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*
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
Department of Molecular Biology, DNAX Research Institute, Palo Alto, CA 94303
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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is required for resistance to Toxoplasma gondii.
To identify the transcription factors involved in this mechanism of
resistance, mice deficient in STAT4, a protein involved in IL-12
signaling, were infected with T. gondii and their immune
responses were analyzed. STAT4-/- mice were unable to
control parasite replication and died during the acute phase of
infection, whereas wild-type mice controlled parasite replication and
survived this challenge. The susceptibility of STAT4-/-
mice to toxoplasmosis correlated with a defect in their ability to
produce IFN- in response to infection, whereas administration of
IFN- to these mice inhibited parasite replication and delayed time
to death. Interestingly, analysis of infected STAT4-/-
mice revealed that these mice did produce low levels of IFN- during
infection, and the ability of splenocytes from infected or uninfected
STAT4-/- mice to produce IFN- was enhanced by the
addition of IL-2 plus IL-18. Moreover, administration of IL-2 plus
IL-18 to STAT4-/- mice resulted in elevated serum levels
of IFN- associated with a decreased parasite burden and delayed time
to death. In vivo depletion studies demonstrated that the ability of
IL-2 plus IL-18 to mediate STAT4-independent resistance to T.
gondii is dependent on NK cell production of IFN-. Together,
these studies identify STAT4 as an important transcription factor
required for development of the innate NK and adaptive T cell responses
necessary for resistance to T. gondii. However, other
signaling pathways can be used to bypass STAT4-dependent production of
IFN- and enhance innate resistance to T.
gondii. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the major mediator of resistance to this
parasite (1, 2). Infection with T. gondii
results in the production of IL-12, which is required to stimulate the
production of IFN- by NK and T cells. Thus, the absence of
endogenous IL-12 during the acute phase of toxoplasmosis prevents the
development of protective IFN- responses and results in unrestricted
parasite replication and death of infected animals (3, 4).
However, IL-12-independent pathway(s) of IFN- production following
vaccination with the attenuated ts-4 strain of T. gondii
have been reported (5, 6).
Although IL-12 is important for the development of resistance to
T. gondii, the molecular basis for this event is not clear.
Signaling through the IL-12 receptor (IL-12R) leads to the activation
of STAT4 (7, 8), and NK and T cells from mice that lack
the STAT4 gene (STAT4-/-) have
defects in their ability to produce IFN-, proliferate, and lyse
target cells in response to IL-12 (7, 9). Nevertheless,
although IL-12-/- or
STAT4-/- mice have been demonstrated to be
deficient in production of IFN- and development of Th1 responses
(7, 10, 11), other studies have shown that Th1 responses
can occur in the absence of IL-12 or STAT4 (12, 13, 14).
Because IL-12 can also signal through NF-
B (15), STAT1
(16), and STAT3 (7, 8), these alternative
pathways may contribute to the development of Th1-type responses in the
absence of STAT4. Moreover, although IFN-
can signal through STAT4
in human T cells (17), in murine systems, other factors
with the capacity to signal through STAT4 have not been identified. It
remains unclear whether STAT4 is required for IL-12-mediated resistance
to T. gondii, or whether there are STAT4-independent
pathways that lead to the development of IFN- responses during
toxoplasmosis.
Although IL-12 is a critical cytokine required for development of a Th1
response, various cofactors (IL-1, IL-2, TNF-, and CD28) are
required for maximal production of IFN- by Th1 cells
(18, 19, 20, 21, 22). Many of these cofactors have been demonstrated
to enhance IL-12-induced production of IFN- during toxoplasmosis
(19, 23, 24). The discovery of IL-18 (also called
IFN--inducing factor, or IGIF), a cytokine functionally similar to
IL-12 (25) but structurally related to members of the IL-1
family (26), has raised important questions about the
relationship between IL-18 and IL-12. Like IL-12, IL-18 stimulates NK
and T cell production of IFN- and enhances T cell proliferation, as
well as NK cell cytotoxicity (27). However, whereas IL-12
drives Th1 differentiation (28, 29), Robinson and
colleagues reported that IL-18 does not drive the development of Th1
responses, but rather enhances the effects of IL-12 (30).
Nevertheless, recent studies have shown that in the absence of IL-18,
Th1 responses are impaired (31, 32, 33, 34), suggesting an
important role for IL-18 during the development of Th1 responses. Thus,
although functionally similar, the relationship between IL-12 and IL-18
in resistance to toxoplasmosis remains unclear, and there are few
reports that address the relationship between these two cytokines
during infection (33). The studies presented here
demonstrate an important role for STAT4 in the development of
protective immunity to T. gondii and demonstrate that
exogenous IL-2 plus IL-18 can partially overcome the requirement for
STAT4 during the innate response to this pathogen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A two-site ELISA was used to measure levels of IFN-, IL-2,
IL-4, and IL-12p40 as previously described (35).
Recombinant IFN- was purchased from Genzyme (Cambridge, MA). Human
IL-2 (HuIL-2)4 was
purchased from Chiron (Emeryville, CA). Recombinant murine (rm) IL-18
was purchased from PeproTech (Rocky Hill, NJ). IL-18 levels were
measured using a rat mAb specific for IL-18 as capture Ab and a
polyclonal goat anti-IL-18 Ab (both Abs were supplied by R&D
Systems, Minneapolis, MN) in combination with a peroxidase-conjugated
donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA) for
detection. The sensitivity of this assay was routinely 39 pg/ml of
rmIL-18. RmIL-12 was supplied by the Immunology Department of Genetics
Institute (Cambridge, MA). Rat mAbs GK1.5 (IgG2b, anti-CD4),
H35-17.2 (IgG2b, anti-CD8), and XMG 6 (IgG1, anti-IFN-) were
purified from ascites. JES5-2A5 (anti-IL-10) was provided by DNAX
(Palo Alto, CA). Anti-asialoGM1 was purchased from Wako Chemicals
(Richmond, VA). Rabbit or rat IgG was purchased from Sigma (St.
Louis, MO).
Mice, infection, and experimental design
STAT4-/- mice (129/Sv/C57BL/6) were
provided by Dr. J. N. Ihle (St. Jude Children’s Research
Hospital, Memphis, TN), wild-type 129Sv/C57BL/6 mice were provided by
Dr. S. Banerjee (BASF Bioresearch Corporation, Worcester, MA). Both
STAT4+/+ and STAT4-/-
mice were derived on a 129/Sv background, crossed once with C57BL/6
mice, and maintained as homozygous breeding pairs. Mice were bred and
maintained in Thoren caging units (Thoren Caging System, Hazleton, PA)
within the University Laboratory Animal Research facilities at the
University of Pennsylvania. Mice were 6–8 wk old when used for
experiments. Swiss Webster and CBA/ca mice were purchased from The
Jackson Laboratory (Bar Harbor, ME) and were used to maintain the ME49
strain of T. gondii and as a source of cysts for infection.
For infection, mice were injected i.p. with 20 cysts. To assess
parasite burden, peritoneal cells from infected mice were collected,
washed twice, counted, and used for cytospin preparations. The
percentage of cells infected were estimated by counting >500
cells/slide. For cytokine treatment, mice were treated i.p. with 2,500
IU IFN-, or 150,000 IU HuIL-2, and/or 500 ng murine IL-18 1 day
before infection and daily thereafter. For depletion of NK cells,
CD4+ T cells, or CD8+ T
cells during toxoplasmosis, groups of STAT4-/-
mice were treated i.p. with 50 µl anti-asialoGM1, 0.2 mg
anti-CD4 (GK1.5), or anti-CD8 (H35-17.2), and control mice were
treated with an isotype Ab (rat or rabbit IgG) 3 days before infection
and every other day thereafter. FACS analysis of splenocytes and
peritoneal exudates cells (PECs) from treated mice showed at least 98%
cell depletion of NK1.1+,
CD4+, or CD8+ T cells. For
depletion of IFN-, IL-4, or IL-10, mice were treated with 2 mg
anti-IFN- (XMG6), anti-IL-10, or 5 mg anti-IL-4 (11B11)
3 days before infection and every 3 days thereafter.
Histological analysis
For analysis of pathological changes following infection, mice were sacrificed at different times after infection, and samples of livers, lungs, and spleens were removed and fixed overnight in Accustain 10% formalin neutral buffered solution (Sigma) and then embedded in paraffin, followed by hematoxylin and eosin staining. For immunohistochemistry, 5-µm paraffin sections were heated for 1 h at 60°C, rehydrated, and incubated for 30 min with 0.3% H2O2/0.2 M NaN3 to quench endogenous peroxidase activity, followed by blocking with 10% goat serum (Vector Laboratories, Burlingame, CA) in HBSS. Sections were then stained for 1 h with a polyclonal rabbit Ab against inducible NO synthase (iNOS; Transduction Laboratories, Lexington, KY), T. gondii (from Dr. F. G. Araujo, Palo Alto Medical Foundation, Palo Alto, CA), or isotype control Ab (Sigma). After washing in HBSS, sections were incubated with biotinylated anti-rabbit IgG Ab (Vector Laboratories). Subsequent incubation of the slides with peroxidase-conjugated avidin-biotin complex (Vectastain Elite ABC kit; Vector Laboratories) was performed according to the manufacturer’s instructions. Specific staining was visualized using 3–3'-diaminobenzidine (Vector Laboratories) as substrate, counterstained with hematoxylin, dehydrated, and mounted in Permount (Fisher Scientific, Fair Lawn, NJ).
Analysis of recall responses
Splenocytes from infected mice were prepared and washed in RPMI
1640 containing 10% heat-inactivated FCS, 2 mM glutamine, 1000 U/ml
penicillin, 10 µg/ml streptomycin, 0.25 mg/ml fungizone, 10 mM HEPES
(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid) (Life Technologies, Grand Island, NY), 1 mM sodium pyruvate, 1%
(v/v) nonessential amino acids (Life Technologies), 5 x
10-5 M 2-ME. Splenocytes were then counted and
plated at 2 x 105/well in a final volume of
200 µl in flat-bottom 96-well plates (Costar, Cambridge, MA). Cells
were stimulated with tachyzoites lysate Ag (TLA) for 48 h, and the
levels of IFN- in the supernatants were measured by ELISA. For
proliferation assays, splenocytes were stimulated with TLA for 48
h and then pulsed with 0.5 µCi
[3H]thymidine/well for another 24 h. Cells
were harvested with a cell harvester (Cambridge Technology, Cambridge,
MA), and incorporation of [3H]thymidine was
determined using a liquid scintillation counter (LKB Wallac, Turku,
Finland).
Analysis of NK cell cytotoxicity
YAC-1 cells (American Type Culture Collection, Manassas, VA) were labeled with 100 µCi 51Cr (Amersham, Arlington Heights, IL) for 1 h at 37°C, washed, and used as targets. PECs or splenocytes from mice were harvested, washed twice, and the number of live cells was estimated based on trypan blue exclusion. These cells were plated at different E:T ratios and incubated at 37°C for 4 h. Supernatants were harvested with a Skatron cell press (Skatron, Sterling, VA), the amount of 51Cr released was estimated using a gamma counter (Packard, Meriden, CT), and specific lysis was calculated as previously described (36).
FACS analysis
The following FITC- or PE-labeled Abs (anti-NK1.1,
anti-CD3
, anti-CD4, anti-CD8, anti-CD25,
anti-CD44, anti-IFN-, mouse IgG2a, and rat IgG2a) were
purchased from PharMingen (San Diego, CA) and were used to study the
expression of these molecules. Splenocytes from uninfected
STAT4-/- mice were stimulated with
104 U/ml HuIL-2 plus 20 ng/ml IL-18 for 4 h.
Cells were harvested, washed, and stained for CD4, CD8, or NK1.1,
washed, fixed with paraformaldehyde, washed, permeabilized with
saponin, and stained for IFN-. Stained cells were analyzed with a
FACScan cytofluorometer (Becton Dickinson, Mountain View,
CA).
RNase protection assay (RPA)
Total RNA was extracted from spleens using Tri-reagent (Sigma)
and was assessed for cytokine mRNA content using the RiboQuant
Multiprobe RPA system (PharMingen). Briefly, 10 µg of RNA from each
sample was hybridized in solution with the appropriate radiolabeled
antisense RNA probe set. mCK-1 (containing IL-4, IL-5, IL-10, IL-13,
IL-15, IL-9, IL-2, IL-6, and IFN-) was used for detection of
cytokine mRNA. Following hybridization, free probe and remaining ssRNA
were digested with RNase A and T1, and the protected probes were
purified and resolved on 5% denaturing polyacrylamide gels using Ultra
Pure Sequagel reagents (National Diagnostics, Atlanta, GA). Dried
gels were exposed to phosphorimaging screens, and protected fragments
were visualized using a Phospho-Imager GS-525 (Molecular Imager system;
Bio-Rad, Richmond, CA).
Statistics
An unpaired Student’s t test was used for the majority of statistical analysis performed in these studies. The only exceptions were the use of a nonparametric Mann-Whitney test to compare the times of death for different experimental groups, and the use of a paired Student’s t test for analysis of activation markers expressed by CD4+ T cells. All statistical analysis was performed using INSTAT software (GraphPad Software, San Diego, CA). A p value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the role of STAT4 in resistance to T.
gondii, STAT4+/+ and
STAT4-/- mice were inoculated i.p. with 20
cysts of T. gondii and survival was monitored.
STAT4-/- mice succumbed to infection between
days 5 and 16 following challenge, whereas all
STAT4+/+ mice survived for >3 mo (Fig. 1
A). The susceptibility of
STAT4-/- mice to T. gondii was
characterized by the presence of a significantly higher percentage of
infected cells in the peritoneal cavity on days 5 and 7 (30 and 50%,
respectively) postinfection, compared with
STAT4+/+ mice (<1%) at these time points (Fig. 1B, p < 0.01). In these experiments, the
total number of PECs recovered from STAT4-/-
and STAT4+/+ mice infected for 7 days was
25.7 x 106 ± 5.4 x
106 and 4.13 x 106 ±
0.82 x 106, respectively (data shown are
mean ± SD from three experiments). Immunohistochemical staining
for parasites and iNOS in the lungs and spleens of infected mice
demonstrated a heavy parasite burden in
STAT4-/- mice compared with
STAT4+/+ mice, associated with a lack of iNOS
(Fig. 1C). Large numbers of parasites were also detected in
the livers, brains, hearts, and small intestines of moribund
STAT4-/- mice, but not
STAT4+/+ mice. These results are similar to
previous studies that characterized IL-12-/-
and IFN--/- mice infected with T.
gondii (5, 37). Given the high parasite burden in the
peritoneal cavity and large numbers of parasites throughout the body,
these results suggest that STAT4-/- mice die as
a consequence of unrestricted parasite replication and associated
tissue damage.
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), STAT4-/-
mice had significantly lower levels of serum IFN- compared with
STAT4+/+ mice (Fig. 2B,
p < 0.01). Because IFN- is the major mediator of
resistance during toxoplasmosis, the reduced levels of serum IFN-
observed following infection of STAT4-/- mice
suggested that the susceptibility of these mice to T. gondii
is likely due to the absence of IFN-. To determine whether IFN-
could provide protection in the absence of STAT4, exogenous IFN- was
administered i.p. to STAT4-/- mice following
infection with T. gondii. Daily administration of 2500 IU
IFN- to STAT4-/- mice infected with T.
gondii resulted in a significant decrease in the percentage of
PECs infected on day 5 postinfection compared with control mice (Fig. 2C, p < 0.01), and the total numbers of
PECs recovered from the peritoneal cavity were 9.92 x
106 ± 2.4 x 106
(PBS) vs 5.6 x 106 ± 0.9 x
106 (IFN-) with p < 0.05
(data shown are the means ± SD from three experiments).
Moreover, administration of IFN- to STAT4-/-
mice led to a significant delay in the time to death of these mice
(Fig. 2D). However, administration of IFN- did not
enhance Ag-specific production of IFN- during recall responses (data
not shown). Though this treatment did not lead to long-term resistance
to T. gondii, this is likely a consequence of the relatively
low levels of IFN- that were used in these experiments. These
results do not exclude the possibility that there is a STAT4-dependent,
IFN--independent mechanism that contributes to control the growth
and replication of T. gondii in wild-type mice.
Nevertheless, in the absence of STAT4, mice are susceptible to
toxoplasmosis due to unrestricted parasite growth associated with
reduced levels of IFN-. Interestingly, low levels of IFN- could
be detected in the serum of STAT4-/- mice on
day 7 postinfection, indicating that there is a STAT4-independent
pathway for production of IFN- during toxoplasmosis.
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Because IL-12 is important for NK cell activation (proliferation,
cytotoxicity, and production of IFN-) as well as development of Th1
responses, the response of NK and T cells following i.p. infection of
STAT4-/- and STAT4+/+
mice with T. gondii were compared. Whereas infection of
STAT4+/+ mice resulted in increased NK cell
activity at the local site of infection as measured by cytolysis of
YAC-1 cells, this was not observed in STAT4-/-
mice (Fig. 3A). However, FACS
analysis revealed that STAT4-/- mice had a
lower percentage of NK cells (38.2 ± 0.39,
STAT4+/+; 3.02 ± 2.5,
STAT4-/-; p < 0.01) and T
cells (9.0 ± 1.2, STAT4+/+; 1.2 ± 1,
STAT4-/-; p < 0.01) (data
shown are the mean ± SD from three mice of a representative
experiment) at the local site of infection on day 5 postinfection
compared with STAT4+/+ mice (Fig. 3B),
whereas this difference was not observed between uninfected
STAT4-/- and STAT4+/+
mice (data not shown). Further analysis of cytospins revealed that the
majority of PECs in STAT4-/- mice on day 5
postinfection were neutrophils and macrophages. Interestingly, whereas
there were similar percentages of NK cells in the spleens of infected
STAT4+/+ and STAT4-/-
mice on day 5 postinfection, STAT4-/- mice
still had a 2- to 4-fold reduction in NK cell cytolytic activity (data
not shown). In addition, total numbers of splenocytes recovered from
STAT4+/+ mice on day 7 postinfection was
increased 
2-fold compared with STAT4-/- mice
(STAT4+/+, 265 x 106
± 58 x 106;
STAT4-/-, 122.4 x
106 ± 16 x 106;
p < 0.01, data shown are mean ± SD from three
experiments), and a marked increase in the percentage of
CD4+ and CD8+ T cells that
expressed activation markers (CD44, CD25) was observed. In contrast,
STAT4-/- mice infected with T.
gondii did not display an expansion of splenocytes, and the
increased expression of activation markers was less pronounced (Fig. 4). These findings suggest that STAT4
and/or IFN- are important for T cell activation following infection
with T. gondii, and that in the absence of STAT4, there may
be a defect in the recruitment and/or expansion of NK and T cells at
the local site of infection.
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and NO, but showed enhanced proliferation
compared with splenocytes from STAT4+/+ mice
(Fig. 5, day 7, p <
0.01). Previous studies have shown that the reduced T cell
proliferation during acute toxoplasmosis was due to the production of
reactive nitrogen intermediates (38). Thus, the enhanced
proliferative responses observed in the
STAT4-/- mice are likely a consequence of the
low levels of NO in these cultures. In addition, the levels of IL-2
produced by cells from STAT4+/+ and
STAT4-/- mice during these recall responses
were low (<1 ng/ml), and there were no significant differences between
these groups.
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response by day 5, but also increased levels of mRNA for IL-10, IL-6,
and IL-15 by day 7 (Fig. 6). In contrast,
STAT4-/- mice lacked an IFN- response on day
5, but developed a mixed Th1 (IFN- and IL-2) and Th2 (IL-4, IL-10,
IL-13, IL-6) type response by day 7 postinfection (Fig. 6). The
increased levels for IFN- mRNA in day 7-infected
STAT4-/- mice, compared with sham-infected
mice, confirm the presence of a STAT4-independent mechanism that leads
to the production of IFN- following infection with T.
gondii. Because previous studies have demonstrated that
STAT4-/- mice default to a Th2 response
(7, 9), and RPA analysis revealed the presence of Th2
cytokines in infected STAT4-/- mice, studies
were performed to determine whether infection of
STAT4-/- mice with T. gondii results
in the development of a Th2 response that antagonizes production of
IFN-. Stimulation of splenocytes from day 7-infected
STAT4+/+ and STAT4-/-
mice with TLA induced low levels of IL-4 (<200 pg/ml), and there was
no significant difference between STAT4+/+ and
STAT4-/- mice. Administration of anti-IL-4
to STAT4-/- mice before challenge with T.
gondii did not enhance serum levels of IFN- or decrease
parasite burden in these mice, and in vitro neutralization of IL-10 or
TGF-ß only slightly enhanced production of IFN- during recall
responses (data not shown). Together, these studies indicate that the
defect in IFN- response in STAT4-/- mice was
not due to a Th2-polarized response.
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The studies described above demonstrate an important role for
STAT4 in the development of optimal IFN- responses during
toxoplasmosis. However, STAT4-/- mice do
produce low levels of IFN- in serum and recall responses following
infection with T. gondii. These data indicate the
presence of a STAT4-independent mechanism for the production of IFN-
during this infection, albeit insufficient to protect against
toxoplasmosis. Nevertheless, these findings led to the hypothesis that
it would be possible to induce a protective IFN- response
independently of STAT4. IL-18 and IL-2 have been shown to be important
for the development of IFN- responses during innate and adaptive
responses (31, 32, 39), suggesting that they may be
candidates to induce IL-12- or STAT4-independent production of IFN-.
In vitro studies using splenocytes from uninfected
STAT4-/- mice showed that IL-2 alone stimulated
proliferation but did not induce production of IFN- (Fig. 7, A and B).
However, when IL-2 was combined with IL-18, production of IFN- and
proliferative responses in these cultures were enhanced. In contrast,
splenocytes from STAT4-/- mice infected for 7
days produced increased levels of IFN- and had higher levels of
proliferation in response to IL-2 alone or in combination with
IL-18 compared with uninfected mice. Splenocytes from
STAT4+/+ mice stimulated with IL-2 plus
IL-18 had at least 2- to 3-fold higher levels of IFN- in the
supernatant than splenocytes from STAT4-/- mice
(data not shown).
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, after 4-h stimulation with IL-2
plus IL-18, revealed that NK cells were the major cell population that
was positive for IFN- in cultures from uninfected and infected
STAT4-/- mice (Fig. 7
). Although 40% of NK
cells from uninfected STAT4-/- mice were
positive for IFN- after stimulation with IL-2 plus IL-18, only low
levels of IFN- protein were detected in culture supernatants at
24 h. In contrast, only 11% of NK cells from infected
STAT4-/- mice were positive for IFN-, but
there were higher levels of IFN- protein in these culture
supernatants compared with uninfected STAT4-/-
mice (Fig. 7). These data have to be interpreted with care as analysis
of NK cell-proliferative responses in these cultures demonstrated that
NK cells from infected STAT4-/- mice
proliferated to a greater extent than did NK cells from uninfected
STAT4-/- mice (data not shown). Nevertheless,
these data demonstrate that IL-2 plus IL-18 could stimulate the
production of IFN- by NK cells from infected mice independently of
STAT4.
Because in vitro studies showed that the combination of IL-2 and IL-18
could enhance production of IFN- by splenocytes from infected
STAT4-/- mice, the ability of these cytokines
to augment resistance to T. gondii in
STAT4-/- mice was tested. Daily administration
of IL-2 (150,000 IU) or IL-18 (500 ng) alone to infected
STAT4-/- mice had no effect on serum levels of
IFN-, and only IL-18 decreased the percentage of infected PECs at
day 5 from 66 to 38.7% (p < 0.035,
n = 3 experiments). However, the most significant
results were obtained when infected STAT4-/-
mice were treated with the combination of IL-2 plus IL-18. This
treatment resulted in a significant decrease in the percentage of
infected PECs (Fig. 8A,
p < 0.01), as well as the total numbers of PECs
recovered from treated mice (PBS, 18.4 x
106 ± 1.9 x 106;
IL-2 plus IL-18, 10.2 x 106 ± 1.15 x
106; p < 0.01, data shown are
mean ± SD from three experiments). This decrease in the parasite
burden was associated with elevated serum levels of IFN- (Fig. 8B, p < 0.05). Moreover, treatment of
STAT4-/- mice with IL-2 plus IL-18 led to a 5-
to 6-day delay in time to death of STAT4-/-
mice (Fig. 8D, p < 0.0001). This treatment
also led to an increase in NK cell cytolytic activity (Fig. 8C) and a 2-fold expansion in the number of splenocytes in
STAT4-/- mice. However, this treatment did not
ultimately protect STAT4-/- mice, and many free
parasites were observed in the peritoneal cavity at the time of death
of STAT4-/- mice treated with IL-2 plus
IL-18.
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, CD4, CD8,
or NK cells. Depletion of IFN- antagonized the protective effects of
this treatment (Fig. 9A,
p < 0.01). However, IL-2- and IL-18-treated,
IFN--depleted mice still had a significantly lower percentage of
infected PECs compared with PBS, isotype control mice
(p < 0.05, Fig. 8A), and the total
numbers of PECs recovered from both groups were not significantly
different (18 x 106 ± 6.1 x
106, IL-2 plus IL-18 and anti-IFN-;
22.3 x 106 ± 4.5 x
106, PBS, isotype; p = 0.42, data
shown are mean ± SD from three experiments). Together, these data
suggest either incomplete neutralization of IFN- or that there is an
IFN--independent mechanism that contributes to the control of
parasite growth at the local site. Depletion of NK cells led to reduced
serum IFN- levels in STAT4-/- mice on day 3
postinfection (Fig. 9B, p < 0.05), and this
approached statistical significance at day 5 postinfection
(p = 0.07). In contrast, depletion of
CD4+ or CD8+ T cells did
not alter serum levels of IFN- on day 3 or 5 postinfection (Fig. 9C). Nevertheless, depletion of NK cells,
CD4+ T cells, or CD8+ T
cells alone had no significant effect on the number of infected PECs in
treated mice on day 7 (all <1% infected PECs). This suggests that, in
mice treated with IL-2 plus IL-18, the levels of IFN- produced in
mice treated with antiasialoGM1 is sufficient to control parasite
growth in the peritoneal cavity. Together, these data demonstrate that
treatment of STAT4-/- mice with IL-2 plus IL-18
results in enhanced resistance to T. gondii associated with
increased production of IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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as well as directing differentiation of T cells to
produce IFN-. The in vivo significance of this is illustrated by
studies in which treatment with anti-IL-12 Ab inhibits the
development of Th1 responses and increases susceptibility to certain
intracellular infections (40). Several members of the STAT
family of transcription factors are involved in IL-12 signaling, but
their role in IL-12-mediated resistance during toxoplasmosis is
unclear. The studies presented here demonstrate that
STAT4-/- mice have a severe defect in their
ability to produce IFN- during toxoplasmosis, and this is associated
with unrestricted parasite growth. These results confirm the importance
of IL-12 in resistance to T. gondii (3, 5, 41)
and identify STAT4 as an important transcription factor involved in
these events.
Interestingly, following infection with T. gondii, low
levels of IFN- were produced in the absence of STAT4. The factors
involved in this process are unclear, but our studies revealed that
infection of STAT4-/- mice resulted in an
up-regulation of serum levels of IL-18 (day 0, 0.017 ± 0.037; day
7, 1.48 ± 0.63; (ng/ml), p < 0.001. Data shown
are mean ± SD from three experiments.). Moreover, infection of
T. gondii also resulted in elevated levels of IL-2 mRNA
(Fig. 6). These results suggest that endogenous IL-2 and IL-18 may
contribute to the low levels of IFN- observed in infected
STAT4-/- mice. Experiments to determine the
significance of endogenous IL-2 and/or IL-18 to the production of
IFN- by STAT4-/- mice are currently
underway. Nevertheless, administration of IL-2 plus IL-18 could enhance
STAT4-independent production of IFN- by NK cells. However, although
this treatment could bypass the requirement for STAT4 for the
production of IFN-, it did not result in long-term resistance to
T. gondii. Thus, although NK cells can be stimulated
nonspecifically with IL-2 plus IL-18 to produce IFN-, in the absence
of signaling through STAT4, T cells fail to develop a protective Th1
response. In agreement with these findings, Murphy and colleagues have
shown that STAT4 is required for the TCR-mediated production of IFN-
by CD4+ T cells, suggesting that STAT4 is
important for the differentiation into Ag-specific producers of
IFN-. However, in those studies, the ability of activated
CD8+ T cells to produce IFN- following TCR
ligation was not impaired in the absence of STAT4 (42).
Similarly, recent studies have shown that following viral infection,
CD8+ T cells that produce IFN- can be
generated in the absence of IL-12 (43), and
IL-12-deficient mice immunized with low doses of a
temperature-sensitive mutant of T. gondii displayed
increased resistance to rechallenge that was dependent on
CD8+ T cells (6). These latter
studies raise the question of why, following infection with T.
gondii, STAT4-/- mice do not develop a
protective CD8+ T cell response? Previous studies
have shown that optimal CD8+ T cell responses
during toxoplasmosis, as well as other pathogens, are dependent on the
ability of CD4+ T cells to produce IL-2
(44, 45, 46). Thus, the lack of a CD4+ T
cell response in STAT4-/- mice would lead to a
defective CD8+ T cell response. If this was the
case, then it might be expected that the administration of IL-2 to
infected STAT4-/- mice would lead to the
development of a protective CD8+ T cell response.
However, administration of IL-2, alone or in combination with IL-18, to
STAT4-/- mice did not lead to enhanced T cell
production of IFN- during recall responses (our unpublished
results), suggesting that this regime could not induce an Ag-specific
Th1-type response. Further studies are required to determine whether
the absence of STAT4 leads to a direct defect in the generation of
protective CD8+ T cell responses during
toxoplasmosis or whether this is simply a consequence of defective
CD4+ T cell responses.
Another possible explanation for the lack of a protective T cell
response in the absence of STAT4 is that these mice have been reported
to be biased toward Th2 differentiation (12). Thus,
challenge of STAT4-/- mice with T.
gondii could lead to the development of a Th2-type response.
Although elevated levels of IL-4 mRNA were observed in spleens from
infected STAT4-/- mice, production of IL-4
protein was not detected during recall responses, suggesting that non-T
cells may be the source of the IL-4 mRNA (47, 48, 49).
Nevertheless, in vivo neutralization of IL-4 in
STAT4-/- mice did not lead to increased
production of IFN- or enhance resistance to T. gondii.
Furthermore, in vitro neutralization of either IL-10 or TGF-ß only
slightly enhanced production of IFN- during recall responses,
indicating that the lack of an IFN- response following infection is
not due to the development of an alternative "suppressive" pathway.
Moreover, in a single experiment, in vivo depletion of IL-10 in
STAT4-/- mice following infection of T.
gondii had no effect on parasite burden, serum IFN-, or
production of IFN- in recall responses (data not shown). These
results are consistent with studies by Stamm et al., which showed that
STAT4-/- mice did not default to a Th2 response
following infection with Leishmania major
(50).
One of the interesting points of these studies is that in
STAT4-/- mice, NK cells, and not T cells,
appear to be the major population that makes IFN- in response to
IL-2 plus IL-18. Similar results were observed when we examined the
responses of splenocytes from wild-type mice (G. Cai and C. A.
Hunter, unpublished studies). The basis for this difference is unclear,
but there are several possible explanations. Stimulation with IL-12
up-regulates receptors for IL-18 and IL-2 on T cells
(51, 52, 53), whereas NK cells express these receptors
constitutively (54, 55). Thus, in the absence of
IL-12-mediated signaling, T cells would be unable to respond to this
cytokine combination whereas NK cells could respond. However,
lymphocytes from infected STAT4-/- mice produce
more IFN- in response to IL-2 plus IL-18 than cells from uninfected
STAT4-/- mice, indicating that infection
results in an activation process that enhances the ability to respond
to IL-2 plus IL-18. The nature of this activation process, and how it
affects NK cell responses, is unknown. Nevertheless, that NK cells are
the major population of lymphocytes that are IFN--positive when
splenocytes from STAT4-/- mice are stimulated
with IL-2 plus IL-18 demonstrates that NK cells do respond more readily
to IL-2 plus IL-18 than T cells. These findings highlight a basic
question about the molecular events that allow NK cells to readily
produce IFN- in response to cytokines, whereas T cell production of
IFN- is largely restricted to Ag-specific responses.
While these studies confirm the importance of IL-12 in resistance to
T. gondii, the identification of STAT4 as a critical
transcription factor in these events adds to our knowledge of signaling
pathways involved in the development of cell-mediated immunity and
resistance to this parasite. For example, like STAT4, the NF-B
family member RelB is also required for the production of IFN-
during toxoplasmosis (56). Thus, both the JAK/STAT and
NF-B signaling pathways have critical roles in the development of
the innate and adaptive responses that lead to the production of
IFN-. Other transcription factors, such as the Tec kinases Rlk and
Itk and the NF-B family member Bcl-3, are not required for early
resistance to T. gondii, but are required for resistance
during the chronic stage of this infection (57, 58). In
contrast, the IFN consensus sequence binding protein (ICSBP)
transcription factor is required for the production of IL-12, essential
for resistance to T. gondii (59). Thus,
there are defined signaling pathways and transcription factors
associated with early and late T cell responses required for immunity
to toxoplasmosis. Understanding which signaling pathways and
transcription factors are involved in the initiation and/or maintenance
of protective immunity to this opportunistic pathogen should be useful
in the design of approaches to stimulate cell-mediated resistance in
immunocompromised patients with defects in IL-12 signaling or T cell
deficiencies (60, 61, 62).
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Acknowledgments |
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Footnotes |
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2 DNAX is supported by Schering Plough Corporation.
3 Address correspondence and reprint requests to Dr. Christopher A. Hunter, Department of Pathobiology, School of Veterinary Medicine, 3800 Spruce Street, University of Pennsylvania, Philadelphia, PA 19104-6008.
4 Abbreviations used in this paper: HuIL-2, human IL-2; iNOS, inducible NO synthase; PECs, peritoneal exudates cells; rm, recombinant murine; TLA, tachyzoites lysate Ag; RPA, RNase protection assay.
Received for publication February 24, 2000. Accepted for publication June 16, 2000.
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