Loss of Phospholipid Asymmetry and Surface Exposure of
Phosphatidylserine Is Required for Phagocytosis of Apoptotic Cells
by Macrophages and Fibroblasts*
Valerie A.
Fadok
,
Aimee
de Cathelineau,
David L.
Daleke§,
Peter M.
Henson, and
Donna L.
Bratton
From the Program in Cell Biology, Department of Pediatrics,
National Jewish Medical and Research Center, Denver, Colorado 80206 and the § Department of Biochemistry and Molecular
Biology/Medical Sciences, Indiana University, Bloomington,
Indiana 47405
Received for publication, April 28, 2000, and in revised form, September 10, 2000
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ABSTRACT |
Removal of apoptotic cells during tissue
remodeling or resolution of inflammation is critical to the restoration
of normal tissue structure and function. During apoptosis, early
surface changes occur, which trigger recognition and removal by
macrophages and other phagocytes. Loss of phospholipid asymmetry
results in exposure of phosphatidylserine (PS), one of the surface
markers recognized by macrophages. However, a number of receptors have been reported to mediate macrophage recognition of apoptotic cells, not
all of which bind to phosphatidylserine. We therefore examined the role
of membrane phospholipid symmetrization and PS externalization in
uptake of apoptotic cells by mouse macrophages and human HT-1080 fibrosarcoma cells by exposing them to cells that had undergone apoptosis without loss of phospholipid asymmetry. Neither mouse macrophages nor HT-1080 cells recognized or engulfed apoptotic targets
that failed to express PS, in comparison to PS-expressing apoptotic
cells. If, however, their outer leaflets were repleted with the
L-, but not the D-, stereoisomer of
sn-1,2-PS by liposome transfer, engulfment by both
phagocytes was restored. These observations directly demonstrate that
loss of phospholipid asymmetry and PS expression is required for
phagocyte engulfment of apoptotic cells and imply a critical, if not
obligatory, role for PS recognition in the uptake process.
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INTRODUCTION |
In tissues undergoing remodeling or during resolution of
inflammation, apoptotic cells are cleared by phagocytes prior to their
lysis, supporting the idea that surface changes promoting recognition
occur early in the apoptotic process. Removal prior to lysis prevents
the release of potentially toxic or immunogenic intracellular contents,
thus maintaining normal tissue structure and function in the face of
considerable cell death. Little is known about the specific surface
markers that trigger recognition by phagocytes. In some cases, exposure
of specific carbohydrates on the apoptotic cell surface may trigger
binding to phagocyte lectins (1-5). In other cases, molecules
such as thrombospondin (6),
collectins,1 or complement
products (iC3b) (7) appear to bridge the apoptotic cell to the
phagocyte; however, the molecules on the apoptotic cell to which they
bind have not been identified. The best studied and most consistent
surface change occurring on apoptotic cells is the exposure of
phosphatidylserine (PS)2
associated with loss of phospholipid asymmetry (8-20). We showed several years ago that exposure of PS during apoptosis appeared to
trigger recognition of apoptotic cells by subsets of macrophages, because uptake of apoptotic bodies could be inhibited in a
dose-dependent and stereospecific manner by liposomes
containing PS and by structural analogues of PS but not by liposomes
containing other anionic phospholipids (8, 21, 22). Using relatively
simple inhibition assays employed by most investigators in the field,
we have previously shown that in mouse thioglycollate-elicited
peritoneal macrophages, a PS-dependent mechanism appears to
be dominant, whereas in unstimulated mouse bone marrow macrophages and
human monocyte-derived macrophages, the
v
3/CD36/thrombospondin
mechanism appears to predominate (6, 23). The latter can be stimulated
to recognize PS by treating them with digestible particulate stimuli,
including
-glucan.
We have recently described a system in which loss of phospholipid
asymmetry and PS expression can be divorced from other changes associated with apoptosis (24). HL-60 cells cultured in the presence of
the ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) to
reduce putrescine and spermidine levels were still able to undergo
apoptosis following treatment with UV irradiation. Thus, they showed
the classic nuclear morphologic changes and plasma membrane
vesiculation associated with apoptosis in these cells. In addition,
untreated and DFMO-treated irradiated cells showed identical DNA
fragmentation, caspase 3 activity, and depression of aminophospholipid
translocase activity. In contrast, apoptotic DFMO-treated HL-60 cells
failed to express PS externally, as measured by annexin V binding, and
they showed impaired membrane phospholipid flip-flop, as measured
by uptake of
1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-caproyl]-sn-glycero-3-phosphocholine (24). These membrane changes were restored by repletion of the cells
with putrescine prior to induction of apoptosis. Therefore HL-60 cells
pretreated with DFMO and induced to undergo apoptosis by UV could
provide an apoptotic cell target for phagocytosis that does not undergo
loss of phospholipid asymmetry, allowing us to test the hypothesis that
loss of phospholipid asymmetry is required for uptake by macrophages
and other phagocytes. Additionally, we found that undifferentiated PLB
985, a human myelomonocytic cell line (25) that undergoes nuclear
apoptosis following exposure to UV, fails to express PS externally
because of defective phospholipid scrambling. This may be contrasted
with PLB 985 cells differentiated toward either neutrophils with
dimethyl sulfoxide or retinoic acid or macrophages with phorbol esters
or vitamin D3; these cells do express PS and show enhanced membrane
phospholipid flip-flop (scrambling) following induction of apoptosis.
These cells provided a second system with which to test the hypothesis
that exposure of PS was required for recognition and uptake of
apoptotic cells by phagocytes.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The human promyelocytic cell line HL-60 and
the human leukemia T cell line Jurkat were obtained from American Type
Culture Collection (Manassas, VA) and cultured in RPMI containing 20% fetal calf serum (Gemini Bioproducts, Calabasas, CA), glutamine, penicillin, and streptomycin. The human myelomonoblastic PLB 985 cells
(25) were a gift from Dr. Christina Leslie and were cultured in the
same medium, except that 10% fetal calf serum was used. For
some experiments, the cells were differentiated into monocytes by
treatment for 5 days with 10
7 M
1,25-dihydroxyvitamin D3. Mouse bone marrow-derived macrophages derived
from C3H/Hej mice were cultured in macrophage colony-stimulating factor in 96-well tissue culture plates as described (21, 26) and used
for phagocytosis after 7 days of culture. To induce the ability to
overtly recognize phosphatidylserine on apoptotic cells, bone
marrow macrophages were treated with 75 µg/ml
-glucan (Accurate Chemical Co.) on day 5 of culture and were used 48 h later (21). HT-1080 human fibrosarcoma-derived cells (27), obtained from the
American Type Culture Collection were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum,
nonessential amino acids, and sodium pyruvate.
Treatment with DFMO and/or Putrescine--
Polyamines were
depleted as described (24). Briefly, HL-60 cells were plated at a
density of 1.25 × 105 cells/ml in culture medium
containing 1 mM 2-(difluoromethyl)-DL-ornithine monohydrochloride monohydrate, a generous gift of Dr. Ekkehard Bohme,
Hoescht Marion Roussel, Inc., Cincinnati, OH. This compound inhibits
ornithine decarboxylase and thereby depletes the cells of polyamines,
in particular putrescine and spermidine (24). The cells were used after
5 days of culture. For reconstitution of polyamines, 10 µM putrescine was added on day 3 of culture. Control
cells were plated at 6.25 × 104 cells/ml, because
they grew faster than DFMO-treated cells. All cultures achieved a final
density of 1-1.25 × 106 cells/ml.
Reconstitution of Plasma Membrane Outer Leaflet with
Phosphatidylserine--
Small unilamellar liposomes containing a 50:50
molar ratio of phosphatidylserine (derived from brain; Avanti Polar
Lipids, Alabaster, AL) to phosphatidylcholine (derived from bovine
liver; Avanti Polar Lipids) were made. For some experiments,
1-palmitoyl-2-oleoyl-sn-3-glycerophospho-L-serine (POP-L-S) and
1-palmitoyl-2-oleoyl-sn-3-glycerophospho-D-serine (POP-D-S) were used instead of brain-derived
phosphatidylserine. POP-L-S was purchased from Avanti Polar
Lipids, and POP-D-S was synthesized from
1-palmitoyl-2-oleoyl-sn-3-glycerophosphocholine (Avanti
Polar Lipids) by phospholipase D (type VII,
Streptomyces species, Sigma)-catalyzed headgroup exchange in
the presence of D-serine (28, 29). The crude product was
purified by CM-52 cellulose chromatography as described (30). The
individual phospholipids, stored in chloroform:methanol (90:10), were
added to glass tubes and dried under nitrogen. PBS was added, and the
lipid mix was vortexed and then sonicated for 3 min as described
previously (8). Ten micromoles of total lipid were added to 10 million apoptotic or viable cells in 1.5 ml of PBS and incubated at 37 °C
end over end for 30 min. The cells were washed twice with PBS and used
immediately. Surface PS was confirmed by determining the ability of the
cells to bind FITC-conjugated annexin, determined by flow cytometry. In
some cases, the added PS was removed by washing the cells three times
in PBS containing 2% bovine serum albumin, and the cells were
incubated in tissue culture medium at 37 °C for 1 h to allow
translocation of remaining PS to the inner leaflet by the
aminophospholipid translocase. Removal of PS was confirmed by assessing
the ability to bind annexin-FITC, as determined by flow cytometry.
Induction of Apoptosis and Its Quantitation--
Apoptosis was
induced in HL-60 cells and PLB 985 cells by exposure to UV irradiation
at 254 nm for 5 min. Jurkat T cells were exposed for 10 min. The cells
were then cultured for 2 h and harvested. For phagocytosis, the
cells were resuspended in Dulbecco's modified Eagle's medium at a
concentration of 0.5 × 106 cells/50 µl. An
aliquot of cells was prepared by cytocentrifugation, and apoptosis was
quantitated by evaluation of nuclear morphology at the light
microscopic level.
Annexin Binding and Flow Cytometry--
Loss of phospholipid
asymmetry and exposure of PS were evaluated by analysis of
annexin-V-FITC binding. Propidium iodide was used as a control to
determine the level of secondary necrosis. The cells were stained
exactly as recommended by the manufacturer of the annexin kit (RD
Systems, Minneapolis, MN). Stained cells were analyzed using a FACScan
cytometer and PCLysys software (Becton Dickinson, Franklin Lakes, NJ).
Phagocytosis Assay--
Targets for uptake (0.5 × 106 cells/well in 96-well plates) were added to
mouse bone marrow-derived macrophages in serum-free medium for 30 min.
For HT-1080 cells, 5 × 106 targets were added per
well in 24-well plates in growth medium containing serum for
1 h. Unphagocytosed cells were washed away with PBS, and then the
wells were stained with a modified Wright's-Giemsa stain (Leukostat,
Fisher). Phagocytosis was counted as described previously by light
microscopy (8, 21, 22). The only modification was that a phagocytic
index was not calculated; all data are reported as percent phagocytes
positive for uptake.
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RESULTS |
HL-60 cells were treated with DFMO to deplete intracellular
polyamines; to some cells, putrescine was added to replete the polyamine stores (24). The cells were then exposed to UV light to induce apoptosis. The percentage of apoptotic cells, as determined by nuclear morphology, was the same (48.7 ± 3.9% for apoptotic HL-60 cells and 51.7 ± 5.2% for apoptotic DFMO-treated HL-60
cells). Exposure of phosphatidylserine was assessed by flow cytometry, using FITC-labeled annexin V, and induction of apoptosis was confirmed by cellular morphology. The population of HL-60 cells previously cultured in DFMO showed a dramatic reduction in PS-positive cells (Fig.
1). In contrast, if DFMO-pretreated cells
had been repleted with putrescine for 2 days prior to induction of
apoptosis, PS exposure was restored (Fig. 1).

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Fig. 1.
Phosphatidylserine exposure is inhibited in
cells treated with DFMO but restored when polyamines are repleted with
putrescine. A, cytometric data from one representative
experiment. The dot plots illustrate annexin binding on the
horizontal axis and propidium iodide binding on the
vertical axis. The numbers in each quadrant
represent the percentage of the total population. The lower left
quadrant represents annexin-low cells, the lower right
quadrant represents annexin-high (i.e. PS exposed
externally), and the upper right quadrant represents cells
that are annexin-high and permeable to propidium iodide.
FL1-H, green fluorescence (annexin V); FL2-H, red
fluorescence (propidium iodide). B, the percentage
of annexin-high (PS-positive) cells in five experiments.
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These populations of HL-60 cells were subsequently fed to mouse bone
marrow-derived macrophages, half of which had been stimulated to
recognize PS overtly, as suggested by PS inhibition of uptake. As
expected, very little phagocytosis was seen when macrophages of either
type were fed viable HL-60 cells (Fig.
2). In contrast, in HL-60 cells
that had been UV-irradiated to induce apoptosis, uptake was
significantly increased. If the cells had been pretreated with DFMO
prior to induction of apoptosis to inhibit loss of phospholipid asymmetry, uptake was significantly reduced. Importantly, uptake into nonstimulated macrophages, which are thought to use primarily the
vb3/CD36/thrombospondin mechanism, was also greatly reduced. Those
HL-60 cells that had been repleted by putrescine prior to induction of
apoptosis and that therefore lost phospholipid asymmetry and exposed PS
externally were recognized by macrophages of either type (Fig. 2).

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Fig. 2.
Phagocytosis of apoptotic HL-60 cells is
correlated with loss of phospholipid asymmetry. Unstimulated bone
marrow-derived macrophages (BMDM) were those cells
derived from the bone marrow after 7 days in macrophage
colony-stimulating factor. Stimulated bone marrow-derived
macrophages were treated for 48 h with -glucan to induce the
ability to recognize PS on apoptotic cells. Put,
putrescine.
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These data suggested that loss of phospholipid asymmetry and exposure
of PS on apoptotic cells were required for macrophage phagocytosis,
regardless of which recognition phenotype was involved. It was then
important to determine whether introduction of PS into the outer
leaflet by liposome transfer might restore phagocytosis of
DFMO-pretreated HL-60 cells. Brain-derived PS was introduced into the
outer leaflet of DFMO-pretreated HL-60 cells after they had undergone
apoptosis. Because aminophospholipid translocase activity is
reduced during apoptosis (24, 31, 32), outer leaflet PS would not be
expected to be transported back into the inner leaflet. As is shown in
Fig. 3, these cells did express PS
externally (assessed by annexin V binding), and they were also recognized and engulfed by macrophages (Fig. 2).

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Fig. 3.
Phosphatidylserine expression on viable and
DFMO-treated apoptotic HL-60 cells can be induced by liposome transfer
of brain-derived PS to the outer leaflet. All populations were
labeled with annexin V-FITC and analyzed by flow cytometry. These dot
plots represent one experiment of a total of five. The upper left
panel shows annexin binding to viable HL-60 cells, the upper
middle panel shows binding to UV-treated HL-60 cells, and the
upper right panel shows the binding to DFMO-pretreated,
UV-treated HL-60 cells. The lower left panel shows annexin
binding to viable HL-60 cells treated with liposomes containing PS, the
lower middle panel shows viable HL-60 cells treated with
liposomes but then washed with bovine serum albumin and cultured for an
additional hour, and the lower right panel shows DFMO-pretreated,
UV-irradiated HL-60 cells treated with liposomes containing PS.
FL1-H, green fluorescence; FL2-H, red
fluorescence.
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Nonapoptotic HL-60 cells were also treated with liposomes to introduce
brain-derived PS into the outer leaflet. As is shown in Fig. 3, these
cells acquired the ability to bind annexin V. If the liposomes were
washed away and the cells were cultured for 1 h, they no longer
bound annexin V, presumably because they translocated any remaining
outer leaflet PS into the inner leaflet (Fig. 3). Viable HL-60 cells
expressing PS in the outer leaflet were recognized and engulfed by bone
marrow-derived macrophages (Fig. 4); if,
however, they had been cultured to allow for PS translocation into the
inner leaflet, they were not recognized and therefore were not engulfed
(Fig. 4).

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Fig. 4.
Mouse bone marrow macrophages phagocytose
viable HL-60 cells that express PS externally via liposome
transfer. Uptake is compared with that for viable cells, apoptotic
HL-60 cells, and viable HL-60 cells treated with liposomes containing
PS derived from brain but then washed and cultured to allow
translocation of PS. Data shown are the means ± S.E. for three
experiments.
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We have recently found a cell line, PLB 985 (25), that fails to
lose phospholipid asymmetry during apoptosis. Although these cells
undergo morphologic changes of apoptosis, they fail to express PS
externally, as is shown in Fig. 5. Uptake
of apoptotic PLB cells, compared with apoptotic Jurkat T cells and
apoptotic HL-60 cells, by unstimulated mouse bone marrow-derived
macrophages was significantly reduced (Fig.
6). Thus, in a second system in which drug manipulation was not required to prevent loss of phospholipid asymmetry, PS exposure was confirmed as a requirement for efficient phagocytosis by macrophages.

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Fig. 5.
Phosphatidylserine expression, as indicated
by annexin V binding, for undifferentiated and vitamin-D3
differentiated apoptotic PLB 985 cells compared with apoptotic Jurkat T
cells and apoptotic HL-60 cells. For all target cells, apoptosis
was induced by irradiation with UV light. These histograms are from one
experiment representative of four. FL1-H, green
fluorescence; FL2-H, red fluorescence.
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Fig. 6.
Macrophages show reduced recognition and
phagocytosis of apoptotic PLB 985 cells compared with apoptotic Jurkat
T cells and apoptotic HL-60 cells. The inset shows the
degree of apoptosis for each irradiated population, as determined by
light microscopy. Each graph illustrates the mean ± S.E. for four
experiments.
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We next tested the hypothesis that loss of phospholipid asymmetry was
required for uptake by phagocytes other than macrophages. HT-1080 cells
are an adherent cell derived from a human fibrosarcoma (27). These
cells were exposed to fresh and apoptotic Jurkat T cells or PLB 985 cells, and uptake was evaluated. Fig. 7
shows that HT-1080 cells were able to phagocytose apoptotic Jurkat T cells but not apoptotic PLB 985 cells, unless the latter were treated
to replete the outer leaflet with brain-derived PS. Furthermore, viable
Jurkat cells were phagocytosed if their outer leaflets were loaded with
PS by liposome transfer. PLB 985 cells, like HL-60 cells, can be
differentiated into monocytic cells with 1,25-dihydroxyvitamin D3 (33).
Apoptosis was induced in the differentiated PLB 985 cells by UV
irradiation, and the cells were assessed by annexin V binding for PS
exposure. Differentiated PLB 985 cells were found to express PS on
their outer leaflets (Fig. 5) and were recognized and engulfed by
HT-1080 cells (Fig. 8).

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Fig. 7.
HT-1080 fibrosarcoma cells recognize those
target cells containing PS in the outer leaflet. Targets were
loaded with either brain-derived PS- or phosphatidylcholine
(PC)-containing liposomes, as described under
"Experimental Procedures," and fed to the phagocytes for 1 h.
Data are mean values ± S.E.; n = 9.
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Fig. 8.
Differentiated PLB 985 cells, in contrast to
undifferentiated cells, are recognized by phagocytes. Fresh or
apoptotic, undifferentiated or differentiated PLB 985 cells were
coincubated with HT-1080 cells, and phagocytosis was evaluated as
described in the legend to Fig. 7. Apoptotic Jurkat T cells were used
as positive controls. Data are mean values ± S.E.;
n = 9. Diff, differentiated;
Undiff, undifferentiated.
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Lastly, it became important to determine whether phosphatidylserine
inserted into the plasma membrane was recognized in a stereospecific
manner, as we have previously reported (8, 9). We used two different
1-palmitoyl-2-oleoyl-sn-3-glycerophosphoserine compounds,
containing either L- (POP-L-S) or
D-serine (POP-D-S), to load apoptotic PLB 985 cells and viable Jurkat T cells; loading of each lipid was confirmed by
annexin V staining and flow cytometry. These targets were exposed to
mouse bone marrow-derived macrophages that were either unstimulated or
stimulated with transforming growth factor-
and
-glucan in
the presence or absence of mAb 217G8E9 (directed against a newly
described receptor for phosphatidylserine; Ref. 9) or its isotype
control. Fig. 9 shows that only PLB cells
loaded with POP-L-S were engulfed and that uptake was
inhibited by Mab 217G8E9, implicating the PS receptor in recognition of these cells. Furthermore, this antibody inhibited uptake of
POP-L-S-loaded targets even by unstimulated macrophages.
The isotype control had no effect on the engulfment of either
macrophage population (data not shown). We also observed that viable
Jurkat T cells loaded with POP-L-S but not
POP-D-S were taken up by both sets of macrophages; 30 ± 5% unstimulated and 42 ± 6% glucan-stimulated macrophages
engulfed POP-L-S-loaded Jurkat T cells, whereas 4.2 ± 0.7% unstimulated and 5.1 ± 1.2% stimulated macrophages
engulfed POP-D-S-loaded cells. These data suggest that
phosphatidylserine is recognized stereospecifically by the receptor for
PS.

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Fig. 9.
Engulfment of apoptotic PLB 985 cells by
macrophages occurs if the cells are loaded with POP-L-S but
not POP-D-S. Targets were loaded with either
POP-L-S or POP-D-S as described under
"Experimental Procedures" and exposed to mouse macrophages that
were stimulated (or not) to up-regulate a new PS receptor. Mab 217G8E9
(designated mab 217) was added at 200 µg/ml 30 min prior to addition
of lipid-loaded targets. Controls were apoptotic PLB cells with
no lipid added to their membranes. Data are mean
values ± S.E.; n = 2. BMDM,
bone marrow-derived macrophages.
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DISCUSSION |
Apoptotic cells that failed to lose phospholipid asymmetry and
that did not express PS externally were not phagocytosed by macrophages
or by fibrosarcoma cells, despite the fact that they showed the
morphological changes of apoptosis and displayed the characteristic
biochemical markers, including DNA fragmentation and caspase 3 activity
(24). These data suggest that loss of phospholipid asymmetry and
external expression of PS are required for recognition of apoptotic
cells by macrophages and other phagocytes.
Phosphatidylserine appears to be a critical ligand required for
uptake of apoptotic cells. It is clear that some, if not all, macrophages recognize this phospholipid specifically, because their
uptake of apoptotic cells and lipid-symmetric red cells can be
inhibited stereospecifically by PS and its structural analogues glycerophosphorylserine and phosphoserine (8, 34-40). The data presented herein suggest that PS exposure is required for uptake by all
macrophages, including those that predominantly use the
v
3/CD36/thrombospondin mechanism. The obvious question that then
arises is why uptake by the latter is not inhibited by PS liposomes.
One likely explanation is that the inhibition assays traditionally used
in analysis of apoptotic cell clearance are relatively insensitive,
even when specific antibodies are used. This hypothesis receives some
support from the observations by us and many other investigators that
complete inhibition is never seen, even when inhibitors of multiple
receptors are combined (for example, see Ref. 41). We have recently
described a new receptor that appears to mediate
phosphatidylserine-specific recognition of apoptotic cells and
phospholipid-symmetric red cell ghosts, as well as the release of
transforming growth factor-
following uptake of apoptotic
cells (9). This receptor is expressed at low levels on macrophages
typically reported to use the
v
3/CD36/thrombospondin mechanism
for uptake but is up-regulated on macrophages that have been stimulated
to recognize PS by
-glucan. In fact, the antibody against this new
PS receptor (Mab 217G8E9) is a poor inhibitor of apoptotic cell uptake
by human monocyte-derived macrophages or mouse bone marrow-derived
macrophages that have not been stimulated to up-regulate the receptor
by a particulate stimulus such as glucan (9). By contrast, we
found that uptake of apoptotic PLB 985 cells and viable Jurkat T cells
loaded with POP-L-S could be inhibited by Mab 217G8E9,
suggesting involvement of the PS receptor on these cells also. We
hypothesize that the disparities between these two systems (apoptotic
cells versus lipid-loaded cells) results from the likelihood
that the lipid loading yields a target cell with more recognizable PS
on its surface than occurs during apoptosis. Additionally, given
that mAb 217G8E9 is an IgM, we believe it to be of relatively low
affinity for both determining expression by flow cytometry and for
inhibition of uptake; we are currently developing new reagents to
better explore this disparity. However, that this receptor is
functional on unstimulated macrophages is supported by our additional
observation that the anti-PS receptor monoclonal antibody stimulates
transforming growth factor-
production and inhibits tumor
necrosis factor-
production,3 as does uptake
of apoptotic cells, as we have reported previously (42). Other
macrophage receptors incriminated in uptake of apoptotic cells (6, 20,
43) have been suggested to bind PS (as well as other phospholipids)
offered in liposomes; these receptors include CD36, CD68, and
CD14 (44-46). Whether these receptors specifically recognize PS
exposed on an apoptotic cell and/or how they cooperate with the
receptor we have described remain to be determined.
This newly described PS receptor is also expressed on HT-1080
cells as well as other fibroblasts and epithelial cells, and anti-PS
receptor antibody inhibits their uptake of apoptotic cells (9). Other
phagocytes, including Sertoli cells, endothelial cells, and smooth
muscle vascular cells have been shown to recognize PS on apoptotic
cells (47-49); whether they express and use the new PS receptor
remains to be determined, although preliminary assessment of human
umbilical vein endothelial cells by flow cytometry was positive for PS
receptor expression.3 These cells have been shown to
utilize scavenger receptors for uptake of apoptotic cells, some of
which may recognize PS. The HT-1080 cells used herein engulf apoptotic
cells poorly when compared with macrophages; similar results were
obtained when primary fibroblasts were used (50).3 The
engulfment assay used herein determines the percentage of phagocytes
containing apoptotic bodies recognizable by light microscopy at any one
point in time. This type of assay does not take into account the rate
of uptake or the rate of digestion. In our hands, a higher percentage
of macrophages can be scored positively at any time point when compared
with fibroblasts or epithelial cells, suggesting to us that macrophages
are more efficient.
Loss of phospholipid asymmetry could also contribute to recognition of
apoptotic cells by facilitating exposure of other ligands that are
cryptic when cells are viable. The loss of phospholipid asymmetry
associated with apoptosis is characterized by changes in membrane
fluidity relative to that seen in cells maintaining normal asymmetric
phospholipid distribution. Alterations in fluidity could contribute to
changes in protein conformation, distribution in the membrane, and/or
function. For example, it was shown that the ligand specificity for the
vitronectin receptor
v
3 (CD51/CD61) was altered depending on the
phospholipid milieu in which the receptor was placed (51). Alternations
in lipid packing have also been implicated in the function of
lymphocyte function antigen-1 (LFA-1) (CD 11a/CD18) (52). It is
intriguing to note that intercellular adhesion molecule-3
(ICAM-3) appears to be a recognition ligand for macrophage uptake of
apoptotic cells but does not facilitate function of viable cells,
suggesting that it undergoes some qualitative change during apoptosis
(53). It is possible that loss of phospholipid asymmetry contributes to
exposure of an otherwise cryptic epitope on this adhesion molecule.
However, introduction of PS into the outer leaflet of viable cells
induced their uptake, as shown in Fig. 4, suggesting that PS alone is a
sufficient signal for uptake by phagocytes, although we cannot rule out
the possibility that introduction of PS into the outer leaflet changes
the membrane in a way that exposes otherwise cryptic recognition ligands.
There is other experimental support that loss of phospholipid asymmetry
is required for macrophage recognition of apoptotic cells. It was
recently reported that preincubation of apoptotic cells and
lipid-symmetric erythrocytes with annexin V inhibited their uptake by
all macrophages tested, whereas it did not inhibit uptake of opsonized
red cells via the Fc receptor (54). In addition, Verhoven
et al. (55) correlated the appearance of PS on the outer
leaflet with recognizability by thioglycollate-elicited peritoneal
macrophages. Using a different approach, Shiratsuchi et al.
(56) looked at uptake of HeLa cells transfected with Fas and induced to
undergo apoptosis with anti-Fas antibody; they found that uptake by
peritoneal exudate macrophages could be inhibited by liposomes
containing phosphatidylserine. They also treated the viable transfected
cells with N-ethylmaleimide, one of the known
sulfhydryl-reactive agents that can inhibit aminophospholipid translocase activity (57-60). Even though
N-ethylmaleimide-treated cells did not undergo apoptosis,
they were recognized and engulfed by macrophages, albeit at very low
levels compared with uptake of apoptotic cells. This uptake appeared to
be PS-dependent, because it was inhibited by PS liposomes,
although the possible effects of N-ethylmaleimide on other
membrane proteins could also have contributed to uptake.
Uptake of apoptotic cells by human macrophages was found to be enhanced
in the presence of serum, and it was suggested that complement
opsonization was responsible for the enhancement (7). Uptake into
macrophages in the experiments reported herein were performed in the
absence of serum. However, uptake of apoptotic cells into HT-1080
fibrosarcoma cells was done in the presence of serum; yet the
requirement for loss of phospholipid asymmetry and exposure of PS was
retained. In our hands also, the presence of serum in the assay
enhances total levels of uptake by macrophages and fibroblasts but does
not abrogate the requirement for PS recognition by either population.
It is possible therefore that the serum either provides additional
molecules to promote adhesion prior to uptake or that factors in serum
stimulate uptake by phagocytes in a ligand-independent manner.
In summary, the data presented herein suggest that loss of phospholipid
asymmetry and exposure of PS are required for recognition and removal
of apoptotic cells by macrophages and other phagocytes. Based on our
observations that only L stereoisomers induce ingestion of
lipid-loaded cells, we suspect that this requirement implicates the
newly reported PS receptor as a key molecule for signaling ingestion,
regardless of which adhesion ligands are initially involved in binding
the target to the phagocyte. We speculate that the ability to inhibit
uptake by PS liposomes or the anti-PS receptor antibody (an IgM
believed to be of relatively low affinity) may require a threshold
level of this receptor on the phagocyte, providing one explanation for
the disparity between the requirement for PS exposure described herein
and the inability to inhibit uptake with PS liposomes in unstimulated macrophages.
 |
ACKNOWLEDGEMENTS |
Mouse bone marrow-derived
macrophages were kindly supplied by Linda Remigio and David Riches, and
human umbilical vein endothelial cells were provided by Lisa Lehman and
Marcella Bilstrom.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants GM 48211, GM 47230, HL 60980, and HL 30343.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: National Jewish
Medical and Research Center, D509, 1400 Jackson St., Denver, CO 80206.
Tel.: 303-398-1281; Fax: 303-398-1381; E-mail:
fadokv@njc.org.
Published, JBC Papers in Press, September 13, 2000, DOI 10.1074/jbc.M003649200
1
C. A. Ogden, D. L. Bratton, V. A. Fadok, and P. M. Henson, unpublished data.
3
V. A. Fadok, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
PS, phosphatidylserine;
DFMO, difluoromethylornithine;
POP-L-S, 1-palmitoyl-2-oleoyl-sn-3-glycerophospho-L-serine;
POP-D-S, 1-palmitoyl-2-oleoyl-sn-3-glycerophospho-D-serine;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
mAb, monoclonal antibody.
 |
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