By
From the Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, EH9 3JT United Kingdom
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Abstract |
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We have examined the role of the immunomodulatory cytokine transforming growth factor
(TGF)- in the resolution and pathology of malaria in BALB/c mice. Circulating levels of
TGF-
, and production of bioactive TGF-
by splenocytes, were found to be low in lethal infections with Plasmodium berghei. In contrast, resolving infections with P. chabaudi chabaudi or P. yoelii were accompanied by significant TGF-
production. A causal association between the
failure to produce TGF-
and the severity of malaria infection was demonstrated by treatment
of infected mice with neutralizing antibody to TGF-
, which exacerbated the virulence of P. berghei and transformed a resolving P. chabaudi chabaudi infection into a lethal infection, but had
little effect on the course of P. yoelii infection. Parasitemia increased more rapidly in anti-TGF-
-treated mice but this did not seem to be the explanation for the increased pathology of infection as peak parasitemias were unchanged. Treatment of P. berghei-infected mice with recombinant TGF-
(rTGF-
) slowed the rate of parasite proliferation and prolonged their
survival from 15 to up to 35 d. rTGF-
treatment was accompanied by a significant decrease in
serum tumor necrosis factor
and an increase in interleukin 10. Finally, we present evidence
that differences in TGF-
responses in different malaria infections are due to intrinsic differences between species of malaria parasites in their ability to induce production of TGF-
.
Thus, TGF-
seems to induce protective immune responses, leading to slower parasite growth,
early in infection, and, subsequently, appears to downregulate pathogenic responses late in infection. This duality of effect makes TGF-
a prime candidate for a major immunomodulatory
cytokine associated with successful control of malaria infection.
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Introduction |
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Experimental infection of mice with various species and
strains of rodent malaria parasites has facilitated dissection of the immunological events associated with both parasite clearance and the pathology of malaria infection. Resolution of a primary infection with the nonlethal parasites
Plasmodium yoelii 17X, P. chabaudi chabaudi, and P. chabaudi
adami is dependent on IFN- (1); TNF-
and IFN-
act
synergistically to optimize nitric oxide production (4, 5),
which is involved in parasite killing (6). The difference between lethal and nonlethal murine malarias can be explained, at least in part, by the ability of the mice to mount
an early IFN-
response (7, 8) and/or an early TNF-
response (2); this may, in turn, be linked to early IL-12 production (4).
However, proinflammatory cytokines can also contribute to the pathology of rodent malaria. In mice infected
with lethal strains of P. berghei, neutralization of IFN- (9),
or blocking IFN-
signaling by disruption of the IFN-
receptor gene (10), delays or completely abrogates mortality,
whereas overproduction, or sustained production, of IFN-
or TNF-
predisposes to severe pathology in both P. chabaudi chabaudi- and P. vinckei-infected mice (2, 9, 11).
Thus, it seems that an early proinflammatory cytokine response mediates protective immunity, whereas a late response contributes to pathology.
In nonlethal infections, inflammatory responses may be
actively downregulated by antiinflammatory cytokines. One
candidate cytokine is IL-10. IL-10-deficient mice infected
with P. chabaudi chabaudi AS showed increased mortality
compared to normal littermates, even though peak parasitemias were not significantly different (12). However,
similar IL-10-deficient mice infected with nonlethal P. yoelii or with the relatively avirulent P. chabaudi adami
556KA showed the same response to infection as wild-type
mice (1). In P. berghei ANKA infections, susceptible strains
of mice show increased expression of IFN- mRNA and
reduced expression of mRNA for TGF-
compared to resistant strains of mice (13), suggesting that TGF-
may play
a role in downregulation of pathogenic proinflammatory cytokines.
TGF-, which is produced by a wide range of cells including macrophages and T cells (14), has both pro- and
antiinflammatory properties, depending on its environment
and concentration (15). Importantly, TGF-
suppresses
production of TNF-
and nitric oxide from macrophages
(16, 17) and suppresses production of IFN-
and TNF-
from NK cells (18). It has recently been proposed that these
effects may be mediated via enhanced IL-10 production by
macrophages (19), eventually leading to a shift in the immune response away from a Th1-like response and towards
a Th2-like response (20).
Although murine malaria models do not replicate all the
features of human malaria, there are strong correlations between the patterns of cytokine production seen in infected
mice and humans. In certain circumstances, IFN- responses are associated with protective immunity to P. falciparum (21, 22), but IFN-
levels are higher in clinical cases
of malaria than in asymptomatic cases (23, 24), and there is
evidence of a causal association between IFN-
secretion
and fever (25). Similarly, TNF-
mediates parasite killing
by macrophages (26, 27), but severe P. falciparum malaria is
accompanied by high levels of circulating TNF-
(28, 29),
and polymorphisms within the promoter region of the
TNF-
gene have been linked to an increased risk of cerebral malaria (30). Together, these observations indicate that
in humans, as in mice, there is a critical balance to be found
in terms of the inflammatory response to malaria infection.
Understanding how this balance is maintained may provide
new approaches to control of malarial parasitemia and prevention of severe disease.
To investigate the role of TGF- in the pathogenesis
of malaria, we have measured TGF-
production from
splenic mononuclear cells of mice infected with both nonlethal (P. chabaudi chabaudi A/J and P. yoelii 17X) and lethal
(P. berghei NK65) rodent malarias and have examined the
effect of neutralizing antibodies to TGF-
, or recombinant
TGF-
, on the course of malaria infections in vivo. We
conclude that levels of TGF-
are inversely correlated with the severity of malaria infections in mice and that TGF-
plays an essential role in downregulating the production of
potentially pathogenic proinflammatory cytokines. Furthermore, differences in TGF-
production in mice infected with different Plasmodium species appear to be due to
intrinsic differences in the ability of parasite antigens to induce TGF-
production from macrophages.
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Materials and Methods |
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Parasites
P. berghei (NK65), P. chabaudi chabaudi (A/J), and P. yoelii (17X) were obtained from Professor David Walliker, WHO Malaria Repository, University of Edinburgh, Edinburgh, UK. P. berghei is highly virulent in mice (31), susceptibility to P. chabaudi chabaudi varies between strains of inbred mice but resolves spontaneously in BALB/c mice (32), and P. yoelii is generally avirulent, although a lethal strain (17XL/YM) has been derived from the avirulent 17X strain (33). Cryopreserved parasites were thawed, injected intraperitoneally into BALB/c mice, and maintained by regular passage into naive mice.
Parasitized mouse erythrocytes (20-40% parasitemia) were purified by layering onto 72% Percoll (Pharmacia Biotech AB, Uppsala, Sweden) and centrifuged at 2,300 rpm for 15 min. Schizonts were recovered from the gradient interface and washed in RPMI (GIBCO BRL, Paisley, UK).
Mice
4-6-wk-old male BALB/c mice were obtained from Harlan (Oxford, UK). The drinking water of experimental mice was supplemented with 2.5 g/liter p-aminobenzoic acid to ensure that parasite growth was not inhibited by a lack of essential nutrients (34).
Experimental Malaria Infections
Mice were infected with either 104 (P. berghei) or 105 (P. berghei, P. chabaudi chabaudi, or P. yoelii) parasite-infected erythrocytes, in 100 µl of PBS by intraperitoneal injection. Control mice received an equal number of uninfected erythrocytes. Parasitemia was monitored every second day by Giemsa-stained thin blood smears obtained from tail bleeds.
For estimation of cytokine production from spleen cells, and to
obtain serum for cytokine assays, test and control mice were killed at regular intervals; blood was obtained by venepuncture and spleens were retained for cellular assays. Blood was allowed to
clot and serum was stored at 20°C until required.
To examine the effect of neutralization of TGF- on the
course of malaria infection, mice were each given 50 µg of an
IgG1 monoclonal antibody to TGF-
, which neutralizes all three
TGF-
isoforms (1835-01; Genzyme Diagnostics, Cambridge,
MA), by intraperitoneal injection 1 d before malaria infection and
on days 2, 5, and 7 after infection. Control mice received 50 µg
of polyclonal mouse IgG1 (Serotec, Oxford, UK).
To examine the effect of increased TGF- levels in malaria-
infected mice, mice were infected with P. berghei and given either 5 ng or 20 ng of recombinant TGF-
1 (R&D Systems Europe
Ltd., Abingdon, UK) in 100 µl of PBS by intraperitoneal injection on the day of infection and then daily for another 4 d. Control mice received PBS only.
Mononuclear Cell Cultures
Mononuclear cells were obtained from macerated spleens by
centrifugation over a 5-ml gradient of mouse lymphocyte separation medium (Harlan-Seralab, Loughborough, UK). After washing in RPMI, cells were resuspended in complete medium
(RPMI containing 2 g/liter NaCO2, 2mM L-glutamine, 100 U/ml
penicillin, and 100 µg/ml streptomycin, all from ICN Biomedicals, Costa Mesa, CA) and allowed to adhere to plastic petri
dishes coated with mouse serum for 90 min at 37°C. Nonadherent cells were washed away, adherent cells (~90% macrophages
and ~10% lymphocytes) were recovered by incubation for 30 min at 4°C in calcium- and magnesium-free saline (HBSS; Sigma
Chemical Co., St. Louis, MO) and counted, and 2.5 × 106 adherent cells were added to each well of a 48-well microtiter plate.
Cells were cultured in 350 µl of complete medium at 37°C in 5%
CO2 in air for up to 48 h either without further stimulation or in
the presence of 5 × 105 live malaria parasites, uninfected red
blood cells, or the mitogen Con A (5 µg/ml; Sigma Chemical
Co.). Cell culture supernatants were collected and stored at
20°C until required.
TGF- Bioassay
This assay, based on the ability of TGF- to inhibit the in vitro
proliferation of mink lung epithelial cells (Mv-I-Lu; 35), measures the combined effects of all TGF-
isoforms. Mv-I-Lu cells (ATCC clone CCL64; European Collection of Cell Cultures,
Wiltshire, UK) were maintained by weekly passage in Eagle's
minimum essential medium (Sigma Chemical Co.) supplemented
with 0.1 M nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin,
and 10% heat-inactivated fetal bovine serum (FBS; all from Sigma
Chemical Co.). Cells were detached from the culture flask by incubation at 37°C with 0.5% trypsin/5.3 mM EDTA, washed, and
resuspended in complete medium, containing 6% serum substitute (Ultroser; GIBCO BRL) instead of FBS, at 2 × 104 cells/ml.
104 cells, in a volume of 50 µl, were aliquoted into each well of a
96-well, flat-bottomed microculture plate and left to adhere for 2 h.
Culture supernatants were tested for TGF- activity either
with or without prior acid-activation. Acid-activation converts the inactive precursor form of TGF-
into biologically active TGF-
, allowing total TGF-
to be measured; in contrast, only biologically active TGF-
is measured in non-acid-activated
samples. For acid activation, 10 µl 1 M HCl was added to 100 µl
supernatant, incubated at 4°C for 30 min, and neutralized by
dropwise addition of 20 µl 1 M NaOH. 50 µl of test sample or
TGF-
1 standard (recombinant murine TGF-
1, IC50 0.03-0.05
ng/ml; Sigma Chemical Co.) and 50 µl fresh medium (with 6%
Ultroser) were added to triplicate wells of Mv-I-Lu cells and incubated for 48 h at 37°C in 5% CO2 in air; [3H-]thymidine (0.5 µCi/well; Amersham Life Sciences, Little Chalfont, UK) was
added for the last 18 h of culture. For cell harvesting, cells were incubated with 50 µl trypsin/EDTA for 15-30 min at 37°C and then
harvested onto glass fiber filters; incorporation of 3H was assessed
by liquid scintillation counting. Concentration of TGF-
in the
sample was calculated by reference to the percentage inhibition of 3H
incorporation caused by different concentrations of the TGF-
1 standard. The specificity of the assay was confirmed by blocking the
inhibition of cell growth with a neutralizing monoclonal antibody to all isoforms of murine TGF-
(20 µg/ml; 1D11.16; Genzyme).
ELISAs
TGF-1.
IFN-, TNF-
, IL-10, and IL-4.
Statistical Analysis
Differences between the means, of groups of five mice, were determined by Student's t test.
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Results |
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TGF- Production during Fatal and Resolving Murine
Malaria Infections
After infection with either 104 or 105 infected erythrocytes, P. berghei infection was universally fatal by day 20 (104) or days 13-15 (105) after infection. In contrast, P. yoelii and P. chabaudi chabaudi infections resolved spontaneously; parasitemia peaked around days 14-16 and was cleared by day 23 after infection.
Mice were killed at various time points after infection
and spleens were removed. Spontaneous production of
both latent and bioactive TGF- by splenic mononuclear
cells was assessed by bioassay. In P. berghei-infected mice
there was a steady and significant decline in total and bioactive TGF-
production from day 10 after infection onwards (day 0 versus day 20, total TGF-
student's t test (t)
= 7.47, degrees of freedom (df ) = 8, P <0.001; bioactive TGF-
t = 81.7, df = 8, P <0.0001), with levels of TGF-
being inversely related to parasitemia (Fig. 1 a). In both P. chabaudi chabaudi- and P. yoelii-infected mice there was a
transient fall in bioactive TGF-
production as parasitemia
increased; this was statistically significant for P. chabaudi chabaudi at day 7 (t = 3.77, df = 8, P = 0.005) and for P. yoelii
at day 12 (t = 4.99, df = 8, P = 0.005), but total TGF-
production increased steadily and significantly throughout
the course of infection (day 0 versus day 23, t >3.9, df = 8, P = 0.004 in both cases; Fig. 1, b and c).
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In a parallel experiment, levels of circulating serum
TGF-1 in infected mice were measured by ELISA (Fig.
2). This assay measures both latent and bioactive TGF-
1.
In P. berghei-infected mice, plasma TGF-
1 levels declined
significantly between days 8 and 20 (t = 6.3, df = 7, P
<0.001). In contrast, plasma TGF-
1 levels in mice infected with P. chabaudi chabaudi or P. yoelii decreased transiently during the first week of infection (day 0 versus day
3, t = 3.7, df = 8, P = 0.006 for P. chabaudi chabaudi; t = 2.34, df = 8, P <0.049 for P. yoelii) but then rose steadily for the remainder of the infection (day 0 versus day 20, t
>5.9, df = 8, P <0.001 in both cases).
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Taken together, these data suggest that TGF- may play
a crucial role in preventing the severe pathology of malaria.
Where TGF-
levels are maintained at normal or above
normal levels, the mice survive, but where TGF-
levels
are suppressed, the mice die.
The Role of TGF- in Murine Malaria Infections
To test the hypothesis that
TGF- is required to prevent the severe pathology associated with some murine malarias, mice were treated with a
neutralizing antibody to all mouse TGF-
isoforms immediately before and during infection with P. berghei, P. chabaudi chabaudi, or P. yoelii. Anti-TGF-
antibody, or isotype-matched control IgG, was administered 1 d before
infection and on days 2, 5, and 7 after infection (Fig. 3).
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In this experiment, P. berghei-infected mice received 105
infected erythrocytes, leading to death between days 10 and 13. Anti-TGF- treatment significantly enhanced the
rate of increase P. berghei parasitemia (Fig. 3 a, treated versus untreated mice, day 7, t = 10.2, df = 8, P <0.0001),
and all the infected mice died on either day 6 or 8 after infection, 4 or 5 d earlier than in mice who received the control antibody (Fig. 3 d). The anti-TGF-
antibody did not,
by itself, cause any observable side-effects, as treated, uninfected mice remained perfectly healthy (data not shown).
Neutralization of TGF- converted the normally nonlethal P. chabaudi chabaudi infection into a rapidly lethal infection. Infected mice began dying at day 6 after infection and
all died by day 12 (Fig. 3 e). Although parasitemia increased
more rapidly in anti-TGF-
-treated mice than in untreated mice (day 10, t = 4.74, df = 8, P <0.001) and the
peak parasitemia occurred ~2 d earlier in treated mice, the
peak was not significantly higher than in untreated mice
(Fig. 3 b; t = 0.92, df = 8, P = 0.39).
In contrast, neutralization of TGF- had little effect on
the overall course of P. yoelii infection. Parasitemia rose
slightly earlier in treated mice and was significantly higher
at day 12 (treated versus untreated, t = 3.58, df = 8, P = 0.007), but the peak parasitemia was not significantly different (day 16, t = 1.31, df = 8, P = 0.23). Parasitemia resolved spontaneously (Fig. 3 c) and all the mice survived
(Fig. 3 f ).
These data show very clearly that TGF- plays a crucial
role in protecting mice with P. chabaudi chabaudi infection
from the severe pathology of malaria and suggest that the
lack of a TGF-
response in mice infected with P. berghei
may explain the severe pathology associated with this parasite. Interestingly, neutralization of TGF-
has little effect
on the outcome of P. yoelii infection, suggesting that this
parasite interacts very differently with the host's immune
system compared with P. berghei or P. chabaudi chabaudi.
The data also suggest that TGF-
may contribute to the control of parasite growth as, in all cases, neutralization of TGF-
led to increased rates of parasite proliferation.
However, the effect of anti-TFG-
antibody on mouse
survival is probably distinct from its effect on parasitemia, as
parasitemia increased more rapidly in all infections, but (a)
death was accelerated only in P. berghei and P. chabaudi chabaudi infections and (b) P. chabaudi chabaudi-infected mice
died at parasitemias that are compatible with survival in
control mice.
To test
the hypothesis that the severity of P. berghei malaria in
BALB/c mice is associated with the failure to upregulate TGF- production during infection, mice were infected
with 105 P. berghei-infected erythrocytes and treated with
either 5 or 20 ng/mouse of rTGF-
1 daily for 5 d.
In mice receiving rTGF-1, the parasitemia rose less
quickly than in control mice (Fig. 4 a). Importantly, death was
significantly delayed in treated mice; mice receiving 20 ng
TGF-
1 died 12 d later than untreated mice, whereas mice
receiving 5 ng/day died ~20 d later (Fig. 4 b). However, it
was not possible to separate the effect of TGF-
on parasite
growth from the effect on mouse survival; in mice treated
with 20 ng rTGF-
, parasitemia rose more quickly, and
death occurred earlier, than in mice receiving 5 ng TGF-
.
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Interaction between TGF- and Other Cytokines
It is known that much of the pathology of malaria is mediated by proinflammatory cytokines such as TNF- and IFN-
(25, 28, 29). TGF-
is a
known antiinflammatory agent that acts to downregulate
the production of proinflammatory cytokines (37). We hypothesized that the severe pathology of P. berghei infection was due to overproduction of proinflammatory cytokines in
the absence of TGF-
. Circulating levels of TNF-
and
IFN-
in the plasma of infected mice were measured by
ELISA.
In mice infected with P. berghei, serum TNF- levels rise
significantly over the first 10 d of infection (Fig. 5 a; day 3 versus day 10, t = 3.26, df = 8, P = 0.012). As predicted,
when P. berghei-infected mice are treated with rTGF-
,
there is a highly significant decrease in circulating levels of
TNF-
(Fig. 5 a; t >4.8, df = 8, P <0.001 at days 3, 7, and 10), and when mice are given anti-TGF-
antibody,
serum TNF-
levels are significantly higher (day 7 after infection, control mice, mean TNF-
= 5.4 ± 0.5 ng/ml;
anti-TGF-
mice, mean = 9.15 ± 0.92 ng/ml; t = 7.92, df = 7, P <0.0001).
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In contrast, rTGF-1 or anti-TGF-
antibody had an
unexpected effect on levels of circulating IFN-
in P. berghei-infected mice. rTGF-
1 treatment lead to a small,
although statistically significant, increase in serum IFN-
levels (Fig. 5 b), whereas anti-TGF-
antibody had no significant effect on IFN-
levels (day 7 after infection, control mice, mean IFN-
= 40.5 ± 3.1 U/ml; anti-TGF-
-treated mice, mean = 44.3 ± 2.25 U/ml; t = 1.97, df = 7, P = 0.1).
An alternative means by which TGF-
may downregulate inflammatory cytokines is by augmentation of the production of other antiinflammatory cytokines.
We therefore measured plasma levels of IL-4 and IL-10 in
P. berghei-infected mice and assessed the effect of rTGF-
or anti-TGF-
antibody. Neither IL-4 nor IL-10 levels
changed significantly over the course of infection with P. berghei (Fig. 5, c and d). Treatment with rTGF-
over 5 d
lead to an apparent steady decline in IL-4 levels (Fig. 5 c),
but the difference between the two groups was statistically
significant only on day 10 (t = 3.7, df = 8, P <0.01). In
contrast, rTGF-
leads to a rapid and highly significant increase in IL-10 production in infected mice (Fig. 5 d) that
persisted until at least day 10 after infection (days 3, 7, and
10, t >2.33, df = 8, P <0.05 in all cases). Anti-TGF-
antibody treatment had no significant effect on IL-4 or IL-10
levels (data not shown).
In Vitro Induction of TGF- by Live Malaria Parasites
The failure of mice infected with P. berghei to sufficiently
upregulate TGF- production was investigated in vitro.
Plastic adherent splenic mononuclear cells from uninfected
mice were incubated in vitro for up to 36 h with parasitized erythrocytes, uninfected erythrocytes, or mitogen, and
the concentration of total and bioactive TGF-
in the culture supernatants was measured by bioassay (Table 1).
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The mitogen Con A induced production of TGF-
within 2-6 h, and TGF-
levels peaked at 18 h. Live P. chabaudi chabaudi parasites were almost as effective as Con
A, inducing TGF-
production within 6 h, with levels also
peaking after 18 h in culture. In contrast, P. berghei parasites
induced little if any TGF-
production.
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Discussion |
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We have shown that murine infection with P. chabaudi
chabaudi or P. yoelii, but not P. berghei, is accompanied by
increased TGF- production, as assessed by raised serum
levels of TGF-
1 in vivo and spontaneous secretion of
TGF-
in vitro by splenic mononuclear cells from infected
mice. We have also shown that TGF-
levels during murine malaria infection are inversely correlated with severity
of disease, in that lethal infections are accompanied by low
levels of TGF-
and self-resolving infections are accompanied by high levels of TGF-
. These findings have been
confirmed by treatment of mice with either neutralizing
antibodies to TGF-
or rTGF-
1. These findings are consistent with the only other data regarding TGF-
in murine malaria, which simply showed that P. berghei (ANKA)-
induced TGF-
mRNA levels were lower in susceptible strains of mice than in resistant strains (13).
Given the wealth of evidence that the pathology of murine malaria is mediated by the inflammatory cytokines
IFN- and TNF-
(2, 9, 11), the most likely explanation
for our observations was that TGF-
acted to downregulate either the production or the activity of these cytokines.
We were able to show quite clearly that rTGF-
1 treatment led to a decrease in circulating levels of TNF-
but
had much less effect on levels of IFN-
. These observations are consistent with the known targets of the antiinflammatory activities of TGF-
, namely its ability to suppress
translation of TNF-
mRNA in macrophages (37) and to
inhibit transcription of genes for cytokines such as IL-8
(38) and macrophage chemoattractant protein (MCP1; 39).
In contrast, there is little evidence that TGF-
has a direct
effect on macrophage-derived IFN-
; rather, TGF-
antagonizes the effects of IFN-
downstream of IFN-
itself, for example by inhibiting the induction of MHC class II
expression (40).
Although our data are consistent with a direct effect of
TGF- on TNF-
production, we cannot rule out an indirect effect via IL-10 (19, 20). IL-10 is induced by TGF-
(19) and has been shown to suppress production of TNF-
in activated macrophages (37). Consistent with this hypothesis, IL-10 levels were not raised in the serum of P. berghei-infected mice, but IL-10 was clearly upregulated
when infected mice were treated with rTGF-
1. A role for
IL-10 in ameliorating the pathology of P. chabaudi chabaudi
infection has been shown in IL-10-deficient mice (12) but,
interestingly, IL-10 does not seem to play a similar role in
avirulent P. yoelii or P. chabaudi adami infections where
IL-10-deficient mice were able to resolve their infections
normally (1). This mirrors our observation that the pathology of P. yoelii was not affected by neutralization of TGF-
activity and suggests that the pathology of P. yoelii infection
has a very different aetiology. However, IL-10 is unlikely
to be the only pathway for TGF-
activity as P. chabaudi
chabaudi infection is much more severe in mice treated with
anti-TGF-
antibody, where all the mice died, than in IL-10-deficient mice, where mortality is restricted to female mice and even then only 50% of the female mice die (12).
However, in both IL-10-deficient and anti-TGF-
-
treated mice, death occurs without a significant increase in
peak parasitemia, indicating that failure to control P. chabaudi chabaudi replication is not the cause of death.
Our demonstration that TGF- upregulates IL-10 production without downregulating IFN-
offers an explanation for the observation that both IFN-
and IL-10 levels
are raised in acute P. falciparum infection in humans (41).
Little is known of TGF-
responses in human malarias; supernatants of human mononuclear cells cocultured with P. falciparum-infected erythrocytes contained high levels of
TGF-
(42), but TGF-
serum levels are lower in patients with acute P. falciparum malaria than in healthy controls
(42a).
In addition to downregulation of TNF- production,
TGF-
may inhibit the development of malarial pathology
by direct effects on parasite sequestration. Adherence of infected erythrocytes to cerebral capillary endothelium, via
cellular adhesion molecules such as intercellular adhesion
molecule (ICAM)-1 and vascular cell adhesion molecule
(VCAM)-1, has been proposed to contribute to the development of cerebral malaria (43, 44). Mice in which the
TGF-
1 genes have been disrupted show enhanced expression of both ICAM-1 and VCAM-1 (45), suggesting that
TGF-
may play a role in downregulating expression of
these adhesion molecules thus reducing the risk of cerebral
malaria.
Although our study provides strong evidence that TGF-
can protect against the severe pathology of P. berghei and P. chabaudi chabaudi malaria, our data also indicate that TGF-
may play a role in controlling parasite growth, at least in
the early stages of infection, as anti-TGF-
antibody led to
more rapid parasite growth and a 5-d course of rTGF-
1
resulted in slower parasite growth. In the case of P. chabaudi
chabaudi infection, the effect of TGF-
on survival is clearly
distinct from its effect on parasite growth; this distinction is
less clear in P. berghei infection. The apparent ability of
TGF-
to help control parasite growth may relate to the
fact that, early in an immune response, low concentrations
of TGF-
actually promote inflammation, recruit monocytes and macrophages to the site of injury, and activate them to become phagocytic (15). In support of this hypothesis, we have shown that TGF-
1 increases phagocytosis of P. falciparum-infected erythrocytes by human peripheral blood mononuclear cells in vitro (Omer, F.M., and
E. Riley, manuscript in preparation). Thus, inhibition of
TGF-
early in the infection may inhibit macrophage activation and nonspecific parasite clearance, as has been
shown for other pathogens such as Candida albicans (46),
pneumococcus (47), and HIV (48). In contrast, high concentrations of TGF-
are antiinflammatory but also inhibit
the activity of inducible nitric oxide synthase (49), thus reducing the ability of macrophages to control the growth of
intracellular pathogens such as Trypanosoma cruzi (50) and
Leishmania amazonensis (51). Thus, the balance between
controlling parasite replication and avoiding immunopathology appears to depend on controlling local and systemic concentrations of TGF-
.
In summary, the bimodal activities of TGF--promoting inflammation and parasite clearance early in infection
while downregulating inflammation and pathology later in
infection
make it a very strong contender for being a major immunoregulatory molecule, maintaining the balance
between the protective and pathogenic effects of other inflammatory cytokines during malaria infections. If this supposition is correct, one would predict that severe malaria in
humans is associated with reduced capacity to produce
TGF-
. Studies are currently underway in our laboratory
to test this hypothesis. However, this theory begs the question as to why some infections are associated with low
TGF-
production. The preliminary data presented here,
indicating that P. berghei parasites fail to induce TGF-
production from normal mouse macrophages, suggests that genetic differences between parasites may be responsible.
Possible explanations for the failure of P. berghei to induce
TGF-
include the failure to induce TGF-
per se, induction of high levels of IL-12 or IFN-
, both of which have
been shown to act as negative regulators of TGF-
production (52), or induction of TGF-
inhibitors such as
2-macroglobulins (49). Identification of the mechanisms of
TGF-
antagonism by P. berghei may provide clues as to
the pathogenesis of severe malaria in humans.
![]() |
Footnotes |
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Address correspondence to Eleanor M. Riley, Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Rd., Edinburgh, EH9 3JT, UK. Phone: 44-131-650-5540; Fax: 44-131-667-3210; E-mail: e.riley{at}ed.ac.uk
Received for publication 15 November 1997 and in revised form 18 February 1998.
We thank David McGuinness for statistical advice, Richard Carter and Niklas Ahlborg for constructive comments on the manuscript, and David Walliker and Pedro Cravo for providing malaria parasites.
This study was funded by the Wellcome Trust. F.M. Omer is supported by a Wellcome Trust International Fellowship.
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References |
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---|
1. | van der Heyde, H.C., B. Pepper, J. Batchelder, F. Cigel, and W.P. Weidanz. 1997. The time course of selected malarial infections in cytokine-deficient mice. Exp. Parasitol. 85: 206-213 [Medline]. |
2. | 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]. |
3. | Favre, N., B. Ryffel, G. Bordmann, and W. Rudin. 1997. The course of Plasmodium chabaudi chabaudi infections in interferon-gamma receptor-deficient mice. Parasite Immunol. 19: 375-383 [Medline]. |
4. |
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-![]() ![]() |
5. | Jacobs, P., D. Radzioch, and M.M. Stevenson. 1996. In vivo regulation of nitric oxide production by tumor necrosis factor alpha and gamma interferon, but not by interleukin-4, during blood stage malaria in mice. Infect. Immun. 64: 44-49 [Abstract]. |
6. | Nussler, A.K., L. Rénia, V. Pasquetto, F. Miltgen, H. Matile, and D. Mazier. 1993. In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria parasites or adjuvants. Eur. J. Immunol. 23: 882-887 [Medline]. |
7. |
Shear, H.L.,
R. Srinavasan,
T. Nolan, and
C. Ng.
1989.
Role
of IFN-![]() |
8. | de Souza, J.B., K.H. Williamson, T. Otani, and J.H.L. Playfair. 1997. Early gamma interferon responses in lethal and nonlethal murine blood stage malaria. Infect. Immun. 65: 1593-1598 [Abstract]. |
9. | Waki, S., S. Uehara, K. Kanbe, K. Ono, M. Suzuki, and H. Nariuchi. 1992. The role of T cells in pathogenesis and protective immunity to murine malaria. Immunology 75: 646-651 [Medline]. |
10. |
Rudin, W.,
N. Favre,
G. Bordmann, and
B. Ryffel.
1997.
Interferon-![]() |
11. |
Kremsner, P.G.,
S. Neifer,
M.F. Chaves,
R. Rudolph, and
U. Bienzle.
1992.
Interferon-![]() |
12. | Linke, A., R. Kuhn, W. Muller, N. Honarvar, C. Li, and J. Langhorne. 1996. Plasmodium chabaudi chabaudi: differential susceptibility of gene-targeted mice deficient in IL-10 to an erythrocytic-stage infection. Exp. Parsitol. 84: 253-263 [Medline]. |
13. |
de Kossodo, S., and
G.E. Grau.
1993.
Profiles of cytokine
production in relation with susceptibility to cerebral malaria.
J. Immunol.
151:
4811-4820
|
14. |
Assosian, R.K.,
B.E. Fleurdelys,
H.C. Stevenson,
P.J. Miller,
D.K. Madtes,
E.W. Raines,
R. Ross, and
M.B. Sporn.
1987.
Expression and secretion of type ![]() |
15. |
Wahl, S.M.,
N. McCartney-Francis, and
S.E. Mergenhagen.
1989.
Inflammatory and immunomodulatory roles of TGF-![]() |
16. |
Espevik, T.,
I.S. Figari,
M.R. Shalaby,
G.A. Lackides,
G.D. Lewis,
H.M. Shepard, and
M.A. Palladino Jr..
1987.
Inhibition of cytokine production by cyclosporin A and transforming growth factor ![]() |
17. |
Ding, A.,
C.F. Nathan,
J. Grayear,
R. Derynck,
D.J. Stuehr, and
S. Srimai.
1990.
Macropahge deactivating factor and
transforming growth factor-![]() ![]() ![]() ![]() |
18. |
Bellone, G.,
M. Aste-Amezaga,
G. Trinchieri, and
U. Rodeck.
1995.
Regulation of NK cell functions by TGF-![]() |
19. |
Maeda, H.,
H. Kuwahara,
Y. Ichimura,
M. Ohtsuki,
S. Kurakata, and
A. Shiraishi.
1995.
TGF-![]() |
20. |
Maeda, H., and
A. Shiraishi.
1996.
TGF-![]() |
21. | Ferreira, A., L. Schofield, V. Enea, H. Schellekens, P. Van der Meide, W.E. Collins, R.S. Nussenzweig, and V. Nussenzweig. 1986. Inhibition of development of exoerythrocytic forms of malaria parasites by IFN-gamma. Science 232: 881-884 [Medline]. |
22. | Herrera, M., F. Rosero, S. Herrera, P. Caspers, D. Rotmann, F. Sinigaglia, and U. Certa. 1992. Protection against malaria in Aotus monkeys immunised with a recombinant blood stage antigen fused to a universal T cell epitope: correlation of serum gamma-interferon levels with protection. Infect. Immun. 60: 154-158 [Abstract]. |
23. | Riley, E.M., P.H. Jakobsen, S.J. Allen, J.G. Wheeler, S. Bennett, and B.M. Greenwood. 1991. Immune responses to soluble exoantigens of Plasmodium falciparum may contribute to both pathogenesis and protection in clinical malaria: evidence from a longitudinal, prospective study of semi-immune African children. Eur. J. Immunol. 21: 1019-1025 [Medline]. |
24. |
Mshana, R.N.,
J. Boulandi,
N.M. Mshana,
J. Mayombo, and
G. Mendome.
1991.
Cytokines in the pathogenesis of malaria: levels of IL-1![]() ![]() ![]() |
25. | Harpaz, R., R. Edelman, S.S. Wasserman, M.M. Levine, J.R. Davis, and M.B. Sztein. 1992. Serum cytokine profiles in experimental human malaria. Relationship to protection and disease course after challenge. J. Clin. Invest. 90: 515-523 [Medline]. |
26. | Bouharoun-Tayoun, H., C. Oeuvray, F. Lunel, and P. Druilhe. 1995. Mechanisms underlying the monocyte-mediated antibody-dependent killing of Plasmodium falciparum asexual blood stages. J. Exp. Med. 182: 409-418 [Abstract]. |
27. | Rockett, K.A. M.M., Awburn, B.B. Aggarwal, W.B. Cowden, and I.A. Clark. 1992. In vivo induction of nitrite and nitrate by tumour necrosis factor, lymphotoxin and interleukin 1: possible roles in malaria. Infect. Immun. 60: 3725-3730 [Abstract]. |
28. | Grau, G.E., T.E. Taylor, M.E. Molyneux, J.J. Wirima, P. Vassalli, M. Hommel, and P.H. Lambert. 1989. Tumor necrosis factor and disease severity in children with falciparum malaria. N. Engl. J. Med. 320: 1586-1591 [Abstract]. |
29. | Kwiatkowski, D., A.V.S. Hill, I. Sambou, P. Twumasi, J. Castracane, K.R. Manogue, A. Cerami, D. Brewster, and B.M. Greenwood. 1990. TNF concentration in fatal, non- fatal cerebral and uncomplicated Plasmodium falciparum malaria. Lancet. 336: 1201-1204 [Medline]. |
30. |
McGuire, W.,
A.V.S. Hill,
C.E.M. Allsopp,
B.M. Greenwood, and
D. Kwiatkowski.
1994.
Variation in the TNF-![]() |
31. | Cox, F.E.G. 1988. Major animal models in malaria research: rodent. In Malaria: Principles and Practice of Malariology. W.H. Wernsdorfer and I. McGregor, editors. Churchill Livingstone, London. 1503-1543. |
32. | Stevenson, M.M., J.J. Lyanga, and E. Skamene. 1982. Murine malaria: genetic control of resistance to Plasmodium chabaudi. Infect. Immun. 38: 80-88 [Medline]. |
33. | Yoelii, M., B. Hargreaves, R. Carter, and D. Walliker. 1975. Sudden increase in virulence in a strain of Plasmodium yoelii. Ann. Trop. Med. Parasitol. 69: 173-178 [Medline]. |
34. | Peters, W.. 1967. Chemotherapy of Plasmodium chabaudi infection in albino mice. Ann. Trop. Med. Parasitol. 61: 52-56 [Medline]. |
35. |
Danielpour, D.,
L.L. Dart,
K.C. Flanders,
A.B. Roberts, and
M.B. Sporn.
1989.
Immunodetection and quantitation of the
two forms of transforming growth factor-![]() ![]() ![]() |
36. |
Lucas, C.,
B.M. Fendly,
F.R. Mukku,
W.L. Wong, and
M.A. Palladino.
1991.
Generation of antibodies and assays for
transforming growth factor ![]() |
37. |
Bogdan, C.,
J. Paik,
Y. Vodovotz, and
C. Nathan.
1992.
Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-![]() |
38. |
Smith, W.B.,
L. Noack,
Y. Khew-Goodall,
S. Isenmann,
M.A. Vadas, and
J.R. Gamble.
1996.
Transforming growth
factor-![]() |
39. |
Kitamura, M..
1997.
Identification of an inhibitor targeting
macrophage production of monocyte chemoattractant protein-1 as TGF-![]() |
40. |
Nandan, D., and
N.E. Reiner.
1997.
TGF-![]() ![]() |
41. |
Wenisch, C.,
B. Parschalk,
E. Narzt,
S. Looareesuwan, and
W. Graninger.
1995.
Elevated serum levels of IL-10 and
IFN-![]() |
42. |
Wahlgren, M.,
J.S. Abrams,
V. Fernandez,
M. Bejarano,
M. Azuma,
M. Torii,
M. Aikawa, and
R.J. Howard.
1995.
Adhesion of Plasmodium falciparum-infected erythrocytes to human cells and secretion of cytokines (IL-1-![]() ![]() ![]() ![]() |
42a. |
Wenisch, C.,
B. Parschalk,
H. Burgmann,
S. Looareesuwan, and
W. Graninger.
1995.
Decreased serum levels of TGF-![]() |
43. | Ockenhouse, C.F., T. Tegoshi, Y. Maeno, C. Benjamin, M. Ho, K. Ei, Khan, Y. Thway, K. Win, M. Aikawa, and R.R. Lobb. 1992. Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. J. Exp. Med. 176: 1183-1189 [Abstract]. |
44. | Berendt, A.R., D.L. Simmons, J. Tansey, C.I. Newbold, and K. Marsh. 1989. Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum. Nature. 341: 57-59 [Medline]. |
45. |
Nakabayashi, T.,
J.J. Letterio,
A.G. Geiser,
L. Kong,
N. Ogawa,
W. Zhao,
T. Koike,
G. Fernandes,
H. Dang, and
N. Talal.
1997.
Up-regulation of cytokine mRNA, adhesion
molecule proteins, and MHC class II proteins in salivary
glands of TGF-![]() |
46. |
Spaccapelo, R.,
L. Romani,
L. Tonnetti,
E. Cenci,
A. Mencacci,
G. Del Sero,
R. Tognellini,
S.G. Reed,
P. Puccetti, and
F. Bistoni.
1995.
TGF-![]() |
47. |
Pfister, H.W.,
K. Frei,
B. Ottand,
U. Koedel,
A. Tomasz, and
A. Fontana.
1992.
Transforming growth factor ![]() ![]() |
48. |
Poli, G.,
A.L. Kinter,
J.S. Justement,
P. Bressler,
J.H. Kehrl, and
A.S. Fauci.
1991.
Transforming growth factor ![]() |
49. |
Webb, D.J.,
J. Wen,
J.L. Lysiak,
L. Umans,
F. Van Leuven, and
S.L. Gonias.
1996.
Murine ![]() ![]() |
50. |
Silva, J.S.,
D.R. Twardzik, and
S.G. Reed.
1991.
Regulation
of Trypansoma cruzi infections in vitro and in vivo by transforming growth factor ![]() ![]() |
51. |
Barral-Neto, M.,
A. Barral,
C.E. Brownwell,
Y.A.W. Skeiky,
L.R. Ellingsworth,
D.R. Twardzik, and
S.G. Reed.
1992.
Transforming growth factor ![]() |
52. |
Marth, T.,
W. Strober,
R.A. Seder, and
B.L. Kelsall.
1997.
Regulation of transforming growth factor-![]() |