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Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2527-2534
Early Phagocytosis of Glucose-6-Phosphate Dehydrogenase
(G6PD)-Deficient Erythrocytes Parasitized by Plasmodium
falciparum May Explain Malaria Protection in G6PD Deficiency
By
Marina Cappadoro,
Giuliana Giribaldi,
Estella O'Brien,
Franco Turrini,
Franca Mannu,
Daniela Ulliers,
Gino Simula,
Lucio Luzzatto, and
Paolo Arese
From the Dipartimento di Genetica, Biologia, Biochimica,
Università di Torino, Torino, Italy; and the Department of
Haematology, Royal Postgraduate School of Medicine,
Hammersmith Hospital, London, UK.
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ABSTRACT |
In population-based studies it has been established that inherited
deficiency of erythrocyte (E) glucose-6-phosphate dehydrogenase (G6PD)
confers protection against severe Plasmodium falciparum (P
falciparum) malaria. Impaired growth of parasites in G6PD-deficient E in vitro has been reported in some studies, but not in others. In a
systematic analysis, we have found that with five different strains of
P falciparum (FCR-3, KI, C10, HB3B, and T9/96), there was no
significant difference in either invasion or maturation when the
parasites were grown in either normal or G6PD-deficient (Mediterranean
variant) E. With all of these strains and at different maturation
stages, we were unable to detect any difference in the amount of P
falciparum-specific G6PD mRNA in normal versus deficient
parasitized E. The rate of 14C-CO2 production
from D-[1-14C] glucose (which closely reflects
intracellular activity of G6PD) contributed by the parasite was very
similar in intact normal and deficient E. By contrast, in studies of
phagocytosis of parasitized E by human adherent monocytes, we found
that when the parasites were at the ring stage (ring-stage parasitized
E [RPE]), deficient RPE were phagocytosed 2.3 times more intensely
than normal RPE (P = .001), whereas there was no difference
when the parasites were at the more mature trophozoite stage
(trophozoite-stage parasitized E [TPE]). Phagocytic removal markers
(autologous IgG and complement C3 fragments) were significantly higher
in deficient RPE than in normal RPE, while they were very similar in
normal and deficient TPE. The level of reduced glutathione was
remarkably lower in deficient RPE compared with normal RPE. We conclude
that impaired antioxidant defense in deficient RPE may be responsible
for membrane damage followed by phagocytosis. Because RPE, unlike TPE,
are nontoxic to phagocytes, the increased removal by phagocytosis of
RPE would reduce maturation to TPE and to schizonts and may be a highly
efficient mechanism of malaria resistance in deficient subjects.
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INTRODUCTION |
THE PROTECTIVE ROLE of erythrocyte (E)
glucose-6-phosphate dehydrogenase (G6PD) deficiency against infection
with Plasmodium falciparum (P falciparum) malaria (see
Luzzatto1 and Greene2 for reviews) is supported
by case-control studies performed in Africa3-5 and
Southeast Asia.6,7 Some studies showed protection for both
hemi/homozygous and heterozygous subjects,6-8 while other
studies indicated that only heterozygous females were protected, while
hemi/homozygous subjects with a more severe degree of G6PD deficiency
were not protected.5,6 In vitro studies have yielded conflicting results. Parasite growth in hemizygous G6PD-deficient E in
short-term cultures without oxidant stress was not inhibited in some
studies9-11 and inhibited in other
studies,12-14 whereas oxidant stress constantly inhibited
growth in deficient E.10,11 Considering those
discrepancies, one wonders whether protection against malaria mortality
and the consequent selective advantage can be accounted for merely by
less favorable growth conditions of the parasite in the deficient host
E.15,16
In this study, we first attempted to reconcile some of the
contradictory data in the literature. In carefully controlled
experiments performed with several strains of P falciparum, we
found that invasion and maturation of the parasite in both the first
and second growth cycle were quantitatively indistinguishable in normal and deficient E, provided exclusively freshly drawn E were used. Similarly, in all strains and at all maturation stages studied, P
falciparum G6PD mRNA was not significantly different in normal and
deficient parasitized E. Next, we investigated directly how the G6PD
status of the host cell impacts on the parasite's metabolism: we found
that the parasite's contribution to 14C-CO2
production from D-[1-14C] glucose (which closely reflects
intracellular activity of G6PD) again was not significantly different
in normal and deficient parasitized E. In an early study of malaria in
deficient heterozygotes, suicidal infection was mentioned as one
possible mechanism of protection.4 As a specific test of
this possibility, we investigated quantitatively the susceptibility of
parasitized E to phagocytosis by periferal blood monocytes. We found
that deficient, ring-stage parasitized E (RPE) had on their surface a
higher density of phagocytic removal markers (such as autologous IgG
and complement C3 fragments) than normal RPE, and that in keeping with
this finding, deficient RPE were phagocytosed more effectively and
almost as effectively as E with more mature forms of the parasite.
Preferential phagocytosis at an early stage of the schizogonic cycle
offers an attractive explanation for the selective advantage of
deficient individuals against P falciparum malaria.
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MATERIALS AND METHODS |
Materials.
Buffers, culture media, substrates, enzymes and coenzymes, mannitol,
heparin, gentamicin, methylene blue, guanidine thiocyanate, cesium
chloride, saponin, Triton X-100,
5-amino-2,3-dihydro-1,4-phthalazinedione (luminol),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were from Sigma (St Louis,
MO); Percoll was from Pharmacia (Uppsala, Sweden); D-[1-14C] glucose, specific activity 53.4 mCi/mmol,
Megaprime DNA labeling system, Hybond-N nylon filters, protein A,
[125I]-labeled with Bolton and Hunter reagent, and
[125I] were from Amersham International (Amersham, UK);
Iodo-beads iodination reagent was from Pierce (Rockford, IL); goat
antibody to human C3c (affinity purified, polyclonal) was from The
Binding Site (Birmingham, UK); Diff-Quik parasite stain was from Baxter Dade AG (Dudingen, Switzerland); sterile plastics were from Costar (Cambridge, MA). All other reagents were purchased from common commercial sources. Sendai virus was a kind gift of Prof H. Ginsburg (Jerusalem, Israel).
Erythrocyte preparation, P falciparum cultivation,
stage-separation of parasitized E, and assessment of parasite growth.
Control E were obtained from hematologically healthy subjects with G6PD
B. Deficient blood was obtained from healthy hemizygote male deficient
(Mediterranean variant) subjects. In all cases informed consent was
obtained. Blood anticoagulated with citrate-phosphate-dextrose with
adenine (CPDA-1) was kept for 12 to 24 hours at +4°C for parasite
growth experiments and metabolic studies, and for 1 to 10 days for mRNA
studies. Normal and deficient E were isolated from plasma and white
blood cells by 85% Percoll gradient in phosphate-buffered saline (PBS)
centrifugation and three washings in wash medium (RPMI 1640 medium
containing 25 mmol/L Hepes, 20 mmol/L glucose, and 32 mg/L gentamicin,
pH 7.30). P falciparum strains FCR-3, KI, C1O, HB3B, and T9/96
were cultivated and synchronized as described17 at 0.5%
hematocrit (parasite growth experiments and metabolic studies) and at
5% hematocrit (mRNA studies). Briefly, normal schizont-stage
parasitized E (SPE) separated on Percoll-mannitol gradient17 (parasitemia >95% SPE) were mixed with normal
or deficient E suspended in growth medium (RPMI 1640 medium containing 25 mmol/L Hepes, 20 mmol/L glucose, 2 mmol/L glutamine, 24 mmol/L NaHCO3, 32 mg/L gentamicin, and 10% AB or A human serum,
pH 7.30) to start synchronous cultures at indicated hematocrit values. For all studies except growth studies, parasitemia of inoculum was
adjusted to 20% normal SPE for isolation of RPE and to 5% normal SPE
for isolation of trophozoite-stage parasitized E (TPE). Fourteen to 18 hours after inoculum (ring-stage in the first cycle), 34 to 38 hours
(trophozoite-stage in the first cycle), and 40 to 44 hours after
inoculum (schizont-stage in the first cycle), RPE, TPE, and SPE were
separated on Percoll-mannitol gradients.17 The parasitemia
was usually 30% to 45% RPE, >95% TPE and >95% SPE. For parasite
growth studies, parasitemia of inoculum was adjusted to 1% to 20%
normal SPE as indicated. Parasite growth in normal and deficient E was
expressed as parasite invasion and parasite maturation. Parasite
invasion was defined as ratio between ring-stage parasitemia measured
14 to 18 hours after inoculum and inoculum parasitemia (first cycle
invasion), or as ratio between ring-stage parasitemia measured 62 to 66 hours after inoculum and schizont-stage parasitemia measured 40 to 44 hours after inoculum (second cycle invasion). Parasite maturation was
defined as ratio between trophozoite-stage parasitemia measured 34 to
38 hours after inoculum and ring-stage parasitemia measured 14 to 18 hours after inoculum (first cycle maturation), or as ratio between
trophozoite-stage parasitemia measured 82 to 86 hours after inoculum
and ring-stage parasitemia measured 62 to 66 hours after inoculum
(second cycle maturation). Nonparasitized and parasitized E were
counted electronically. To assess total parasitemia and relative
contribution of RPE, TPE, and SPE, slides were prepared from cultures
at indicated times, stained with Diff-Quik parasite stain, and 400 to
1,000 cells examined microscopically. Significance of differences in parasitemia between normal and deficient parasitized E was assessed by
t-test for paired samples.
Opsonization of erythrocytes.
Washed nonparasitized and stage-separated parasitized E were incubated
in Hepes-glucose wash medium (10 mmol/L Hepes, 10 mmol/L glucose, 140 mmol/L NaCl, pH 7.30) supplemented with 50% fresh autologous serum at
10% hematocrit for 15 minutes at 37°C. Cells were then washed
twice in the same medium, resuspended at 10% hematocrit, and used for
phagocytosis assay or for measurement of bound autologous IgG and
complement C3 fragment.
Extraction of P falciparum total RNA.
Total RNA was extracted from synchronized cultures 14 to 18 hours
(RPE), 34 to 38 (TPE), and 40 to 44 hours (SPE) after inoculum during
the first cycle of parasite growth using the guanidine thiocyanate
method.18 For the extraction of total RNA from the peripheral blood of patients infected by P falciparum
malaria, 2 to 3 mL of blood was collected in CPDA-1 and RNA was
extracted as mentioned above.18
Northern blot analysis and quantitation.
Total RNA was fractionated on a 1.2% agarose formaldehyde denaturing
gel and blotted.19 The P falciparum G6PD probe used was K2O2, a 1.6-kb fragment of the P falciparum
G6PD gene.20 A 2.2-kb EcoR1 fragment of
P falciparum calmodulin gene21 was used as an
internal control to normalize the expression of P falciparum G6PD gene, as the regulation of the calmodulin gene is considered to be
independent of the G6PD status of host E. Probes were labeled using the
Amersham Megaprime DNA labeling system and hybridized as described
previously.20 Quantitation values for P falciparum G6PD and calmodulin mRNA were obtained from autoradiographs of the same
Northern blot filters hybridized with both probes. The hybridization
signals obtained with both probes from the same lane were quantified by
densitometry using a 2D analytical scanner (Biomed Instruments,
Fullerton, CA). The K2O2 signals were normalized using calmodulin
signals, and G6PD mRNA levels were then compared in normal and
deficient parasitized E.
Measurement of 14C-CO2 production from
D-[1-14C] glucose.
14C-CO2 production from D-[1-14C]
glucose was measured in nonparasitized and parasitized E according to
Pescarmona et al.22 Maximal activation of
14C-CO2 production was achieved by adding
methylene blue (MB) dissolved in PBS-glucose, pH 7.40 (final
concentration, 135 µmol/L). 14C-CO2
production in samples incubated at 37°C was linear for 60 minutes
under all conditions studied. Nonparasitized E were submitted to the
same culture conditions and to the same treatment as parasitized E. 14C-CO2 production values obtained with
cultures that contained 30% to 45% RPE were extrapolated to 100% RPE
parasitemia using the following calculation: I = (TOT  N × n)/(1 n), where I = amount of 14C-CO2
produced by 100% RPE; TOT = amount of 14C-CO2
produced by the whole culture; N = amount of
14C-CO2 produced by nonparasitized E; n = fraction of nonparasitized E.
Sendai virus treatment of RPE and TPE.
Sendai virus specifically lyses E membrane leaving the parasite
membrane intact.23 RPE were treated with Sendai virus as follows: RPE-enriched pellet was resuspended in wash medium at 2%
hematocrit and incubated with Sendai virus (40 to 100 µg protein/mL, equivalent to 800 to 1,200 hemagglutination units) for 20 minutes at
37°C. Virus-treated and nontreated RPE were then washed twice and
resuspended in the same volume of wash medium. Nontreated RPE were
counted electronically. Because microscopic inspection of virus-treated
samples indicated extensive lysis, reference was done to counts of
nontreated cells. Residual 14C-CO2 production
observed in virus-treated E was subtracted. Isolated TPE and
nonparasitized E were treated with half the amount of Sendai virus used
to lyse RPE. Nontreated TPE were counted electronically; for
virus-treated TPE, reference was done to counts of nontreated cells, as
indicated above. In all cases, a known amount of cells was used for
measurement of 14C-CO2 production. When applied
to nonparasitized E, virus lysis eliminated hexokinase and lactate
dehydrogenase totally and hemoglobin to 98%. G6PD and glyceraldehyde
3-phosphate dehydrogenase were consistently retained (approximately
40% and 50% retained, not shown). Because D-[1-14C]
glucose must be first phosphorylated by parasite hexokinase and
adenosine triphosphate (ATP) to give
D-[1-14C] glucose-6-phosphate, the presence of retained
host G6PD alone without hexokinase should not alter our results. Only
the recycling of labeled glucose-6-phosphate to host cytoplasm or loss
of nicotinamide adenine dinucleotide phosphate (NADP),
ATP, or Mg++ from the parasite compartment could
artefactually increase or decrease 14C-CO2
production, respectively. We checked those possible artifacts by
supplementing virus-treated TPE with hexokinase/ATP, NADP, Mg++, or with various combinations of the above substances.
As expected, supplemented hexokinase/ATP alone increased
14C-CO2 production, while none of the other
additions was effective (not shown).
Measurement of reduced glutathione (GSH).
GSH levels were measured as described24 in nonparasitized E
and in stage-separated parasitized E. GSH levels obtained with separated RPE that contained 30% to 45% RPE were extrapolated to
100% RPE parasitemia using the calculation described above.
Preparation of monocytes and measurement of phagocytosis.
Human mononuclear cells were separated from fresh normal or deficient
(Mediterranean variant) blood and plated as indicated.25 Phagocytosis was quantified as monocyte-ingested hemoglobin by measuring heme-enhanced luminescence as described.26
Phagocytosis was expressed as number of ingested E per monocyte making
use of a calibration curve prepared with each specific E sample as indicated.26
Binding of autologous IgG and complement C3 fragments.
Erythrocyte-bound autologous IgG and complement C3 fragment was
measured as described25 in nonparasitized E and in
stage-separated parasitized E. Data obtained with separated RPE that
contained 30% to 45% RPE were extrapolated to 100% RPE parasitemia
using the calculation described above.
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RESULTS |
Parasite growth in normal and deficient E.
In a first series of experiments performed with the FCR-3 strain,
invasion and maturation were measured in the first and second cycle of
parasite growth in normal and deficient E, with 4% parasitemia at the
time of inoculum and 0.5% final hematocrit. We found no difference in
both parameters during the first cycle
(Table 1). In the second cycle, we see some
degree of inhibition of both invasion and maturation in deficient E. However, t-test for paired samples indicates that differences
were not significant. Similar results were obtained with strains KI,
C10, HB3B, and T9/96 (data not shown). In a second series of
experiments performed with the FCR-3 strain, a larger number of growth
experiments was performed at higher (10% to 20%) parasitemia of the
inoculum-parasitized culture and 0.5% final hematocrit. A
t-test for paired samples of those experiments again indicated
no significant difference in invasion (significance of difference,
P = .79, n = 8) and maturation (significance of difference,
P = .40, n = 7) between normal and deficient parasitized E
during the first cycle of parasite growth.
P falciparum G6PD mRNA in normal and deficient RPE and
TPE.
Total RNA was harvested from cultures of several strains (FCR-3, KI,
C10, HB3B, T9/96) of P falciparum grown in either normal or
deficient E, and analyzed by Northern blot using K202 as a P
falciparum G6PD probe. As shown in Fig
1A, G6PD gene was found to be constitutively expressed in normal and
deficient RPE and TPE. Total RNA was extracted from peripheral blood of
two G6PD-normal patients infected with P falciparum
malaria and probed with the P falciparum G6PD K202 probe. A
single mRNA was revealed, similar in size to the one detected in RPE
(Fig 1B). To compare the amount of P falciparum G6PD mRNA in
parasites grown in normal and deficient E, the densitometry values
obtained for G6PD mRNA were normalized with those of calmodulin mRNA.
The ratio of normal to deficient G6PD mRNA was then calculated for ring
and trophozoite stages for all strains studied, and schizont stage for
FCR-3. At all maturation stages studied, the amount of P
falciparum G6PD mRNA was similar in normal and deficient
parasitized E (Table 2). The size of
trophozoite G6PD mRNA corresponded to the size of G6PD mRNA of 5.1 kb
previously reported.20 However, the size of G6PD mRNA in
normal and deficient RPE was approximately 200 bp longer than that
found in normal or deficient TPE (Fig 1).
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| Fig 1.
(A) Expression of the P falciparum G6PD gene in
parasites grown in normal and deficient E. Total P falciparum
RNA (strain HB3B) was analyzed by Northern blot using K2O2 as a probe
(see Materials and Methods). Approximately 4 µg of total RNA
extracted from parasites grown in normal (N) and deficient (D) E at
ring (RPE) and trophozoite (TPE) stage of the first growth cycle.
Illustrative lanes are labeled at the top with the respective G6PD
status. (B) Expression of the P falciparum G6PD gene in
peripheral blood of patients infected with P falciparum
malaria. Total RNA was extracted from the peripheral blood of two P
falciparum-infected G6PD normal patients and analyzed as indicated
above. Patient 1: Total RNA extracted from 2 mL of peripheral blood
from a patient with a parasitemia of 0.25% RPE, corresponding to
approximately 1.8 × 107 parasitized E. Patient 2: Total
RNA extracted from 3 mL of peripheral blood from a patient with a
parasitemia of 0.6% RPE, corresponding to approximately 8 × 107 parasitized E. RPE: 4.5 µg of total RNA extracted
from in vitro culture (ring stage) of strain C1O.
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Table 2.
Comparison of P falciparum G6PD mRNA Expression
in Different Parasite Strains Grown in Normal and Deficient E
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Production of 14C-CO2 from
D-[1-14C] glucose in normal and deficient RPE.
Production of 14C-CO2 from
D-[1-14C] glucose, which closely reflects intracellular
activity of G6PD, is remarkably stimulated by MB and other endogenous
or exogenous redox compounds.27 Because P
falciparum produces oxidant metabolic products,28 host
14C-CO2 production is likely to be activated in
parasitized E. Thus, we preferred to compare net maximal
parasite-dependent 14C-CO2 production (briefly,
net maximal parasite production) obtained after MB stimulation. Net
maximal parasite production (Table 3, column D-B) was calculated by subtracting
14C-CO2 production of MB-stimulated (normal or
deficient) nonparasitized E from 14C-CO2
production of MB-stimulated (normal or deficient) parasitized E. Net
maximal parasite production was very similar in normal and deficient
RPE. Thus, ring forms appear to possess the same maximal G6PD activity
irrespective of the G6PD activity of their host cell. Basal
14C-CO2 production in deficient RPE was half
that in normal RPE (Table 3, column C), indicating lower protection
against oxidative stress compared with normal RPE.
14C-CO2 production in Sendai virus-treated
normal and deficient RPE was considerably reduced in both (80%
and 85%, respectively). However, residual activity was very
similar in normal and deficient RPE (not shown).
Reduced GSH in normal and deficient RPE.
As shown in Table 4, culture conditions per
se led to a decrease in GSH levels. After 14 to 18 hours in culture,
GSH decrease was more pronounced in nonparasitized deficient E
(33%) compared with nonparasitized normal E (25%). The
presence of ring-stage parasites caused a further decrease in GSH
level. In absolute terms, the level of GSH in deficient RPE was very
low (0.21 ± 0.25 µmol/1010 cells, n = 6),
corresponding to 21% of time-matched nonparasitized deficient E.
Production of 14C-CO2 from
D-[1-14C] glucose in normal and deficient TPE and SPE.
As shown in Table 5, basal production of
14C-CO2 was remarkably increased in TPE
compared with RPE. By contrast, basal production of
14C-CO2 was decreased in deficient SPE
(44%) and in normal SPE (31%) compared with TPE.
Differences in basal or MB-stimulated 14C-CO2
production between normal and deficient mature forms (TPE, SPE) were
not significant. Virus-treated normal and deficient TPE retained 84%
and 89% of 14C-CO2 production, respectively.
Net maximal parasite 14C-CO2 production (see
Table 3, column D-B) was not calculated in TPE because of the good
retention after virus treatment. As observed in RPE, addition of MB to
virus-treated TPE increased 14C-CO2 production
by 38% in normal TPE and by 54% in deficient TPE.
Phagocytosis of normal and deficient RPE and TPE by adherent human
monocytes.
As shown in Fig 2, deficient RPE were
phagocytosed 2.3 times more intensely than normal RPE (9.25 ± 1.25 deficient RPE per monocyte, n = 5 v 4 ± 0.75 normal RPE per
monocyte, n = 5, P = .001). The number of phagocytosed RPE per
monocyte was close to the maximal erythrophagocytic capacity of
cultured monocytes according to the method used here.26 The
transition of ring-forms to trophozoite-forms was accompanied by
irrelevant increase in phagocytosis in the case of parasitized
deficient E (from 9.25 ± 1.25 RPE per monocyte to 10.5 ± 1.5 TPE per monocyte, n = 5, P = .190). By contrast, normal TPE
were phagocytosed 2.2 times as intensely compared with normal RPE (8.8 ± 2.5 normal TPE per monocyte v 4 ± 0.75 normal RPE per
monocyte, n = 5, P = .003). Normal and deficient TPE were phagocytosed maximally and similarly (8.8 ± 2.5 normal TPE per monocyte, n = 5; 10.5 ± 1.5 deficient TPE per monocyte, n = 5, difference not significant). Phagocytosis of nonparasitized and parasitized E was also tested with monocytes prepared from deficient blood and found to be very similar to phagocytosis by normal monocytes (not shown).
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| Fig 2.
Phagocytosis of normal and deficient nonparasitized
(NPE), RPE, and TPE by adherent human monocytes. Parasitized E were
fractionated from asynchronous cultures by the Percoll-mannitol method.
After opsonization with fresh autologous serum and washings,
parasitized E and nonparasitized controls were subjected to
phagocytosis. Phagocytosis is expressed as number of ingested cells per
monocyte. Mean values ± SD of five separate experiments. Significance
of differences between normal and deficient RPE: P = .001;
between normal and deficient TPE: P = .229. Significance of
differences was assessed by t-test for paired samples.
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Binding of autologous IgG and complement C3 fragments to normal and
deficient RPE and TPE.
As shown previously,25 phagocytosis of parasitized E is
dependent on opsonization with fresh autologous serum and is mediated by binding of autologous IgG and complement factors. As shown in
Fig 3, binding of autologous IgG (expressed
as number of [125I] Protein A molecules bound per cell)
and complement C3 fragments (expressed as cpm of [125I]
anti-C3c antibodies bound per 1,000 cells) was significantly higher in
deficient RPE compared with normal RPE (P = .001 and P = .006, respectively). As for phagocytosis, deposition of autologous IgG was not significantly different in normal and deficient TPE.
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| Fig 3.
Binding of autologous IgG (A) and complement C3 fragments
(B) in normal and deficient NPE, RPE, and TPE. Normal and deficient
parasitized E were fractionated from asynchronous cultures by the
Percoll-mannitol method. After opsonization with fresh autologous serum
and washings, parasitized E and nonparasitized controls were incubated
with [125I] Protein A, or with [125I]
anti-C3c antibodies and their binding assayed as indicated. Binding of
molecules is expressed as number of [125I] Protein A
molecules per cell (as measure of autologous IgG binding) or as cpm of
[125I] anti-C3c antibodies bound per 1,000 cells (as
measure of complement C3 fragments binding). Mean values ± SD of five
separate experiments. Data obtained with separated RPE fractions that
contained 30% to 45% RPE were extrapolated to 100% RPE parasitemia
as detailed in Materials and Methods. Data obtained with separated TPE
fractions (>95% TPE) were not extrapolated to 100% TPE parasitemia.
Binding of autologous IgG binding: difference of normal RPE versus
deficient RPE: P = .001; normal TPE versus deficient TPE:
P = .39. Binding of complement C3 fragments: difference of
normal RPE versus deficient RPE: P = .006; normal TPE versus
deficient TPE: P = .004. Significance of differences was
assessed by t-test for paired samples.
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DISCUSSION |
Epidemiological studies (reviewed by Luzzatto1 and
Greene2) clearly show that G6PD deficiency confers
protection against P falciparum malaria. Clinical
field work performed in Africa4,5 has demonstrated
protection in heterozygotes for G6PD deficiency, but other studies have
shown protection in heterozygotes and in homo/hemizygous
subjects.6-8
Growth of P falciparum in deficient E.
Culture studies seemed to provide a rationale for protection based on
impaired growth in E with the African A-12,13 and the
Mediterranean G6PD variant14 during the first cycle of
growth. However, data presented here show that growth of P falciparum in synchronized cultures was not significantly impaired in fresh deficient E with the Mediterranean variant during the first
and second cycle of growth. Differences in terms of invasion and
maturation were never significant over a wide range of parasitemias of
the inoculum-parasitized culture. Methodologic differences may explain
discrepancies in the culture results. For instance, we found that blood
samples stored for longer than 24 hours were frequently unable to
sustain optimal parasite growth possibly in relation to E membrane
damage (Turrini F., unpublished results, 1997).
Expression and activity of parasite G6PD in parasitized deficient E.
It has been shown that P falciparum after some cycles in
G6PD-deficient E seems to adapt to this type of host
cells.13 It has been hypothesized therefore that inhibition
and adaptation might be related to induction of the parasite's own
G6PD.13,20 As to the level of P falciparum G6PD
mRNA, in all five strains studied (FCR-3, KI, C10, HB3B, and T9/96), no
difference was found between normal and deficient parasitized E within
each maturation stage during the first cycle of growth (Table 2). This
finding was in agreement with data showing that the P
falciparum G6PD activity could be detected in normal and deficient
parasitized E at all stages of the intraerythrocytic
cycle.29,30
To ascertain activity of parasite G6PD in normal and deficient E, we
compared basal and MB-stimulated 14C-CO2
production in stage-separated normal and deficient parasitized E and
used Sendai virus permeabilized parasitized E. In late forms (TPE and
SPE), both approaches indicated that basal and MB-stimulated 14C-CO2 production was very similar in normal
and deficient TPE and SPE and was largely accounted for by parasite
G6PD, whereby host G6PD contributed to approximately 1% of total
14C-CO2 production. In RPE, assessment of
parasite G6PD activity by the Sendai virus technique was not possible,
because Sendai virus treatment abrogated 80% to 85% of
14C-CO2 production. Thus, parasite G6PD
activity in RPE was inferred by comparing net maximal
14C-CO2 production in normal and deficient RPE.
Using this indirect approach, net maximal
14C-CO2 production was very similar in normal
and deficient RPE (Table 3, column D-B). Taken together, these data
suggest that the expression of the parasite G6PD gene is comparable in
normal and deficient E at all maturation stages examined and the same amount of 14C-CO2 is produced.
Preferential phagocytosis of deficient RPE: An alternative mechanism
of protection in G6PD deficiency.
The explanation we are offering for protection against malaria by G6PD
deficiency is based on preferential phagocytosis of deficient RPE. This
appears to be the ultimate consequence of very low parasite G6PD
activity in RPE, accompanied by inherently low G6PD activity and very
low GSH levels in the deficient host E. This combination makes
deficient RPE peculiarly prone to oxidative membrane modifications
leading to opsonization and phagocytic removal. Specifically, we
suggest that as a consequence of vulnerability to oxidants, deficient
RPE bind higher amounts of removal markers and are therefore
phagocytosed more effectively than normal RPE. Previous work has
determined that deposition of autologous IgG with specificity to
aggregated or clustered band 3, and deposition of complement C3
fragments are essential for phagocytosis.25 It has also
been shown that oxidative clustering of band 3 is originated by
oxidation of specific cysteine residues located on the cytoplasmic
domain,31 whereby the process is more likely to occur in
deficient E, because of their inability to regenerate GSH and thiols.
We have shown previously that phagocytosed ring-forms are digested
rapidly by cultured monocytes, and the process repeated without loss of
efficiency.32 By contrast, more mature forms of the
parasite, although actively phagocytosed, hinder repetition of the
process because the malarial pigment hemozoin, abundantly present in
mature parasite forms, is indigestible and severely affects functions
of the monocyte such as oxidative burst and ability to perform multiple
cycles of phagocytosis.32 Increased phagocytosis of
ring-forms is certainly advantageous to the host, because it will keep
parasitemia down (see Luzzatto et al4 and Bienzle et
al5), and because less parasitized E mature to trophozoite
or schizont, which adhere to cerebral and renal postcapillary
venules.33 Adhesion to cerebral endothelia may cause
cerebral malaria, a major cause of malaria mortality.34 Also, if deficient RPE in the double E population in heterozygotes are
preferentially removed, we may expect to find less parasitized deficient E in circulation, in agreement with the findings of Luzzatto
et al.4 Other methods of host defense may
depend on direct parasite killing by cytokines, oxygen, or nitrogen
radicals, produced by adherent or adjacent host cells, such as
leuko/monocytes, endothelial cells, and platelets.35 Due to
oxidant vulnerability of deficient RPE, enhanced intracellular killing
of early parasite forms may cooperate with increased phagocytosis to
elicit better resistance.
Preferential phagocytosis of RPE: Common mechanism of protection in
widespread protective E mutations.
The three major E mutations conferring protection against
malaria36 are characterized by increased generation of
noxious oxygen radicals (thalassemias, sickle cell
anemia37,38) or by decreased ability to cope with oxidant
damage (G6PD deficiency). Because the presence of developing parasite
with its remarkable metabolic demands in a limited time period imposes
an additional oxidative stress upon the host cell leading to its
enhanced demise,25,28 it is conceivable that similar
oxidant-mediated membrane modifications leading to enhanced
phagocytosis occur in RPE in all three mutations in question. Data
obtained by some of us (Ayi K., Turrini F., Mannu F., Arese P.,
unpublished results, 1998) indicate that heterozygous beta-thalassemic E behave quite similarly as deficient E, showing the
same parasite invasion and maturation as nonthalassemic controls, enhanced deposition of removal markers, and enhanced phagocytosis at
ring-stage. Other studies have also indicated increased
opsonization39 and enhanced susceptibility to phagocytosis
in parasitized E with thalassemic trait.40 In sickle cell
anemia heterozygotes, a suicidal infection, ie, preferential
phagocytosis of parasitized E with sickle cell trait has been
previously described by one of the authors.41
The present model does not exclude and is compatible with other defense
mechanisms possibly operating in parallel in vivo: preferential damage
of parasites harbored in G6PD-deficient parasites by cytokines, toxic
oxygen, or nitrogen metabolites secreted by activated phagocytes or
endothelia; preferential pitting of the parasite from G6PD-deficient
cells in the spleen42 reduced rosetting.43 The
recently proposed model of protection in
alpha+-thalassemia,44 based on increased
susceptibility to P vivax and subsequent induction of
cross-protection against P falciparum malaria, also appears to
be compatible with this model, as increased splenomegaly was present in
alpha+-thalassemics with P falciparum infection,
indicating enhanced phagocytic removal in those subjects. Finally, we
cannot exclude, despite present clear-cut results and absence of ex
vivo evidence, that parasite growth might be impaired in mutant E under
in vivo conditions of fluctuating oxygenation and pH values, mechanical and osmotical stress, or in the presence of increased amounts of fetal
hemoglobin.45 Future research will allow investigators to
dissect the relative weight of outlined mechanisms of resistance and,
possibly, suggest new ones.
In conclusion, we can visualize the mechanism of relative resistance
against P falciparum as resulting from the following stepwise
sequence: (1) Invasion of G6PD-normal and deficient E by the parasite
is equally efficient; apparently the level of G6PD activity is not
relevant for this process. (2) During its ring stage of
intraerythrocytic development, the parasite has very little G6PD
activity compared with the host cell (although production of
parasite-specific G6PD mRNA has started). Therefore, in deficient host
cells, the total G6PD (from host plus parasite) is very low; this is
associated with GSH depletion, which makes the deficient parasitized
cell very vulnerable to damage even in the absence of any deliberately
imposed oxidative stress. These damaged cells are highly susceptible to
phagocytosis. (3) By the time the parasite has developed into a
trophozoite (and subsequently into a schizont), its own G6PD activity
is high; in a deficient host cell it is, in fact, much higher than that
of the host cell itself. At this stage of development the physical
properties of the parasitized E are sufficiently different from those
of nonparasitized E that they are susceptible to phagocytosis
regardless of their G6PD status. (4) As a result of the above, the
stage at which parasitized E are likely to undergo phagocytosis is
brought forward in the case of deficient E, thus limiting the level of
parasitemia. The above model of resistance against malaria is
applicable, in principle, to both males who are hemizygous and females
who are heterozygous for G6PD deficiency.
| |
FOOTNOTES |
Submitted February 6, 1998;
accepted May 19, 1998.
Supported by World Health Organization/United Nations Development
Programme/World Bank Special Programme for Research and Training in
Tropical Diseases (WHO TDR IMMAL); Compagnia di San Paolo, Torino;
Italian Ministry of University, Roma; and British Council, Roma. M.C.
was a recipient of a research grant from the European Community
(Contract No. BIO2-CT93-6917).
M.C. and G.G. contributed equally to this work.
Address reprint requests to Paolo Arese, MD, PhD, Dipartimento di
Genetica, Biologia, Biochimica, Via Santena 5 bis, 10126 Torino, Italy;
e-mail: arese{at}molinette.unito.it.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract
