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Infection and Immunity, November 1999, p. 5579-5586, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Endogenous Balance of Soluble Tumor Necrosis Factor Receptors
and Tumor Necrosis Factor Modulates Cachexia and Mortality in Mice
Acutely Infected with Trypanosoma cruzi
Carine
Truyens,1
Faustino
Torrico,1
Rudolf
Lucas,2,
Patrick
De
Baetselier,2
Wim A.
Buurman,3 and
Yves
Carlier1,*
Laboratory of Parasitology, University of Brussels
(ULB)1 and Institute for Molecular
Biology, Unit of Cellular Immunology, University of Brussels
(VUB),2 Brussels, Belgium, and
Department of Surgery, Faculty II, University of Limburg,
Maastricht, The Netherlands3
Received 11 May 1999/Returned for modification 22 June
1999/Accepted 9 August 1999
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ABSTRACT |
To better understand the role of tumor necrosis factor (TNF) during
Trypanosoma cruzi infection in BALB/c mice, we have
investigated the kinetics of circulating tumor necrosis factor (TNF),
soluble TNF receptor 1 (sTNR1), and sTNFR2 levels, as well as the
interactions between such factors, in relation to parasitemia,
cachexia, and mortality of acutely infected animals. Our data show that
the parasitemic phase of T. cruzi infection in mice is
associated with high levels of circulating TNF and sTNFR2, resulting in
the formation of cytokine-receptor complexes and some degree of
neutralization of TNF bioactivity. Although sTNR2 levels always
exceeded TNF levels, low sTNFR/TNF circulating ratios were associated
with cachexia in all infected mice, whereas the lowest ratios were observed in dying animals harboring the highest parasitemia. We also
studied the modulation of sTNFR/TNF ratios induced by anti-TNF antibodies administered to infected animals and their consequences on
the outcome of the infection. The injection of anti-TNF monoclonal antibody (MAb) TN3 into infected mice resulted in a paradoxical overproduction of TNF (associated with a higher parasitemia), lowered
the sTNFR/TNF circulating ratios, and considerably worsened cachexia
and mortality of animals. Another anti-TNF MAb (1F3F3) decreased the in
vivo availability of TNF as well as parasite levels and reduced
cachexia. Altogether, such results highlight that, besides playing a
beneficial role early in infection, TNF also triggers harmful effects
in the parasitemic phase, which are limited by the in vivo simultaneous
endogenous production of soluble receptors.
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INTRODUCTION |
Tumor necrosis factor (TNF) includes
two related molecules, termed TNF-
(found in membrane and soluble
forms) and lymphotoxin alpha (produced only in soluble form), which
transduce their activities through two membrane TNF receptors (TNFRs)
with apparent molecular masses of 55 kDa (TNFR1, CD120a) and 75 kDa
(TNFR2, CD120b) (2, 56). The extracellular domains of these
receptors are released in the circulation of healthy individuals by
proteolytic cleavage (4, 26, 41). Such soluble TNFRs (sTNFR)
retain the ability to bind TNF, acting either as antagonist or agonist
of TNF bioactivity (18, 39, 42). Among its numerous
biological activities, TNF is involved in the killing of tumor cells
and in the control of intracellular pathogen multiplication (19,
20, 46), and it limits the extent and duration of inflammatory
processes (37). Besides these beneficial effects, it induces
cachexia associated with cancer and various infectious diseases
(38) and is involved in the pathogenesis and lethality of
septic shock (2, 56) and cerebral malaria (20,
36).
Trypanosoma cruzi is the protozoan parasite causing Chagas'
disease, a highly prevalent infection in Latin America. In vitro infection of human and murine cells with T. cruzi increases
TNF mRNA levels and TNF release (10, 51, 55). This cytokine has been detected in situ and in the supernatants of splenic cells as
well as in the blood of some infected mice (28, 31, 49, 51,
58). Studies using sTNFR1-deficient mice (12),
transgenic mice expressing high levels of sTNFR1-Fc
3 fusion protein
(33), or mice in which TNF-specific antibodies (Abs) were
injected in vivo (1, 28, 48) suggested a beneficial role of
TNF in the control of the acute T. cruzi infection in mice.
However, in vivo reduction of TNF levels, which would support such
conclusions, was not demonstrated in these latter studies. Moreover, no
information is available on the production of sTNFR during T. cruzi infection. Though the ability of TNF to enhance the in vitro
NO-dependent trypanocidal activity of gamma interferon (IFN-)- or
lipopolysaccharide (LPS)-activated macrophages has been clearly
demonstrated (8, 21, 40, 48, 57), we have shown TNF to
mediate a harmful effect by inducing cachexia associated with murine
T. cruzi acute infection (54). In addition, in
vivo administration of exogenous TNF (8) or of potent TNF
inducers such as LPS (30) or anti-CD3 Abs (29)
resulted in higher mortality in animals acutely infected with T. cruzi.
To better define the role of TNF during T. cruzi infection
in mice and considering that sTNFR can considerably modulate the bioactivity of TNF, we have investigated the kinetics of circulating TNF, sTNFR1, and sTNFR2 levels, as well as the interactions between such factors, in relation to parasitemia, cachexia, and mortality of
acutely infected animals. We also investigated the modulation of
sTNFR/TNF ratios induced by anti-TNF antibodies administered to
infected animals and their consequences on the outcome of the infection.
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MATERIALS AND METHODS |
Mice, T. cruzi infection, and blood processing.
Two-month-old male BALB/c mice were purchased from B&K Universal (Hull,
United Kingdom). Mice were infected by intraperitoneal (i.p.)
inoculation of 100 blood trypomastigotes of the Tehuantepec strain of
T. cruzi maintained in our laboratory. Parasitemia was determined in tail blood every 3 to 4 days, as previously described (11). Mortality and weight of mice were regularly recorded. The body weight changes were expressed as (weight on experimental day 
weight on day 0) × 100/weight on day 0. Blood was
obtained from tail or by cardiac puncture (in mice anesthetized by
ether), using special precautions to avoid cytokine proteolysis and
unexpected release: after being collected on heparin with LPS-free
material, blood was immediately kept on ice, mixed with 1 volume of 13 mM sodium citrate containing protease inhibitors (1 mM TCLK
[N-p-tosyl-L-lysine-chloromethyl ketone]
hypochloride [Sigma, St. Louis, Mo.] and 1,000 KIU of aprotinin
[Boehringer Mannheim, Mannheim, Germany] per ml), and centrifuged.
Plasma samples were stored at 70°C until use.
Treatment of mice with TNF-specific MAbs.
Two kinds of
monoclonal Abs (MAbs) recognizing specifically mouse TNF were
administered to mice: a chimeric construction of F(ab')2
from hamster MAb TN3 (clone 19.12) with mouse Fc1 (47, 50) (kindly provided by M. Bodmer, Celltech, Slough, United Kingdom) and MAb 1F3F3 (a rat IgM [35]), used as
ascites fluid. Purified unrelated mouse monoclonal immunoglobulin G1
(IgG1) and ascites fluid containing an irrelevant rat monoclonal IgM
(IR968) (both kindly provided by H. Bazin, Unit of Experimental
Immunology, University of Leuven, Brussels, Belgium) were used as
controls. The material to be injected to mice contained less than 10 pg of endotoxin per injection, as measured with the Limulus
amoebocyte lysate assay (detection limit, 1 pg/ml; Coatest endotoxin;
Chromogenix, Mölndal, Sweden). T. cruzi-infected mice
received i.p. injections of TN3-Fc1 (200 µg), 1F3F3 (11 µg), or
control MAb (at the same amount) in phosphate-buffered saline twice a
week during 2 weeks, the first injection being given the day before
T. cruzi inoculation. The choice of Ab amount to be injected
was based on the previously described capacity of the Ab to bind TNF
and to neutralize its biological activity (35, 47, 50).
ELISA for murine TNF, sTNFR, and TNF-sTNFR complexes.
Enzyme-linked immunosorbent assay (ELISA) for murine TNF was performed
as described elsewhere (16). Briefly, 96-well Immunomaxisorp plates (Nunc, Roskskilde, Denmark) were coated with rabbit polyclonal IgG directed against murine TNF-. After blocking nonspecific binding
with 1% bovine serum albumin and washing, 50-µl aliquots of plasma
samples (half-diluted in protease inhibitor solution as described
above) or of serial dilutions of recombinant murine TNF- (Boehringer
Mannheim) were added to each well. After washing, the plates were
incubated successively with a polyclonal rabbit IgG anti-mouse TNF-,
peroxidase-conjugated donkey F(ab')2 anti-rabbit IgG
(Jackson Immunoresearch, West Grove, Pa.), and
H2O2 and 3,3', 5,5'-tetramethylbenzidine
(Kirkegaard & Perry Laboratories, Gaithersburg, Md.) as substrate and
chromogen, respectively. The reaction was stopped after 15 min before
absorbances at 450 nm were read. Such ELISA detected free TNF as well
as TNF bound to sTNFR1 and -2 (or MAb TN3), with a detection threshold
of 20 pg of TNF per ml.
In some studies, MAb TN3 was used as capture Ab instead of the anti-TNF
polyclonal Ab (the other ELISA steps remaining identical), since it was
verified that the TNF-TN3 Ab complexes were not detected with such
ELISAs. The differences between the levels of TNF obtained in both
ELISAs allowed to appreciate the amounts of TNF-TN3 complexes.
ELISAs for sTNFR were performed as described earlier (3).
Soluble receptors were captured from plasma samples (diluted 1/15 and
1/25 for sTNFR1 and R2, respectively) with polyclonal rabbit IgG
anti-murine sTNFR1 or -R2 and revealed with the same biotinylated
antibodies and streptavidin-peroxidase (Dako, Glostrup, Denmark).
Saturation, washings, incubation times, and substrate were as for TNF
ELISA. Standard titration curves were obtained by making serial
dilutions of recombinant murine sTNFR1 or R2. These sTNFR ELISAs
recognized both unbound receptors and receptor-ligand complexes. The
detection thresholds were 40 and 390 pg/ml, respectively for sTNFR1 and
-R2.
Another ELISA was developed for the detection of complexes between TNF
and its soluble receptors. The complexes, captured
by rabbit polyclonal
IgG against murine TNF-
from mouse plasma
samples (diluted twofold),
were detected by successive incubation
with biotinylated polyclonal
rabbit IgG anti-murine sTNFR1 or
R2 and streptavidin-peroxidase.
Incubations, saturation, washings,
and substrate were as described
above. Results are expressed as
absorbances at 450
nm.
sTNFR/TNF molar ratios were calculated as C1 × M2/C2 × M1,
where C1 and C2 are the concentrations in nanograms per milliliter
of
sTNFR and TNF, respectively, and M1 and M2 are the molecular
masses of
sTNFR (sTNFR1, 30,000; sTNR2, 40,000) and monomeric
TNF (17,000),
respectively.
Bioassay for TNF-neutralizing activity.
The assay measured
the effect of mouse plasma samples on the bioactivity of standard TNF,
using the previously described TNF bioassay (17). Briefly, 5 pg of standard murine recombinant TNF TNF (Boehringer Mannheim), the
amount able to lyse 50% of 3 × 104 WEHI cells, was
incubated for 1 h at 37°C with mouse plasma samples (1/4
diluted) before being added to WEHI cells (3 × 104
cells/well in a final volume of 100 µl). After incubation for 20 h, cell viability was assessed by a colorimetric method (incubation with 12 mM MTT (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide; Sigma) during 3 h followed by solubilization of crystals with acid isopropanol containing 1% Triton X-100). The
TNF-neutralizing activity was estimated by the decrease of the
percentage of lysed cells. It was verified that the protease inhibitors
added into the blood samples did not interfere with the bioassay.
ELISA for murine interleukin-6 (IL-6) and IFN-.
The
cytokines were detected in mouse plasma samples (fourfold diluted),
using commercially available assays (Intertests; Genzyme, Cambridge,
Mass.) as described by the manufacturer. The detection limit was 5 pg/ml for both ELISAs.
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RESULTS |
The acute T. cruzi infection resulted in high
circulating levels of both TNF and sTNFR.
As shown in Fig.
1A, TNF was not found in the blood of
noninfected mice, whereas it became detectable in T. cruzi-infected mice from day 11 postinfection (p.i.). Mean levels
peaked on day 28 p.i. (mean ± standard error of the mean
[SEM], 2.1 ± 0.5 ng/ml), as did parasitemia, before decreasing
and remaining detectable in the circulation of 53% of mice in the
chronic phase of infection, when circulating parasites were
undetectable (from day 48 p.i.), at levels ranging between 0.2 and
0.5 ng/ml.
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FIG. 1.
Parasite, TNF, and sTNR circulating levels in T. cruzi-infected mice. BALB/c mice were inoculated with 100 parasites at day 0 (n = 28); 52% died during the
parasitemic phase (mean survival time, 24 ± 0.7 days).
Parasitemia is expressed as geometric means. TNF and sTNFR levels,
detected by ELISA, are given as arithmetic means ± SEM. Asterisks
indicate that TNF or sTNFR levels are significantly different from
initial levels before infection (*, p < 0.05; **, p < 0.005; Mann-Whitney-Wilcoxon U test).
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As shown in Fig.
1B and C, uninfected mice displayed basal levels of
sTNFR2 about 20 times higher than those of sTNFR1 (mean
± SEM,
49.3 ± 2.8 versus 2.8 ± 0.6 ng/ml).
T. cruzi
infection
induced a moderate increase of circulating amounts of TNFR1,
which
reached maximal mean value around four times above the basal
level
on day 34 p.i. By contrast, sTNFR2 exhibited a major
increase
during the acute phase of infection, peaking at day 28 p.i. with
levels 14 times above the basal mean level (681.7 ± 63.7 ng/ml).
Its kinetics closely paralleled those of TNF and
parasitemia.
During the chronic phase of the infection (from day
48 p.i.),
the levels stabilized between 4 and 8 ng/ml for sTNFR1
and 200
to 300 ng/ml for
sTNFR2.
As shown in Table
1, calculation of molar
ratios between circulating levels of sTNFR and TNF highlights the large
excess
of sTNFR2 over TNF during the course of infection, with mean
ratios
ranging from 135 to 1,700, whereas sTNFR1 barely (1.1- to
32-fold)
exceeded TNF. Interestingly, both sTNFR/TNF ratios were
particularly
reduced during the ascending phase of parasitemia (days 18 to
28 p.i.). Altogether, these data suggest in vivo interactions
between sTNFR and TNF that may modulate circulating TNF bioactivity
during the acute phase of infection.
TNF-sTNFR complexes and TNF-neutralizing activity are detectable in
blood of mice acutely infected with T. cruzi.
As shown in
Fig. 2A, complexes between TNF and both
soluble receptors could be detected in plasma of acutely infected mice at day 28 p.i. but not in those of noninfected animals. Moreover, plasma of infected mice displayed a TNF-neutralizing activity (Fig.
2B), since they could significantly (by 63%) inhibit the cytotoxic
activity of standard TNF on WEHI cells. Such neutralizing activity was
significantly correlated with the levels of sTNFR2 but not with those
of sTNFR1 (Spearman correlation coefficients, 0.664 [P < 0.001] and 0.173 [P > 0.05], respectively;
n = 15). These results strongly argues for the
involvement of sTNFR2 (rather than another unknown factor) in the
neutralizing activity found in plasma from infected mice.
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FIG. 2.
Detection of TNF-sTNFR complexes (A) and
TNF-neutralizing activity (B) in the plasma of T. cruzi-infected ( ) or noninfected ( ) mice. (A) TNF-sTNFR
complexes were detected by ELISA. Results are expressed as absorbances
at 450 nm (n = 11 noninfected and 35 infected mice,
respectively, at day 28 p.i.). Asterisks indicate significant
differences between noninfected and infected groups (P < 0.005; Mann-Whitney-Wilcoxon U test). (B)
TNF-neutralizing activity was measured by testing the biological
activity of exogenous TNF on TNF-sensitive cells (at a concentration
able to lyse 50% of WEHI cells), in the presence of plasma from
noninfected or infected (day 28 p.i.) mice. Results are expressed
as the percentage of WEHI cells lysed after overnight incubation with
TNF plus mouse plasma (n = 15 in each mouse group). A
reduction of this value denotes an inhibition of TNF bioactivity.
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Mortality of T. cruzi-infected mice is associated with
low sTNFR/TNF ratios.
Table 2
compares the data for surviving and nonsurviving mice. The results
indicate that on day 21 p.i., mice that died (mean survival time,
24 ± 0.7 days p.i.) displayed significantly (3.3-fold) higher
circulating mean levels of TNF and of both sTNFR (1.7- and × 1.5-fold for sTNFR1 and -R2, respectively), but lower sTNFR/TNF molar
ratios, than surviving mice.
A more detailed analysis of data obtained for mice 1 or 2 days before
or on the day of death (
n = 6) shows that nonsurviving
mice always exhibited circulating TNF levels above 2.8 ± 0.31
ng/ml, associated with sTNFR1 and -R2/TNF ratios below 0.81 ±
0.19 and 170 ± 37, respectively. Such profiles were never
observed
in surviving mice. These data suggest that the endogenous
balance
between sTNFR and TNF, as well as a threshold level of TNF,
determines
the outcome of
infection.
Moreover, nonsurviving mice always displayed higher parasitemia than
surviving mice, indicating that the high TNF levels and
subsequent low
sTNFR/TNF ratios were not related to the control
of infection, at least
during the ascending phase of parasitemia
(between days 14 and 28 p.i.).
Cachexia in T. cruzi-infected mice is also associated
with low sTNFR/TNF ratios.
The features of cachexia occurring in
mice acutely infected with T. cruzi have been detailed in a
previous study (54). As shown in Fig.
3, such cachexia, observed in all acutely
infected mice from days 18 to 39 p.i., was associated with reduced
sTNFR/TNF ratios (between 2.5 and 5.8 for sTNFR1/TNF and 150 and 170 for sTNFR2/TNF). A time lag of roughly 1 week was noted between weight and ratio variations.
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FIG. 3.
Circulating sTNFR/TNF molar ratios and cachexia in
T. cruzi-infected mice. Results are from four representative
mice inoculated on day 0 with 100 parasites and expressed as arithmetic
means ± SEM. Molar ratios sTNFR/TNF (bars) are calculated from
circulating TNF and sTNFR levels measured by ELISA. Body weight changes
(line) are expressed as the percent variation of mouse weight at each
experimental point compared to the weight on day 0 of infection.
Asterisks indicate that the weight is significantly different from the
weight at day 11 p.i. (P < 0.05; Student
t test).
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Interestingly, the reduction in molar ratios were not so drastic as
those observed in dying mice (Table
2), suggesting that
an increasing
concentration of bioactive TNF accounts for the
evolution from
nonlethal cachexia to
mortality.
Administration of anti-TNF MAb TN3 to T. cruzi-infected
mice increased TNF levels, cachexia, and mortality through decreasing
the sTNFR/TNF ratios.
To better study the dynamic relationship
between sTNFR/TNF ratios, mortality and cachexia, MAbs documented to be
able to neutralize TNF in vivo were administered to infected mice.
Mice infected with
T. cruzi received MAb TN3 or an unrelated
control MAb by the i.p. route on days
1, 2, 6, 9, 13, and 16
relative
to parasite inoculation. The two experiments performed
(with 18 and 11 mice per group) gave similar results. As shown
in Fig.
4A (displaying results obtained from one
representative
experiment), TNF levels increased drastically in the
blood of
TN3-treated mice compared to control animals. This could not
be
due to a direct effect of MAb TN3 since previous work showed it
did
not induce TNF release in noninfected mice (
5). An effect
related to endotoxin contamination of injected reagents may also
be
ruled out since (i) only traces of LPS were found in the injected
material and (ii) the control Ab, which contained similar traces
of
LPS, did not trigger TNF overrelease in
T. cruzi-infected
mice
(comparison between Fig.
1A and
4A). The TNF increment could also
result from the prolongation of TNF half-life in the circulation
by its
complexation with the injected MAb TN3, as previously observed
with
other anticytokine MAbs (
6,
53). Since the TNF ELISA
used,
based on an anti-TNF polyclonal Ab, does not discriminate
between free
and TN3-complexed TNF, we constructed TNF ELISA using
MAb TN3 as
capture Ab, that detects only free TNF. As shown in
Table
3, if TNF-TN3 complexes could be detected
in the plasma
at day 17 p.i., their amounts were considerably
reduced later
in the infection, on days 21 and 24 p.i., indicating
the presence
of higher amounts of free TNF, resulting from a higher
endogenous
production in TN3-treated mice. Since TNF is known to
stimulate
the production of IL-6, a cytokine previously documented to
be
present in the blood of
T. cruzi-infected mice
(
53), levels
of this cytokine were determined in blood of
TN3-treated infected
mice as a potential marker of TNF bioactivity. As
shown in Fig.
5, IL-6 production at days
21 and 24 p.i. was higher in TN3-treated
mice than in control
animals, suggesting that the TNF detected
in the former group of
animals is more bioactive.
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FIG. 4.
TNF, sTNFR, and parasite blood levels and mortality
rates in T. cruzi-infected mice treated with anti-TNF MAb
TN3. Mice were inoculated with 100 parasites on day 0 and received
either TN3 (n = 25 for parasitemia and mortality
determinations; n = 10 for TNF and sTNFR
determinations) or unrelated Ab (n = 18 for parasitemia
and mortality determinations; n = 9 for TNF and sTNFR
determinations). Arrows indicate time points of Ab injections.
Asterisks indicate time points at which differences between groups were
statistically significant (*, P < 0.05; **, P < 0.005; Student t test for TNF and sTNFR levels,
Mann-Whitney-Wilcoxon U test for parasitemia, and Yates
corrected chi-square test for mortality rates).
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TABLE 3.
Circulating levels of total and TN3-complexed TNF in
T. cruzi-infected mice treated with anti-TNF TN3 or
control Aba
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FIG. 5.
Circulating IL-6 levels in T. cruzi-infected
mice treated with the TN3 anti-TNF MAb TN3. IL-6 was detected by ELISA
in plasma of infected mice treated with TN3 or control MAb
(n = 5 per group). Levels are given as arithmetic
means ± SEM. Asterisks indicate significant differences between
TN3-treated and control mice (P < 0.05, Student
t test).
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However, the treatment of mice with MAb TN3 did not affect the
circulating levels of sTNFR1 and -R2 (Fig.
4B and C), which
underwent
variations similar to those described for untreated
T. cruzi-infected mice (Fig.
1). Subsequently, the molar ratios
sTNFR/TNF were significantly decreased on days 17, 21, and 24
p.i.
in TN3-treated mice in comparison to animals receiving the
control Ig
(Fig.
6). Such reduction of sTNFR/TNF
ratios was associated
with considerably worsened cachexia, which
started earlier (Fig.
7), as well as with
significantly shortened survival times (20.5
± 2.5 versus
26.1 ± 8.1 days,
P < 0.005) and enhanced
mortality
rates, since all TN3-treated mice died (Fig.
4D), harboring
extremely
high parasitaemia (Fig.
4E).
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FIG. 6.
Molar ratios between circulating levels of sTNFR and TNF
in T. cruzi-infected mice treated with the anti-TNF MAb TN3.
Mice were inoculated with 100 parasites on day 0 and treated with the
anti-TNF TN3 or control MAb during the 2 first weeks of infection (Fig.
4). Results are expressed as arithmetic means ± SEM (n = 5 to 9). Asterisks indicate significant differences between
groups (P < 0.05, Student t test).
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FIG. 7.
Cachexia in T. cruzi-infected mice treated
with the anti-TNF MAb TN3. T. cruzi-infected mice were
treated as for Fig. 4 with anti-TNF TN3 or control MAb. Changes in body
weight of 10 mice per group are expressed as percent variation of mouse
weight at each experimental point compared to the weight on day 0 of
infection (arithmetic means ± SEM). Asterisks indicate
significant differences between the two groups (P < 0.05, Student t test).
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Administration of anti-TNF MAb 1F3F3 to T. cruzi-infected mice reduced cachexia but did not affect survival
of animals, through slightly increasing the sTNFR/TNF ratios.
The
two experiments performed (with 11 and 7 mice per group) gave similar
results. As shown in Table 4 (results
obtained from one representative experiment), TNF as well as IL-6
levels were roughly reduced by half when MAb 1F3F3 was administered to infected mice on days 1, 2, 5, 8, 11, and 14 relative to parasite inoculation, indicating lower bioavailability of TNF in blood of these
mice. As for TN3 MAb, 1F3F3 treatment did not modify the levels of
sTNFR compared to animals receiving the control Ab; consequently, the
sTNFR/TNF ratios were increased slightly, 1.5- to 2-fold. Although
parasitemia was reduced by half during the experiments, the mortality
rates remained similar in the two groups of mice (mean survival
time ± SEM, 22.0 ± 0.9 versus 23.1 ± 1.1), suggesting
that the induced increases of sTNFR/TNF ratios were not sufficient
enough to improve survival. However, as previously published
(29), mice treated with MAb 1F3F3 underwent a significant reduction of cachexia.
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TABLE 4.
Circulating levels of TNF and sTNFR/TNF ratios in
T. cruzi-infected mice treated with 1F3F3 anti-TNF or
control MAbsa
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DISCUSSION |
Our data show that the parasitemic phase of T. cruzi
infection in mice is associated with high levels of circulating TNF and sTNFR2, resulting in the formation of cytokine-receptor complexes and
some degree of neutralization of TNF bioactivity. Low sTNFR/TNF circulating ratios were associated with cachexia in all infected mice,
and the lowest ratios were observed only in dying animals harboring the
highest parasitemia. The administration of anti-TNF MAb TN3 to infected
mice lowered the sTNFR/TNF circulating ratios (through a paradoxical
overproduction of TNF) and considerably worsened cachexia and mortality
in the animals while increasing parasite levels. Another anti-TNF MAb
(1F3F3) reduced blood amounts of TNF and parasites, as well as
cachexia. Collectively, these results indicate that during the
parasitemic phase of infection, TNF triggers harmful effects
(contrasting with its beneficial role earlier in the infection), which
are limited by the simultaneous in vivo production of endogenous
soluble receptors.
The close relationship between the kinetics of blood parasites, TNF
levels, and sTNFR2 levels during the parasitemic phase of T. cruzi infection argues for a direct effect of parasites on the
release of TNF and sTNFR2. This may be due to the action of membrane
glycoproteins of T. cruzi, which have recently been shown to
trigger TNF production in vitro (10). Since TNF and sTNFR2
are cleaved from cell membrane by the same protease (43), these parasitic glycoproteins might at the same time account for the
TNF and sTNFR2 release. TNF itself, known to trigger its own release
(2) as well as that of sTNFR2 (4, 32), and other TNF-inducing cytokines produced during T. cruzi infection
such as IFN- and IL-12 (45, 52), may also enhance the
parasitic effect. In addition, the renal lesions occurring during
T. cruzi infection (14) might increase the blood
levels of TNF and sTNFR2 further by reducing their clearance
(3).
Plasma from acutely infected mice displayed some capacity to neutralize
TNF, associated with the presence of cytokine-receptor complexes
involving mainly sTNFR2 (present at much higher amounts than sTNFR1).
On the other hand, our results suggest that variations of sTNFR/TNF
ratios modified the amount of bioactive TNF. Indeed, mice harboring
reduced sTNFR/TNF ratios (treated with MAb TN3) displayed increased
levels of IL-6, a cytokine previously detected in mouse T. cruzi infection (53) and that may be considered a
functional marker of TNF bioactivity (27). Conversely,
animals presenting higher sTNFR/TNF ratios (treated with MAb 1F3F3)
lowered their IL-6 amounts. These observations strongly suggest that
during murine T. cruzi infection, TNF bioactivity is
regulated by sTNFR.
We have previously established the role of TNF in the cachexia
associated with T. cruzi acute murine infection
(54). Indeed, infected mice exhibited a severe weight loss
that could be reduced by the administration of MAb 1F3F3. In the
present work, we confirm our previous results by showing that MAb 1F3F3
limits the in vivo TNF bioactivity and that, conversely, the increase
of TNF levels (observed in the TN3-treated mice) exacerbated the weight
loss of infected animals. A relationship was established between
sTNFR/TNF ratios and the intensity of cachexia during the course of the parasitemic phase of infection, indicating the capacity of sTNFR to
limit the TNF effects on weight loss. Moreover, the occurrence of
cachexia in all mice acutely infected with T. cruzi 1 week after the changes in sTNFR/TNF ratios indicates that only low levels of
bioactive endogenous TNF are required during a prolonged time to induce
such harmful effects, in full agreement with previous studies
(13).
The present results also indicate that high levels of bioactive TNF are
lethal in acutely infected mice and that a massive production of TNF,
as paradoxically induced by the administration of MAb TN3, kills all
mice. Though not currently studied in T. cruzi infection,
the mechanisms of TNF-dependent mortality have been described for two
models: the low-dose endotoxin septic shock (24) and the
generalized Shwartzman reaction (22). Both are mediated
through TNFR1 and require sensitizing factors
(D-galactosamine and an initial injection of a low dose of
LPS, respectively). Acutely T. cruzi-infected mice are known
to be sensitized to the lethal action of TNF, since administration of
exogenous TNF (at concentrations not lethal for control mice) kills
them (8). Such sensitization in T. cruzi
infection might be related to an overexpression of TNFR1 on host cell
membrane, since they are released only at low levels in the
circulation. A role for IFN- should not be excluded since this
cytokine was documented to trigger lethality during septic shock
(24) and Shwartzman reactions (22) in association
with TNF and was increased in blood of our acutely infected mice (data
not shown).
The harmful effects of TNF (cachexia and mortality), observed during
the parasitemic phase of the infection, are associated with parasite
levels. This observation completes previous studies highlighting a
protective role of TNF in T. cruzi infection (12, 33,
48). In addition, different in vitro studies clearly showed a
protective effect of TNF in the control of cell infection with parasites (8, 21, 40, 48, 57). This apparent contradiction can be resolved by considering TNF as differently involved in two
evolutive steps of the acute phase of infection: the incubation period
(roughly the first week after parasite inoculation), with undetectable
levels of parasites in the blood, and the parasitemic phase (around 1 to 6 weeks p.i.). The TNF initially produced in the first days of
infection in response to T. cruzi inoculation could
contribute to initiate the control of parasite multiplication, in
synergy with IFN- produced under IL-12 stimulation (9). Parasites having escaped this initial control continue to multiply actively in the vertebrate tissues till being detected in blood (parasitemic phase). At this step, TNF production, highly stimulated by
the circulating parasites, may reach dangerous concentrations. The
simultaneous release of a large excess of sTNFR2 in the circulation, also induced by infection, might partially inhibit the harmful effects
of TNF, allowing the survival of some animals, though enough bioactive
TNF remains available to trigger cachexia. Surviving mice develop
specific immune responses, improving the control of infection and
thereby ensuring the continuation of the parasite cycle. This view is
strengthened by the results obtained when the TNF-neutralizing MAb TN3
was administered into mice before and early after infection (between
days 1 and 16 p.i.). This Ab might efficiently neutralize the
low levels of TNF produced in vivo during this early step of infection,
resulting in a more intense tissue parasite multiplication and
ultimately extremely high parasitemia and overproduction of TNF (this
should explain the paradoxical TNF increase observed in these animals).
These high cytokine amounts may overcome the neutralization capacities of soluble receptors, causing extreme cachexia and death of all mice.
Why another MAb such as 1F3F3 has an inverse effect remains unclear,
whereas its capacity to neutralize TNF has clearly been demonstrated in
vitro and in vivo (35). Besides isotype-related differences
(IgM for 1F3F3 versus IgG1 for TN3) in their pharmacokinetics and
capacities to bind FcR and C1q, 1F3F3 displays a higher avidity than
TN3 and neutralizes TNF in another way. Indeed, 1F3F3 binds to a TNF
sequence situated outside the site responsible for its recognition by
the receptors (34), whereas TN3 interacts with an amino acid
sequence located within this region (47). Therefore, MAb
1F3F3 might neutralize only soluble TNF (since it might be more
difficult for a conformational change of membrane-anchored TNF to
occur), whereas MAb TN3 would inhibit the activity of both forms. As
membrane and soluble TNF differ in the ability to bind to TNFR1 and -R2
(23, 44) and are differently involved in the control of
other infections with intracellular pathogens (7, 15), we
might further hypothesize that the use of MAb 1F3F3 rather than TN3 has
also a different impact on T. cruzi infection. Moreover,
1F3F3, as an IgM MAb, might bridge several molecules of TNF on host
cell membranes (the TNF sequence recognized by 1F3F3 is extracellular),
thereby triggering a better control of parasite multiplication through
higher IFN- production, as observed in human T-cell leukemia virus
type 1-infected T cells treated with an anti-TNF MAb (25).
In conclusion, our data highlight the role of the endogenous balance
between TNF and its soluble receptors and the subsequent harmful versus
beneficial effects of TNF on the outcome of T. cruzi infection.
| |
ACKNOWLEDGMENTS |
We thank Francine Keruzore for diligent technical assistance, H. Collet for LPS determinations, M. Bodmer for providing the anti-TNF
chimeric MAb TN3, and H. Bazin for providing an unrelated MAb.
This work was supported by grants from the Ministry of Scientific
Policy and the Université Libre de Bruxelles, Brussels, Belgium
(Concerted Research Actions).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Parasitologie, Faculté de Médecine, ULB, Route de Lennik
808, CP 616, B-1070 Brussels, Belgium. Phone: 32 2 555 62 55. Fax: 32 2 555 61 28. E-mail: ycarlier{at}ulb.ac.be.
Present address: Department of Pharmacology, University Medical
Centre of Geneva, Geneva, Switzerland.
Editor:
J. M. Mansfield
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Abstract
Introduction
