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PHAGOCYTES
From the Coagulation and Fibrinolysis Laboratory,
Thrombosis Research Institute, Emmanuel Kaye Building, London; Leukemia
Research Fund Centre, Institute of Cancer Research, London; Cell Centre
for Cell and Molecular Biology, Institute of Cancer Research, London;
Academic Department of Haematology and Cytogenetics, The Royal Marsden
Hospital, London; FACS Laboratory, Imperial Cancer Research Fund,
London; and Tumour Biology and Metastases Section of Cancer
Therapeutics, Institute of Cancer Research, Sutton, United Kingdom.
During cell death of human cultured leukocytes (Jurkat, HL-60,
THP-1, U937) and freshly prepared leukocytes, we observed a greater
than 100-fold increase in the affinity of apoptotic and necrotic cells
for fluorescein isothiocyanate (FITC)-heparin in comparison with live
cells. Binding of FITC-heparin was reversed in the presence of high
ionic strength, unlabeled heparan sulfate, and heparin and pentosan
polysulfate, but not in the presence of chondroitin and dermatan
sulfates. During the course of cell death, the increase in the
percentage of cells positive for annexin V binding correlated with the
increase in the population positive for binding FITC-heparin. Confocal
microscopy demonstrated that heparin binding to dead cells was
restricted to 1 or 2 small domains on the surfaces of apoptotic cells
and to larger, but still discrete, areas that did not localize with
chromatin on ruptured necrotic cells. The heparin-binding domains
originated from the nucleus and may correspond to the
ribonucleoprotein-containing structures that have recently been shown
to segregate within the nucleus of cells and to move onto the cell
membrane. We observed that phagocytosis of dead Jurkat cells by
monocyte-derived macrophages was blocked when the heparin-binding
capacity of the dead cells was saturated by the addition of pentosan
polysulfate. From this we concluded that the ability of dead cells to
bind to heparan sulfate proteoglycans on the surfaces of macrophages
may assist in phagocytic clearance.
(Blood. 2002;99:2221-2227) The first critical step in the clearance of
apoptotic cells is the recognition by phagocytes of changes occurring
on the surface of the dying cell. These "eat me" signals trigger
binding and engulfment.1 A repertoire of receptors on the
phagocytic surface are used to bind to these eat me signals, a prime
example of which is a specific receptor for phosphatidylserine (PS)
that is exposed on the outer membranes of cells early in apoptosis.
One group of cell surface receptors for which a role has yet to be
established in the phagocytosis of apoptotic cells is the heparan
sulfate proteoglycans (HSPGs), whose synthesis is up-regulated during
macrophage differentiation.2-7 HSPGs have an established function in cell adhesion, and recent reports demonstrate involvement in the binding step during phagocytosis of latex beads by a number of
cell lines.8,9 This opsonic property coincides with the well-documented ability of HPSGs to act as receptors for pathogenic bacteria, viruses, and protozoan parasites, and binding is often associated with the eventual internalization of the infective agent
(reviewed in Dehio et al9).
The structural feature responsible for the action of HPSGs as receptors
is the heparan sulfate chains. These provide a linearly arranged,
high-negative charge capable of binding tightly to a wide variety of
extracellular protein ligands.10 In the current study, we
investigated whether HPSGs could be involved in the clearance of dying
leukocytes by determining whether ligands for HPSGs occur on the cell
surface during apoptosis. Fluorescein isothiocyanate (FITC)-labeled
heparin was used as a probe because of the close structural similarity
between heparin and heparan sulfate. We have observed that heparin does
not bind with high affinity to live cells (defined as the dissociation
constant (Kd) < 1 µM) but only to
apoptotic and necrotic cells. Dying cells exhibit an enhanced affinity
because of binding to discrete domains that arise within the nucleus
during apoptosis, and they are released onto the plasma membrane during
cell death. Finally, we have established that the phagocytosis of dead
cells by monocyte-derived macrophages is blocked by pentosan
polysulfate, a high-affinity ligand for the heparin-binding sites.
Materials
Unfractionated porcine heparin (Mr, 15 kd) was obtained from Pharmacia
Upjohn (Sweden), and pharmaceutical preparations of low-molecular-weight heparin (LMWH) were obtained as follows: Certoparin (7 kd), Novartis; Fragmin (6 kd), Pharmacia Upjohn; Fraxiparin (4.5 kd) and CY222 (3.5 kd), Sanofi (France); and Bemiparin (3 kd), Laboratorios Farmaceuticos ROVI S.A. (Spain). Heparan sulfate
(30 kd; Laboratorio Derivati Organici, Italy) and pentosan polysulfate
(3.5 kd, SP 54, Benechemie, Germany) were kindly supplied by Dr B. Mulloy (NIBSC, Herts, United Kingdom). Chondroitin sulfate A and C were
purchased from Sigma, and dermatan sulfate was obtained from Mediolanum
Farmaceuti (Italy). FITC-labeled unfractionated heparin and FITC-LMWH
(labeled Bemiparin) were prepared by Dr F. Wusteman (School of
Molecular Medicine and Biosciences, University of Wales, Cardiff). In
brief, free amino groups on the heparin were acetylated, and free
reducing ends were coupled to diaminoethane using pyridine:borohydride
as a reducing agent. This material was labeled with FITC and was
purified by repeated purification and ion exchange chromatography on
Dowex 1. No loss in anticoagulant activity was observed after the
labeling. All other chemicals used were of analytical grade and were
obtained from Sigma.
Cell culture
For flow cytometric experiments involving freshly prepared leukocytes, blood was obtained from human unmedicated volunteers after informed consent. Approximately 50 mL blood was taken by venipuncture into 0.38% sodium citrate and was mixed with 6% dextran (1:4 vol/vol). After 60 minutes at room temperature, the supernatant was removed and an equal volume of HEPES buffer was added. Cells were removed by centrifugation at 400g for 8 minutes. Lysis was performed by adding 1 mL 0.25% NaCl to the pellet and was followed immediately by 1 mL 1.6% NaCl. Cells were washed with HEPES buffer and resuspended in RPMI 1640 medium before treatment with apoptotic agents as described above. The separated cells were mostly granulocytes during FACS analysis. Cell morphology Cytospin preparations of cells were fixed in absolute methanol and stained with Giemsa-May-Grünwald. Cell morphology was evaluated by light microscopy using Olympus AH-2 equipment (objective ×20, ×40, ×60, ×100).Flow cytometry Cells were washed twice with complete RPMI 1640 medium and were resuspended at an approximate density of 1 × 106 cells/mL. Aliquots (100 µL) of the cell suspension were diluted 10-fold with HEPES buffer (20 mM HEPES, 137 mM NaCl, 2.0 mM KCl, 5.6 mM glucose, 1.0 mM MgCl2, 1 g/L bovine serum albumin, pH 7.2). Various concentrations of FITC-heparin were prepared with the cells, and samples were incubated in the dark at 4°C for 10 minutes before washing once in annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). PE-annexin V and 7-AAD were added according to the manufacturer's instructions, and samples were immediately analyzed using a Becton Dickinson FACSCalibur flow cytometer calibrated using fluorescent beads. Acquisition processing of data from 10 000 cells was carried out during each experiment. The Kd for binding to dead cells was determined from triplicate values of relative fluorescence intensity (RFI) obtained at dilutions of FITC-heparin from 0.04 to 1.3 µM that were fit to a single-site ligand-binding equation using Enzfitter (Sigma).Cell sorting Cells were labeled with annexin V and 7-AAD, as described above, and were sorted into live, apoptotic, and necrotic fractions by a FACSVantage SE cell sorter (Becton Dickinson). The minimum number of cells collected into RPMI 1640 medium was 40 000 in each confocal microscopy sample.Sorted cells were resuspended in HEPES buffer before the addition of FITC-heparin (1 µM). After 10 minutes on ice, cells were washed once in annexin V binding buffer and were relabeled with PE-annexin V and 7-AAD. The cell suspension was then mixed with Mowiol in a 1:5 (vol/vol) proportion before they were mounted onto glass slides. Confocal laser scanning microscopy was performed using a Bio-Rad MRC 1024 equipped with a krypton-argon laser in conjunction with a Nikon Eclipse 600 microscope (objective ×60). Eight thin horizontal planes of sections were made, each 1 µm apart (zoom factor, 3.23). A standard FITC filter set combined with the 488-nm laser line was used to visualize FITC-labeled heparin-binding sites, whereas a Texas Red filter set combined with the 588-nm laser line enabled localization of areas that bound PE-annexin V (cell membrane) and 7-AAD (intracellular distribution). Fluorescence microscopy Cells were sorted as described above and were labeled with FITC-heparin at a final concentration of 1 µM for 10 minutes. After one wash with HEPES buffer, the cells were stained with DAPI, and glass slides were prepared. Fluorescence microscopy was performed using a Zeiss Axioshop, and images were captured using Quips-FISH application (Vysis, United Kingdom).Heparin binding to isolated nuclei Jurkat cells were treated with Camptothecin and were isolated according to the procedure of Martelli et al.11 In brief, cells were resuspended to 1.5 × 107/mL in TM-2 buffer (10 mM Tris, pH 7.4, 2 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin) and were incubated for 5 minutes on ice. Triton X-100 was added at 0.5% (wt/vol), and the cell suspension was sheared by one passage through a 21-gauge needle. Nuclei were sedimented at 400g for 6 minutes, washed once in TM-2 buffer, and resuspended in 1 mL HEPES buffer before FITC-heparin (final concentration, 1µM) was added. After 10 minutes, the nuclei were washed once in buffer containing 10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 5 mM MgCl2, and 1 µg/mL aprotinin and leupeptin. A sample of nuclei in suspension was transferred to a glass slide, stained with DAPI, and examined by fluorescence microscopy as described above.Influence of the enhanced heparin affinity of dead cells on phagocytosis Monocyte-derived macrophages (MDMs) were separated from human fresh blood using Lymphoprep density gradient medium (Nycomed, Oslo, Norway) and were cultured on Permanox chamber slides (Lab-Tek, United Kingdom) in the presence of 10% autologous serum.7 Live Jurkat cells were incubated with TAMRA as described previously.12 Half the Jurkat cells labeled with TAMRA (JurkatT) were treated with Camptothecin for 30 hours. Live and dead cell preparations were added (4 × 105cells/mL) to the chambers culturing MDMs. After 60-minute incubation at 37°C, the supernatant was removed and MDM monolayers were washed with PBS containing 0.3% immunoglobulin G-free bovine serum albumin and 0.15% NaN3 at 4°C. MDMs were then labeled with FITC-labeled anti-human CD14 at 4°C in the dark for 45 minutes. The supernatant was removed, and cells were washed in PBS and were subjected to laser scanning cytometry using a laser scanning cytometer (CompuCyte, MA) connected to a BX50 fluorescence microscope (Olympus, NJ).13 Slides containing MDM monolayers were moved through a 488-nm laser line on a computer-controlled motorized stage. Differentially treated areas on the microscope slide were scanned individually by using the ×20 objective and 5-mW laser output. Fluorescence from FITC-labeled CD14 and TAMRA were collected using photomultiplier tubes protected by, respectively, a 530/30 bandpass filter and a 580/30 bandpass filter. Cells were defined on the basis of their FITC-staining and threshold level (fluorescence above background), which were optimized so that as many single cells as possible could be contoured without losing fluorescence information. For each event, fluorescence integral, area, and maximum pixel were calculated. Cell clumps were excluded based on a plot of cell area versus maximum pixel, and a plot of green versus orange integral fluorescence was used to quantify the percentages of phagocytosed TAMRA-labeled Jurkat cells. At least 1000 contoured events were recorded for each treatment area.
Data obtained during flow cytometry were analyzed using WinMDI version 2.8 computer software (Scripps Research Institute, La Jolla, CA), where RFI was calculated as a Gmean. Microsoft Excel was used to calculate mean values and standard error of the mean (SEM). Each experiment was repeated at least 3 times. Data for laser scanning cytometry were analyzed on a Hewlett Packard (Palo Alto, CA) computer using WinCyte 3.4 software (CompuCyte).
Binding of heparin to live, apoptotic, and necrotic cells Heparin binding by leukocytic cells undergoing cell death induced by a number of agents was determined with each of 4 cell lines, namely, Jurkat, THP-1, U937, and HL-60. Live, apoptotic, and secondary necrotic cells were discriminated according to the binding of PE-annexin V and the uptake of 7-AAD, as described in "Materials and methods." When these parameters were used to sort the cells, the typical morphology of live, apoptotic, and necrotic cells was observed by light microscopy as shown in the inserted micrographs in Figure 1.
The binding of FITC-heparin to live, apoptotic, and necrotic Jurkat
cells is shown in Figure 2. No binding
was observed to live cells except for a slight increase in RFI at 5 µM FITC-heparin. In contrast, enhanced binding was observed to
apoptotic and necrotic cells at all concentrations of FITC-heparin
tested. The RFI reached a maximum at 5 µM FITC-heparin (tested up to
20 µM FITC-heparin, data not shown), and in comparison with live
cells, it was an order of magnitude higher for apoptotic cells and more
than 2 orders of magnitude higher for necrotic cells. The
Kd for binding of FITC-heparin to dead
cells was determined as 259 nM (data not shown). Binding was rapid
(complete within 1 minute), reversed by the inclusion of 0.8 M NaCl in
the heparin binding buffer, and independent of calcium (data not
shown).
A summary of the results obtained with each of the cell lines is shown
in Table 1. RFI values were determined
for each of the populations at a single concentration of FITC-heparin
(500 nM). The mean increase in RFI for apoptotic cells over live cells was 10-fold; for necrotic cells, it was more than 100-fold. A similar
increase in RFI was observed during apoptosis of leukocytes prepared
from fresh blood, as shown in Figure 3.
The appearance of FITC-heparin-positive cells and PE-annexin
V-positive cells was compared in timed samples taken during apoptosis
of Jurkat cells (Figure 4). At later time
points, good agreement was observed between the percentage of cells
found to be PE-annexin V positive and the percentage of FITC-heparin
positive cells. Correlation was not as good in the early phase of the
study, possibly because of a degree of overlap between the binding of
FITC-heparin by apoptotic and live cells (Figure 2).
Figure 5 depicts the observations made on
the ability of unlabeled heparan sulfate, pentosan polysulfate, and
various molecular weights of heparin to block the binding of
FITC-heparin to necrotic cells. FITC-LMWH was used for these
experiments because it was observed in preliminary experiments that
this has a weaker affinity for cells than FITC-heparin. As a block for
the binding of FITC-LMWH, IC50 decreased with increased
molecular weight (Table 2). Affinity for
dead cells was reduced as the molecular weight of heparin was
decreased, consistent with findings of previous reports regarding the
binding of heparin to proteins, such as platelet factor
4.14 The IC50 of pentosan polysulfate (3.5 kd)
was similar to that of unfractionated heparin. The ability of pentosan
polysulfate to bind with high affinity despite its low molecular weight
is attributed to the high-charge density of pentosan polysulfate (3.5 sulfate groups per disaccharide as opposed to 2.4-2.8 sulfate groups
per disaccharide for heparin). Heparan sulfate, which has a lower
degree of sulfation than heparin (0.8-1.8 sulfate groups per
disaccharide) gave an IC50 value of 0.3 µM.15 Chondroitin sulfates A and C and dermatan sulfate
had no effect on the binding of FITC-LMWH when tested up to 15 µM.
These experiments demonstrate the specificity of heparin binding and
rule out a contribution to the binding of the single FITC reporter
moiety.
Localization of binding sites for FITC-heparin by confocal and fluorescence microscopy Confocal microscopy of the FITC-heparin-labeled live, apoptotic, and necrotic Jurkat cells prepared by sorting confirmed that live cells do not bind heparin (Figure 6 [1A]). On apoptotic cells, 1 or 2 discrete domains of heparin binding were observed at the cell surface (Figure 6 [1B]). In cells that had membranes that were damaged but unruptured, a broader region of heparin binding was seen, though this was still discrete (Figure 6 [1C]). Heparin binding to ruptured necrotic cells was to discrete domains that exhibited high fluorescence intensity that did not coincide with the redness of chromatin present as lobules (Figure 6 [1D]) or as a residue (Figure 6 [1E]). Further histologic investigation by fluorescence microscopy confirmed that the sites responsible for heparin binding arose as discrete domains that were not present in live cells (Figure 6 [2A]) but that developed within the nucleus (stained with DAPI) during apoptosis. (Figure 6 [2B-C]). During the course of cell death, these domains became separated from the chromatin (Figure 6 [2C]) and eventually were attached to the cell membrane (Figure 6 [2B-D]) or appeared alongside the residual chromatin on the membrane of necrotic cells (Figure 6 [2E]). These domains were also observed in nuclei from cells harvested at earlier time points. Staining of the nuclei with FITC-heparin and DAPI medium showed no binding to nuclei from live cells (Figure 6 [3A]). In the first sample taken at 30 minutes, 1 or 2 larger areas of relatively less intense binding of heparin were observed (Figure 6 [3B]). In later samples at 1 and 2 hours, several discrete smaller domains of higher intensity were more common (Figure 6 [3C-D]). Nuclear material isolated at 4 and 6 hours showed lobulated nuclei that exhibited high-intensity binding to large domains that were separated from the DAPI-positive material (Figure 6 [3E-F]). During this series of experiments, the addition of excess unlabeled heparin blocked the fluorescence signal.
Influence of the heparin affinity of dead cells on phagocytosis Phagocytosis was determined by the incorporation of TAMRA-labeled Jurkat cells into monolayers of MDM using laser scanning cytometry. When incubated with dead Jurkat cells, 31.9% of MDM were positive for TAMRA (Figure 7C) compared with only 2.1% in the presence of live cells (Figure 7B). When the dead cells were preincubated with annexin V (400 µg/mL for 15 minutes), the incorporation was reduced to 8.5% in agreement with the known importance of PS exposure as a ligand for the phagocytosis of dead cells (Figure 7D). The incorporation was reduced to 4.2% when cells were preincubated with pentosan polysulfate at a concentration that would block available heparin binding sites (2 µM) (Figure 7E). The observed reduction shows that the heparin-binding sites play a significant role in the uptake of dead cells, presumably through the interaction with macrophage HPSGs. The level of incorporation was reduced to 1% when pentosan polysulfate and annexin V were combined (Figure 7F).
We have observed that in comparison with healthy leukocytes,
apoptotic and necrotic leukocytes have a high affinity for
heparin When we investigated the binding of heparin by confocal microscopy, we observed that binding occurred to discrete areas on the membrane of apoptotic cells (Figure 6 [1B]) or were separated from chromatin in necrotic cells. The development of focused, highly intense regions of heparin binding was confirmed by conventional fluorescence microscopy. The source of the heparin binding material appears to be the nucleus because the nuclei in live cells do not bind heparin. Early in the apoptotic process, however, a large binding domain was formed within the nucleus (Figure 6 [3B]). The intensity of heparin binding was considerably enhanced when it occurred outside the nucleus, indicating a lack of accessibility to binding proteins when located within the nucleus, perhaps because of the presence of other ligands. This finding of discrete domains of nuclear material segregating during apoptosis corresponds closely to structures recently described as clusters of ribonucleoproteins,25,26 for which the term HERDS (heterogeneous ectopic ribonucleoprotein-derived structures) has been coined.27 These proteins are normally distributed throughout the nucleus in the nuclear matrix, but during apoptosis they form into small fibrillogranular structures that pass into the cytoplasm and are eventually extruded at the cell surface in membrane-bound blebs.28-32 The ribonucleoproteins are obvious candidates as heparin-binding proteins because many are highly basic proteins and contain sequences that confer high affinity for heparin.33 Our findings demonstrate that the heparan sulfate-binding property of apoptotic cells may be of physiological significance in assisting phagocytic clearance. The exposure of PS is well established as a signal for the phagocytosis of apoptotic cells, a mechanism that can be blocked by the presence of annexin V.34 Macrophages are known to express HSPGs that have been observed to play a role in the phagocytosis of latex beads.8,9 We have now shown that the phagocytosis of dead cells can be effectively blocked by saturation of the heparin-binding sites with pentosan polysulfate, a highly potent inhibitor of heparin binding to nonviable cells. Moreover, because the exposure of areas with high affinity for heparin occurs early in apoptosis, coincidentally with the exposure of PS (Figure 4), HPSG binding may make a significant contribution to the clearance mechanism. If this is the case, then heparin treatment may reduce the rate of removal of dying cells.
We thank Dr M. Ormerod (Institute of Cancer Research, Sutton, United Kingdom) for helpful discussions and for critical reading of the manuscript, Dr F. S. Wusteman (School of Molecular Medicine and Biosciences, University of Wales, Cardiff) for synthesis of fluorescein-labeled heparin, Dr Barbara Mulloy (Laboratory for Molecular Structure, National Institute for Biological Standards and Control, Potters Bar, United Kingdom) for gifts of heparan sulfate and pentosan polysulfate, and Dr T. R. Hawthorne (Novartis) for the gift of recombinant annexin V.
Submitted March 15, 2001; accepted November 9, 2001.
Supported by a grant from the British Heart Foundation (PG/98/82). No commercial support has been received for this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael F. Scully, Thrombosis Research Institute, Emmanuel Kaye Building, Coagulation and Fibrinolysis Laboratory, 1B Manresa Rd, Chelsea, London SW3 6LR, United Kingdom; e-mail: mscully{at}tri-london.ac.uk.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
-hydroxycholesterol were obtained from
Sigma (Poole, United Kingdom); CD95 (Fas antibody, R and O Systems, United Kingdom); phycoerythrin (PE)-annexin V and viability dye 7-AAD
(7-amino-actinomycin D, Via-Probe, CA) and other reagents for
fluorescence-activated cell sorter (FACS) analysis were obtained from
Becton Dickinson, CA; TAMRA
5-6-carboxytetramethyl-rhodamine-succinimidyl-ester was obtained from
Molecular Probes (Eugene, OR); Vectashield mounting medium for
fluorescence with DAPI was obtained from Vector Laboratories (Burlingame, CA), and Mowiol from Calbiochem (United Kingdom). Recombinant unlabeled annexin V was obtained from Dr T. R. Hawthorne (Novartis, Basel, Switzerland); FITC-labeled anti-human CD14
and CD45 and the respective isotype controls were obtained from Sigma.
-
-
heparan
sulfate, pentosan polysulfate, unfractionated heparin (average Mr, 15 kd), and pharmaceutical preparations of depolymerized heparin of
various average molecular weights (as listed in "Materials and
methods"). After 60 minutes, FITC-LMWH was added at 1 µM, and
incubation continued for another 10 minutes. Cells were then labeled
with PE-annexin V and 7-AAD, and the RFI of the necrotic population
was determined by FACS analysis as in Figure 
-
-
-
-
-
