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Production by T Cells in Experimental Chagas’ Disease Is Mediated by IL-181
*
Max Planck Institute for Immunobiology and
Department of Pathology, Freiburg, Germany;
Department of Special Zoology and Parasitology, Ruhr-University, Bochum, Germany;
Institute of Immunology, University of Leipzig, Leipzig, Germany;
¶ Department of Immunology, Institut Pasteur, Paris, France; and
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Department of Immunology, University of Cape Town, Cape Town, South Africa
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-
blood levels as in wild-type mice were
detected 2 wk postinfection. In vivo and in vitro analysis demonstrated
that T cells, but not NK cells, were recruited to infected organs.
Analysis of mice double deficient in the recombinase-activating gene 2
(RAG2) and IL-12p35, as well as studies involving T cell
depletion, identified CD4+ T cells as the cellular source
for IL-12-independent IFN- production. IL-18 was induced in
IL-12p35-/- mice and was responsible for IFN-
production, as demonstrated by in vivo IL-18 neutralization studies. In
conclusion, evidence is presented for an IL-12-independent IFN-
production in experimental Chagas’ disease that is T cell and IL-18
dependent. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Host resistance during experimentally induced Chagas’ disease is
dependent on both innate and acquired cell-mediated immune responses,
requiring the combined efforts of a number of cells, including NK
cells, CD4+ and CD8+ T
cells, as well as Ab production by B cells (1, 2, 3).
Cytokines play key roles in regulating both parasite replication and
immune responses in infected animals. IFN- has been most closely
associated with host resistance during the acute phase of infection.
This cytokine inhibits both in vivo and in vitro parasite replication
through the induction of NO synthesis by IFN--activated macrophages
(4). IFN- is believed to be produced by NK cells at the
onset of infection and by CD4+ and
CD8+ (5, 6) T cells later during
infection. Synergistically with TNF-, IFN- is controlling growth
of the parasite (7), and mice deficient in the receptor
for IFN- (IFN-R-/-) or the inducible NO
synthase
(iNOS-/-)4
are highly susceptible to infection. This results in an exacerbating
parasitemia and mortality due to defective macrophage activation and NO
production (8). In contrast to IFN-, the suppressive
cytokines, including IL-10 and TGF-
, have been associated with
susceptibility to T. cruzi infection (9, 10, 11) by
inhibiting IFN--mediated macrophage activation
(12).
Production of IFN- and TNF- during experimental Chagas’ disease
is induced by IL-12, leading to a protective cell-mediated immune
response (13). Release of IL-12 by macrophages is
triggered by invasion of these cells by blood trypomastigotes of
T. cruzi early after infection (14, 15, 16).
IFN- and TNF- induce NO synthase (iNOS), which then leads to the
production of macrophage-derived NO required to control acute infection
with T. cruzi (7, 8, 13). The early and T
cell-independent production of IFN- by NK cells is an important
innate element of the host resistance to T. cruzi infection
(5). Ag-specific CD8+ and
CD4+ T cells are required to control parasitemia
during acute infection (17) by MHC class I-restricted
cytotoxic T cells (18), the production of inflammatory
cytokines (19), and Th1 cell responses (20, 21).
Since IL-12 release and subsequent IFN- production by NK and T cells
during the acute phase of experimental Chagas’ disease are important
for mediating protective cellular immune responses, the objective of
our study was to better define the role of IL-12 in these responses. In
this study, we report that IL-12p35-/- mice are
highly susceptible to infection with T. cruzi despite the
presence of a delayed IFN- and TNF- response. We identified
CD4+ T cells as the cellular source for the
IL-12-independent IFN- production, which was driven by an
IL-18-dependent mechanism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Young adult (7- to 8-wk-old) IL-12p35-/- mice on 129Sv/Ev background (22) backcrossed five times to C57BL/6 and C57BL/6 wild-type mice were maintained under specific pathogen-free conditions. By intercrossing with C57BL/6 mice deficient in the recombinase-activating gene 2 (RAG2) (23), double-deficient mice were generated (IL-12p35-/-RAG2-/-).
A cloned population of the reticulotropic T. cruzi strain Tulahuen (WHO reference MHOM/CH/00/Tulahuen C2) was routinely maintained in mice (24), and trypomastigotes were isolated by differential centrifugation of blood from infected mice. Groups of five mice were infected i.p. with 50 or 500 trypomastigotes, and the resulting survival and parasitemia were monitored by hemacytometer counting of blood samples. For determination of cytokines and reactive nitrogen intermediates (RNI), serum from infected animals was prepared using serum separator tubes (Microtainer; BD Biosciences, Franklin Lakes, NJ).
For preparation of inactivated T. cruzi (iTC), monolayers of LLC-MK2 cells (CCL7.1; American Type Culture Collection, Manassas, VA) were infected and cultured in IMDM (Life Technologies, Paisley, U.K.) supplemented with 10% FBS (Life Technologies), 0.05 mM 2-ME (Roth, Karlsruhe, Germany), and penicillin and streptomycin (100 U/ml and 100 µg/ml, respectively; Biochrom, Berlin, Germany). Inactivation of culture trypomastigotes was performed by 10 freeze-thaw cycles, as described previously (25).
Histopathological analysis
Day 14 infected mice were killed by cervical dislocation. Tissue specimen were collected and fixed in paraformaldehyde (4% in PBS) for further processing. Tissues were dehydrated in ethanol, and paraffin-embedded sections were stained with H&E and subjected to microscopic analysis. Studies were done with a Zeiss microscope (Jena, Germany).
Quantitation of cytokine transcripts by RNase protection assay
For RNA extraction, spleens were isolated from mice at days 0, 7, 10, and 14 after infection. After homogenization in solution D (4 M guanidinium thiocyanate (Sigma, Deisenhofen, Germany), 0.5% N-laurosylsarcosine (Sigma), 1 M sodium citrate (Merck, Darmstadt, Germany), 0.1 M 2-ME (Serva, Heidelberg, Germany)), suspensions were acidified in 2 M sodium acetate, purified by extractions with phenol/chloroform, precipitated with isopropanol, and washed twice in 70% ethanol. Air-dried RNA was dissolved in RNase-free H2O and stored at -70°C until used. To determine the concentration and purity of extracted RNA, the absorption was determined at 260 and 280 nm, and possible degradation was examined after migration on a 1.2% formaldehyde/agarose gel. For hybridization with radiolabeled antisense RNA, extracted RNA was completely dried in a vacuum evaporator centrifuge and dissolved in hybridization buffer (RiboQuant; BD PharMingen, San Diego, CA).
Unlabeled sense RNA for IL-18, IFN-, and GADPH and all reagents were
supplied by BD PharMingen (RiboQuant). For the synthesis of
radiolabeled antisense RNA probes, the final reaction mixture (20 µl)
contained 120 µCi of [-32P]UTP (3000
Ci/mmol; Amersham, Arlington Heights, IL); UTP (61 pmol), GTP, ATP, and
CTP (2.75 nmol each); DTT (100 nmol); transcription buffer (1x) RNasin
(40 U); T7 RNA polymerase (20 U); and an equimolar pool of mCK-2b
multiprobe template set. After 1 h at 37°C, the reaction was
terminated by incubation with DNase (2 U) for 30 min at 37°C. Probes
were purified by extractions with phenol/chloroform and precipitated
with ethanol. Air-dried probes were dissolved (3 x
105 cpm/µl) in hybridization buffer and added
(2 µl) to tubes containing extracted RNA from spleens of T.
cruzi-infected mice dissolved in 8 µl of hybridization buffer.
Samples were overlaid with mineral oil, heated to 90°C, and incubated
at 56°C for 16 h. Single-stranded RNA was then digested
by addition (100 µl) of RNase A (80 ng/µl) and RNase T1 (250
U/µl) in RNase buffer. After incubation for 45 min at 30°C, samples
were treated for 15 min at 37°C with 18 µl of a mixture of
proteinase K (0.3 mg/ml), yeast RNA (0.06 mg/ml), and proteinase K
buffer to stop digestion. dsRNA was isolated and precipitated as above,
dissolved in loading buffer, and electrophoreses was performed in
standard 6% acrylamide/8 M urea sequencing gel. Dried gels were placed
on BioMax film (Kodak, Rochester, NY) with intensifying screens and
were developed at -70°C for 72 h.
To quantify the relative amount of IL-18 and IFN- expression, gels
were scanned and the respective bands were densitometrically evaluated
with a Macintosh microcomputer-based Image Analysis System (NIH 1.52;
National Institutes of Health, Bethesda, MD). Resulting densities of
IL-18 and IFN- were standardized against GADPH, and the mean
cytokine induction was calculated by normalization against the relative
expression of IL-18 and IFN- in spleens from uninfected
mice.
Cultivation of APCs
Peritoneal cell suspensions were prepared from uninfected and infected IL-12p35-/- mice 14 days after infection by peritoneal lavage with IMDM. Resuspended cells were cultured in complete IMDM in 96-well U-bottom plates (Nunc, Naperville, IL) at 2 x 106/ml for 4 h, and nonadherent cells were removed. Adherent peritoneal macrophages from infected IL-12p35-/- mice were tested for the presence of intracellular parasites after cultivation in chamber slides (Nunc) and staining with DiffQuick (Baxter Scientific, Mundelein, IL).
Cultivation of spleen cell suspensions
Spleen cell suspensions were prepared from uninfected and infected mice at days 7, 10, and 14 after infection, depleted of erythrocytes, and resuspended in IMDM. Isolated cells were cultured in complete IMDM in 48-well flat-bottom plates (Nunc) at 2 x 106/ml. The cultures were incubated with either medium alone, iTC (4 x 106/ml), or LPS (5 µg/ml, Escherichia coli 0111:B4; Sigma). Cell supernatants from the cultures were harvested after 48 h of culture and stored at -80°C.
For depletion of CD4+ T cells, spleen cell
suspensions were incubated with CD4 Dynabeads (Dynal,
Robbins-Scientific, Mountain View, CA). Depleted cell suspensions from
spleen cells contained 5% CD4+ T cells, as
determined by flow cytometric analysis. Nondepleted and
CD4+ T cell-depleted cells were cultured in
complete IMDM in 96-well flat-bottom plates (Nunc) at 2 x
106/ml and incubated with medium alone or iTC
(4 x 106/ml). Supernatants were examined
for the production of IFN- after 48 h.
For enrichment of CD4+ T cells, spleen cell
suspensions were incubated with CD4 Dynabeads (Dynal,
Robbins-Scientific) and CD4+ T cells were
detached from the beads with CD4 DETACHaBEAD (Dynal). Enriched cell
suspensions from spleen cells contained >90%
CD4+ T cells, as determined by flow cytometric
analysis. Enriched CD4+ T cells from infected
animals were added to infected adherent peritoneal macrophages at
4 x 106/ml. Cell supernatants from the
cultures were harvested after 48 h of culture and stored at
-80°C. Nondepleted and CD4+ T cell-depleted
cells were cultured in complete IMDM in 96-well flat-bottom plates
(Nunc) at 2 x 106/ml and incubated with
medium alone or iTC (4 x 106/ml).
Supernatants were examined for the production of IFN- after
48 h.
For in vitro supplementation studies with IL-12 or IL-18, 2 x
105 spleen cells from naive
IL-12p35-/- mice were infected in 100 µl
complete IMDM in 96-well flat-bottom plates (Nunc) with 2 x
106 culture trypomastigotes of T.
cruzi in vitro. Noninfected and infected cells were incubated with
either medium alone, rIL-12 (100 U/well; BD PharMingen), rIL-18 (24
ng/well; BD PharMingen), or IL-12 and IL-18 (100 U and 24 ng/well,
respectively), and the release of IFN- was measured after
48 h.
Flow cytometric analysis
At days 0, 7, 10, and 14 after infection, spleens of single mice were isolated for FACS analysis. Single-cell suspensions were depleted of erythrocytes, and 5 x 105 cells were resuspended in PBS containing 3% heat-inactivated and dialyzed FBS (Life Technologies) and 0.01% NaN3. To avoid unspecific Ab binding, cells were preincubated for 20 min with a dilution (1/40) of heat-inactivated mouse and rat serum and anti-CD32 mAb (2.4G2; American Type Culture Collection). For detection of different lymphocyte populations, cells were conducted with a two-color combination of fluorescein- and PE-conjugated mAbs (BD PharMingen) against specific cell surface markers (NK cells, NK1.1/CD11b; CD4+ T cells, CD90.2/CD4; CD8+ T cells, CD90.2/CD8). For analysis of NK, CD4, and CD8 cell phenotypes on a FACScan (BD Biosciences), the lymphocyte-rich region was analyzed using CellQuest software (BD Biosciences).
Determination of IFN-, TNF-, and RNI levels in blood and
supernatants
IFN- levels in sera and culture supernatants were analyzed in
3-fold serial dilutions using a two-site sandwich ELISA utilizing
purified and biotinylated Abs for IFN- (BD PharMingen). After
incubation with alkaline phosphatase coupled to streptavidin (Southern
Biotechnology Associates, Birmingham, AL) and developing with
p-nitrophenyl phosphate (Sigma), the absorbance was read on
a microplate reader (Dynatech MR 600; Dynatech Scientific, Cambridge,
MA). Using a test wavelength of 405 nm and a reference wavelength of
495 nm, samples were compared with appropriate standards (BD
PharMingen). The detection limit was 0.015 ng of IFN-/ml.
The quantification of NO2 and NO3 in sera of mice was performed using the Griess reaction (8). Briefly, 50-µl volumes of serial dilutions of serum samples and sodium nitrate (1 µM to 1 mM; Sigma) as standard were made in normal mouse serum in 96-well microtiter plates (Nunc). Twenty microliters of freshly prepared reaction solution (1.8 µg/ml NADPH and 2.5 µU/ml nitrate reductase; Boehringer Mannheim, Mannheim, Germany) were added, and plates were incubated at room temperature for 20 min, after which 50 µl of Griess reagent was added. The absorbance was read as described above. The detection limit of NO2 and NO3 was 1.5 µM/ml.
Detection of biologically active TNF- in serum was performed
by a cytotoxic assay on L929 cells, as described previously
(26). Briefly, 50 µl of L929 cell suspension (4 x
105/ml; kindly provided by C. Galanos, Max Planck
Institute for Immunobiology, Freiburg, Germany) in exponential growth
was cultured for 24 h in 96-well microtiter plates (Nunc) in RPMI
1640 (Life Technologies) supplemented with 10% heat-inactivated
FBS, 2 mM L-glutamine, 30 mM HEPES (Life Technologies), and
penicillin and streptomycin (100 U/ml and 100 µg/ml, respectively;
Biochrom). To each well, serial dilution of the samples in complete
medium containing 2 µg/ml mitomycin (Sigma) was added in duplicates.
After 48 h of incubation at 37°C, viability of cells was
assessed with a colorimetric (MTT; Sigma) method, as described by
Espevik and Nissen-Meyer (27). Murine rTNF- (BD
PharMingen; sp. act., 4 x 107 U/mg protein)
was included as a standard. The number of units per millilter of
activity was defined as the reciprocal of the dilution required to
induce a 50% decrease in absorbance to control cells exposed to
mitomycin alone. The detection limit of the assay was 0.01 U of
TNF-/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo IL-12 neutralization (14) or supplementation
(13) studies during experimental Chagas’ disease
suggested an important role of IL-12 for protective IFN- production
and subsequent type 1 responses controlling the infection. To further
investigate the role of IL-12, infection studies using
IL-12p35-/- mice were performed and compared
with C57BL/6 wild-type mice using infectious doses of 50 T.
cruzi blood trypomastigotes (Fig. 1
). Wild-type mice controlled parasitemia
in the blood during acute infection. In contrast,
IL-12p35-/- mice showed an uncontrolled
parasitemia that typically began 2 wk postinfection (Fig. 1A). Moreover, most wild-type mice survived infection
(68.2 ± 17.7%, four independent experiments), while all mutant
mice succumbed to infection by the third week (Fig. 1B).
Mortality in wild-type mice was accompanied by increased parasite
burden in infected target organs, such as the heart and liver (Fig. 1C, amastigote nests in the insets) and severe
tissue destruction with large necrotic lesions. In contrast, only poor
inflammatory infiltrates were found in the organs of moribund mutant
mice (Fig. 1C, liver), indicating a reduced cellular immune
response in the absence of IL-12. To investigate the cellular immune
responses, in vivo production of inflammatory cytokines and RNI was
determined during acute infection by increasing the infectious doses to
500 blood trypomastigotes, which allows for direct monitoring of
inflammatory cytokine secretion into the blood in mice
(8). As shown in Fig. 2A, increasing levels of
IFN-, TNF-, and RNI were found in the blood of infected wild-type
mice early during infection. In the absence of endogenous IL-12 blood
levels of IFN-, TNF- as well as RNI were significantly reduced at
day 10, but increased by day 14 postinfection, reaching similar
concentrations of IFN- and TNF- as observed in wild-type mice.
These results show that substantial IFN- and TNF- production is
present in the absence of endogenous IL-12 in response to infection
with T. cruzi.
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Since IL-12 is the major factor for IFN- production by NK and T
cells, we did not expect the observed rather normal IFN- blood
concentration in the absence of endogenous IL-12. Consequently, we
investigated the cellular source of IFN- production in
IL-12p35-/- mice. Spleen cells were isolated at
various time points after infection, restimulated in vitro with either
iTC or LPS, and T or NK cell-derived IFN- secretion was measured by
ELISA. As shown in Fig. 2
B,
IL-12p35-/-, but not wild-type splenocytes
showed an impaired IFN- production in response to LPS, indicating a
defective NK cell response. In contrast, Ag-specific restimulation led
to a substantial, but delayed release of IFN- by
IL-12p35-/- splenocytes, reflecting the course
of IFN- production in vivo. These results indicated that T cells
rather than NK cells were the main cellular source of IFN-
production in infected IL-12p35-/- mice. As
expected, increasing IFN- secretion in wild-type mice was paralleled
by increasing cell numbers of NK cells, CD4+, and
CD8+ T cell subpopulations in the spleen of
infected wild-type mice, determined by FACS analysis (Fig. 2C). In contrast, IL-12p35-/- mice
showed no NK cell recruitment in infected organs, which could explain
the observed impaired release of IFN- by LPS-restimulated
splenocytes. On the other hand, a delayed increase of both
CD4+ as well as CD8+ T cell
subpopulations was present at 14 days postinfection, which correlated
with the delayed, but substantial IFN- production observed in vivo
and in vitro. Taken together, these data suggest an impaired IFN-
production and recruitment into infected organs of
IL-12p35-/- NK cells, but substantial and
delayed responses of CD4+ and
CD8+ T cells.
CD4+ T cells are a main cellular source of IFN-
production in T. cruzi-infected
IL-12p35-/- mice
To investigate whether CD4+ T cells in
T. cruzi-infected wild-type mice were the source of
IL-12-independent IFN-, CD4+ T cell-depleted
or T cell-enriched splenocytes were stimulated by Ag, and IFN-
production was measured. As shown in Fig. 3A, CD4+
T cell depletion caused a drastic reduction of IFN- production after
restimulation of wild-type or IL-12p35-/-
spleen cells isolated 14 days after infection. In addition, primed and
enriched IL-12p35-/- CD4+
T cells cocultured with syngenic macrophages showed similar increased
IFN- production, which was even more pronounced when cocultured with
Ag-presenting macrophages (Fig. 3B). Naive control
CD4+ T cells, cocultured with infected
macrophages, produced only marginal amounts of IFN- (data not
shown). These results demonstrate substantial T.
cruzi-specific IFN- production by
IL-12p35-/- CD4+ T cells,
although this delayed IFN- production could not control infection
in vivo.
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production was beneficial for T. cruzi-infected
IL-12p35-/- mice, we intercrossed
IL-12p35-/- with
RAG2-/- mice and compared the outcome of
infection between IL-12p35-/- and
IL-12p35-/-RAG2-/-
double-deficient mice. The latter were unable to produce any IFN-
(Table I). Moreover, compared with
IL-12p35-/- mice, a 10-fold increased
parasitemia was found 10 days postinfection, showing an
IL-12-independent residual resistance of T. cruzi-infected
IL-12p35-/- mice, which was dependent on the
presence of T cells.
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production by T
cells
IL-18, a recently identified IFN--inducing factor (28, 29), is known to compensate for at least some of the functional
activities of IL-12 in certain models of intracellular infections
(30, 31, 32). IL-18, together with IL-12, is able to
synergistically induce IFN- expression in experimental Chagas’
disease (33). Therefore, it was interesting to determine
whether IL-18 is induced in the absence of endogenous IL-12 and
functions as a possible mediator for IL-12-independent IFN-
production. Kinetic expression studies in T. cruzi-infected
IL-12p35-/- and wild-type mice were performed
using an RNase protection assay (Fig. 4).
Low constitutive IL-18 was present in splenocytes from naive mice of
both strains (data not shown). Infection with T. cruzi
induced expression of IL-18 in both mouse strains with somewhat
distinct kinetics. Whereas the expression of IL-18 in wild-type mice
was increasing until day 14 postinfection, IL-18 was strongly induced
in IL-12p35-/- mice at the onset of infection,
but decreased after a peak at day 10 after infection. The induction of
IL-18 was paralleled by increased expression of IFN- in both mouse
strains and was delayed in the absence of IL-12 (Fig. 4), indicating
endogenous IL-18 as a likely candidate for the observed IFN-
production. Consequently, its potential to activate IFN- production
in IL-12p35-/- splenocytes was analyzed. Spleen
cells of naive mice were isolated and infected in vitro with
culture-derived T. cruzi trypomastigotes or left uninfected.
After stimulation with IL-12, IL-18, or both, the production of IFN-
was determined (Fig. 5). Uninfected cells
secreted only marginal amounts of IFN- when stimulated with either
cytokine alone. Infection of IL-12p35-/- spleen
cells with T. cruzi, however, resulted in a substantial
IFN- production with either IL-12 or IL-18, which was comparable
with the synergistic effect of both cytokines. No IFN- production
was measured in the supernatants of uninfected or infected spleen
cells, which have been incubated with medium alone. Since IL-18 along
with T. cruzi was able to induce substantial amounts of
IFN- independently of IL-12, it was likely that IL-18 is a mediator
of the observed delayed IFN- production in T.
cruzi-infected IL-12p35-/- mice. This
hypothesis was confirmed by in vivo neutralization of endogenous IL-18.
Treatment with neutralizing anti-IL-18 Abs led to a significant
reduction of IFN- and an increase in parasitemia in
IL-12p35-/- mice when compared with mice
treated with an isotype control Ab (Table II). In conclusion, these data provide
evidence for an IL-18-mediated T cell-dependent production of IFN-
in T. cruzi-infected IL-12p35-/-
mice, contributing to the residual resistance in the absence of
IL-12.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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that in
turn activates macrophage trypanocidal mechanisms by iNOS-dependent
production of RNI. We have recently demonstrated that
IFN-R-deficient mice as well as iNOS-deficient mice are unable to
survive even low doses of infection with T. cruzi due to
defective RNI effector mechanisms (8). In the present
study, the in vivo function of IL-12 during experimental Chagas’
disease was characterized in mice deficient for IL-12. Our main
findings show evidence for the importance of IL-12 during infection
with T. cruzi: 1) IL-12 is necessary for an effective immune
response; 2) IL-12 is important for the early IFN- production; 3)
during the later course of the acute phase of infection, specific
CD4+ T cells are able to produce IFN-
independently of endogenous IL-12; and 4) IL-18 is able to mediate this
T cell-dependent IFN- production in the absence of endogenous
IL-12.
IL-12p35-deficient mice were susceptible to infection with T.
cruzi and demonstrated increased parasitemia and mortality.
Impaired immunity in the absence of IL-12 was accompanied by reduced
production of inflammatory cytokines, including IFN- and TNF-, as
well as a reduced NO release. These responses are known to be crucial
for controlling parasite replication by IFN--activated macrophages
(7, 13). Our results obtained in
IL-12p35-/- mice are in agreement with in vivo
IL-12 neutralization studies showing increased susceptibility
(14) or in vivo supplementation studies, resulting in
increased serum IFN- and TNF- levels and reduced parasitemia
after T. cruzi infection (13). A similar
phenotype has also been observed in mice deficient for the p35 or p40
subunit of IL-12 infected with other intracellular pathogens, including
Leishmania major (22), and Leishmania
donovani (34), Toxoplasma gondii
(35), Mycobacterium tuberculosis
(36), or Listeria monocytogenes
(37). Interestingly, the production of IFN- and TNF-
was not completely absent in T. cruzi-infected
IL-12p35-/- mice and increased 14 days after
infection, demonstrating an IL-12-independent pathway of IFN-
production, also observed in L. donovani infection
(34, 38). A similar mechanism may operate during infection
with influenza (39), mouse hepatitis virus
(40), systemic lymphatic choriomeningitis virus
(41), or pulmonary adenovirus (42). In
addition, low-dose infection of IL-12p35-/-
mice with L. monocytogenes also leads to considerable
IFN- production and survival of mice (37). Thus, our
report on IL-12-independent IFN- production in T. cruzi
infection extends these observations to a protozoan infection
model.
Consequently, we identified the cellular source of IL-12-independent
IFN- production after infection with T. cruzi. Since
IL-12 activates IFN- production in NK and T cells (43),
these cell types were potent candidates for the cellular source of
IFN-. NK cells are usually involved in the early T cell-independent
production of IFN-, which is triggered by macrophage-derived IL-12
(5, 44). However, NK cells from mutant mice were not
expanded in the spleens from infected
IL-12p35-/- mice, and LPS could not activate
the release of IFN- by mutant splenocytes in vitro (in
contrast to wild-type mice). We concluded from these data that NK cell
recruitment into infected organs and efficient IFN- production are
dependent on IL-12 activation in experimental Chagas’ disease. The
observation of a delayed, but eventual IFN- response at day 14 was
more consistent with a T cell source of IL-12-independent IFN-
production. This was confirmed by flow cytometry analysis, which showed
that CD4+ and CD8+ T cell
expansion in infected spleens was delayed, but increased in parallel
with blood IFN- levels in T. cruzi-infected
IL-12p35-deficient mice. Moreover, Ag-specific IFN- responses were
also present in culture supernatants of
IL-12p35-/- splenocytes restimulated 14 days
postinfection, but not at earlier time points of infection. Finally, ex
vivo depletion of the CD4+ T cell subpopulation
reduced Ag-specific IFN- levels, providing convincing evidence that
CD4+ T cells were the major IL-12-independent
IFN- producers. Since a completely defective IFN- production was
accompanied by an even more exacerbated parasitemia in
IL-12p35-/-RAG2-/-
mice infected with T. cruzi, the T cell-dependent IFN-
production most likely contributed to a residual resistance of
IL-12p35-/- mice. B cell Ab responses are also
mediating some resistance to T. cruzi (45) and
may have contributed to the overall susceptibility observed in
IL-12p35-/-RAG2-/-
mice. The delayed inflammatory response, however, could not compensate
for the increased parasitemia in IL-12p35-/-
mice early during infection, showing that an IL-12-mediated optimal
inflammatory response during innate immunity is crucial during
experimental Chagas’ disease.
We finally addressed the mechanism involved in
CD4+ T cell-dependent IFN- production in the
absence of IL-12. In particular, we investigated the potential role of
IL-18, a recently identified IFN--inducing factor (28, 29). IL-18 is known to induce IFN- in T cells synergistically
with IL-12 (46), but also independently of IL-12
(47). However, recent infection studies in IL-18-deficient
(IL-18-/-) mice resulted in contradictory
conclusions, which depended on the capacity of IL-18 to induce IFN-
and therefore to facilitate resistance in murine infection models.
L. major infection in independently generated
IL-18-/- C57BL/6 mice resulted in either a
resistant (48) or susceptible (49) phenotype,
depending on the ability of the respective mouse model to produce
IFN-. The latter outcome was also observed in a cryptococcal model,
which highlighted the capacity of IL-18 to induce IFN-
(50). IL-18 can compensate for some of the functional
activities of IL-12 at least in some models of intracellular infections
(31, 32, 47), due to its ability to induce IFN-
production synergistically with IL-12 (46). In
experimental Chagas’ disease, IL-12 and IL-18 gene expression is
believed to synergistically induce expression of IFN-
(33), which made it a promising candidate to investigate.
Indeed, IL-18 was induced during infection with T. cruzi in
wild-type as well as in IL-12p35-/- mice, and
neutralization of endogenous IL-18 reduced the delayed IFN-
production in IL-12p35-/- mice in vivo, with a
resulting increased susceptibility. Recently performed preliminary
T. cruzi infection studies in mice deficient for the IL-18R
(51) resulted in increased mortality compared with
wild-type mice (our unpublished data), supporting a beneficial role of
IL-18. In addition, we could show that IL-18 was able to stimulate
T. cruzi-infected IL-12p35-/-
splenocytes to produce IFN- in vitro, indicating that T.
cruzi itself is able to induce IFN- production synergistically
with IL-18. Taken together, these data strongly suggest that IL-18
partly compensates for IL-12 deficiency as a mediator for IFN-
production by CD4+ T cells in response to
infection with T. cruzi. A similar IL-18-dependent mechanism
has also recently been found in studies of adenovirus-infected
IL-12-deficient mice (42). Our data do not exclude the
possibility of other factors involved in IL-12-independent production
of IFN-, which may also be indicated by the different kinetics of
IL-18 expression compared with IFN-, observed in T.
cruzi-infected IL-12-deficient mice (see Fig. 4). Recently,
Cousens et al. (52) identified IFN- as an
IFN--inducing factor acting alternatively to IL-12 in lymphatic
choriomeningitis virus infection. However, in experimental Chagas’
disease, IFN- does not appear to have an important role since we
were unable to detect IFN--specific messages in infected
wild-type or IL-12p35-/- mice by RNase
protection assay (our unpublished data). The observation that
infection with T. cruzi itself promotes IFN-
production may indicate that unique properties of the parasite
contribute to these responses. This has been shown for T.
cruzi-derived glycosyl-phosphatidylinositol-anchored mucin-like
glycoproteins, which are able to initiate the synthesis of
proinflammatory cytokines in macrophages (16), and for
TolA-like parasite surface proteins, which are also able to facilitate
the generation of a CD4+ T cell response
(53).
In conclusion, we have shown that IL-12 is a central molecule in an
early protective cell-mediated immune response to infection with
T. cruzi. In the initial interaction with the host, the
parasite promotes the production of IFN- in NK cells by the
induction of IL-12. Our studies in IL-12p35-deficient mice demonstrated
the presence of an alternative mechanism of IFN- production by
CD4+ T cells, which is IL-12 independent and
induced by endogenous IL-18 in response to T. cruzi.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Frank Brombacher, Department of Immunology, Faculty of Health Science, University of Cape Town, Groote Schuur Hospital, OMB H47, Observatory 7925, South Africa. E-mail address: fbrombac{at}uctgsh1.uct.ac.za
3 Current address: Molecular Infection Biology, Research Center, Borstel, Germany.
4 Abbreviations used in this paper: iNOS, inducible NO synthase; iTC, inactivated T. cruzi; RAG, recombinase-activating gene; RNI, reactive nitrogen intermediates.
Received for publication February 14, 2001. Accepted for publication July 10, 2001.
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) mice were infected with 50 blood
trypomastigotes, and the subsequent parasitemia (A),
survival (B), and histopathology (C) were
monitored. Results in A are expressed as the means
± SDs of five mice per group. The asterisks indicate statistically
significant differences between wild-type and IL-12p35-/-
mice (**, p < 0.01, Student’s
t test). For histological examination, H&E-stained
sections were prepared at day 17 postinfection. In contrast to heart
sections of infected wild-type mice (upper left), hearts
of IL-12p35-/- (upper right) mice revealed
amastigote nests (inset), but poor inflammatory cell
infiltrates (arrow). Whereas liver tissue from wild-type mice
(lower left) showed small foci of mononuclear cell
infiltration with minimal tissue damage, liver tissue from infected
IL-12p35-/- mice (lower right) showed
increased numbers of intracellular parasites (inset)
accompanied by severe destruction of the liver parenchyma with
confluent necrosis (arrow). Shown are representative sections from five
individually analyzed mice per group (original magnification x230).
Comparable results were obtained in four independent experiments.
) and IL-12p35-/- (
)
mice was analyzed, as described in Materials and
Methods. Results are expressed as means ± SD. The
asterisks indicate statistically significant differences compared with
the corresponding nondepleted cells (**, p <
0.01, Student’s t test). Similar results were obtained
in an independent experiment. After enrichment, CD4+ T
cells were cocultivated with Ag-presenting peritoneal macrophages
obtained from uninfected or infected IL-12p35-/- mice
(B). After 48 h, the amount of IFN-
