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Infection and Immunity, February 1999, p. 513-519, Vol. 67, No. 2
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Therapy with a Combination of Low Doses of
Interleukin 12 and Chloroquine Completely Cures Blood-Stage Malaria,
Prevents Severe Anemia, and Induces Immunity to
Reinfection
Karkada
Mohan,
Hakeem
Sam, and
Mary M.
Stevenson*
Centre for the Study of Host Resistance,
McGill University, and The Montreal General Hospital Research
Institute, Montreal, Quebec H3G 1A4, Canada
Received 13 August 1998/Returned for modification 14 October
1998/Accepted 11 November 1998
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ABSTRACT |
The immunoregulatory cytokine interleukin 12 (IL-12) induces host
resistance against experimental malaria. In this study, we tested the
feasibility of using IL-12 in combination with chloroquine (CQ) to
rescue susceptible A/J mice from lethal blood-stage Plasmodium chabaudi AS infection. Combined treatment with low doses of CQ and IL-12 resulted in a >15-fold reduction in the parasite load and
100% survival of A/J mice with established infections. Compared to
control mice, which succumbed to severe anemia, CQ-plus-IL-12-treated mice had significantly higher early- and late-stage erythroid-cell progenitors in the bone marrow and spleen, resulting in significantly higher hematocrits, erythrocyte counts, and percentages of
reticulocytes. Production of parasite-specific gamma interferon
(IFN-
) by splenocytes from these mice was upregulated >20-fold
relative to controls in parallel with enhanced IFN- mRNA expression.
Further, enhanced responsiveness to IL-12 and increased downstream
IFN- production in CQ-plus-IL-12-treated mice was evident from
increased mRNA expression for the 
1 and 2 subunits of IL-12
receptor in the splenocytes. Moreover, this combined therapy induced
higher levels of anti-malaria antibodies than did CQ alone as well as
sterile immunity against reinfection. Because IL-12 can be used at low doses and is effective even in established infections, it may be
feasible to use this immunochemotherapeutic approach in human malaria.
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INTRODUCTION |
Malaria remains a major public
health problem in most tropical countries, particularly sub-Saharan
Africa. It has been estimated that between 300 million and 500 million
individuals are infected annually and between 1.5 million and 2.7 million people die of malaria every year (2). Despite
decades of frustrating research, an effective vaccine against this
deadly disease is still not a reality (2, 5). In the
meantime, however, we must rely on effective therapeutic strategies for
treating acute infections to prevent malaria-associated complications
and mortality, especially in patients with malaria due to
Plasmodium falciparum. Chloroquine (CQ) has been both an
affordable and well-tolerated drug for use in third-world countries,
but this drug now faces severe limitations because of the widespread
emergence of CQ-resistant P. falciparum strains and, more
recently, P. vivax strains (20, 29). To overcome
this problem, different combinations of antimalarial drugs have been
used, but in most instances, multidrug-resistant P. falciparum strains have emerged (28). Thus, intensive
investigations directed toward finding an effective method to
successfully treat acute malaria infections are under way.
Interleukin 12 (IL-12), a potent immunomodulatory cytokine, has been
proven to be effective in conferring protection against bacterial,
viral, and intracellular parasitic infections (15, 27). This
pleiotropic cytokine not only enhances cell-mediated immune responses
but also influences humoral immunity by inducing isotype switching
through both gamma interferon (IFN-)-dependent and -independent
mechanisms (17). IL-12 also appears to stimulate enhanced
antibody (Ab) production in switched B cells (17). Both mice
and nonhuman primates can be protected against preerythrocytic malaria
infections following IL-12 treatment (8, 24). Our laboratory
has demonstrated the effectiveness of IL-12 in inducing protective
immunity against blood-stage infection in the murine model of P. chabaudi AS malaria (26). In addition to its NK cell-activating, IFN--stimulatory, and Th1-polarizing effects early
during P. chabaudi AS blood-stage infection, IL-12 induces remarkable upregulation of splenic erythropoiesis, thereby preventing the fatal anemia associated with this infection (18, 19,
26). However, the dose of IL-12 appears to be critical, given the
potential toxic effects of this cytokine (8, 22).
Although IL-12 can induce protective Th1-type immunity against
experimental malaria infections, its therapeutic value is limited, given the need to begin treatment prior to or on the day of
establishing infection (8, 24, 26). The main goal of this
study was to improve the efficacy of IL-12 treatment, especially in
terms of its efficacy in established infections. We examined the
possibility of using IL-12 as a therapeutic agent, in combination with
CQ, for treating established P. chabaudi AS infection in
susceptible A/J mice. Our findings demonstrate that low-dose CQ plus
IL-12 treatment of mice with established blood-stage infection induced a protective Th1 immune response and efficient upregulation of erythropoiesis during primary infection and higher anti-malaria Ab
production following reinfection.
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MATERIALS AND METHODS |
Mice, parasites and infection protocol.
Male A/J mice, 8 to
12 weeks old, were purchased from Jackson Laboratory (Bar Harbor,
Maine). The mice were infected intraperitoneally with 106
P. chabaudi AS parasitized red blood cells (PRBC) in
pyrogen-free saline, and parasitemia and survival rate were monitored
as described previously (26). To assess reinfection
immunity, mice were challenged with the same dose of parasites 4 weeks
after recovery from the primary infection and parasitemia was monitored
for 2 weeks.
IL-12 and CQ treatment.
Murine recombinant IL-12 (rIL-12)
was a gift from S. Wolf, Genetics Institute (Cambridge, Mass.). CQ
diphosphate was purchased from Sigma (St. Louis, Mo.). To establish an
optimal subcurative dose of CQ, mice were treated orally with 25 mg of
CQ per kg of body weight (the therapeutic dose) or 12.5 and 6.25 mg/kg,
divided according to World Health Organization recommendation
(31). For the therapeutic dose, an initial dose of 10 mg/kg
was given on day 3 postinfection (p.i.) followed by 5 mg/kg at 6, 24, and 48 h. For 12.5 and 6.25 mg/kg, the dose of CQ used was
one-half and one-quarter of the therapeutic dose, respectively, at each treatment time point. Since our intention was to use lower doses of
IL-12 in combination with a subcurative dose of CQ, the dose of IL-12
was decreased from our previously reported (26) protective dose of 0.6 µg/mouse (0.1 µg per day for 6 days) administered i.p.
starting on the day of infection, to 0.3 µg/mouse (0.05 µg for 6 days). Further, to test the protective efficacy of IL-12 in an
established infection in combination with a subcurative dose of CQ, the
dose of IL-12 was further decreased to 0.2 µg/mouse (0.05 µg per
day for 4 days) and treatment was started on day 3 p.i., once
parasitemia was established at 0.5 to 1%. Infected mice receiving CQ
alone, IL-12 alone, or CQ plus IL-12 combinations were monitored daily
for parasitemia and survival.
Hematology and erythropoietic progenitor assays.
Hematocrit,
total RBC counts, and percentage of reticulocytes were determined for
heparinized blood from individual mice by using standard hematological
procedures. Single-cell suspensions were obtained from the bone marrow
and spleen, and the number of RBC bursts (BFU-E) and CFU (CFU-E) were
determined in colony forming assays by using methylcellulose semisolid
medium in Iscove modified Dulbecco minimal essential medium as
previously described (19). The number of RBC progenitor
colonies were counted after 48 h for CFU-E and after 7 days for
BFU-E, and data are presented as mean ± standard error of the
mean (SEM) per organ. The number of BFU-E in the peripheral blood was
determined by using blood mononuclear cells separated by density
gradient centrifugation and is expressed as mean ± SEM per 5 × 106 total cells.
Quantitation of IFN- and anti-malaria Abs.
Single-cell
suspensions of unfractionated spleen cells were plated at 2 × 106 cells per well and incubated for 48 h with either
medium alone, 5 µg of concanavalin A per ml, (ConA) or P. chabaudi AS antigen (mpAg) equivalent to 2 × 106
PRBC. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was
used to measure IFN- in the culture supernatants as described previously (26). Malaria-specific, total immunoglobulin G
(IgG) in sera obtained 2 weeks after reinfection was determined by
ELISA. Total anti-malaria IgG was captured by using soluble mpAg-coated plates, and the level of malaria-specific IgG in the sera was estimated
by using goat anti-mouse IgG-horseradish peroxidase conjugate (Bio-Rad
Laboratories, Richmond, Calif.) and
2,2'-azinobis(3-ethylbenzthiazolinesulfonate) substrate (Boehringer
Mannheim Canada, Laval, Canada). The levels of Ab are expressed as the
optical density at 405 nm. Sera from uninfected, normal A/J mice served
as negative controls.
RT-PCR for IFN-, IL-12R1, and IL-12R2 mRNA
expression.
Reverse transcriptase PCR (RT-PCR) was performed
essentially as described previously to determine the relative
quantities of mRNA for IFN- and the two IL-12 receptors, 1 and
2 (11). The primers and probe used for IFN- have been
described previously (1). For IL-12R subunits, primers, and
probes were designed based on the recently cloned cDNA sequences,
GenBank accession no. U23922 and U64199, for the 1 and 2
subunits, respectively (3, 23). The sequences of primers and
probes used were as follows: IL-12R1, sense primer,
5'-TGA-AGA-CGG-CGC-GTG-GGA-GTC-A-3'; antisense primer,
5'-TCG-CGG-GTA-CAA-CAC-CTC-CGG-G-3'; probe, 5'-GCG-AGC-GGA-CAC-TGC-GAG-CG-3' (product size, 412 bp);
IL-12R2, sense primer, 5'-GGT-TGC-TGG-CTC-CTC-ACC-AGG-3';
antisense primer, 5'-ATG-CAG-CCC-CTT-TGC-TCC-GGG-3';
probe, 5'-TCC-CCC-ACA-CTG-GCT-GCG-GA-3' (product size,
424 bp). Both positive and negative controls were included in
each assay to ensure the efficacy of the reaction and to rule out
possible genomic DNA contamination. The primers and probe for
glyceraldehyde 6-phosphate dehydrogenase (G6PDH), the housekeeping gene
used in this study, have been described previously (11). For
RT-PCR, 1 µg of total RNA, isolated from unfractionated spleen cells
with TRIzol reagent (Gibco BRL, Grand Island, N.Y.), was reverse
transcribed with Moloney murine leukemia virus RT (Gibco). The reaction
mixture was diluted 1:8, and 10 µl was used for PCR amplification of
IFN- (30 cycles), IL-12 receptor subunit 1 and 2 mRNA (30 cycles), and G6PDH (26 cycles) with Taq DNA polymerase
(Gibco). Following electrophoresis and Southern transfer onto nylon
membranes (Hybond-N; Amersham, Arlington Heights, Ill.), PCR products
were hybridized with internal cytokine-specific oligonucleotide probes
labeled with [32P]ATP and visualized by autoradiography.
The intensity of bands corresponding to specific cytokine-cytokine
receptors was analyzed by high-resolution optical densitometry
(SciScan; United States Biochemical, Cleveland, Ohio) and normalized to
those of G6PDH.
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RESULTS AND DISCUSSION |
Effect of combined CQ-plus-IL-12 treatment on the course of
infection and survival.
To determine a subcurative dose of CQ
suitable for use in combination with IL-12 in later experiments, we
first performed a CQ dose-response study in P. chabaudi
AS-infected A/J mice. As demonstrated previously by our laboratory,
mice of this strain are extremely susceptible to P. chabaudi
AS infection and experience fulminant parasitemia and severe anemia
with 100% mortality within a few days of peak parasitemia
(26). Mice were treated beginning on day 3 p.i. with
the curative dose of 25 mg of CQ per kg (body weight) or with two
subcurative doses of 12.5 and 6.25 mg/kg. Since the parasite strain
used is CQ sensitive, as expected, the curative dose of CQ did not
allow the parasitemia to exceed 0.5% and no parasites were detectable
from day 7 through day 28 p.i. (Fig.
1A). Untreated mice showed high
parasitemia of more than 50%, and all the mice succumbed to infection
by day 11 p.i. (Fig. 1C). Similarly, mice given 6.25 mg of CQ per
kg of body weight developed mean peak parasitemia in excess of 40% and
all the mice in this group succumbed to infection. On the other hand,
mice receiving half the curative dose, that is, 12.5 mg/kg, showed a
modest reduction in peak parasitemia to around 20% and the mortality rate was about 30%. We selected this latter dose of CQ to be used in
combination with IL-12 to explore the possibility of reducing the
parasite burden and rescuing all of the infected animals.
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FIG. 1.
Dose response to CQ (A) and the effect of CQ plus IL-12
combined therapy (B) on the course of P. chabaudi AS
infection (A and B) and survival rate (C) of A/J mice. Mice were
infected i.p. with 106 PRBC and were treated with CQ alone
(12.5 mg/kg) or IL-12 alone (0.05 µg/day for 4 days) or both,
starting on day 3 p.i., after the parasitemia was established at
0.5 to 1.0%. Pooled parasitemia data represent mean ± SEM from 9 to 12 individual mice studied in two (A) or three (B) experiments. The
mean percent survival data shown are pooled from 10 to 12 mice in each
group.  , 100% mortality.
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In the preliminary experiments, we identified the optimum dose of IL-12
suitable for use with half the therapeutic dose of
CQ. Initially, 0.05 µg of IL-12 treatment per mouse was given
for 6 days, starting on the
day of infection (a total of 0.3 µg/mouse).
Using this combination
therapy, it was possible to reduce the
peak parasitemia to about 3%
and all the mice survived infection
(data not shown), while the mice
receiving 0.3 µg of IL-12 alone
did not survive infection, suggesting
the need for combined treatment
for survival. Since our aim was to test
the efficacy of the CQ
plus IL-12 treatment combination in established
infection, we
conducted experiments with 0.05 µg of IL-12 per mouse
per day
starting on day 3 p.i. and continued until day 6 p.i., so as to
deliver a total IL-12 dose of 0.2 µg/mouse along with
12.5 mg
of CQ per kg. Untreated mice, mice treated with CQ or IL-12
alone,
and CQ-plus-IL-12-treated mice had an initial mean parasitemia
of 0.6% ± 0.08% (range, 0.3 to 1.6%) on day 3 p.i., before
treatment
was initiated. As shown in Fig.
1B, following CQ plus IL-12
treatment,
parasite proliferation was markedly suppressed and the mean
peak
parasitemia was only 3.1% and was reached only on day 10 p.i.
In contrast, untreated mice had a mean peak parasitemia of 63.5%
by day 7 p.i. and all the mice were dead by day 11 p.i. (Fig.
1C). Untreated mice appeared severely anemic and hypothermic and
were
very lethargic for 3 to 4 days prior to death. Similarly,
mice given a
total dose of 0.2 µg of IL-12 alone had a mean peak
parasitemia of
>60% by day 7 p.i., and all the mice succumbed
to infection by
day 9 p.i. (Fig.
1B and C). Mice receiving 12.5
mg of CQ per kg of
body weight alone had a mean peak parasitemia
of 33.5% by day 9 p.i. (Fig.
1B), showed a survival rate of 70%
(Fig.
1C), and were also
lethargic, although these mice did not
show signs of severe anemia or
hypothermia. Parasites were detectable
in these mice most of the time
during the first 4 weeks of infection,
with a recrudescent parasitemia
on day 21 p.i. (Fig.
1B).
We also tested the curative efficacy of combining 6.25 mg of CQ per kg
of body weight with or without a total IL-12 dose of
0.2 µg/mouse
(days 3 through 6 p.i.). These mice developed severe
infection and
anemia and succumbed to infection during the second
week of infection
(data not shown). In contrast to mice receiving
lower doses of CQ and
IL-12, either alone or in combination, mice
on combined therapy with
12.5 mg of CQ per kg of body weight and
0.2 µg of IL-12 per mouse
remained healthy and active throughout
the course of infection.
Parasites were not detectable after day
12 p.i., and all the mice
survived infection in repeated experiments
(Fig.
1B and C). Thus, this
dose of IL-12, in combination with
half the curative dose of CQ, is as
efficient as using 0.3 µg
of IL-12 per mouse and is effective even in
established infections.
Moreover, this dose also represents the
critical curative level
required in combined therapy, since lowering
the total dose of
IL-12 further to 0.15 µg results in higher parasite
burden (>30%),
even when combined with half the curative dose of CQ
(data not
shown). Because a total IL-12 dose of 0.2 µg/mouse given
along
with 12.5 mg of CQ per kg markedly reduces the parasite load and
rescues 100% of infected mice, we consider this combination therapy
to
be optimal for successful treatment of established
P. chabaudi AS
infection.
Effect of combination therapy on the development of malarial
anemia.
Having established the protective efficacy of 12.5 mg of
CQ per kg in combination with 0.2 µg of IL-12, we explored the
possible mechanism(s) involved. Once infected, P. chabaudi
AS-susceptible A/J mice suffer from severe anemia and shock prior to
death (33). We previously showed that severe anemia is a
main factor contributing to mortality, since blood transfusions given
to these mice after the development of peak parasitemia rescued up to
90% of the mice (34). Our recent study examining the
erythropoietic role of IL-12 in infected A/J mice showed that 0.1 µg
of IL-12 for 6 days, starting on the day of infection, results in more
than a sevenfold increase in splenic erythropoiesis (19). In
the present study, we examined mice receiving CQ plus IL-12 therapy,
using a substantially lower dose of IL-12 in an established infection,
for possible inhibition of infection-induced development of anemia. The
peripheral blood picture of CQ-plus-IL-12-treated animals was compared
with that of untreated mice and mice receiving CQ or IL-12 alone. As shown in Table 1, mice receiving combined
therapy had a significantly higher hematocrit and total RBC count on
day 7 p.i. compared to untreated or mice treated with 12.5 mg of
CQ per kg or 0.2 µg of IL-12 alone. Signs of severe anemia were
apparent in untreated or IL-12-treated mice, while mice treated with
12.5 mg of CQ per kg retained hematocrit and RBC counts at
significantly higher levels than did untreated mice. However, mice in
the combined treatment group alone had significantly higher levels of
both hematocrit and RBC counts after 7 days of infection compared to controls and showed no signs of anemia.
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TABLE 1.
Effect of CQ and IL-12 treatment on hematological
parameters in A/J mice with established P. chabaudi
AS infectiona
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It is possible that since the mice receiving optimum combined therapy
had >15-fold lower parasitemia, the absence of anemia
was due to
decreased direct RBC destruction by the developing
parasites. On the
other hand, it is also possible that the extent
of upregulation of
erythropoiesis following infection could have
been of several
magnitudes higher in the mice receiving combined
therapy. To address
this question, we first examined the percentage
of reticulocytes in the
peripheral blood. On day 7 p.i., mice
treated with 12.5 mg of CQ
per kg, with or without IL-12 treatment,
showed significantly higher
percentages of reticulocytes than
did untreated mice (Table
1).
However, mice treated with IL-12
alone failed to show a significantly
higher percentage of reticulocytes
compared to untreated mice. Since
reticulocytosis is apparent
during weeks 2 and 3 of infection in
P. chabaudi AS-resistant
C57BL/6 mice (
33), we
also examined blood films from surviving
mice on day 10 p.i. At
this time, A/J mice receiving CQ plus IL-12
treatment had more than a
twofold increase in the percentage of
reticulocytes compared to mice
receiving 12.5 mg of CQ per kg
alone.
Next, we examined the effect of CQ and IL-12 treatment on
erythropoiesis by determining the number of erythroid progenitors
in
the bone marrow, spleen, and blood. Treatment of infected A/J
mice with
0.05 µg of IL-12 per mouse for 4 days, starting on day
3 p.i.,
along with half the curative dose of CQ significantly
enhanced bone
marrow and splenic erythropoiesis compared to no
treatment or treatment
with CQ or IL-12 alone (Table
2). A
marked
increase was seen in the extent of splenic erythropoiesis in
terms
of both BFU-E and CFU-E. Mice receiving the combined treatment
had a nearly twofold increase in the numbers of splenic BFU-E
and more
than a threefold increase in the numbers of splenic CFU-E
compared to
untreated animals or mice treated with CQ alone. Mice
treated with 12.5 mg of CQ per kg alone showed a small but significant
increase only in
the bone marrow CFU-E compartment. However, mice
receiving IL-12
monotherapy had significantly enhanced CFU-E numbers
in bone marrow and
spleen and higher BFU-E in the spleen compared
to untreated controls.
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TABLE 2.
CQ-plus-IL-12 combination therapy markedly upregulates
erythropoiesis in A/J mice with established P. chabaudi
AS infectiona
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Our earlier study showed that IL-12 treatment of normal, but not
infected, A/J mice results in bone marrow suppression (
19),
possibly due to the ability of this cytokine to enhance the
mobilization
of bone marrow precursors to the spleen (
9).
These studies
suggest a role for IL-12 in enhancing extramedullary
erythropoiesis,
especially in the spleen. To examine the mobilization
of bone
marrow erythroid precursors to the spleen following low-dose CQ
plus IL-12 treatment, we investigated the frequency of BFU-E in
the
peripheral blood. We observed a nearly threefold increase
in the blood
BFU-E numbers in mice treated with CQ plus IL-12
compared to untreated
mice and nearly a twofold increase compared
to mice receiving either CQ
or IL-12 alone (Table
2).
It is interesting that even though the dose of IL-12 used was similar
in mice receiving either 12.5 or 6.25 mg of CQ per kg,
the extent of
erythropoiesis was higher in the former group of
mice, which had a
lower parasite load. This indirectly suggests
that high parasitemia may
inhibit efficient erythropoietic upregulation,
possibly due to impaired
levels of other hematopoietic cofactors
such as IL-3, IL-6, IL-11, and
steel factor in mice with high
parasite burdens. Thus, the use of IL-12
treatment along with
half the curative dose of CQ not only suppresses
parasitemia and
limits the extent of direct RBC destruction by the
parasites but
also initiates a more efficient erythropoietic response.
The erythropoietic
stimulatory effect of IL-12 is not restricted to the
combination
of CQ plus IL-12, since we observed similarly enhanced RBC
genesis
when we used IL-12 in combination with clindamycin to treat
infected
A/J mice (unpublished observations). This finding raises the
possibility
of using IL-12 in combination with other antimalarial drugs
to
prevent fatal anemia. In human situations, this is particularly
important in areas with widespread CQ resistance, where one has
to rely
on other antimalaria drugs for the treatment of acute
infections.
However, this strategy should be tested in infections
due to
drug-resistant parasite strains, because such strains are
usually
resistant to higher-than-therapeutic doses of the
drugs.
Development of a novel therapeutic strategy to prevent malarial anemia
is critical, considering the importance of anemia in
malaria morbidity
and mortality. Studies of Gambian children with
severe
P. falciparum malaria and presenting with severe anemia
showed marked
dyserythropoietic changes, including erythroblast
multinuclearity,
karyorrhexis, incomplete and unequal mitosis,
and cytoplasmic bridging
(
30). While blood transfusion has been
proven to be
beneficial in correcting malarial anemia (
12),
the risks
involved are considerable, especially because of the
high levels of
human immunodeficiency virus infection in many
malaria-endemic
populations. This calls for a method of treatment
which can correct
hyperparasitemia as well as infection-induced
anemia without the need
for transfusion. The combination therapy
used in this study appears to
be promising in this direction.
Endogenous upregulation of
erythropoiesis enhances reticulocyte
numbers in the peripheral blood
and prevents the risk of prolonged
parasite patency observed following
transfusion (
34), since
reticulocytes are refractory to
infection by most normophilic
malaria
parasites.
Combination therapy significantly upregulates
parasite-specific IFN- production and IL-12R expression.
We next examined the level of IFN- production by splenocytes, in
terms of both protein and gene expression, following combination therapy of infected A/J mice. We have previously demonstrated the
essential role of IFN- in the protective host response against P. chabaudi AS in resistant C57BL/6 mice (10, 18,
25) as well as in IL-12-treated, susceptible A/J mice (18,
26). Anti-IFN- treatment of IL-12-treated A/J mice resulted in
a significant increase in parasite load and high mortality
(26). Our recent study also suggested that a defect in the
production of IFN- by NK cells early during infection contributes to
the extreme susceptibility of A/J mice to blood-stage malaria
(18). This finding is consistent with an earlier observation
in resistant C57BL/6 mice that tissue-specific expression of IFN-
mRNA in the spleen is significantly higher by day 3 p.i. compared
to that in A/J mice, and higher expression of this cytokine mRNA
persists for 1 week p.i. (10). Furthermore, both in vivo
IL-12 treatment of A/J mice and in vitro IL-12 supplementation of
enriched NK cell cultures from infected A/J mice significantly enhance
the levels of spontaneous NK cell-derived IFN- production
(18). To achieve a protective response in A/J mice, our
previous study used 0.1 µg of IL-12/mouse/day for 6 days starting on
the day of infection, for a total dose of 0.6 µg of IL-12
(26), while the present study used 0.05 µg of IL-12 daily
for only 4 days starting on day 3 p.i., for a total dose of 0.2 µg of IL-12, in combination with CQ, once the parasitemia was
established between 0.5 and 1.0%. Hence, we asked whether inclusion of
low, subprotective doses of IL-12 in combined treatment influences the
immune response in A/J mice by inducing a protective Th1 response. In
response to nonspecific stimulation with ConA, splenocytes from
untreated mice, mice treated with CQ or IL-12 alone, or those receiving CQ plus IL-12 combined treatment secreted comparable amounts of IFN-
on day 7 p.i. (Fig. 2A). In
contrast, spleen cells from mice treated with both CQ and IL-12
secreted significantly higher levels of IFN- in response to specific
stimulation with mpAg than did those from untreated mice or mice
treated with CQ or IL-12 alone. Following mpAg stimulation, splenocytes
from mice receiving IL-12 alone produced more than a fourfold-higher
concentration of IFN- than did those from untreated or CQ-treated
mice. The magnitude of upregulation of IFN- in mice treated with CQ
plus IL-12 was more than 20-fold higher than in untreated or CQ-treated groups of mice and was nearly 5-fold higher than in mice given IL-12
alone. In parallel, on day 7 p.i., IFN- mRNA expression was
also upregulated in mice receiving combined treatment (Fig. 2B). This
suggests the development of efficient antiparasitic immunity in mice
given combination therapy during the early phase of infection by an
enhanced IFN- production. Further, this finding has an important
bearing on the development of the adaptive immune response, since it
has been shown that during murine Leishmania major
infection, IFN-, along with IL-12, acts as an essential cofactor in
the development of parasite-specific, Th1 type protective immunity
(7).
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FIG. 2.
Effect of CQ plus IL-12 combined therapy on the levels
of IFN- secretion (A) and the expression of mRNA for IFN-,
IL-12R1, and IL-12R2 (B) by splenocytes obtained from P. chabaudi AS-infected A/J mice on day 7 p.i. IFN- secretion
by unfractionated spleen cells was measured in the culture supernatants
by ELISA after 48 h of stimulation with ConA or mpAg as indicated.
Data represent mean ± SEM from four individual mice from two
experiments. Levels of IFN- and IL-12R mRNA expression were measured
by RT-PCR, and the results shown are from two representative mice in
each group. Lanes 1 and 2, untreated mice; lanes 3 and 4, mice given
12.5 mg of CQ per kg alone; lanes 5 and 6, mice given CQ+IL-12 combined
therapy. *, P < 0.001 by Student's unpaired
t test.
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Responsiveness to IL-12 and subsequent downstream signal transduction
events, IFN-
production, and Th-cell development has
recently been
shown to be dependent on the levels of IL-12R
1
and IL-12R
2
expression on the responder cells (
6,
32). Coexpression
of
these two IL-12R subunits results in the expression of both
high- and
low-affinity IL-12 binding sites and confers IL-12 responsiveness
in
transfected cells (
23). Hence, we questioned whether
enhanced
IFN-
production and development of a protective Th1 immune
response
in mice receiving combined therapy correlate with enhanced
IL-12R
expression. For this, we analyzed the expression of
1 and
2
IL-12R subunit mRNA expression in splenocytes isolated on day
7 p.i. from mice given CQ plus IL-12 combined treatment. Compared
to untreated mice, CQ-plus-IL-12-treated mice had a higher expression
of both IL-12R
1 and
2 (Fig.
2B), indicating a role for IL-12
in
upregulating its own receptors on responder cells during
P. chabaudi AS infection. Higher levels of IL-12R expression on
responder
cells may explain why spleen cells from mice receiving
combined
treatment produced significantly higher levels of IFN-
than
did
cells from control mice following specific Ag stimulation. We
recently observed that on day 7 p.i., spleens from IL-12-treated,
infected A/J mice contain more than fourfold greater absolute
numbers
of asialo-GM1
+ NK cells and more than twofold greater
numbers of B cells than
do spleens from uninfected mice
(
18). While NK cells expressing
increased IL-12R could
contribute to higher levels of protective
IFN-
secreted during the
early course of infection, IL-12 may
also influence B-cell production
of parasite-specific Ab. To test
this latter possibility, we
quantitated anti-malaria Ab levels
as well as the development of
immunity against reinfection in
the surviving A/J mice in CQ-treated
and CQ-plus-IL-12-treated
groups.
CQ plus IL-12 treatment results in higher anti-malaria Ab levels
and protects against reinfection.
Mice surviving primary infection
were reinfected after 4 weeks of parasite clearance. The course of
infection was monitored, and sera were collected 14 days after
reinfection. Anti-malaria Ab in terms of total IgG in serum was
significantly higher in mice receiving combined therapy than in those
treated with CQ alone (Fig. 3A). High
anti-malaria Ab levels correlated with protection, since sterile
immunity was found after reinfection of mice treated with CQ plus IL-12
during primary infection whereas low-grade parasitemia was observed in
mice given CQ alone (Fig. 3B).
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|
FIG. 3.
Effect of CQ plus IL-12 combined treatment on the levels
of anti-malaria Ab (total IgG) production (A) and the course of
P. chabaudi AS reinfection (B) in A/J mice. Mice were
reinfected with 106 PRBC 4 weeks after recovery from
primary infection, and the total anti-malaria IgG level was measured in
1:256-diluted sera by ELISA 2 weeks after reinfection. (A) Mean ± SEM data for optical densities (O.D.) representing Ab levels from six
individual mice; and (B) mean ± SEM percent parasitemia from six
individual mice. *, P < 0.001, by Student's
unpaired t test.
|
|
Our present understanding of immunity against blood-stage
P. chabaudi AS infection in resistant mice is that control of primary
infection is dependent on an early Th1 response involving NK-cell
and
macrophage activation, followed by a Th2 response involving
protective
Ab during the chronic phase (
10,
13,
18,
25,
26), which
eventually clears parasites from the circulation.
The present study
revealed that IL-12 used in the combined therapy
of A/J mice, in
addition to providing protection against acute
infection, induces
better Ab responses during reinfection. Although
both cell- and
Ab-mediated immune mechanisms are believed to be
important in
protection against malaria, the results of the present
study do not
distinguish between the relative importance of the
two arms of the
immune system, since we had to evaluate IFN-
and antibody production
at different phases of infection. In practice,
unlike in murine
malaria, "semi-immune" or nonimmune humans are
vulnerable to
reinfection after successful treatment of primary
infection with
antimalaria drugs. By using CQ plus IL-12 combination
treatment, it may
be possible not only to reduce the primary parasite
load and the
associated complications but also to induce a protective
Ab response
and immunity to reinfection. Our findings raise the
question of the
role of IL-12 and IFN-
in influencing Ab responses
during human
malaria infections where long-lasting immunity in
populations in areas
of endemic infection is associated primarily
with anti-malaria Ab
levels (
16).
In areas with endemic malaria infection, the use of antimalarial drugs
has an inhibitory effect on the acquisition of anti-malaria
Ab, both in
children and, more particularly, in adults on chemoprophylaxis
(
16). It is not clear yet whether this is due to direct
immunosuppressive
effects of drugs like CQ or to decreased exposure to
malaria parasite
antigens. In the present study, we observed lower
levels of expression
of both IFN-
and IL-12R mRNA in splenocytes of
mice treated with
CQ alone compared to untreated mice. These mice had
lower anti-malaria
Ab levels than did CQ-plus-IL-12-treated mice,
although it was
not possible to compare the Ab levels in mice treated
with CQ
alone with those in untreated mice, which did not survive till
our reinfection studies. However, the issue of possible
immunosuppression
by CQ could be resolved by studying the effect of CQ
treatment
in
P. chabaudi AS-resistant C57BL/6 mice. In any
event, as shown
by the present study, any immunosuppressive effects of
CQ can
be effectively overcome by using CQ plus IL-12 combined
treatment,
which resulted in marked upregulation of IFN-
and IL-12R
mRNA
levels as well as that of anti-malaria Ab
levels.
IL-12, in combination with amphotericin B, has been used as
immunotherapy in
Histoplasma capsulatum-infected SCID mice
(
35)
and, more recently, in combination with antibiotics for
bacterial
clearance in
Mycobacterium avium-infected SCID
mice (
4). Furthermore,
successful therapy of chronic,
nonhealing murine cutaneous leishmaniasis
with the combination of
sodium stibogluconate and IFN-
was found
to be dependent on
continued production of IL-12 (
14). Cure
of established
Leishmania major infection in mice following combined
therapy with Pentostam and IL-12 involves a switch from a Th2-
to a
Th1-type immune response (
21). However, in this study,
IL-12
alone appeared unable to enhance Th1 cell expansion in vivo,
which was
thought to be due to high parasite loads in mice receiving
only IL-12
compared to mice given combined treatment with the
drug and IL-12.
Similarly, A/J mice could be rescued from lethal
malaria by using IL-12
when the treatment was given on the day
of infection (
26)
but not after the infection was established
(unpublished observations).
In contrast, as observed in the present
study, IL-12-induced
development of an early Th1 response appears
possible even in
established infections when CQ is given along
with IL-12 to
substantially reduce the parasite load. Moreover,
in this treatment
regimen, doses of both CQ and IL-12 could be
reduced to one-half and
one-third, respectively, compared to the
doses required to induce
parasite suppression when either of these
is used alone. In particular,
cure of established infections with
this combined treatment and the
feasibility of using IL-12 at
low, possibly nontoxic, doses suggest the
usefulness of IL-12
in combination with antimalarial drugs in treating
human
malaria.
| |
ACKNOWLEDGMENTS |
This work received grant support from the National Institutes of
Health (AI35955) and the Medical Research Council of Canada (MT12638
and MT14663). H. Sam is a recipient of an M.D./Ph.D. studentship from
Medical Research Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Montreal General
Hospital Research Institute 1650 Cedar Ave., Montreal, Quebec H3G 1A4,
Canada. Phone: (514) 937-6011 ext. 4507. Fax: (514) 934-8332. E-mail:
mcev{at}musica.mcgill.ca.
Editor:
J. M. Mansfield
| |
REFERENCES |
| 1.
|
Allen, R. D.,
T. A. Stanley, and C. L. Sidman.
1993.
Differential cytokine expression in acute and chronic murine graft-versus-host disease.
Eur. J. Immunol.
23:333-339[Medline].
|
| 2.
|
Butler, D.
1997.
Time to put malaria control on the global agenda.
Nature
386:535-541[Medline].
|
| 3.
|
Chua, A. O.,
V. L. Willinson,
D. H. Presky, and U. Gubler.
1995.
Cloning and characterization of a mouse IL-12 receptor-beta component.
J. Immunol.
155:4286-4294[Abstract].
|
| 4.
|
Doherty, T. M., and A. Sher.
1998.
IL-12 promotes drug-induced clearance of Mycobacterium avium infection in mice.
J. Immunol.
160:5428-5435[Abstract/Free Full Text].
|
| 5.
|
Facer, C. A., and M. Tanner.
1997.
Clinical trials of malaria vaccines: progress and prospects.
Adv. Parasitol.
39:1-68[Medline].
|
| 6.
|
Guler, M. L.,
N. G. Jacobson,
U. Gubler, and K. M. Murphy.
1997.
T cell genetic background determines maintenance of IL-12 signaling: effect on BALB/c and B10.D2 T helper cell type 1 phenotype development.
J. Immunol.
159:1767-1774[Abstract].
|
| 7.
|
Heinzel, F. P.,
D. S. Schoenhaut,
R. M. Rerko,
L. E. Rosser, and M. K. Gately.
1993.
Recombinant interleukin 12 cures mice infected with Leishmania major.
J. Exp. Med.
177:1505-1509[Abstract/Free Full Text].
|
| 8.
|
Hoffman, S. L.,
J. M. Crutcher,
S. K. Puri,
A. A. Ansari,
F. Villinger,
E. D. Franks,
P. P. Singh,
F. Finkelman,
M. K. Gately,
G. P. Dutta, and M. Sedegah.
1997.
Sterile protection of monkeys against malaria after administration of interleukin-12.
Nat. Med.
3:80-83[Medline].
|
| 9.
|
Jackson, J. D.,
Y. Yan,
M. J. Brunda,
L. S. Kelsey, and J. E. Talmadge.
1995.
Interleukin-12 enhances peripheral hematopoiesis in vivo.
Blood
85:2371-2376[Abstract/Free Full Text].
|
| 10.
|
Jacobs, P.,
D. Radzioch, and M. M. Stevenson.
1996.
A Th1-associated increase in tumor necrosis factor alpha expression in the spleen correlates with resistance to blood-stage malaria in mice.
Infect. Immun.
64:535-541[Abstract].
|
| 11.
|
Kichian, K.,
F. P. Nestel,
D. Kim,
P. Ponka, and W. S. Lapp.
1996.
IL-12 p40 messenger RNA expression in target organs during acute graft-versus-host disease.
J. Immunol.
157:2851-2856[Abstract].
|
| 12.
|
Lackritz, E. M.,
C. C. Campbell,
T. K. Ruebush,
A. W. Hightower,
W. Wakube,
R. W. Steketee, and J. B. O. Were.
1989.
Effect of blood transfusion on survival among children in a Kenyan hospital.
Lancet
340:524-528.
|
| 13.
|
Langhorne, J.
1989.
The role of CD4+ T cells in the immune response to Plasmodium chabaudi.
Parasitol. Today
5:362-365.
|
| 14.
|
Li, J.,
S. Sutterwala, and J. P. Farrell.
1997.
Successful therapy of chronic, nonhealing murine cutaneous leishmaniasis with sodium stibogluconate and gamma interferon depends on continued interleukin-12 production.
Infect. Immun.
65:3225-3230[Abstract].
|
| 15.
|
Locksley, R. M.
1993.
Interleukin 12 in host defense against microbial pathogens.
Proc. Natl. Acad. Sci. USA
90:5879-5880[Free Full Text].
|
| 16.
|
McGregor, I. A., and R. J. M. Wilson.
1988.
Specific immunity: acquired in man, p. 559.
In
W. H. Wernsdorfer, and I. A. McGregor (ed.), Malaria: principles and practice of malariology. Churchill Livingstone, Ltd., Edinburgh, United Kingdom.
|
| 17.
|
Metzger, D. W.,
R. M. McNutt,
J. T. Collins,
J. M. Buchanan,
V. H. Van Cleave, and W. A. Dunnick.
1997.
Interleukin-12 acts as an adjuvant for humoral immunity through interferon--dependent and -independent mechanisms.
Eur. J. Immunol.
27:1958-1965[Medline].
|
| 18.
|
Mohan, K.,
P. Moulin, and M. M. Stevenson.
1997.
NK cell cytokine production not cytotoxicity contribute to resistance against blood-stage Plasmodium chabaudi AS infection.
J. Immunol.
159:4990-4998[Abstract].
|
| 19.
|
Mohan, K., and M. M. Stevenson.
1998.
Interleukin 12 corrects severe anemia during blood-stage Plasmodium chabaudi AS in susceptible A/J mice.
Exp. Hematol.
26:45-52[Medline].
|
| 20.
|
Murphy, G. S.,
H. Basri,
Purnomo,
E. M. Andersen,
M. J. Bangs,
D. L. Mount,
J. Gorden,
A. A. Lal,
A. R. Purwokusumo,
S. Harjosuwarno,
K. Sorensen, and S. L. Hoffman.
1993.
Vivax malaria resistant to treatment and prophylaxis with chloroquine.
Lancet
341:96-100[Medline].
|
| 21.
|
Nabors, G. S.,
L. C. C. Afonso,
J. P. Farrell, and P. Scott.
1995.
Switch from a type 2 to type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam.
Proc. Natl. Acad. Sci. USA
92:3142-3146[Abstract/Free Full Text].
|
| 22.
|
Orange, J. A.,
T. P. Salazar-Mather,
S. M. Opal,
R. L. Spencer,
A. H. Miller,
B. S. McEwen, and C. A. Biron.
1995.
Mechanisms of interleukin 12-mediated toxicities during experimental viral infections: Role of tumor necrosis factor and glucocorticoides.
J. Exp. Med.
181:901-914[Abstract/Free Full Text].
|
| 23.
|
Presky, D. H.,
H. Yang,
L. J. Minetti,
A. O. Chua,
N. Nabavi,
C. Y. Wu,
M. K. Gately, and U. Gubler.
1996.
A functional interleukin 12 receptor complex is composed of two beta-type cytokine receptor subunits.
Proc. Natl. Acad. Sci. USA
93:14002-14007[Abstract/Free Full Text].
|
| 24.
|
Sedegah, M.,
F. Finkelman, and S. L. Hoffman.
1994.
Interleukin 12 induction of interferon -dependent protection against malaria.
Proc. Natl. Acad. Sci. USA
91:10700-10702[Abstract/Free Full Text].
|
| 25.
|
Stevenson, M. M., and M. F. Tam.
1993.
Differential induction of helper T cell subsets during blood-stage Plasmodium chabaudi AS infection in resistant and susceptible mice.
Clin. Exp. Immunol.
92:77-83[Medline].
|
| 26.
|
Stevenson, M. M.,
M. F. Tam,
S. F. Wolf, and A. Sher.
1995.
IL-12-induced protection against blood-stage Plasmodium chabaudi AS requires IFN- and TNF- and occurs via a nitric oxide-dependent mechanism.
J. Immunol.
155:2545-2556[Abstract].
|
| 27.
|
Trinchieri, G., and P. Scott.
1994.
The role of interleukin 12 in the immune response, disease and therapy.
Immunol. Today
15:460-463[Medline].
|
| 28.
|
White, N. J.
1992.
Antimalarial drug resistance: the pace quickens.
J. Antimicrob. Chemother.
30:571-585[Abstract/Free Full Text].
|
| 29.
|
White, N. J.
1996.
Treatment of malaria.
N. Engl. J. Med.
335:801-806.
|
| 30.
|
Wickramasinghe, S. N.,
S. Abdalla, and D. J. Weatherall.
1982.
Cell cycle distribution of erythroblasts in P. falciparum malaria.
Scand. J. Hematol.
29:83-87[Medline].
|
| 31.
|
World Health Organization.
1990.
Practical chemotherapy of malaria.
W. H. O. Tech. Rep. Ser.
805:1-124.
|
| 32.
|
Wu, C. Y.,
J. Ferrante,
M. K. Gately, and J. Magram.
1997.
Characterization of IL-12 receptor 1 chain (IL-12R1)-deficient mice: IL-12R1 is an essential component of the functional mouse IL-12 receptor.
J. Immunol.
159:1658-1665[Abstract].
|
| 33.
|
Yap, G. S., and M. M. Stevenson.
1992.
Plasmodium chabaudi AS: erythropoietic responses in resistant and susceptible mice.
Exp. Parasitol.
75:340-352[Medline].
|
| 34.
|
Yap, G. S., and M. M. Stevenson.
1994.
Blood transfusion alters the course and outcome of Plasmodium chabaudi AS infection in mice.
Infect. Immun.
62:3761-3765[Abstract/Free Full Text].
|
| 35.
|
Zhou, P.,
M. C. Sieve,
R. P. Tewari, and R. A. Seder.
1997.
Interleukin-12 modulates the protective immune response in SCID mice infected with Histoplasma capsulatum.
Infect. Immun.
65:936-942[Abstract].
|
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Abstract
Introduction
