1. Art and Diseño

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Natural Protection Against Severe Plasmodium falciparum Malaria
Due to Impaired Rosette Formation
By Johan Carlson, Gerard B. Nash, Vilma Gabutti, Fadwa AI-Yaman, and Mats Wahlgren
Genes for two lethal diseases, thalassemia and sickle cell
anemia, are favored by evolutionbecause, in their heterozygous form, they protect against cerebral malaria. Rosette
formation, the binding of uninfected redcells (RBCsI to Plssmodium fabiparuminfected RBCs (PRBCsI, has previously
been foundto be associatedwith cerebral malaria,the most
important sovere manifestation of P fa/ciparum malaria. We
show here that thalassemic RBCs and, under certain conditions, even hemoglobinS (HMI-containing RBCs possess an
impaired abilityto bind to PRBCs, forming small and weak
erythrocyte rosettes compared
with rosettes formed by normal RBCs. This decreasedrosetting a b i l i is associated with
the small size of the thalassemic RBCs and with distortion
of the mechanical properties of HbS-containing RBCs. The
impairment of rosette formation may hinder the development of cerebral malaria by abatement of sequestration.
0 7994 by The American Society of Hematology.
E
cerebral malaria exhibit an impaired rosette-forming ability,
with data suggesting that this is a mechanism that could
mediate natural protection against the disease.
XCESSIVE BINDING of Plasmodium falciparum-infected erythrocytes [parasitized red blood cells
(PRBCs)] to endothelial cells and to uninfected red blood
cells (RBCs) seems crucial for the occurrence of microcirculatory obstruction in human cerebral malaria, but the underlying processes that lead to this state of infection are comp l e ~ . "One
~ attractive approach to the understanding of the
pathophysiologic mechanism is the study of the red cell
defects known to protect against severe malaria. It has been
shown that individuals with a- and &thalassemia and with
sickle cell trait (HbAS) can acquire P fakiparum malaria,
but that they experience reduced fatality and seventy of
the disease;"" eg, the protection against cerebral malaria in
children with HbAS is more than 90%, but the effect on
parasite densities is less pronounced." Blood group 0 was
recently found also to be associated with significant resistance to cerebral malaria as compared with blood group A
or B (Hill" and Hill et al, personal communication, 1992).
It has been suggested that the hampered growth and an increased tendency to sickling of HbAS-infected cell^'^"^ and
neoantigen expression on thalassemic RBCS'~.'~
are the reasons for the protective effects. However, while these mechanisms may play roles in the resistance to malaria, they do
not fully explain the absence of microcirculatory obstruction
seen in individuals with aberrant red cells. Could it be that
these RBCs are less prone to forming rosettes and to binding
to endothelial cells and, therefore, hinder the development
to coma and death?
Increased frequency of spontaneous erythrocyte rosetting
around PRBCs has been shown to be associated with cerebral
m a l a ~ i a .At
~ . ~autopsy Hidayat et a l l 8 observed "rosetting of
the parasitized erythrocytes . . . within the partially occluded lumens," and in exvivo experiments, the obstruction
of the blood flow was found to be considerably more pronounced with a rosette-forming parasite than with a nonrosetting parasite that merely bound to the vascular endothelium." Moreover, anti-rosetting antibodies were frequent in
sera of children with mild malaria but absent or only present
at low levels in those with severe di~ease.4.~
Thus, rosetting
has been proposed to play a keyrole in the excessive sequestration of PRBCs and RBCs in the microvasculature and in
the pathogenesis of cerebral disea~e.4.'.'~-~~
Therefore, it
could be postulated that the protective effect of certain red
cell disorders against cerebral malaria is mediated via impaired rosette formation. We investigated a number of red
cell disorders with respect to their impact on rosetting and
report that RBCs from individuals naturally protected against
Blood, Vol 84, No 11 (December l ) , 1994: pp 3909-3914
MATERIALS AND METHODS
P falciparum culture. The P falciparum R'PA1 parasite, a
cloned, rosetting parasite obtained from the Palo Alto Ugandan
strain, was cultured according to standard procedures**with10%
h
'serum added to the buffered malaria culture menormal AB+ R
dium (MCM).
Erythrocytes. Blood was drawn into heparinized tubes or tubes
containing citrate phosphate dextrose (CPD), and the RBCs were
washed three times in TRIS-Hanks' solution. AB0 blood group
typing was performed by hemagglutination with monoclonal antibodies specific for the different blood group antigens (BioCarb,
Lund, Sweden). RBC morphology was evaluated by light microscopy, and the erythrocyte mean cell volume (MCV) and hemoglobin
concentration were measured by a Coulter S Plus (Coulter Electronics Ltd, Luton, UK). Hemoglobin electrophoresis and electrofocusing were used to quantify the hemoglobin type of the RBCs. RBCs
were obtained from four individuals with sickle cell disease (HbSS),
from three that were of the sickle cell trait (HbAS), and from one
exhibiting the hemoglobin SC (HbSC) phenotype. Samples from
nine Papua New Guineans with Southeast (SE) Asian ovalocytosis
contained 11.8% to 89.2% ovalocytic cells as counted in 500 RBCs
with a 40X objective lens. Of the 19 individuals with P-thalassemia
From the Microbiology and Tumor Biology Center, Karolinska
Institute, and the Swedish Institute for Infectious Diseases Control,
Stockholm, Sweden: the Department of Infectious Diseases, Uppsala
University9 Akademiska sjukhuset, Uppsala, Sweden: the Department of Haematology, University of Birmingham, Birmingham, UK;
the Department of Pediatrics, University of Torino, Torino, Italy:
and the Institute of Medical Research, Madang, Papua New Guinea.
Submitted January 18, 1994; accepted July 27, 1994.
Supported by grants from the United Nations Development Program/World BanVWorld Health Organization Special Programme
for Research and Training in Tropical Diseases (TDR), The Maud
and Birger Gustavsson Foundation, the Swedish Medical Research
Council, the Swedish Agency for Research Cooperation with Developing Countries (SAREC), and the Swedish Society of Medicine.
Address reprint requests to Mats Wahlgren, MD, Microbiology
and Tumor Biology Center, Karolinska Institute, Box 780, S-l71 77
Stockholm, Sweden.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 W.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8411-0041$3.00/0
3909
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CARLSON ET AL
3910
trait, 14were ItaliansorWestAfricanswith
8'. one was a Thai
with the b/p-thalassemia type. and three were West Africans with
combined p+/HbS. All the 0-thalassemic RBCshad a lowMCV.
Three of four individuals with a-thalassemia trait were of the athal I phenotype, while one (Thai) was of the Hb Constant Spring
phenotype. All of these RBC samples exhibited a moderate microcytosis. RBCs werealso obtained from three patients with microcytic
anemia(secondary to chronicirondeficiency
or severebacterial
infection), and from six samples of cord blood with high HbF contents (67% to 85% HbF).
Assessment of rosette formationand disruption of rosettes. The
R+PAI strain grown in 0' RBCs was subcultivated in blood from
donors with normal (HbAA), &thalassemia trait, or HbAS RBCs,
and assessment for rosette formation was made as described previously.'.' The rosetting rate was defined as the number of PRBCs
in rosettes, expressed as a percentage of the total number of latestage (trophozoite and schizont) PRBCs. The individual rosette size
was defined as the mean number of uninfected RBCs bound to each
PRBC." Rosettes in culture were disrupted mechanicallyby drawing
the erythrocyte mixture through a narrow gauge injection needle six
to eight times.', Assessment of rosette formation was made after
spontaneous reformation of rosettes, and different aliquots from the
same culture were compared after different pretreatments.
Carboq-Juorescein diacetate (C-FDA)-labelingof RBCs and assessment of rosette-forming capacip. The relative rosette-forming
capacity of RBCs from patients with various red cell disorders was
measured as described before, by a method where C-FDA-labeled
RBCs were allowed to
compete for rosetting with unlabelled RBCs."
The R'PAI strain was cultured in blood group 0 (HbAA) RBCs,
and rosettes were disrupted by addition of heparin (50 IUlmL; Kabi
Pharmacia AB, Stockholm, Sweden).
Fractionation
and
deoxvgenation
of HbS-containing
RBCs.
HbSS or HbSC RBCs were fractionated ona Ficoll-Isopaque continuous density gradient (Pharmacia AB, Uppsala, Sweden), andthe
less dense cells were harvested as described." Cyclical deoxygenation-reoxygenation was performed for 15 hours with a Cyclical Gas
Exchanger(WolfsonResearchLaboratories,Birmingham,
UK),'s
causing changes in cell hydration and deformability that mimic the
deterioration that occurs for dense cells in vivo.'" In separate experiments, HbAS, HbSS, or HbSC RBCs were deoxygenated by adding
a mixture of sodium dithionite and disodium hydrogen phosphate
(2 v01 0.1 14mol& Na2S20, + 3 v01 0. I 14 molL Na'HPO,;pH
6.8) to the erythrocyte suspension in proportion 1 : I .''
The morphologyof the RBCs was studied before and
after cyclical
deoxygenation-reoxygenation or treatment with dithionite, after fixation with I % glutaraldehyde.HbASRBCsandlessdenseHbSS
RBCs were essentially discocytic, except that the latter contained a
few distorted, boat-shaped cells (irreversibly sickled cells, ISCs).
Treatment with dithionite led to the formation of distortedcells and
of ISCs, and few discocytes remained (about 30% for HbAS cells
and less for HbSS cells). Cyclical deoxygenation of the less dense
HbSSRBCs also led to formation of distortedRBCs, as well as
some ISCs(8% on average). A distinction shouldbe made, however,
between dithionite-treated cells, which are distortedby the formation
of polymerized HbS, and cells subject to cyclical deoxygenationreoxygenation, which dehydrate and accumulate membrane damage
but do not contain polymer when reoxygenated
for further study.
No changes were seen in HbAARBCs treated with either ofthe
above-mentioned methods.
Measurement of eythrocyte binding strength bv a micropipette
in
method. Samples of malaria cultures werediluted100-fold
MCM,andthesuspensionwasplaced
in a micropipette chamber
where the binding strength of individual RBCs within rosettes was
studied by a dual-micropipettemethod (Fig l), as describedpreviously.*' The aspiration pressure (P) required to detach cells from
Fig 1. The dual-micropipettetechnique.Theparasitized
RBC
(right) is held by the larger holding pipette, and a bound RBC is
detached by increasingthe aspiration pressurein the smaller pipette
(left).
rosettes by a micropipette (internal diameter, D) was recorded, and
theforcewascalculated
[F = (n/4)(D2)P]. In someexperiments,
after all the cells had been stripped froma rosetting, parasitizedcell,
other cells in the chamber were picked up with one of the pipettes
and brought into contact with the stripped parasitized cell, allowing
cell-cell binding to occur. After a delay of 3 minutes, the force to
detach the nonparasitized cell was measured, as described.2R
RESULTS
RBCs from individuals with various red cell disorders
were examined for their capacity to form rosettes in competition with normal(HbAA) 0,Rh' RBCs. Whereas cord RBCs
with a high HbF content or RBCs from individuals with SE
Asian ovalocytosis bound equally well or only slightly less
as compared with normalRBCs, thalassemic RBCs and other
microcytic RBCs exhibited a reduced rosette-forming capacity, as did HbS-containing RBCs under deoxygenated conditions (Fig 2).
Thus, both a- and 0-thalassemic RBCs exhibited a reduced rosette-forming capacity when competing with RBCs
from normal (HbAA) individuals (Fig 2). When RBCs from
four individuals with 0-thalassemia trait were comparedwith
RBCs fromfivenormal (HbAA) individuals, a significant
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391 1
IMPAIRED ROSETTE FORMATION IN MALARIA
reduction in mean binding strengthwas found forthe smaller
,&thalassemic RBCs (Fig 3). These studies also confirmed
thepreviouslyreportedfinding
of astrain-specific A B 0
blood group preferencein rosette-forming capacity. Ahigher
bindingcapacityandlarger
rosetteswere foundfor the
R'PA1 clone with blood group A/AB RBCs compared with
group O/B RBCS,'~ and similarly, when using the same P
5
4
I
HbAA
I
T
6
T T
3
HbF
2
8
Ovalocytosis
I
1
HbAs
0
I
&thalassemia
HbAA
Fig 3. Force required to detach individual uninfected RBCs bound
i n a spontaneous rosette aroundP falciparum-infected RBCs. Values
given are mean values 2 SD. The binding strength wascompared by
measuring 50 t o 68 RBCs from every patient. The 0-thalassemia trait
was P+-type1. AB0 blood group
of donors: A, A; 0,
B; 0 0.Statistical
analysis was performed between themean detachment force of the
t w o groups of patients using the nonparametric Mann-Whitney U
test (P= .0121.
Hbss
HbSC
HbEF
HbAA
microcytosis
0 20 40 60 80 100 120
Rosette forming capacity
(% of control)
Fig 2. Relative rosetting capacity of various C-FDA-labeled RBCs
to HbAA infected RBCs. The tested RBCs were from nine individuals
of normal hemoglobin phenotype Inorrno-or microcytic) or from 35
individuals with various red cell disorders (CS, Hb Constant Spring;
S / p , mixed HbS/p-thal trait). m, RBCs under normal ambient 0,;
0, RBCs treated by cyclical deoxygenationlreoxygenation;a, RBCs
deoxygenated by sodium dithinonite.The binding of C-FDA-labeled
RBCs from an 0 Rh+ donor wasused as index (100%). and the blood
group A/AB preference of the strain was compensated for as described p r e v i o ~ s l y . ~ ~
falciparum clone here, stronger binding forces were seen for
RBCs from the HbAAhlood group
A donors than for RBCs
from the HbAAhiood group B or 0 donors (Fig 3). Also
with the P-thalassemic individuals, small ABO-differences
could be noted (Fig 3). When the R'PA1 clone was cultured
in RBCs from 12 individualswith ,&thalassemia traitand
in RBCs from three normal HbAA controls,asignificant
correlation between the rosette-forming capacityof the abberant RBCs and the MCV was found (Fig 4A), imposed on
the blood group ABO-related differences already known to
exist. A similar correlation was also seen between the erythrocyte MCV and the mean individual rosette size: the lower
the MCV, the smaller the rosettes (Fig 4B). No significant
difference wasseen inthe rosetting rate orin the parasitemias
of the cultures (data not shown).
RBCsfromindividuals withmicrocytic anemiadue to
other disorders (eg, severe chronic bacterial infection, iron
deficiency, or HbE disease) were also found to bind poorly
in rosettes when competing with normocytic cells (Fig 2).
A significant reduction ( P < .01, Mann-Whitney U test) in
binding strength for microcytic cells compared with normocyticred cells wasfound when RBCsfromtwo patients
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3912
CARLSON ET AL
with microcytic anemia secondary to iron deficiencykhronic
bacterial infection were compared with ABO-matched controls (binding force: mean, 1.4 X IO"" and 1.6 X 10 -li) N,
respectively, for the microcytic RBC populations; 2.2 X
IO-^'" and 2.5 X 10 "l N for the normocytic).
HbSS or HbAS RBCs bound normally to P falcipururninfected RBCs when competing withHbAA RBCs under
normal oxygen pressure, but after exposure of HbSS cells
to cyclical deoxygenation-reoxygenation or when deoxygenated with dithionite before the assay, the binding capacity
A
4
h
I2O]
50
1
60
.
I
70
.
I
80
MCV
.
I
After
Reformation
Deoxy
Control
HB/HbAS
3.3 It_ 0.8
3.5 2 0.7
2.4 2 0.6
3.3 't 0.6
P < ,0001
NS
Deoxy
Control
NS/HbSS
4.2 f 1.1
4.5 It_ 1.2
3.3 2 0.9
4.2 t_ 1.1
P < ,0001
Deoxy
Control
4.2 f 1.2
4.2 f 1.2
3.2 2 0.9
4.0 2 0.9
P
NNHbAS
NS
'r. ,0001
NS
I
90
100
90
100
2
50
Before
Disruption
+
51
'
Donor
Initials/
Phenotype
The P fakiparum clone R
PA1 was cultured in untreated RBCs
and RBCs deoxygenated by dithionite (see Materials and Methods)
from three different donors. The cultures were assessed for mean
individual rosette size (the number of uninfected RBCs bound toparasitized RBCs f SD) by counting 50 rosettes, and each determination
was performed twice. Statistical analysis was performed between the
individual rosette sizes before and after reformation using the nonparametric Mann-Whitney U test.
Abbreviation: NS, not significant.
0
601".
Table l.Mean Individual Rosette Size Before Disruption and After
Spontaneous Reformation of Rosettes in P falciparum Cultures
Grown in HbAS and HbSS RBCs
60
70
80
MCV
Fig 4. The relationship betweenMCV and the relative erythrocyterosetting capacity or mean individual rosette size. RBCs were obtained from 12 individuals with @thalassemia trait (/3'-type 1) or
from three individuals with normal (HbAA) RBCs and with different
AB0 blood group: A, A;
AB; U, B; 0, 0. Patients with normal
(HbAA) hemoglobin phenotypeare indicated as AA. In both analyses,
50 rosettes wereassessed t w i c e and ontwo independent occasions.
(A) MCV versus erythrocyte-rosetting capacity of C-FDA-labeled
RBCs (when the blood group phenotype
has been compensated
for)?' The statistical analysis was performedwith simple regression
analysis: r = 0.95; 9 = 0.91; P < ,0001.(B)MCV versus mean individual
rosette size. Statistical analysis was performedwith multipleregresparasion analysis, taking intoaccount that, when using the R+PAI
site, RBCs of the AlAB phenotypes form larger rosettes than 018
phenotypes, independent of other parameters": r = .90 8 = .81; P
< .001.
+,
was reduced (Fig 2). When P falciparum (clone R+PAl)
was cultured in REKs from two individuals with sickle cell
trait (HbAS) and one with sickle cell disease (HbSS), no
significant difference was seen in the rosetting rate, the mean
individual rosette size, or the parasitemia compared with
cultures withHbAA RBCs of corresponding A B 0 blood
group (data not shown). However, chemical deoxygenation
of the blood, followed by mechanical disruption and spontaneous reformation of rosettes, induced a significant drop in
mean individual rosette size in the HbAS and HbSS cultures
compared with untreated HbAS/SS controls (Table 1). By
using the micropipette technique, it could also be shown that
HbSS discocytes had a mean binding force not differing
from that of discocytes from a normal (HbAA) individual
(Fig 5). After cyclical deoxygenation-reoxygenation of the
HbSS RBCs, significantly impaired binding capacity was
found in the ISCs and also in the moderately distorted, dehydrated RBC population (Fig 5).
DISCUSStON
Using three independent assays we report that certain aberrant RBCs form small and weak erythrocyte rosettes and
hypothesize that this reduced ability to form erythrocyte rosettes may protect against severe P fakiparum malaria, ie,
by preventing the formation of large stable red cell aggregates and obstructing the cerebral microvasculature otherwise seen in cerebra1 malaria.
Low cell volume, a common denominator of the a- and
P-thalassemias, is associated with a reduced rosette-forming
capacity.29Here we also show that the reduction in rosetting
seems to be related to the low red cell volume (MCV) per
se, irrespective of the origin of microcytosis, as small erythrocytes from HbAA patients with other causes of microcytosis as wellof those with HbE and HbC form weak
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391 3
IMPAIRED ROSETTE FORMATION IN MALARIA
Fig 5. Force required to remove individualuninfected RBCs attached to a rosetting P fakiparumparasitized cell for 3 minutes. Values given are
mean attachmentforce (? SD) as measured by micromanipulation of 28 to 36 uninfected RBCs per
assessed cell population. Cyclical deoxygenation/
reoxygenation was performed with a Cyclical Gas
Exchanger (see MaterialsandMethods). Donors
(initials and hemoglobin phenotype):U, BCIHbAA;
a, MS/HbAA; 0,BT/HbSS; 0 NS/HbSS; @, C S /
HbSS. Statisticalanalysis was performed with Student's ttest for each HbSS donor between thediscocyte and the distorted RBC population and betweenthe
discocyte andthe
ISC populations,
respectively. All differences in mean detachment
forces between the various RBC populations were
statistically significant (all P values < .001).
01
I
HbAA H ~ S Sdiscocytes
discocytes before cyclical
de-/reoxygenation
rosettes. Other investigators have studied features of microcytic cells that might protect against
malaria, such as restriction of parasite growth or impairment of cytoadherence of
infectedcells,but
have not foundconvincingcorrelaa common
tions.'"."" Restricted rosetteformationmaybe
protective mechanism for microcytic RBCs, thus conferring
significant clinical protection against cerebral malariain disorderswithprofound
microcytosis likethe thalassemias.
This hypothesis is supported also by the recentreport by
other investigators of impaired rosette formationof thalassemic RBCs.?' The possible protective effect of impaired rosette formation in other disorders might be moredifficult to
assess (eg, iron deficiency, with a mixed pattern of microcytosis)although malnutrition-andespecially
iron deficiency-has
in factbeenclaimedtoconfer
protection
against malaria. However, this issue is highly controversial,
and polar views have been
A diminished rosette-formingcapacity wasevident already for deoxygenated, slightly distorted HbAS, HbSC, or
HbSS cells. In addition, after repeated sickling of HbSC or
HbSS cells, rosetting was inhibited even when the cells were
oxygenated. Thus, already moderate changes in cellularmechanical properties, knowntooccurnotonly
with HbSS
R B C S ~but
~ also withbothinfected and uninfected HbAS
RBCsunder unfavorableconditions,"j cause asignificant
loss of rosette-binding capacity. The obstruction of the microvasculature and the concomitant lower oxygen
tension
seen in cerebral malaria seems likely to be such an unfavorof inducing changes
able or even extreme condition capable
in the rheologic properties of a significant number of HbAS
cells. Acknowledging thewell-known difficulties in extrapolating in vitro data to in vivo conditions, we nevertheless
suggest that the impaired rosette-forming ability expressed
even in moderatelychangedHbASRBCs,inconjunction
with other potential protective phen~mena,'.""~ may
contribute to the protection against cerebral malaria. Paradoxically,
thispotential mechanism of resistance, aswellasothers
previously de~cribed,'~.~'
seemstoinvolve
all HbS-con-
Discocytes
Distorted RBcS
I
IsCS
HbSS cells after cyclical
deoxygenationheoxygenation
taining RBCs, despite the factthat individuals with homozygoussicklecelldisease
oftensuffer more severely from
malarial disease with high mortality. However, this fact may
mostlikely be attributed to the sickle cell disease per se
despiteaninnateresistanceto
malarialinfection existing
also in homozygote^.^'^^^
Rosette formation is mediated by protein ligands, rosettins, on the infected RBCs that bind to carbohydrate receptors on the uninfected R B C S . ~ ~ , *Whether
~ . ~ " the decreased
rosette binding with microcytotic RBCs is due to a lower
availability and/or a lower expression of the carbohydrate
structures remains to be investigated. For HbSS and HbAS
RBCs, on the other hand, it is not lack
of receptors but
the accessibility of them, influenced by the distortion or
rigidification of the cells, that abatesbinding, as therosetting
capacity is dependent on the ambient oxygen pressure.
Some studies also give support for
a modifying role of SE
Asian and Melanesian ovalocytosisand HbF in P falciparum
malaria.41,42However, the protection conferred by SE Asian
ovalocytosis or a high HbF content does not seem to involve
erythrocyte rosetting.
ACKNOWLEDGMENT
We thank all blooddonorsthatparticipated
in thisstudyand
Drs Rosa Bachetta (DNAX Research Institute, Palo Alto, CA) and
Michael Alpers (Institute of Medical Research, Madaug, Papua, New
Guinea)forinvaluablehelpwiththebloodsamples.Thehelpful
advice on the statistics givenby Johan Bring (Department of Statistics, Uppsala University, Uppsala, Sweden) is also acknowledged.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1994 84: 3909-3914
Natural protection against severe Plasmodium falciparum malaria
due to impaired rosette formation
J Carlson, GB Nash, V Gabutti, F al-Yaman and M Wahlgren
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