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Laboratories of
*
Immunology and
Parasitology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
(IL-4R-/-), we have observed different disease
outcomes to Leishmania major infection depending on the
parasite substrain. Infection with L. major LV39 caused
progressive, nonhealing ulcers and uncontrolled parasite growth in both
IL-4-/- and IL-4R-/- mice. In contrast,
infection with L. major IR173 was partially controlled
in IL-4-/- mice but efficiently controlled in
IL-4R-/- mice. Both IL-4-/- and
IL-4R-/- mice infected with either substrain displayed
reduced Th2 responses. Surprisingly, IFN-
secretion was not
up-regulated in the mutant mice, even in the IL-4R-/-
mice, which were resistant to L. major IR173. The lack
of increased IFN- production suggests that cytokine cross-regulation
may not be operating in this model and that the effective ratios of
Th1/Th2 cytokines become more indicative of disease outcome. The
partial vs complete resistance to IR173 in IL-4-/- or
IL-4R-/- mice implies that, in addition to IL-4, IL-13
may be involved in disease progression during L. major
infection. The results with LV39 infection indicate that yet another
unidentified factor is capable of causing susceptibility to L.
major in the absence of IL-4 or IL-4
signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
Th1-associated cytokines (2, 3). Neutralization of IFN- activity in
vivo either by Ab treatment (4) or gene-targeting of IFN- (5),
IFN- receptor (6), or IL-12p40 loci (7), induces susceptibility in
normally resistant mouse strains. Similarly, a single injection of
anti-IL-4 Ab at the time of infection reverses the disease outcome
in otherwise susceptible BALB/c mice (8, 9). Because of these reliable
outcomes, infection with L. major is considered the
prototypic model to study the regulation of Th1/Th2 responses in vivo. From this model, it would be anticipated that the genetic disruption of IL-4 would consequently permit healing in BALB/c mice. Unexpectedly, we have found that genetically pure BALB/c IL-4-deficient mice remain as susceptible to L. major LV39 infection as the BALB/c wild-type controls (10). These findings imply that factors other than IL-4 contribute to disease progression in leishmaniasis and have challenged the Th1/Th2 paradigm in general.
As an extension of these studies, we have used BALB/c
IL-4R-deficient mice (11) in parallel infections with the BALB/c
IL-4-/- mice. The IL-4 receptor is a heterodimer complex
comprised of the IL-4R -chain in association with the common
(c) chain (12, 13, 14, 15). IL-13, a cytokine with similar properties to
IL-4 (16), also uses IL-4R -chain for signaling, along with the
ligand-specific chain IL-13R1 (17, 18, 19, 20). Therefore,
IL-4R-/- mice should be defective in both IL-4 and
IL-13 signaling, while IL-4-/- mice would retain IL-13
function. By comparing parallel infections in these mice, we could
indirectly assess the contribution of IL-13 in promoting susceptibility
to L. major infection. In addition, we compared the disease
progression of two different L. major substrains, LV39 and
IR173. Both parasite substrains cause cutaneous lesions in BALB/c mice
and are used by several groups studying mouse models of leishmaniasis
(1, 21, 22, 23, 24). Both LV39 and IR173 induce the TCR Vß4, V8 subset of
CD4+ T cells implicated in rapid IL-4 transcription and
driving Th2 responses (25, 26). More importantly, both infections are
prevented to progress if a single dose of IL-4 neutralizing Ab is
delivered at the time of challenge (8, 9, 10).
In these studies, we confirm the absence of any effect of IL-4 deletion
on the outcome of infection with L. major LV39, and find
that even with the IR173 substrain, the IL-4-/- mice
remain far more susceptible than C57BL/6 mice. In contrast,
IL-4R-/- mice were fully capable of resolving
IR173-driven lesions, while parasite numbers in LV39-infected
IL-4R-/- mice remained as high as in wild-type
controls. These data strongly suggest that regardless of L.
major substrain, the genetic absence of IL-4 alone does not
convert BALB/c mice to a truly resistant phenotype. The results
obtained with IL-4R-/- mice imply a previously
undiscovered role for IL-13 in susceptibility to L. major
infection, and that in addition to IL-13, another disease-promoting
factor or pathway can be induced by L. major infection that
is independent of IL-4 or IL-4R signaling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c IL-4-/- and
IL-4R-/- mice were generated as described (11, 27) and
bred under specific pathogen-free conditions in the National Institute
of Allergy and Infectious Diseases (NIAID) Animal Care Unit. Where
indicated, IL-4R+/- littermates were used as controls.
BALB/c and C57BL/6 mice were purchased from the Division of Cancer
Treatment, National Cancer Institute (Frederick, MD).
Parasite infection, lesion measurement, and parasite quantitation
L. major substrains LV39 (MRHO/SU/59/P) and IR173 (WHOM/IR/-173) promastigotes were cultured at 26°C in 199 medium supplemented with 20% HI-FCS (HyClone laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 40 mM HEPES, 0.1 mM adenine (in 50 mM HEPES), 5 mg/ml hemin (in 50% triethanolamine), and 1 mg/ml 6 biotin (M100S). Infective-stage metacyclic promastigotes were isolated from stationary culture (5–6 day old) by negative selection using peanut agglutinin (28) (Vector Laboratories, Burlingame, CA). Mice were infected with 105 purified metacyclics in the left hind footpad. Lesion size was measured with a metric caliper and calculated by subtracting the size of the contralateral noninfected footpad. Parasites from the infected footpad lesions were quantitated by homogenizing the tissue using a Teflon-coated microtissue grinder in a microfuge tube containing 200 µl of M199/S. The tissue homogenates and cell suspensions of draining lymph node cells were serially diluted in a 96-well flat-bottom microtiter plate containing biphasic medium prepared using 50 µl NNN medium with 30% defibrinated rabbit blood and overlaid with 50 µl M199/S. The number of viable parasites was determined from the reciprocal of the highest dilution at which promastigotes could be detected after 7 days of incubation at 26°C.
Culture medium
Complete RPMI (cRPMI)2 consisted of RPMI 1640 medium (Biofluids, Rockville, MD) supplemented with 10% FBS (Life Technologies, Rockville, MD), 1 mM sodium pyruvate, 2 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin.
Cytokines and Abs
Anti-IL-4 (11B11) (29) was prepared by Verax (Lebanon,
NH). Anti-CD3 mAb (2C11) was purchased from PharMingen (San Diego, CA).
Anti-IFN- (XMG-6, Rat IgG1) and anti-ß-galactocidase isotype
control (GL113, Rat IgG1) were purified from ascites, and kindly
provided by Sara Hieney (Laboratory of Parasitic Diseases, NIAID,
Bethesda, MD). Monoclonal rat ant-mouse IL-4R (M1) was purchased
from Genzyme (Cambridge, MA).
Cell stimulation
For Ag-specific responses, draining lymph node cells
were cultured at 3 x 106/ml in 24-well plates in
cRPMI containing 25 µg/ml of soluble leishmania Ag (SLA) obtained
from LV39 or IR173 promastigotes. Supernatants were collected at
72 h and assayed for cytokine production. CD4+ T cells
were isolated from draining lymph nodes by incubation with FITC-labeled
anti-CD8, anti-CD45R, and MHC anti-IAd or
anti-IAb mAbs (PharMingen), followed by negative
selection using sheep anti-fluorescein magnetic beads (PerSeptive
Biosystems, Framingham, MA). CD4-enriched cells (85–97% purity) were
plated in 200 µl at 1 x 106/ml in cRPMI on 96-well
plates coated with anti-CD3 (10 µg/ml). Supernatants were
harvested at 48 h and assayed for IL-4, IL-10, IL-13, and IFN-
by ELISA.
Cytokine and Ig ELISAs
IL-4 (Endogen, Woburn, MA), IL-13 (R&D Systems,
Minneapolis, MN), and IL-10 (PharMingen) ELISAs were performed
according to manufacturer directions. IFN- was measured in a
two-site ELISA (30, 31). IgG1 and IgG2a isotyping reagents were
purchased from Southern Biotechnology Associates (Birmingham, AL). IgE
ELISA was performed as described (32). Briefly, 96-well plates were
coated with 2 µg/ml each of two monoclonal anti-IgE Abs (02131D
from PharMingen, and AMI2501 from BioSource, Camarillo, CA). After
blocking and overnight incubation with serum samples, plates were
developed with HRP-conjugated goat anti-IgE Abs (Southern
Biotechnology Associates) followed by peroxidase substrate (Bio-Rad,
Hercules, CA).
RNA isolation and RT-PCR
Draining lymph nodes from individual mice were removed
and immediately homogenized in RNAzolB (Biotecx, Friendswood, TX), and
RNA was extracted according to manufacturer directions. A total of 5
µg of total RNA was reverse-transcribed using oligo(dT)15
primers (Novagen, Madison, WI). For cytokine transcript quantitation, a
constant amount of cDNA was PCR-amplified in the presence of serial
dilutions of the competing plasmid pMus3 (kindly provided by David
Shire, Sanofi Recherché, Labége, France). Samples were
adjusted to equivalent amounts by comparing the band intensities of
ß2-microglobulin products. Amounts of input cDNA for the
PCR reaction were 10 ng for B2µ amplification and 200 ng
for the cytokine PCR amplification. Four-fold dilutions of the pMus3
plasmid ranged from 106 to 15625 copies for B2µ, 15600 to
244 copies for IFN- and IL-12p40, and 960 to 15 copies for IL-4,
IL-13, and IL-10. The cycling conditions were 94°C for 20 s,
55°C for 20 s, and 72°C for 30 s for 33 cycles. Product
sizes ranged from 200 to 300 bp for cDNA products and 440 bp for pMus3.
Band intensities were quantitated by a Kodak (Rochester, NY) Digital
Science Analysis System. The number of molecules of each cytokine was
based on the point where PCR products were equivalent to the products
of pMus3 and were calculated graphically. The log (cDNA template/pMus3
template) was calculated and plotted vs the log (pMus3 copy number)
with the aid of a Microsoft Excel program kindly provided by Charles
Chu (North Shore University Hospital, Manhasset, NY) (33). Inducible
nitric oxide synthase (iNOS) transcripts (primers from Clontech, Palo
Alto, CA) were measured semiquantitatively by reducing the number of
cycles to 26, which was within the linear range of PCR amplification.
The relative number of iNOS transcripts was represented as net band
intensities of the PCR products after ethidium bromide staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
remain susceptible to L. major LV39
We have previously shown that BALB/c IL-4-/-
mice remain susceptible to infection with L. major LV39
(10). A plausible explanation for this unanticipated result is that a
similar cytokine, such as IL-13, is able to replace IL-4 activities by
signaling through IL-4 receptors. To test this possibility, we infected
BALB/c, BALB/c IL-4-/-, and BALB/c
IL-4R-/- mice with L. major LV39. As shown
in Fig. 1
A, lesion sizes and
disease progression in the BALB/c IL-4-/- and
IL-4R-/- mice were indistinguishable. Lesion
ulceration was also comparable among the groups, with four out of five
BALB/c, four out of five IL-4-/- and five out of five
IL-4R-/- mice developing open ulcers during the 8-wk
infection. Infection of IL-4R heterozygous littermates produced lesion
sizes comparable to wild-type BALB/c mice and did not show any effects
of gene-dosage (data not shown).
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B). In contrast, when the
IL-4-/- and IL-4R-/- mice were treated
with 11B11, both knockouts remained susceptible to LV39 infection. The
absence of an effect of 11B11 in the IL-4-/- mice
reinforces the specificity of the Ab. The result in the 11B11-treated
IL-4R-/- mice discounts the possibility that the
effect of Ab treatment in the wild-type mice might be due to macrophage
activation by immune complexes of 11B11 and IL-4. More generally, the
results reveal a disturbing incongruity regarding the effects of Ab
neutralization vs gene deletion.
IL-4-/- and IL-4R-/- mice show
differences in susceptibility to L. major substrains
While the results using LV39 confirm previous results
regarding the behavior of this parasite strain in IL-4-/-
mice, others have reported that IL-4-/- mice are
resistant to infection with another L. major strain
(MHOM/IL/81/FEBNI) (34). In addition, L. major strain IR173
was found to be controlled in IL-4-/- mice (S. Reiner and
D. Brown, personal communication). In parallel infections, LV39 was
compared with IR173 in IL-4-/-,
IL-4R-/-, BALB/c, BALB/c mice treated with 11B11, and
C57BL/6 mice (Fig. 2). In the two
experiments shown, LV39 infections in these mice were similar to those
described in Fig. 1, with slight variations among the experiments. The
lesions were nonetheless still nonhealing and ulcerative, and were
fundamentally different in size and pathology when compared with those
observed in C57BL/6 mice. In IL-4-/- mice, IR173 produced
a somewhat variable outcome. From a compilation of experiments, 6 of 21
IL-4-/- mice developed severe ulcers and were clearly as
susceptible as wild-type mice to IR173 infection. In the majority of
IL-4-/- mice infected with L. major IR173,
lesion sizes were intermediate between those of susceptible BALB/c and
resistant C57BL/6 mice, and mimicked the outcome seen in 11B11-treated
BALB/c mice. Thus, in these parallel infection studies, the
inconsistencies in the published results are reconciled by differences
clearly related to the parasite substrain used for infection. It needs
to be emphasized, however, that the IL-4-/- mice infected
with IR173 still do not display the resistant phenotype of C57BL/6
mice.
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-/- mice infected with L. major IR173
appeared to contain lesion development better than both the
IL-4-/- and the 11B11-treated BALB/c mice, and in some
cases even better than C57BL/6 mice. In no case was ulceration observed
in the IR173-infected IL-4R-/- mice (0/15). Based on
their comparison with C57BL/6 mice, BALB/c IL-4R-/-
mice show a genuinely resistant phenotype to IR173.
To more accurately assess disease progression and initiation of
parasite growth control, parasite numbers were quantitated from footpad
lesions and draining lymph nodes at two time points after infection
(Fig. 3). In the LV39 infections,
parasite growth was clearly not contained in either
IL-4-/- and IL-4R-/- mice; in experiment
1, parasite concentrations in the footpad tissue were even greater than
in BALB/c wild-type controls. The only LV39-infected groups that gave
an indication that parasites were being contained within the
inoculation site were the 11B11-treated BALB/c mice, which showed a
log-fold reduction in parasite load between days 38 and 56, and the
C57BL/6 mice, which showed a 1000-fold reduction. In contrast, all of
the groups infected with IR173, with the exception of the BALB/c
controls, had significant reductions in tissue parasite concentrations
when these time points were compared. An 
100-fold reduction was
observed in each of the mutant strains and in the 11B11-treated BALB/c
mice in experiment 1. The presumed killing of the parasite was most
impressive in the C57BL/6, which showed a 10,000-fold reduction in
amastigote concentration in the footpad. It should be noted that the
IL-4R-/- group infected with IR173 had significantly
reduced parasite numbers in the lesions at the earlier time point in
both experiments, suggesting that in these mice, control of parasite
load was initiated much earlier or that the numbers of parasites never
approached those observed in even the resistant C57BL/6 mice.
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, right panel), suggesting that the
kinetics and/or mechanisms of parasite control differ in these sites.
In addition, when the lymph node parasite burdens of IR173-infected
IL-4-/- mice were compared with wild-type mice, no
difference was seen at either time point examined, and in each case,
the parasite numbers in this metastatic site were 100- to 1000-fold
greater than in the resistant C57BL/6 mice. Furthermore, even the
IL-4R-/- mice infected with IR173 had 50- to 250-fold
higher parasite loads in their draining nodes than the C57BL/6 mice.
Thus, by the criteria of parasite dissemination and growth within
primary draining lymph node, the effect of both the IL-4 and IL-4R
deletions are minimized.
Th2 responses are defective in IL-4-/- and
IL-4R-/- mice
We next asked whether T cell cytokine profiles would predict the
outcomes of LV39 and IR173 infections, particularly in the
IL-4R-/- group, which showed the most dramatic
acquisition of a resistant phenotype. Cytokine levels were measured in
the supernatants after Ag stimulation of total lymph node cells and
from anti-CD3-stimulated CD4+ T cells obtained from
draining lymph nodes 41 days postinfection (Fig. 4). Within each individual group, LV39
and IR173 infections induced similar amounts of the cytokines shown,
with no consistent increases of one particular cytokine by either
L. major substrain. As anticipated, a general Th2 defect in
IL-4R-/- and IL-4-/- mice was evidenced
by 2- to 10-fold lower IL-4, IL-13, and IL-10 secretion from
CD4+ cells as compared with the BALB/c controls (Fig. 4B). This was paralleled in the SLA-stimulated cultures,
with the exception of unexpectedly high amounts of IL-4 in the
IL-4R-/- supernatants (Fig. 4A). These
levels of IL-4 are most likely attributable to a lack of IL-4
consumption by the IL-4R-/- cells and the subsequent
accumulation of IL-4 during the 72-h culture. The addition of
anti-IL-4R mAb (M1) to parallel cultures increased the levels of
IL-4 >6-fold in BALB/c mice (from 406 pg/ml to 2755 pg/ml), but had
little effect on IL-4R-/- cultures (538 pg/ml to 505
pg/ml with M1). The lack of such IL-4 accumulation in the
CD4+-purified cultures may be due to the presence of other
IL-4-producing cells, such as mast cells or basophils, in the SLA
cultures.
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secretion from CD4+ T cells would be predicted to
be increased in the IL-4-/- and IL-4R-/-
mice. While responses to SLA and anti-CD3 stimulated
CD4+ cells from C57BL/6 mice produced significant amounts
of IFN-, none of the BALB/c mutants, including the
IL-4R-/- group that controlled infection with
IR173, produced levels of IFN- above those observed in the BALB/c
control mice.
To directly measure cytokine levels without ex vivo stimulation, mRNA
was prepared from draining lymph nodes 56 days after infection and
analyzed for IFN-, IL-12p40, IL-4, IL-13, and IL-10 transcripts by
quantitative RT-PCR using the competitive plasmid pMus3. Transcripts
for the iNOS were measured by semiquantitative RT-PCR (Fig. 5). Comparing the heterozygous controls
(IL-4R+/-) and IL-4R-/- in the two
parasite infections, we found no apparent up-regulation of any specific
cytokine transcript measured between the LV39 or IR173 infections.
IFN- transcripts showed a slight increase (1.2- to 1.5-fold) in the
IL-4R-/- mice. The number of IL-12p40 molecules was
increased 2-fold in the IL-4R-/- in both LV39 and
IR173 infections. As predicted from the CD4+ cytokine
data, the number of IL-4 and IL-13 transcripts were reduced 2- to
3-fold in the IL-4R-/- infections. Unexpectedly, IL-10
transcripts were not altered in the knockouts, even though the amount
of IL-10 secreted from CD4+ cells was decreased (Fig. 4).
This may be due to the production of IL-10 by non-CD4+,
such as macrophages or B cells (35).
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). This may be due to the lack of restraint in parasite
growth in the lymph node, as opposed to the footpad, in the
IR173-infected IL-4R-/- mice (Fig. 3). This is
especially apparent when the levels of parasites in the lymph nodes are
compared with a genetically resistant C57BL/6 strain.
Anti-IFN- abrogates resistance in IR173-infected
IL-4R-/- mice
Since there was no apparent up-regulation of IFN- in
the IL-4R-/- mice infected with IR173, we wanted to
determine a role for IFN- in their resistance. Wild-type and
IL-4R-/- mice were treated with anti-IFN- Ab at
the time of infection with IR173. As shown in Fig. 6, the IL-4R-/- mutants
were exquisitely sensitive to anti-IFN- treatment, as reflected
by increased footpad swelling. This result suggests that while
decreased Th2 cytokines do not result in an increase in the amount of
IFN- that is released by CD4+ T cells in response to
infection with IR173, the complete absence of IL-4 may permit even low
levels of IFN- to mediate potent effector activities.
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Because cytokine quantitation after ex vivo stimulation or
mRNA measurements from an isolated tissue may not be indicative of
imbalanced Th1/Th2 ratios occurring in vivo, we measured total serum
IgE, IgG1, and IgG2a 38 days after infection (Fig. 7). IL-4 promotes isotype switching to
IgE and IgG1 isotypes, while IgG2a levels indicate IFN- activity in
vivo (36). The isotype patterns were comparable between LV39 and IR173
infections, and did not reveal a preferential IFN- response in the
IR173-infected IL-4-/- or IL-4R-/- mice.
Total serum IgE levels in infected IL-4-/- and
IL-4R-/- mice were 1000-fold lower than in the BALB/c
control mice. Total IgE was only reduced 10-fold in the 11B11-treated
BALB/c mice. Although IL-4 has been shown to be critical for induction
of IgE transcripts, using a sensitive ELISA (detection limit 1.5
ng/ml), we were able to detect as much as 80 ng/ml of IgE in
IL-4-/- or IL-4R-/- mice after
infection. Infections with Plasmodium spp. (37),
retroviruses (MAIDS) (38), Schistosoma mansoni (39), and
Nippostrongylus brasiliensis (11) have also been reported to
induce serum IgE or productive IgE transcripts in IL-4-/-
mice.
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-/- groups, and 2-fold less in the 11B11
groups, compared with infected BALB/c controls. Values were not
significantly different between the LV39 or IR173 infections in the
mutant mice. Total serum IgG2a levels, however, were increased 3- to
9-fold in the IL-4-/- and IL-4R-/- mice
compared with BALB/c mice at 38 days after infection, indicating that
there was an imbalance toward the Th1 response at this time point. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
receiving either one of the two L. major substrains, LV39 or
IR173. The results reconcile the conflicting findings of previous
independent studies, in which the IL-4 deletion was found to have
either no effect (LV39) or an appreciable inhibitory effect (IR173) on
the growth of L. major in BALB/c mice, and clearly
attributes these differences to parasite-related factors. More careful
analysis, however, indicates that even for the IR173 substrain, the
complete absence of IL-4 did not convert BALB/c mice into a healing
phenotype resembling that seen in a genetically resistant mouse strain,
C57BL/6 (Fig. 2). We observed only a modest reduction of parasites in
the footpad, and parasite numbers within the draining lymph node
remained as high as in the wild type mice (Fig. 3).
In striking contrast, IL-4R-/- mice were highly
resistant to the IR173 substrain; they displayed a genuine healing
phenotype with respect to the size and progression of their footpad
lesions and parasite burden in the lesion. This finding is novel and
suggests that, in addition to IL-4, IL-13 may be responsible for
promoting nonhealing infections in BALB/c mice. There is mounting
evidence that IL-13 plays an active role in the immune response to
other infections, including N. brasiliensis (40)
Trichuris muris (41), and S. mansoni (42) (D.
Jankovic, personal communication).
However, this finding must also be qualified by the fact that for
the LV39 substrain, the deletion of the IL-4R gene had relatively
little effect on parasite growth, either in the inoculation site or
in the draining lymph node. Furthermore, even for the IR173 substrain,
the parasite load within the draining lymph node was not nearly so well
contained as in C57BL/6 mice. In addition to IL-13R1, another
receptor for IL-13 has been recently cloned, IL-13R2, which binds
IL-13 with a high affinity independently of IL-4R (43, 44). Although
information regarding the nature of IL-13R2 function in vivo is
lacking, chronic stimulations such as L. major infection may
permit IL-13 to function through IL-13R2 in the absence of IL-4R.
If so, then differential expression of IL-13R2 in inflammatory
macrophages infected with LV39 compared with IR173 might explain the
different outcomes of infection in these mutant mice. Alternatively,
additional factor(s) other than IL-4 and/or IL-13 must be present in
LV39-infected IL-4-/- and IL-4R-/-
BALB/c mice that distinguish them from IR173-infected mice in the
manner in which the host response either promotes parasite growth or
mediates parasite killing. This factor is most likely produced by
CD4+ T cells, since BALB/c IL-4-/- mice
infected with LV39 will control infection if transiently depleted of
CD4+ T cells in vivo (45).
A striking observation in our studies was that the healing or
nonhealing phenotypes exhibited by the knockout mice were not
correlated with the levels of IFN- produced during infection,
assayed as either concentrations of secreted IFN- in culture
supernatants of draining lymph node cells following stimulation with Ag
or anti-CD3, or levels of draining lymph node IFN- mRNA. We also
measured IFN- production by cytoplasmic staining after stimulation
with PMA and ionomycin (data not shown). An increased frequency of
IFN--producing CD4+ cells at 41 days after infection was
only observed in the C57BL/6 groups infected with LV39 or IR173, with
5-fold more IFN--staining cells over noninfected C57BL/6 mice.
Interestingly, the frequency of CD4+ IFN--producing
cells remained at levels near the BALB/c controls, even in the
IL-4R-/- group infected with IR173 that have healed
(data not shown).
It should be noted that our studies have not addressed the possibility
that a much higher frequency of IFN--producing cells might have been
found within the inoculation site in the IR173-resistant
IL-4R-/- mice, or from CD8+ T cells (46).
Nonetheless, from the models of reciprocal regulation between IFN-
and IL-4 (47), we anticipated that a lack of IL-4 would lead to a
vigorous IFN- response. What we observed was that limiting IL-4
signaling had little or no effect on the amount of IFN- produced by
CD4+ T lymphocytes. Remarkably, the
IL-4R-/- mice that healed their footpad infections
with IR173 made far less IFN- than the C57BL/6 mice, and even less
than the wild-type controls. Furthermore, there was no detectable
difference in these assays in the IFN- responses between
LV39-susceptible and IR173-resistant IL-4R-/- mice. An
absence of apparent cross talk between Th1 and Th2 cells in the
L. major mouse models has been noted by others. In the
studies by Kopf et al. (34), the ability of IL-4-/-
BALB/c mice to control infection with their L. major
substrain was also not associated with an early increase in the IFN-
response. C3H mice expressing an IL-4 transgene continued to produce
high levels of IFN- despite their inability to control infection
(48). Recombinant parasite Ags presented in immune-stimulating
complexes generated a strong, but mixed, Th1 and Th2 response that was
not protective, despite the activation of large numbers of
CD4+ T cells secreting IFN- (49). In each of these
studies it was concluded that IL-4 is the key player in L.
major susceptibility, and that its role in exacerbating infection
is not necessarily related to it capacity to down-regulate a
host-protective Th1 response. Our studies generally support this
conclusion, with the proviso that in addition to IL-4, other Th2
cytokines can promote infection without affecting the magnitude of
the IFN- response. These cytokines could theoretically act by
interfering with macrophage activation or by recruiting mononuclear
phagocytes to the inoculation site, supplying a reservoir of host cells
that are permissive to parasite growth.
While the IFN- levels were not up-regulated in the draining lymph
node of IL-4-/- or IL-4R-/- mice, the
residual levels were critical insofar as the IR173-resistant
IL-4R-/- mice failed to control infection when treated
with anti-IFN- Abs (Fig. 7). The absence or diminution of IL-4
and other type 2 cytokines means that the ratio of IFN- to type 2
cytokines was nonetheless skewed in favor of a Th1 response, and even
low levels of IFN- became sufficient to mediate control of parasite
growth within the inoculation site. The absence of elevated IFN-
responses in the mutant mice supports the argument proposed by Murphy
and colleagues (50) that the defective Th1 responses in BALB/c mice can
be explained, at least in part, by IL-4-independent, cell-intrinsic
differences, specifically the inability to maintain IL-12Rß2
expression during Th phenotype development. We extend this argument by
suggesting, based on the results with the LV39 substrain, that in the
face of intrinsically low levels of IFN-, an effect of other
disease-promoting Th2 cytokines can be more easily revealed.
While it is difficult to resolve the inconsistency between treatment with IL-4 Ab vs the gene knockout in the mice infected with LV39, the explanation that we favor is that in the knockout animals, compensatory cytokines are induced to replace the critical functions that even low levels of IL-4 might mediate during the development of innate and adaptive immune responses. These compensatory mechanisms would not be expected to operate in 11B11-treated mice, which produce residual amounts of bioactive IL-4, as evidenced by the fact that IgE and IgG1 class switching was not as ablated as in the knockout mice.
An alternative explanation is that the residual amounts of IL-4 in the
11B11-treated mice enhance an effector activity that is especially
critical to the control of lesions in mice infected with LV39. Previous
reports that small amounts of IL-4 may be necessary for development of
Th1 responses in vivo (51) or that IL-4 can synergize with IFN- to
activate macrophages for leishmanicidal activity in vitro (52, 53) and
can promote localized healing when administered intralesionally with
adjuvant (54), support a protective role for the residual amounts of
IL-4 in the 11B11-treated mice. The studies by Kamogawa et al. (55)
also suggest that IL-4 is required for the development of T cells that
can eventually produce IFN-. While the results of the
CD4+ depletion argue that LV39-infected IL-4 knockouts can
control infection in the absence of any IL-4 (45), it is possible that
the global depletion of Th2 cytokines afforded by anti-CD4
treatment might sustain a high enough Th1/Th2 ratio to control
infection without the contribution of any putative IL-4-dependent
effector mechanism.
In summary, we have found alternative pathways for L. major
disease exacerbation that are independent of IL-4 or IL-4R
signaling. These alternative pathways exert their effects without any
obvious influence on the levels of IFN- produced, and are in all
likelihood revealed as compensatory pathways in mice with genetic
deficiencies in conventional IL-4-mediated mechanisms of disease
exacerbation. The identification of such a factor and the
mechanisms of induction by one parasite vs another will provide a
better understanding of the full range of L. major
escape mechanisms and possible interventions.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: cRPMI, complete RPMI; SLA, soluble leishmania Ag; iNOS, inducible nitric oxide synthase.
Received for publication December 9, 1998. Accepted for publication February 25, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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