Published November 1, 1995

Early Redistribution of Plasma Membrane
Phosphatidylserine Is a General Feature of Apoptosis
Regardless of the Initiating Stimulus: Inhibition by
Overexpression of Bcl-2 and Abl
B y Seamus J. M a r t i n , * C h r i s E M . R e u t e l i n g s p e r g e r , ~
A n n e J. M c G a h o n , * James A. R a d e r , * R o b C. A. A. van Schie,w
D r a k e M . LaFace,* a n d D o u g l a s tL. G r e e n *
From the *Division of Cellular Immunology, La Jolla Institutefor Allergy and Immunology, La Jolla,
California 92037; ~.Department of Biochemistry, University of Limburg, Maastricht, The
Netherlands; and w
of Immunology, University College London Medical School, London,
United Kingdom

A critical event during programmed cell death (PCD) appears to be the acquisition of plasma
membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they
rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes
that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this
phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we
show that PS externalization is an early and widespread event during apoptosis of a variety of
murine and human cell types, regardless of the initiating stimulus, and precedes several other
events normally associated with this mode of cell death. We also report that, under conditions
in which the morphological features ofapoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the
PM was similarly prevented. These data are compatible with the notion that activation of an
inside-outside PS translocase is an early and widespread event during apoptosis.
cell death (PCD) 1 is a fundamental feature
p ,rogrammed
of many important biological processes (1-4). Mammalian cells, as well as many other cell types, typically undergo
a series of distinct morphological changes as they die; this
form of PCD has been termed apoptosis (5). This subset of
PCD appears to account for the majority of PCD in higher
organisms.
A critical stage of apoptosis involves the acquisition of
surface changes by the dying cell that result in the recognition and uptake of these cells by phagocytes (6-9). The nature and kinetics of these surface changes are the subject of
active investigation but are still largely unknown (10). A series of studies has shown that apoptotic human neutrophils,
as well as some other cell types, are recognized by bone
marrow- and blood-derived macrophages by means of a
~Abbreviationsused in this paper:Act D, actinomycinD; CHX, cyclohexamide; Dex, dexamethasone;PA, phosphatidicacid; PC, phosphatidylcholine; PCD, programmed cell death; PE, phosphatidylethanolamine;PI,
phosphatidylinositol;PM, plasmamembrane;PS, phosphatidylserine;SM,
sphingomyehn; TSP, thrombospondin;VnR, vitronectinreceptor; VP16, etoposide.
1545

thrombospondin (TSP)/otd33 (vitronectin receptor [VnR])/
CD36 complex, which interacts with an as yet unidentified
"charge-sensitive" moiety on the apoptotic cell (9-14).
There is also evidence to suggest that phosphatidylserine
(PS), which is normally almost totally confined to the inner
leaflet of the plasma membrane (PM), may act as as a membrane "flag" on apoptotic cells (15-17). Macrophages elicited from the peritoneal cavities of mice specifically recognized apoptotic cells in a manner that was inhibited by
liposomes containing PS, but not by liposomes containing
other aminophospholipids (15, 16). However, because of
the lack of a specific probe for PS, changes in the distribution of this aminophospholipid during apoptosis have yet to
be demonstrated directly.
A recently discovered family of proteins, the annexins,
has been found to have high affinity for aminophospholipids in the presence of Ca 2+ ions (18-20). A member of this
family, annexin V, has been shown by several groups to
preferentially bind PS (18, 19, 21) and to inhibit PS-dependent procoagulant reactions (22). Annexin V has also been
used successfully as a probe of PS exposure during platelet

J. Exp. Med. 9 The Rockefeller University Press 90022-1007/95/11/1545/12 $2.00
Volume 182 November 1995 1545-1556

Downloaded from jem.rupress.org on February 10, 2015

Summary

T. pH 7. as assessed by the increased annexin V-binding properties o f these cells. Cycloheximide (CHX). Cell Cycle Analysis. 23). respectively. For inhibition experiments. dexamethasone (Dex). La Jolla. propidium iodide dye was added to a final concentration of 10 txg/ml before analysis.org on February 10. Cells were grown at 37~ in a humidified 5% CO2 atmosphere and were seeded at a density of 0. repressors o f apoptosis such as Bcl-2 and Abl were also found to inhibit PS externalization. Cells were then selected for 5 d in G418 and cloned by limiting dilution. a major source o f p r o c o a g u l a n t activity (21. Thus. phosphatidylinositol (PI). sphingomyelin (SM). Here we show that apoptosis. To distinguish cells that had lost membrane integrity. 150 mM NaC1. to a final concentration of 10 -3 M. Green. tK. Scoring of Apoptosis. gas. Propidium Iodide Uptake Assay. The decreased binding of DNA-binding dyes to apoptotic cells (these cells appear as a distinct peak below the G0/G~ peak) allows the discrimination ofapoptotic cells from their healthy counterparts by conventional cell cycle analysis (26). The scale bar is in arbitrary units. 2015 activation. Cell membrane permeability was assessed by determining the ability of cells to exclude the 1546 DNA-binding fluorescent dye propidium iodide as previously described (25). phosphatidic acid (PA). P. was accompanied by dramatic changes in PS distribution at the PM. S. purified phospholipids. staurosporine. Bcl-2 and Abl-transfected Cell Lines. and phosphatidylethanolamine (PE). (A) Cells were cultured in either medium alone or medium containing 250 ng/rnl of anti-Fas IgM for 7 h and viewed under phase contrast microscopy. manuscript submitted for publication). Pure preparations (10 mg/ml) of lipids in chlorofoml/methanol (90:10) were added to glass tubes in appropriate combinations and were evaporated to dryness under N. Cells were assessed for binding of annexin V by incubating for 5 rain in 0. CA). Martin. MO).. Briefly. Bissonnette. CEMneo and CEMbcl_2 cells.000 cells from a representative experiment. etoposide (VP-16). were prepared as follows. (Thousand Oaks. (C) Cell death. the kind gift of Dr. Apoptotic cells were discriminated from healthy cells by the condensed and fragmented appearance of their nuclei under light microscopy. A. induced by a variety o f agents. John Reed (La Jolla Cancer Research Foundation. G. Liposomes were then prepared by sonicating the lipid mixtures on ice for 3-5 rain Figure 1. (B) Detection of internucleosomal DNA fragmentation in Jurkat cells treated with the indicated concentrations ofanti-Fas IgM for 7 h. CA). N-acetyl sphingosine (C2-ceramide) was purchased from BIOMOL Research Laboratories.5-1 • 106 cells/ml in most experiments. . (Plymouth Meeting. Jean Wang (University of California. Transfectants were recloned by limiting dilution and were screened for Bcl-2 expression by Western blot (data not shown). Significantly. Isolation of Human Neutrophils. providing further evidence that this event is tightly coupled to apoptosis. liposomes composed of70% phosphatidylcholine (PC) and 30% of each of the phospholipids PS.8 mM CaC12).Published November 1. cytospin preparations of cells were stained with Leukostat (Fisher Scientific Co. Inc. (St Louis. HL-60 cells were transfected with a plasmid containing the Abl temperature-sensitive mutant. Thymi were removed from 6-16-wk-old mice and were disrupted between sterile glass slides to liberate thymocytes. hydrogen peroxide. according to standard procedures (9). and D. Cotter. San Diego. followed by screening for Abl expression by Western blot (McGahon. 1995 Materials and M e t h o d s Cell Culture and Reagents. Recombinant annexin V was produced and purified as previously described (27). Notably. 1. followed by flow cytometry. after a 24-h exposure to the indicated concentrations of anti-Fas IgM. or 100% PC. Each data point was derived from an analysis of 10. PA). as assessed by trypan blue exclusion assay. Isolation ofMurine Thymocytes. NY) to reveal nuclear morphology.rupress. annexin V provides a convenient tool with which to determine directly whether changes in membrane PS distribution occur as a general feature o f apoptosis and to measure the kinetics o f these changes on a single-cell basis. a recent report demonstrated increased annexin V binding during serum w i t h d r a w a l . the changes preceded loss o f membrane integrity by several hours. 5 mM KC1.4. Human neutrophils were purified from heparinized blood drawn from normal healthy volunteers.i n d u c e d apoptosis o f murine germinal center B cells as well as a B cell line (24). J. Cell cycle analysis was performed as previously described (25). and Percoll were all purchased from Sigma Chemical Co. Apoptotic cells were identified using previously published criteria (5). Thymocytes prepared in this way were routinely 99% viable. 1K.1-1 Ixg/ml FITC-conjugated annexin V (1:1 stoichiometric complex) in Hepes buffer (10 mM Hepes-NaOH. Redistribution of Phosphatidylserine during Apoptosis Downloaded from jem. Annexin V-FITC Preparationand Binding Assays. Anti-Fas IgM mAb (CH-11) was purchased from Kamiya Biomedical Co. In this regard. or a control plasmid (plKP4). stably transfected with the retroviral vectors pZipNeo and pZipBcl-2. 1 mM MgC12. as assessedby propidium iodide uptake. Induction of apoptosis by ligation of Fas (CD95) on Jurkat cells. PS redistribution was detected during apoptosis o f all cells o f h u man and mouse origin tested and in response to all stimuli that resulted in apoptosis. were kindly provided by Dr.J. All cell lines were cultured in RPMI 1640 containing 5% FCS and were routinely subcultured every 2-3 d. Lipid mixtures were then resuspended in Hepes buffer. as described (24).. CA). actinomycin D (Act D). by vortexing. changes in PS asymmetry were detected before the morphological changes associated with apoptosis had occurred. Thymocytes were then washed three times in HBSS. plKK-160.

As in previous reports (29). Changesin plasmamembranePS distributionduring apoptosis induced by Fas ligation. prepared as described in Materials and Methods. Fas Aminophospholipid Redistribution at the PM Is Rapidly Detected upon Ligation of Fas on Jurkat Cells. followed by stainingwith annexin V-FITC in the presence or absence of the indicated liposomes. (/3) annexin V binding to anti-Fas IgM-treated Jurkat cells in the presence or absence of the indicated concentrationsof liposomes. 30). 1 B). constitutively expresses Fas and is rapidly killed u p o n treatment o f these cells with anti-Fas antibody (Fig..= 2oo ]00 == Log annexin V binding = Figure 2. and. (n~/ml) ~ A anti-F~Ls AxVhi 300 (%) 57.rupress. Typically.7 3s. DNA Isolation and Electrophoresis.. as described in Materials and Methods. as assessedby the binding of annexin V-FITC. Fas-FasL interactions are likely to be important physiological triggers of cell death in the i m m u n e system (31) and possibly elsewhere (32).. T o determine whether PS externalization occurred during apoptosis induced by Fas ligation. (B) Jurkat cellswere exposed to the indicated concentrations ofanti-Fas IgM for 7 h. features of apoptosis. liposomes were added to the annexin V-FITC solution at various final concentrations (see Results). 5. the inner PM leaflet contains essentially all PS.000 cells were analyzed in each condition. 1). CT) on low output.org on February 10. such as propidium iodide (Fig.1 1 10 100 Total lipid qaM) Figure 3.000 cellswere analyzedin each condition. because of its high affinity for PS. ultimately.. Cells were then stained with the annexin V-liposome mixture in the usual manner.5 0 9. uptake of vital dyes.0 75 53. and P C distributed equally between both leaflets (33). Inhibition of annexin V-FITC binding to apoptotic cells by PS liposomes. Results o 0 .4 none + PE + PS + PI + PA + SM J pc Log annexin V binding B 400 49. Fig. a T lymphocytic leukemia cell line. (A) Time course of PS externalizationin Jurkat cells exposed to 200 ng/ml anti-FaslgM.2 150 57.5 53.4 I 3001 46. in a sonifier (model 250. cleavage o f D N A into nucleosomal multiples (Fig. Annexin V binding was assessedby flow cytometry.6 = 9.2 37. Results are representativeof six independent experiments. followed by incubation for 10 rain at room temperature to allow binding of the annexin V. Jurkat cells were treated with anti-Fas antibody and probed for changes in surface PS expression at various times 1547 Martin et al.1 ) is a cell-surface molecule belonging to the T N F / n e r v e growth factor receptor family. Before staining cells with annexin V-FITC. Branson Ultrasonics Corp.01 . Data are representativeof two separate experiments. including dramatic P M blebbing and collapse of cells into numerous intact vesicles (apoptotic bodies. As outlined above. Jurkat cells were cultured in medium alone or medium containing 100 ng/ml anti-FasIgM for 6 h. 10. Danbury. most PE and PI. 1 C). cells killed under these conditions were found to exhibit all of the characteristic cell generally exhibits an asymmetric distribution o f its maj o r phospholipids.000 cells were analyzedin each case.3 . 1 A). Liposomes were used within 24 h of preparation.0 . 10. Jurkat. which has previously been shown to transduce a death signal u p o n ligation by either anti-Fas mAbs (29) or its specific ligand (FasL. Downloaded from jem. 2015 == . Induction of Apoptosis in Jurkat Cells by Fas Ligation. + + . (A) Annexin V binding to untreated versus anti-Fas IgM-treated Jurkat cells in the presence or absence of 5 p~Mof the indicatedliposomes. and binding was assessed by flow cytometry.o AxV h. with SM largely confined to the outer leaflet.Published November 1. annexin V is a convenient probe for m o n i t o r i n g changes in the distribution o f this phospholipid in the PM during apoptosis. 1995 A A anti-Fas Lipid none J J = = Log annexin V binding B AxV. DNA was isolated and subjected to electrophoresis as previously described (28). The P M o f a healthy ( C D 9 5 / A p o .

000 cells at each time point. during Fas-induced apoptosis. 3 B). Quantitation of annexin V binding was perfomled on 5. versus loss of PM integrity. Jurkat cells were either left untreated (top) or were exposed to 300 ng/ml ofanti-Fas IgM (bottom)for 5 h. as assessed by annexin V-FITC binding. In the dose-response experiment (right). T o confirm that a n n e x i n V b i n d - PS Exposure on Apoptotic Cells Precedes Increases in Membrane Permeability and Nuclear Condensation. PS exposure on the external PM leaflet precedes some of the other features of apoptosis. PA. anti-Fas I g M .F I T C (AxVI~ H o w e v e r . The proportion of cells with apoptotic nuclei was assessed on cytospun preparations of cells made at the same rune points. Similar t i m e . a p o p u l a t i o n o f cells w i t h dramatically increased a n n e x i n V . anti-Fas m A b .hibited by PS but not Other Phospholipids.Published November 1. cells were exposed to anti-Fas lgM for 7 h. and SM. thereafter. Apoptotic cells (R2) can be discriminated from norreal cells (RI) on the basis of their light scattering properties (left). (A) Kinetics of PS externalization. Annexin V Binding to Apoptotic Cells Is In. 1995 A i II- Log annexin V binding B 100 n g / m l anti-Fas m A b 50 ---'O-'9 PM changes nuclear changes D 3o [3 Nuclear changcs [] PM changes 2O 10~ 00 l 2 3 4 5 6 7 anti-Fas mAb (ng/ml) Time (h) C.t r e a t e d cells was almost completely inhibited in the presence o f 5 btM PS liposomes. Half-maximal i n h i b i t i o n o f a n n e x i n V b i n d i n g was observed with 2 ~xM PS liposomes (Fig.000 cells were analyzed at each time point. with a nnnimum of 100 cells scored per field. (C) Detection ofannexin V binding before cell shrinkage. only o n e p o p u l a t i o n o f cells was detected that displayed a u n i f o r m l o w b i n d i n g to a n n e x i n V . as indicated. as assessed by changes in light scattering properties of the cells. As s h o w n in Fig. ~ or~ Forward Scatter Forward Scatter Figure 4. These data indicate that a n n e x i n V b i n d i n g is highly specific for PS.F I T C in the presence or absence o f liposomes prepared from a panel o f phospholipids (see Materials and Methods). 3 B). w i t h i n 1 h o f Fas ligation.rupress. in Jurkat cells treated with 1 ~g/ml of anti-Fas lgM. 2 A. Fig. PI. 2015 40 . b u t r e m a i n e d u n changed in the presence o f equal a m o u n t s o f liposomes prepared from PC. A l t h o u g h o u r initial ing to apoptotic cells reflected the exposure o f PS o n these cells. before t r e a t m e n t w i t h anti-Fas antibody. as assessed by propidium iodide uptake. Partial i n h i b i t i o n o f a n n e x i n V b i n d ing was observed with high concentrations o f PA liposomes.t r e a t e d Jurkat cells were stained w i t h a n n e x i n V . 10.b i n d i n g properties (AxV hi) began to emerge in these cultures and increased rapidly thereafter. 3 A demonstrates that a n n e x i n V b i n d i n g to experiments revealed that externalization o f PS a c c o m p a nied Fas-induced apoptosis o f Jurkat cells. 2 B). PE.d e p e n d e n t i n creases in A x V hi cells were observed over a wide range o f anti-Fas a n t i b o d y concentrations (Fig. Results are the mean of four randomly seiected rid&. (B) Correlation between the appearance of PS surface-positive cells and cells with morphological features of apoptosis after exposure to anti-Fas mAb. b u t n o t other phospholipids (Fig. it r e m a i n e d a possibility that the appearance o f AxV hi cells coincided with the appearance o f cells with "leaky" m e m b r a n e s in these cultures. thereby allowing a n n e x i n V to gain access to 1548 Redistribution of Phosphatidylserine during Apoptosis Downloaded from jem.org on February 10.

Morphological data represent the means o f four randomly selected fields. 36). In contrast. 5 /3). As shown in Fig. Fig. We considered this unlikely since increases in PM permeability typically occur late during apoptosis. 10 IxM Dex. 5.cells appeared in anti-Fas antibody-treated cultures (lower right quadrants). These experiments (Fig. A x V hi cells in anti-Fas IgM-treated Jurkat cultures were found in greater abundance at each time point than cells that were apoptotic on morphological criteria. well before any increases in cell permeability occurred. Very few cells in cultures undergoing necrosis exhibited increased annexin V-binding properties in the absence of vital dye uptake. PS externalization on Jurkat cells during apoptosis induced by diverse stimuli.org on February 10. based on morphological criteria. 6 illustrates that Jurkat cells undergoing apoptosis in response to low-dose ethanol treatment bound annexin V-FITC well before any increase in membrane permeability. using another parameter of apoptotic cell death-cell shrinkage (which can be measured by flow cytometery as a change in the light scattering properties of these cells). Jurkat cells undergoing necrosis at a higher dose of ethanol. as before (Fig.Published November 1. 4 A) demonstrated that a major population of AxVhi/pro pidium iodide. (B) Correlation between the appearance of cells with changes in PM phosphofipid distribution and cells with morphological features of apoptosis (assessed on stained cytospun cell preparations) inJurkat cultures exposed to the indicated stimuli at the concentrations described in A. Because membrane changes are fundamental features of apoptosis. these data suggest that redistribution of PS during apoptosis may precede the nuclear condensation and cell shrinkage events also seen during this process. 4 C. . arrow). Alternatively. 6). as previously described for a variety of ceil types (38). 100 ~ M CHX. 2015 PS inside the cell. it was again found that changes in PM phospholipid distribution consistently preceded the nuclear changes associated with apoptosis (Fig. To determine whether apoptosis-associated changes in PM lipid asymmetry occurred coincident with or before the classical morphological features ofapoptosis. The appearance of AxV hi cells in these cultures also preceded propidium iodide dye uptake. these changes should occur regardless of the initiating stimulus. exhibited increases in annexin V-FITC binding only when membrane integrity was lost (Fig. Fig. and 2.000 cells were analyzed in each condition. An essential difference between apoptosis and necrosis is the kinetics of loss of membrane integrity during these forms of cell death. thus confirming that annexin V was indeed binding to the external PM leaflet of these cells. to exclude this possibility. To confirm that increases in annexin V binding occur during necrosis coincident with loss of membrane integrity.5 p. At each time point. as in previous experiments (data not shown). Jurkat cells treated with anti-Fas IgM were stained with annexin V-FITC along with propidium iodide dye. with the proportion of AxV hi cells in each culture at the same time point. and the appearance of cells with morphological features of apoptosis in these cultures was monitored over time by directly enumerating these cells on stained cytospun cell preparations. to reveal cells that bound annexin V and have PM damage. region 1. as indicated. which implies that increased annexin V binding to necrotic cells can be attributed solely to entry of annexin V into the cell and binding to PS on the inner leaflet of the plasma membrane. 1995 Annexin V Binding to Necrotic Cells Occurs Only as a Consequence of Membrane Rupture. the proportion of A x V hi cells in the same cultures was assessed by flow cytometry. Jurkat cells were exposed to apop1549 Martin et al. 4 A). 4 B. A untreated anti-Fas Dex i m Sta r UV ] C~-ier Log annexin V binding B 80 84 O {WI]~ Nuclear changes PM changes 40 84 20 0 ~- r Treatment 5. because PM disruption during necrosis is extensive. Furthermore. A x V hi cells were also found in abundance in the cell population that had not yet undergone any reduction in cell size in anti-Fas IgM-treated cultures (Fig. i00 ~zM etoposide (VP-16). However. 5 A demonstrates that PS exposure accompanied apoptosis of Jurkat cells in response to all of the agents tested. To determine whether this was true with respect to PS externalization.M staurosporine (Staur) after irradiation for 15 min on a UVB transilluminator (UV). Whereas apoptotic cells retain the ability to exclude vital dyes for several hours. membrane lipids are likely to redis- Downloaded from jem. Jurkat cells were induced to undergo apoptosis by Fas ligation. or because of 50~ heat shock. 5. Taken together. PS Exposure during Apoptosis Is Stimulus Independent. Figure tosis-inducing concentrations of ethanol as well as necrosisinducing concentrations of ethanol or heat shock. Correlating the proportion of apoptotic cells. or 50 txM Cg-ceramide (Cg-cer). (A) Cells were cultured for 7 h in either medium alone or in the presence of 300 tag/ml anti-Fas IgM. we exposed Jurkat cells to a range of apoptosis-inducing agents (34-37).rupress. necrotic cells undergo rapid swelling and lysis within minutes (1. 10 IxM Act D. with a m i n i m u m of 100 cells counted per field.

!(9. Oh 7h 15h I . The percentage of apoptotic or necrotic cells was also assessed at the same time point by direct morphological assessment on cytospun preparations of cells from each culture..000 ceils were analyzed under each condition. Redistribution of PS during apoptosis of nmrine thymocytes is protein and R. .tM Dex or I0 IxM VP-16. Numbers in parentheses refer to the percentage of apoptotic cells detected in each condition.~ Ethanol (5%) ~'-"- "~. 50~ Heat-Shock ~_ . T h e A1. as shown.6 apoptosis: 2% "4 i +CHX Annexin VoFITC +Act D Figure 6.i/~ " 7o.:. 7o. ~o. Changes in PS asymmetry during activation-induced cell death ofa murine T cell hybridoma. w e e x t e n d e d o u r studies to o t h e r cell types. Cells (10.1 cells has also recently b e e n s h o w n to be a F a s . 2015 apoptosis: 0% .. ~11 9 .t.. U s i n g this system.<'..M CHX or 10 IxM Act D. with a minimum of 100 cells counted per field. 2. .. Redistribution of Phosphatidylserine during Apoptosis Downloaded from jem. for a similar period. 10. w e o b s e r v e d that A1. as indicated. T o explore further w h e t h e r loss o f PS a s y m m e t r y is a general a c c o m p a n i m e n t to apoptosis. Results are derived from the means of tbur randomly selected fields. 7). no addition +CHX m +Act D tribute rapidly to b o t h sides o f the P M by simply f l o w i n g around rupture points.NA synthesis dependent. PS Externalization Is a Widespread Phenomenon during Apoptosis of Cells of Human and Murine Origin. in the presence or absence of 50 p.000 per treatment) were then analyzed by flow cytometry.. Numbers in parentheses refer to the percentage of cells exhibiting increased annexin V binding in each condition. Apoptotic cells (A0) appear below the G0/G1 peak of cells.and PS redistribution (bottom). DNA content B Ctrl 71)j~ Dex VP-16 t(97. ! ' ! 99 necrosis: 100% Figure 7.1 cells were cultured for the indicated times in the presence of plate-immobilized anti-CD3 mAb (1452C11) and were then analyzed for apoptosis-associated changes in light scattering properties (top).'~" .. ~. Jurkat cells were induced to undergo apoptosis by exposure to 5% ethanol for 90 rain or induced to undergo necrosis by exposure to 10% ethanol or 50~ heat shock.~ Ethanol (10%) ~L2] -'!. Annexin V binding and propidium iodide uptake of Jurkat cells undergoing apoptosis versus necrosis. Numbers within the dot plots represent the percentage of cells in each quadrant.C D 3 c o m p l e x (39).1 cells also externalized PS c o i n c i d e n t w i t h their entry into apoptosis (Fig. .. _ apoptosis: 4% -~0 16~ 162 16a 16~ .Published November 1.%~. A c t i v a t i o n i n d u c e d cell death o f A1.d e p e n d e n t p h e n o m e n o n (40)./~!~: t .. Freshly isolated thymocytes were cultured for 16 h in medium alone (Ctrl) or medium containing 1 I.1 m u r i n e T cell h y b r i d o m a has previously b e e n f o u n d to u n d e r g o apoptosis as a c o n s e q u e n c e o f activation via the T C R . A1.. followed by staining with annexin V-FITC and propidium iodide... there was a dra1550 Log annexin V binding Figure 8.:" ..~ Untreated necrosis: 1% Z " '" :~"-~2"5 %] . ~ 87 necrosis: 97% A Ctrl Dex VP-16 (22o/0)1 (88o/0)1 (91%: no addition r '.org on February 10. 1995 Cell death by morphological assessment . . As in the previous experiments w i t h Jurkat cells. 7o. Results are representative of six separate experiments. (A) Assessment ofapoptosis by cell cycle analysis. (B) Detection of cells with PS redistribution during exposure to the same treatments as indicated in A.rupress. . ~ necrosis: 7% 14 Log annexin V binding apoptosis: 93% ! II .

10 A. whereas CEMbcl_2 (bcl-2-transfected) cells were protected under the same conditions. normal murine thymocytes (34). Because thymocyte apoptosis is one case where apoptosis can be blocked by protein or 1KNA synthesis inhibitors (34). a T lymphoblastoid line (28). Several genes with the ability to repress apoptosis have been described in recent years.or VP-16-induced thymocyte apoptosis.i] CTRL (Oh) 1_i CTRL (12h) t (1. apoptosis repressor proteins fall into two main groups: the bd-2 and abl gene families. we sought to determine whether overexpression o f these gene products blocked apoptosisassociated membrane changes as effectively as they blocked the morphological features o f apoptosis. (vector-transfected) cells readily underwent apoptosis in response to several stimuli. . Log annexin V binding Figure 9. B and C). and human peripheral blood neutrophils (9. 42). Freshly isolated peripheral blood neutrophil populations contained few sub-G0/G I (apoptotic) cells (Fig. as assessed by the appearance of cells with sub-G0/G1 D N A content (25). Because the protection from death afforded by Bcl-2 and Abl is generally assessed by either enumerating morphologically apoptotic cells or conducting vital dye assays. HL-60 cells were transfected with a temperature-sensitive v-abl construct (HL-60. 11-13).1 cultures exposed to immobilized anti-CD3 antibody. the induction ofapoptosis in all of these cell types correlated with the rapid appearance o f A x V hi cells in these cultures (Figs. suggesting that Bcl-2 acts upstream of the effector elements that induce these changes. In each case. Significantly. 9 A) or AxV hi cells (Fig. T o determine whether overexpression of Abl also blocked membrane changes during apoptosis. Act D !]Ceramide ~-! anti-Fas DNA content B ~]CTRL (Oh) ~tCTRL (12h) 96%! 1 ~ICHX ~ ~iAct D ~Ceramide ~(33%) ~]anti-Fas I83%) 9 o. C H X ) the spontaneous apoptosis ofneutrophils induced concomitant decreases or increases in the proportion of A x g hi cells in these cultures (Fig. Under all conditions. Fig.5%~ i t (50~) -illCHX !. Results are representative of four separate experiments. 9). 50 IxM C2-ceramide.000 cells were analyzed under each condition. Several other cell types that have been extensively studied in relation to apoptosis were also examined: the HL-60 human promyelocytic leukemia cell line (35.abl ~ cells in response to C H X treatment was blocked at 32~ but not at 39~ (Fig.rupress. . As shown in Fig. 38). Act D.Published November 1. or 500 ng/ml anti-Fas IgM. CEMbd_ 2 cells were also found to be protected from externalizing PS under conditions in which PS externalization was readily detected in CEM~eo cells (Fig. Freshly isolated periperal blood neutrophils were incubated for 12 h in either medium alone (untreated) or medium containing 50 IxM CHX. Thus far. was blocked by macromolecular synthesis inhibition.r o 1551 Martin et al. Ceils were induced to die by treatment with a variety of stimuli k n o w n to trigger apoptosis in these cells (see legends to Figs. 8 B). the majority of thymocytes that exhibited increased annexin V-binding characteristics excluded propidium iodide dye (data not shown). 8-10). 11 A).org on February 10. 8 and 9 and data not shown).abl ~) which is active at 32~ but inactive at 39~ These cells were found to be resistant to the induction o f apoptosis at the permissive temperature for Abl but readily died at the restrictive temperature (Fig. 11 B). regardless of the cell type or apoptosisinducing stimulus. CEM. 10. 2015 matic and rapid increase in A x V hi cells concomitant with the appearance of apoptotic cells in AI.tM Act D. T o address this question. 8 A shows that D e x . cell lines stably transfected with bd-2 or abl were compared with their vector-transfected counterparts for their ability to resist changes in PM pbospholipid distribution during treatment with various apoptosis-inducing stimuli. (A) Assessmentofapoptosis by cell cycle analysis. As with thymocytes. 1995 Inhibition of Apoptosis and Membrane Phospholipid Redistribution by Bd-2 and Abl. PS externalization during spontaneous as well as Fas. (B) Detection of cells with externalized PS during exposure to the same treatments as indicated in A. Bcl-2 and Abl have both been shown to protect against apoptosis induced by a wide range of stimuli (41. agents that either delayed (C2-ceramide) or accelerated (Fas ligation. Similarly. 9 B). 10 I.or druginduced apoptosis of human peripheraI blood neutrophils. we tested whether these inhibitors could also effectively block the appearance of A x V hi ceils in thymocyte cultures treated with dexamethasone or VP-16. PS externalization on HL-60. CEM. 10. Annexin V binding in cultures undergoing apoptosis preceded propidium iodide uptake in all cases (data not shown). These data provide compelling evidence for a tight association between apoptosis and exposure o f PS on the external PM leaflet. whereas PS externalization in response to C H X treatment Downloaded from jem. as was the appearance of AxV hi cells in the same cultures (Fig.

2015 ] Log annexin V binding i Concentration (p-M) CEMneo CEMbci-2 ~36% . In addition.Published November 1. O u r results also suggest that PS exposure precedes the nuclear changes that define apoptosis and also precedes the loss of membrane integrity by several hours. The nature of this moiety is still unknown. suggesting that fibroblasts use the V n R as well as a mannose/fucose-specific lectin to bind apoptotic ceils (43). CD69. 16) have provided convincing evidence for the involvement of PS in the recognition of apoptotic cells by certain macrophage populations. It is not yet known whether these changes have any role in the process of thymocyte deletion/recognition in vivo. i ~ect~ 20 40 0 9 0 0 10 20 30 40 50 Concentration (laM) 2~ r i . macrophages derived from the peritoneal cavity of mice (an inflammatory site) used a putative PS receptor to bind apoptotic cells.000 cells were analyzed in each condition. Fadok and colleagues (15. leagues have shown that apoptotic human neutrophils are recognized by blood. 1995 A 100 60 UV 5 0 Act D 30 40 ~/ 30' 20' 10 ~ ~ 0 60 10 20 i 9 i - i - i . (e) Other workers have recently implicated a 75-kD cell-surface receptor. Each data point was derived from an analysis of 5. rather.. 12). this protein should prove to be a very useful general probe for apoptosis. Inhibition of apoptosis-associated PM changes by Bd-2. suggesting that this event is tightly coupled to apoptosis. Discussion In this study we have shown that PS. after a 10 re. Although this study provides direct evidence that redistribution o f P M phospholipids is a widespread event during apoptosis. whereas bone marrow. Redistribution of Phosphatidylserine during Apoptosis Downloaded from jem. or 50 lU. in the recognition ofapoptotic neutrophils and lymphocytes by macrophages (44). (c) O f particular relevance to this study. and CD25. 8). (A) Death of CEM. however. T o date.rupress. N o t all macrophage populations recognized apoptotic cells in a PS-dependent manner. Cell death was assessed by uptake of propidium iodide. suggesting that these are mutually exclusive recognition mechanisms (17). (at) H u m a n fibroblasts have also been found to recognize a TSP-binding moiety on apoptotic cells (43). implicating a sugar lectin-type interaction in this process (7. defined by the 61D3 antibody. i - i 9 0 i 10 20 30 40 9 D . recent studies have found that thymocytes undergoing spontaneous as well as steroid-induced apoptosis up-regulate T C R [3/CD3. 5.~o (vector-transfected) compared with CEMbd_2 cells after exposure to the indicated treatments for 24 h. Results shown are representative of six separate experiments. PS externalization was efficiently blocked under conditions (macromolecular synthesis inhibition. 0 20 40 60 80 100 .000 cells. particularly because annexin V appears to detect apoptosis-associated membrane changes on live cells before the nuclear condensation events that occur during this process. (C) Bcl-2 inhibits apoptotic morphology. However. Cells were exposed for 8 h to the same treatments as indicated in B. (a) Early studies indicated that changes in membrane carbohydrate structures accompanied apoptosis of murine thymocytes. normally confined to the internal leaflet of the PM. acquisition of the ability to recognize PS on apoptotic cells was associated with a loss of the ability to recognize cells via the VnP.org on February 10. Because increased annexin V binding was detected as an early event in every model of apoptosis tested. Interestingly. as assessedby annexin V binding.ted C I ~11 cCEEI~Ie~ I Treatment occurred on HL-60vector cells at both temperatures (data not shown).in exposure to UV light. is externalized during apoptosis in a stimulus-independent manner. it remains likely that macrophages "see" apoptotic ceils using a variety of receptors. these cells also appear to bind apoptotic cells using a mechanism that can be inhibited by mannose or fucose sugars. as well as apoptosis-associated PS externalization.or blood-derived macrophages used the T S P / V n R / C D 3 6 system (15. (B) PS externalization detected in CEM~. reports in the literature have described at least five separate membrane changes that may lead to recognition of apoptotic cells by phagocytes (Fig. 16). insofar as macrophage uptake o f these cells could be inhibited by N-acetyl glucosamine or N-acetyl galactosamine..M CHX. 25 p.o versus CEMbd_2 ceils cultured for 8 h in either medium alone (untreated).or bone marrow-derived macrophages because of the appearance of a "charge-sensitive" TSP-binding moiety on these cells (9-13). (b) Studies by Savill and col1552 9 i Figure 10. and down-regulate C D 4 and CD8 on their surface (45). Numbers in parentheses represent the percentage of AxVhi cells detected under each condition. i 9 i Exposure time (min) ~ untre~. overexpression of Bcl-2 or Abl) in which the morphological features of apoptosis were prevented.M Act D. Results are from a representative experiment.

We are currently investigating the possibility that PS exposure and fodrin proteolysis during apoptosis are linked phenomena. as indicated. The existence of a PC translocase (Mdr2) suggests that a family of similar flippases. and 6). It is possible that some of the membrane changes that stimulate macrophage recognition occur only on particular cell types and are not in widespread use. Information from the Caenorhabditus elegans model of PCD has already suggested that the recognition and engulfment phases of this process are likely to be complex (2). which is active at 32~ and inactive at 39~ were cultured in medium alone (untreated) or medium containing 50 ~M CHX. The reasons for the apparent use of such a diverse array of receptors for the recognition and uptake of apoptotic cells are not yet understood.rupress. The nonfacilitated passage of charged lipids with polar head groups. N-acetyl galactosamine.. A and B. on the order of many hours or even days (46). which appears to be responsible for transporting PC across the PM.Published November 1. which would explain the extremely rapid kinetics of PS externalization on apoptotic cells (Figs.6%) Phagocyte 9 Untreated (lectin?) mannose/ \ / tucose Figure 12. ced-5. N-acetyl glucosamine. this would require the spontaneous flip of PS from the inner to the outer PM leaflet. disruption of these cytoskeletal components has been shown to facilitate redistribution of membrane phospholipids (50).ts). one explanation for the loss of PM phospholipid asymmetry during apoptosis could be the activation of a specific inside-outside PS translocase. In this context.org on February 10. and ced-lO) are involved in the engulfment phase (2). .ts 80 GluNAC nT_) "~ 40. cecl-2. Studies have also suggested that PM-associated phospholipid-binding cytoskeletal proteins. the nonerythroid form of spectrin. such as spectrin. 10. (B) PS externalization on HL-60am. given the critical nature of this interaction in the PCD process. 12. it is also likely that there is some redundancy in the macrophage-apoptotic cell interaction. a process that would require several hours. cells treated as indicated in A. 7 of these (ced-1. i 9 C H X 32oC (9"5%) . Furthermore. and 6). across the PM is extremely slow.ts cells after culture for 6 h under the indicated conditions. case. 49). p-glycoprotein) have been previously implicated in the ATP-dependent export of a diverse array of hydrophobic chemotherapeutic drugs from malignant cells. In view of the rapid externalization of PS observed on apoptotic cells in this study (Figs. Of the 11 genes known to be required for normal cell death in this worm. much like the scheme depicted in Fig. forms ofapoptosis (37). However. However. . 1995 A ~ / eceptor HL-60vector HL-60v-abl. 2 A. ced-6. has now been identified (47). cecl-7.3%) ~ ~ ~ . Evidence suggests that transport (flip-flop) of phospholipids from the internal to the external leaflet of the PM is an active process. cecl-8. The numbers in parentheses refer to the percentage of cells exhibiting increased annexin V-binding properties. may play a role in limiting the movement of phospholipids across the PM under normal circumstances (50). HL-60 cells expressing a temperature-sensitive v-abl (abl. 4 A. i n o n e 9 . Apoptosis was assessed by enumerating apoptotic cells on cytospun cell preparations. one such transloMartinet al. 2015 Log annexinV binding 1553 ~D3_antigen . members of which (mdrl. such as PS. . It is plausible that this group of genes form several receptorligand pairings. A and B. ~ (17. Therefore. as described in Materials and Methods. and 4.ather surprisingly. . Fadok and colleagues (15) suggested that the hypothetical exposure of PS on the outer PM leaflet of apoptotic cells may be due to the failure of an outside-inside PS translocase that has been implicated in the maintenance of normal PM asymmetry in erythrocytes and platelet membranes (48. Results shown are the means o f three randomly selected fields with a minimum of 100 cells counted per field. specific for other phospholipids. may also exist (46). becomes proteolytically cleaved during many. Schematic representation ofphagocyte-apoptotic cell interactions that have been described to date (see text for details). ~ 2 0 - 0 I 9 J n o n e Hi. if not all. this PC translocase is a member of the mdr (multidrug resistance) gene family. (A) Induction ofapoptosis in HL-60vector versus HL-60abl. we have recently found that fodrin. P. Downloaded from jem. 1(85%) /~ I Figure 11. Inhibition of apoptosis-associated PM changes by Abl. 2. facilitated by an ATP-dependent membrane translocase. we tend to favor an active model of PS transport during apoptosis.. GIuNAC. Previously.000 cells were analyzed in each condition. Although elusive for several years. GalNAC. ~ P CHX kk 39oc 1 (11.

Exp. However.. Dransfield. Rev.. J. Wyllie. 56:351-358. Vi- Redistribution of Phosphatidylserine during Apoptosis Downloaded from jem. 84:1518-1527. 11149 N.J. Fadok. and C. Martin is the recipient of Wellcome Trust International Travelling Fellowship 041080. Morris.F. This possibility remains to be tested. Wyllie. Rev. Haslett. J. the component(s) of the cell death machinery responsible for triggering membrane changes is regulated in a Bcl-2-independent fashion in these cells. Dr. Morris. J. because neutrophils do not normally express Bcl-2.G. 1993. R.S.. Cohen.. John Reed for providing Bcl-2 transfectants and plasmids. 1992. 1985. P.J. and H. similar to the recently discovered PC translocase Mdr2 (47). and Dr. J. R. and Bcl-2-expressing neutrophils were still recognized and engulfed by peritoneal macrophages. J. Horvitz. 3. neutrophil numbers in bcl-2-transgenic mice remained normal. R. We thank Dr. 14:131-136. 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La Jolla Institute for Allergy and Immunology. Phagocyte recognition of cells undergoing apoptosis.S. Med. 8 B) or overexpression o f Bcl-2 or Abl (Figs. and A. Br. as compared with neutrophils from nontransgenic control littermates. Phagocytosis of senescent neutrophils by human monocytederived macrophages and rabbit inflammatory macrophages. Apoptosis and programmed cell death in immunity. N. Fadok.E. Wyllie. S. V. and C. 4. 1995 These data are provocative. 6. Cell death: the significance of apoptosis.S. 11. Pathol. 1982. Duval. WyUie. Walport. Raft. D. and C. A. van Schie is supported by the Leukaemia Research Fund (UK).. Am. Clin. LaJolla. Kerr.R. Annu. 11). because these agents also blocked all other apoptosis-associated changes. it may also be the case that some membrane changes associated with neutrophil senescence are indeed uncoupled from the apoptotic changes (Fig. M.. 1991..I<. Kerr.L. 356:397-400.H. A. 9. 7:663-698. M. 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