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Prepublished online as a Blood First Edition Paper on November 14, 2002; DOI 10.1182/blood-2002-07-2016.
RED CELLS
From the Microbiology and Tumor Biology Center
(MTC), Karolinska Institutet, Stockholm, Sweden; the
Swedish Institute for Infectious Disease Control (SMI), Stockholm,
Sweden; the Department of Biochemistry, University of
Makerere, Kampala, Uganda; and the Department of Medical Biochemistry
and Microbiology, Biomedical Center, Uppsala University, Uppsala,
Sweden.
Plasmodium falciparum may cause severe forms of malaria
when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates
of P falciparum-infected erythrocytes (parasitized red
blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such
as heparin has been identified as a marker for severe disease. Here we
report that pRBCs of the parasite FCR3S1.2 and wild-type clinical
isolates from Uganda adhere to heparan sulfate (HS) on endothelial
cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by
HS/heparin or enzymes that remove HS from cell surfaces.
35S-labeled HS extracted from HUVECs bound directly
to the pRBCs' membrane. Using recombinant proteins corresponding to
the different domains of P falciparum erythrocyte
membrane protein 1 (PfEMP1), we identified Duffy-binding-like
domain-1 Mature trophozoites, the later
intraerythrocytic form of Plasmodium falciparum, are bound
in the deep microvasculature, primarily to endothelial cells
(cytoadherence) and to erythrocytes (rosetting), whereas ring-stage
trophozoites circulate in the peripheral blood. The sequestration of
parasitized red blood cells (pRBCs) and RBCs is suggested to be
mediated by P falciparum erythrocyte membrane protein 1 (PfEMP1), a parasite-derived polypeptide expressed at the surface of
the infected pRBCs. The resulting binding of pRBCs in the inner
organs1 helps the parasite to hide from the immune system
and withdraw from splenic clearance. Yet binding of the pRBCs may be
lethal to the parasite because excessive sequestration may cause severe
malaria and the death of its human host.
PfEMP1 is a high-molecular weight (200 to 350 kDa) transmembrane
polypeptide consisting of 4 to 7 extracellular domains encoded by the var gene family.2,3 PfEMP1 mediates the
sequestration of pRBCs through interactions with receptors such as
CD36, platelet endothelial cell adhesion molecule-1
(PECAM-1)/CD31, intercellular adhesion molecule-1
(ICAM-1), immunoglobulin G (IgG), and
IgM.4-12 Lectinlike interactions13 of PfEMP1
have been described with glycans such as the blood group A
antigen14,15; heparan sulfate (HS)-like
glycosaminoglycans (GAGs) present on uninfected
erythrocytes16,17; and chondroitin sulfate (CS), a
galactosaminoglycan. Indeed, CS of the A type (CSA), containing
4-O-sulfated glucosamine units, has been found to act as a
receptor for pRBC binding in the placenta18 and in the
microvasculature.19
GAGs are long, linear carbohydrate chains attached to a core protein,
forming a proteoglycan. HS is one such GAG, composed of alternating
glucosamine and uronic acid residues in a repeating disaccharide unit
(-4GlcA About 50% of rosettes formed by cultured strains or fresh isolates
have been found to be sensitive to the disruption by HS and
heparin.13,16,17 Furthermore, a heparinase III-sensitive receptor has been suggested to be involved in the formation of rosettes.16 The capacity to bind heparin to the pRBC
surface has also been found to be relatively common among clinical
samples and associated with the severity of disease.23
Duffy-binding-like domain-1 Parasites
The P falciparum clinical isolates U10, U11-1, U11-2, U14,
U15, U18, U22-1, U22-2, and U26 were obtained from venous blood sample
taken from malaria patients in Kampala, Uganda. In all cases,
informed consent was obtained from the patients and/or their parents.
The erythrocytes were immediately separated from mononuclear cells on
Polymorphprep (Axis-Shield, Oslo, Norway) and washed in RPMI
1640. Nonimmune AB Rh+ serum to a final hematocrit
of 40% and freezing media (28% glycerol, 3% sorbitol, 0.65%
NaCl) in a 1:1 ratio were added before freezing the cells
in liquid N2. For the assays, the freshly frozen isolates were thawed and cultured in malaria culture medium in their own blood
containing 15% human serum (blood group AB Rh+) until
they had matured into trophozoites.26
Cells
Polysaccharides GAGs were radiolabeled by N-3H-acetylating free amino groups to a specific activity of 125 000 cpm/µg, 70 000 cpm/µg, and 475 000 cpm/µg for bovine lung heparin, swine liver HS, and swine intestine HS, respectively, as described.29 Selective chemical modification of bovine lung heparin was prepared as described previously.29,30 The proteoglycan pool from HUVECs was purified from Na235SO4 (NEN)-metabolically labeled cells essentially as described.31 To isolate the GAGs HS and CS from the intact proteoglycan, a proteolytic digestion of the core proteins with pronase E (0.3 mg/mg protein) was performed. To purify HS from the GAG pool, CS was cleaved off with chondroitinase ABC followed by anion-exchange chromatography as described.31 CS was isolated by treating the GAG pool with nitrous acid at pH 1.5 to cleave HS, followed by separation on anion-exchange chromatography. Heparin, HS, and CSA from porcine intestine used in rosetting, cytoadherence, and immunofluorescence assays were obtained from Løvens Kemiske Fabrik (Ballerup, Denmark). Bovine lung heparin was a gift from Pharmacia (Kalamazoo, MI) and was purified as described.32 HS from swine liver and swine intestine was purified as previously described.20Expression of recombinant PfEMP1 domains Gene constructs encoding 3 PfEMP1 domains (DBL1 ,
cysteine-rich interdomain region 1 [CIDR1], and
DBL2 ) of FCR3S1.2 were expressed as glutathione
S-transferase (GST) fusion proteins and purified as
described.16 Briefly, the pGEX-4T-1 system was used, and
individual fragments were inserted downstream of a GST sequence. The
proteins were expressed in Escherichia coli (BL21) and
induced with 0.1 mM isopropyl- -D-thiogalactoside, following a 4-hour incubation at 30°C. The fusion proteins were purified on
glutathione-sepharose according to the manufacturer (GST Gene Fusion
System; Pharmacia-Upjohn, Uppsala, Sweden). The fusion proteins were
extensively dialyzed against PBS before use.
Cytoadherence assay The cytoadherence assay was performed as described27 with some modifications. Briefly, HUVECs or HLECs were seeded out onto gelatin-treated Thermion coverslips (13 mm2) (Nunc, Labassco, Sweden). The cells were seeded at a density of 30 000 cells per well and incubated at 37°C for 24 to 48 hours before use to allow for attachment. Then, pRBCs freed of rosettes by mechanical disruption with a syringe were resuspended in binding medium (RPMI 1640; 25 mM HEPES, pH 6.8; 25 µg/mL gentamicin) with or without 10% human serum and added to the RPMI 1640-prewashed target cells. The cultures were allowed to incubate for 1 hour with intermediate resuspension of sedimented erythrocytes every 15 minutes. After incubation, unbound pRBCs were removed by washes in binding medium, and the remaining cells were fixed with 1% glutaraldehyde in PBS for 1 hour at room temperature. The cells were stained with 1% Giemsa for 1 hour at room temperature. To estimate the number of bound pRBCs per cell, 300 to 500 endothelial cells were counted by means of light microscopy (Optiphot-2; Nikon, Tokyo, Japan) at magnification × 1000.Inhibition of adhesion was tested by addition of different GAGs (HS,
heparin, and CSA at 0.01 to 10 mg/mL); antibodies (antihuman PECAM-1/CD31 monoclonal antibodies, and clone JC/70A [DAKO,
Copenhagen, Denmark] at 0.001 to 10 µg/mL); or proteins (DBL1 Rosette disruption assay The assay was performed essentially as described.27,33 Briefly, GAGs (0.001 to 1 mg/mL) were added to 25 µL aliquots of a rosetting FCR3S1.2 culture in a microtiter plate. The mixture was incubated for 30 minutes at 37°C. The rosetting rate was subsequently estimated after staining with acridine orange and compared with mock-treated controls. The level of rosetting was expressed as the number of rosette-forming late-stage pRBCs per total number of late-stage pRBCs × 100.Immunofluorescence HUVECs or HLECs were detached from the culture flasks with a "rubber policeman" and resuspended in PBS to a cell density of 2 to 4 × 106. HUVECs were used with or without 0.3% bovine serum albumin (BSA). Fusion proteins, consisting of GST-DBL1, GST-CIDR1, or GST alone, were added at 50, 100, or 200 µg/mL to the cells and incubated for 1 hour. The cells
were washed 3 times in PBS, and the binding of the fusion proteins was
visualized by incubating the cells with mouse anti-GST monoclonal
antibody (clone GST-2, IgG2b, diluted 1:100) (Sigma) following
incubation with fluorescein isothiocyanate (FITC)-conjugated
antimouse immunoglobulin antibody (diluted 1:20) (DAKO). All
incubations were for 1 hour at room temperature and with slow rotation.
Surface fluorescence was studied in incident ultraviolet (UV)
light by means of a Nikon Optiphot-2 microscope. In some of the
experiments, 1 mg/mL HS was added together with the ligand, or the
cells were treated with 10 µg/mL anti-PECAM-1/CD31 antibodies or
with 0.2 IU/mL heparinase III.
Binding of GAGs to pRBCs Trophozoite-infected erythrocytes (2 × 108) were incubated with 0.01 nM [3H]HS or 3000 cpm [35S]HS and [35S]CS in PBS for 1 hour at room temperature with intermediate resuspension every 15 minutes. After 3 washes in PBS, the cells were lysed in 1 mM Tris (tris(hydroxymethyl)aminomethane), pH 7.4, and 0.1 mM EDTA (ethylenediaminetetraacetic acid). Samples were centrifuged, and membranes were separated from the supernatant. Radioactivity bound to the membranes was analyzed by liquid scintillation counting.Binding of GAGs to recombinant PfEMP1 domains DBL1-GST, CIDR1-GST, DBL2-GST, or GST alone was coupled
to a 1 mL NHS-Hi-trap-column (Pharmacia-Upjohn) as suggested by the
manufacturer. Radiolabeled [35S]HS and
[35S]CS, purified from HUVECs, or
[3H]heparin purified from bovine lung were added to the
column equilibrated in Tris-buffered saline (TBS) (50 mM Tris,
pH 7.4; 150 mM NaCl) and incubated for 20 minutes. Unbound material was
washed out with 5 mL TBS. Elution of bound GAGs was performed with a
stepwise gradient of 0.2 to 2 M NaCl in TBS. Fractions were analyzed
for radioactivity by liquid scintillation counting.
pRBCs bind to HS available on the endothelial cell surface To evaluate the function of GAGs as receptors for pRBCs on human endothelial cells, FCR3S1.2 pRBC adhesion to HUVECs and HLECs was studied as described. An average of 487 ± 44 FCR3S1.2 pRBCs bound per 100 HUVECs and 342 ± 62 FCR3S.2 pRBCs bound per 100 HLECs, as estimated from 3 individual experiments (Figure 1A-B). When the HUVECs were pretreated with heparinase III to remove HS from the cell surface, adhesion decreased by more than 70% (73.5%) (Figure 1C), and an average of 129 ± 37 pRBCs bound per 100 HUVECs. Adhesion was not affected when HUVECs were pretreated with neuraminidase, chondroitinase ABC, or hyaluronidase (Figure 1C), indicating that neither sialic acid, CS, nor hyaluronan participated in the binding of infected erythrocytes to the cells. The binding capacity to HLECs decreased by more the 40% upon heparinase III treatment, and an average of 189 ± 56 pRBCs bound per 100 HLECs (44%; data not shown).
To confirm that the use of an HS receptor is true not only for the
cloned parasite FCR3S1.2, wild-type clinical isolates from Uganda were
analyzed. Of 9 isolates, 8 grow into mature trophozoites, with
parasitemia ranging between 2.2% and 7.2%. These were tested for
binding to HUVECs. Four of these 8 isolates, U10, U11-2, U14, and U18,
showed adhesion over 100 pRBCs per 100 cells (140, 207, 101, and 232 pRBCs per 100 cells, respectively) (Table
1). These were considered to be binders
to HUVECs and are discussed further. Isolates generating lower
cytoadherence than 100 pRBCs per 100 cells were considered to
be nonbinders to HUVECs in this study (U11-1, U15, U22-1, and U22-2
with 51, 11, 6, and 38 pRBCs per 100 cells, respectively). After
heparinase III treatment of the target cells, U10, U11-2, U14, and U18
decreased in binding by 44, 29, 94, and 10%, respectively (79, 147, 6, and 209 pRBCs per 100 cells, respectively) (Table 1).
HS blocks the adhesion of pRBCs to endothelial cells In a complementary assay, we tested whether soluble GAGs could compete for the binding of pRBCs to endothelial cells. To estimate the competing effect of the different GAGs, HS, heparin, and CSA (0.1 to 10 mg/mL) were added together with the infected erythrocytes (FCR3S1.2) to HUVECs and HLECs in the cytoadherence assay. HS and heparin inhibited adhesion to HUVECs in a dose-dependent manner, whereas CSA had no effect even at the highest concentrations tested (Figure 2A). Cytoadherence of pRBCs to HLECs was also affected by the addition of the GAGs HS and heparin in a dose-dependent manner (Figure 2B). On HUVECs, HS was found to be more effective than heparin in the cytoadhesion assay with a 50% inhibitory concentration (IC50) of 0.3 mg/mL as compared with 1.2 mg/mL for heparin. The opposite was found on HLECs, on which heparin, with an IC50 of 1 mg/mL, was a more effective inhibitor than HS. The IC50 for HS could not be determined at the concentrations tested (Figure 1B). In parallel, the same GAG preparations were examined for inhibition of rosette formation (Figure 2C). For rosette disruption, heparin was a better inhibitor than HS. Heparin inhibited rosetting with an IC50 of 0.02 mg/mL, whereas HS did not completely inhibit rosetting even at the highest concentration tested.
The effect of GAGs on cytoadherence of wild-type clinical isolates from Uganda was analyzed on the isolates according to the same criteria (U-10, U11-2, U14, and U18). Isolates U10 and U14 decreased by 44% and 54% after adding 1 mg/mL HS and by 54% and 94% upon addition of 1 mg/mL heparin, respectively (Table 1). Heparin (1 mg/mL) affected cytoadhesion of sample U11-2 and binding decreased by 36%, whereas double HS concentration was needed to exert a modest effect. No loss of adhesion from the use of any of the GAGs was seen in sample U18. Two different domains of PfEMP1 mediate binding to HS and PECAM-1/CD31 Because PECAM-1/CD31 is a known receptor involved in cytoadherence of pRBCs and is expressed by HUVECs, we wanted to examine the relative involvement of PECAM-1/CD31 and HS as receptors for pRBCs of FCR3S1.2. The interaction between recombinant domains of PfEMP1 and the receptors when they are located on the human endothelial cell surface was studied. The 2 most N-terminal domains of PfEMP1var1, DBL1 and CIDR1, were tested
for binding to HUVECs by means of an immunofluorescence assay as
described. Recombinant GST-fusion proteins of DBL1 or CIDR1 were
incubated with HUVEC, and the adhesion was detected by means of a
fluorescent antibody to GST. Strong fluorescence was observed when
DBL1 and CIDR1 bound to normal HUVECs, whereas GST alone did not
bind to the cells (Figure 3A-B, F-G, and
data not shown). When the HUVECs were pretreated with heparinase III to
remove cell surface HS, no binding was detected with DBL1 (Figure
3C), whereas CIDR1 continued to bind well to the HS-free cells
(Figure 3H). In contrast, incubation of the cells with an
anti-PECAM-1/CD31 antibody blocking PECAM-1/CD31 on the cell surface
abolished binding of CIDR1 (Figure 3I) but not binding of DBL1
(Figure 3D). DBL1 also bound to HLECs in an HS-sensitive
manner (Figure 3K-M). With these results, we show for the first time
that DBL1 acts as a ligand to an endothelial receptor and add HS to
the receptor map of DBL1 for cytoadherence, but also confirm earlier
data with PECAM-1/CD31 as a CIDR receptor.15 The role of
HS as a receptor for DBL1, but not for CIDR1, could be further
confirmed by competition of adhesion with soluble HS (Figure 3E,J,
respectively). These findings together suggest that the 2 domains are
binding to distinct receptor molecules at the cell surface. On HUVECs,
the DBL1 domain binds to an HS receptor, and CIDR1 binds to
PECAM-1/CD31. No difference was seen in the absence or presence of
BSA.
The role of the 2 receptor molecules was subsequently tested in the
cell adhesion assay. When the endothelial cells were incubated with
anti-PECAM-1/CD31 antibodies (0.001 to 10 µg/mL), 100% inhibition of cytoadherence was reached at a concentration of 10 µg/mL antibody (Figure 4A). Total inhibition could be
achieved at a 100 × lower concentration of antibody when the cells
were pretreated with heparinase III (0.2 IU/mL) (Figure 4A).
Preincubation of HUVECs with recombinant GST-DBL1
35S-labeled human endothelial HS binds to infected
erythrocytes and to DBL1 In the next step, the purified [35S]HS and
[35S]CS fractions obtained from HUVECs were examined for
direct binding to either of the 3 different recombinant PfEMP1 domains
(DBL1
Adhesion of P falciparum-infected erythrocytes to
uninfected erythrocytes and to endothelial cells in deep
microvasculature is thought to play a major role in the fatal outcome
of severe malaria.1 Multiple receptors, which include both
proteins and carbohydrates, are known to be involved in
sequestration.1 Disruption of these interactions
with adhesion antagonists may therefore be one of the strategies to
treat severe malaria. Here, we demonstrate the involvement of human
endothelial HS as receptor for infected erythrocytes via its
interaction with the N-terminal domain DBL1 The present study was conducted to gather more insight into the
role of GAGs as potential endothelial receptors for cytoadherence because HS was suggested as a cytoadhesion receptor for pRBCs on the
basis of results obtained with an epithelial cell line (Chinese hamster
ovary [CHO] cells).15 We show for the first time that DBL1 Our finding of HS as an endothelial receptor for pRBCs may at first glance seem to be in conflict with previous work that suggested that HS does not influence cytoadherence.34 The authors of the previous work argued that an HS receptor could not be an endothelial receptor for pRBCs because heparinase-treated target cells did not show decreased cytoadherence. Their study used heparinase I, an enzyme that is specific for IdoA2S within an N-sulfated sequence as found in highly sulfated sequences of HS and heparin yet rare in HS of human endothelial cells.35 Heparinase I treatment will therefore leave the HS chains on endothelial cells only partially digested, probably with enough sites remaining on the cell surface for the parasite to attach. The parasite lines used were HB3EC-6 and HB3C32, both known to bind CD36 but of an unknown heparin- or HS-binding status. HS is expressed as protein-bound sugar chains in a tissue- and species-specific pattern, providing binding sites for a wealth of endogenous molecules32 and exogenous invaders such as protozoa, bacteria, or viruses.36 The biosynthetically controlled fine structure of HS creates a sophisticated tissue pattern as demonstrated by the tissue-staining pattern with the use of diverse anti-HS antibodies21,22,37,38 or growth factor molecules.39,40 GAGs such as heparin and CSA have previously been implicated in the reversion of sequestration in vitro and ex vivo by affecting either cytoadherence41,42 or rosetting13,16,23,25 of pRBCs. With this substantiation, it is important to investigate GAGs from different origins. Both HS and heparin were found to block cytoadherence to HUVECs and
HLECs. The higher concentrations of HS and heparin needed to affect
adhesion to HLECs as compared with HUVECs (Figure 2A-B) are most likely
due to the different receptor ensembles available on their cell
surfaces. For example, CD36, a receptor involved in
cytoadherence,5 is present on HLECs but is lacking on the HUVECs. HS was found to effectively block cytoadherence to HUVECs at an
approximately 10-fold lower concentration than in rosette formation,
whereas the opposite was true for heparin16 (Figure 2A,C).
This may suggest a distinct binding specificity of DBL1 PECAM-1/CD31, another receptor that contributes to cytoadherence of
FCR3S1.2,6 is present on HUVECs.44 In
agreement with previous results, CIDR1 Fresh wild-type clinical P falciparum isolates have
been demonstrated to bind to multiple receptors.23 The
PfEMP1var1 expressed by the parasite used in
this study, FCR3S1.2, has also been shown to mediate adherence to
multiple receptors15 and is therefore a good model
parasite for studying receptor recognition. The N-terminal head
structure (DBL1 Malaria parasites from patients are not clonal, yet a large number of patient blood samples contain GAG-binding parasites.17,23 As many as 50% of rosettes formed by fresh isolates have been shown to be sensitive to the disruption by HS and heparin.13,16,17 In another study using fresh wild-type isolates, it was found that more then 80% of the isolates tested (90 of 111 samples analyzed) bound to heparin, which could be associated to severity of disease.23 Clinical isolates in our study were analyzed for the use of HS receptor in cytoadherence. At least 2 of the 4 analyzed samples definitely contained parasites binding to HS for adhesion to HUVECs. For this reason, glycans are interesting candidates for the design of competitors against a broad range of strains also suggested by studies in which heparin was used as adjunct therapy in cerebral malaria.46,47 An improved understanding of the molecular mechanisms operative in sequestration may well provide a means to treat vascular clogging without affecting host tissues. The success of an antiadhesion drug therapy must be based on an action directed toward as many parasite determinants as possible. The finding that HS from different sources (highly sulfated from intestine, relatively poorly sulfated from endothelial cells) compete more potently than heparin in the cytoadhesion of infected erythrocytes opens an opportunity for selective design of competitors. Erythrocyte rosetting seems to have a preference for N-sulfated rather than O-sulfated groups, which differs from the specificity of many other endogenous and exogenous heparin/HS-binding molecules.17 These efforts should open novel possibilities in the development of carbohydrate-based drugs that could function as antiadhesive molecules in the treatment of malaria.
We thank Robert Kisilevsky for critical reading of the manuscript. Collecting of clinical isolates from Uganda by Berit Schmidt Aydin is very much appreciated.
Submitted July 5, 2002; accepted October 23, 2002.
Prepublished online as Blood First Edition Paper, November 14, 2002; DOI 10.1182/blood-2002-07-2016.
Supported by a grant from the program "Glycoconjugates in Biological Systems" (GLIBS) sponsored by the Swedish Foundation for Strategic Research, the Swedish Research Council, a grant from the European Commission (QLRT-PL-1999-30109), and the Swedish International Development Authority (Sida/SAREC).
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: Mats Wahlgren, Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Box 280, SE-171 77 Stockholm, Sweden; e-mail: mats.wahlgren{at}smi.ki.se.
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domain of PfEMP1
Top
Abstract
Introduction
),
chondroitinase ABC (
), hyaluronidase (
), or heparinase
III (
) for 2 hours before incubation with FCR3S1.2 for 1 hour
at 37°C. All samples are compared with controls in which pRBCs alone
were incubated with untreated HUVECs. All data are expressed as the
mean of 3 independent experiments ± SD.
) or after
pretreatment (
) was incubated with HUVECs for 1 hour before pRBCs were
added to HUVECs and allowed to bind for 1 hour. All data are expressed
as the mean of 3 independent experiments ± SD.
