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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1293-1300
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGYAU#0
Adherence of phosphatidylserine-exposing erythrocytes to
endothelial matrix thrombospondin
Annamaria B. Manodori,
Gilda A. Barabino,
Bertram
H. Lubin, and
Frans A. Kuypers
From the Children's Hospital Oakland Research Institute, Oakland,
CA; and Department of Chemical Engineering, Northeastern University,
Boston, MA.
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Abstract |
Phospholipid asymmetry is well maintained in erythrocyte (RBC)
membranes with phosphatidylserine (PS) exclusively present in the inner
leaflet. The appearance of PS on the surface of the cell can have major
physiologic consequences, including increased cell-cell interactions.
Because increased adherence of PS-exposing RBCs to endothelial cells
(ECs) may be pathologically important in hemoglobinopathies such as
sickle cell disease and thalassemia, we studied the role of PS exposure
in calcium ionophore-treated normal RBC adherence to human umbilical
vein endothelial cell (HUVEC) monolayers. When HUVEC monolayers were
incubated with these PS-exposing RBCs, the ECs retracted and the RBCs
adhered primarily in the gaps opened between the ECs. A linear
correlation was found between the number of PS-exposing RBCs in
the population and the number of adhering RBCs to the monolayer.
Pretreatment of RBCs with annexin V significantly decreased
adherence by shielding PS on the RBCs. Similarly, PS-containing lipid
vesicles decreased RBC binding by competing for the PS binding sites in
the monolayer. PS-exposing RBCs and PS-containing lipid vesicles
adhered to immobilized thrombospondin (TSP) and matrix TSP,
respectively, and adherence of PS-exposing RBCs to EC monolayers was
reduced by antibodies to TSP and to its EC receptor,
 v 3. Together, these results indicate a
role for PS and matrix TSP in the adherence of PS-exposing RBCs to EC
monolayers, and suggest an important contribution of PS-exposing RBCs
in pathologies with reported vascular damage, such as sickle cell anemia.
(Blood. 2000;95:1293-1300)
© 2000 by The American Society of Hematology.
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Introduction |
The distribution of phospholipids in the normal
erythrocyte (RBC) membrane is highly asymmetrical, with
phosphatidylserine (PS) located exclusively in the inner
leaflet.1 The loss of membrane asymmetry and the exposure
of PS can affect the hemostatic balance,2 and leads to
recognition and removal of aging red cells by macrophages.3
In patients with sickle cell anemia or thalassemia, a small but
significant subpopulation of RBCs can be found that expose PS on their
surface.4-6 Adherence of RBCs to the endothelium may play a
significant role in vasoocclusive events in sickle cell
disease,7 and PS exposure in RBCs could be important in
this process.1,8 Although previous reports on abnormal
adherence of PS-exposing RBCs to endothelial cell (EC) monolayers
suggest a role for PS in adherence,9-11 the mechanism remains to be elucidated.
Phagocytotic macrophage recognition of PS-exposing cells has been
extensively studied. Recognition of aged neutrophils was shown to be
charge dependent and blocked in a stereo-specific and dose-dependent
manner by PS-containing liposomes.12 Similarly, phagocytosis of mouse lymphocytes was inhibited by PS-containing liposomes,13 suggesting that macrophages can specifically
recognize PS. Phagocytosis of apoptotic, PS-exposing neutrophils was
found to be mediated by macrophage  v3
integrin. However, blocking experiments suggested that the recognition
mechanisms involving v3 and PS were
mutually exclusive.14 Subsequently, the participation of
thrombospondin (TSP) as a molecular bridge binding to both CD36 and
v3 was shown.15
Although many mechanisms have been implicated in the binding of sickle
red cells to the endothelium,16,17 one of the most documented, and probably significant, interactions between sickle RBCs
and the endothelium is mediated by TSP.18,19 This large multifunctional adhesive protein is synthesized and secreted by many
different cell types, including ECs and activated platelets. TSP is
present in soluble form in the plasma and can be found in the basement
membrane of ECs and in the extracellular matrix of cultured ECs, where
it functions in cell-cell and cell-matrix interactions. The versatility
of TSP lies in its many domains, with binding sites for heparin,
calcium, fibrinogen, fibronectin, collagen V, plasminogen,
histadine-rich glycoproteins, and sulfated glycolipids.20
Calcium ions regulate the ligating properties of TSP, the transition
from its adhesive to its nonadhesive conformer being modulated by
calcium depletion. In addition, the physical state of TSP affects its
conformation, influencing its interaction with other
molecules.21
Abnormal levels of soluble plasma TSP have been found in sickle
patients.22 This soluble TSP appears to be instrumental in
bridging sickle RBC adherence to ECs.18,23 On RBCs, TSP interacts with CD36, sulfated glycolipids,24 and a normally cryptic domain of the dominant membrane protein, band 3, which is
subject to rearrangement in hematologic disorders.11,25 TSP
binding to ECs occurs through interactions with the integrin, v3,26 heparin
sulfate,23,27 and an integrin-associated protein.26 In addition to a role for soluble TSP, sickle
RBC adherence to immobilized TSP 28-30 suggests a role for
matrix TSP, exposed by vascular injury, in sickle cell pathology.
On the basis of these observations, we postulated that PS exposure in
RBCs leads to PS-mediated RBC adherence to matrix TSP in damaged
endothelial monolayers. In this study we used calcium-loaded RBCs to
show a linear increase of RBC adherence with PS exposure and to
elucidate a mechanism that explains this phenomenon.
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Materials and methods |
Reagents
Gelatin, fibronectin from human plasma, bovine serum albumin (BSA),
glutaraldehyde, Hanks' buffered saline solution (HBSS), HEPES buffer,
thrombin, histamine, EDTA, EGTA, calcium ionophore A23187,
phosphatidylcholine (PC) from egg yolk, and L--phosphatidyl-L-serine from bovine brain were obtained from Sigma Chemicals Co (St. Louis, MO). Calcium, magnesium-free phosphate buffered saline (PBS) was from
the University of California San Francisco cell culture facility. Endothelial Cell Growth Medium (EGM) was from Clonetics Corp (San Diego, CA). Dispase II was obtained from Boehringer Mannheim Corp (Indianapolis, IN). NBD (7-nitro-2-1,3-benzoxadiozol-4-yl)-labeled phosphatidylcholine was purchased from Avanti Polar Lipids, Inc (Alabaster, AL). Nucleopore filters were from Corning Costar Corp (Cambridge, MA). Eight-chambered culture slides were obtained from NUNC
(Naperville, IL). Biomeda Gel Mount was from Biomeda Corp (Foster City,
CA), and Vectashield Mounting Medium was from Vector Labs (Burlingame,
CA). TSP was a gift from Dr Jack Lawler, Beth Israel Deaconess Medical
Center, Boston, MA; band 3 peptides and monoclonal antibody (MAb) 1F4
were gifts from Dr Irwin W. Sherman, University of California
Riverside; polyclonal antibody to CD59 was a gift from Dr Samuel Test,
Children's Hospital Research Institute, Oakland, CA. Monoclonal
antibodies, OKM5 to CD36 were from Coulter Immunotech, Inc (Westbrook,
ME); L230 to v and AP3 to 3 were from
American Type Culture Collection (Rockville, MD); polyclonal antibodies
to thrombospondin were from Calbiochem (San Diego, CA). Phycoerythrin
conjugated goat antimouse IgG and phycoerythrin conjugated donkey
antirabbit IgG were purchased from Jackson ImmunoResearch Laboratories,
Inc (Westgrove, PA); Biotinylated antirabbit IgG and Streptavidin Texas
Red were from Amersham Pharmacia Biotech (Piscataway, NJ). FITC-labeled
annexin V was prepared as described previously.4
Erythrocytes
Blood samples were collected in citrate after informed consent from
healthy donors or sickle cell patients of Children's Hospital Oakland.
Red cells were isolated by centrifugation, washed 3 times in HBSS, and
the buffy coat was removed after each wash. Phospholipid organization
was scrambled in normal RBCs by incubation in 0.5 mmol/L
CaCl2 in the presence of 2 µmol/L calcium ionophore
A23187.4 After incubation, the calcium ionophore was
removed by back extraction with BSA. These PS-exposing RBCs were mixed
with untreated red cells to obtain samples with 5% to 40% PS-exposing
red cells. Alternatively, back extraction of ionophore was omitted, the
PS-exposing cells were mixed with normal cells to a mixture of 20%
treated, 80% untreated RBCs and subsequently incubated in 2.5 mmol/L
MgCl2 and 8 mmol/L EGTA, followed by extraction of the
ionophore with BSA. This treatment reverses the exposure of
PS.31 The percentage of PS-exposing cells in erythrocyte
samples was measured by annexin V-FITC labeling, using flow cytometry
on a Becton Dickinson FACScan.4
Phospholipid vesicles
Phosphatidylcholine (PC) from egg yolk and phosphatidylserine (PS)
from bovine brain were mixed, in a 4:1 molar ratio in
chloroform/methanol 2/1, dried under nitrogen and resuspended in HBSS
to produce liposomes with a final concentration of 100 mmol/L lipid.
Alternatively, pure PC liposomes were made at the same final
concentration of phospholipid. Unilamellar vesicles with a mean
diameter of 100 nm were generated using extrusion.32 The
liposomes were passed 6 times through a Thermobarrel Extruder (Lipex
Biomembranes, Vancouver, BC) equipped with two 0.1 mm pore size
Nucleopore filters. NBD-labeled vesicles consisting of 10% NBD-labeled
PC and 90% PC, or 10% NBD-labeled PC, 40% PC and 50% PS, were
prepared as follows. The labeled and unlabeled phospholipids were mixed
in chloroform/methanol, dried under nitrogen, and resuspended in HBSS.
Subsequently, the lipid mixtures were sonicated at 4°C for 5 minutes at 30W using a microtip Branson sonifier (Branson Sonic Power,
Danbury, CT). The supernatant was used after centrifugation at
245 000g for 30 minutes.
RBC receptor labeling
To examine for coexpression of the cytoadherent receptors, CD36 or
band 3 peptide 3d, on PS-exposing red cells, PS-exposing RBCs, normal
RBCs, or sickle RBCs were resuspended to an hematocrit of 4% in PBS,
1% albumin (PBSA), and labeled with either 100 ng/mL OKM5 (MAb to
CD36) or 1 mg/mL 1F4 (MAb to band 3 peptide 3d) for 45 minutes. at
4°C, and subsequently with goat antimouse secondary antibody
conjugated to phycoerythrin for flow cytometric analysis.
Endothelial cell culture
Umbilical cords were obtained from anonymous donors at the Labor and
Delivery Unit of Alta Bates Medical Center (Berkeley, CA) or San
Francisco General Hospital (San Francisco, CA) with approval from the
respective Human Research Committees. Human umbilical vein endothelial
cells (HUVECs) were isolated within 3 days of cord collection,
according to the method of Jaffe et al33 with the following
modifications. Enzymatic digestion to free the HUVECs was for 10 minutes at 37°C with 0.15% dispase in M199 medium. Cells were
grown at 37°C in 5% CO2 / 95% air in gelatin-coated
flasks in EGM. Cobblestone morphology and positive staining by an
antibody to von Willebrand factor identified the resulting culture as
ECs. At 85% confluence, the cells were subcultured to
fibronectin-coated 8-chambered slides. The second and third passages
were used for adherence assays no later than 24 hours after HUVECs had
reached confluence.
Matrix exposure
To generate exposure of the matrix, HUVECs were grown to confluence
in chambered slides as described previously, and treated for 5 minutes
with either 10 mmol/L histamine or 0.1 NIH U/mL thrombin, or for 10 seconds with 0.5 mmol/L EDTA in HBSS, 1% BSA, 50 mmol/L HEPES pH 7.4 (HAH). Similar incubation times of HUVECs with HAH served as control.
Microscopic observation of gap formation was used to characterize the
retraction of the ECs and the exposure of the matrix. The HUVEC
monolayers were washed with HAH and subsequently tested for their
ability to bind RBCs.
To generate a surface completely devoid of endothelial cells, but with
the matrix still present, HUVEC monolayers were washed with PBS and
incubated in a modification of a buffer described by Wu et
al,34 0.01 mol/L phosphate, 0.15 mol/L NaCl, 5 mmol/L NaHCO3, 10 mmol/L EDTA, 0.1% BSA, 1 mmol/L
phenylmethylsulfonyl fluoride, pH 7.2. After 30 minutes at 37°C,
ECs were gently washed away with HAH.
TSP exposure
To visualize the exposure of TSP in the matrix, histamine-treated
HUVEC monolayers were fixed for 10 minutes with 5.7% paraformaldehyde in PBS, washed twice with PBSA, and incubated for 1 hour at RT in PBSA.
The cells were incubated for 3 hours at room temperature with
saturating solutions of polyclonal antibodies to TSP. After three
5-minute washes in PBSA with vigorous shaking, the cells were incubated
with biotinylated antirabbit IgG for 45 minutes. The monolayers were
washed again 3 times as above and subsequently incubated with Texas
red-conjugated streptavidin. Controls with primary antibody omitted
were included in each experiment. The slides were washed briefly with
PBSA, mounted with Vectashield mounting medium, and photographed at
magnification ×400 with a Zeiss Axiovert 135TV microscope (Carl
Zeiss, Inc, Thornwood, NY). A quantitative image processing system
based on the MicroImager 1400 digital camera (Xillis Technologies,
Vancouver, BC) and image analysis software, Xphoto (University of
California, Berkeley, CA), was used to acquire and analyze 170 µm
optical sections of cells.
Gravity adherence assays
RBC adherence to HUVECs was measured using a modification of the
methods described by Sugihara et al.18 Packed RBCs were resuspended to a hematocrit of 1% in HAH. Confluent HUVEC monolayers were washed with MAH to remove traces of serum, covered with RBC suspensions, and incubated at 37°C for 25 minutes. The wells were then filled completely with MAH, sealed with packing tape, and inverted
at 37°C for 20 minutes. While still inverted, the well walls and
gaskets of the slide chambers were removed. The slides were rinsed in
HBSS under standard conditions to remove nonadherent RBCs, fixed in 3%
glutaraldehyde in PBS, stained, and mounted. RBC adherence was
monitored visually by microscopy. RBCs adherent to HUVEC monolayers in
15 fields marked by a grid (same random fields for each sample) were
counted at magnification ×200 and the mean adherence and variance
(n = 15) were calculated. The mean of the means and standard error of
the means from at least 4 experiments, analyzed in parallel, are
plotted as ratios of test relative to control. Absolute values were
analyzed for statistical significance by the Student t test.
To evaluate the effect of different components on the adherence of
PS-exposing cells to endothelium, RBC suspensions were incubated for 5 minutes with 50 mg/mL 3d peptide or 50 mg/mL 3dS (band 3 control
peptide) or with 10 mg/mL annexin V in MAH. Alternatively, RBCs were
incubated for 5 minutes in HBSS with phospholipid vesicles to a final
concentration of 4.8 mol/L phospholipid before addition of the
RBC/vesicle mixture to the endothelial cells. The vesicles were
composed of either egg phosphatidylcholine (PC) or a mixture of egg PC
and bovine brain PS at a molar ratio of 4:1 (PC:PS).
RBC adherence to immobilized proteins
Flow adherence assays were performed according to the method
described by Barabino et al.29 In short, 2.5 µg purified
proteins were immobilized on a glass plate at 37°C for
1 hour. After washing each slide with HBSS, a parallel-plate flow
chamber was mounted on the glass slide using vacuum to maintain the
assembly. RBC suspensions of 1% Hct, sustained at 37°C, were drawn
over the slide using a syringe pump (Harvard Apparatus, South Natick,
MA) at a controlled flow rate to give a venular wall shear stress of 1 dyne/cm2. Adherent RBCs were visualized using an
inverted-phase contrast microscope (DIAPHOT-TMD, Nikon, Garden City,
NY) equipped with a charge-coupled device (CCD) video
camera (Model 72, Dage-MTI, Michigan City, IN) All experiments were
recorded. For each experiment, the protein layer was washed for 2 minutes with HBSS, followed by a 10-minute perfusion with the RBC
suspension. The number of adherent RBCs remaining after a 10-minute
rinse period were counted in a minimum of 12 fields. The mean of the
means and standard error of the means from at least 4 experiments,
analyzed in parallel, are plotted as ratios relative to control RBCs to
BSA. Statistical comparison of absolute adherence values between
PS-exposing and normal RBCs to each of the proteins was by the Student
t test.
Phospholipid vesicle adherence to TSP
Confluent EC monolayers were washed and treated with 0.01 mol/L
phosphate, 0.15 mol/L NaCl, 5 mmol/L NaHCO3, 10 mmol/L
EDTA, 0.1% BSA, 1 mmol/L phenylmethylsulfonyl fluoride, pH
7.234 to detach the ECs. The matrix was then incubated with
fluorescent phospholipid vesicles in HBSS, 50 mmol/L HEPES, pH 7.4 in
the presence or absence of 0.5 mmol/L CaCl2, for 3 hours at
4°C on a rotator. Nonadherent vesicles were washed off with HAH,
and the slides were mounted with Vectashield mounting medium. Vesicle adherence to TSP was monitored by fluorescence microscopy at
magnification ×400.
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Results |
To investigate the adherence of ionophore-treated PS-exposing RBCs
to HUVEC monolayers, we generated mixtures of PS-exposing RBCs and
control RBCs as described in "Materials and Methods." The number
of PS-exposing cells in these mixtures was determined by annexin V
labeling and flow cytometry, and the RBC mixtures were tested for
adherence to confluent HUVEC monolayers by our static adherence assay.
The correlation between the number of adherent RBCs in these mixtures
relative to control and the percentage PS-exposing RBCs in the
population is shown in Figure 1. The linear correlation (r = .94, P = .001) shows a strong
dependence of RBC adherence on the percentage of calcium
ionophore-induced PS-exposing cells in the suspensions. Interestingly,
the incubation of confluent HUVEC monolayers with these RBC mixtures
resulted in a mild retraction of the ECs, which was more pronounced as
the number of PS-exposing cells increased. Figure
2 shows a typical result of the adherence of RBCs from a mixture that contained 40% PS-exposing cells. The adherence of RBCs seemed to occur preferentially at the edges of the
endothelial cells and in the gaps between cells. Although gap formation
was less pronounced when the number of PS-exposing cells in the
population decreased, RBCs were preferentially found at the edges of
the ECs in all mixtures tested. For all subsequent experiments, we used
mixtures of RBCs with 5% to 10% PS-exposing cells.
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| Fig 1.
Correlation of PS exposure with adherence to EC
monolayers.
Ionophore-treated RBCs were mixed at varying proportions with untreated
RBCs. The percentage of PS-exposing RBCs was measured by FACS analysis
of annexin binding. Adherence to HUVECs was by a static assay. The
coefficients of the linear regression are r = 0.94,
P = .001, n = 4.
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| Fig 2.
HUVEC monolayers incubated with normal or PS-exposing
RBCs.
(A) Normal RBCs. (B) PS-exposing RBCs. EC retraction was caused by an
incubation with an RBC population containing 40% PS-exposing cells.
Images were obtained at a magnification ×400; the final scale is
indicated.
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Because ionophore treatment will induce many changes in the red cell
membrane, the role of PS needed to be confirmed. Since annexin V binds
to PS-exposing RBCs, we incubated mixtures containing 5% to 10%
PS-exposing RBCs with annexin V before the static adherence assay.
Pretreatment of PS-exposing RBCs with annexin V significantly reduced
adherence to ECs by 55% (P = .03, n = 6, Figure
3), indicating that annexin V shields PS on
the RBCs from interacting with the HUVEC monolayer. We hypothesized
that, if PS on the surface of the RBCs served as a recognition site for
the HUVEC monolayer, the presence of phospholipid vesicles that
contained PS would interfere with this recognition. Figure 3 shows that
the presence of phospholipid vesicles that contain 80% PC and 20% PS
significantly blocked adherence by 73% (P = .007, n = 5)
compared with control experiments with vesicles that contain PC only.
We also tested for the effect of these components on the adherence of
sickle RBCs to HUVEC monolayers. The sickle cell samples contained up to 5% PS-exposing cells, and adherence was similarly and significantly reduced by the presence of PC/PS vesicles and annexin V. The presence of PC vesicles had no effect on adherence (data not shown).
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| Fig 3.
Effect of blocking agents on PS-exposing RBC adherence to
EC monolayers.
PS-exposing RBC suspensions were incubated for 5 minutes with 1 of the
following before layering over ECs in a static assay: buffer (Control),
annexin V, PS vesicles (20% PS, 80% PC, 5 mmol/L lipid), PC vesicles
(100% PC, 5 mmol/L lipid). The mean of the means and standard error of
the means of 5 experiments are plotted as percentage of the
buffer-treated control adherence, which is set to 100%. *
Indicates significant differences from the control.
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It has been reported that PS exposure in ionophore-treated RBCs can be
reversed without changing other effects that the ionophore treatment
has on cell size, shape, and surface qualities.31 Following
this method, ionophore-treated RBCs were incubated with MgCl2
and EGTA before removal of the ionophore with BSA. This resulted
in a 76% decrease of PS-exposing cells from an RBC population that
originally contained 12% PS-exposing cells (P = .01,
n = 3, Figure 4A). This dramatic decrease
in PS exposure paralleled a 76% decrease in adherence, from
89.7 ± 15.0 (SEM) to 21.2 ± 9.5 RBCs/mm2,
(P = .04, n = 3, Figure 4B), suggesting that the reversal
of PS exposure was an important factor in the decrease of adherence. Taken together, these data strongly suggest a role for PS on the surface of the RBCs in the recognition by and adherence to HUVEC monolayers.
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| Fig 4.
Reversing PS exposure on ionophore-treated RBCs reduces
RBC adherence to ECs.
Percentage annexin V positive RBC (A) and static RBC adherence to
HUVECs (B) were measured using ionophore-treated (PS), or
ionophore-treated and reversed RBCs (PSR). * indicates a significant
reduction by the Student t test (n = 3). Error bars
depict the standard error.
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Other factors may also play a role in adherence of these cells, in part
indicated by the inability to completely block adherence under
conditions that decrease the availability of PS, as described previously. We investigated potential ionophore treatment-induced exposure of the known cytoadhesive molecules, CD36 and the band 3 peptide 3d. Normal RBCs were used as negative control, and because CD36
is expressed on sickle reticulocytes, we used a sickle RBC sample with
a high reticulocyte count as positive control. RBCs were labeled with
MAb OKM5 to CD36, to test for the presence of this cell surface
molecule. Figure 5 shows that CD36 was
present in the sickle RBC samples only. No CD36 could be observed on
either control RBCs or ionophore-treated, PS-exposing RBCs. Similarly, 1F4 prepared against peptide 3d (residues 547-553 of band 3) was used
to test for the presence of this cryptic band 3 site on
ionophore-treated cells. Fluorescent labeling of band 3 occurred only
in the ionophore-treated, PS-exposing RBCs, and not on the sickle RBCs
or control RBCs (Figure 5). Importantly, no nonspecific fluorescent
labeling was observed when cells were labeled with secondary
phycoerythrin-conjugated antibody only. These data suggest some
relationship between the expression of the band 3 cytoadherence peptide
and PS exposure on ionophore-treated RBCs. No indication as such could
be found for CD36.
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| Fig 5.
Exposure of PS, CD36, and band 3 peptide 3d on normal-,
PS-exposing-, or sickle RBCs.
Normal untreated RBCs (Normal), RBCs treated with 0.5 mmol/L
CaCl2, and 2 µmol/L A23187 (PS), or sickle RBCs (SS) were
labeled with annexin V-FITC (solid bars), or with MAb OKM5 against CD36
(striped bars) or 1F4 against band 3 peptide 3d (shaded bars) and
subsequently with antimouse/PE, and analyzed by FACS. The percentage of
cells in the population positive for either of these surface markers
are indicated as percentage of gated events. *Indicates significant
differences by the Student t test from normal RBC labeling.
Error bars depict the standard error; n = 4.
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To evaluate the role of band 3 in the adherence of ionophore-treated
PS-exposing RBCs, we investigated whether the 3d peptide could block
adherence of ionophore-treated PS-exposing RBCs to HUVEC monolayers. A
similar peptide, 3dS, consisting of the same amino acids as 3d but in
scrambled order was used as control. The 3dS peptide had no effect on
adherence in any experiment. The potential blocking effect of the 3d
peptide differed greatly from experiment to experiment. Of 6 independent experiments, blocking was observed in 3 cases with a
maximum in 1 case of 60% decrease in adherence; however, in the other
3 experiments no effect was observed. These data do not indicate a
clear role for band 3 in the adherence of ionophore-treated PS-exposing
cells to HUVEC monolayers. We repeated these experiments using RBCs
from sickle cell patients that contained PS-exposing cells with similar
results. The 3dS control peptides had no effect, whereas adherence was slightly but inconsistently reduced in some cases by 3d peptides (data
not shown).
These results indicated a role for PS in the adherence of RBCs to HUVEC
monolayers, and given the preferential binding to the edges of the
cells and gaps between cells, suggested the importance of the
endothelial matrix in the binding. We therefore decided to evaluate the
adherence of PS-exposing RBCs to purified adhesive molecules of the
endothelial matrix, TSP, and fibronectin. Purified proteins were
immobilized on glass slides and tested for their ability to support RBC
adherence under dynamic flow conditions (1 dyne/cm2). The
results shown in Figure 6 were expressed
relative to the adherence of normal RBCs to immobilized BSA, set
arbitrarily to 1. Normal RBCs adhered only slightly to BSA and
fibronectin, and significantly more so to TSP (201 ± 44,
317 ± 128, and 1822 ± 157 RBCs/mm2,
respectively). PS-exposing RBCs adherence was significantly greater to
BSA (405 ± 61 RBCs/mm2, P = .01) and to TSP
(2895 ± 192 RBCs/mm2, P = .005) than normal
RBC adherence to these proteins, and binding to fibronectin was
inconsistent as indicated by the relatively large standard error. These
results suggested that TSP may be an important factor in the adherence
of PS-exposing cells to the edges of ECs and in the gaps of HUVEC
monolayers.
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| Fig 6.
Adherence by shear flow of normal- or PS-RBCs to
immobilized matrix proteins.
Purified BSA, fibronectin, or thrombospondin were immobilized onto
glass slides for 1 hour before being exposed to normal (solid
bars) or PS-exposing (shaded bars) RBCs at 1 dyne/cm2 in a
dynamic flow assay. RBC adherence is reported as a ratio, relative to
normal RBC adherence to BSA, which was arbitrarily set to 1. Error bars
represent standard error of the means calculated from the
absolute values and normalized relative to normal RBC adherence to BSA.
*Indicates significant differences between normal- and PS-exposing RBC
adherence to BSA (P = .01, n = 5) and between normal- and
PS-exposing RBC adherence to TSP (P = .005, n = 5).
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To test whether PS-exposing RBC adherence would be further enhanced by
increasing the TSP exposure, we treated ECs with agents to cause the
cells to retract, as indicated in "Materials and Methods." Panels
A and B of Figure 7 show buffer- or
histamine-treated EC monolayers immunolabeled with polyclonal
antibodies to TSP. Panel A shows that in confluent monolayers, TSP is
entirely covered with ECs and unavailable to the antibody. In contrast,
histamine treatment resulted in abundant matrix TSP exposure (panel B). Similar results are found after treating ECs with thrombin or EDTA,
agents that also lead to retraction of endothelial cells (data not
shown). When ECs were pretreated with thrombin, histamine, or EDTA,
adherence of PS-exposing RBCs increased significantly, approximately
2-fold (Figure 7, panel C).
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| Fig 7.
Effect of EC retraction on matrix TSP exposure and
PS-exposing RBC adherence.
EC monolayers were pretreated with buffer (A) or histamine (B), labeled
with antibodies to TSP, and viewed by fluorescent microscopy.
Histamine-induced EC contraction exposed TSP (B), which is normally
cryptic (A). Pretreatment with thrombin (white bar), histamine (striped
bar), or EDTA (shaded bar) causes enhanced adherence of PS-exposing
RBCs (C). Adherence is reported relative to the adherence of the
untreated control (solid bar), which was arbitrarily set to 1. Error
bars represent standard error of the means calculated from the absolute
values from 4 experiments and normalized relative to the adherence of
the buffer-treated control. *Indicates significant differences between
PS-exposing RBC adherence to ECs treated with buffer, and thrombin,
histamine, or EDTA.
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When ECs were layered with PS-exposing RBCs in the presence of
polyclonal antibodies to TSP, the adherence was reduced significantly by 52% (P = .04, n = 4, Figure
8). Because anti-TSP did not completely block adherence, we also tested for an inhibiting effect of antibodies to its receptor, v3. PS-exposing RBC
adherence to ECs was reduced 50% (P = .05, n = 4) by L230
a monoclonal antibody to v3. Both antibodies together did not additionally block adherence, suggesting a
synergistic rather than an additive effect, and that the receptor-bound TSP conformer might be necessary to mediate PS-exposing RBC adherence to EC monolayers. Control antibodies had no effect on adherence.
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| Fig 8.
Effect of blocking antibodies on PS-exposing RBC
adherence to EC monolayers.
Confluent EC monolayers were washed and layered in a static assay with
PS-exposing RBC suspensions in the presence of 1 of the following:
polyclonal antibodies to CD59 or TSP, monoclonal antibodies to
v (L230), or a nonblocking monoclonal antibody
to 3 (AP3), or in buffer alone (control). Results are
reported as percentage of the control adherence, which is set to 100%.
Error bars represent standard error of the means calculated from the
absolute values from 4 experiments and normalized relative to the
adherence of the buffer-treated control. *Indicates significant
differences from the control by the Student t test.
|
|
To test for the interaction between PS and TSP in the matrix, we
prepared fluorescent phospholipid vesicles that contained 50% PS. Pure
PC vesicles served as control. The ECs were gently removed from the
matrix and the presence of TSP in the remaining surface was confirmed
by using TSP antibody and fluorescence microscopy. The EC-depleted
matrix was then incubated with lipid vesicles in the presence or
absence of calcium, and subsequently labeled for immunofluorescence by
polyclonal antibodies to TSP. Figure 9
shows that PS vesicles (A), but not PC vesicles (B), bound to matrix
proteins. Panels C and D are micrographs of the matrix incubated first
with PS vesicles and then with polyclonal antibodies to TSP. These 2 micrographs are of the same field showing that the PS vesicles (green
fluorescence, panel C) bound to TSP (red fluorescence, panel D).
Although much of the lipid vesicles in panel C had been
displaced by the antibody labeling (note the difference between panels
A and C), this data shows the interaction between PS vesicles and TSP,
which was not detected between PC vesicles and TSP. No difference in PS
vesicle binding to TSP was found due to the presence or absence of
calcium.
View larger version (182K):
[in this window]
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| Fig 9.
PS vesicles adhere to matrix TSP.
EC-depleted matrix was incubated with lipid vesicles and subsequently
labeled for immunofluorescence by polyclonal antibodies to TSP and
streptavidin-Texas Red. Panel A was labeled with PS vesicles; panel B
was labeled with PC vesicles. Panels C and D are of the same fields
labeled with both PS vesicles and TSP; C was photographed with
excitation for green fluorescence to detect the phospholipid labeling,
and D was photographed with excitation for red fluorescence to detect
TSP.
|
|
| |
Discussion |
Phospholipid asymmetry is well maintained in normal plasma
membranes, and the loss of this asymmetry with the exposure of PS at
the outer surface of the cell has significant physiologic consequences.1 The importance of PS availability at the
cell surface as a docking site for factors in the hemostatic system has
been very well characterized.2 In addition, it has become apparent that exposure of PS in the early stages of programmed cell
death is seminal for the recognition and removal of the apoptotic cell.35 Hence, the presence of PS on the surface may be a
trigger for cell-cell interaction, and in this case, RBC-endothelial interaction.
This interaction seems of particular physiologic consequence in those
disorders where PS exposure on RBCs as well as vascular damage or blood
flow complications are indicated. These conditions include sickle cell
disease, thalassemia, diabetes, and malaria. Subpopulations of
PS-exposing RBCs have been reported in patients with sickle cell and
thalassemia4-6 and a correlation was found between the risk
for stroke and PS exposure in sickle disease.8 Before the
presence of PS-exposing sickle cells in vivo was
established,4,5,36 it was suggested that the increased
propensity of sickle RBCs to adhere to the vascular wall was related to
the abnormal exposure of aminophospholipids in the external leaflet of
the RBC membrane.9 This was based on the loss of normal
membrane structure of sickle cells incubated under low oxygen tension
in vitro. A similar loss of phospholipid asymmetry under low oxygen
tension in vivo could in turn increase the interaction with endothelial
cells and play a role in vaso-occlusive crisis. We argued that sickling
was not necessary for this interaction between RBCs and endothelium,
given the presence of a subpopulation of PS exposing RBCs under normal oxygen tension. To test this hypothesis we evaluated the in vitro adherence of PS-exposing RBCs (obtained by calcium loading normal RBCs)
to endothelial monolayers.
Our data indicate not only a role for PS but also show the involvement
of factors in the matrix, in particular TSP, in the binding of
PS-exposing RBCs. The strong correlation between adherence and the
number of exposing PS RBCs supports the hypothesis made previously by
others9-11,37 that loss of membrane asymmetry may lead to
RBC-EC binding. A role for PS in this binding process is indicated by
the reduction of adherence when PS on the RBCs is blocked with annexin
V. In addition, a significant reduction in adherence was assessed by
incubation in a magnesium/EGTA environment, which reversed the PS
exposure in ionophore-treated RBCs.31 Moreover, the
presence of PS-containing vesicles competed with PS-exposing RBCs for
the apparent binding sites on the HUVEC monolayer. In these
experiments, it also became apparent that the adherence of RBCs
occurred preferentially at the edges of the endothelial cells in the
gaps between the cells. This localized interaction was confirmed by the
increase of adherence in these areas when endothelial cells were
retracted by other factors such as histamine, thrombin, or EDTA before
addition of PS-exposing RBCs. Taken together, these data strongly
suggest a role for PS in the adherence of PS-exposing RBCs to (damaged) endothelium.
Our data do not exclude that other factors may be additionally
important for binding of RBCs to EC monolayers. In particular, RBC
binding to sites of exposed TSP may point at factors such as CD36 or
band 3 peptide 3d. The thrombospondin receptor CD36, found
predominantly on reticulocytes, has been shown to mediate sickle RBC
adherence to ECs.18 However, we did not find the appearance
of CD36 on ionophore-treated RBCs. In Plasmodium
falciparum-infected RBCs, the principal integral protein of the
membrane, band 3, aggregates in the membrane bilayer. Structural
modifications occur and normally cryptic residues become exposed.
Peptides designed to the exposed epitopes of band 3 block adherence of
malaria infected RBCs to ECs.38 Clustering of band 3 also
occurs in sickle RBCs and the same synthetic 3d peptides were reported
to block adherence of sickle- and calcium-loaded RBCs.11,25
Antibody binding to ionophore-treated RBCs indeed confirmed the
presence of this site on PS-exposing RBCs. Under our conditions,
however, the addition of the synthetic 3d peptide seemed to reduce
adherence of ionophore-treated cells only in some cases, with rather
inconsistent results. Although our data do not exclude a role for other
factors such as band 3, they indicate a major role for PS in the
adherence of PS-exposing cells to HUVEC monolayers, and in particular
to factors in the matrix.
The role of matrix TSP was suggested by the binding of PS-exposing RBCs
to immobilized purified TSP. The presence of antibodies to TSP and its
receptor v3, decreased PS-exposing RBC
adherence to HUVEC monolayers. And the presence of PS in pure lipid
vesicles led to the binding of these vesicles to EC denuded matrix.
These data indicate a direct interaction between PS-containing lipid surfaces and TSP. Such an interaction of TSP and PS-exposing cells had
not been previously demonstrated. On the contrary, PS- and TSP-mediated
recognition of apoptotic neutrophils by macrophages has been shown to
be mutually exclusive.14 But, in the studies so far
undertaken on the recognition of PS-exposing cells, a bridging mechanism of soluble TSP was sought. Because the functional properties of TSP depend on its conformation, dictated by the particular environment in which it is found,21 the interaction between PS and TSP apparently necessitates binding properties characteristic of
immobilized TSP, either on glass or in the extracellular matrix. In
addition, the reduction in adhesion by both polyclonal antibodies against TSP and a monoclonal antibody against the active binding site
of v3 suggest that the interaction of
both components is important in the binding of PS-exposing RBCs to
matrix TSP. This may explain the preferential binding of the RBCs at
the edges of the retracted ECs.
This finding seems relevant in pathologies in which vascular injury is
prevalent and in which a subpopulation of PS-exposing RBCs is a
characteristic, such as sickle cell disease. A number of studies
indicate endothelial damage in this disease. Circulating ECs have been
observed,39 suggesting vascular injury and matrix exposure.
Abnormal levels of inflammatory agents, interleukin-1 (IL-1), TNF,
thrombin, or histamine are often found in patients with sickle cell
disease.22,40 In vitro, these agents cause EC activation,
barrier dysfunction due to EC retraction, and increased RBC
adherence,17,41-43 presumably to the exposed matrix. Sickle RBC adherence to matrix components has been studied by dynamic flow of
RBCs over immobilized purified proteins. A role for von Willebrand
factor, laminin, and collagen I is supported by these works.28,29,44-47 Moreover, sickle RBCs adhere
unequivocally to immobilized TSP,29,30,48 an interaction
influenced by fibronectin, von Willebrand factor and anionic
polysaccharides,24 and recently, a site that binds sickle
RBCs has been mapped within the C-terminal cell-binding domain of
TSP.48
Our studies suggest that the PS-exposing subpopulation of cells may
play an additional important role in adherence of RBCs by causing EC
retraction. The mechanism by which PS-exposing RBCs cause barrier
dysfunction can only be speculated. One option is the fact that
ionophore treatment results in the release of lipid breakdown products
that modulate EC barrier properties. RBCs contain a phospholipase D,
that is activated under conditions in which PS is exposed on the
surface of the cell generating phosphatidic acid,49 and it
has recently been reported that phosphatidic acid disrupts barrier
integrity.50 Although it may be tempting to suggest a role
of phosphatidic acid in EC activation, this remains to be proven.
In conclusion, our data indicate a role for PS on the surface of the
RBCs in adherence to endothelial cell monolayers. This interaction
preferentially occurs in areas where the normal confluent cell
monolayer is disrupted, involves matrix proteins such as thrombospondin, and is relevant in pathologies in which vascular injury
occurs in the presence of subpopulations of PSexposing RBCs.
| |
Acknowledgments |
We would like to thank Dr Narla Mohandas, Dr Dan Callahan, and Kevin
Benson for their assistance with fluorescence microscopy, and Eileen
Finnegan for technical assistance.
| |
Footnotes |
Submitted May 10, 1999; accepted October 5, 1999.
Sponsored by grants no. HL55213, DK32094, HL20985, and
M01RR01271 from the National Institute of Health.
Reprints: Annamaria Manodori, Children's Hospital Oakland
Research Institute, 5700 Martin Luther King Jr Way, Oakland, CA 94609.
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.
| |
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Top
Abstract
Introduction