatidylserine (PS), that is normally restricted to the inner membrane layer, is externalized and subsequently recognized by phagocytes. However, it has been unclear whether PS externalization is sufficient for phagocytosis induction. In a cultured cell line undergoing Fas-mediated apoptosis, PS externalization preceded other apoptotic events. When transbilayer movement of membrane phospholipids was analyzed, a decrease of the uptake of PS and phosphatidylethanolamine and an increase of phosphatidylcholine incorporation were observed upon apoptosis induction. Apoptotic cultured cells were phagocytosed by macrophages in a manner dependent on externalized PS before plasma membrane permeability increased. Moreover, a N-ethylmaleimide treatment caused PS externalization independent of apoptosis, and such cells underwent PS-mediated phagocytosis. These results suggested that PS is externalized as a result of membrane phospholipid redistribution and externalized PS by itself induces apoptosing cell phagocytosis. q 1998 Academic Press
Apoptotic cells are engulfed by phagocytes, and this event has been presumed to prevent inflammation and damage to surrounding tissues caused by the noxious contents of dead cells (1,2). Apoptosing cells most likely expose a marker molecule(s), such as proteins, sugars, and phospholipids, which phagocytic cells recognize to capture target cells for phagocytosis (3,4). Among them, a membrane phospholipid PS has been the most extensively characterized in terms of both how it becomes 1 To whom correspondence should be addressed at Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920-0934, Japan. Fax: (81) 76-234-4480. E-mail: [email protected]. Abbreviations used: FITC, fluorescein isothiocyanate; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NBD, 4-nitrobenzo-2-oxa-1,3-diazole; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine.
expressed on the surface of apoptosing cells and how it is involved in phagocytosis (3-7). A variety of apoptosis triggers presumably cause redistribution of the plasma membrane phospholipid at early apoptotic stages; for example, PS and PE, which are normally confined to the inner leaflet, are transferred to the outer leaflet. With several different apoptotic cells such as lymphocytes (8), erythrocytes (9), vascular smooth muscle cells (10), and spermatogenic cells (11), externalized PS has been shown to be recognized by phagocytic cells leading to subsequent phagocytosis. However, it has been unclear whether apoptotic cells are phagocytosed before plasma membrane permeability increases and PS externalization by itself is sufficient for inducing phagocytosis. We here assessed such issues. MATERIALS AND METHODS Cell culture and apoptosis analysis. HeLa cells were maintained in minimal essential medium supplemented with 10% fetal bovine serum at 37 7C in a humidified atmosphere with 5% CO2 in air. The cells were transfected with an expression vector pSRa (12) containing human Fas cDNA (13) using lipofectin (Gibco BRL, MD, U. S. A.). Cell clones resistant to G418 (1 mg/ml) were examined for the expression of cell surface Fas by staining them with an anti-human Fas monoclonal antibody, CH-11 (14) (MBL, Nagoya, Japan). One of the clones that gave a strong signal was selected and named HF1. The presence of cell surface Fas was determined by flow cytometry using an anti-human Fas monoclonal antibody, UB2 (MBL) and fluorescein-conjugated anti-mouse IgG (Immunotech, Marseilles, France). Normal mouse IgG (Zymed Laboratories, CA, U. S. A.) was used as a negative control. HF1 cells were induced to undergo apoptosis by cultivating them in the presence of CH-11 (0.5 mg/ml). Plasma membrane permeability, activity of mitochondrial enzyme, chromatin condensation, and DNA fragmentation were determined by standard procedures; a dye exclusion test with trypan blue, an enzyme assay with MTT (15, 16) using a commercial kit (Chemicon, CA, U. S. A.), staining with Hoechst33342, and agarose gel electrophoresis, respectively. PS externalization was determined in a flow cytometer (EPICS XL; Coulter, FL, U. S. A.) as described previously (17, 18). Briefly, FITC-labeled annexin V was added to test cells simultaneously with a plasma membrane-non-permeable dye, propidium iodide, and cells less intensely stained with propidium iodide were analyzed for the binding of FITC-annexin V. Phospholipid movement assay. Transbilayer movement of phospholipids was monitored with NBD-labeled phospholipid analogues
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(Avanti Polar Lipids, AL, U. S. A.); (palmitoyl, C6-NBD)-PS, (palmitoyl, C6-NBD)-PE, and (palmitoyl, C6-NBD)-PC, essentially as described previously (19). Briefly, NBD-phospholipids were dried down and dissolved with small amounts of ethanol. HF1 cells were suspended (1 1 106 cells/ml) in phosphate-buffered saline containing 1 mM MgCl2 and 2 mM CaCl2 , and supplemented with NBD-phospholipids (1 mM). After a 1-min incubation on ice, the cells were spun down and resuspended in the same buffer. NBD-phospholipids loaded on the outer leaflet of the plasma membrane were allowed to be transferred to the inner leaflet by incubating the cells at 25 7C for 5 min unless otherwise stated. The cells were then briefly treated with fatty acid-free bovine serum albumin (Sigma, MO, U. S. A.) (5 mg/ml) to remove the phospholipid analogues remaining on the outer leaflet; that is, ‘back exchange’ (20). These cells were then supplemented with propidium iodide and subjected to a flow cytometric analysis. The amounts of NBD-phospholipids transferred to the inner membrane leaflet were determined with cells less intensely stained with propidium iodide. Macrophage preparation and phagocytosis assay. BDF1 (female, 8-week old) mice were injected with thioglycollate-containing medium (fluid thioglycollate medium; Difco, MI, U. S. A.) (2 ml or 1.5 mg sodium thioglycollate per animal), and peritoneal fluids were obtained after 4 days. The cells in the fluid were collected by centrifugation, washed with Hank’s balanced salt solution (Nissui Pharmaceutical, Tokyo, Japan) and cultivated on a coverslip (øÅ15 mm) with RPMI1640 medium containing 10 mM Hepes, pH 7. Cells unattached to the coverslips were washed out and the remaining cells were analyzed for the identity of macrophage; the cells were mostly mononuclear and more than 90% of them showed incorporation of fluorescence-labeled latex beads (Polybead Microparticles, øÅ0.75 mm, Polysciences, PA, U. S. A.), indicating that they were at least 90% pure as macrophages. A phagocytosis assay was performed essentially as described (11). Briefly, target cells were labeled with biotin, mixed with macrophages (at a ratio of ten target cells against one macrophage), and incubated at 37 7C for 2-3 h. The reacted cells were washed with phosphate-buffered saline and subsequently treated with trypsin (0.5 mg/ml). They were then fixed and supplemented with FITC-conjugated avidin, and the extent of phagocytosis was determined under a fluorescence/phase contrast microscope (BX50; Olympus, Tokyo, Japan). The number of macrophages containing engulfed apoptotic cells was determined and shown relative to the total number of macrophages, taken as 100; that is, the phagocytic index. The mean and standard deviations of a typical example from at least three independent experiments were presented. Liposomes were prepared according to standard procedures. In brief, phospholipids (Avanti Polar Lipids) were dried as films, suspended in phosphate-buffered saline, and sonicated. The resulting liposomes were used in experiments without further fractionation at a total phospholipid concentration of 1 mM. They were composed of either PC only (PC-liposomes) or a combination of PC and PS at a molar ratio of 7:3 (PS-liposomes).
RESULTS AND DISCUSSION Apoptosis of HF1 cells. The cell surface expression of Fas in HF1 cells was evident by flow cytometric analysis while control cells containing an empty vector showed minimal fluorescence intensities (Fig. 1A). The over expression of Fas did not cause significant changes in cell growth but a slight change in morphology was noticeable; HF1 cells looked more spherical than parent HeLa cells (not shown). HF1 cells were induced to undergo apoptosis by the addition of an apoptosis-agonistic antibody, CH-11, and biochemical changes during an apoptosis course were determined (Fig. 1B). The extent
of chromatin condensation was determined by staining DNA with a fluorescence dye, Hoechst33342. Condensation was evident soon after the antibody addition and reached a maximal level at about 8 h. The integrity of mitochondria was examined by a calorimetric MTT assay, and the activity to cleave MTT began to decrease at 6 h reaching a maximal level at 12 h. Membrane permeability, assessed by a dye exclusion test with trypan blue, continued to increase up to 36 h. Internucleosomal DNA fragmentation became detectable after a 12-h incubation with CH-11 (not shown). The presence of PS on the surface of apoptotic HF1 cells was analyzed by staining the cells with FITClabeled annexin V and propidium iodide. The cells that were not stained with propidium iodide were analyzed for the amount of cell surface PS in a flow cytometer. Mean fluorescence of a main peak shifted at 3 h after apoptosis induction and did not significantly change for the next 5 h (right panels in Fig. 1B), indicating that PS externalization was maximized by 3 h. The above results showed that apoptotic events examined so far took place with different time courses and the externalization of PS reached maximal level earlier than other biochemical changes. Change of transbilayer movement of phospholipid analogues during apoptosis. Verhoven et al. reported that the efficiency by which fluorescence-labeled phospholipid analogues are transferred from the outer to the inner leaflet of the plasma membrane bilayer changes in apoptotic cells; that is, the incorporation of NBD-PS decreased and that of NBD-PC increased in a T-cell line upon apoptosis caused by an anti-CD3 antibody treatment (19). We adopted a similar method to determine the change of phospholipid analogue movement in cells undergoing Fas-dependent apoptosis. The mode of NBD-phospholipid translocation in normal HF1 cells was first examined. The cells were incubated with fluorescence-labeled phospholipid analogues, NBD-PS, -PE and -PC, for 1 min on ice; this should result in the phospholipid analogues being incorporated into the outer leaflet of the plasma membrane bilayer. In fact, more than 80% of the fluorescence signals disappeared from the labeled cells after extraction with bovine serum albumin (not shown). NBD-phospholipids loaded on the cell surface were then allowed to be transferred to the inside by raising the incubation temperature to 25 7C. The amounts of NBD-phospholipids remaining in the cells after ‘back exchange’ were determined in a flow cytometer. The incorporation of all three phospholipid analogues occurred time dependently, and NBD-PS and -PE were more efficiently transferred than NBD-PC (Fig. 2A). We then examined changes in phospholipid analogue movement in HF1 cells upon apoptosis induction with CH-11. The cells were recovered at various time points after the antibody addition and subjected to a phospho-
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FIG. 1. Apoptosis of HF1 cells by anti-Fas antibody. (A) Cell surface expression of Fas. HF1 cells and control HeLa/SRa cells (containing a vector alone) were treated with UB2 (solid lines) or control IgG (broken lines) and analyzed in a flow cytometer. (B) Time course of apoptosis. Left panels; HF1 cells were treated with CH-11 (open circles) or left untreated (closed circles), and the progress of apoptosis, in terms of chromatin condensation (top), inactivation of a mitochondrial enzyme (middle), and plasma membrane permeabilization (bottom) was analyzed. Right panels; PS externalization in apoptotic HF1 cells. The cells at each time point after apoptosis induction were analyzed for the binding of propidium iodide and FITC-annexin V in a flow cytometer. Shown on the far right are FITC profiles of cells less intensely stained with propidium iodide.
lipid movement assay. The amounts of NBD-phospholipids initially loaded on the cells needed to be determined with each cell preparation since they decreased with the progress of apoptosis (not shown). The efficiency of incorporation of NBD-PS and -PE started to decrease soon after apoptosis induction and reached 60-80% that of control cells at 3.5 h, while that of NBDPC increased up to 150% (Fig. 2B). This was explained by the appearance of cells with less NBD-PS or -PE and of cells with more NBD-PC incorporated (Fig. 2C). These results indicated that the movement of NBD-PS and -PE into the inner leaflet of the membrane bilayer decreased and that of NBD-PC contrarily increased in HF1 cells undergoing Fas-dependent apoptosis. All these changes became evident synchronously at 2-3 h after apoptosis induction (bottom panels in Fig. 2C), and this is the time point when PS externalization was observed (Fig. 1B). The above results were consistent with those obtained by Verhoven et al. (19).
PS and PE are restricted to the cytoplasmic side presumably through the action of an enzyme called aminophospholipid translocase (5-7), which was recently identified as a P-type adenosine triphosphatase (21). Verhoven et al. (19) interpreted their results as that aminophospholipid translocase is inactivated and scramblase, an enzyme responsible for the two-way movement of phospholipids between the plasma membrane leaflets (5-7), is activated in cells undergoing apoptosis, and that these changes lead to the cell surface exposure of PS. We here showed that similar changes in transbilayer movement of membrane phospholipids occurred in cells undergoing Fas-mediated apoptosis. We observed a decrease of NBD-PE incorporation in apoptotic cells, which was not investigated by Verhoven et al. This finding further supported the above speculation that aminophospholipid translocase is influenced upon apoptosis induction. All such changes may disrupt asymmetric localization of the
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FIG. 2. Transbilayer movement of fluorescence-labeled phospholipid analogues in apoptotic HF1 cells. (A) Time course with normal HF1 cells. The mean fluorescence of NBD in cells less intensely stained with propidium iodide (see MATERIALS AND METHODS) was determined at each time point in a flow cytometer and is shown relative to that in cells before ’back exchange’, which is taken as 100%. (B) Change of phospholipid analogue movement upon apoptosis induction. Cells at each time point after apoptosis induction were subjected to a NBD-phospholipid movement assay. The extent of NBD-phospholipid transfer was determined as in (A) and is shown relative to that in untreated cells (time 0), which is taken as 100%. (C) Appearance of cells with altered levels of phospholipid analogue movement upon apoptosis induction. Top nine panels; staining profiles with propidium iodide and NBD of cells after apoptosis induction. Cells with altered NBD levels are circled. Bottom panels; the number of the circled cells relative to that of total cells is shown with the mean and standard deviations from three independent experiments.
plasma membrane phospholipid and bring about its redistribution on the two membrane leaflets. Another recent study with a similar approach suggested that an increase in scramblase activity was more important than the inactivation of the other enzyme for the occurrence of PS externalization (22). Although the mechanism for asymmetric localization of the plasma membrane phospholipid has not yet been fully elucidated, the involvement of membrane-integrated enzymes with ATP-hydrolyzing activity is presumed. Tang et al. have cloned a gene coding for a Ptype adenosine triphosphatase and shown that its yeast homologue is responsible for the transport of NBD-PS from the outer to the inner leaflet of the yeast plasma membrane (21), and multidrug resistance proteins have been suggested to function as lipid flippases (23). It is naive but important to ask whether the activ-
ity and/or the concentration of such enzymes is altered upon apoptosis induction. A possible regulation is that aminophospholipid translocase is proteolytically inactivated by a caspase since PS externalization is influenced in the presence of caspase inhibitors (24). Phagocytosis of apoptotic HF1 cells by mouse peritoneal macrophages. We next asked if the apoptosing HF1 cells are susceptible to PS-mediated phagocytosis. As seen in Fig. 1B, a population of cells more intensely stained with propidium iodide appeared as apoptosis proceeded, and these were considered to be cells at late apoptotic stages with increased plasma membrane permeability. Cells became detached from the culture containers at about 4 h after apoptosis induction, and we found that such cells were mostly propidium iodide positive while cells remaining attached to dishes were negative (Fig. 3A). The attached cells were thus considered
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FIG. 3. Phagocytosis of HF1 cells undergoing Fas-mediated apoptosis by mouse peritoneal macrophages. (A) Separation of propidium iodide-positive (top square) and -negative (bottom square) cells. HF1 cells were treated with CH-11 for 10 h, and cells detached from (floating) and those remaining attached to (attaching) culture dishes were separated and analyzed for the binding of propidium iodide and FITC-annexin V in a flow cytometer. (B) Phagocytosis of the attached HF1 cells at 0 and 26 h after apoptosis induction. (C) Effect of liposomes on phagocytosis. Phagocytosis reactions were conducted in the presence of PS- and PC-liposomes, and extents of phagocytosis are shown relative to those in the reaction with no added liposomes, which is taken as 100%.
to be at early apoptotic stages with intact plasma membrane permeability. We collected the attached cells at 0 (control) and 26 h after a CH-11 treatment and analyzed them in a phagocytosis assay with peritoneal macrophages prepared from thioglycholate-treated mice. The antibody-treated HF1 cells were significantly more phagocytosed than control cells by macrophages (Fig. 3B), and all the phagocytosis reactions were inhibited in the presence of PS-liposomes while PC-liposomes showed a minimal effect (Fig. 3C). These results indicated that cells at early stages of apoptosis were phagocytosed by macrophages, at least in part, in a PS-mediated manner. Cells undergoing Fas-mediated apoptosis show PS externalization (24-27), but as to whether these cells
FIG. 4. PS externalization and phagocytosis of N-ethylmaleimide-treated HF1 cells. (A) PS externalization. HF1 cells were treated with various concentrations of N-ethylmaleimide for 6 min at 37 7C and PS externalization was analyzed by flow cytometry. Numbers are percentages of propidium iodide-negative cells. (B) Phagocytosis. The drug-treated HF1 cells were subjected to a phagocytosis assay with mouse peritoneal macrophages. (C) The effect of liposomes on phagocytosis was determined as described in the legend for Fig. 3C.
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are engulfed by phagocytes dependent on the externalized PS remained to be investigated. We here provide evidence that they are. It is thus presumed that other cells in which apoptosis is induced through the Fas system, such as T lymphocytes under activation-induced death (28-30), acute lymphocytic leukemia cell lines treated with an anti-cancer drug, doxorubicin (31), and influenza virus-infected cultured cells (27), undergo heterophagic elimination depending on the presence of PS on the cell surface. All these apoptotic cells could be cleared before their lysis, thus causing no damage to the organism. Phagocytosis of N-ethylmaleimide-treated HF1 cells. The above results suggested that PS externalization is solely responsible for the phagocytosis of apoptotic cells by macrophages. We then asked if PS externalization that occurs independent of apoptosis can be a trigger of phagocytosis. Since the activity to define the translocation of aminophospholipids is inhibited by SH-blockers such as N-ethylmaleimide (32), we determined PS externalization in HF1 cells treated with the drug and found that it induced PS externalization in a dose-responsive manner (Fig. 4A). The drug-treated cells did not seem to apoptose since most were not intensely stained with Hoechst33342 (not shown) or propidium iodide. When such cells were subjected to a phagocytosis assay with macrophages, they were phagocytosed almost in parallel with the appearance of PS on the cell surface (Fig. 4B). The extent of phagocytosis was not as large as that with apoptosing HF1 cells (see Fig. 3B), but the reaction was inhibitable with PS-liposomes (Fig. 3C). These results indicated that non-apoptotic cells were phagocytosed by macrophages in a PS-mediated manner. All the above results suggested that the externalization of PS on the surface of apoptotic cells has an essential role in their subsequent elimination by phagocytosis. It is likely that macrophages, and other phagocytic cells as well, recognize target cells through their receptors specific to PS and rapidly phagocytose them. This may prevent the surrounding tissues from being exposed to the noxious contents of lysed cells. Although members of the scavenger receptor family have been strong candidates for the PS receptor (33-37), its identity is still a matter of debate. Elucidation of the molecular basis for recognition of apoptosing cells by phagocytes is necessary for a better understanding of the fate of apoptotic cells. Such efforts should lead to the development of new therapeutics to overcome diseases for which effective medical treatment is not yet available. ACKNOWLEDGMENTS We thank R. Schlegel and K. Hanada for their important suggestions on experimental procedures and critical comments on the manuscript. We also thank M. Beppu, H. Kizaki, and F. Amano for their valuable suggestions. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education,
Science and Culture of Japan, by the Sasakawa Scientific Research Grant from the Japan Science Society and by OFUKAI (The Alumnae Association of Japan Women’s University).
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27. Fujimoto, I., Takizawa, T., Ohba, Y., and Nakanishi, Y. (1998) Cell Death Differ. in press. 28. Dhein, J., Walczak, H., Ba¨umler, C., Debatin, K.-M., and Krammer, P. H. (1995) Nature 373, 438–441. 29. Brunner, T., Mogil, R. J., LaFace, D., Yoo, N. J., Mahboubi, A., Echeverri, F., Martin, S. J., Force, W. R., Lynch, D. H., Ware, C. F., and Green, D. R. (1995) Nature 373, 441–444. 30. Ju, S.-T., Panka, D. J., Cui, H., Ettinger, R., El-Khatib, M., Sherr, D. H., Stanger, B. Z., and Marshak-Rothstein, A. (1995) Nature 373, 444–448. 31. Friesen, C., Herr, I., Krammer, P. H., and Debatin, K.-M. (1996) Nature Med. 2, 574–577.
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