| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Instituto Oswaldo Cruz, FIOCRUZ (Fundaçao Oswaldo Cruz), and
Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) monolayers was
investigated. Long term (5 to 13 day) replication of infective
(trypomastigote) T. cruzi forms was blocked by supernatants
from activated (anti-TCR) CD4+ T cells of infected mice
or by rIFN-
. However, when CD4+ T cells from infected
mice were cocultured with M and activated by anti-TCR, marked
exacerbation of trypomastigote growth in M ensued. The deleterious
effect required contact between T cells and infected M. Both
anti-Fas and TCR activation killed a proportion of CD4+
T cells. Ly-6 activation did not induce AICD and did not exacerbate
parasite growth. However, Fas-mediated killing of T cells before Ly-6
activation led to exacerbated parasite growth. Although a minor
population, Fas-susceptible cells were the major source of IFN-
production by activated T cells. Addition of a neutralizing
anti-Fas ligand antibody blocked 50 to 60% of CD4+ T
cell AICD and reduced trypomastigote growth in T/M cocultures
stimulated by anti-TCR. The results demonstrate that in
CD4+ T cells from infected mice, the onset of AICD
selectively ablates IFN- production and up-regulates parasite
replication in M in vitro. These findings suggest a deleterious role
for AICD in T. cruzi infection. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
)4
to control intracellular parasite replication (4, 5). Supernatants from
T cells activated by T. cruzi Ags or mitogens contain
cytokines that restrict the intracellular growth of T. cruzi
in infected M cultures (6, 7). The cytokine IFN- plays a central
role in M microbicidal activity against intracellular forms of
T. cruzi in vitro (8, 9) and also during the initial phase
of infection in vivo (10, 11). Depending on parasite strain and host
susceptibility in different models, CD4+ T cells can
secrete large amounts of IFN- and TNF-
, but little or no IL-4
upon activation (12). Nonetheless, activation through the TCR results
in suppressed T cell responses (12), a feature that could help the
parasite to escape complete destruction and persist in the host.
Different mechanisms of CD4+ T cell suppression in vitro
have been described (12, 13). Infection with highly virulent blood form
trypomastigotes suppresses CD4+ T cell responses through
IFN- production and secretion of nitric oxide (NO) metabolites by
cocultured activated M (12). On the other hand, in infection with
metacyclic trypomastigotes, a form of suppression was found that is
independent of M products (13) and results from activation-induced
cell death (AICD) (14), a regulatory response of activated
CD4+ T cells to TCR;CD3 engagement (15, 16). Both
susceptibility of CD4+ T cells to AICD in vitro and
CD4+ T cell apoptosis in vivo have been described in the
course of experimental Chagas’ disease induced by metacyclic
trypomastigotes (17). AICD is regarded as a mechanism for the
elimination of activated T cells, reducing damage to host tissues (15, 16). However, in the case of T. cruzi infection, it has been
suggested that AICD could play a deleterious role for the host by
killing activated T cells and restricting the effectiveness of
antiparasite effector responses (18). To investigate whether AICD plays
a role in T cell modulation of parasite growth within infected M, we
developed an in vitro system of coculture of T.
cruzi-infected M monolayers and purified CD4+ T
cells from infected donors. Activation of these T cells was elicited
under various controlled conditions. We found that while addition of
CD4+ T cell supernatants or IFN- reduced the load of
infective T. cruzi forms, direct activation through the TCR
of cocultured CD4+ T cells from infected donors exacerbated
parasite accumulation in M monolayers. In this investigation we
demonstrate that the onset of CD4+ T lymphocyte AICD is
responsible for exacerbation of parasite growth, with potentially
deleterious consequences for the host. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male BALB/c and C57.Bl.6 (B6) mice, 6 to 8 wk of age, were
obtained from the Oswaldo Cruz Institute (IOC-FIOCRUZ, Rio de Janeiro,
Brazil) animal facility. Mice were infected s.c. with
105 chemically induced metacyclic trypomastigotes from
the T. cruzi clone Dm28c in 0.1 ml, as previously described
(17). Uninfected littermates were used as controls and as a source of
normal peritoneal M. Chemically induced metacyclic forms were
obtained with triatomine artificial urine-proline medium as originally
described (19). Parasitemia peaked between 18 and 26 days of infection.
All T cell preparations used in this study were obtained between 18 and
26 days of infection.
Infection of M monolayers with T. cruzi
Normal resident M from uninfected BALB or B6 mice were
obtained by peritoneal lavage with DMEM. Cells were washed and cultured
at 37°C in 7% CO2 in a humid atmosphere at 3
x 105 cells/well in 24-well vessels (Corning Glass Works,
Corning, NY) in 1 ml of complete culture medium containing DMEM (Sigma
Chemical Co., St. Louis, MO), supplemented with 10% heat-inactivated
FCS, 2-ME (5 x 10-5 M), MEM nonessential amino
acids, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate,
and 10 µg/ml gentamicin sulfate. After overnight incubation,
nonadherent cells were discarded, and adherent cells were washed,
leaving approximately 2 x 105 M. These M
monolayers were infected by overnight incubation with Dm28c metacyclic
trypomastigotes (19) at a 10:1 parasite: cell ratio in culture medium
at 37°C in 7% CO2. After 18 h, monolayers were
extensively washed to remove extracellular parasites.
Coculture of M monolayers with purified T cell subsets
Splenocytes obtained from either T. cruzi-infected or
control mice syngeneic with the M donors were depleted of
erythrocytes by treatment with Tris-buffered ammonium chloride and
filtered through nylon wool columns. Nonadherent cells were subjected
to negative selection by magnetic cell sorting using a mixture of
anti-CD8 (or anti-CD4, in the case of purified CD8+
T cells), anti-MHC class II, anti-B220, anti-MAC-1, and
anti-CD16/CD32 mAbs as previously described (17) plus
anti-TCR
to yield highly purified CD4+ or
CD8+ T cells after three rounds of separation with
anti-rat Ig-coated magnetic beads (Bio-Mag, Perseptive Diagnostics,
Cambridge, MA). By flow cytometry, CD4+ T cells had <1%
contaminating CD8+ and MHC class II+ cells, in
both control and infected groups.
The M monolayers provided an efficient source of accessory cells for
anti-TCR activation of purified T cells. Since addition of phorbol
ester does not interfere with CD4+ T cell AICD in cells
from infected mice (14), and since T cell activation by anti-Ly-6
strictly requires phorbol ester as a cofactor (20), most experiments
were conducted in the presence of phorbol ester (0.5 ng/ml PMA; Sigma).
Anti-TCR activation of T cells in the absence of phorbol ester yielded
similar results (see Table II
).
|
. Therefore,
CD4+ T cells were obtained by treatment of nylon
wool-nonadherent cells with anti-CD8 mAb and C, followed by a
second cycle of indirect cytotoxicity as previously described (17).
Second, in all experiments involving anti-Ly-6 mAb, purified T
cells were supplemented with 10% irradiated (3000 rad) normal
syngeneic splenocytes regardless of the stimulus used. This treatment
restored anti-Ly-6-mediated T cell activation, which was deficient
with adherent M.
Purified CD4+ T cells (2 x 106) in 1 ml
of complete culture medium, were added to prewashed, T.
cruzi-infected syngeneic M monolayers (2 x
105) in the presence of PMA and in the presence or the
absence of 10% (v/v) culture supernatant of anti-TCRß mAb
H57.597 (21). Additional Abs added along with anti-TCR included
mouse (gld strain) anti-mouse FasL (B6 strain)
mAb K10 (10 µg/ml), a neutralizing IgG2b/k anti-FasL Ab that
reacts with an allotypic determinant on mouse FasL that is present in
B6- but not in BALB-activated T cells (22); and mouse isotype control
mAb 156-8 (10 µg/ml), an IgG2b/k Ab specific for hepatitis virus B
(AYW3 strain, produced at FIOCRUZ, Rio de Janeiro, Brazil). Cultures
were set up in duplicate and maintained for up to 13 days. On the
indicated days, individual wells were gently pipetted up and down, and
20-µl aliquots of the supernatant were removed for assessment of
viable extracellular T. cruzi trypomastigote numbers. Motile
trypomastigotes were easily identified in the counting chamber, based
on established morphologic criteria (23). Results are presented as the
mean and SE of trypomastigote number in the duplicate cultures.
In experiments comparing the effect of anti-Fas treatment (using
the Fas agonist mAb Jo2) (24) on Ly-6 activation, a two-step culture
procedure was used to avoid coincidence of both CD4+ T cell
AICD and the onset of lymphokine secretion (25). Purified
CD4+ T cells (2 x 106, plus 10%
irradiated splenocytes) were cultured alone in 1 ml of complete culture
medium containing PMA plus 2 µg/ml hamster anti-mouse Fas mAb Jo2
or 2 µg/ml of control hamster IgG mAb. After overnight incubation,
the entire content of individual wells was aspirated and transferred to
wells containing decanted T. cruzi-infected M monolayers.
Cocultures were then initiated in the absence or the presence of newly
added activating anti-TCR or 0.5% (v/v) ascites of anti-Ly-6
A/E mAb D7 (20). Extracellular trypomastigote accumulation was measured
as described above. Controls included infected M cultured with PMA
in the absence of T cells and in the presence or the absence of
anti-Fas or anti-Ly-6. No changes in M viability or number
of trypomastigotes recovered were noticed with any of these treatments.
Suspensions of CD4+ T cells from infected mice contained a
limited (<102) number of motile trypomastigotes. Following
activation in the absence of adherent M, no detectable parasites
were recovered after 5 days or more, even when partially purified T
cells were strongly activated.
Quantitation of T cell viability
Either partially or highly purified CD4+ T
cells were cultured alone or added to infected M at 2 x
106/ml in replicates in 24-well vessels in the absence (PMA
alone) or the presence of anti-TCR (10% supernatant),
anti-Ly-6 (0.5% ascites), anti-Fas, or control hamster IgG (2
µg/ml) and anti-FasL or control mouse IgG2b (10 µg/ml). After
20 h, cells were gently dispersed with a Pasteur pipette or
detached with EDTA and washed, and viable cell counts for each
individual well were determined by trypan blue exclusion. The mean
viable cell recovery in unstimulated cultures was taken as a reference.
The percent cell loss (the reciprocal of remaining viable cells) was
calculated for each individual well according to the formula: % cell
loss = 100 - (viable cell number in stimulated culture) x
100/(viable cell number with PMA alone). The mean and SE of duplicate
or triplicate cultures are shown.
Assay for IFN- production
Purified CD4+ T cells were cultured overnight
with anti-Fas or isotype control as described above and were
stimulated, or not, with anti-TCR the next day. Supernatants were
collected 48 h after TCR activation and stored frozen until use.
The IFN- content in supernatants was evaluated by a sandwich ELISA
technique, using two rat anti-mouse IFN- mAbs (PharMingen, San
Diego, CA), one of which was biotinylated. The reaction was revealed
with 2.5 µg/ml avidin-peroxidase conjugate (Sigma) using ABTS
substrate, according to a protocol provided by the manufacturer
(PharMingen). Dilutions of the supernatants were compared with a
standard curve of murine rIFN- (PharMingen), ranging from 1.0 to
10.0 ng/ml.
Abs and cytokines
Anti-CD8 mAb 53-6.7, anti-B220 mAb 6B2, anti-MHC class
II mAb AMS 32-1, anti-MAC-1 mAb M1/70, anti-CD16/CD32 mAb
2.4G2, anti-Fas mAb Jo2, anti-FasL mAb K10 (22), and control
hamster IgG mAb were purchased from PharMingen. Anti-CD4 mAb GK1.5,
anti-TCR mAb 13D5, anti-rat 
chain mAb MAR 18.5, and
anti-Ly-6 A.2/E.1 mAb D7 (20) were gifts from Dr. Ethan Shevach,
National Institutes of Health (Bethesda, MD). Anti-TCRß mAb
H57.597 (21) was a gift from Dr. Maria Bellio, Institute Pasteur
(Paris, France). A control mouse IgG2b mAb (mAb 156-8 against HVB) was
obtained from Bio-Manguinhos, FIOCRUZ. Anti-IFN- mAb R4-6A2 and
biotinylated anti-IFN- mAb XMG1.2 were obtained from PharMingen.
The murine recombinant cytokines used were rIFN- (20 ng/ml), rGM-CSF
(1 ng/ml), rTNF- (40 ng/ml), rIL-1ß (20 ng/ml), rIL-2 (50 U/ml),
rIL-4 (10 ng/ml), and rIL-10 (40 ng/ml) and were obtained from
PharMingen.
Statistical analysis
Comparisons between control and experimental groups were made using Student’s t test. p < 0.05 or lower was considered significant. Since trypomastigote accumulation in culture is exponential, data were normalized by taking the logarithmic transformation before the t test was applied.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and by supernatants from activated
CD4+ T cells
Peritoneal resident M monolayers from BALB/c mice were infected
by 18-h exposure to T. cruzi metacyclic trypomastigotes,
washed, and recultured for up to 13 days. Extracellular motile
trypomastigote (infective) forms were first observed after 3 days in
culture, and their number continuously increased thereafter. At the
M density used, trypomastigotes comprised the predominant parasite
form in culture. There was variability in the number of trypomastigotes
produced between different experiments, probably related to the period
of time that epimastigotes are maintained in the laboratory before
induction of metacyclogenesis. However, trypomastigote growth in
replicate cultures within the same experiment was quite similar.
Several recombinant murine cytokines were tested to determine the
kinetics of extracellular trypomastigote accumulation in M cultures
(Fig. 1). Among all cytokines tested
(rIL-1ß, rIL-2, rIL-4, rIL-10, rTNF-, rGM-CSF, and rIFN-), only
rIFN- had a clear protective and long term effect against
replication of T. cruzi in M monolayers, as detected
after 5 to 8 days in culture (Fig. 1). On the other hand, rGM-CSF
consistently increased the number of accumulated T. cruzi
trypomastigotes (Fig. 1). It should be noted that at earlier time
points, such as 3 days in culture, IFN- completely blocked the
emergence of motile parasites in the medium (not shown). We also tested
the effect of supernatants from anti-TCR-activated CD4+
T cells from either control or T. cruzi-infected mice (Fig. 1). Only CD4+ T cells from infected hosts produced
supernatants active against T. cruzi replication. In
agreement with previous studies (12), we found that CD4+ T
cells from acutely infected mice produced 18 times more IFN- than
control CD4+ T cells following activation with anti-TCR
mAb (not shown).
|
monolayers
Given the above results, we expected to find significant
containment of parasite replication in M following activation of
cocultured CD4+ T cells with anti-TCR mAb.
However, coculture of purified CD4+ T cells from infected
mice by itself increased trypomastigote replication 2-fold in the
absence of exogenous stimulation (Table I). Moreover, anti-TCR activation of
these T cells resulted in a marked increase (12-fold in this
experiment) in the number of T. cruzi trypomastigote forms
recovered (Table I). Enhancement of parasite replication was observed
at all time points investigated throughout a 13-day period. Growing
parasites were derived from infected M, since activation of
CD4+ T cells alone did not result in detectable parasite
growth (data not shown). The ability to potentiate parasite replication
was a characteristic of CD4+ T cells from T.
cruzi-infected donors, since activation of CD4+ T
cells from control mice did not result in enhanced parasite replication
or resulted in much less replication compared with that in T cells from
infected mice (data not shown). Exacerbation of parasite replication
required CD4+ T cell activation by anti-TCR, but
addition of PMA was not necessary even though the number of
trypomastigotes increased (Table II
).
However, since we studied accessory activation pathways that required
phorbol ester, addition of PMA was maintained in the remaining
experiments.
|
monolayers (Fig. 2A). Activation of
CD4+ T cells markedly potentiated trypomastigote growth,
while activation of CD8+ T cells resulted in much less
trypomastigote replication in M (Fig. 2A).
However, while CD4+ T cells invariably exacerbated
trypomastigote growth, use of activated CD8+ T cells gave
variable results. In some experiments, CD8+ T cells had
little effect on trypomastigotes and increased the number of
noninfective epimastigotes, while in other experiments,
CD8+ T cells actually suppressed trypomastigote growth. The
reason for this variable behavior of CD8+ T cells is being
investigated. In any case, CD8+ T cells were clearly
distinct from CD4+ T cells in the ability to help
trypomastigote growth.
|
in culture, we tested the need for direct T/M cell contact for
exacerbation of parasite replication (Fig. 2B).
Infected M monolayers and CD4+ T cells were cultured in
the same compartment or separated by a cell-impermeable membrane in
culture inserts. Splenic CD4+ T cells contained endogenous
accessory cells to allow full activation when separated from M.
Separation of CD4+ T cells and M markedly reduced
parasite growth compared with that in unseparated cocultures (Fig. 2B). These results demonstrate the need for direct
cell contact between activated CD4+ T cells and M to
potentiate the growth of T. cruzi.
Modulation of CD4+ T cell activation by
Fas-induced death regulates parasite replication in M monolayers
Activation of CD4+ T cells from T.
cruzi-infected mice with anti-TCR, but not with anti-Ly-6,
results in both AICD by apoptosis and down-regulated proliferation (14, 17). We then investigated the role of CD4+ T cell AICD in
exacerbation of parasite growth. Following coculture with infected M
monolayers in the presence of different stimuli, AICD was evaluated in
recovered CD4+ T cells. Addition of anti-TCR killed a
substantial proportion of the T cells, while addition of anti-Ly-6
did not result in T cell death and, instead, increased T cell viability
after 1 day in culture (Fig. 3A), presumably because
of lymphokine-mediated rescue from spontaneous cell death. The Fas/FasL
death pathway has been implicated in mediating CD4+ T cell
AICD (16). Addition of an agonist anti-Fas mAb killed
CD4+ T cells, and when combined with anti-Ly-6,
anti-Fas abrogated the protective effect of anti-Ly-6, leading
to cell death (Fig. 3A). Susceptibility to both
anti-TCR- and anti-Fas-mediated death was restricted to
CD4+ T cells from infected, but not from control, mice (not
shown). The same results were observed in a repeat experiment. The data
indicate that AICD is preserved in CD4+ T cell interaction
with infected M and confirm that the Ly-6 activation pathway is
deficient in inducing AICD. We then explored this differential behavior
by evaluating the role of Fas-induced death in the control of parasite
replication (Fig. 3, B and C). Contrasting
with the exacerbated trypomastigote growth elicited by TCR activation,
Ly-6 activation of CD4+ T cells from infected mice did not
result in appreciable enhancement of parasite replication in M (Fig. 3, B and C) compared with that in
unstimulated T cells. Strikingly, when Ly-6 activation was combined
with prior treatment with anti-Fas, a marked enhancement of
parasite replication was seen, similar to that with TCR activation
(Fig. 3, B and C). The experiment was
repeated with identical results. Treatment with a control hamster IgG
mAb instead of anti-Fas did not result in enhanced parasite growth
(not shown). In the absence of T cells, neither anti-Fas nor
anti-Ly-6 mAbs had any effect on M viability or parasite
replication. Both killing and activation of host CD4+ T
cells are required for exacerbating parasite growth, since treatment
with anti-Fas alone was far less effective (Fig. 3, B
and C) despite inducing comparable T cell killing in
the presence or the absence of anti-Ly-6 (see Fig. 3A).
|
A). Anti-FasL Ab
had little or no effect on proliferation or viability of
CD4+ T cells from control B6 mice, but consistently
increased the otherwise depressed anti-TCR;CD3 proliferative
responses of CD4+ T cells from infected mice (not shown).
When added to cocultures of infected B6 M and TCR-activated
CD4+ T cells from infected B6 mice, anti-FasL mAb, but
not an isotype control, markedly reduced the growth of T.
cruzi trypomastigotes in culture (Fig. 4, B and
C) in a manner consistent with the blockade of AICD
achieved in CD4+ T cells (Fig. 4A).
Blockade of parasite replication by anti-FasL was confirmed in two
additional experiments. On the other hand, anti-FasL mAb had no
effect on trypomastigote replication in M cultured in the absence of
T cells (data not shown). Together, these results indicate the
involvement of CD4+ T cell AICD in up-regulation of
parasite replication in M monolayers.
|
is critically involved in the control of
parasite replication in M, it was of interest to investigate the
effect of Fas-mediated death in IFN- production by CD4+
T cells from T. cruzi-infected mice. To avoid the
coincidence of IFN- secretion and the onset of AICD (25), T cells
were first treated overnight with either anti-Fas or a control
hamster mAb and subsequently stimulated with anti-TCR, and
resulting supernatants were assayed for IFN- content (Fig. 5). Prior overnight treatment with
anti-Fas results in killing of 25 to 30% of CD4+ T
cells from infected hosts (see Fig. 3A), but
eliminated 70.6% of the IFN- produced upon subsequent TCR
activation (Fig. 5). Killing with anti-Fas also eliminated 67.0%
of IFN- production upon Ly-6 activation (not shown). These results
indicate that although they are a minor population, Fas-susceptible
CD4+ T cells comprise the majority of IFN--producing
cells or contain most of IFN- high producer CD4+ T cells
in T. cruzi-infected hosts.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Assessment
of extracellular accumulation of infective trypomastigotes in culture
was used to evaluate the roles of T cell activation and AICD in
modulation of parasite replication in M. Activation of
CD4+ T cells led to surprisingly deleterious effects,
helping parasite replication and spreading in M monolayers.
Extracellular trypomastigote accumulation strictly required the
presence of adherent M, since it was not seen in cultures of
partially purified and activated CD4+ T cells (data not
shown), even though these cultures initially contained a few motile
trypomastigotes. Moreover, supernatants from activated CD4+
T cells did not potentiate trypomastigote accumulation, arguing against
an unlikely effect of T cell products on extracellular trypomastigote
replication. In fact, it is well established that growth of T.
cruzi as trypomastigotes requires a suitable host cell for
invasion and replication as amastigotes before trypomastigotes are
released to the medium (23). In the absence of host cells,
extracellular trypomastigotes do not divide and differentiate into
noninfective epimastigote forms capable of extracellular growth
(23). The ability of activated CD4+ T cells to potentiate
trypomastigote growth in M was unexpected. It is known that IFN-
is able to kill intracellular forms of T. cruzi through
activation of inducible NO synthase and NO production (8, 9). Studies
have demonstrated intense IFN- production in vivo (27) and by
activated CD4+ T cells ex vivo (12) during acute T.
cruzi infection. We have confirmed that CD4+ T cells
from infection secrete large amounts of IFN- upon activation (not
shown), and that their supernatants reduced trypomastigote growth in
M. Despite this antiparasite effector potentiality, TCR activation
of intact CD4+ T cells exacerbated trypomastigote growth in
cocultured M. The potentiating effect on parasite replication
required physical association with infected M, as demonstrated in
experiments of separation by a cell-impermeable membrane. A small, but
discernible, deleterious effect was also seen in the absence of
exogenous stimulation. This effect could reflect ongoing activation of
CD4+ T cells by M-presenting parasite Ags, but this
possibility needs to be tested.
A critical role for AICD in exacerbation of parasite growth was
established from several evidences. First, anti-TCR activation of
CD4+ T cells potentiated parasite growth in culture, while
anti-Ly-6 activation had little or no effect. We have previously
demonstrated that, different from TCR activation, Ly-6 activation of
CD4+ T cells from infected mice does not induce AICD (14).
Similar results were obtained in the presence of cocultured M.
Possibly, Ly-6 activation delivers weaker activation signals to T
cells, insufficient to induce AICD. In previous studies with T cell
hybridomas, Ly-6 activation also failed to induce AICD to the same
extent as direct TCR;CD3 engagement (28).
Second, deliberate mimicking of AICD by killing with anti-Fas
coupled to Ly-6 activation exacerbated trypomastigote replication in a
manner comparable to the deleterious effect of anti-TCR. Different
from control CD4+ T cells, which are resistant to killing
by either anti-Fas or anti-TCR (data not shown), a proportion
of CD4+ T cells from T. cruzi-infected mice
(between 25–30%) are susceptible to death after ligation with
anti-Fas mAb. Exacerbated parasite growth, however, required both
Fas-mediated killing and Ly-6 activation. One possibility to explain
these results could be that exacerbation of parasite growth results
from summation of two opposing effects by distinct CD4+ T
cell subpopulations. One could be a contact-dependent effect of
Fas-resistant T cells, increasing parasite replication. The second
effect would be AICD, which appears to kill Fas-susceptible T cells
protective against intracellular parasite growth. However, alternative
possibilities, such as release of parasite-helping mediators by
Fas-stimulated cells, cannot be ruled out. The nature of the cell
contact-dependent signal for parasite growth is unknown, but
Fas-mediated M killing is unlikely to play a role. Anti-Fas is not
cytotoxic for M and has no effect on parasite growth in M alone
(not shown). Moreover, M killing should restrict, rather than
potentiate, trypomastigote growth by reducing the number of target
cells available for replication. On the other hand, several studies
have demonstrated T cell-mediated M activation (29, 30, 31) or
deactivation (32) through cell contact-mediated signals such as
membrane TNF- (29, 32) or CD40 ligand (31). Close physical
association mediated by cell adhesion molecules could also be necessary
for delivery of regulatory cytokines at high local concentrations.
Cytokines such as TGF-ß or IL-10, which regulate acute T.
cruzi infection (33, 34, 35), could be involved. Further investigation
is necessary to clarify the role of cell adhesion molecules, regulatory
cytokines, and signal transduction pathways involved in exacerbation of
parasite growth within M monolayers.
Finally, additional evidence for the deleterious role of AICD on
parasite replication was obtained by blockade of AICD with
anti-FasL mAb. While anti-Fas killing of CD4+ T
cells potentiated parasite growth by Ly-6-activated T cells, blockade
of TCR-mediated CD4+ T cell AICD by anti-FasL reduced
trypomastigote replication in M. The extent of reduction in parasite
growth mirrored the extent of protection from AICD achieved. On the
other hand, addition of either anti-Fas or anti-FasL to M
cultured alone had no effect on parasite replication (data not
shown).
We observed that Fas-mediated killing of CD4+ T cells
reduced subsequent IFN- production in a manner disproportionate to
the amount of cell death. This result implies that Fas-susceptible
cells contribute the majority of IFN- production in CD4+
T cells from T. cruzi-infected mice. The finding is perhaps
not surprising, since susceptibility to Fas-mediated death is only
acquired after chronic activation of T cells (36) and correlates with T
cells bearing memory/activated phenotype in man (37). In fact,
memory/activated T cells secrete severalfold more IFN- than naive T
cells (38, 39). The onset of AICD could regulate parasite growth by
selectively ablating those effector T cells showing intense and
continued IFN- secretion. These results might have implications for
infection in vivo. Infection with T. cruzi leads to intense
polyclonal lymphocyte activation (40), which could result in induction
of both Fas susceptibility and FasL expression by T cells. We found
increased Fas and FasL expression by CD4+ T cells in the
course of T. cruzi infection in vivo (M. F. Lopes, N.
Giese, H. R. Morse III, and G. A. DosReis, manuscript in
preparation). Therefore, CD4+ T cells from infected mice
are in many aspects similar to chronically activated T cells
susceptible to propriocidal regulation by AICD (15, 41). Our results
suggest that CD4+ T cells could play a dual role in host
protection against T. cruzi, being able to either control or
exacerbate the parasite load in tissues depending upon whether AICD is
induced following encounters with infected M. However, since AICD
was studied in vitro with anti-TCR mAb, any extrapolation to
infection in vivo must be interpreted with caution, waiting for studies
with transgenic mice and defined T. cruzi Ags. Regarding
involvement of distinct T cell subsets, our results are not
incompatible with the suggested role of type 2 cytokines in
exacerbating infection by metacyclic trypomastigotes (42). In fact, the
increase in parasite burden we observe in vitro could have resulted
from more than one mechanism, for example Th1 T cell AICD plus
Th2-dependent down-regulation of M activation. The culture system
employed here could be useful in investigating molecular mechanisms of
anti-parasite defense as well as down-regulatory signals involved
in antimicrobial responses of host T lymphocyte subsets.
|
Acknowledgments |
|---|
, and anti-Ly-6 mAbs. |
Footnotes |
|---|
2 M.P.N. is D.Sc. student of Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro.
3 Address correspondence and reprint requests to Dr. George A. DosReis, Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS Bloco G, Ilha do Fundão, Rio de Janeiro RJ 21944-970, Brazil.
4 Abbreviations used in this paper: M, macrophage/macrophages; NO, nitric oxide, AICD, activation-induced cell death; FasL, Fas ligand; rGM-CSF, recombinant granulocyte-macrophage CSF.
Received for publication July 18, 1997. Accepted for publication October 16, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-ß. Eur. J. Immunol. 22:2501.[Medline]
and interferon- on macrophage activation for the killing of intracellular Trypanosoma cruzi through a nitric oxide-dependent mechanism. Eur. J. Immunol. 22:301.[Medline]
-interferon induces macrophage activation and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections. J. Immunol. 140:4342.[Abstract]
is required for resistance to acute Trypanosoma cruzi infection in mice. J. Immunol. 146:3626.[Abstract]
ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
ß T cell receptors. J. Immunol. 142:2736.[Abstract]
. J. Immunol. 158:3673.[Abstract]
production. J. Immunol. 140:1401.[Abstract]
by subsets of Lyt-2+ and L3T4+ T cells identified by expression of Pgp-1. J. Immunol. 138:3583.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L. V. C. Guillermo, E. M. Silva, F. L. Ribeiro-Gomes, J. De Meis, W. F. Pereira, H. Yagita, G. A. DosReis, and M. F. Lopes The Fas death pathway controls coordinated expansions of type 1 CD8 and type 2 CD4 T cells in Trypanosoma cruzi infection J. Leukoc. Biol., April 1, 2007; 81(4): 942 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Silva, L. V. C. Guillermo, F. L. Ribeiro-Gomes, J. De Meis, R. M. S. Pereira, Z. Wu, T. C. Calegari-Silva, S. H. Seabra, U. G. Lopes, R. M. Siegel, et al. Caspase-8 Activity Prevents Type 2 Cytokine Responses and Is Required for Protective T Cell-Mediated Immunity against Trypanosoma cruzi Infection J. Immunol., May 15, 2005; 174(10): 6314 - 6321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Alcaide and M. Fresno The Trypanosoma cruzi membrane mucin AgC10 inhibits T cell activation and IL-2 transcription through L-selectin Int. Immunol., October 1, 2004; 16(10): 1365 - 1375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-B. Voisin, D. Buzoni-Gatel, D. Bout, and F. Velge-Roussel Both Expansion of Regulatory GR1+ CD11b+ Myeloid Cells and Anergy of T Lymphocytes Participate in Hyporesponsiveness of the Lung-Associated Immune System during Acute Toxoplasmosis Infect. Immun., September 1, 2004; 72(9): 5487 - 5492. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Yan, M Wang, W J Lemon, and M You Single nucleotide polymorphism (SNP) analysis of mouse quantitative trait loci for identification of candidate genes J. Med. Genet., September 1, 2004; 41(9): e111 - e111. [Full Text] [PDF]
|
|
|
|
|
|
F. L. Ribeiro-Gomes, A. C. Otero, N. A. Gomes, M. C. A. Moniz-de-Souza, L. Cysne-Finkelstein, A. C. Arnholdt, V. L. Calich, S. G. Coutinho, M. F. Lopes, and G. A. DosReis Macrophage Interactions with Neutrophils Regulate Leishmania major Infection J. Immunol., April 1, 2004; 172(7): 4454 - 4462. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Gavrilescu and E. Y. Denkers Apoptosis and the Balance of Homeostatic and Pathologic Responses to Protozoan Infection Infect. Immun., November 1, 2003; 71(11): 6109 - 6115. [Full Text] [PDF]
|
|
|
|
 |
|
 M. d. L. Higuchi, L. A. Benvenuti, M. Martins Reis, and M. Metzger Pathophysiology of the heart in Chagas' disease: current status and new developments Cardiovasc Res, October 15, 2003; 60(1): 96 - 107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lucas, L. M. Stuart, J. Savill, and A. Lacy-Hulbert Apoptotic Cells and Innate Immune Stimuli Combine to Regulate Macrophage Cytokine Secretion J. Immunol., September 1, 2003; 171(5): 2610 - 2615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Miyahira, M. Katae, S. Kobayashi, T. Takeuchi, Y. Fukuchi, R. Abe, K. Okumura, H. Yagita, and T. Aoki Critical Contribution of CD28-CD80/CD86 Costimulatory Pathway to Protection from Trypanosoma cruzi Infection Infect. Immun., June 1, 2003; 71(6): 3131 - 3137. [Abstract] [Full Text] [PDF]
|
|
|
|
