Phosphatidylserine Receptors: Role of CD36 in Binding of Anionic Phospholipid Vesicles to Monocytic Cells*

Jonathan F. TaitDagger §parallel and Christina SmithDagger

From the Departments of Dagger  Laboratory Medicine, § Medicine, and  Pathology, University of Washington, Seattle, Washington 98195-7110

    ABSTRACT
Top
Abstract
Introduction
References

Exposure of phosphatidylserine (PtdSer) has been implicated in the recognition and phagocytosis of senescent and apoptotic cells, and CD36 has been proposed as one receptor protein that recognizes PtdSer and other anionic phospholipids. We investigated the binding of phospholipid vesicles to the monocytic leukemia cell lines THP-1 and J774A.1 with a flow cytometric assay; vesicles contained 50 mol% PtdSer, phosphatidylinositol (PtdIns), or phosphatidylglycerol (PtdGro), with the balance being phosphatidylcholine. Specific, high affinity binding was observed for vesicles containing PtdSer, PtdIns, or PtdGro. Specificity of the assay was confirmed by control experiments with erythrocytes, which showed minimal vesicle binding, and with annexin V, which blocked the binding of PtdSer, PtdGro, and PtdIns vesicles to the THP-1 cells. However, O-phospho-L-serine (to 1 mM) had no effect on the binding of PtdSer vesicles, indicating that high affinity binding requires a surface containing multiple phosphoserine groups rather than a single molecule. A monoclonal antibody to CD36 blocked up to 60% of the specific binding of PtdSer vesicles but had minimal to no effect on the binding of PtdGro or PtdIns vesicles. This antibody also selectively inhibited the phagocytosis of PtdSer-containing vesicles as measured by fluorescence microscopy, indicating that CD36 is functionally significant for phagocytosis of this vesicle type. In addition, collagen and thrombospondin, two other putative ligands of CD36, were unable to inhibit the binding of PtdSer vesicles. We conclude that CD36 is the primary protein responsible for the high affinity binding of PtdSer vesicles to these monocyte-like cells. In addition, CD36 appears to be specific for PtdSer among anionic phospholipids, and non-phospholipid ligands of CD36 do not share binding sites with PtdSer on CD36.

    INTRODUCTION
Top
Abstract
Introduction
References

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.

    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.

    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.


View larger version (25K):
[in this window]
[in a new window]
 
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 (diamond , total binding) or presence (triangle , nonspecific binding) of 500 µM unlabeled PtdSer vesicles. Specific binding (black-square) 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.

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.


View larger version (27K):
[in this window]
[in a new window]
 
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.

                              
View this table:
[in this window]
[in a new window]
 
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.

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.


View larger version (18K):
[in this window]
[in a new window]
 
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).

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).


View larger version (14K):
[in this window]
[in a new window]
 
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 (black-square) 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. black-square, PtdSer; open circle , PtdGro; triangle , PtdIns.

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).


View larger version (19K):
[in this window]
[in a new window]
 
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. black-square, PtdSer; open circle , PtdGro; triangle , 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.

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.

                              
View this table:
[in this window]
[in a new window]
 
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.

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.


View larger version (92K):
[in this window]
[in a new window]
 
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.


    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.


View larger version (9K):
[in this window]
[in a new window]
 
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. bullet , CD36 molecule; open circle , receptor protein for other vesicle types.

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.

    ACKNOWLEDGEMENTS

We thank Donald Gibson for assistance with experimental work, the staff of the Hematopathology Laboratory for assistance with flow cytometry, Kathy Hutchinson for assistance with fluorescence microscopy, Dr. Wim Hol and Stewart Turley for use of the dynamic light scattering apparatus, and Drs. Wenzhe Li, Brad Cookson, Brent Wood, and Daniel Sabath for helpful advice and comments.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL-47151.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.

parallel To whom correspondence should be addressed: University of Washington, Dept. of Laboratory Medicine, Room NW-120, Box 357110, Seattle, WA 98195-7110. Tel.: 206-548-6131; Fax: 206-548-6189; E-mail: tait{at}u.washington.edu.

The abbreviations used are: PtdSer, phosphatidylserine; C7PtdCho, 1,2-diheptanoyl-sn-3-phosphatidylcholine; NBD-PtdCho, 1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-3-phosphatidylcholine; PtdCho, phosphatidylcholine; PtdGro, phosphatidylglycerol; PtdIns, phosphatidylinositol.
    REFERENCES
Top
Abstract
Introduction
References

  1. Devaux, P. F., and Zachowski, A. (1994) Chem. Phys. Lipids 73, 107-120[CrossRef]
  2. Williamson, P., and Schlegel, R. A. (1994) Mol. Membr. Biol. 11, 199-216[Medline] [Order article via Infotrieve]
  3. Zwaal, R. F., and Schroit, A. J. (1997) Blood 89, 1121-1132[Free Full Text]
  4. Tang, X., Halleck, M. S., Schlegel, R. A., and Williamson, P. (1996) Science 272, 1495-1497[Abstract]
  5. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) J. Immunol. 148, 2207-2216[Abstract/Free Full Text]
  6. Fadok, V. A., Savill, J. S., Haslett, C., Bratton, D. L., Doherty, D. E., Campbell, P. A., and Henson, P. M. (1992) J. Immunol. 149, 4029-4035[Abstract/Free Full Text]
  7. Martin, S. J., Reutelingsperger, C. P., McGahon, A. J., Rader, J. A., van Schie, R. C., LaFace, D. M., and Green, D. R. (1995) J. Exp. Med. 182, 1545-1556[Abstract]
  8. Bennett, M. R., Gibson, D. F., Schwartz, S. M., and Tait, J. F. (1995) Circ. Res. 77, 1136-1142[Abstract/Free Full Text]
  9. Verhoven, B., Schlegel, R. A., and Williamson, P. (1995) J. Exp. Med. 182, 1597-1601[Abstract]
  10. Savill, J., Fadok, V., Henson, P., and Haslett, C. (1993) Immunol. Today 14, 131-136[CrossRef][Medline] [Order article via Infotrieve]
  11. Rigotti, A., Acton, S. L., and Krieger, M. (1995) J. Biol. Chem. 270, 16221-16224[Abstract/Free Full Text]
  12. Ryeom, S. W., Silverstein, R. L., Scotto, A., and Sparrow, J. R. (1996) J. Biol. Chem. 271, 20536-20539[Abstract/Free Full Text]
  13. Tandon, N. N., Kralisz, U., and Jamieson, G. A. (1989) J. Biol. Chem. 264, 7576-7583[Abstract/Free Full Text]
  14. Asch, A. S., Barnwell, J., Silverstein, R. L., and Nachman, R. L. (1987) J. Clin. Invest. 79, 1054-1061[Medline] [Order article via Infotrieve]
  15. Endemann, G., Stanton, L. W., Madden, K. S., Bryant, C. M., White, R. T., and Protter, A. A. (1993) J. Biol. Chem. 268, 11811-11816[Abstract/Free Full Text]
  16. Abumrad, N. A., El-Maghrabi, M. R., Amri, E.-Z., Lopez, E., and Grimaldi, P. A. (1993) J. Biol. Chem. 268, 17665-17668[Abstract/Free Full Text]
  17. Oquendo, P., Hundt, E., Lawler, J., and Seed, B. (1989) Cell 58, 95-101[Medline] [Order article via Infotrieve]
  18. Acton, S., Rigotti, A., Landschulz, K. T., Xu, S., Hobbs, H. H., and Krieger, M. (1996) Science 271, 518-520[Abstract]
  19. Ramprasad, M. P., Fischer, W., Witztum, J. L., Sambrano, G. R., Quehenberger, O., and Steinberg, D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9580-9584[Abstract]
  20. Ramprasad, M. P., Terpstra, V., Kondratenko, N., Quehenberger, O., and Steinberg, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14833-14838[Abstract/Free Full Text]
  21. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T., and Tada, K. (1980) Int. J. Cancer 26, 171-176[Medline] [Order article via Infotrieve]
  22. Tsuchiya, S., Kobayashi, Y., Goto, Y., Okumura, H., Nakae, S., Konno, T., and Tada, K. (1982) Cancer Res. 42, 1530-1536[Abstract]
  23. Ralph, P., Prichard, J., and Cohn, M. (1975) J. Immunol. 114, 898-905[Abstract]
  24. Wood, B. L., Gibson, D. F., and Tait, J. F. (1996) Blood 88, 1873-1880[Abstract/Free Full Text]
  25. Gabriel, N. E., and Roberts, M. F. (1984) Biochemistry 23, 4011-4015[Medline] [Order article via Infotrieve]
  26. Tait, J. F., Gibson, D., and Fujikawa, K. (1989) J. Biol. Chem. 264, 7944-7949[Abstract/Free Full Text]
  27. Lim, T. K., Bloomfield, V. A., and Nelsestuen, G. L. (1977) Biochemistry 16, 4177-4181[Medline] [Order article via Infotrieve]
  28. Astion, M. L., Orkand, A. R., Olsen, G. B., Pagliaro, L. J., and Wener, M. H. (1993) Lab. Med. 24, 341-344
  29. Lee, K. D., Nir, S., and Papahadjopoulos, D. (1993) Biochemistry 32, 889-899[Medline] [Order article via Infotrieve]
  30. Tait, J. F., and Gibson, D. (1992) Arch. Biochem. Biophys. 298, 187-191[Medline] [Order article via Infotrieve]
  31. Fadok, V. A., Laszlo, D. J., Noble, P. W., Weinstein, L., Riches, D. W., and Henson, P. M. (1993) J. Immunol. 151, 4274-4285[Abstract/Free Full Text]
  32. Ren, Y., Silverstein, R. L., Allen, J., and Savill, J. (1995) J. Exp. Med. 181, 1857-1862[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.