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*
Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, United Kingdom;
Wellcome Trust Research Laboratories, College of Medicine, University of Malawi, Blantyre, Malawi;
Department of Microbiology and Immunology, MCP Hahnemann University School of Medicine, Philadelphia, PA 19129;
Institut National de la Santé et de la Recherche Médicale Unité 430, Hôpital Broussais, Paris, France;
¶ Kenya Medical Research Institute-Wellcome Trust Programme, KEMRI Centre for Geographical Medicine Research Coast, Kilifi, Kenya;
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Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;
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Department of Medicine, Division of Rheumatology, Washington University School of Medicine, St. Louis, MO 63110; and
**
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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60 amino acids containing four invariant cysteines and a number
of other conserved residues (2). The extracellular domain
of the most common allelic variant of CR1 is composed of 30 SCRs, the
first 28 of which are arranged in tandem repeats in homologous groups
of 7, with each group of 7 being known as a long homologous repeat
(LHR) (Ref. 3 ; see Fig. 1
A). The binding sites
on CR1 for its ligands C3b and C4b have been mapped, with SCRs 8–12
and SCRs 15–18 preferentially binding to C3b and SCRs 1–4
preferentially binding to C4b (Refs. 3, 4, 5 ; see Fig. 1A).
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This study set out to further characterize the role of CR1 in rosetting by identifying the region of CR1 required for interaction with infected erythrocytes and by determining the requirement for CR1 in the rosetting of P. falciparum field isolates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CR1 deletion mutants LHR-A, LHR-B, and
LHR-D+ were produced from culture supernatants of
transiently transfected COS-7 cells as described previously
(9, 10, 11). LHR-A comprises SCRs 1–7, LHR-B comprises SCRs
8–14, and LHR-D+ comprises SCRs 22–30 (Fig. 1
A). The first three SCRs of LHR-C (SCRs 15–17) were
expressed in Pichia pastoris (12) and C3b
dimers were produced from human plasma C3 as described elsewhere
(11). Soluble recombinant CR1 (13) and mAb
YZ-1 were obtained from D. Fearon (University of Cambridge, Cambridge,
U.K.). The CR1 mAbs J3B11, J8B10, and J3D3 were as described previously
(14); 1B4, 3D9, 7G8, 9H3, and HB8592 were obtained from R.
Taylor (University of Virginia School of Medicine, Charlottesville,
VA); 1F11.G12, 3C6.D11, and 6B1.H12 were obtained from T Cell Sciences
(Needham, MA); and E11 was purchased from Serotec (Oxford, U.K.).
Additional amounts of mAb J3B11 were also obtained from J. Cohen
(University of Reims, Reims, France). The CD36-specific mAb OKM5 was a
gift from T. Mercolino (OrthoDiagnostic Systems, Raritan, NJ), isotype
control mAbs were purchased from PharMingen (San Diego, CA) and rabbit
Ig (X0936) and rabbit polyclonal Ab to human IgA/G/M (A1090) were
purchased from Dako (Carpinteria, CA).
Parasites and parasite culture
The P. falciparum-rosetting laboratory strains used were clone R29 (15), clone PAR+ (16), and lines TM180 and TM284 (17). Rosetting in these parasite strains is 40–80% of infected erythrocytes in rosettes and is stable within a given cycle, but varies from one cycle to the next. Parasites were cultured in group O human erythrocytes in RPMI 1640 medium with sodium bicarbonate (Life Technologies, Rockville, MD) supplemented with 2 mM L-glutamine, 25 mM HEPES, 20 mM D-glucose, 25 µg/ml gentamicin, and 10% normal human serum (pooled from at least four donors), and gassed with a mixture of 96% nitrogen, 3% carbon dioxide, and 1% oxygen. Parasites were selected once a week by sedimentation in gelatin to maintain the rosetting phenotype (18) and were synchronized once a week by sorbitol lysis (19). Field isolates from malaria patients in Kenya and Malawi were collected as described previously (7) and cultured in supplemented RPMI 1640 as above except that 10% pooled human AB serum was used to avoid ABO incompatibility. Isolates were grown for at least 16 h in vitro and maturity assessed by Giemsa-stained thin films. Only those isolates in which the majority of parasites matured into pigmented trophozoites and had a rosette frequency of >10% infected erythrocytes in rosettes were used in Kenya and of >5% infected erythrocytes in rosettes were used in Malawi.
Rosette reversal by CR1 deletion mutants
Parasitised cells were stained with 20 µg/ml ethidium bromide
for 5 min at 37°C, washed twice with incomplete RPMI 1640
(supplemented as above but without serum), and resuspended at 4%
hematocrit in binding medium (RPMI 1640 medium without sodium
bicarbonate, supplemented as above but with 10% pooled human serum
that had been heat-inactivated at 56°C for 30 min). Ten microliters
of each LHR CR1 deletion mutant was added to a 10-µl aliquot of
culture suspension to give a final concentration of 50 µg/ml deletion
mutant (1 µM) and then incubated for 1 h at 37°C and
assessed for rosetting as described below. The negative control culture
was incubated with 10 µl of COS-7 cell culture supernatant from
mock-transfected cells, and the positive control culture was incubated
with 50 µg/ml soluble CR1 (250 nM), which had been shown
previously to cause 50% inhibition of rosetting in clone R29
(8). SCRs 15–17 were tested at 50 µg/ml and 500 µg/ml
as described above, and rosetting was compared with a control culture
with no added recombinant protein.
Assessment of rosetting
Ten microliters of culture suspension, prestained with ethidium bromide as described above, was placed on a clean glass slide and covered with a 22-mm x 22-mm coverslip. Slides were blinded before counting and then were viewed on an epifluorescence microscope using a combination of fluorescence and bright field. The illumination was adjusted so that both ethidium bromide-stained infected erythrocytes and unstained uninfected erythrocytes could be viewed simultaneously. At least 200 infected cells were counted and assessed for rosetting, with the binding of two or more uninfected erythrocytes constituting a rosette. The rosette frequency is the percentage of mature (pigmented trophozoite) infected erythrocytes forming rosettes.
Rosette reversal by CR1 mAbs
Parasite cultures were stained with 20 µg/ml ethidium bromide as described above and resuspended in binding medium at 2% hematocrit. mAbs were added to 20-µl aliquots of culture suspension to give a final concentration of 1 µg/ml and 10 µg/ml and were incubated at 37°C for 30 min before assessment of rosetting. Isotype controls were tested at the same concentration. mAb J3B11 was also tested at 0.1 µg/ml, 0.5 µg/ml and 100 µg/ml .
Inhibition of rosette reformation by mAbs J3B11, 1B4, and 3D9
The mAbs were added to 100-µl aliquots of R29 culture suspension at 2% hematocrit to give a final concentration of 1 µg/ml; then rosettes were mechanically disrupted by passing the suspension 10 times through a 26-gauge needle (17). A negative control culture with no additive and a culture with 1 µg/ml IgG1 isotype control were treated in the same way. The cultures were incubated at 37°C for 1 h to allow rosettes to reform, then assessed for rosetting as described above.
Inhibition of rosette reformation by C3b dimers
C3b dimers were added to culture suspension at 2% hematocrit to give a final concentration of 100, 10, and 1 µg/ml, then mechanically disrupted, incubated, and assessed for rosetting as above.
Rosetting in C3-deficient serum
Serum from a patient genetically deficient in C3 was obtained from Mark Walport (Imperial College, London, U.K.). Parasites were grown for one full cycle (from mature trophozoite stage through to mature trophozoite stage in the next cycle) in complete RPMI 1640 containing 10% C3-deficient serum or 10% normal human serum, and rosetting was assessed as described above.
Rosette reversal by mAbs and polyclonal Abs in laboratory strains and field isolates
J3B11 and an isotype control were tested at 1 µg/ml, and rosetting was compared with that in a control culture with no added Ab. The CD36 mAb OKM5 and an isotype control were tested at 10 µg/ml, a concentration shown previously to abolish rosetting in the parasite line Malayan Camp (20). The rabbit polyclonal Abs to human IgA/IgG/IgM were tested at 1/50 dilution and compared with rosetting in the presence of an equivalent concentration of Ig derived from normal rabbit serum (1/100 dilution). The latter control was not available for the testing of Kenyan field isolates. All incubations with Ab were for 30 min at 37°C, and assessment of rosetting was as described above. Malawi experiments were conducted by S.J.R., who was supplied with blinded J3B11 and IgG1 isotype control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To localize the region of CR1 required for P.
falciparum rosetting, we tested the ability of soluble CR1
deletion mutants (Fig. 1A) to
reverse rosettes with parasite clone R29. A soluble CR1 recombinant
protein consisting of the entire extracellular domain of CR1-reversed
rosetting (Fig. 1B), as had been shown previously
(8), and a deletion mutant consisting of LHR-B also
reversed rosetting to 50% of the control value (Fig. 1B,
control mean rosette frequency, 46.5% (SE, 1.4); LHR-B mean rosette
frequency, 24.0% (SE, 1.8), p < 0.001, Student’s
t test). No statistically significant reversal of rosetting
was seen with LHR-A or LHR-D+ (Fig. 1B). The entire domain of LHR-C was not tested, however, we
did test a recombinant protein comprising the first three SCRs of LHR-C
(SCRs 15–17). This region includes the C3b binding site and is
virtually identical (three-amino acid differences) to the first three
SCRs of LHR-B (SCRs 8–10). The recombinant protein SCRs 15–17 also
significantly reversed rosetting compared with the control culture
(Fig. 1C, control mean rosette frequency, 60.1% (SE, 2.7);
SCRs 15–17 (500 µg/ml) mean rosette frequency, 36% (SE, 5.6),
p < 0.01 Student’s t test; SCRs 15–17 (50
µg/ml) mean rosette frequency, 39.0% (SE, 7.5), p <
0.05, Student’s t test). These data suggest that the region
of CR1 that interacts with infected erythrocytes to form rosettes is in
LHR-B and LHR-C and includes the first three SCRs of LHR-C (the C3b
binding site). On a molar basis, the full-length soluble CR1 reversed
rosettes more effectively than LHR-B and SCRs 15–17, presumably
reflecting the presence of multiple C3b binding sites in the
full-length protein.
Rosette reversal by mAbs to CR1
Further elucidation of the region of CR1 required for P.
falciparum rosetting came from studies of rosette reversal by mAbs
to CR1. The epitopes recognized by the various mAbs have been mapped to
particular SCRs within CR1 (Table I; data
from Ref. 9). Most of the mAbs bind at several locations
due to the repetitive nature of CR1. In most cases, it is not known
whether the mAbs that group together have identical activities, because
the fine specificity of their epitopes has not been determined;
therefore, all mAbs in Table I were tested independently. Each Ab was
tested at 1 and 10 µg/ml for its ability to reverse rosetting in
clone R29 and compared with isotype control mAbs and control cultures
with no added Ab. J3B11 was the only mAb to reverse rosetting (Table I), giving maximum reversal at 1 µg/ml (Fig. 2A, control mean rosette
frequency, 55.5% (SE, 6.7), J3B11 (1 µg/ml) mean rosette frequency,
20.4% (SE, 4.6), p < 0.01, Student’s t
test). Higher concentrations of J3B11 (10 and 100 µg/ml) caused
microscopic agglutination of erythrocytes in some experiments;
therefore, rosette reversal at these concentrations was not studied
further. Agglutination was not seen with any other Ab or reagent tested
in this study. The epitope recognized by J3B11 has been mapped to SCRs
3, 10, and 17 (Fig. 1A), and this mAb is known to inhibit
the rosetting of C3b-coated erythrocytes to CR1 on lymphocytes
(14). Two other mAbs, 1B4 and 3D9, also map to the C3b
binding site on CR1 (9) and block C3b-mediated rosetting
(21). We considered the possibility that these mAbs may
have been ineffective at reversing rosettes due to a lower affinity for
CR1 than J3B11, or due to their epitopes being masked when infected
erythrocytes are bound to CR1 in rosettes. We therefore conducted an
alternative assay in which rosettes were mechanically disrupted in the
presence of the mAbs to allow access of the Ab to CR1 and then assessed
inhibition of the reformation of rosettes (the rosettes reform
spontaneously after such mechanical disruption). It was found that mAbs
1B4 and 3D9 gave similar rates of inhibition to J3B11 when tested for
their ability to inhibit rosette reformation after rosettes had been
mechanically disrupted (Fig. 2B). The differences between
the activities of these Abs may therefore reflect the precise epitope
recognized by each mAb (the fine specificity of their epitopes is not
known, and it is unknown whether these mAbs cross-inhibit each other)
or may simply reflect differing affinities for CR1. In a single
experiment, the other mAbs in Table I were also assessed for their
ability to inhibit rosette reformation in clone R29. No inhibition was
seen with any of the other mAbs.
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Because the inhibition of the rosette reformation assay appeared
to be more sensitive than the rosette reversal assay, we used the
former to examine the ability of C3b dimers to inhibit rosette
reformation in clone R29. Consistent with the other data, rosetting was
inhibited by 50% in the presence of 100 µg/ml (300 nM) of C3b
dimer (Fig. 3). Taken together, the
results from the deletion mutants, the mAbs, and the C3b dimers all
indicate that the region of CR1 required for P. falciparum
rosetting maps to the C3b binding site in LHR-B and LHR-C, and
particularly involves SCRs 10 and 17.
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One interpretation of the above data is that rosetting might occur
by C3b bridging between the infected erythrocyte and CR1. To examine
this hypothesis, we grew parasites R29, TM180, and
PAR+ in complete RPMI 1640 containing 10%
C3-deficient serum and compared rosetting to control cultures in RPMI
1640 with 10% normal human serum. Parasites in C3-deficient serum
invaded erythrocytes as efficiently as the control cultures (data not
shown) and were morphologically normal. Rosetting in C3-deficient serum
was the same as that seen in the control cultures (Table II), indicating that C3b is not required
for P. falciparum rosetting.
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The above experiments with parasite clone R29 show that
approximately half to two-thirds of the rosettes are inhibited by CR1
mAbs and deletion mutants. Clearly other erythrocyte molecules may also
contribute to rosetting in this parasite, and other candidate receptors
that have been identified include CD36 (20), ABO blood
group oligosaccharides (17), and heparan sulfate-like
molecules (22). Serum factors such as Igs in normal human
serum also play a role in rosetting in some parasite lines
(23, 24, 25), possibly by stabilizing the interaction between
the infected and uninfected erythrocytes (25). The extent
to which these various rosetting mechanisms operate in different
parasite isolates is not well understood. Therefore, we set out to
study the role of CR1-mediated rosetting in a range of laboratory
strains and natural parasite populations. We tested the ability of the
CR1 mAb J3B11 to reverse rosettes in various isolates, and, in some
experiments, we also studied the effect of mAb OKM5 that reverses
CD36-mediated rosetting (20) and the effect of a rabbit
polyclonal antihuman Ig reagent. In three of four laboratory strains
(including R29), rosettes were reversed by J3B11, none were reversed by
the CD36 mAb OKM5, and three of four were reversed by the anti-Ig
Abs (Fig. 4A), including clone
PAR+ which has been shown previously to be highly
dependent on IgM for the formation of rosettes (23, 24).
In 14 of 15 Kenyan field isolates, rosetting was reduced to <75% of
the control value in the presence of J3B11, and in several of these
isolates rosetting was virtually abolished (Fig. 4B). The
antihuman IgA/IgG/IgM polyclonal Ab also reversed rosetting in many of
these isolates, although to a lesser extent than J3B11 (Fig. 4B). No rosette reversal occurred with the CD36 mAb,
confirming that CD36 only rarely plays a role in rosetting
(26). In the Malawi isolates, in 5 of 10 isolates,
rosettes were reversed by J3B11 (other Abs not tested) (Fig. 4B). These data show that CR1 is important in rosetting in
the majority of field isolates and confirm that Igs also have a role to
play in many isolates.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results also confirm the reports of others (23, 24, 25) that Igs found in normal human serum are required for rosetting, as rosetting was inhibited by polyclonal Abs to human Igs in 3 of 4 laboratory strains and 11 of 15 field isolates. It is unclear whether the requirement for Ig is linked in any way to the CR1-dependent rosetting mechanism. One hypothesis that would combine the two phenomena is that binding of a natural Ab to the surface of infected erythrocytes (possibly to PfEMP1) leads to complement activation and the deposition of C3b on the infected erythrocyte surface, which then contributes to rosetting by binding to CR1. However, as described above, in this report we have excluded a role for C3b in rosetting, and current data also indicate that Ig of defined specificity is not involved in the rosette-enhancing effect. In one report, two different monoclonal IgM preparations derived from patients with Waldenström’s macroglobulinemia were able to support rosetting (23), suggesting that the effect of Ig is not due to recognition of a particular Ag, but occurs through a nonspecific aggregating effect.
Despite the apparent heterogeneity in rosetting mechanisms described to date (8, 17, 20, 22, 23, 24, 25), the data described in this report indicate a CR1-dependent mechanism of rosetting that occurs commonly and could, therefore, be a target for rosette-reversing therapies to treat severe malaria. The mapping studies, however, indicate that the region of CR1 required for rosetting includes the C3b binding site, and the only effective rosette reversing mAb, J3B11, is known to inhibit binding of C3b to CR1 (14). Therefore, although interventions aimed at inhibiting CR1-dependent rosetting may be feasible, they could have the dual effect of interfering with other CR1 functions such as phagocytosis and clearance of immune complexes (1). Due to the intricate host-parasite interactions seen in malaria infection, such intervention could have harmful as well as beneficial effects and clearly should not be embarked upon without considerable forethought and further research.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alex Rowe, Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JT, U.K.
3 Abbreviations used in this paper: CR, complement receptor; LHR, long homologous repeat; PfEMP1, P. falciparum erythrocyte membrane protein 1; SCR, short consensus repeat.
Received for publication July 3, 2000. Accepted for publication August 30, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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