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From the Gonçalo Moniz Research Center - Fundaçào Oswaldo Cruz, Salvador, Bahia, Brazil
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The DTH-like aspect of the inflammatory lesions in CChC suggests the
participation of T helper (Th) 1 (Th1) cells in its pathogenesis, as
this lymphocyte subset strongly promotes the activation of macrophages
by the secretion of cytokines such as interferon-
(IFN-). The
expansion and function of Th1 cells are regulated by cytokines produced
by Th2 cells, such as interleukin (IL) -4, -10 and -13, potent
promoters of humoral immune responses.12
In several
disease models, it has been shown that cross-regulations between these
two T-cell subsets are critical for the determination of disease
outcome. In fact, certain pathogens can induce the preferential
expansion of one Th subset, while suppressing the other.13
T. cruzi infection, however, induces a non-polarized
response: in different murine experimental models, both resistant and
susceptible mice had a mixed pattern of response to the
parasite.14,15
On the other hand, an association between
progression to severe CChC and high IFN- levels has been
demonstrated in human beings.16,17
If Th1 responses indeed
mediate disease in T. cruzi-infected individuals, cytokines
such as IL-4 could play a beneficial, anti-pathogenic role during
T. cruzi infection. Thus, we investigated a possible
modulating role of IL-4 in a CChC model in which IL-4-deficient mice
are infected with T. cruzi, as described below.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific-pathogen-free, 6- to 8-week-old female or male wild-type, IL-4-/- and nu/nu BALB/c mice were raised and maintained at the animal facilities at the Gonçalo Moniz Research Center-FIOCRUZ, and provided with rodent diet and water ad libitum.
Parasites, Infection and T. cruzi Antigen Preparation
Trypomastigotes of Colombian-strain18 T. cruzi were obtained by infection of LCC-MK2 cell line. Infection was performed by inoculation of 100 trypomastigotes by intraperitoneal route. Parasitemia was evaluated at different time points after infection by counting the number of trypomastigotes in peripheral blood aliquots contained between a glass slide and a coverslip, with a determined blood volume per microscopic field.19 Epimastigotes of Colombian and PF strain20 were obtained by axenic culture in liver infusion tryptase medium. T. cruzi antigen was prepared by subjecting epimastigotes to five cycles of freezing and thawing. T. cruzi lysates were centrifuged at 30,000 x for 30 minutes; supernatants were then aliquoted and stored at -70°C until use.
Histopathological Evaluation
Groups of IL-4+/+ or IL-4-/- mice were sacrificed at different time points after infection. Heart, liver, spleen, and striated muscle were removed and fixed in buffered 10% formalin. Sections were analyzed by optical microscopy after paraffin embedding followed by standard hematoxylin/eosin staining. Inflammatory cells infiltrating heart tissue were counted using digital morphometric evaluation. Images were digitalized using a JVC TK-1280 color video camera adapted to an Axioskop 2 microscope (Carl Zeiss, Göttingen, Germany). The images were analyzed using the AxioVision II program (Carl Zeiss), with which the inflammatory cells were manually counted and integrated by area. The number of parasite nests was also determined by counting in the acquired images. One 100 fields per section were counted in five to ten sections per heart.
Immunohistochemistry
Sections of 5 µm from frozen heart fragments were fixed with cold acetone and incubated with one of the following antibodies: rat anti-mouse CD4 or CD8, (PharMingen, San Diego, CA); rat anti-mouse Fc receptor (2.4G2 cell culture supernatant); peroxidase-conjugated goat anti-mouse IgM (Sigma, St. Louis, MO); T. cruzi-hyperimmunized rat antiserum. The sections pre-incubated with rat antibodies were incubated with horseradish-peroxidase conjugated to anti-rat IgG (Sigma). Reaction was developed using the peroxidase substrate diaminobenzidine (Vector, Burlingame, CA), followed by counterstaining with hematoxylin. The percentage of CD4+ or CD8+ cells was determined by counting the number of positive cells over the total number of inflammatory cells determined as described above.
Reconstitution and Infection of BALB/c nu/nu Mice
BALB/c nu/nu mice received adoptive transfer of thymocytes obtained from newborn wild-type mice or IL-4-/- mice (one thymus per mouse). After 15 days, reconstituted and non-reconstituted control nude mice were infected with 100 Colombian strain trypomastigotes. Mice were sacrificed 30 days later for histopathological evaluation of the hearts, as described above.
In Vitro Stimulation and Measurement of Proliferative Response
Spleen cell suspensions were prepared in RPMI medium (Life Technologies, GIBCO-BRL, Gaithersburg, MD) supplemented with 10% fetal calf serum (Hyclone, Logan, Utah), L-glutamine (2 mmol/L), vitamins, sodium pyruvate (1 mmol/L), Hepes (10 mmol/L), 5 x 10-5 mol of 2-mercaptoethanol, and gentamicin (50 µg/ml) (Sigma). For cytokine determination, spleen cells were cultured in 24-well plates and stimulated with 1 µg/ml of concanavalin A (Con A) (Sigma) or Colombian strain T. cruzi antigen (50 µg/ml). Cell-free supernatants were collected after 72 hours and stored at -20°C for cytokine analysis. To evaluate the proliferative response, splenocytes were plated in 96-well plates at 4 x 105/well in 200 µl and triplicate wells were stimulated with Con A or T. cruzi antigen for 120 hours, as described in the figure legends. After pulsing with 1 µCi of methyl[-3H]thymidine (Amersham, Little Chalfont, England) for 14–18 hours, proliferation was assessed by measurement of [methyl-3H]thymidine uptake in a ß-plate counter (Packard, Meriden, CT).
Cytokine and Anti-T. cruzi Antibody Quantification
Supernatants of splenocyte cultures were tested for IFN-, IL-2,
and IL-4 contents by ELISA, using antibody pairs from PharMingen and
following the manufacturer’s instructions. Reaction was developed
using the 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate
(Kinkergaard & Perry Laboratories, Gaithersburg, MD) and read at 450
nm.
Anti-T. cruzi isotype production was evaluated by ELISA using PF-strain epimastigote lysate as antigen and biotinylated isotype-specific anti-mouse IgM, IgG1, IgG2a, IgG2b, and IgG3 antibodies (PharMingen), followed by streptovidin-peroxidase conjugate (Sigma). Reaction was developed using TMB substrate as described above.
Statistical Analyses
Data were analyzed using Student’s t-test, Wilcoxon’s rank sum test, or Fisher’s exact probability test, as indicated in the text. Differences were considered significant when P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Parasitemia levels in T. cruzi-infected wild-type
BALB/c mice were about five times higher than in IL-4 knockout mice
(Figure 1A
; P < 0.02, <
0.01 and < 0.002 on the 15th, 21st, and 25th or 30th days
postinfection, respectively; Wilcoxon’s rank sum test; statistical
analysis was not meaningful after the 30th day postinfection due to
bias arising from the death of many wild-type animals). Moreover,
whereas 55% of the animals in the wild-type mouse group died before or
on the 40th day of infection, only 15% of infected IL-4-/- mice did
not reach the 60th day after infection (Figure 1B
; P =
0.019 on the 60th day postinfection; Fisher’s exact probability test).
Tissue parasitism in spleen, liver and striated muscle was also lower
in IL-4-/- mice than in IL-4+/+ mice. Heart tissues from IL-4-/-
mice had three- to fourfold less parasite nests than those of wild-type
mice (P < 0.05, Wilcoxon’s rank sum test;
Figure 1C
). After the fourth month of infection, no intact parasite,
and only a single macrophage containing parasite antigenic material,
was detected in those heart tissues by immunohistochemistry.
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Inflammatory infiltrates in tissues of all infected mice were composed mainly of mononuclear cells. In the acute phase of the disease (up to 40 days after infection), foci of inflammatory infiltration of similar intensity were found in the liver and in striated muscles of animals from both groups, causing intense myocytolysis, in the presence of parasites. Large spleens, with intense cell proliferation and myeloid metaplasic reaction with megakariocytes were found in mice from both groups. Infected wild-type mouse spleens, however, were significantly larger than those of IL-4-/- mice (mean weights ± SD were 625 ± 63 and 373 ± 60 mg, respectively; P < 0.0001, Student’s t-test).
Interestingly, the intensity of myocarditis was higher, with 3- to
10-fold more mononuclear cells, in IL-4-deficient than in wild-type
mice, despite the lower heart parasitism (P <
0.05, Wilcoxon’s rank sum test; Figures 1D and 2, A and B
). Inflammation was multifocal
and mononuclear cells were frequently found attached to myocardial
fibers.
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. In contrast, a
conspicuous multifocal inflammatory reaction, with or without
associated fibrosis, was observed in hearts from IL-4-/- mice (Figure 2D)
.
Myocarditis increased in severity from the fourth month to the seventh
month of infection in mice from both groups. Wild-type mice, however,
still had a mild disease, with little fibrosis (Figure 2E)
, whereas
IL-4-/- mice had severe multifocal myocarditis, with mononuclear
cells frequently adhering to cardiac fibers undergoing myocytolysis
(Figure 2F)
. In the latter mice fibrosis was very prominent,
particularly in the atria, both around intact heart fibers and in areas
of active inflammation.
Increased Proportion of CD4+ T Cells in Hearts of IL-4-/- Mice
The mononuclear infiltrate in hearts of mice from both groups was
mainly composed by macrophages (Fc receptor+ and
surface IgM- cells), B cells (surface
IgM+ cells; these constituted no more than 5% of
the infiltrate), and T lymphocytes (CD8+ or
CD4+ cells; these constituted no more than 25%
of the infiltrate), with higher numbers of CD8+
cells in relation to CD4+ cells 30 days after
infection (Figure 3)
. At four and seven
months after infection, however, CD4+ cells
predominated, inverting the
CD8+/CD4+ ratio. This was
significantly more intense in IL-4-/- mice than in wild-type mice at
seven months after infection (Figure 3
, P < 0.05,
Wilcoxon’s rank sum test). The majority of CD8+
cells in the acute phase of infection surrounded parasite nests (Figure 2G)
, whereas CD4+ cells were frequently found in
intimate association with damaged fibers (Figure 2H)
.
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At 30 days after infection, IL-4-/- mouse splenocytes produced
two to three times more IFN- in response to T. cruzi
antigen in vitro than those of wild-type mice (Figure 4A)
. Moreover, non-specific stimulation
by concanavalin A (Con A) led splenocytes from IL-4-/- mice to
produce IL-2 (not shown) and proliferate at normal levels, whereas
splenocytes from wild-type mice had markedly reduced responses (Figure 4C
; P < 0.0357, Student’s t-test).
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and data not shown). However, splenocytes of IL-4-/- mice still
produced higher levels of IFN- on in vitro T.
cruzi-antigen stimulation than wild-type mouse splenocytes (Figure 4A
; P < 0.0309, Student’s t-test). This
was associated with increased IgG3 (P <
0.0207, Student’s t-test) and reduced IgG1
(P < 0.0021, Student’s t-test)
anti-T. cruzi antibody levels (Figure 4D)
. The production of
IL-2 by splenocytes obtained from both groups of mice on stimulation
with T. cruzi antigen was similar in all time points
analyzed (Figure 4B)
. T. cruzi Infection Causes Intense Myocarditis in nu/nu BALB/c Mice Reconstituted with IL-4-/- Thymocytes
T. cruzi-infected BALB/c nu/nu mice had intense
parasitemia and succumbed to the acute infection. Their hearts, 30 days
after infection, had a remarkable high parasite load without any
inflammation (Figure 5A)
. In contrast,
hearts from infected nu/nu mice, previously reconstituted with
thymocytes from both wild-type and IL-4-/- mice, had inflammation
(Figure 5B)
. The inflammation, which was diffuse and composed by
mononuclear cells, similar to that observed in infected wild-type or
IL-4-/- mice, was more intense in nu/nu mice reconstituted with
IL-4-/- than with wild-type mouse thymocytes (two-fold more
inflammatory cells). In addition, the number of parasite nests was
lower in IL-4-reconstituted nu/nu mice (Figure 5)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to T. cruzi antigens. The infective inoculum
used, however, by being higher than that which occurs in
nature,21
may not reproduce closely what happens in
natural infection. Moreover, T. cruzi strains differ
markedly in their biological behavior,24
a fact that has
to be taken into account when interpreting conflicting observations in
experimentally or naturally infected mammals.
As described in the literature,25
wild-type mice were
immunosuppressed in the acute phase of T. cruzi infection,
as demonstrated by their reduced lymphoproliferative response to Con A
in vitro. T. cruzi-infected IL-4-/- mice, with reduced
parasitism, on the other hand, had more intense in vitro
lymphoproliferative response to Con A (Figure 4C)
and less marked
splenomegaly than wild-type mice. Whether these two latter phenomena
could be ascribed to the reduction in parasitism or could result from a
putative effect of IL-4 on the non-specific immunosuppression or on the
polyclonal B-cell activation of T. cruzi-infected mammals is
open to speculation.
The reduced acute-phase parasitemia and mortality of T.
cruzi-infected IL-4-/- mice, in relation to infected normal
mice, can be ascribed to an enhanced parasite-specific Th1 immune
response. This enhanced response was demonstrated in this paper by the
detection of increased amounts of IFN- in supernatants of
antigen-stimulated splenocytes and increased levels of IFN-
-dependent IgG3 anti-T. cruzi antibodies in
vivo. As expected, while splenocytes from wild-type mice
produced IL-4 on in vitro stimulation with Con A and
T. cruzi antigen in all time points analyzed, no IL-4 was
produced in vitro by IL-4-/- splenocytes (not shown) and
IL-4-/- mice had reduced levels of IL-4-dependent IgG1 anti-T.
cruzi antibodies. In fact, IFN- has been shown to control
parasitism in vivo and in
vitro.26-31
On the other hand, the development of heart inflammatory foci in T. cruzi infection was intensified in IL-4-/- mice. This, to our knowledge, has not been previously reported. That it was not due to a putative abnormality in the target organ, and indeed depended on T-cell activity was demonstrated by the passive transfer of the severe myocarditis-susceptibility trait to athymic nude mice by IL-4-/- thymocytes. In addition, the development of inflammation in thymocyte-reconstituted athymic mice, as also reported for the first time herein, is probably the most conclusive demonstration that the heart disease in T. cruzi-infected mammals is immune mediated.
The inverse relationship between tissue parasitism and inflammatory response in hearts of IL-4-/- mice was striking. In fact, only a single macrophage containing T. cruzi antigen could be observed in several sections of heart tissue obtained from four mice four months after infection. These findings, however, cannot be used as a conclusive evidence that CChC is mediated by non-parasite-specific immune responses,32 since very little parasite antigen, undetectable by immunohistochemical analysis, could theoretically maintain T cells specifically activated and recruiting large numbers of effector macrophages. Evidence for autoimmune phenomena in the pathogenesis of CChC comes from elsewhere.4-9
At the acute phase of the infection, when parasitism was brought under
control, heart-infiltrating lymphocytes were predominantly
CD8+. These lymphocytes, rather than
CD4+ cells, were frequently found in the vicinity
of parasites or parasite antigens. These findings indicate that
CD8+ cells may be directly involved in parasite
control, either by releasing IFN- and/or by lysing infected cells
before the full differentiation of amastigotes into trypomastigotes,
and are consistent with data showing severe, lethal infection in
ß2-microglobulin or TAP-1 knockout mice.33
Later on,
during the intermediary and chronic phases of the infection, the
percentage of CD4+ cells increased and supplanted
the percentage of CD8+ cells, mainly in
IL-4-/- mice. Contrasting to what was found for
CD8+ cells in the acute phase, these cells were
frequently found in close association with myocytes undergoing
degenerative changes. These findings are consistent with the hypothesis
that CD4+ T cells mediate CChC and with the fact
that anti-CD4-antibody treatment, and not anti-CD8, cures experimental
CChC in mice.5,22
As for the reduction of parasitism, the enhanced heart alterations in
IL-4-/- mice could be easily ascribed to an intensified Th1 response,
since Th1 responses have been shown to be more aggressive than Th2
responses to host tissues in several situations.13
In
fact, an association of severity of myocarditis and increased IFN-
levels has been described in human beings.16,17
IL-4, by regulating an IFN--producing response, may play an
important role in preventing the development of incapacitating heart
disease in T. cruzi-infected animals. In fact, the intense
carditis and extensive fibrosis found in hearts of IL-4-/- mice
during the chronic phase of T. cruzi infection closely
resembles the severe CChC found in human patients.2
The
modulating activity of IL-4-producing T cells may maintain a balance
between parasitism and tissue integrity in the indeterminate phase of
the infection, during which mild inflammatory foci resolve into focal
fibrosis and perhaps a contained Th1 response would keep parasitism
under relative control.3
The progressive destructive
process in CChC could therefore result from a failure of a pathogenic
Th1 response to be down-regulated by IL-4. This failure could, in its
turn, depend on host genetic characteristics, on age-dependent changes
of the immune system,34
superposition of infections by
unrelated microorganisms, and/or by T. cruzi
re-infection.35
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Acknowledgements |
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Footnotes |
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Supported by grants from the Brazilian National Research Council (CNPq) and FIOCRUZ.
Accepted for publication April 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is required for resistance to acute Trypanosoma cruzi infection in mice. J Immunol 1991, 146:3626-3632[Abstract]
treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by IL-10 and TGF-ß. Eur J Immunol 1992, 22:2501-2506[Medline]
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