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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 674-682
RED CELLS
Multiple human serum components act as bridging molecules in
rosette formation by Plasmodium falciparum-infected
erythrocytes
Elizabeth A. Somner,
Julie Black, and
Geoffrey Pasvol
From the Department of Infection and Tropical Medicine, Imperial
College School of Medicine, Middlesex, United Kingdom.
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Abstract |
Rosetting, the binding of parasitized erythrocytes to 2 or more
uninfected erythrocytes, is an in vitro correlate of disease severity
in Plasmodium falciparum malaria. Although cell ligands and
receptors have been identified and a role for immunoglobulin M has been
suggested, the molecular mechanisms of rosette formation are unknown.
The authors demonstrate unequivocally that rosette formation by P
falciparum-infected erythrocytes is specifically dependent on
human serum, and they propose that serum components act as
bridging molecules between the cell populations. Using heparin
treatment and Percoll density gradient centrifugation, they have
developed an assay in which parasitized erythrocytes grown in
serum-containing medium and optimally forming rosettes are stripped of
serum components. These infected cells were no longer able to form
rosettes when mixed with erythrocytes and incubated in serum-free
medium. Rosette formation was restored by the addition of serum or
certain serum fractions obtained by concanavalin A (conA) affinity,
anti-IgM affinity, anion exchange, and gel filtration chromatography.
The authors clearly demonstrate that multiple serum components IgM and
at least 2 othersare involved in rosette formation. Those others
consist of 1 or more acidic components of high-molecular mass that
binds to conA (but that is not thrombospondin, fibronectin, or von
Willebrand's factor) and of at least 1 more basic, smaller component
that does not bind to conA. Data on the size and number of
rosettes formed support the authors' hypothesis that multiple bridges
are involved in this complex cellular interaction. These findings have
important implications for the understanding of pathogenic adhesive
interactions of P falciparum and host susceptibility to severe malaria.
(Blood. 2000;95:674-682)
© 2000 by The American Society of Hematology.
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Introduction |
Plasmodium falciparum malaria is responsible
for approximately 2 million deaths annually, most of which occur among
children in sub-Saharan Africa. The devastating pathologic conditions
that result in severe malaria are caused by complex cellular and
molecular interactions between the human host and the parasite, after
parasite invasion of erythrocytes. Serious clinical complications occur when these parasitized red blood cells (PRBC) sequester in the microvasculature of a variety of organs1 as a result of
cytoadhesion and rosette formation. Cytoadhesion is a receptor-ligand
interaction between the parasite protein P falciparum
erythrocyte membrane protein 1 (PfEMP-1) and receptors on the
surface of endothelial cells,2 whereas rosetting is the
binding of PRBC to 2 or more uninfected red blood cells (RBC).
Rosetting, occurring in conjunction with cytoadhesion, is believed to
contribute to the development of cerebral
malaria.3,4
Investigations to elucidate the molecular basis of rosette formation
have been fueled by a number of studies demonstrating a correlation
between rosette formation and disease severity in Africa,5-7 though studies carried out in
Thailand8 and Papua New Guinea9 failed to show
such a correlation. The parasite molecules first implicated as
rosetting ligands were the low-molecular-mass polypeptides known as
rosettins.10,11 Proteins of approximately 22 kd were found
at the surface of 2 strains of rosetting parasites, whereas their
nonrosetting counterparts lacked these molecules, and antibodies to
these antigens were able to disrupt rosettes. However, the genes for
these proteins have not yet been cloned. Recently, 2 separate studies
using parasite clones R2912 and FCR3S1.213 have
identified PfEMP-1 as a parasite ligand involved in rosetting.
Various molecules on uninfected erythrocytes have also been implicated
as rosetting receptors. One study, using the Malayan Camp parasite,
showed that CD36 was the erythrocyte receptor for this parasite
isolate.14 Other studies using different parasites have
described the involvement of the ABH blood group
determinants.15 Recently, complement receptor 1 (CR1) has
been shown to be involved in the rosetting of a number of parasite
lines and clones.12 Another study, using parasite clone
FCR3S1.2, suggested that heparan sulfate is a rosetting
receptor,13 though its presence on the RBC surface is
unconfirmed. Overall, little is known about the precise molecular
mechanisms of interaction between infected and uninfected erythrocytes
of a rosette. The role that serum components could play, as bridging
molecules between the 2 cell populations (ie, as promoters of the
cell-to-cell interaction) has not been evaluated in the previous
studies that aimed to identify the parasite ligands and red cell
receptors involved in rosetting.
There is now accumulating, but indirect, evidence that serum IgM and
possibly IgG are involved in rosette formation by some parasite clones
and isolates.4,16 In the first of these studies, Scholander
et al4 examined transmission electron micrographs of a
brain autopsy specimen from a patient who died of cerebral malaria and
observed the presence of fibrillar strands on the PRBC surface,
apparently forming bridges to adherent RBC. Fibrils were subsequently
found on a number of PRBC from parasite isolates and clones propagated
in vitro, and antibodies to human immunoglobulins were found by
immunogold transmission electron microscopy to bind to the fibrillar
strands. Clough et al16 established the requirement for
serum to enable the formation of rosettes in culture and demonstrated that IgM may play a role in rosetting by P falciparum clone
Palo Alto 1 (PA1). In this study the parasites were grown in the
presence or absence of serum, to which IgM or antibody to IgM was added to the culture. However, the requirement that serum components act as
bridging molecules between the PRBC and the RBC of a rosette was not
demonstrated. The reason was that it was possible the lack of parasite
rosettes grown in the absence of serum was the result of a failure of
the PRBCs to express the rosetting phenotype under these conditions.
The purpose of the current study was to determine whether there is an
absolute requirement for human serum components to act as bridging
molecules between P falciparum-infected and P
falciparum-uninfected RBC in rosettes. We describe a serum-free
assay system that used parasites grown in serum-containing medium and
capable of rosetting to enable us to establish the absolute requirement
for human serum and to test the ability of serum fractions produced by
various chromatographic techniques to support rosetting. We establish that multiple serum molecules in addition to IgM are involved in
rosette formation, and we define some of the biochemical
characteristics of the bridging molecules involved.
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Materials and methods |
Chemicals and reagents
RPMI 1640, gentamicin, glutamine, hypoxanthine, and Albumax I (a
lipid-rich bovine serum albumin) were obtained from GibcoBRL (Life
Technologies, Paisley, UK). Pefabloc SC was obtained from S. Black
(Import and Export, Hertford, UK). Antihuman IgM (µ-chain specific)
agarose was obtained from Sigma Chemical (Dorset, UK). Thrombospondin,
fibronectin, and von Willebrand's factor were obtained from Calbiochem
(Nottingham, UK). All other chromatography matrices and Percoll were
obtained from Amersham Pharmacia Biotech (St Albans, UK), and all other
reagents were obtained from Sigma Chemical.
Serum and erythrocytes
Nonimmune human AB serum (NHS) and O Rh-positive erythrocytes were
obtained from the North London Blood Transfusion Service (Colindale,
London, UK). NHS was used from a single donor for each experiment.
Animal sera were obtained from Sigma. C1q-deficient serum17
and C3-deficient serum18 were kindly provided by Dr M. Botto (Hammersmith Hospital, Imperial College School of Medicine, London, UK). All sera were kept frozen at  20°C before use.
Heat-inactivated NHS was obtained by heating the serum to 56°C for
30 minutes. All sera were used at a final concentration of 10%
vol/vol.
Parasite culture
The cloned P falciparum parasites PA1(from the Palo Alto
line, Uganda), previously well characterized in terms of its rosetting properties19 and also referred to as FCR3S by some
authors,13 was used for all experiments. Parasites were
cultured continuously in RPMI 1640 supplemented with 11 mmol/L glucose, 2 mmol/L glutamine, 200 µmol/L
hypoxanthine, and 20 µg/mL gentamicin (complete medium, CM)
containing NHS (10% vol/vol) (CM-NHS). Washed erythrocytes were added
to the culture medium at a hematocrit level of 2%. Parasites were
maintained tightly synchronized according to the methods of Lambros and
Vanderberg20 and Holder and Freeman,21 so that
the age span of the parasites was less than 6 hours for all
experiments. Maintenance of the rosetting phenotype (R+) above 50% of
the parasitized erythrocytes in rosettes, and typically above 65%, was
achieved by Percoll gradient centrifugation using 63%
Percoll.22 Rosetting levels were determined as described below.
Assay for assessment of the role of serum and serum fractions in
rosette formation
Cultures (25-50 mL) of late-stage parasites (early schizonts) grown
in CM-NHS and containing between 5% and 10% parasitemia were
harvested and PRBC purified using a modification of the method of
Handunnetti et al.22 Briefly, the cells were treated with 13.4 µg/mL heparin (sodium salt, grade 1A from porcine intestinal mucosa) to disrupt the rosettes, and they were incubated for 15 minutes
at 37°C. The completeness of rosette disruption was assessed by
fluorescence microscopy with an MC100 spot microscope (Axioshop; Zeiss,
Oberkoden, Germany) × 1000 objective, in incident
ultraviolet light, after the addition of ethidium bromide (50 µg/mL)
to stain the parasite nucleic acid. The heparin-treated cells (0.5-1 mL) were layered onto a cushion of 70% Percoll (1.5-3 mL) containing 13.4 µg/mL heparin and prepared as follows: 9 parts undiluted Percoll
were diluted with 1 part of ×10 concentrated, phosphate-buffered saline (PBS) (137 mmol/L NaCl, 3 mmol/L KCl, 10 mmol/L
Na2HPO4, 1.76 mmol/L
KH2PO4, pH 7.2). To provide a 70%
Percoll solution, 78 mL of this stock Percoll solution was mixed with
22 mL RPMI 1640 containing 20 µg/mL gentamicin (RPMI-gentamicin),
and heparin (13.4 µg/mL) was added. The sample was spun at
1900g for 10 minutes at 20°C, and the interface containing
the mature PRBC was removed and washed twice with RPMI-gentamicin.
After assessing the percentage parasitemia by Giemsa staining of an
aliquot of the pelleted cells (the parasitemia varied between 55% and
95%), the pellet was diluted in RPMI-gentamicin, and 2-µL aliquots
were added to Eppendorf tubes containing 30 µL complete medium with
Albumax I (0.5% wt/vol) in place of the NHS (CM-Albumax I). This
CM-Albumax I was either unsupplemented (negative control), supplemented
with NHS (10% vol/vol; positive control), or supplemented with varying
amounts of serum fractions obtained after chromatographic separation
procedures (see below). Eppendorf tubes containing parasitized cells in
the various media were then incubated at 37°C for 30 minutes to
allow the parasites to recover before they were transferred to a
96-well, flat-bottomed plate, to which was added equal volumes (30 µL) of the same supplemented CM-Albumax medium containing
erythrocytes (2.2 µL) that had been previously washed in
RPMI-gentamicin. Cell densities were determined: hematocrit levels of
the cultures were between 4% and 5%, and parasitemia levels were
between 5% and 10% for all assays. Ethidium bromide (50 µg/mL) was
added to all samples, the plate was placed into a gas box with a
mixture of 5% O2, 7% CO2, and 88%
N2, and samples were incubated at 37°C for 45 minutes.
For each aliquot, rosetting was assessed by placing duplicate 10 µL
samples on a slide under coverslips and counting 200 parasitized cells
per coverslip using a fluorescence microscope ( × 1000
objective, studied in incident ultraviolet light). The rosetting rate
was expressed as the percentage of parasitized cells that bound 2 or
more uninfected erythrocytes to their surfaces. To assess the average
size of rosettes formed, the number of uninfected RBC in at least 100 rosettes was counted per sample, and the mean was calculated. The
significance of differences between 2 mean rosette sizes was assessed
at a confidence limit of P = .05.
Chromatographic techniques
NHS (5-10 mL) was spun at 10,000g for 10 minutes to remove
any particulate matter, filtered through a 1.2 µmol/L filter, and loaded onto the various columns as described below. All columns were
packed and run according to the manufacturer's instructions. Soybean
trypsin inhibitor (10 µg/mL) was added to each of the eluted
fractions from all columns. The fractionation of serum on each column
was repeated using at least 2 different batches of serum.
Concanavalin A-Sepharose affinity chromatography
An equal volume of binding buffer (containing 20 mmol/L Tris-HCl,
0.5 mol/L NaCl, pH 7.4) was added to the spun and filtered serum, and
the sample was loaded onto a 5-mL conA-Sepharose affinity column (XK
1.6 cm × 20 cm; Pharmacia, Uppsala, Sweden)
equilibrated in the same binding buffer. After the column was washed
with 10 vol binding buffer, the bound fraction was eluted with 0.2 mol/L methyl  -D-mannopyranoside.
Antihuman IgM-agarose affinity chromatography
An equal volume of binding buffer (containing 10 mmol/L phosphate,
0.5 mol/L NaCl, 1 mmol/L EDTA, pH 7.2) was added to the spun and
filtered serum, and the sample was loaded onto a 5-mL antihuman IgM
agarose (IgM affinity) column (econo-column, 1 cm×10 cm; Bio-Rad,
Hemel Hempstead, UK) equilibrated in the same binding buffer. After the column was washed with 5 to10 vol binding buffer, the
bound fraction was eluted with 100 mmol/L glycine-HCl, 150 mmol/L NaCl,
pH 2.4. After the addition of 1 mol/L Tris-HCl, pH 9 (100 µL/mL of
eluted fraction), pH neutrality was immediately restored.
Ion exchange chromatography on Sepharose Q
An equal volume of binding buffer (containing 20 mmol/L Bis-Tris
propane, 35 mmol/L Na2SO4, pH 7.2) was added to
the spun and filtered serum, and the sample loaded onto a 5-mL
Sepharose Q, quaternary ammonium anion exchange column (1.6 cm × 20 cm; Pharmacia XK) equilibrated in the same binding
buffer. After the column was washed in 5 vol binding buffer, the bound
fraction was eluted with a 200-mL linear salt gradient between 0 and
0.5 mol/L NaCl in the same buffer, and 4-mL fractions were collected.
Gel filtration chromatography on Sephacryl S-200 HOURs
Pefabloc (1 mmol/L) and EDTA (0.5 mmol/L) were added to the spun and
filtered serum, and the sample was loaded onto a 500-mL Sephacryl S-200
HOURs column (Pharmacia XK 2.6 cm × 100 cm) equilibrated and
eluted with phosphate-buffered saline. Fractions (6 mL) were collected.
Analysis and storage of serum fractions obtained by chromatography
Absorbance at 280 nm (A280) of the fractions
from each of the columns was determined, and fractions were pooled and
concentrated to approximately the original loading volume. Gradient
fractions from the ion exchange column, however, were concentrated to
2.5 times less than the loading volume, using an Amicon stir cell (YM
10 filters; Amicon, Beverly, MA). To prevent interference in the rosetting assay, NaCl, glycine, and methyl -D-mannopyranoside were diluted to low levels (<100 mmol/L, 5 mmol/L, and 5 mmol/L, respectively) in eluted fractions. Aliquots (up to 20 µg protein) of
the fractions before and after pooling and concentration, in the
absence (nonreduced) or presence (reduced) of 0.1 mol/L dithiothreitol, were resolved by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) (10% acrylamide) according to the method of
Laemmli.23 Gels were stained with Coomassie brilliant blue.
Aliquots of the pooled and concentrated fractions were fast frozen as
soon as possible after preparation (within 2 days of running the
column) using liquid nitrogen, and they were stored at 70°C
until they were used in the rosetting assay when they were fast thawed
at 37°C. Fractions were used in the rosetting assay at
concentrations between 10% and 15% vol/vol.
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Results |
Rosette formation is serum dependent
Initially, an assay was developed to assess the ability of serum
components to act as bridging molecules between the PRBC and uninfected
erythrocytes of PA1 rosettes. Parasites were grown in culture in
complete medium containing NHS (10% vol/vol) to ensure that the
parasites were capable of expressing the rosetting phenotype.
Preliminary experiments demonstrated that the rosetting rate of these
cultures was greatest at the early schizont stage (~30 hours after
the invasion of RBC). Therefore, all subsequent assays were conducted
with early schizonts. To remove the serum components effectively from
the cells and to separate the PRBC from the uninfected erythrocytes, a
combination of heparin treatment and Percoll density gradient
centrifugation was used. Heparin completely disrupts the rosettes of
PA1 at a concentration of approximately 10 µg/mL, allowing serum
components that could act as bridging molecules to be released from
physical entrapment within the rosette. Subsequent density gradient
treatment partially separated PRBC from the uninfected cells. A typical
parasitemia of the PRBC-enriched layer was 65%. Mixing these washed
PRBC with freshly washed erythrocytes in complete medium without serum
resulted in crenation of the cells, which prevented the visualization
of rosettes. Therefore, Albumax-I, which prevents crenation, was added
to the complete medium. CM-Albumax I could not support rosetting of the
PRBC in the absence of serum, and typically no rosettes were observed
(the rosetting rate was always less than 5%). This suggested that
either there was an absolute requirement for serum component(s) to act
as bridging molecules and to promote the interaction between the 2-cell
populations of a rosette, that the PRBC had been damaged by the
treatment, or that Albumax I inhibited rosette formation. PRBC-RBC
mixes were incubated in CM-Albumax I medium containing NHS (10%
vol/vol). The rosetting rate in these samples was similar to that
observed before heparin and Percoll treatment, which typically
consisted of more than 60% rosetting (rosetting rates of the culture
before and after treatment were within 10% of each other). This
indicated that there was an absolute requirement for serum component(s)
to act as bridging molecules and to promote interaction between the
cell populations. In studies in which the culture was treated with
heparin, washed thoroughly instead of undergoing Percoll treatment, and
placed in the CM-Albumax I medium, a dramatic reduction in rosetting to
approximately 10% to 15% also resulted. All the studies presented
here used the Percoll-based assay method because it appeared to be a
more effective treatment for the removal of serum (background rosetting
rate of 0%-5% for the heparin-Percoll method versus 10%-15% for the heparin-washing method), and it enabled the addition of uninfected RBC
not present in the original culture. Time-course studies indicated that
rosette reformation in the presence of serum was rapid (within 10 minutes). However, the incubation time was increased to 45 minutes to
ensure optimum rosette formation in all assays. A serum titration curve
showing that at least 5% (vol/vol) serum was required for optimum
rosette formation is presented in Figure 1.
At low serum concentrations (1%), the rosettes formed were small and easily disrupted, but at 10% serum concentrations, the rosettes were
large and robust (ie, the cells appeared firmly adhered to each other)
(average number of uninfected RBC in the rosettes, 4.3). Plasma was no
more effective at promoting rosette formation than serum (data not
shown). A parasite isolate obtained from a patient from Uganda also
demonstrated serum-dependent rosetting at high levels (approximately
80%), and a serum titration curve similar to that shown in Figure 1
was obtained (data not shown). The negative control without serum
(CM-Albumax I) and the positive control with 10% vol/vol serum
(CM-Albumax I-NHS) were included in all subsequent experiments
designed to assess the effect of serum and serum fractions as bridging
molecules in rosette formation.
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| Fig 1.
Effect of serum concentration on rosette formation by
P falciparum PA1-infected RBC.
PRBC cultured in CM-NHS and rosetting at high levels (more than 60%)
were stripped of serum components using combination heparin treatment
to disrupt the rosettes and Percoll density gradient centrifugation to
separate the PRBC from uninfected cells and medium components. The PRBC
were mixed with freshly washed, uninfected erythrocytes in a serum-free
medium (CM-Albumax I) in the absence or presence of increasing
concentrations of serum, and rosetting was assessed by fluorescence
microscopy (×1000 objective in incident ultraviolet light). The
relative percentage rosetting is defined as ×100 the rate of
rosetting in a culture in which the CM-Albumax I medium is supplemented
with the indicated amount of serum/the rate of rosetting in a culture
in which the CM-Albumax I medium is supplemented with 10% (vol/vol)
NHS. Results are presented as the mean of 3 separate experiments, each
using a different batch of human nonimmune AB serum ± SD.
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Heat-inactivated NHS and human serum from an individual deficient in
either C1q17 or C318 supported the formation of
rosettes similarly to that of NHS. Various animal sera, including adult bovine, fetal calf, chicken, sheep, and guinea pig, were tested for
their ability to support rosette formation by PA1 in the assay system
(all used at a final concentration of 10% vol/vol). However, none of
those tested could support rosette formation to more than 15% of that
of NHS.
After showing the requirement for human serum components as bridging
molecules between the PRBC and uninfected erythrocytes of PA1 rosettes,
we sought to characterize the molecule(s) involved. Nonimmune human AB
serum was fractionated using a variety of chromatographic techniques.
Molecules were separated based on differences in affinity, size, and
charge, and the fractions obtained were tested in the biologic assay
system for their ability to support rosette formation.
Multiple serum components are involved in bridging parasite-infected
erythrocytes to uninfected erythrocytes
Three different techniquesconA affinity, anion exchange, and gel
filtration chromatographyindependently demonstrated that multiple
components are involved in PA1 rosette formation. ConA is a lectin with
affinity for glucose and mannose residues; therefore, it binds to many
glycoproteins and glycolipids in serum, including IgM. The unbound
material contains nonglycosylated proteins (eg, albumin) and
glycosylated molecules (eg, IgG). The SDS-PAGE profile of the 2 fractions is shown in Figure 2A, and it
demonstrates the mutual exclusion of components into either the conA
unbound (C1) or bound (C2) fractions. As shown in Figure 2B, neither
the unbound nor the bound fractions alone were able to support PA1 rosette formation to more than 15% of the levels in whole serum. However, when the 2 fractions were combined, the rosette formation rate
was restored to levels similar to those of 10% whole serum. Furthermore, as shown in Figure 3 and Table
1, in the presence of either fraction
alone, the rosettes were small and fragile, and the average size was
significantly smaller than the size of rosettes in 10% whole serum
(2.7 uninfected RBC/rosette in C1 and 2.3 uninfected RBC/rosette in C2
versus 4.3 uninfected RBC/rosette in 10% whole serum)
(P = .05). However, when the 2 fractions were combined, the
rosette size was similar to that in 10% whole serum.
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| Fig 2.
At least 2 serum components are required for rosette
formation, as demonstrated using conA affinity chromatography.
(A) SDS-PAGE profile of fractions eluted from a conA-Sepharose
affinity column. Aliquots of whole serum (WS) and conA-unbound (C1) and
conA-bound, eluted (C2) fractions (up to 20 µg protein, corresponding
to the same relative amount of each fraction) were resolved by SDS-PAGE
(10% acrylamide) in the absence (nonreduced) or presence (reduced) of
0.1 mol/L dithiothreitol. The gel was stained with Coomassie brilliant
blue. Molecular mass markers (M) are 30, 46, 66, 97, and 220 kd from
bottom to top, respectively. (B) Effect of the conA fractions on
rosette formation. The rosetting assay was conducted as described in
the legend to Figure 1, and the fractions were used at 10% (vol/vol)
each, for all experiments. Two different batches of serum were
fractionated, and the assay was conducted in duplicate for each batch
(mean ± SD for 4 experiments).
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| Fig 3.
Size and quality of rosettes are determined by multiple
serum components.
Rosettes visualized by immunofluorescence in the presence of ethidium
bromide to stain parasite nucleic acid (× 1000 objective in
incident ultraviolet light) showing the difference between the effect
of individual and combined serum fractions. (A) Small rosettes formed
in the presence of individual serum fractions. The adhesion between the
cells was weak, and the rosettes sometimes fell apart. Panel 1, conA-unbound fraction, C1; panel 2, anion exchange-unbound fraction,
A1; panel 3, IgM affinity-unbound fraction, M1. (B) Large rosettes
formed in the presence of whole serum or combined fractions. These
rosettes were robust and stable, the interaction between the cells was
strong, and the rosettes did not fall apart. Panel 1, conA-unbound
fraction C1 + bound fraction C2; panel 2, anion exchange-unbound
fraction A1 + gradient fraction GF4; panel 3, IgM affinity-unbound
fraction M1 + bound fraction M2 (ie, IgM).
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Anion exchange chromatography, which separates molecules based on
differences in isoelectric point (PI), was initially carried out at pH
8, and the bound fraction was eluted with a 0 to 0.5 mol/L salt
gradient. Under these conditions, most serum components bind to the
column, and only the most basic components (eg, IgG) do not bind.
Rosetting activity was mediated by the bound material only in fractions
that were eluted after albumin, suggesting that the PIs of the
components involved in rosetting were lower than those of albumin
(PI = 4.8) (data not shown). We then sought to provide additional
evidence for the requirement of multiple components in rosette
formation by using a pH for the chromatography that would allow
separation of the rosetting components into an unbound, more basic
fraction containing albumin (higher PI) and a bound, more acidic
fraction (lower PI). Chromatography was carried out at pH 7.2, and the
bound material was eluted with a 0 to 0.5 mol/L NaCl gradient. Gradient
fractions with similar protein profiles, as determined by SDS-PAGE
(Figure 4A) were pooled into 4 gradient fractions (G1 to G4) (Figure 4B). As shown in Figure 4C, the unbound fraction (A1) alone supported a high level of rosette formation (61%
relative rate). However, as shown in Table 1 and Figure 3, the average
sizes of the rosettes were significantly smaller than they were in 10%
whole serum (2.6 versus 4.4 uninfected RBC) (P = .05). Both
cell populations appeared to be less strongly bound together than they
were with whole serum (Figure 3, panel A2). Of the bound fractions,
only GF4 was able to support rosetting when tested alone, albeit at
much lower levels than with A1 (31% relative rate), and these rosettes
were also small and fragile (Table 1). However, when combined, the
rosetting rate and the rosette size with A1+GF4 were similar to those
of whole serum, indicating that components in both A1 and the gradient
fraction GF4 were involved in rosette formation (Figures 3 [panel
B2], 4; Table 1). Gradient fraction GF3 also markedly increased the rosetting rate when combined with A1, but to a lower level than GF4;
this probably results from the spanning of serum components between
adjacent fractions.
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| Fig 4.
At least 2 serum components are involved in rosette
formation, as demonstrated using anion exchange chromatography.
SDS-PAGE profile of fractions eluted from a Sepharose Q anion exchange
column. Aliquots of the unbound fraction (A1) and gradient fractions
(GF) before (A) and after (B) pooling and concentration (aliquots of up
to 10 µg protein, corresponding to 10 times more gradient fraction
than the WS and A1 fractions), were resolved by SDS-PAGE (10%
acrylamide) as described in the legend to Figure 2. (C) Effect of the
fractions on rosette formation. The rosetting assay was conducted as
described in the legend to Figure 1, and the fractions were used at
15% (vol/vol) of A1 and 10% (vol/vol) of GF1-4 for all experiments.
Two different batches of serum were fractionated, and the assay was
conducted in duplicate for each batch (mean ± SD for 4 experiments).
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Gel filtration chromatography of serum on a Sephacryl S-200 column was
performed to provide information on the size range of the components
involved in rosette formation. The elution profile of the serum from
this column is shown in Figure 5A. The 3 peaks of absorbance correspond with the presence of IgM (S1, higher molecular mass), IgG (S2, intermediate molecular mass), and albumin (S3, lower molecular mass). The SDS-PAGE profile of the 3 fractions of
pooled eluate is provided in Figure 5B. Although the size differential is clear, it is also apparent that some components spanned more than 1 fraction. The activity of each of these fractions in the assay, tested
individually and in combination, is shown in Figure 5C. When all 3 fractions were added, rosette formation was restored to 85% of the
rate obtained with 10% whole serum, and the average size of rosettes
was not significantly different from the size of rosettes in whole
serum (4.0 versus 4.3 uninfected RBC) (Figure 3). Fraction 2 alone
supported rosette formation at high levels (65% relative rate), as did
any combination containing fraction 2. In contrast, fractions 1 and 3 individually provided only 20% of the activity of whole serum.
However, when fractions 1 and 3 were combined, they also supported
rosette formation at high levels (70% relative rate). It should be
noted, however, that each of the individual fractions and each of the
paired fractions tested resulted in rosettes that were significantly
smaller than rosettes formed with whole serum (Table 1).
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| Fig 5.
Different-sized serum components are required for rosette
formation.
(A) Sephacryl S-200 elution profile. Six-microliter fractions were
collected, and the A280nm was determined for each.
Fractions were pooled (S1-S3) and concentrated to the original volume
of serum loaded. (B) SDS-PAGE profile of proteins in pooled fractions
eluted from Sephacryl S-200. Aliquots of the whole serum (WS) and the
pooled fractions (S1-S3) (up to 20 µg protein loaded, corresponding
to the same relative volumes of each) were resolved by SDS-PAGE (10%
acrylamide) as described in the legend to Figure 2. Molecular mass
markers (M) are 19, 30, 46, 66, 97, and 220 kd from bottom to top,
respectively. (C) Effect of the individual and recombined S-200
fractions on rosette formation. The rosetting assay was conducted as
described in the legend to Figure 1, and the fractions were used at
15% (vol/vol) each for all experiments. The column was run using 2 different batches of serum, and the assay was conducted in duplicate
for each batch (mean ± SD for 4 experiments).
|
|
Because we had established that there were multiple serum components
involved in rosette formation of PA1 using affinity for conA, PI, and
size as criteria, we then sought to determine the relationship among
these 3 criteria in the context of the various serum components
involved in rosette formation. Fractions from the 3 columns were tested
in combination, and the results are provided in Table
2. Combinations of the conA fractions with the anion exchange fractions showed that the conA unbound fraction (C1)
was able to combine with the anion exchange gradient fraction (GF4) to
restore rosette formation to a similar relative rate, 10% whole serum.
Rosette sizes were not significantly different than those formed in
10% whole serum. A similar result was obtained with the conA-bound
fraction (C2) and the anion exchange-unbound fraction (A1). Conversely,
the unbound fractions from the 2 columns (C1 + A1) were not active
together (40% relative rate), and rosette sizes were significantly
smaller than those of rosettes formed in whole serum were (2.8 vs 4.3;
P = .05). A similar result was obtained with the bound
fractions from the 2 columns. These data suggest that C1 and A1 contain
similar rosetting components, as do C2 and GF4. Combination of the conA
fractions with the S-200 higher and lower molecular mass fractions
provides some idea of the relative size of the rosetting components in
the 2 fractions. C1 complements S1 (higher molecular mass fraction),
suggesting that because higher and lower molecular mass components are
important for stable rosette formation, the C1 predominantly contains
the lower molecular mass activity. Similarly, C2 complements S3 (lower molecular mass fraction), suggesting that C2 predominantly contains the
higher molecular mass component(s) involved in rosetting. Combination
of the anion exchange fractions with the S-200 fractions supports this
proposition because A1 complements S1 relative to S3 and GF4
complements S3 relative to S1.
Previously, a role for IgM in PA1 rosette formation had been
proposed,4,16 though the direct involvement of IgM as a
bridging molecule had not been demonstrated. We therefore sought to
establish directly whether IgM was one of the multiple components
involved in PA1 rosette formation.
IgM is one of the serum components involved in PA1 rosette formation
IgM was purified from nonimmune human AB serum by affinity
chromatography. This provided 2 fractions, an unbound fraction (M1)
with a profile identical to that of whole serum that was depleted of
IgM and a bound IgM fraction (M2) that was devoid of other proteins, as
shown by SDS-PAGE and immunoblotting (data not shown). The unbound,
IgM-depleted fraction supported rosette formation between 50% and 65%
of the level achieved with whole serum, whereas the IgM fraction alone
had a low level of assay activity (less than 10%), even when it was
tested at a high concentration (30% vol/vol) (data not shown).
Furthermore, the rosettes formed in the IgM-depleted serum were
significantly smaller than those formed in whole serum (average number
of uninfected RBC per rosette, 2.7 versus 4.3, respectively)
(P = .05) (Figure 3, panel A3; Table 1). Rosette formation
increased to a rate similar to that of 10% whole serum when the 2 fractions were combined. IgM levels were similar to those in the 10%
whole serum control, and the average size of the rosettes increased to
3.9 uninfected RBC per rosette, which is not significantly different
than that in the 10% whole serum control (Figure 3, panel B3; Table
1).
To provide additional data on the involvement of IgM in rosette
formation, the fractions from the conA, anion exchange and S-200
columns that contained IgM were replaced with affinity-purified IgM
(M2). Western blot experiments confirmed the presence of IgM exclusively in the conA-bound fraction, predominantly in the anion exchange GF4 (with a much lower but detectable level in GF3), and
predominantly in S1 with a just detectable amount in S2 (data not
shown). When the IgM (M2) was combined with C1, A1, or S2 + S3, the
rosette formation rate did not increase significantly compared with the
level achieved with these fractions alone. In addition, as shown in
Table 1, the combination of C1 + M2 and S2 + S3 + M2 did not
increase the size of rosettes over those obtained with C1 or S2 + S3,
which remained significantly smaller than the size of rosettes in whole
serum (P = .05). Other components than IgMhigh molecular
mass, glycosylated component(s), IgMin the S200 S1 and the conA C2
fractions must therefore be essential for IgM to play its role in
rosette formation. In contrast, the addition of IgM to A1 increased the
average size of rosettes from 2.6 uninfected RBC (significantly less
than whole serum) to 4.2 uninfected RBC (not significantly different to
10% whole serum). These data suggest that a major contributor to
rosette formation in GF4 is IgM.
To explore the possibility that the other high molecular mass
glycosylated components important in rosette formation in the conA C2
fraction were adhesive glycoproteins involved in cell-cell or
cell-matrix adherence systems, von Willebrand's factor, fibronectin, and thrombospondin were each added to conA C1 + M2 (IgM) individually or in combination. Each adhesive glycoprotein was tested on 2 separate
occasions, at a range of concentrations similar to those in medium
containing 10% serum or plasma. von Willebrand's factor (0.5-10 µg/mL), fibronectin (3-100 µg/mL), and thrombospondin (0.2-4 µg/mL) did not support rosette formation; the number and size of
rosettes formed was similar to those formed with conA M2 alone. For
each adhesive glycoprotein and at all concentrations tested, the
relative rosette formation rate was within 10% of the relative rosette
formation rate of conA M2 alone (25%; mean of 3 separate experiments).
The average size of rosettes formed was also similar to conA M2 alone
and was significantly less than the average size of rosettes formed in
whole serum in all experiments.
| |
Discussion |
We have shown unequivocally that rosette formation of P. falciparum is dependent on the presence of specific serum
components that appear to act as bridging molecules between infected
and uninfected cells of a rosette. Serum fractionation using a variety of chromatographic techniques indicated that multiple components are
involved in the adhesion interaction, including IgM and at least 2 other molecules. The current study was conducted with a parasite clone.
A parasite isolate rosetting at high levels and obtained from a patient
from Uganda was shown to be serum dependent. Furthermore, most
rosetting parasites obtained from children with P. falciparum
malaria in Malawi, East Africa have been found to be dependent on serum
for rosette formation (Rogerson personal communication). This indicates
that the serum requirement for rosetting is a widespread phenomenon in
P. falciparum malaria.
It is interesting that rosette formation of P. falciparum-infected erythrocytes is dependent on human serum.
Rosette formation was not supported to any extent by serum obtained
from adult bovine, fetal calf, chicken, sheep, or guinea pig. We
observed a similar ability to support rosette formation between batches
of individual human sera (Figure 1). These batches were all from
non-malaria immune persons in the UK. Sera obtained from
malaria-endemic regions might be variable in their ability to support
rosette formation. Because rosetting has been found to correlate with
disease severity,5-7 polymorphisms in the serum molecules
involved in rosetting are possible. In addition, sera from persons in a
malaria-endemic region are likely to contain antibodies to parasite
proteins on the surface of the infected cell, and these may inhibit
rosette formation.
The severe pathologic conditions of P. falciparum malaria are
associated with the sequestration of parasites in the microvasculature of a variety of organs, a process believed to be the result of cytoadherence and rosetting. The finding that serum components are
critical for rosette formation has important implications in the
understanding of these adhesive interactions. PfEMP-1, an
antigenically variant protein product of the var gene family, appears to be the parasite ligand involved in
cytoadherence24 and rosetting.12,13 It is
possible that a population of these proteins adheres to endothelial
cells and uninfected erythrocytes with serum components as bridging
molecules, which emphasizes the importance of identifying the serum
molecules involved in bridge formation and the molecular basis for this
adhesion interaction.
A number of adhesive interactions involve plasma components as bridging
molecules between cell populations and provide precedents for the
rosetting process. These interactions occur under physiological or
pathologic circumstances and include the adhesion of leukocytes to
endothelium through fibrinogen, a process involved in leukocyte trafficking and recirculation,25 the adhesion of apoptotic
neutrophils to macrophages through thrombospondin,26 and
the invasion mechanism of pathogenic mycobacteria in which the
complement cleavage product C2a associates with the mycobacterial
surface and cleaves C3, resulting in macrophage
recognition.27 It has been suggested that P. falciparum-infected erythrocytes attach to endothelial cells
through several receptor-ligand pairs. In addition to PfEMP-1, modified erythrocyte band 3 may play a role in cytoadherence; it has
been proposed that this interaction with the endothelium occurs through
thrombospondin.28 A number of adhesive glycoproteins involved in cell-cell and cell-matrix interactions, including thrombospondin, von Willebrand's factor, and fibronectin, present in
the conA-bound fraction (C2) were tested for their ability to support
rosette formation in combination with the conA-unbound fraction (C1)
and IgM. However, each of these high-molecular-mass adhesive
glycoproteins supported rosette formation similarly to C1+ IgM alone,
suggesting either that they were not involved in the bridging
interaction between the parasitized and the uninfected cells or that
additional components in the bound fraction (C2) were required for
bridge formation.
One study that implicated PfEMP-1 as the rosetting ligand on
the parasitized cell also identified CR1 as a rosetting receptor on the
uninfected cell.12 This may suggest that complement
components are the bridging molecules. Both C3b and C4b, opsonins that
are produced by the activation of the complement cascades, bind to CR1.
However, our studies provide no evidence that complement activation is
important in rosette formation of PA1. The use of heat-treated serum
and of C1q- and C3-genetically deficient sera in the assay did not
affect rosetting. Other workers have also reported that heat-treated
serum supports rosetting just as effectively as fresh serum (Rowe JA,
DPhil Thesis, University of Oxford, 1994). Because most of the CR1
studies were carried out with a different parasite clone, R29, it is
possible that different mechanisms are responsible for rosette
formation in the different parasites or that CR1 is able to bind to
other serum factors that do not involve complement activation for their formation.
Multiple serum components are involved in rosette formation,
highlighting the complexity of this interaction, as evidenced by the
results of 3 different fractionation techniquesconA affinity, anion
exchange, and gel filtration. It is therefore possible that in the
presence of whole serum at optimum levels, there are individual, unrelated, parallel bridges involving single serum components that span
the 2 cell populations. Alternatively, there may be serial bridges in
which more than 1 molecule is involved or a combination of these 2 types of bridge. The conA data (Figure 2) show that both unbound (C1)
and bound (C2) serum components are essential for rosette formation
because only a very low level of rosetting is observed with either
fraction alone. This may indicate that there are no parallel bridges,
that all bridges involve more than 1 serum component and these are in
different fractions, or that, perhaps more likely, multiple bridges are required to achieve great enough strength of interaction to allow stable rosette formation. The anion exchange data are somewhat different (Figure 4). The unbound material (A1) is able to support rosetting to a relatively high level, though the rosettes are small and
fragile. Thus, although the combination data (Table 2) suggest that C1
and A1 are similar, A1 must contain additional component(s) that allow
rosettes to form. It is possible that the components form individual
parallel bridges so that the strength of interaction, though relatively
weak, is strong enough to allow small rosettes to form. Alternatively,
a serial bridge formed by the components in A1 may allow the cells
to interact.
The S-200 gel filtration data also provide evidence for the involvement
of multiple serum components in rosetting, and they provide additional
understanding of the relative sizes of the molecules involved (Figure
5). These data suggest that there is a rosetting component(s) of higher
molecular mass, probably more than 150 kd, that spans fractions F1 and
F2 and a rosetting component(s) of lower molecular mass, probably more
than 60 kd, that spans fractions F2 and F3. Hence, F2 is active alone,
and F1 and F3 combined support rosetting. The fact that components span
fractions probably accounts for the small size of rosettes for all
fraction combinations, except S1 + S2 + S3 because it is likely
that 1 or more of the components responsible for rosette formation is limiting in all single and paired fraction samples.
We have provided clear evidence that IgM is 1 of the bridging molecules
involved in rosette formation and that it is a major contributor to
rosette size and stability. Removal of IgM from the serum resulted in
the formation of rosettes that were significantly smaller than those in
whole serum (Figure 3, Table 1). The addition of IgM to the depleted
serum resulted in rosettes that were not significantly different in
size to those in whole serum. It is possible that IgM forms independent
parallel bridges that stabilize the rosettes and allow more uninfected
cells to bind. Alternatively, IgM may contribute to the strength of an
interaction. It is clear that IgM alone cannot support rosette
formation, even when added at high concentration (30% vol/vol). This
suggests that IgM cannot span the 2 cell populations and another
molecule(s) is required for binding (ie, it is involved in a serial
bridge) or that the strength of interaction brought about by IgM alone
is insufficient to hold together the 2 cell populations. In the latter
case, other bridges would be required to achieve the cumulative
strength needed. When IgM was added back to the conA C1 fraction, it
was unable to increase the percentage of rosette formation or the size
of rosettes, and it was unable to replace the high molecular mass fraction of the gel filtration column. This supports the notion that
something else is present in the C2 and S1 fractions that either forms
a serial bridge with the IgM or contributes a parallel bridge to
strengthen the interaction. When the IgM was added back to the anion
exchange-unbound fraction (A1), it was able to increase the size of the
rosettes significantly, but it did not restore the percentage rosetting
to control levels. Thus, a major component in the anion exchange
gradient fraction (GF4) that contributes to rosetting is IgM. However,
it is possible that other component(s) in GF4 have a role in rosette
formation because this fraction alone supports a low level of rosette
formation, whereas IgM alone cannot. Whatever its bridging mechanism,
it appears that the intact IgM molecule is required because IgM
monomers do not increase the size of rosettes formed when they are
added back to IgM-depleted serum (Black J, unpublished observation).
The data on the combination of the different column fractions (Table 2)
provide initial understanding of the types of molecule involved in
addition to IgM, though the identity of these molecules is unknown. One
or more of the molecules is of lower molecular mass, does not bind to
conA, and is acidic (with a lower PI than albumin). However, it is less
acidic than the other molecule(s) involved, which are of higher
molecular mass and glycosylated. Plasma and serum are extremely complex
mixtures of molecules, and many of these have not been identified in
terms of their sequence or function. This, together with the emerging
understanding of the complexity of the rosetting interaction, makes
identification of the molecules involved a difficult but achievable
task. It should now be possible to combine the fractionation approaches presented here, to produce a purification strategy that enables further
resolution of the components. Additional sophisticated methods of
analysis are required, including mass spectroscopy, to identify the
important molecules involved in rosetting. Polymorphisms in the serum
molecules responsible for this cell interaction may result in
individuals with differing abilities to support rosette formation.
Because rosette formation in vitro has been shown to be correlated with
disease severity in a number of studies carried out in
Africa,5-7 such polymorphisms may dramatically affect the
likelihood of developing severe P. falciparum malaria. The propensity of a particular parasite to form rosettes is therefore governed by the interplay of a number of different factors,
including parasite genotype, host RBC receptor, and serum component polymorphisms.
| |
Acknowledgments |
The authors thank Dr A. A. Holder for helpful advice and for critical
reading of the manuscript. They also thank Professor C. Green for
support throughout this work.
| |
Footnotes |
Submitted May 10, 1999; accepted September 13, 1999.
Supported by Northwick Park Institute for Medical Research, The
Wellcome Trust, and the British Infection Society.
Reprints: Geoffrey Pasvol, Department of Infection and Tropical
Medicine, Imperial College School of Medicine, The Lister Unit,
Northwick Park Hospital, Watford Road, Harrow, Middlesex, HA1 3UJ,
United Kingdom; e-mail: g.pasvol{at}ic.ac.uk.
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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Abstract
Introduction