Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3116-3119
Impairment of Plasmodium falciparum Growth in Thalassemic Red
Blood Cells: Further Evidence by Using Biotin Labeling and Flow
Cytometry
By
Kovit Pattanapanyasat,
Kosol Yongvanitchit,
Pongsri Tongtawe,
Kalaya Tachavanich,
Wanchai Wanachiwanawin,
Suthat Fucharoen, and
Douglas S. Walsh
From the Center of Excellence for Flow Cytometry, Office for Research
and Development; the Department of Pediatrics, the Department of
Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University,
Bangkok, Thailand; the Department of Immunology and Medicine, US Army
Medical Component, Armed Forces Research Institute of Medical Sciences
(AFRIMS), Bangkok, Thailand.
 |
ABSTRACT |
Certain red blood cell (RBC) disorders, including thalassemia, have
been associated with an innate protection against malaria infection.
However, many in vitro correlative studies have been inconclusive. To
better understand the relationship between human RBCs with thalassemia
hemoglobinopathies and susceptibility to in vitro infection, we used an
in vitro coculture system that involved biotin labeling and flow
cytometry to study the ability of normal and variant RBC populations in
supporting the growth of Plasmodium falciparum malaria
parasites. Results showed that both normal and thalassemic RBCs were
susceptible to P falciparum invasion, but the parasite
multiplication rates were significantly reduced in the thalassemic RBC
populations. The growth inhibition was especially marked in RBCs from
-thalassemia patients (both -thalassemia1/-thalassemia2 and
-thalassemia1 heterozygote). Our observations support
the contention that thalassemia confers protection against malaria and
may explain why it is more prevalent in malaria endemic areas.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
THE HIGH FREQUENCIES of genetically
abnormal red blood cells (RBCs) in persons from endemic areas of
Plasmodium falciparum malaria are thought to have evolved
through balanced polymorphisms resulting from protection of RBC
variants against this parasite infection.1,2 Various forms
of thalassemia, an inherited autosomal recessive hemolytic anemia
associated with diminished or absent expression of either the
- or 
-globin genes, are common throughout tropical
countries, including Thailand.3-5 Several clinical and
epidemiologic studies argue that thalassemia genes may confer
protection against malaria.6-8 The in vitro culture of
P falciparum in RBCs developed by Trager and
Jensen9 has been adapted to describe the resistance of
thalassemic RBCs to P falciparum infection.2,10,11
However, some studies were unable to show inhibited parasite growth in
thalassemic RBCs.12,13 This limitation could be due to
subtle variations in culture conditions or the requisite manipulation
of RBC parasite cultures because relative rates of parasite growth were
measured with normal and abnormal RBCs in separate dishes or wells. To
overcome this obstacle, we have recently developed a culture system in
which parasites are simultaneously cultured in two different RBC
populations, one of which is biotinylated.14 This method
offers a direct simultaneous comparison of parasite growth over time in
two RBC populations and minimizes the inherent variability among
dish-to-dish or well-to-well comparisons. We used this novel coculture
system to compare the level of in vitro growth of the P
falciparum parasite in RBCs from persons with various forms of -
and -thalassemia syndromes with normal RBCs.
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MATERIALS AND METHODS |
Blood samples.
After informed consent, 5 mL of venous blood was obtained from each
volunteer, preserved in sterile citrate dextrose solution, and used
within 1 week. Blood samples were collected from healthy control
volunteers (n = 35) and persons with abnormal RBCs (n = 139),
consisting of 28 classical HbH disease
(-thalassemia1/-thalassemia2;o+;
/), 17 HbH with Constant Spring
(-thalassemia1/CS; /CS), 15 heterozygous -thalassemia1, 28 nonsplenectomized
o-thalassemia with HbE disease (-thalassemia/HbE),
30 splenectomized -thalassemia/HbE, 11 heterozygous -thalassemia,
and 10 HbE heterozygotes. A diagnosis of Hb types for all subjects was
made by standard hematologic techniques and gel
electrophoresis.15 All thalassemic subjects had normal G6PD
levels, no evidence of concurrent infection, and none had received a
blood transfusion for at least 3 months. RBCs from a group of 10 frequent blood donors known to support robust-malaria parasite growth
were used as a reference standard.
Parasite culture.
A P falciparum strain (TM267TR) from Thailand was maintained in
normal group O RBC suspensions at 37°C, 5% CO2
atmosphere in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented
with 10% heat-inactivated AB-positive serum with 2 mmol/L L-glutamine (Flow Laboratories, Herts, UK), and 25 mmol/L HEPES buffer (Calbiochem, San Diego, CA). A cyanmethemoglobin method for measuring hemoglobin leakage indicated that this culture medium was nontoxic for normal and
thalassemic RBCs. The medium was changed daily to maintain optimal pH
and nutrient levels. The sorbitol lysis method was used to synchronize
parasite growth.16
Culture of parasite in two different RBC populations.
We used a modified coculture system in which parasites were
simultaneously grown in a mixture of two distinct RBC
populations.14 Briefly, a synchronous collection of
parasites at 90% ring (young) stage in either normal control RBCs or
thalassemic RBCs was used for initiating culture. The parasitemias at
the initiation of incubation were nearly equal in all experimental and
control cultures. An aliquot containing an equal number of infected
RBCs of either group were mixed and added into 1 mL of a coculture
containing 200 × 106 RBCs each of normal and abnormal
RBCs. One of the two RBC populations was prelabeled with biotin
(sulfosuccinimidyl 6-biotinamido hexanoate) (Pierce & Warriner, Ltd,
Rockford, UK) at a concentration of 0.3 pg/cell, an amount that does
not affect the growth rates or intraerythrocytic development of
parasites in either normal or thalassemic RBCs.14 Three
replicates of 200 µL each of RBC coculture were transferred into
96-well coster flat-bottomed microtiter plates. Aliquots of 5 × 106 cultured RBCs were taken from the cocultures at the end
of the first or second schizogonic cycles and incubated with 10 µL of titrated streptavidin fluorescein isothiocyanate (FITC) (Amersham, Arlington Heights, IL) for 30 minutes at 4°C in the dark. The cells
were washed twice in cold phosphate-buffered saline (PBS), and the cell
pellet was mixed with the vital stain hydroethidine (Polysciences, Inc,
Warrington, PA) at a concentration of 5 µg/mL in PBS for at least 30 minutes at 37°C before flow cytometric analysis. Parallel control
experiments were conducted simultaneously by culturing normal control
and thalassemic RBCs alone with malaria parasites.
Flow cytometric analysis.
Analysis of the RBCs for biotin/streptavidin-FITC and parasite DNA
content was performed by using a FACScan flow cytometer (Becton
Dickinson, San Jose, CA) equipped with a 15-mW argon ion laser tuned at
488 nm. Logarithmic green and red fluorescences of FITC and ethidine
were measured through 530/30 and 585/42 band pass filters,
respectively. RBCs were gated on the basis of their logarithmic
amplification of the forward scatter and 90° light scatter signals.
Instrument fluorescence calibration and sensitivity were calibrated
using Calibrite beads (Becton Dickinson). A total of 30,000 RBCs in
replicate wells were analyzed for each sample.
Data were analyzed with CellQuest software (Becton Dickinson). Results
were expressed as percent parasitemia of both unbiotinylated and
biotinylated RBCs containing parasite DNA by using a two-parameter cytogram analysis. The multiplication rate data from each paired experiment were presented as a relative percent parasitemia ratio between thalassemic and normal RBCs. In addition, all experimental results were compared with a reference standard established from 10 frequent blood donors. Parasite morphology and the number of parasites
in each culture were also determined from Giemsa blood smears.
Statistical analysis.
The statistical significance of difference between results was
determined by the Mann-Whitney U-test. P values of .05 or less were considered significant.
| |
RESULTS |
The use of biotin/streptavidin-FITC and the DNA fluorochrome
hydroethidine enabled simultaneous flow cytometric analysis of the two
different RBC populations and the parasitemias.
First, to verify that biotinylation would not affect the invasion or
growth of malaria parasites, P falciparum-infected
biotinylated and unbiotinylated RBCs stained for both surface biotin
and intraerythrocytic parasite DNA were compared. Figure
1, a two-parameter dot plot of the
unbiotinylated normal and biotinylated normal RBCs, indicates that
growth rates were similar (Fig 1A). Similar levels of parasitemias and
growth rates were also observed in unbiotinylated and biotinylated thalassemic RBCs.
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| Fig 1.
Representative two-parameter dot plot of P
falciparum-infected unbiotinylated red blood cells (RBCs) and
biotinylated RBCs stained with biotin (x-axis) and hydroethidine
(y-axis). (A) Unbiotinylated normal RBCs and biotinylated normal
control RBCs; (B) unbiotinylated thalassemia RBCs and biotinylated
normal control RBCs. Upper left quadrant shows unbiotinylated RBCs with
stained parasite DNA, upper right quadrant shows double staining of
both biotinylated RBCs and parasite DNA. Lower left quadrant represents
noninfected unbiotinylated RBCs and lower right quadrant shows
noninfected biotinylated RBCs. Percent parasitemia in unbiotinylated
and biotinylated normal control RBCs are 17.3 and 17.9, respectively
(A); percent parasitemia in unbiotinylated thalassemic RBCs and
biotinylated normal control RBCs are 1 and 17.7, respectively (B).
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Then, thalassemic RBCs were cocultured with normal RBCs. The relative
multiplication rates of parasites cultured in all thalassemic genotypes
tested were significantly lower than that of normal RBCs
(P < .0001). Figure 1B shows a comparison of unbiotinylated HbH RBCs containing a lower parasitemia than the biotinylated normal
RBCs. A similar pattern was also seen for HbH/CS
(-thalassemia1/CS) and other thalassemias of both
nonsplenectomized and splenectomized -thalassemia/HbE subjects.
RBCs from nonsplenectomized -thalassemia/HbE supported a parasite
multiplication rate that was 0.61 ± 0.32 of normal control and 0.72 ± 0.26 for splenectomized -thalassemia/HbE (Fig
2). No significant difference in
multiplication rate between nonsplenectomized- and splenectomized-
-thalassemia/HbE was found (P = .17). For -thalassemia
trait and HbE trait, the multiplication rates were 0.68 ± 0.16 and
0.66 ± 0.14 of control, respectively. A similar decrease in the
multiplication level was also seen in -thalassemia RBCs. HbH was
least able to support parasite growth. The multiplication rates when
compared to normal control were 0.44 ± 0.29, 0.58 ± 0.20, and 0.50 ± 0.22 for HbH, HbH/CS, and -thalassemia1 trait, respectively. There was a significantly lower multiplication rate in
-thalassemia when compared with splenectomized -thalassemia/HbE, particularly -thalassemia1 trait and HbH RBCs
(P < .004 and .002, respectively). For nonsplectomized
-thalassemia/HbE (P < .05 and P = .28 for HbH
and -thalassemia1 trait). There was no significant difference between the parasite multiplication rate in normal control
RBCs and the reference standard group.
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| Fig 2.
P falciparum-multiplication rates in normal
control and thalassemic red blood cells expressed as a relative
parasitemia in normal controls. Dashed line represents a reference
standard established from frequent blood donors.
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| |
DISCUSSION |
To better understand the "malaria hypothesis" in which
hemoglobinopathies may confer protection against
infection,1 we have recently developed a novel technique in
which malaria parasites are simultaneously cultured in two RBC
populations.14 This is achieved by the biotinylation of one
RBC population that is then mixed with another unbiotinylated RBC
population together with P falciparum parasites. By using this
coculture system, we found that RBCs from normal and thalassemic
subjects were equally susceptible to merozoite invasion as indicated by
a measurable parasitemia after the first growth cycle (schizogony).
However, in subsequent growth cycles, thalassemia RBCs were
significantly less supportive of parasite growth than were normal RBCs
(Fig 2). These in vitro findings indicate that parasite growth in
thalassemia RBCs is significantly diminished, consistent with recent in
vitro findings that poor re-invasion rates are noted in the second and
third cycles of parasites in thalassemic RBCs.17 These data
are also consistent with clinical observations that describe fewer or
milder P falciparum malaria infections in people with
thalassemia.6-8
In comparison with parasite growth in the cocultured normal RBCs and
thalassemic RBCs, the level of inhibition of growth support of P
falciparum among the abnormal RBCs varied (Fig 2).
However, the mean multiplication rate in each type of thalassemic RBCs was lower than that obtained in normal RBCs. Variability in RBCs from
the same type of thalassemia suggested that the severity of each
individual's disease (anemia/Hb content) may be involved. Moreover,
other factors such as RBC age,17 RBC deformability, as well
as individual membrane properties may affect growth rates of P
falciparum.18,19 A protective role of relatively less surface area of microcytic RBCs available for parasite invasion was
also suggested.20,21 Accumulation of unmatched - and
-globin chains in the cell and in the membrane cytoskeleton could
also lead to abnormal linkage Hbs resistant to parasite
protease,22 associated with membrane damage by increased
generation of free oxygen radical.23,24
Interestingly, our in vitro study showed that parasite growth was
especially low in -thalassemia RBCs, especially from HbH RBCs and in
-thalassemia1 trait, indicating that differences in
thalassemic genotypes may confer different levels of protection against
malaria. The -thalassemia RBCs were more resistant to parasite
growth than -thalassemia RBCs, a finding that may relate to
inclusion bodies, known to accumulate in vivo from excess -globin chain.4 However, in vitro demonstration of
this phenomenon would require addition of redox dyes or elevation of
temperature,4,25,26 manipulations that would severely alter
the established culture conditions and potentially lead to aberrant
parasite growth. Overall, that less severe disease among persons with
-thalassemia is associated with a selective advantage against
malaria infection may account for the relatively high prevalence of
-thalassemia in comparison with -thalassemia in Southeast
Asia.3,5
In summary, the combination of biotin/streptavidin-FITC enabled
simultaneous flow cytometric analysis and parasite growth rate of the
two distinct RBC populations. With this approach, the inhibitory effect
of several different forms of thalassemia has been shown in vitro. The
mechanism of this protection is unclear but may be because of the
interaction between thalassemia phenotype, modifications of the RBC
membrane, and abnormal intracellular environment. The biotin-labeled
RBC coculture method may be useful in defining these mechanism(s).
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FOOTNOTES |
Submitted May 8, 1998; accepted December 14, 1998.
Supported in part by Siriraj-China Medical Board, Grant No. 75-348-221 and by the Malaria Program of the US Army Medical Research and Materiel
Command, Ft Detrick, MD.
The opinions or assertions contained herein are the private views of
the authors and are not to be construed as official or as reflecting
the views of the Department of the Army or the Department of Defense.
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 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Kovit Pattanapanyasat, PhD, Office for
Research and Development, Faculty of Medicine, Siriraj Hospital,
Bangkok 10700, Thailand.
| |
REFERENCES |
1.
Weatherall DJ:
Common genetic disorders of the red cell and the "malaria hypothesis."
Ann Trop Med Parasitol
81:539, 1987[Medline]
[Order article via Infotrieve]
2.
Nagel RL, Roth EF Jr:
Malaria and red cell genetic defects.
Blood
74:1213, 1989[Abstract/Free Full Text]
3.
Wasi P, Pootrakul S, Pootrakul P, Pravatmung P, Winichagoon P, Fucharoen S:
Thalassemia in Thailand.
Ann NY Acad Sci
344:352, 1980[Medline]
[Order article via Infotrieve]
4.
Weatherall DJ, Clegg JB:
Thalassaemia Syndromes (ed 3). Oxford, UK, Blackwell Scientific Publication, 1981.
5.
Wasi P:
Haemoglobinopathies in Southeast Asia, in
Bowman J
(ed):
Distribution and Evolution of the Hemoglobin Gene Loci. New York, NY, Elsevier Science, 1983, p 179.
6.
Willcox M, Bjorkman A, Brohult J:
Falciparum malaria and -thalassemia trait in northern Liberia.
Ann Trop Med Parasitol
77:335, 1983[Medline]
[Order article via Infotrieve]
7.
Flint J, Hill AVS, Bowden DK, Oppenheimer SJ, Sill PR, Serjeantson DJ, Bana-Koiri J, Bhatia K, Alpers MP, Boyces AJ, Weatherall DJ, Clegg JB:
High frequencies of -thalassemia are the result of natural selection of malaria.
Nature
321:744, 1986[Medline]
[Order article via Infotrieve]
8.
Oppenheimer SJ, Hill AVS, Gibson FD, Macfarlane SB, Moody JB, Pringle J:
The interaction of alpha thalassaemia with malaria.
Trans R Soc Trop Med Hyg
81:332, 1987
9.
Trager W, Jensen JB:
Human malaria parasites in continuous culture.
Science
193:673, 1976[Abstract/Free Full Text]
10.
Friedman MJ, Roth EF, Nagel RL, Trager W:
The role of hemoglobin C, S and Bart in the inhibition of malaria parasite development in vitro.
Am J Trop Med Hyg
28:777, 1979
11.
Ifediba TC, Stern A, Ibrahim A, Rieder RF:
Plasmodium falciparum in vitro: Diminished growth in hemoglobin H disease erythrocytes.
Blood
65:452, 1985[Abstract/Free Full Text]
12.
Friedman MJ:
Oxidant damage mediates variant red cell resistance to malaria.
Nature
280:245, 1979[Medline]
[Order article via Infotrieve]
13.
Santiyanont R, Wilairat P:
Red cells containing hemoglobin E do not inhibit malaria parasite development in vitro.
Am J Trop Med Hyg
30:541, 1981
14.
Pattanapanyasat K, Yongvanitchit K, Heppner DG, Tongtawe P, Kyle DE, Webster HK:
Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.
Cytometry
25:287, 1996[Medline]
[Order article via Infotrieve]
15.
Winichagoon P, Adirojnanon P, Wasi P:
Levels of haemoglobin H and proportions of red cells with inclusion bodies in the two types of haemoglobin H disease.
Br J Haematol
46:507, 1980[Medline]
[Order article via Infotrieve]
16.
Lambros C, Vanderberg JP:
Synchronization of Plasmodium falciparum erythrocytic stage in culture.
J Parasitol
65:428, 1979
17.
Senok AC, Li K, Nelson EAS, Yu LM, Tian LP, Oppenheimer SJ:
Invasion and growth of Plasmodium falciparum is inhibited in fractionated thalassaemic erythrocytes.
Trans R Soc Trop Med Hyg
91:138, 1997[Medline]
[Order article via Infotrieve]
18.
Schrier SL, Rachmilewitz E, Mohandas N:
Cellular and membrane properties of alpha and beta thalassemic erythrocytes are different. Implication for differences in clinical manifestations.
Blood
74:2194, 1989[Abstract/Free Full Text]
19.
Schrier SL:
Thalassemia: Pathophysiology of red cell changes.
Annu Rev Med
45:211, 1994[Medline]
[Order article via Infotrieve]
20.
Nurse GT:
Iron, the thalassaemia, and malaria.
Lancet
3:938, 1979
21.
Luzzi GA, Torri M, Aikawa M, Pasvol G:
Unrestricted growth of Plasmodium falciparum in microcytic erythrocytes in iron deficiency and thalassemia.
Br J Haematol
74:519, 1990[Medline]
[Order article via Infotrieve]
22.
Geary TG, Delaney EJ, Klotz IM, Jensen JB:
Inhibition of the growth of Plasmodium falciparum in vitro by covalent modification for hemoglobin.
Mol Biochem Parasitol
9:59, 1983[Medline]
[Order article via Infotrieve]
23.
Rachmilewitz EA, Shinar E, Shalev O, Galili V, Schrier SL:
Erythrocyte membrane alterations in -thalassaemia.
Clin Haematol
14:163, 1985[Medline]
[Order article via Infotrieve]
24.
Scott MD, Eaton JW:
Thalassaemic erythrocytes: Cellular suicide arising from iron and glutathione-dependent oxidation reactions?
Br J Haematol
91:811, 1995[Medline]
[Order article via Infotrieve]
25.
Scott GL, Rasbridge MR, Grimes AJ:
In vitro studies of red cell metabolism in haemoglobin H disease.
Br J Haematol
18:13, 1970[Medline]
[Order article via Infotrieve]
26.
Wickramasinghe SN, Hughes M, Higgs DR, Weatherall DJ:
Ultrastructure of red cells containing haemoglobin H inclusions induced by redox dyes.
Clin Lab Haematol
3:51, 1981[Medline]
[Order article via Infotrieve]
TOP
ABSTRACT
INTRODUCTION
