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Department of Immunology and Infectious Diseases, and
Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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than those from
STAT6+/+ mice as measured by ELISA. However,
there was no significant difference in IL-4 production between the two
groups. Semiquantitative RT-PCR of transcript levels in intact draining
lymph nodes and skin from inoculation sites confirmed a similar pattern
of cytokines in vivo as that observed in stimulated lymph node cells in
vitro. These results indicate that STAT6-mediated IL-4 signaling is
critical for progression of L. mexicana infection in
genetically susceptible mice and demonstrate that in the absence of
STAT6, susceptible mice default toward a Th1-like response and control
cutaneous L. mexicana infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many studies have indicated that control of L. major lesion
growth in genetically resistant mice such as C3H/HeN and C57BL/6
strains is associated with the expansion of the CD4+ Th1
cell subset and the production of cytokines such as IL-12, IFN-, and
IL-2 (4, 5). On the other hand, nonhealing responses in susceptible
BALB/c mice have been related to the expansion of the CD4+
Th2 cell subset and the production of cytokines such as IL-4 and IL-10
(4, 5). The disease-exacerbating role of IL-4 has been shown to be due
to its ability to inhibit macrophage leishmanicidal activity and
down-regulate the development of a Th1-like response (6, 7). This has
been evident in studies that demonstrated that genetically susceptible
mice lacking IL-4 are protected from cutaneous infection with L.
major (8) as well as L. mexicana (9, 10). However,
other studies suggest that the inability of the host to generate an
IL-12-initiated Th1-like response and produce IFN- rather than the
induction of a Th2-like response and IL-4 production may be the crucial
factor in determining susceptibility to L. major (11),
Leishmania amazonensis (12), L. mexicana (13),
and Leishmania panamensis (13).
IL-4 signals through its receptor using two pathways (14). One pathway responsible for IL-4-mediated growth involves phosphorylation of IL-4-induced phosphotyrosine substrate (also termed insulin receptor substrate-2 (IRS-2)3) or the antigenically related IRS-1 and its association with phosphatidylinositol 3-kinase (14, 15). The other pathway responsible for IL-4-mediated differentiation events involves phosphorylation of Janus kinases JAK1 and JAK3 and subsequent activation of STAT6, a signal transducer and activator of transcription (14, 16). Recent studies show that STAT6 is essential in the IL-4 signaling mechanism for development to the Th2 subset (17, 18). Furthermore, IL-13, a cytokine closely related to IL-4 in biologic function (19), has been shown to share receptor components with IL-4 (20) and also signal through the STAT6 pathway (21).
The purpose of this study was to explore the role of STAT6-mediated IL-4 signaling in cutaneous lesion formation and in the immune response following L. mexicana infection. To approach this question, we compared cutaneous lesion growth following local L. mexicana inoculation in C57BL/6 x 129/Sv mice homozygous for the disrupted STAT6 gene (STAT6-/-) with that in wild-type (STAT6+/+) counterparts of matched age and sex. In addition, we analyzed Ab profiles in sera and cytokine responses in the draining lymph nodes and skin from inoculation sites in L. mexicana-infected STAT6+/+ and STAT6-/- mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Breeding pairs of STAT6-/- (C57BL/6 x 129/Sv) mice generated by gene disruption, as described previously (22), were provided by Dr. James Ihle (St. Jude Children’s Research Hospital, Memphis, TN). The mice were bred and maintained in the facility at the Harvard School of Public Health (Boston, MA) according to the guidelines for animal research. Wild-type STAT6+/+ of the same strain combination, age, and sex were used as controls in all experiments.
Parasites
L. mexicana parasites (MYNC/BZ62/M379) obtained from Dr. James Alexander (University of Strathclyde, Glasgow, U.K.) were maintained in the shaven rumps of BALB/c mice. Amastigotes for use in experimental studies were isolated from lesions and enumerated using a Neuebauer hemocytometer (Reichert-Jung, Horsham, PA) as previously described (23).
Infection
Eight- to 12-week-old, sex-matched STAT6+/+ and STAT6-/- mice were infected with 5 x 106 amastigotes of L. mexicana by s.c. inoculation into the shaven rump. Disease progression was monitored by measuring lesion diameter at 2-wk intervals up to 12 wk postinfection. At this time, lesions from STAT6+/+ mice and inoculation sites from STAT6-/- mice were excised, and the histopathology was examined using hematoxylin and eosin stains on paraffin sections.
Preparation of soluble L. mexicana Ag
Soluble L. mexicana Ag (LmAg) for use in ELISA and T cell proliferation assays was prepared from stationary phase promastigotes of L. mexicana. Promastigotes were washed twice in ice-cold PBS and resuspended in a hypotonic buffer consisting of 10 mM Tris-HCl and 2 mM EDTA, pH 7.8. Following a 20-min incubation on ice, the promastigotes were disrupted using a sonicator and then were centrifuged for 30 min at 10,000 x g at 4°C. The protein concentration was determined using a Bradford assay (24).
Leishmania-specific ELISA
Peripheral blood was collected at 2-wk intervals from tail snips of the L. mexicana-infected STAT6+/+ and STAT6-/- mice. Blood was centrifuged at 200 x g, and serum was collected and tested for specific Ab content. The Leishmania-specific levels of the Th2-associated Ab IgG1 and the Th1-associated Ab IgG2a (25) were measured by ELISA as described previously (10). Briefly, each well of a polystyrene microtiter plate (Corning, Corning, NY) was coated with 0.5 µg of LmAg in PBS, pH 9.4, by overnight incubation at 4°C. Plates were washed with PBS and 0.05% Tween 20 (PBS/Tween 20; Sigma, St. Louis, MO) and were blocked with nonfat powder milk for 1 h at 37°C. Serially diluted serum samples (1/100 starting dilution in PBS/Tween 20) were added to the plates and incubated for 2 h at 37°C. Bound Abs were detected by incubation with either goat anti-mouse IgG1 or goat anti-mouse IgG2a horseradish peroxidase conjugate (1/5000 dilution in 25% goat serum and 75% PBS; Southern Biotechnologies, Birmingham, AL). After a further 1-h incubation at 37°C, H2O2 substrate solution (Kirkegaard & Perry, Gaithersburg, MD) was added to the plates. The reaction was stopped by 5% phosphoric acid after approximately 10 min, and the A450 was read on a microplate reader (Molecular Devices, Menlo Park, CA).
For determining Leishmania-specific and total serum levels of the Th2-associated Ab IgE (26), flat-bottom Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 0.5 µg/well of rat anti-mouse IgE mAb (clone 4818-01R; BioSource, Camarillo, CA) diluted in carbonate buffer, pH 9.6. The plates were washed four times with PBS/Tween 20 and were blocked with 10% FCS in PBS/Tween 20 incubated for 2 h at 37°C. After washing, 100 µl of diluted serum samples and purified mouse IgE standards (2 µg/ml starting concentration; clone 27-74; PharMingen, San Diego, CA) were added to the plates in duplicate and incubated overnight at 4°C. Plates were washed three times, and 0.05 µg of biotinylated anti-mouse IgE mAb (clone R35-72; PharMingen, San Diego, CA) was added per well to determine total serum IgE. Similarly, 0.01 µg of biotinylated LmAg was added per well to determine Leishmania-specific IgE. For biotinylation, LmAg (2 mg/ml) in sodium bicarbonate buffer, pH 8.5, was incubated with biotin (long arm) N-hydroxy succinimide ester (Vector, Burlingame, CA) for 2 h at room temperature. The reaction was stopped by addition of 5 µl of ethanolamine, and the complex was dialyzed overnight with PBS/0.05% sodium azide. After a 2-h incubation at 37°C with the appropriate secondary Ab, plates were incubated with 100 µl of streptavidin-peroxidase conjugate (1/1000 dilution in 10% FCS in PBS/Tween 20; Sigma) for 1 h at 37°C. Finally, the plates were washed three times, and H2O2 substrate solution (Kirkegaard & Perry) was added. Again, the reaction was stopped by 5% phosphoric acid after approximately 10 min, and the A450 was read on a microplate reader (Molecular Devices, Menlo Park, CA).
T cell proliferation assay
T cell proliferation assays were performed as previously described (10). STAT6+/+ and STAT6-/- mice were sacrificed at 12 wk postinfection, and the spleens and draining inguinal lymph nodes were removed aseptically. Single cell suspensions were prepared by gentle teasing in RPMI 1640 medium supplemented with 10% FCS (heat inactivated; HyClone, Walkersville, MD), 10,000 U/ml penicillin and 10,000 µg/ml streptomycin (Life Technologies, Grand Island, NY), and 0.05 mM ß-ME (Life Technologies). The cells were centrifuged at 200 x g for 5 min. Erythrocytes from the spleen were lysed by resuspending cells in Boyle’s solution (0.17 M Tris and 0.16 M ammonium chloride). Following two washes, the viable lymphocytes and splenocytes were counted by trypan blue exclusion with a Neuebauer hemocytometer. Lymph node cell suspensions were adjusted to 3 x 106 cells/ml, and spleen cell suspensions were adjusted to 5 x 106 cells/ml. Aliquots (100 µl) of the adjusted cell suspension were added in triplicate to the wells of sterile 96-well flat-bottom tissue culture plates (Costar, Cambridge, MA) containing 100 µl of LmAg (20 µg/ml), Con A (1 µg/ml) as a positive control, or supplemented medium as a negative control. Following incubation at 37°C for 72 h in 5% CO2, cells were pulsed with 1 µCi of [3H]thymidine and further incubated at 37°C for 12 h. Pulsed cells were harvested onto filter paper (Tomtec, Hamden, CT), and [3H]uptake was measure by liquid scintillation on a beta scintillation counter (Wallac, Gaithersburg, MD). Supernatants were collected from parallel cultures after 72 h of incubation for ELISA quantification of cytokine production (see below).
Cytokine ELISA
IL-12, IFN-, and IL-4 production by Con A- and
LmAg-stimulated cells and nonstimulated cells from L.
mexicana-infected STAT6+/+ and
STAT6-/- mice were measured by
capture ELISA as previously described (10). Maxisorp multititer plates
(Nunc) were incubated overnight at 4°C with 2 µg/ml of capture mAb
(rat anti-mouse IL-12, clone C15.6, or rat anti-mouse IFN-,
clone R4-6A2 (both from PharMingen); or rat anti-mouse IL-4, clone
1D11 (from Endogen, Cambridge, MA)) in PBS, pH 9.0. The plates were
blocked with 10% FCS in PBS, pH 7.4, for 1 h at 37°C, after
which murine recombinant standards of IL-12 (0–10 ng/ml; PharMingen),
IFN- (0–30.0 ng/ml; PharMingen), or IL-4 (0–1.5 ng/ml; Endogen)
and the cultured supernatant samples were added in duplicate and
incubated overnight at 4°C. The plates were washed three times in
PBS/Tween 20 and incubated for 1 h at 37°C with 1 µg/ml
biotinylated anti-IL-12 (clone C17.8; PharMingen), anti-IFN-
(clone XMG1.2; PharMingen), or anti-IL-4 (clone 24G2; Endogen). To
detect the biotinylated Abs, streptavidin-linked alkaline phosphatase
(1/5000 dilution in 10% FCS in PBS; PharMingen) was added after
washing and incubated for 45 min in the dark at 37°C. After a final
washing in PBS/Tween 20, 100 µl of
p-nitrophenylphosphatase substrate (Sigma) in glycine buffer
was added to each well. The A405 values of the plates were
measured on a microplate reader (Molecular Devices), and the
concentration of the samples was calculated using the standard curve.
RT-PCR
Total cellular RNA was extracted from snap-frozen lymph nodes
and skin sections from STAT6+/+ and
STAT6-/- mice using RNAzol
(Tel-Test, Friendswood, TX). The quality and the quantity of the RNA
were confirmed by running 2.0 µg on formaldehyde gels. cDNA synthesis
using 2.5 µg of RNA was performed according to the manufacturer’s
protocol (SuperAmp II system cDNA kit, Life Technologies). A
published RT-PCR technique was used to measure relative differences in
transcript levels of IL-12, IL-2, IFN-, TNF-
, IL-1ß, TGF-ß,
IL-10, and IL-4 against levels of the reference gene
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (27, 28, 29). The GeneAmp
9600 system was used to establish logarithmic ranges of PCR
amplification as a function of cycle number and cDNA dilution, and the
hot start technique was used to increase specificity (30). Reaction
conditions included 1.25 µl of cDNA, 1 µM of each 5' and 3' primer,
10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v)
gelatin, 800 µM deoxynucleotide triphosphates (200 µM of each), and
0.625 U of AmpliTaq DNA polymerase in a total volume of 25 µl.
[32P]dCTP (150,000 cpm) was included for quantitative PCR
studies. The thermal cycling parameters were denaturation at 94°C
for 15 s, annealing at 50–70°C for 20 s, and extension at
72°C for 60 s (with a final extension of 7 min at the end of all
cycles). For IFN- and IL-4 primers the touchdown PCR technique (28)
was employed, with a gradual decrease in the annealing temperature from
70 to 60°C over 10 cycles and an additional 22 or 28 cycles,
respectively, with a subsequent annealing temperature of 56°C.
Accession numbers, primer sequences, annealing temperatures, and number
of cycles were previously reported (28, 29) or were as follows: GAPDH
(M32599): sense, 5'-CAT CAA GAA GGT GGT GAA GCA GGC; antisense, 5'-TTG
TGA GGG AGA TGC TCA GTG TTG G (56°C, 23 cycles); IL-1ß (M13177):
sense, 5'-TAA TGG TGG ACC GCA ACA ACG C; antisense, 5'-TCC CAG ACA GAA
GTT GGC ATG GTA G (55°C, 28 cycles); and TGF-ß (L03799): sense,
5'-TTA CTG CTA TGG ACA AGG CAC GGG; antisense, 5'-ATT GAG GGC AAG ACG
TGT ACG AGT G (56°C, 28 cycles).
PCR products (10 µl) were separated on 1–2% agarose gels, and incorporation of [32P]dCTP into PCR product bands was quantified from dried gels on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), as previously described (27). PCR amplification with the GAPDH reference gene was performed to assess variations in cDNA or total RNA loading between samples. Normalized values were derived by dividing the mean of the triplicate 32P values measured for the transcript of interest by the mean of triplicate GAPDH values for the sample. Mean relative transcript levels per group were then determined from cDNA panels that included a negative control in which water was used for the PCR instead of cDNA.
Statistical significance
Student’s unpaired t test was used to determine the statistical significance of the values obtained. Differences in Ab endpoint titers were determined using the Mann-Whitney U prime test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Following s.c. inoculation of 5 x 106 L.
mexicana amastigotes, STAT6+/+
mice developed progressive nonhealing lesions, reaching a size of
almost 1 cm in diameter (Fig. 1
,
A and B). In sharp contrast,
STAT6-/- mice developed either no
lesions during the period of study or slight lesions that were
completely healed by 12 wk postinfection (Fig. 1, A and
B). Similar results were obtained in five separate sets of
experiments.
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At 12 wk postinfection, skin lesions from
STAT6+/+ mice demonstrated extensive
s.c. tissue destruction with diffuse inflammatory infiltrate consisting
of heavily parasitized macrophages, eosinophils, and lymphocytes (Fig. 2A). On the other hand, skin
from the inoculation sites of
STAT6-/- mice at the same time point
displayed preserved skin structure with some inflammatory foci
comprised primarily of lymphocytes and macrophages and only a few
parasites (Fig. 2B).
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Serum IgG1, IgG2a, and IgE levels were determined in L.
mexicana-infected STAT6+/+ and
STAT6-/- mice at 4, 6, 8, and 10 wk
postinfection. Similar results were found at all time points. Ab data
from serum collected at 8 wk postinfection are shown and are
representative of the results (Fig. 3).
At 8 wk postinfection, the STAT6-/-
mice produced two-log fold less IgG1 compared with wild-type
STAT6+/+ mice
(p < 0.001; Fig. 3A). Furthermore,
the STAT6-/- mice produced
significantly lower levels of total IgE (p <
0.0005; Fig. 3C) and threefold less L.
mexicana-specific IgE (p < 0.0005; Fig. 3D) compared with STAT6+/+
mice. Conversely, the STAT6-/- mice
produced nearly two-log fold higher IgG2a titers than
STAT6+/+ mice
(p < 0.002; Fig. 3B).
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, and IL-4 production in vitro by
LmAg-stimulated lymph node cells from
STAT6+/+ and
STAT6-/- mice following L.
mexicana infectionAt 12 wk postinfection, lymph node cells from L. mexicana-infected STAT6+/+ and STAT6-/- mice displayed significant LmAg-induced proliferative responses. There was no significant difference in LmAg-specific proliferation of lymph node cells between the two groups (p < 0.375), indicating that differences in cytokine levels in vitro measured by ELISA were not reflective of a higher proliferative response of one group over the other (data not shown).
The supernatants from the LmAg-stimulated lymphocyte proliferation
assays were analyzed by ELISA for the Th1-like cytokines IL-12 and
IFN-, and the Th2-like cytokine IL-4. LmAg-stimulated lymphocytes
from STAT6-/- mice produced over
fivefold higher levels of IL-12 protein than
STAT6+/+ mice
(p < 0.005; Fig. 4A). Furthermore, the
unstimulated production of IL-12 from lymphocytes of the
STAT6+/+ and
STAT6-/- mice was not significantly
different from that of the LmAg-stimulated lymphocytes of each group,
indicating comparable constitutive secretion (p
> 0.375 and p > 0.10, respectively). On the other
hand, unstimulated lymphocytes from both
STAT6+/+ and
STAT6-/- mice produced undetectable
levels of IFN-. LmAg-stimulated lymphocytes of the
STAT6-/- mice produced significantly
higher levels of IFN- than those of the similarly infected
STAT6+/+ mice
(p < 0.005; Fig. 4B). Although the
LmAg-stimulated lymph node cells from the
STAT6+/+ mice produced higher
quantities of the IL-4, on the average, than those of the
STAT6-/- mice (Fig. 4C),
the difference was not statistically significant
(p < 0.10).
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: 3,681.00 ± 1,862.00
pg/ml in STAT6+/+ mice, 15,070.00
± 1,230.00 pg/ml in STAT6-/- mice
(p < 0.025); IL-4: 97.17 ± 50.44 pg/ml
in STAT6+/+ mice, 33.88 ± 33.41
pg/ml in STAT6-/- mice
(p < 0.375)). Analysis of in vivo cytokine transcript levels in draining lymph nodes and skin from STAT6+/+ and STAT6-/- mice infected with L. mexicana
To determine the in vivo cytokine expression after cutaneous
L. mexicana infection, total RNA from the draining inguinal
lymph nodes and skin from cutaneous inoculation sites was extracted at
12 wk postinfection for semiquantitative RT-PCR analysis. Compared with
those from STAT6+/+ mice, draining
lymph nodes from STAT6-/- mice had
sixfold higher relative transcript levels of IL-12
(p = 0.009), over twofold higher relative
transcript levels of IL-2 (p = 0.049), nearly
threefold higher relative transcript levels of TNF-
(p = 0.002), and threefold higher relative
transcript levels of IFN- (p = 0.004; Fig. 5, B, C,
D, and E, respectively). However, both
STAT6+/+ and
STAT6-/- mice displayed similar
levels of IL-4 transcripts in their lymph nodes
(p = 0.500; Fig. 5F). No significant
differences were found in comparing the normalized relative transcript
levels of IL-1ß, TGF-ß, and IL-10 in the draining lymph nodes from
STAT6+/+ and
STAT6-/- mice (data not shown).
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transcripts (p = 0.007), and eightfold
higher levels of IFN- transcripts than that from
STAT6+/+ mice
(p = 0.045; Fig. 6, B, C, and
D, respectively). Although relative IL-4 transcript levels
in cells from STAT6+/+ skin lesions
appeared to be higher than in cells from
STAT6-/- skin, the difference did
not reach statistical significance when normalized against the
reference gene GAPDH (p = 0.147; Fig. 6E). In addition, there was no significant difference in the
relative gene transcript levels of IL-1ß, TGF-ß, and IL-10 in cells
from the skin of STAT6+/+ and
STAT6-/- mice (data not shown).
Hence, the local immune response in the skin was similar to the immune
response in the draining lymph nodes.
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) with no significant
changes in the Th2-like cytokine, IL-4. This default toward a Th1-like
response may have prevented lesion progression in
STAT6-/- mice. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The proposed mechanisms underlying the development of nonhealing lesions in genetically susceptible mice following L. major infection have included the presence of an IL-4-driven Th2-like response suppressing Th1 cell development (31, 32) and a failure to produce IL-12 (33, 34) and mount an IL-12-induced Th1-like response (11). Studies in IL-4-deficient mice have failed to resolve this controversy. Some investigators have shown IL-4-deficient BALB/c mice to be resistant to L. major infection (8), while others have found these mice to maintain susceptibility (35). Despite the absence of IL-4 in the former study, these mice did not default to a Th1-like response, suggesting that the lack of IL-4 and an IL-4-induced Th2-like response protected the mice (8).
On the other hand, genetically resistant mice lacking IL-12 (36, 37) or
IFN- (38) defaulted to a Th2-like response and were highly
susceptible to cutaneous L. major infection. Interestingly,
IFN-R-deficient 129/Sv/Ev mice were also susceptible to L.
major, but did not develop a Th2-like response (39). The
susceptible mice lacking IFN-R defaulted toward a Th1-like response
implying that IFN-, although important in resistance to L.
major, was not necessary for a Th1-like response and that IL-12,
instead, may be the critical cytokine responsible for Th1 cell
development.
Previous studies have clearly demonstrated that protective immunity
against the L. mexicana complex, which includes L.
mexicana and L. amazonensis strains, is ultimately
dependent upon generation of a Th1-like response and IFN- production
(10, 12, 13). Lymph node cells from genetically susceptible mice
produced little or no IFN- and low levels of IL-4 following L.
amazonensis (12) and L. mexicana infection (10, 13).
Nonetheless, IL-4-deficient C57BL/6 x 129/Sv mice develop a
Th1-like response, as measured by an increase in IFN- production,
and cure L. mexicana infection (10). Our results with
STAT6-/- mice extend earlier
findings and indicate that increased Th1-like responses in L.
mexicana-infected STAT6-/- mice
may be due to the absence of IL-4-mediated suppression of IFN-
production. The implications of IFN- in the development of a
Th1-like response and resistance to L. major are based upon
observations that have showed impaired Th1-like responses following
treatment of genetically resistant C3H/HeN mice with anti-IFN-
Abs (40). However, treatment with recombinant IFN- failed to promote
Th1 cell expansion and cure L. major infection in
susceptible BALB/c mice (41).
Recent studies have shown that IL-12 is critical for the development of
Th1-like CD4+ T cell responses following L.
major infection in resistant mice (36, 37) and that treatment of
susceptible BALB/c mice with rIL-12 cures cutaneous L. major
infection (42). Furthermore, in previously reported studies,
anti-IFN- Ab had no effect on IL-12-induced Th1 cell
differentiation in vitro (43), whereas addition of rIL-12 during
specific priming of CD4+ T cells from transgenic mice
expressing an Ag-specific TCR-ß resulted in the development of the
Th1-like phenotype (44). Previous studies using the L. major
model have indicated that genetic susceptibility of BALB/c mice to this
strain of Leishmania is due to a loss of the ability to
generate an IL-12-induced Th1-like response (11, 45). In the present
study STAT6-/- mice produced
significantly higher levels of IL-12 than
STAT6+/+ and subsequently developed a
Th1-like response. Therefore, these results indicate that diminished
levels of IL-12, and not unresponsiveness to this cytokine, may be the
mechanism responsible for susceptibility to L. mexicana.
In addition to its ability to down-regulate IL-12 and IFN-
production, IL-4 has been shown to also inhibit the production of the
inflammatory cytokines IL-1ß and TNF- from macrophages (46).
Several studies have demonstrated that TNF- plays a protective role
in immunity against L. major infection (47). For example,
lymph node cells from mice resistant to L. major produce
high levels of TNF- when stimulated in vitro, whereas cells from
susceptible strains under the same conditions produce low levels (48).
TNF- has been shown to induce parasite killing by macrophages in the
presence of IFN- by increasing nitric oxide production (49).
Recently, it was shown that mice deficient in both TNF- receptors,
p55 and p75, were able to control L. major infection, but
failed to resolve lesions (50). Although the role of the p75 TNF-
receptor was not found to be essential in L. major
infection, it was concluded that the p55 receptor may be required for
optimal macrophage activation (50). In previous studies IL-4-deficient
mice infected with L. major displayed similar levels of
TNF- transcripts as wild-type mice (8, 35). In contrast, we found
that L. mexicana-infected
STAT6-/- mice displayed
significantly higher relative levels of TNF- in the skin and lymph
nodes compared with wild-type mice. These differences are most likely
due to the ability of IL-13 to inhibit the production of TNF- using
the STAT6 pathway in IL-4-deficient mice and the inability of IL-13 to
do so in STAT6-/- mice (21, 51). The
different species of parasite used in the experiments may also play a
part in the observed differences.
IL-4 signals through two distinct pathways, one of which involves
phosphorylation of IRS-1 and IRS-2 (14, 15) and the other of which
involves JAK1 and JAK3 and subsequent activation of STAT6 (14, 16).
IRS-1 and IRS-2 are interchangeable in the former pathway and play an
important role in proliferative responses to IL-4 (15, 52). However,
IRS-2 couples more sensitively to the IL-4R system than IRS-1 (52, 53).
The IL-4R system is comprised of the IL-4R -chain that governs the
nature of the signal and the common -chain that is necessary for
generation of the signal (14). The proximal region of the IL-4R
-chain includes the tyrosine residues that when phosphorylated
signal the IRS pathway for IL-4-mediated proliferative responses (14, 15, 52). On the other hand, the more distal region of the IL-4R
-chain contains the STAT6 binding sites that are responsible for
IL-4-mediated differential events (14, 53). Previous studies have
demonstrated that although B and T cells from
STAT6-/- mice maintain the ability
to proliferate in response to IL-4, presumably through the IRS pathway,
they have lost other functions of IL-4, such as inducing Th2
development, up-regulation of MHC class II, and CD23 and Ab class
switching to the IgE isotype (17, 18, 22). The current study suggests
that the STAT6-mediated pathway for IL-4 signaling, not the
IRS-mediated pathway, plays the critical role for suppression of the
Th1-like response and consequent lesion growth following L.
mexicana infection.
IL-13 is a cytokine that exhibits similar functions as IL-4 (19, 54, 55). IL-13 also shares the IL-4R -chain and signaling pathway
through STAT6 with IL-4 (20, 56, 57). Mice lacking STAT6 have been
shown to have impaired IL-13-mediated functions, including
up-regulation of MHC class II expression and inhibition of nitric oxide
production by activated macrophages (21). However, it is unlikely that
IL-13 is important in the down-regulation of Th1-like responses, since
IL-4-deficient mice on the same genetic background have been shown to
develop Th1-like responses and control cutaneous L. mexicana
infection despite the presence of IL-13 and an intact signaling pathway
(10).
IL-4 and, to a lesser extent, IL-13 (58) have been demonstrated to enhance Ab class switching to the IgE isotype (26) by a mechanism involving STAT6 (17, 18, 22, 59). Consistent with observations made in other studies (17, 18, 22, 59), the present study showed that STAT6-/- mice infected with L. mexicana also fail to produce IgE. IgG1 production has also been shown to be regulated by, although not completely dependent upon, IL-4 and IL-4 signaling (25, 60). In this study, data showed decreased levels of IgG1 in STAT6-/- mice compared with wild-type mice. This finding extended the findings of previous studies (17, 18, 22, 59), suggesting that STAT6-mediated IL-4 signaling is also important in Ab class switching to IgG1 following L. mexicana infection.
A recent study reported that the immunosuppressive drug leflunomide
pharmacologically inhibits phosphorylation of the tyrosine residues of
JAK3 and STAT6 (61). Following treatment with leflunomide, the JAK3 and
STAT6 proteins remain inactive upon IL-4 binding to IL-4R, and STAT6
fails to bind subsequently to the IL-4-responsive genes, including
those required for class switching to IgG1 (61). Therefore, treatment
with leflunomide inhibits IgG1 production, a result similar to that in
STAT6-/- mice in this study (61).
These observations suggest that protection of L.
major-infected BALB/c mice following leflunomide pretreatment as
reported previously (62) may be due to the ability of this drug to
inhibit STAT6-mediated IL-4 signaling pathway.
Whereas the IgE and IgG1 isotypes are associated with the development of a Th2-like response, switching to the IgG2a isotype has been shown to be increased during Th1-like responses (25). Some studies previously report only a slight increase in Ab IgG2a production in STAT6-/- mice compared with that in wild-type mice (17, 18). However, this and other studies demonstrate significantly higher levels of IgG2a in serum from STAT6-/-deficient mice (22, 59). This latter finding suggests that lack of IL-4 signaling in STAT6-/- mice facilitates class switching to the Th1-associated IgG2a Ab isotype.
In conclusion, L. mexicana-infected STAT6-/- mice on a genetically susceptible C57BL/6 x 129/Sv background are protected from cutaneous lesions and produce significantly higher amounts of the Th1-like cytokines in draining lymph nodes and in skin from inoculation sites than STAT6+/+ mice. In addition, STAT6-/- mice have higher levels of the Th1-associated Ab IgG2a than the STAT6+/+ mice, which, conversely, have higher levels of the Th2-associated Abs IgG1 and IgE. These findings suggest that the STAT6-mediated IL-4 signaling is responsible for the suppression of a protective Th1-like immune response in susceptible mice following L. mexicana infection.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Abhay R. Satoskar, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
3 Abbreviations used in this paper: IRS-2, insulin receptor substrate-2; LmAg, Leishmania mexicana antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication May 8, 1998. Accepted for publication July 23, 1998.
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