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Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104
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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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Experimental murine infections with Leishmania amazonensis
provide a different view of susceptibility to leishmaniasis. This
parasite, as well as the related organism Leishmania
mexicana, induces a nonhealing infection in mouse strains that are
resistant to L. major, such as C3H, C57BL/6, and C57BL/10
(13, 14, 15, 16, 17). Although these mice have a susceptible
phenotype, the immune response cannot be readily defined as a Th2
response. For example, immune cells from C57BL/10 mice chronically
infected with L. amazonensis produce low amounts of IL-4 and
IFN-
during infection. In addition, neutralization of endogenous
IL-4 fails to promote lesion resolution, suggesting that the
susceptible phenotype is not due to the induction of an IL-4-driven Th2
response (15). These results support the hypothesis that
susceptibility to L. amazonensis is due to a failure to
mount a Th1 response, rather than the development of a Th2
response.
Th1 cell development is dependent on the presence of IL-12, as well as
the ability of T cells to respond to IL-12 (18, 19, 20, 21). The
IL-12 responsiveness of the T cell population has been shown to be
dependent on the expression of functional IL-12 receptors. The IL-12R
is composed of two subunits, termed IL-12Rß1 and IL-12Rß2,
which are both required for IL-12 signaling (22, 23, 24).
Recent studies using cells from DO11.10 TCR transgenic mice indicate
that after activation Th1 cells express both subunits of the IL-12R,
whereas Th2 cells express the IL-12Rß1 chain but not the IL-12Rß2
chain of the receptor (18, 19). In this in vitro model of
T cell activation, IL-12 and IFN- promote maintenance of IL-12R
expression, whereas IL-4 inhibits expression of the IL-12Rß2 chain of
the receptor, although IFN- can prevent the loss of the IL-12Rß2
subunit in the presence of IL-4 (18, 19, 20).
In this study, we investigated whether a deficit in IL-12 production or IL-12 responsiveness is associated with susceptibility in C3H mice to L. amazonensis. We found that cells from L. amazonensis-infected mice produced low concentrations of IL-12 when compared with those from L. major-infected mice and that they fail to express a functional IL-12R. Furthermore, we found that IL-4 deficient (IL-4-/-) mice on a C57BL/6 background had reduced expression of the IL-12Rß2 subunit and failed to heal after infection with L. amazonensis, demonstrating that during L. amazonensis infection, decreased IL-12R expression was not linked to IL-4 production. Thus, these studies suggest that the susceptibility of mice to L. amazonensis is due to the absence of a functional IL-12R on the CD4+ T cell population and that this response is mediated in an IL-4-independent manner.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C3HeB/FeJ mice (5 and 6 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free facility. IL-4-/- mice were generated on a C57BL/6 background by Dr. Manfred Kopf (25) and generously provided by Dr. Edward Pearce (Cornell University, Ithaca, NY). Mice were inoculated with 5 x 106 stationary phase promastigotes in 50 µl PBS in the hind footpad. Lesion size was monitored with a dial micrometer and expressed as the difference in footpad thickness between the uninfected foot and the infected foot.
Parasite and Ags
L. amazonensis parasites (MHOM/BR/00/LTB0016) and L. major parasites (MHOM/IL/80/Friedlin) were grown to stationary phase in Grace’s insect cell culture medium ( Technologies, Gaithersburg, MD) with 20% FBS, 2 mM glutamine, 100 U penicillin per ml, and 100 µg streptomycin per ml. Stationary phase parasites were washed three times in PBS before use in infections. Freeze-thaw leishmanial Ag (FT-ag)4 was obtained from stationary phase promastigotes washed four times in PBS and adjusted to a concentration of 109 organisms/ml. Parasite suspensions were then submitted to three freeze-thaw cycles of -70°C and 37°C and then stored at -20°C. The parasite burden of infected footpads was determined using a limiting dilution assay as described (15) and expressed as the negative log parasite titer.
IL-12 and anti-TGF-ß treatment
IL-12 was a generous gift from Genetics Institute (Cambridge,
MA) and was used at 0.2 µg in 50 µl PBS per injection in the
infected footpad. Mice received injections of 0.2 µg three times per
week. This treatment regimen promotes resistance to L. major
in BALB/c mice (Fig. 2
) and leads to more rapid healing in resistant
C57BL/6 mice (our unpublished observations). Some mice were treated
with IL-12 for the first 2 wk of infection, for the second 2 wk of
infection, or for the first 4 wk of infection. Some mice were also
treated with anti-TGF-ß (Genzyme, Cambridge, MA; a generous gift
of Dr. Chris Hunter, University of Pennsylvania, Philadelphia, PA) by
three intralesional injections (80 µg/injection) during the first 2
wk of infection as previously described (26). Control
animals received a similar amount of normal mouse Ig.
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Draining LN cells were obtained from mice at the end of the
course of infection, as indicated in the figure legends. Cells were
plated in complete tissue culture medium (Dulbecco’s minimal essential
medium containing 4.5 mg of glucose/ml, 2 mM L-glutamine,
100 U penicillin-6-potassium/ml, 100 µg streptomycin/ml, 25 mM HEPES,
and 5 x 10-5 2-ME)
as described previously (27), and the cells were incubated
in medium only or stimulated with 1 x 106
parasite equivalents of FT-ag per ml. Supernatants were harvested after
72 h, and IFN- and IL-4 levels were measured by ELISA. ELISA
assays and IL-12p40 and IFN- enzyme-linked immunospot (ELISPOT)
assays were performed as described (27).
Nuclear translocation assays
Single cell suspensions of draining LN cells from mice infected for 2 wk with L. amazonensis or L. major were harvested and plated in complete tissue culture medium at 5 x 106 cells/ml with FT-ag at 1 x 106 parasite equivalents/ml. Cells were incubated for 3 days at 37°C and then replated at 10 x 106 cells/ml with 1 ml/well and a total of 3 wells/group. The cells were then rested at 37°C for at least 1 h. Staurosporine (Sigma, St. Louis, MO) was added at 0.5 µM final concentration to one well for 1 additional h. The staurosporine-treated well and one additional well were then treated with IL-12 at a concentration of 1 ng/ml. Cells were incubated for 30 min at 37°C. Nuclear isolation was then done on all wells using a procedure based on the isoosmotic/Nonidet P-40 lysis protocol by Dyer et al. (28). Briefly, cells were washed in PBS and resuspended in 100 µl wash buffer (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris (pH 8.0), 5 mM NaF), and then 100 µl lysis buffer was added (wash buffer plus 1% Nonidet P-40). Nuclei were pelleted at 2500 rpm, and the supernatant was collected (cytoplasmic extract); the pellet of nuclei was washed once with 500 µl wash buffer and then resuspended in 40 µl low salt buffer (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl2, 0.02 M KCl, 0.2 mM EDTA, and 5 mM NaF). High salt buffer, 15 µl (same as low salt with 2.0 M KCl), was then added, and the nuclei were rocked at 4°C for 20 min. Diluent buffer, 55 µl (20 mM HEPES (pH 7.9), 25% glycerol, 0.1 mM EDTA), was then added, and the nuclei were pelleted by centrifugation at 12,000 rpm in a microfuge for 20 min and the supernatant was then frozen at -80°C. All buffers had a final concentration of 10 µg/ml leupeptin, 1.0 mM DTT, 0.5 mM PMSF, and 0.1 mM sodium orthovanadate. All chemicals were purchased from Sigma.
Nuclear and cytoplasmic extracts were then run on a 7% PAGE and transferred to nitrocellulose membrane using the semidry blotting apparatus from Bio-Rad (Hercules, CA). The membrane was blocked with 5% nonfat dry milk in PBS 0.1% Tween. Anti-Stat4 Ab (C-20, Santa Cruz Biotechnology, Santa Cruz, CA) was hybridized to the membrane overnight at 4°C at a 1:10,000 dilution. The membrane was then washed and hybridized to a secondary donkey anti-rabbit Ab conjugated to hydrogen peroxidase (Amersham, Piscataway, NJ), and the signal was detected using the Pierce Supersignal Reagents (Pierce, Rockford, IL) as directed. The membrane was then stripped with 2-ME/SDS at 57°C, rehybridized with anti-Sp1 Abs (Santa Cruz Biotechnology) as above, and then hybridized with anti-MEK1 Abs (Santa Cruz Biotechnology).
RNase protection assay (RPA)
CD4+ T cells were enriched from the LN
using positive selection at 4°C with Dynabeads (mouse CD4 (L3T4)) as
directed by the manufacturer (Dynal, Lake Success, NY). The cells
recovered were 
70% CD4+ T cells. RNA was
isolated from this cell population using RNA STAT-60 (Tel-Test B,
Friendswood, TX) as directed by the manufacturer, and 5–10 µg of
total RNA were used for RPA analysis with the mRC-3 probe from
PharMingen (San Diego, CA) using the RiboQuant kit as directed
(PharMingen). The signal density of the RPA-generated bands was
determined with the Molecular Imager system and MultiAnalyst software
from Bio-Rad. Variances in sample loading were determined by
differences in the L32 signal when compared with the uninfected control
sample. The signal densities of the IL-12Rß1 and -ß2 bands were
then multiplied by the difference to obtain a normalized value, which
was represented as a graph of arbitrary units.
Competitive quantitative PCR
Competitive quantitative PCR was conducted with the pQRS
competimer (provided by Dr. S. Reiner, University of Pennsylvania)
using the protocol by Reiner et al. (29). Briefly, total
RNA (isolated as above) was reverse transcribed with random primers and
the samples were normalized for 0.0625 pg hypoxanthine
phosphoribosyltransferase cDNA per 5 µl reverse transcription
reaction. The amount of IFN- cDNA was then determined using a
constant amount of competimer and varying dilutions of the normalized
cDNA samples.
Flow cytometry and proliferation assay
Cell division was assessed by flow cytometry using the dye
carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular
Probes, Eugene, OR) as described previously (30). Briefly,
the LN cells draining the site of infection were washed in PBS,
resuspended at 1 x 107 cells/ml, and
incubated at room temperature with an equal volume of 2.5 µM CFSE
diluted in PBS. After 5 min, 0.1 ml of FBS was added to quench the
reaction. The cells were then washed in PBS followed by a wash in
complete tissue culture medium. Cells were cultured in 96-well U-bottom
plates with and without FT-ag; a separate Ag-stimulated well was
incubated with 25 µg/ml anti-TGF-ß Ab. Anti-TGF-ß Abs were
from Genzyme (Boston, MA) and kindly provided by Dr. C. Hunter
(University of Pennsylvania). After 4 days, the cells were stimulated
with PMA (50 ng/ml), ionomycin (500 ng/ml) for 4 h in the presence
of brefeldin A. The cells were then transferred to 12- x 75-mm
polystyrene tubes and washed with 2 ml FACS buffer (PBS with 0.1% BSA
and 0.1% sodium azide). Pelleted cells were resuspended in the
residual volume of FACS buffer and nonspecific binding of Ab to FcR was
blocked with Fc block (anti-FcRIII/II (2.4G2) and rat IgG) for
10 min. Cells were then surface stained with CyChrome-labeled
anti-CD4 Ab or isotype control. Intracellular IFN- was then
detected by fixing the cells in 1% formaldehyde, washing in FACS
buffer containing 0.1% saponin, staining with PE-labeled
anti-IFN- Ab or isotype control in the residual volume, and then
washing in FACS buffer with saponin and again with FACS buffer only.
Acquisition of CD4+ lymphocytes was performed
using a FACScaliber flow cytometer (Becton Dickinson, San Jose, CA).
Proliferation and IFN- expression on the CD4+
gated population was analyzed with CellQuest software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production during L.
amazonensis infection
Our previous studies indicated that susceptibility to L.
amazonensis might be associated with the lack of a Th1 response,
rather than a dominant Th2 response (15). To determine
whether the deficiency of the Th1-type immune response and the
susceptibility of C3H mice to L. amazonensis were associated
with decreased IL-12 production, we measured the number of
IL-12-producing cells in LNs draining the site of infection with
L. amazonensis. To compare our results with those obtained
during a healing response, we also assessed the number of
IL-12-producing cells after L. major infection. As shown in
Fig. 1A, 2 wk after L.
amazonensis infection the number of IL-12-producing cells in the
LN was not significantly greater than the levels observed in uninfected
animals. In contrast, the frequency of IL-12-producing cells from
L. major-infected C3H mice was twice that seen in LNs from
uninfected mice. As a comparison, when we examined the numbers of
IL-12-producing LN cells from the susceptible BALB/c strain infected
with L. major, we found that at 2 wk there was a deficit in
IL-12 production similar to that seen after L. amazonensis
infection of C3H mice (27). The low number of
IL-12-producing cells detected during L. amazonensis
infection correlated with low numbers of IFN--producing cells, low
levels of IFN- in the recall response, and low levels of IFN-
mRNA in the draining LN cell population (Fig. 1, B–D). In
addition, the Ag-specific recall response at 2 wk postinfection
demonstrated that low levels of IL-4 were produced during L.
amazonensis and L. major infections (Fig. 1E).
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We hypothesized that the lack of IL-12 production during L.
amazonensis infection was responsible for the inability of these
mice to mount a Th1 response and heal. To test this hypothesis, we
administered IL-12 to L. amazonensis-infected mice. In
infected BALB/c mice, we found that IL-12 treatment leads to Th1 cell
development and lesion resolution, as previously reported (Fig. 2
E) (9, 10). In
contrast, IL-12 administration had little effect on the course of
lesion development in L. amazonensis-infected C3H mice (Fig. 2A). Moreover, we found that the parasite burden in L.
amazonensis-infected C3H mice remained high over the course of the
infection, with or without the administration of IL-12 (Fig. 2B). In vitro cytokine analysis of Ag-stimulated LN cells
from L. amazonensis-infected C3H mice showed that at 14 wk
postinfection LN cells produced only low amounts of IFN-, when
compared with LN cells from L. major-infected C3H mice,
despite treatment with IL-12 (Table I).
An analysis of IL-4 production from these same cells demonstrated that
LN cells after L. amazonensis and L. major
infections produced relatively low levels of IL-4 (Table I).
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C, no difference was observed in the course
of infection in control mice, and mice treated for either the first 2
or the second 2 wk of infection. We also assessed whether longer
treatment with IL-12 would promote healing. Thus, some mice were given
IL-12 for the first 4 wk of infection. No differences in lesion
progression were observed (Fig. 2D). IL-12Rß2 mRNA is not expressed in CD4+ T cells from L. amazonensis-infected C3H mice
Differential expression of the IL-12Rß2 subunit is one mechanism
involved in regulation of the response to IL-12 (18, 19).
We wanted to determine whether the inability of IL-12 to promote
healing of a L. amazonensis infection correlated with
differences in the expression of IL-12R subunits. Therefore, we
measured mRNA expression levels of the ß1 and ß2 subunits of the
IL-12R in CD4+ T cells enriched from mice
infected with L. amazonensis for 2 wk. For comparison, we
measured the IL-12R mRNA expression in cells from L.
major-infected mice. The results show that infection with either
L. amazonensis or L. major is associated with
enhanced expression of the ß1 subunit (Fig. 3). However, whereas IL-12Rß2 mRNA
levels in cells from L. major-infected mice were
substantially increased relative to cells from uninfected mice,
IL-12Rß2 levels were low in cells from L.
amazonensis-infected mice. Thus, after L. amazonensis
and L. major infection, there was a 9- to 12-fold increase
in the expression of IL-12Rß1 mRNA in CD4+-
enriched cells. In contrast, cells from mice infected with L.
amazonensis exhibited only a 2- to 3-fold increase in IL-12Rß2
mRNA, whereas cells from L. major-infected animals had a
12-fold increase in IL-12Rß2 mRNA expression relative to cells from
uninfected mice (Fig. 3).
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To determine whether differences in IL-12R mRNA expression
correlated with IL-12 responsiveness, we compared the level of IL-12
signaling in LN cells from animals infected with L. major or
L. amazonensis. Cells were harvested at 2 wk postinfection
and cultured in vitro with Ag for 3 days. The cells were then assessed
for their IL-12 signaling capacity by adding IL-12 to the cells for 30
min, followed by isolation of nuclear extracts and assessment of Stat4
levels by Western blot analysis. We found that although Stat4 is
present in cytoplasmic extracts of cells from both infections, only
cells from L. major-infected animals were able to
translocate Stat4 to the nucleus after the addition of IL-12 (Fig. 4). Inhibition of Stat4 nuclear
translocation by staurosporine, a protein kinase inhibitor, indicated
that the nuclear translocation detected in cells from L.
major-infected mice was phosphorylation dependent. The detection
of Sp1, a nuclear protein, demonstrated that nuclear proteins are
present in all extracts, and the absence of the cytoplasmic protein,
MEK1, in the nuclear extracts, demonstrates that there is no
contamination of the nuclear extracts with cytoplasmic proteins. Thus,
based on IL-12R mRNA expression and the nuclear translocation of Stat4,
we conclude that there are significant differences in expression of the
IL-12R in C3H mice infected with L. amazonensis and L.
major.
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Because we do detect some IL-4 production in the recall response
of LN cells from L.
amazonensis-infected mice, we wanted to test whether the presence
of IL-4 is responsible for the susceptible phenotype seen in L.
amazonensis-infected mice. Therefore, we compared the L.
amazonensis infection and the L. major infection in
IL-4-/- mice. As shown in
Fig. 5A,
IL-4-/- mice and the
C57BL/6 control mice were both susceptible to L. amazonensis
as assessed by lesion development. Moreover, nonhealing infections were
associated with the presence of substantial numbers of parasites within
the lesions (Fig. 5B). In contrast, both
IL-4-/- mice and C57BL/6
mice were resistant to L. major infection (data not shown).
The lack of IL-4 did not substantially change the cytokine response
seen in infected mice. Thus, when assayed at the termination of the
experiment, the Ag-specific IFN- recall responses of cells from both
IL-4-/- and C57BL/6 mice
infected with L. amazonensis were low (4.74 ± 1.86
ng/ml and 3.24 ± 2.84 ng/ml (mean ± SD), respectively),
whereas cells from L. major-infected
IL-4-/- and C57BL/6 mice
produced higher levels of IFN- (21.85 ± 14.41 and 29.90
± 12.94 ng/ml, respectively).
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levels detected in the recall
response at the termination of the experiment (data not shown), and
more importantly, exogenous IL-12 had little effect on lesion
development during L. amazonensis infection in
IL-4-/- mice (Fig. 5A) and high parasite burdens were associated with the
persistent lesions (Fig. 5B). These results are similar to
those seen in the C3H mice (Fig. 2A). IL-4-/- mice have decreased expression of the IL-12Rß2 subunit
The inhibition of the IL-12Rß2 subunit expression during
L. major infection in BALB/c mice has
been shown to be dependent on the expression of IL-4 during the early
immune response (6, 7). The correlation of the presence of
IL-4 and the inhibition of IL-12Rß2 expression is consistent with in
vitro data demonstrating a central role of IL-4 in inhibiting IL-12
responsiveness (19). Therefore, it was important to
exclude a role for IL-4 in the L. amazonensis model, which
we accomplished by comparing the increase of the IL-12Rß1 and -ß2
subunits during L. amazonensis and L. major
infection in IL-4-/- mice
(Fig. 6, A and B).
After L. major or L. amazonensis infection of
IL-4-/- mice and C57BL/6
control mice, the IL-12R mRNA expression pattern in the
CD4+ T cells was similar to that seen in C3H mice
(Fig. 3), although during both infections the overall increase in
IL-12R expression over uninfected levels was less than that detected in
C3H mice. Nevertheless, after L. amazonensis and L.
major infection, there was a similar increase in the expression of
IL-12Rß1 mRNA in CD4+-enriched cells, whereas
cells from mice infected with L. amazonensis demonstrated a
much smaller increase in IL-12Rß2 expression compared with L.
major-infected animals. Taken together, these results indicate
that IL-4 is not involved in susceptibility to L.
amazonensis and that it is not responsible for modulating the
expression of the IL-12R in vivo during L. amazonensis
infection.
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One explanation for the differences in the IFN- production, as
well as IL-12R expression, between cells from L. major- and
L. amazonensis-infected mice may relate to decreased
leishmanial specific T cell responses in mice infected with L.
amazonensis compared with L. major. To define the
CD4+ T cell response after infection, we
harvested draining LN cells 2–3 wk postinfection, stained the cells
with CFSE to uniformly label intracellular proteins, and assessed
proliferation to leishmanial Ag during 4 days of stimulation by
following the decrease in CFSE fluorescence intensity
(30). Cells were also stained for intracellular IFN-,
which allowed us to examine the capacity of the Ag-reactive cells to
produce IFN-. We found that both infections were associated with
induction of an Ag-specific CD4+ T cell
population in the draining LN of infected mice, although
CD4+ T cells from the LN of L.
major-infected animals proliferated twice as well in response to
Ag as cells from L. amazonensis-infected mice (Fig. 7). Similarly, a lower frequency of
IFN--producing cells was detected after L. amazonensis
infection when compared with cells from L. major-infected
animals. However, by normalizing for the cells that proliferate in
response to Ag, we found that the differences in the ability of the LN
cells from L. major and L. amazonensis-infected
animals to produce IFN- was not directly due to differences in the
percentage of antigenic responsive CD4+ T cells.
Thus, 34.4% of the proliferating CD4+ T cells
produce IFN- after L. major infection, whereas only 10%
of the proliferating cells from L. amazonensis-infected mice
produce IFN- (Fig. 7).
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production after L. amazonensis infection, because
TGF-ß has been shown to influence IFN- production during L.
major, L. amazonensis, and Leishmania
chagasi infection of BALB/c mice (27, 31, 32).
Neutralization of TGF-ß during the recall response led to an increase
in the percentage of CD4+ T cells from the LN of
infected animals that proliferated in response to Ag, suggesting that
it may partially contribute to the immunosuppression seen in L.
amazonensis-infected mice. However, the percentage of Ag-specific
CD4+ T cells that produce IFN- did not
increase in the presence of anti-TGF-ß (Fig. 7). Finally, to
further explore the potential role of TGF-ß in modulating IL-12
responsiveness in L. amazonensis-infected mice, we treated
mice with a neutralizing anti-TGF-ß mAb during the first 2 wk of
infection and assessed IL-12R expression and cytokine production at 2
wk. No differences in the immune responses were observed (data not
shown). Thus, we have not been able to obtain evidence for a role of
TGF-ß in the regulation of the IL-12R in L.
amazonensis-infected mice, leaving this an open issue. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data, demonstrating IL-4-independent susceptibility to L.
amazonensis infection, are in contrast to reports showing that
IL-4-/- mice are
resistant to infection with L. mexicana and some L.
major strains (33, 34, 35). Nevertheless, recent studies
show that IL-4-/- mice
remain susceptible to L. major strain LV39 (11, 35, 36) and recent experiments using IL-4 receptor 
-deficient
mice have shown that the susceptibility to L. major in the
IL-4-/- mice can be only
partially explained by the actions of IL-13 (35, 36). We
have recently found that Stat6 knockout mice still fail to heal after
infection with L. amazonensis, suggesting that IL-13 is not
contributing to susceptibility (data not shown). Thus, there appear to
be immunomodulatory factors that promote susceptibility to several
Leishmania species independent of the cytokines IL-4 and
IL-13. Whether these factors are similar in L. major
infections in BALB/c mice and L. amazonensis infections in
C3H or C57BL/6 mice will need to be determined. However, it is quite
possible that they differ, because susceptibility to L.
major in susceptible BALB/c and BALB/c
IL-4-/- mice can be
overcome with IL-12 administration, whereas this is not the case with
L. amazonensis infection (9, 10, 37).
Expression of the IL-12Rß2 chain was a critical factor in determining the phenotype of differentiating T cells and that IL-4 was a major inhibitor of IL-12Rß2 expression (18, 19, 20). Although recent studies indicate that expression of the IL-12Rß2 subunit in the presence of IL-4 does not by itself switch a population of cells to a Th1 phenotype (38, 39), the expression of a functional receptor is required for Th1 cell development in leishmaniasis (40, 41). Indeed, we and others have seen differential expression of the IL-12R during the first few days of L. major infection in BALB/c and resistant strains of mice (5, 6, 7). Decreased IL-12R expression appears to be due to enhanced production of IL-4 in BALB/c mice, because anti-IL-4 treatment of BALB/c mice led to an increase in IL-12Rß2 expression and IL-12 responsiveness (5, 6). Our current results demonstrate that during L. amazonensis infection, factors other than IL-4 can also regulate the expression of the IL-12Rß2 subunit and subsequent responsiveness to IL-12.
Recent work in the mouse has shown that IL-4-independent regulation of
the IL-12Rß2 subunit is not unique to L. amazonensis
infection. For example, administration of soluble Ags before
immunization with the same Ag in CFA suppressed cellular proliferation
and IFN- production (42). This loss of the Th1 response
was accompanied by loss of IL-12Rß2 mRNA expression on the
CD4+ T cell population and was not dependent on
the presence of IL-4 (42, 43). In another experimental
system, retroviral expression of the transcription factor GATA-3 in
DO11.10 CD4+ T cells during in vitro T cell
activation inhibited IL-12Rß2 expression and IFN- production in an
IL-4 independent manner (44). Studies are in progress to
examine whether GATA-3 is expressed during L. amazonensis
infection.
Several factors have been linked with susceptibility to cutaneous
leishmaniasis that might contribute to the regulation of IL-12R
expression after L. amazonensis infection. For example,
PGE2 production correlates with susceptibility to
L. major in BALB/c mice (45, 46), and is also
reported to inhibit IL-12R expression (47), but the role
PGE2 plays in L. amazonensis has yet
to be investigated. Another factor that might contribute to
susceptibility to L. amazonensis is IL-10, which is known to
inhibit IL-12 production (48). However, IL-10 is probably
not involved in susceptibility to L. amazonensis, because we
found that IL-10 knockout mice were as susceptible to L.
amazonensis as wild-type controls (our unpublished data).
Alternatively, the inability to respond to IL-12 and the absence of the
IL-12Rß2 subunit mRNA could be a result of insufficient T cell
activation following L. amazonensis infection. In vitro data
has demonstrated that L. amazonensis amastigotes may avoid
CD4+ T cell recognition by preventing MHC class
II presentation of parasite derived peptides (49, 50, 51). An
absence of CD4+ T cell stimulation during
L. amazonensis infection could account for the decreased
levels of both IFN- and IL-4 (Figs. 1 and 7, Table I, and (15, 52)). However, other investigators have shown that during
L. amazonensis infection, CD4+ T cells
are activated and contribute to lesion development (53).
More importantly, we found similar increases in IL-12Rß1 expression
in CD4+ T cells from L. amazonensis-
and L. major- infected mice (Figs. 3 and 6), suggesting that
there was an active immune response after L. amazonensis
infection which is associated with a specific defect in IL-12Rß2
expression. Furthermore, our studies demonstrate that the differences
in IFN- production from LN cells after L. major and
L. amazonensis infection is not primarily due to differences
in the numbers of Ag-reactive T cells, although the percentage of these
cells is less during L. amazonensis infection than during
L. major infection (Fig. 7). However, after normalizing for
the percentage of proliferating CD4+ T cells, the
results show that Ag-reactive cells from L. amazonensis-
infected animals have a decreased tendency to produce IFN- when
compared with Ag-reactive cells from L. major-infected
animals. Our data suggest that L. amazonensis infection has
a direct effect on the ability of Ag-specific
CD4+ T cells to produce IFN- and express a
functional IL-12R.
The cytokine TGF-ß has been reported to inhibit IL-12R expression,
IL-12 signaling, and IL-12 responsiveness in vitro, although the extent
of the regulation of these parameters varies with the dose of TGF-ß
and the species or strain of the immune cells that are studied
(54, 55, 56, 57). TGF-ß has also been shown to suppress
resistance to L. amazonensis infection in BALB/c mice
(31), and during the early immune response, LN cells from
L. major-infected BALB/c mice increased production of
IFN- when treated with anti-TGF-ß (27).
Similarly, IFN- production was limited by TGF-ß during the recall
response of liver-derived immune cells from L.
chagasi-infected mice (32). More recently, both
IFN- production and the percent of T cells expressing IL-12Rß1 and
-ß2 were regulated by TGF-ß during the recall response of PBMCs
isolated from tuberculosis patients (58). Interestingly,
in vivo depletion of TGF-ß during L. major infection of
BALB/c x C57BL/6 F1 mice did not alter
IFN- levels, but resistance to the parasite was accompanied by
increased inducible NO synthase expression (26). In our
system, neutralization of TGF-ß during the in vitro recall response
shows that this cytokine may limit the percentage of cells that
proliferate in response to Ag during both L. amazonensis and
L. major infections but that neutralization of TGF-ß was
unable to alter the percentage of proliferating
CD4+ T cells that produce IFN-, and we did not
observe an increase in IL-12 responsiveness when mice were treated with
anti-TGF-ß in vivo. These are negative results and should be
interpreted cautiously. Nevertheless, our results suggest that TGF-ß
may not play a central role in limiting IFN- production during
L. amazonensis infection in C3H mice. We have also found
that concomitant infection with both L. major and L.
amazonensis leads to healing of both infections, suggesting that
the factors responsible for a depressed immune response to L.
amazonensis do not actively inhibit or suppress resistance to
L. major (our unpublished observations).
The findings presented here show that L. amazonensis can evade an effective host immune response by preventing endogenous IL-12 production and preventing the development of an IL-12-responsive Ag-specific T cell population. Furthermore, in contrast to experimental L. major infections in which host genetics control susceptibility, these studies demonstrate that the lack of IL-12 responsiveness may be dictated by the pathogen, rather than the host. Indeed, human L. amazonensis infection can be associated with several forms of chronic leishmaniasis (59). Our results suggest that the ability to promote a protective immune response in patients with chronic disease may sometimes be difficult, due to a parasite-mediated IL-12 nonresponsiveness. Overall, these results indicate that during infection, the IL-4-independent regulation of the IL-12Rß2 subunit is a possible point of control for the immune response; and our studies provide an impetus to define IL-4-independent mechanisms that regulate IL-12R expression during chronic infectious diseases.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011.
3 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
4 Abbreviations used in this paper: FT-ag, freeze-thaw leishmanial Ag; LN, lymph node; ELISPOT, enzyme-linked immunospot; RPA, RNase protection assay; CFSE, carboxyfluorescein diacetate succinimidyl ester.
Received for publication October 18, 1999. Accepted for publication April 12, 2000.
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J. Ji, J. Sun, and L. Soong Impaired Expression of Inflammatory Cytokines and Chemokines at Early Stages of Infection with Leishmania amazonensis Infect. Immun., August 1, 2003; 71(8): 4278 - 4288. [Abstract] [Full Text] [PDF]
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M. Colmenares, P. E. Kima, E. Samoff, L. Soong, and D. McMahon-Pratt Perforin and Gamma Interferon Are Critical CD8+ T-Cell-Mediated Responses in Vaccine-Induced Immunity against Leishmania amazonensis Infection Infect. Immun., June 1, 2003; 71(6): 3172 - 3182. [Abstract] [Full Text] [PDF]
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F. Aguilar Torrentera, J. D. Laman, M. Van Meurs, L. Adorini, E. Muraille, and Y. Carlier Endogenous Interleukin-12 Is Critical for Controlling the Late but Not the Early Stage of Leishmania mexicana Infection in C57BL/6 Mice Infect. Immun., September 1, 2002; 70(9): 5075 - 5080. [Abstract] [Full Text] [PDF]
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R. B. Smeltz, J. Chen, R. Ehrhardt, and E. M. Shevach Role of IFN-{gamma} in Th1 Differentiation: IFN-{gamma} Regulates IL-18R{alpha} Expression by Preventing the Negative Effects of IL-4 and by Inducing/Maintaining IL-12 Receptor {beta}2 Expression J. Immunol., June 15, 2002; 168(12): 6165 - 6172. [Abstract] [Full Text] [PDF]
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D. E. Jones, M. R. Ackermann, U. Wille, C. A. Hunter, and P. Scott Early Enhanced Th1 Response after Leishmania amazonensis Infection of C57BL/6 Interleukin-10-Deficient Mice Does Not Lead to Resolution of Infection Infect. Immun., April 1, 2002; 70(4): 2151 - 2158. [Abstract] [Full Text] [PDF]
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H. Qi, V. Popov, and L. Soong Leishmania amazonensis-Dendritic Cell Interactions In Vitro and the Priming of Parasite-Specific CD4+ T Cells In Vivo J. Immunol., October 15, 2001; 167(8): 4534 - 4542. [Abstract] [Full Text] [PDF]
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