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INTRODUCTION |
Phospholipids are distributed asymmetrically between the two faces
of the plasma membrane (1-3). In particular,
PtdSer1 is nearly absent from
the extracellular face of the plasma membrane in resting cells.
Membrane phospholipid asymmetry is maintained by the aminophospholipid
translocase, an ATP-dependent enzyme that transports PtdSer
and phosphatidylethanolamine from the outer face to the inner face of
the plasma membrane. This protein was recently cloned and shown to be a
member of a new family of ATP-dependent transporters (4).
Under some circumstances disruption of membrane asymmetry results in
PtdSer exposure at the outer face of the membrane. This is an essential
part of the normal platelet procoagulant response, and increased PtdSer
exposure may also be a signal for clearance of damaged or senescent
cells. More recently, increased exposure of PtdSer has emerged as a key
early event in cells undergoing apoptosis (5-9). Given the earlier
work suggesting a role for PtdSer exposure in triggering phagocytic
removal of cells, exposure of PtdSer may mark apoptotic cells for
phagocytic removal, either by specialized phagocytic cells or by their
healthy neighbors.
There are several means by which apoptotic cells are marked for
phagocytosis; one of these mechanisms involves recognition of exposed
PtdSer (10). The receptors responsible for recognizing cells with
exposed PtdSer are not well known, but several candidate proteins have
been proposed. Two structurally related members of the scavenger
receptor family, CD36 and SR-BI, are implicated in binding of anionic
phospholipid vesicles to some cell types (11, 12). However, CD36 has
also been proposed as a receptor for collagen (13), thrombospondin
(14), oxidized low density lipoprotein (15), long-chain fatty acids
(16), and Plasmodium falciparum-infected erythrocytes (17).
Also, recent work has identified SR-BI more plausibly as a receptor for
high density lipoprotein, and its expression is largely restricted to
liver and steroidogenic tissues (18). Another candidate protein is CD68
(macrosialin), which appears to be involved in the binding of PtdSer
vesicles and apoptotic thymocytes to mouse peritoneal macrophages (19,
20). Given these considerations, additional work is needed to clarify
the role of CD36 as a possible PtdSer receptor in comparison with other
proteins. In this study, we have characterized the binding of
phospholipid vesicles to THP-1 and J774A.1 cells. These cell lines were
chosen because they are monocyte-like with phagocytic properties
(21-23). We have developed a flow cytometric procedure for measuring
the binding of phospholipid vesicles to cells. We find that CD36
appears to be highly specific for PtdSer vesicles and cannot explain
the binding of other anionic phospholipids to these cells.
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EXPERIMENTAL PROCEDURES |
Materials--
All phospholipids were from Avanti Polar Lipids
(Alabaster, AL) and were of the following composition:
C7PtdCho, synthetic 1,2-diheptanoyl-sn-3-phosphatidylcholine; NBD-PtdCho,
1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-3-phospha-tidylcholine; PtdCho, synthetic
1-palmitoyl-2-oleoyl-sn-3-phosphatidylcholine; PtdGro,
synthetic
1-palmitoyl-2-oleoyl-sn-3-phosphatidyl-glycerol; PtdIns,
bovine liver phosphatidylglycerol; PtdSer, bovine brain phosphatidylserine. The THP-l and J774A.1 cell lines were from the
American Type Culture Collection. Normal human red blood cells were
from anonymous donors. Anti-CD36 and goat antibody against mouse IgG
(affinity purified, F(ab')2 fragment, fluorescein
isothiocyanate-labeled) were from Immunotech (Westbrook, ME).
O-Phospho-L-serine and collagen (calf skin,
acid-soluble) were from Sigma. Human thrombospondin was from Life
Technologies, Inc. Recombinant human annexin V was expressed and
purified as described (24). Quantum 24 and Quantum 25 fluorescent beads
were purchased from Flow Cytometry Standards Corp. (San Juan, Puerto Rico).
Cell Culture--
THP-1 cells were grown and passaged 1:3 every
3-4 days in a 5% CO2 atmosphere at 37 °C in RPMI 1640 medium plus L-glutamine (Life Technologies, Inc. Catalog
No. 11875-093), supplemented with 10% fetal bovine serum, 50 units/ml
penicillin, and 50 µg/ml streptomycin. J774A.1 cells were grown in a
5% CO2 atmosphere at 37 °C in Dulbecco's modified
Eagle's medium (Life Technologies, Inc. Catalog No. 11965-092)
supplemented with 1.2 g/liter HEPES, 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin
sulfate, and 50 µM 2-mercaptoethanol. Prior to use in
experiments cells were harvested by centrifugation (5 min at 500 × g), washed once with binding assay buffer, and
resuspended in binding assay buffer (described below).
Preparation and Sizing of Phospholipid Vesicles--
Small
unilamellar phospholipid vesicles were prepared according to Gabriel
and Roberts (25) and quantitated by phosphorus assay as described
previously (26). In all figures, concentrations of phospholipid
vesicles are expressed as the total concentration of monomeric
phospholipids. The molar composition of PtdSer vesicles was 50%
PtdSer, 10% NBD-PtdCho, 20% PtdCho, and 20% C7PtdCho. Unlabeled PtdSer vesicles, used for competition studies or to determine
nonspecific binding, contained 50% PtdSer, 30% PtdCho, and 20%
C7PtdCho. Other types of labeled or unlabeled phospholipid vesicles were made in identical fashion by substituting either PtdIns,
PtdGro, or PtdCho for the 50% PtdSer.
The size of the phospholipid vesicles was determined by dynamic light
scattering on a Model SM-200 apparatus with a BI9000AT autocorrelator
card and version 6.0 software (Brookhaven Instrument Corp.).
Measurements were made at 23 °C with a 488 nm laser line at 90°
scattering angle; vesicle stock solutions were diluted to 0.1-0.2
mM in 50 mM HEPES, pH 7.4, 100 mM
NaCl; data were analyzed by cumulant analysis with a linear fit. The
number of phospholipid molecules per vesicle was then calculated from
the measured diameter assuming a bilayer thickness of 5 nm and an area
per phospholipid molecule of 0.68 nm2 (27). The measured
average vesicle diameter (in nm) and calculated number of phospholipid
molecules per vesicle were as follows: PtdSer, 112 nm and 1.11 × 105; PtdGro, 117 nm and 1.21 × 105;
PtdIns, 113 nm and 1.13 × 105; PtdCho, 156 nm and
2.18 × 105.
Flow Cytometry Binding Assay with Fluorescent Phospholipid
Vesicles--
The vesicle binding assay was performed in
0.160 ml of
buffer consisting of 10 mM HEPES-Na, pH 7.4, 133 mM NaCl, 5.8 mM KCl, 0.7 mM
NaH2PO4, and 5 mM glucose; in
experiments requiring calcium, the buffer was supplemented with either
1 or 2.5 mM CaCl2, as indicated in the text,
and 2 mM MgCl2. NBD-labeled vesicles were added
at 10 µM final concentration or other concentrations as noted, and either 0 or 500 µM unlabeled vesicles were
added to determine nonspecific binding. Cells resuspended in assay
buffer were then added to the tubes at a final concentration of
106/ml. Samples were incubated on ice for 120 min; cells
were pelleted at 500 × g, washed once in assay buffer,
and then resuspended in 0.4 ml of fresh assay buffer containing 0.01 mg/ml propidium iodide (Molecular Probes, Eugene, OR) to allow
detection of any necrotic cells present. Samples were analyzed on a
Coulter XL flow cytometer, with forward and side scatter gates set to
include cells but exclude cellular debris and phospholipid vesicles.
NBD fluorescence was monitored in the FL1 channel and propidium iodide in the FL4 channel. The amount of fluorescent vesicles bound per cell
was then calculated from the mean fluorescence intensity of the gated
cell population. Specific binding was calculated by subtracting
nonspecific binding from total binding.
Competition studies were performed by adding the indicated competitor
at the same time as the NBD-PtdSer vesicles and carrying out the assay
as described above. Blocking experiments were carried out by adding the
blocking agent to the cells, incubating for 30 min on ice, and then
adding 10 µM PtdSer vesicle and 0 or 500 µM
unlabeled PtdSer vesicles and continuing the incubation for 120 min on ice.
Fluorescence Microscopy--
Samples were prepared as described
above with 10 µM NBD-labeled vesicles and incubated for
120 min on ice. Cells were washed three times and suspended in 200 µl
of assay buffer, then incubated either on ice or at 37 °C (to allow
endocytosis to occur) for an additional 30 min, fixed for 15 min on ice
with a final concentration of 0.75% paraformaldehyde in assay buffer,
and washed twice. A wet mount of the cells in 15 µl of assay buffer
was observed under a total magnification of 400× on a Nikon Labophot-2
fluorescent microscope, which had a B-2A filter in place (excitation:
450-490 nm; dichromatic beam splitter: 505 nm; barrier filter: 520 nm). The images were captured with a CCD camera 72S series that was interfaced to an 80486-based microcomputer through a video imaging board as described (28). Prior to printing, all images received a
uniform contrast enhancement with Adobe Photoshop (version 4.0).
Determination of Binding Constants--
To determine binding
constants from the flow cytometric data, the following additional
measurements were made. The fraction of free (unbound) vesicles was
determined over the range of added vesicle concentrations by
fluorometric analysis of the supernatants of binding reactions after
removal of cells by centrifugation. To convert the mean fluorescence
intensity per cell as determined by flow cytometry to the number of
phospholipid molecules bound per cell, a conversion factor was
determined as follows. Cells were incubated with different
concentrations of fluorescent vesicles, washed by centrifugation, and
dissolved in cell binding buffer containing 0.1% Triton X-100, and
their fluorescence intensity was measured by fluorometry and flow
cytometry. The number of molecules bound per cell could then be
calculated by reference to a standard curve prepared with known amounts
of pure NBD-PtdCho dissolved in the same buffer. The binding constant
(Kd) and number of vesicle binding sites per cell
(Bmax) were determined by fitting the specific
binding data to a model of non-cooperative binding to homogeneous
sites. Curve-fitting was performed by iterative non-linear
least-squares analysis (SOLVE function of Microsoft Excel version 5.0).
Fitting was performed with the data of bound phospholipid monomer
versus concentration of free phospholipid monomer. The
fitted Kd and Bmax values
were then expressed in terms of the molarity of vesicles by dividing by
the number of phospholipid molecules per vesicle as determined above.
Determination of Molecules per Cell of CD36--
THP-1 and
J774A.1 cells (2 × 105 cells) were incubated for 30 min on ice in 200 µl of phosphate-buffered saline with 1 mg/ml bovine
serum albumin and 0, 0.5, 1, 3, 10, or 20 µg/ml anti-CD36. Cells were
washed twice and incubated for 30 min on ice in 50 µl with 7.5 µg/ml fluorescein isothiocyanate-antimouse antibody; cells were then
washed twice more and suspended in 300 µl of buffer, and their
fluorescence intensity was measured in the FL1 channel of the flow
cytometer. Quantum 24 and Quantum 25 fluorescein beads were used to
convert observed fluorescence intensity to molecules of equivalent
soluble fluorochrome; this was then converted to molecules of CD36 per
cell by dividing by the manufacturer's stated value for molecules of
fluorescein per molecule of second antibody, assuming one molecule of
second antibody bound per CD36 molecule.
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RESULTS |
Flow Cytometric Assay for Binding of Phospholipid Vesicles to
Cells--
A flow cytometric procedure was developed to measure
binding of vesicles to cells. To isolate the step consisting of binding of vesicles to the cell surface prior to their possible internalization by endocytic processes, assays were performed at 4 °C. Previous work
has shown that binding of vesicles to cells occurs at 4 °C, but
internalization occurs only at higher temperatures (29). To keep the
labeled phospholipid component constant, vesicles of all types were
labeled with 10% NBD-PtdCho; use of NBD-PtdCho also prevented cell
labeling due to possible transmembrane transport of the labeled
phospholipid by the aminophospholipid translocase. When PtdSer vesicles
were added to THP-1 cells, a nearly homogeneous population of labeled
cells was observed, and the mean fluorescence of this population was
reduced by addition of excess unlabeled PtdSer vesicles (Fig.
1). In most experiments, a single
population of cells was observed; significant heterogeneity of the
number of binding sites per cell was only observed when necrotic cells were present, as judged by high propidium iodide uptake; these necrotic
cells had increased uptake of PtdSer vesicles that could not be blocked
by excess unlabeled vesicles. The lower half of Fig. 1 shows a
titration of THP-1 cells with increasing amounts of PtdSer vesicles, in
the absence (total binding) and presence (nonspecific binding) of a
large excess of unlabeled PtdSer vesicles. The results show a
saturable, high affinity component of the binding reaction, as well as
a low affinity, nonsaturable component.

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Fig. 1.
Flow cytometric analysis of PtdSer vesicle
binding to THP-1 cells. Top panel, typical
flow cytometer histograms. The assay was performed with 10 µM PtdSer vesicles in the absence (left) or
presence (right) of 500 µM unlabeled PtdSer
vesicles as described under "Experimental Procedures."
Gate E indicates the region analyzed to determine
the mean fluorescence of the cell population. Lower
panel, titration of THP-1 cells with PtdSer vesicles. The
cells were titrated with the indicated amount of PtdSer vesicles in the
absence ( , total binding) or presence ( , nonspecific binding) of
500 µM unlabeled PtdSer vesicles. Specific binding ( )
was then calculated by subtracting nonspecific binding from total
binding. Shown are means of duplicates from a representative
experiment, with S.D. smaller than symbol size.
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The specific binding of vesicles containing different anionic
phospholipids to THP-1 cells was measured with the same procedure; all
vesicles were labeled with identical amounts of fluorescent phospholipid, i.e. 10% NBD-PtdCho (Fig.
2). As shown in Table I, the apparent affinity and capacity of
binding were very similar for vesicles containing PtdSer, PtdGro, and
PtdIns. However, the capacity for PtdCho binding was approximately
5-fold less than for vesicles containing anionic phospholipids. These
results were confirmed with another monocyte-like cell line, J774A.1.
As shown in Table I, these cells had a very similar affinity for
phospholipid vesicles, but their capacity for binding anionic vesicles
was 3-fold greater.

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Fig. 2.
Specific binding of vesicles to THP-1 cells
and red blood cells. THP-1 cells (solid
symbols) or red blood cells (open
symbols) were incubated with NBD-labeled vesicles of the
indicated type in the presence or absence of 500 µM
unlabeled vesicle of the same type, and specific binding was
calculated. Results are means ± S.D. of duplicates from a
representative experiment.
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Table I
Binding parameters for vesicle-cell interaction
For each cell and vesicle type, results are the means of two
experiments, each performed in duplicate. The average uncertainty
(S.D.) in the binding parameters is ±18%, based on replicate
determinations performed on different days. The dissociation constants
are expressed in terms of the molarity of vesicles.
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As a control to rule out possible nonspecific interaction of vesicles
with any cell membrane, normal human erythrocytes were also titrated
under the same conditions as THP-1 cells (Fig. 2). The observed binding
of anionic vesicles was 10- to 20-fold less than to THP-1 cells, far
less than could be explained by the slightly smaller size of the
erythrocytes. Also, in contrast to results obtained with the THP-1
cells, the binding of PtdCho vesicles to erythrocytes was not
appreciably different from the binding of PtdSer, PtdGro, or PtdIns
vesicles. These data also confirm that the fluorescent labeling of
cells detected by this assay is not due to transfer of individual
NBD-PtdCho molecules from vesicles to the cell membrane. If this were
occurring, one would expect uniform cellular labeling regardless of the
phospholipid head group (anionic or neutral) present in the labeled
vesicles, and the addition of excess unlabeled vesicles would not alter the observed cellular labeling. In addition, labeling of THP-1 cells
and erythrocytes would be equal, which was not observed.
Effect of Annexin V--
Annexin V was used to verify that binding
of anionic phospholipid vesicles to THP-1 cells was not due to a
nonspecific adsorption process for any particles of the size of the
vesicles. (Annexin V binds with high affinity to vesicles containing
anionic but not neutral phospholipids; a concentration of 1 µM annexin V at 2.5 mM CaCl2 is
sufficient to saturate the available binding sites on 50% PtdSer
vesicles at 50 µM phospholipid concentration (26, 30).)
The binding of the three anionic phospholipid vesicles was inhibited by
annexin V (Fig. 3). In contrast, the
binding of PtdCho vesicles was minimally affected.

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Fig. 3.
Effect of annexin V on binding of vesicles to
THP-1 cells. Binding was measured with 10 µM
NBD-labeled vesicles of the indicated type in binding buffer
supplemented with 2.5 mM calcium and in the absence or
presence of annexin V (1 µM). Results are expressed as a
percentage of specific binding observed in the absence of annexin V. Shown are mean ± S.E. for the following number of independent
experiments: PtdCho (n = 11), PtdSer (n = 13), PtdGro (n = 4), PtdIns (n = 4).
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Competition Assays with Unlabeled Vesicles and
Phosphoserine--
Monomeric phospho-L-serine has been
used as a reagent to determine whether binding of vesicles or cells to
a given cell type is due to a putative PtdSer receptor
versus receptors with specificity for other ligands (5, 6,
31, 32). However, monomeric phospho-L-serine up to a
concentration of 1 mM was unable to inhibit the binding of
PtdSer vesicles to THP-1 cells (Fig.
4A). Similarly, glycerophospho-L-serine had no effect up to a concentration
of at least 100 µM (not shown). In contrast, unlabeled
PtdSer vesicles inhibited binding of PtdSer vesicles in a
dose-dependent manner. Similarly, binding of PtdSer
vesicles was inhibited by unlabeled PtdGro and PtdIns vesicles (Fig.
4B).

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Fig. 4.
Effect of competitors on binding of PtdSer
vesicles. A, competition with unlabeled PtdSer vesicles
and O-phospho-L-serine. THP-1 cells were
incubated with 10 µM PtdSer and the indicated
concentrations of unlabeled PtdSer vesicles ( ) or
O-phospho-L-serine ( ). Results are expressed
as specific binding in the presence of competitor divided by specific
binding in the absence of competitor. Results are mean ± S.D. of
triplicates from a single experiment representative of four separate
experiments. B, competition with unlabeled vesicles. THP-1
cells were incubated with 10 µM PtdSer vesicles in the
presence or absence of unlabeled vesicles at the indicated
concentration. Results are expressed as specific binding relative to a
control with no unlabeled competitor vesicles. , PtdSer; ,
PtdGro; , PtdIns.
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Effect of Antibodies and Other Ligands of CD36--
The expression
of CD36 on the cell surface was confirmed as described under
"Experimental Procedures"; these measurements yielded a value of
2.5 × 104 molecules per cell for THP-1 cells and
2.2 × 104 molecules per cell for J774A.1 cells. To
determine whether CD36 could explain some or all of the specific
binding of anionic phospholipid vesicles, binding assays were then
performed in the presence or absence of a monoclonal antibody to CD36
(Fig. 5A). The antibody blocked PtdSer vesicle binding in a dose-dependent manner;
up to 60% of the specific binding of PtdSer vesicles could be blocked at a concentration of 20 µg/ml. In contrast, the antibody had no
effect on the binding of PtdGro, PtdIns, or PtdCho (not shown) vesicles under the same conditions. To rule out possible nonspecific effects of the antibody, control experiments were performed with a
mouse monoclonal antibody of the same isotype to CD71 (the transferrin receptor); this antibody had no effect on PtdSer binding (not shown).
The selective effect of anti-CD36 on PtdSer binding was also confirmed
with J774A.1 cells (Fig. 5B).

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Fig. 5.
Effect of anti-CD36 on vesicle binding.
A, THP-1 cells were preincubated for 30 min at 4 °C with
the indicated concentration of anti-CD36; specific binding was then
determined by measurements with 10 µM NBD-labeled vesicle
in the presence or absence of 500 µM unlabeled vesicles
of the same type. Results are expressed as a percentage of the specific
binding observed in the absence of antibody. Shown are mean ± S.E. of six independent experiments, each with duplicate
determinations. , PtdSer; , PtdGro; , PtdIns. B,
J774A.1 cells were preincubated for 30 min at 4 °C with 20 µg/ml
anti-CD36; specific binding was then measured as described for
panel A. Shown are the mean ± S.E. of 5 to 7 independent experiments, each with duplicate determinations.
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Collagen (13) and thrombospondin (14) have been reported to be ligands
for CD36. We therefore tested their effect on the binding of PtdSer
vesicles under the same conditions used for the experiments with
anti-CD36 described above. Collagen up to 100 µg/ml did not inhibit
the binding of PtdSer vesicles (Table II). Similarly, thrombospondin up to 25 µg/ml did not inhibit binding.
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Table II
Effect of ligands of CD36 on PtdSer vesicle binding to THP-1 cells
Cells were preincubated for 30 min at 4 °C with the indicated
concentration of competitor; specific binding was then determined by
measurements with 10 µM NBD-labeled PtdSer vesicle in the
presence or absence of 500 µM unlabeled PtdSer vesicles.
Assays with thrombospondin were performed in the presence of 1 mM CaCl2. Results are expressed as a percentage of
the specific binding observed in the absence of competitor.
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Effect of Anti-CD36 on Phagocytosis of Vesicles--
Fluorescence
microscopy was performed to determine whether anti-CD36 inhibited
phagocytosis of PtdSer vesicles, as well as their initial binding to
the cell surface. Assays performed entirely at 4 °C confirmed that
all vesicle types bound to the cell surface and that anti-CD36
selectively inhibited cell surface binding of PtdSer vesicles,
consistent with the flow cytometric data. When cells were first
incubated at 4 °C and then warmed to 37 °C to allow endocytosis
to occur, all vesicle types were internalized, as judged by cytoplasmic
and perinuclear staining. However, anti-CD36 selectively inhibited the
endocytosis of PtdSer vesicles (Fig. 6,
panel B versus panel A), whereas it did not
inhibit the endocytosis of PtdGro vesicles (Fig. 6, panel D
versus panel C) or PtdIns vesicles (not shown). Thus, the
component of PtdSer binding inhibited by anti-CD36 is functionally
significant for endocytosis.

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Fig. 6.
Effect of anti-CD36 on binding and
endocytosis of phospholipid vesicles. J774A.1 cells were
preincubated with 0 or 20 µg/ml anti-CD36 for 30 min at 4 °C,
incubated with 10 µM NBD-labeled vesicles for 120 min at
4 °C, washed, incubated for 30 min at 37 °C to allow endocytosis
to occur, and fixed with paraformaldehyde. Cells were observed with a
Nikon fluorescent microscope at 400× magnification and photographed.
A, PtdSer vesicles; B, PtdSer vesicles plus 20 µg/ml anti-CD36; C, PtdGro vesicles; D, PtdGro
vesicles plus 20 µg/ml anti-CD36.
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DISCUSSION |
Properties of Phospholipid Vesicle Binding Sites on Monocytic
Cells--
Phagocytic removal of necrotic or apoptotic cells involves
an initial step of recognition of the target cell, followed by subsequent steps leading to its internalization and digestion. To focus
on the initial step involved in cellular recognition of membranes
containing exposed anionic phospholipids, the present study was
performed under conditions that prevent phagocytosis. Under these
conditions, both THP-1 and J774A.1 cells have saturable, high affinity
membrane binding sites for anionic phospholipid vesicles. The affinity
(Kd = 0.05 to 0.1 nM) and capacity (1400 sites per THP-1 cell, 4600 sites per J774A.1 cell) measured by the flow
cytometric technique are comparable with values obtained by others with
traditional fluorometric or radioligand techniques. For example, Lee
et al. (29) studied binding of PtdSer/PtdCho/cholesterol vesicles to J774A.1 cells at 4 °C and found a population of high affinity sites with a Kd of 0.3 nM and
about 3000 sites per cell for suspension-cultured cells. Similarly,
Rigotti et al. (11) reported a Kd of 0.18 nM for binding of PtdSer/PtdCho/cholesterol liposomes at
4 °C to a Chinese hamster ovary cell line transfected with a
cDNA for scavenger receptor BI.
The vesicle binding is specific by several criteria. Comparable binding
sites are essentially absent on erythrocytes, a cell without phagocytic
or endocytotic capability. Addition of annexin V, which will coat the
surface of anionic phospholipid vesicles, prevented binding, indicating
that binding was not due to a nonspecific adsorption of any particles
of the size of the vesicles. Although binding of PtdCho vesicles could
be demonstrated, the amount of binding was about 5-fold to 10-fold less
than for vesicles containing anionic phospholipids. Vesicle binding
could not be attributed to uptake by necrotic cells, as these cells
could be detected and gated out on the flow cytometer.
Molecular Requirements for Ligand Binding to PtdSer
Receptors--
Some previous studies have used competition assay with
monomeric phosphoserine and its derivatives as a diagnostic criterion for the involvement of a PtdSer receptor in binding and/or phagocytosis of apoptotic cells (5, 6, 31, 32). Fadok et al.
(5) showed that phospho-L-serine and
glycerophospho-L-serine, but not
phospho-D-serine, inhibited the phagocytosis of apoptotic thymocytes by mouse peritoneal macrophages; 50% inhibition of uptake
was observed at about 10 µM phospho-L-serine.
Similarly, phospho-L-serine and
glycerophospho-L-serine, but not
phospho-D-serine, partially inhibited the rosetting of red
blood cells with exposed PtdSer on murine bone-marrow-derived
macrophages (31).
However, in the present study these compounds had no effect on the
binding of PtdSer vesicles to THP-1 cells. This suggests that the
PtdSer receptor requires a surface containing multiple molecules of
phosphoserine to bind vesicles with high affinity. Thus, phosphoserine
may not be useful as a reagent for identifying the involvement of the
PtdSer receptor in the initial step of cell-cell or cell-vesicle
binding. The reason for the discrepancy with earlier studies is not
clear. It is possible that monomeric phosphoserine inhibits a later
step in the adherence or phagocytosis of apoptotic bodies rather than
their initial binding to the cell surface, which is the step measured
in the present study.
Properties of CD36 as a Putative PtdSer Receptor--
An
unexpected finding of this study was the observation that CD36 may have
greater selectivity for PtdSer among anionic phospholipids than has
been inferred from previous studies. In this study, binding of
PtdSer-containing vesicles was inhibited by antibody to CD36 (Fig. 5).
In addition, fluorescence microscopy (Fig. 6) showed that antibody to
CD36 selectively inhibited the endocytosis of PtdSer vesicles,
indicating that CD36 is functionally significant for phagocytosis. In
COS cells transfected with human CD36, Rigotti et al. (11)
showed that PtdIns and PtdSer, but not PtdCho, inhibited the binding of
125I-acetyl-low density lipoproteins to cells at 4 °C;
PtdIns was a better inhibitor than PtdSer. In a system consisting of
primary cultures of rat retinal pigment epithelial cells incubated at 37 °C, Ryeom et al. (12) found that anti-CD36 inhibited
78% of the binding of labeled PtdSer vesicles and 72% of the binding of PtdIns vesicles, whereas it inhibited only 14% of the binding of
PtdCho vesicles.
In addition to its putative role as a PtdSer receptor, CD36 has been
shown to bind several other ligands, including collagen and
thrombospondin. However, these ligands did not compete for PtdSer
vesicle binding to the THP-1 cell surface in this study (Table II).
This suggests that the binding site for PtdSer vesicles on CD36 is not
shared with the binding site for these other ligands. CD36 has been
reported to bind to collagen with an apparent Kd of
3 µg/ml (13) and to thrombospondin with an apparent
Kd of 22 µg/ml (14). Thus, the concentrations of
competitor proteins used should have caused a measurable decrease in
the binding of PtdSer vesicles if these substances shared binding sites
on CD36 with PtdSer vesicles.
Effect of Steric Crowding on Vesicle Binding--
It may seem
paradoxical that PtdGro and PtdIns vesicles compete with PtdSer
vesicles for binding to the cell, yet antibody to CD36 only inhibits
the binding of PtdSer vesicles. These findings can be reconciled in a
model that considers the relative sizes of the vesicles and their
binding proteins (Fig. 7). Initially, vesicles bind to the cell surface via a specific receptor or docking protein; bound vesicles may then collect in coated pits prior to
endocytosis. Among cell-surface proteins, CD36 has a strong preference
for binding PtdSer vesicles, whereas other unidentified protein(s)
provide binding sites for PtdGro and PtdIns vesicles. However, the cell
surface has limited capacity to bind vesicles of any type due to steric
crowding effects; thus, binding of large amounts of one vesicle type
can limit the binding of another vesicle type, even though they may be
binding to different receptor proteins. In contrast, an antibody
molecule is far smaller than a vesicle, and thus binding of anti-CD36
to CD36 would not prevent binding of other vesicle types to their
cognate receptor protein(s). This model can also explain why the number
of high affinity binding sites for PtdSer vesicles (1400-4600
sites/cell) is considerably less than the number of cell-surface CD36
molecules (22,000-25,000). As the surface area of the cell
(d = ~10 µm) is about 104 times the
surface area of the vesicle (d = 0.1 µm),
saturation of cell surface binding due to steric crowding might
be expected once roughly 103-104 vesicles are
bound to the cell surface.

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Fig. 7.
Model for effect of steric hindrance on
vesicle binding to cell surface receptors. A PtdGro or PtdIns
vesicle (diameter 100 nm) is shown bound to its hypothetical receptor
protein (diameter 5 nm) in a pit on the cell surface prior to
endocytosis. This prevents PtdSer vesicles from binding to CD36
molecules in this region of the membrane. , CD36 molecule; ,
receptor protein for other vesicle types.
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In conclusion, CD36 appears to be the major PtdSer-binding protein on
the surface of THP-1 and J774A.1 cells. In addition, the CD36 binding
site for PtdSer vesicles is highly specific and does not also bind
other anionic phospholipids or non-phospholipid ligands.