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World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland;
Institute of Microbiology, University of Lausanne, Lausanne, Switzerland,
Departments of Medicine and Microbiology/Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, CA 94143; and
§
Department of Dermatology, Ludwig Maximilian University, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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8 CD4+ T cells of
BALB/c mice within 1 day of infection. Here we demonstrate that
treatment of Vß4-deficient BALB/c mice with IL-4 during the first
64 h after infection instructs Th2 cell development and
susceptibility to infection. The demonstrated inability of IL-4 to
reverse the resistant phenotype of BALB/c mice treated with
anti-CD4 mAb the day before infection suggest that these effects of
IL-4 require its interaction with CD4+ T cells. In contrast
to draining lymph node cells from BALB/c mice, cells from
Vß4-deficient BALB/c mice remain responsive to IL-12 following
infection. Strikingly, administration of IL-4 to Vß4-deficient BALB/c
mice renders their lymph node cells unresponsive to IL-12 by
down-regulating IL-12R ß2-chain expression. This study directly
demonstrates that in BALB/c mice IL-4 is necessary and sufficient to
initiate the molecular events steering Th2 cell maturation and
susceptibility to L. major. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among the several factors possibly influencing the development of
functionally polarized CD4+ Th effector
responses, cytokines are the most important stimulus. Results from
studies performed in vitro using naive CD4+ T
cells from TCR ß transgenic mice have clearly implicated IL-12 in
the differentiation of Th1 cells (3, 4, 5) and IL-4 in the
differentiation of Th2 cells (6, 7, 8).
The importance of IL-12 for Th1 cell development has been substantiated
in vivo using the murine model of infection with L.
major (9, 10). Several experimental results also
strongly support a requisite role of IL-4 in mediating both Th2 cell
differentiation and susceptibility to L. major in BALB/c
mice (11, 12). In this context, prior studies from this
laboratory have shown that susceptible BALB/c mice, in contrast to mice
from resistant strains, exhibited a burst of IL-4 mRNA expression in
CD4+ T cells from the draining lymph nodes (LN)
within 1 day after infection with L. major
(13). This early burst of IL-4 expression occurred in a
restricted population of Vß4-V8 CD4+ T cells
after cognate recognition of a single epitope of the
Leishmania homologue
(LACK)5
(14) of mammalian RACK1 (15). BALB/c mice
rendered deficient in Vß4+ T cells as a result
of neonatal infection with the mouse mammary tumor virus (MMTV(SIM)),
encoding a superantigen leading to systemic deletion of
CD4+ T cells expressing the Vß4 TCR chain
(16), did not generate early IL-4 transcripts in
CD4+ T cells within the first day of infection,
developed a Th1 response, and were resistant to infection
(15). These results combined with those showing that
BALB/c mice tolerant to LACK as a result of the transgenic expression
of this molecule in the thymus were resistant to infection
(17) clearly indicate that Vß4-V8
CD4+ LACK-reactive cells are required for
subsequent Th2 cell development and susceptibility to L.
major in BALB/c mice. The critical role of these LACK-reactive
cells in the development of aberrant Th2 response in BALB/c mice after
infection with L. major was interpreted as a consequence of
the IL-4 they rapidly produce during the initial stage of infection.
The role of IL-4 in Th2 cell maturation in vivo has also been
substantiated in other model systems where the supply of exogenous IL-4
at the time of priming in vivo has clearly been shown to induce
maturation of specific CD4+ T cells toward the
Th2 phenotype (18, 19).
Recent results challenged a requisite role of IL-4 in Th2 cell maturation and disease progression by showing that the genetic absence of IL-4 did not modify the susceptible phenotype of BALB/c mice (20). However the possibility exists that in BALB/c mice with a disruption of the IL-4 gene, the critical function of IL-4 in Th2 cell maturation and susceptibility to infection is substituted by an alternative pathway not operational in normal BALB/c mice.
Therefore, we initiated studies to directly assess whether IL-4 alone, during the initial phase of infection, was sufficient to instruct Th2 cell development and susceptibility to L. major in BALB/c mice. In this report we show that treatment of otherwise resistant Vß4-deficient BALB/c mice with rIL-4 during the first 2.5 days of infection is sufficient to render them fully susceptible to infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c, C57BL/6, and B10.D2 mice were purchased from IFFA Credo (St. Germain sur l’Arbresle, France). BALB/c mice infected with either MMTV(SIM) (BALB/c-SIM) or MMTV(SW) (BALB/c-SW), were obtained, as described, from litters nursed within 24 h after birth on MMTV(SIM)- or MMTV(SW)-infected BALB/c mothers, respectively (15). This neonatal exposure to the retrovirus results in a progressive decline in the numbers of designated Vß+ CD4+ T cells, which is almost completed by 20 wk of age (16, 21). Depletion was confirmed by flow cytometric analysis using the appropriate anti-TCR Vß-chain mAbs with PBL obtained from mice at the initiation of the experiments. All mice neonatally exposed to MMTV and control mice were used at the age of at least 20 wk.
Parasites and treatment of mice
L. major LV 39 (MRHO/Sv/59/P strain) were maintained in vivo and grown in vitro as previously described (22). For infection, mice were injected s.c. in one hind footpad with 3 x 106 stationary phase L. major promastigotes in a volume of 50 µl. The footpad lesions were measured with a vernier caliper and compared with the thickness of the uninfected footpad. Starting the day of infection with L. major, some mice were injected i.p. eight times with 0.1 µg of murine rIL-4 in PBS-1% normal mouse serum (NMS) every 8 h. Highly purified, murine IL-4 was a gift from Dr. A. Levine (Monsanto, St. Louis, MO) (23). Before injection, the biological activity of the rIL-4 (2 x 106 U/mg) was confirmed with the IL-4-dependent cell line CTLL-44 (24). Transient depletion of CD4+ T cells was accomplished by a single injection of 600 µg of anti-CD4 mAb GK 1.5 i.p. 2 days before parasite inoculation (25).
Abs, FACS analysis, and cell sorting
FITC-conjugated anti-Vß4 (KT4–10) and anti-Vß6 (44-22.1) TCR chains mAbs and PE-conjugated L3T4 (Becton Dickinson, Mountain View, CA) were used to monitor the proportion of cells expressing the corresponding marker using the FACScan and the LYSIS II program (Becton Dickinson).
CD4+ T cells were purified from the draining LN using magnetic activated cell sorting (Miltenyi Biotech, Bergischgladbach, Germany) according to the manufacturer’s directions as previously described (26).
RNA extraction and semiquantitative PCR
Total RNA was isolated from popliteal LN cells and from
CD4+ T cells purified from LN as previously
described (13). Briefly, first-strand cDNA synthesis was
performed on total RNA using a first-strand cDNA synthesis kit
(Pharmacia, Uppsala, Sweden). The semiquantitative PCR developed by
Reiner et al. (27) was performed using the competitor
construct containing sequences for multiple cytokines, the primers for
hypoxanthine guanine phosphoribosyltransferase (HPRT), IL-4 and IFN-
and the PCR conditions described by the authors. The first-strand cDNA
was used directly as a template in the presence of 5-fold serial
dilutions of the competitor. After separation of the PCR products by
electrophoresis in agarose gel containing ethidium bromide, the ratio
of the relative concentration of the IL-4 or IFN- gene to the
relative concentration of HPRT was calculated (27).
Results are expressed as the fold increases in RNA expression in mice
infected with L. major compared with that in
noninfected mice.
Expression of the IL-12R ß1- and ß2-chain mRNA was monitored using a qualitative PCR assay as previously described (28). In brief, all samples were normalized with respect to their HPRT content as determined by the method described above (27). Using normalized samples and control plasmids containing the respective cDNA (29, 30), IL-12R ß1- and ß2-specific PCR reactions were performed using the conditions described previously (28) and the following primers: ß1 3', 5'-ATT CTT GGG GTT CTT GGA GGC-3'; ß1 5', 5'-GCC AAG ATT AAG TTC TTG GTG-3'; ß2 3', 5'-GCG TCG GTA CTG AAT TTC GCA-3'; and ß2 5', 5'-GGG AGT ACA TAG TGG AAT GGA-3'. PCR products were visualized on 1% agarose gels stained with ethidium bromide and scored for expression or lack of expression. All reactions were repeated two or three times to ensure reproducibility.
Lymphocyte cultures and detection of cytokines in supernatants
Popliteal LN cells (5 x 106) were
stimulated with UV-irradiated L. major promastigotes (1
x 106) in a final volume of 1 ml. In one
experiment, exogenous rIL-12 (10 ng/ml; provided by Hoffmann-La Roche,
Nutley, NJ) was also added. Cells were cultured in DMEM supplemented
with 5% heat-inactivated FCS, L-glutamine (216
mg/ml), 5 x 10-5 M 2ß-ME, and 10 mM
HEPES in an atmosphere of 7% CO2. Culture
supernatants were collected after 72 h and stored at -20°C
until use. IFN- was measured in supernatants by ELISA as previously
described (31). Mouse rIFN- (supernatant of L1210 cells
transfected with the murine IFN- gene, gift from Y. Wanabe, Kyoto
University, Kyoto, Japan) was used as the standard. The limit of
detection of the assay was 10 U/ml. IL-4 was measured by a bioassay
using the CTLL-44 cell line (gift from P. Erb, University of Basel,
Basel, Switzerland) as described previously (24).
Recombinant murine IL-4 expressed in X63Ag-653 cells (gift from F.
Melchers, Basel Institute of Immunology, Basel, Switzerland) was used
as the standard. The limit of detection of the assay was 20 pg/ml.
Statistical analysis
Data were analyzed statistically by Student’s t test for unpaired data.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice rendered Vß4 CD4+ T cell
deficient by prior infection with MMTV(SIM) are incapable of generating
the IL-4 transcriptional burst normally occurring in BALB/c mice within
the first day of infection with L. major (15).
In contrast to control BALB/c or BALB/c mice rendered Vß6
CD4+ T cell deficient as a result of infection
with MMTV(SW), Vß4-deficient mice subsequently develop a Th1 response
in the CD4+ T cell population and express a
resistant phenotype (15). Therefore, experiments were
designed to determine whether the administration of exogenous IL-4 to
otherwise resistant Vß4-deficient BALB/c mice during the initial
period of infection with L. major was capable of reversing
their resistant phenotype. The timing of administration of exogenous
IL-4 to Vß4-deficient BALB/c mice used in the present study was
chosen based on previous results showing that neutralizing
anti-IL-4 Ab was no longer able to redirect Th1 cell development in
intact BALB/c mice if administered 72 h after the initiation of
infection (26). The results in Fig. 1
confirm that BALB/c mice made Vß4
CD4+ T cell deficient (BALB/c-SIM), but not
control BALB/c or Vß6-deficient (BALB/c-SW) mice, were resistant to
infection with L. major. Administration of IL-4 only during
the first 64 h after infection to Vß4-deficient BALB/c mice
(BALB/c-SIM) fully reversed their resistant phenotype, because these
mice developed progressive lesions not substantially different from
those in concurrently infected BALB/c or Vß6
CD4+ T cell-deficient control mice (Fig. 1).
Furthermore, the numbers of viable parasites recovered after culture in
vitro of footpad tissue under limiting dilution condition
(32) confirmed that although parasite growth was
controlled in Vß4 CD4+ T cell-deficient BALB/c
mice comparably to that in resistant C57BL/6 mice (
200
parasites/footpad) 6 wk after infection, footpad lesions of Vß4
CD4+ T cell-deficient BALB/c mice treated with
IL-4 during the first 64 h of infection contained numbers of
parasites (108) similar to those found in
control BALB/c mice.
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Treatment with IL-4 during the initial stage of infection with L. major redirects Th2 cell development in Vß4 CD4+ T cell-deficient BALB/c mice
The development of an aberrant polarized Th2 response following infection with L. major underlies the exquisite susceptibility of BALB/c mice (2). Prior studies from this laboratory have shown that BALB/c mice rendered Vß4-deficient developed a Th1 response to infection with L. major that closely resembles that of genetically resistant C57BL/6 mice (15). Therefore, experiments were designed to correlate the ability of exogenous IL-4, given only during the initial phase of infection, to reverse the resistant phenotype of Vß4-deficient BALB/c mice with the ultimate development of polarized Th2 response weeks later.
Together with resistant C57BL/6 mice, groups of BALB/c, BALB/c-SIM, and
BALB/c-SW mice were inoculated with L. major in the hind
footpads. An additional group of BALB/c-SIM mice was treated with rIL-4
during the initial 2.5 days of infection. After 6 wk, a time when
polarized Th differentiation can be demonstrated, mice were sacrificed,
and their draining LN were harvested for cytokine mRNA and protein
assays. Results in Fig. 2 confirm that
Vß4-deficient BALB/c-SIM mice developed a Th1 response similar to
that seen in genetically resistant C57BL/6 mice, with almost
undetectable levels of IL-4 transcripts. In sharp contrast, compared
with uninfected control mice, BALB/c and Vß6-deficient BALB/c-SW mice
had an important increase in the amounts of IL-4 transcripts.
Remarkably, Vß4-deficient BALB/c-SIM mice treated with IL-4 during
the initial phase of infection with L. major had increased
amounts of IL-4 transcripts comparable to those occurring spontaneously
in similarly infected BALB/c or Vß6-deficient BALB/c-SW mice (Fig. 2). It is noteworthy that the IL-4 mRNA observed in BALB/c-SIM mice
treated with rIL-4 during the initial 2.5 days of infection with
L. major was produced by CD4+ T cells
expressing a wide range of Vß TCR chains (results not shown).
Comparable results were observed when the supernatants of cultures of
specifically stimulated, designated LN cell populations were analyzed
for the accumulation of IL-4 (Fig. 2). As previously observed, it is
noteworthy that the responses of the various groups of mice could not
be discriminated on the basis of either the amounts of IFN-
transcripts or the IFN- produced in vitro. Analogous results were
observed using purified CD4+ T cell populations
for cytokine mRNA assays (data not shown). Thus, as assessed by
analysis of both mRNA and protein production, administration of IL-4 to
Vß4-deficient BALB/c SIM mice during the initial 2.5 days of
infection with L. major is sufficient to instruct the
development of an IL-4-producing Th2 response.
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and 2), anti-CD4
treated BALB/c mice neither became susceptible (Fig. 3) nor developed a Th2 response (Fig. 4) following administration of 0.8 µg
of IL-4 during the first 2.5 days of infection. Together, these results
show that to be effective in instructing Th2 cell development IL-4
requires sufficient numbers of CD4+ T cell
precursors, not necessarily expressing the Vß4 TCR chain.
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We have recently documented in vivo the induction of a state of
unresponsiveness to IL-12 in BALB/c mice within 48 h after
infection with L. major (26). These findings
were extended by showing that CD4+ T cells from
susceptible BALB/c mice, in contrast to those from resistant C57BL/6
mice, rapidly lose their capacity to respond to IL-12 in terms of
IFN- production in vitro. This extinction of IL-12 signaling, which
was due to down-regulation of IL-12R ß2-chain expression, was
indirectly demonstrated to be a consequence of the early burst of IL-4
production occurring in BALB/c mice in response to infection with
L. major (28). Abolition of the early IL-4 mRNA
burst produced in response to L. major in
Vß4-deficient BALB/c mice (15) allowed us to directly
correlate 1) the absence of early IL-4 production with the maintenance
of LN cell responsiveness to IL-12 and IL-12R ß2-chain expression
and, conversely, 2) the effect of treatment with exogenous IL-4 during
the early stage of infection on redirecting Th2 cell development and
susceptibility of these mice to infection with the loss of
responsiveness to IL-12 and down-regulation of the IL-12R ß2-chain
expression.
Groups of BALB/c, Vß6-deficient, and Vß4-deficient BALB/c mice were
inoculated in the hind footpads with L. major promastigotes.
One additional group of Vß4-deficient BALB/c mice was also treated
with a total of 0.8 µg of rIL-4 during the first 64 h after the
inoculation of parasites. Five days later, draining LN cells were
stimulated with L. major in vitro in the presence or the
absence of exogenous IL-12, and the levels of IFN- were determined
in supernatants after 3 days of culture. In parallel, analysis of
IL-12R ß1- and ß2-chain mRNA expression in freshly harvested LN
cells was performed using RT-PCR. The results in Fig. 5 show that specifically stimulated LN
cells from either BALB/c or Vß6-deficient BALB/c-SW mice do not
respond to IL-12 in terms of enhanced IFN- production in vitro.
RT-PCR analysis also revealed that the IL-12R ß2-chain mRNA was
absent in LN cells from these mice (Fig. 6). In sharp contrast, similar analysis
in Vß4-deficient BALB/c-SIM mice showed that LN cells were responding
to IL-12 and expressed IL-12R ß2-chain mRNA at high levels (Figs. 5 and 6). Strikingly, treatment of Vß4-deficient BALB/c-SIM mice with
IL-4 during the first 2.5 days of infection resulted in a loss of LN
cell responsiveness to IL-12 and IL-12R ß2-chain mRNA expression.
Comparable results were obtained with LN cells harvested 8 days after
infection (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8 CD4+ LN cells
within the first day after the s.c. inoculation of parasites
(15). The use of Vß4-deficient BALB/c mice, besides
having made possible the present demonstration of the essential role of
IL-4 during the early stage of infection in instructing Th2 cell
development and susceptibility to L. major in BALB/c mice,
should also permit us to define the boundaries in time that limit this
effect of IL-4. Experiments are currently in progress to define the
window of time after infection during which exogenous IL-4 redirects
Th2 cell development in Vß4-deficient BALB/c mice. The results
presented in this paper, obtained using Vß4-deficient BALB/c mice,
complement those showing that treatment with neutralizing anti-IL-4
Abs was capable of redirecting Th1 cell development in intact BALB/c
mice (12). In this context, we have pinpointed a short
time, between 16 and 48 h after infection, during which IL-4 must
be present to instruct susceptibility through Th2 cell development in
intact BALB/c mice, because neutralization of IL-4 after that time no
longer impeded Th2 cell maturation (26).
The rapid IL-4 response to the parasite of Vß4-V8
CD4+ BALB/c cells is focused on a single dominant
I-Ad-restricted T cell epitope of the LACK Ag of
L. major (15, 35). The present demonstration
that exogenous IL-4, only transiently supplied at the initiation of
infection, is able of acting in Vß4-deficient BALB/c mice as a
substitute for the Vß4 V8 CD4+ T cells
reactive to the LACK I-Ad epitope implies that an
essential role of these cells is to provide the IL-4 necessary for Th2
cell maturation. This conclusion is supported by prior results showing
that the specific induction of an unresponsive state in the LACK
I-Ad epitope-reactive Vß4 V8
CD4+ T cells following treatment of BALB/c mice
with altered LACK proteins that differ by a single amino acid from the
natural I-Ad-restricted epitope was capable of 1)
antagonizing the early IL-4 burst in response to LACK and 2)
redirecting the otherwise ineffective Th2 response to a fully
protective Th1 response with long term protection (35).
Thus, in the absence of the IL-4 produced during the early stage of
infection by LACK-reactive Vß4 V8 CD4+ T
cells, Th cell development defaults to the Th1 pathway, with the
resulting establishment of protective immunity to this otherwise lethal
infection.
Observations showing that deletion of LACK-reactive Vß4 V8
CD4+ T cells, either through superantigen
(MMTV(SIM))-mediated deletion of Vß4-expressing
CD4+ T cells (15) or by thymic
expression of LACK as a transgene (17), led to a resistant
phenotype in BALB/c mice already strongly supported the essential role
of these cells in susceptibility to infection with L. major.
These cells have also been shown to expand in the draining LN as late
as 3 wk after infection (36). The results of the present
study, showing that their absence can be fully substituted by exogenous
IL-4 given only during the early stage of infection, further suggest
that their role in the development of progressive disease might be
limited to the production of the IL-4 necessary for Th2 cell
development and that they are not essential at the effector stage of
the Th2 response. This contention is also supported by our recent
observation that infection of BALB/c mice with MMTV(SIM) 3 days after
parasite inoculation, i.e., a time when the early burst of IL-4 mRNA
expression had occurred, neither modified their susceptible phenotype
nor inhibited the magnitude of the Th2 response in their draining LN
despite drastically reduced numbers of Vß4 CD4+
T cells as tested in their blood and draining LN (H. Himmelrich,
unpublished observations).
Similarly to Vß4-deficient BALB/c mice, BALB/c mice transiently
depleted of CD4+ T cells by treatment with
anti-CD4 mAb GK 1.5 the day before infection mount a Th1 response
and are resistant to L. major. The early burst of IL-4 mRNA
expression normally seen in the draining LN of BALB/c mice in response
to infection was also not induced in CD4+ T
cell-depleted BALB/c mice (13). However, early IL-4 mRNA
expression, Th2 cell development, and progressive disease did occur
when parasites were injected 25–30 days after anti-CD4 mAb
treatment at a time that coincides with repopulation of lymphoid organs
with a significantly sized pool of CD4+ T cells
(13) containing sufficient numbers of Vß4 V8
CD4+ T cells rapidly producing IL-4 in response
to the LACK Ag of L. major (P. Launois, unpublished
observations). Thus, in the absence of IL-4 differentiation of the
CD4+ T cell precursors with specificities for
several parasite-derived epitopes gradually repopulating the lymphoid
tissues of BALB/c mice treated with anti-CD4 mAb defaults to the
Th1 pathway, leading to resistance to infection. In contrast to its
unambiguous effect in Vß4-deficient BALB/c mice, treatment with
exogenous IL-4 during the early phase of infection induced neither Th2
cell development nor susceptibility to L. major in otherwise
resistant anti-CD4 mAb-treated BALB/c mice. Inasmuch as the
exogenous IL-4 was given during the first 2.5 days of infection, a time
of maximal depletion of CD4+ T cells in
anti-CD4 mAb-treated mice and given the short half-life of IL-4 in
vivo, these results strongly suggest that these effects of IL-4 require
its direct interaction with parasite-specific
CD4+ T cell precursors.
Previous results have documented, on the one hand, the rapid induction
of a state of unresponsiveness to IL-12 in CD4+ T
cells from susceptible BALB/c mice and, on the other hand, the
maintenance of responsiveness to IL-12 in CD4+ T
cells from resistant C57BL/6 mice (26). The extinction in
BALB/c mice and the maintenance in C57BL/6 mice of IL-12 signaling
correlated with down-regulation and maintenance of IL-12R ß2-chain
mRNA expression, respectively (28, 37). This demonstration
in vivo of the importance of IL-12R ß2 subunit expression for IL-12
signaling confirmed results obtained in vitro using TCR ß
transgenic CD4+ T cells (38). The
results in this report showing that in contrast to wild-type BALB/c
mice, LN cells from Vß4-deficient BALB/c mice maintain IL-12R ß2
subunit expression and their capacity to respond to IL-12 in vitro in
terms of IFN- production support the concept that in vivo loss or
maintenance of IL-12 signaling is an important step in the stable
commitment of naive CD4+ T cell precursors to the
Th2 or Th1 pathway of maturation, respectively (28).
Importantly, the presently demonstrated capacity of exogenous IL-4 to
interfere with maintenance of IL-12R ß2-chain expression and IL-12
signaling in LN cells of Vß4-deficient BALB/c mice provides definite
evidence for the essential role of the IL-4 rapidly produced in intact
BALB/c mice by Vß4 V8 CD4+ T cells in the
induction of a state of unresponsiveness to IL-12. These data provide
direct support for the view that overproduction of IL-4 in the early
stage of infection with L. major, secondarily inducing
extinction of IL-12 signaling, underlies the genetic tendency of BALB/c
mice to mount an aberrant Th2 response to L. major.
It is worth mentioning that administration of exogenous IL-4 to C57BL/6
mice at the dose used in this study resulted in neither modification of
their resistant phenotype nor redirection of the effector cells to the
Th2 phenotype. In addition, similarly to control infected C57BL/6 mice,
LN cells from C57BL/6 mice treated with IL-4 during the early stage of
infection maintained IL-12R ß2-chain mRNA expression and
responsiveness to IL-12 in vitro at least up to 8 days after infection
(data not shown). These results probably do not stem from differences
in sensitivity to IL-4 between CD4+ T cells from
susceptible and resistant mice, since it has been previously reported
that administration of extremely high amounts of IL-4 to C3H mice did
not reverse their resistant phenotype (11). IFN- has
been shown to override the IL-4-induced down-regulation of IL-12R
ß2-chain mRNA expression both in vitro (38) and in vivo
(28). It is thus likely that either
CD4+ T cells from C57BL/6 mice are more sensitive
to IFN- than BALB/c CD4+ T cells or the levels
of endogenous IFN- or that rapidly produced in response to infection
with L. major are higher in C57BL/6 than in BALB/c mice. The
first hypothesis might also be the basis for the results of elegant
experiments using TCR ß transgenic CD4+ T
cells that have shown that B10.D2 cells retained IL-12 responsiveness
after specific activation in vitro even in the presence of an excess of
BALB/c T cells (39) and the IL-4 they produce.
Results showing that genetically pure BALB/c mice deficient in IL-4
neither resolved their cutaneous lesions nor redirected their
CD4+ T cell response toward the Th1 phenotype
(20) have recently disputed the essential role
of IL-4 in susceptibility to L. major and Th2 cell
maturation. However, independent studies also performed with IL-4
knockout BALB/c mice have generated conflicting results
(40). These differences, although still largely
unexplained, were recently attributed at least in part to the use of
different strains of parasites (41). Recent extension of
these investigations using both IL-4-deficient and IL-4R
-chain-deficient BALB/c mice did not permit this controversy to be
settled. In addition to confirming the previously reported conflicting
results obtained using IL-4-deficient BALB/c mice, the data obtained
using IL-4R -deficient BALB/c mice suggested, on the one hand, that
IL-13, signaling through the IL-4R -chain, might be involved in
disease progression (41) and, on the other hand, that
IL-13 could be involved in controlling dissemination of parasites
during the late and chronic phase of infection (42).
Nevertheless, confronted with the present observation directly showing
the requisite role of IL-4 during the early phase of infection for Th2
cell maturation leading to progressive disease in genetically intact
BALB/c mice, an unimpaired Th2 cell maturation and susceptibility to
L. major in IL-4 knockout BALB/c mice could suggest the
induction in these mice of a compensatory factor(s) able to replace the
normally essential function of IL-4. Such a factor(s), able to
substitute IL-4 in IL-4-deficient mice, probably also originates from
CD4+ T cells, because IL-4 knockout BALB/c mice
transiently depleted of CD4+ T cells at the
beginning of infection controlled parasite growth
(43).
In summary, this study directly indicates that IL-4 during the initial
period of infection with L. major is necessary and
sufficient to set in motion the molecular events, including
down-regulation of the IL-12R ß2 expression in
CD4+ T cells, ultimately resulting in Th2 cell
maturation and susceptibility to infection in BALB/c mice. These
results give further strength to the data that have implicated
LACK-reactive Vß4 V8 CD4+ T cells as
initiators of the events ultimately mediating the susceptible phenotype
of BALB/c mice infected with L. major. Further analysis of
the molecular basis for their rapid activation and IL-4 production
following parasite inoculation will significantly contribute to
understanding of the mechanism(s) underlying Th2 cell development in
this model system.
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Acknowledgments |
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Footnotes |
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2 On leave from the Institut Pasteur (Paris, France) and presently at the Pasteur Institute of Cayenne, French Guyana.
3 Address correspondence and reprint requests to Dr. M. Röcken, Department of Dermatology and Allergology, Ludwig-Maximilians University, Frauenlobstr. 9–11, 80337 Munich, Germany.
4 Address correspondence and reprint requests to Dr. J. A. Louis, World Health Organization Immunology Research and Training Center, Institute of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland.
5 Abbreviations used in this paper: LACK, Leishmania homologue of mammalian RACK1; MMTV, mouse mammary tumor virus; NMS, normal mouse serum; LN, lymph node; HPRT, hypoxanthine guanine phosphoribosyltransferase.
Received for publication December 2, 1999. Accepted for publication February 24, 2000.
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production and diminishes interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065. independent mechanism. J. Exp. Med. 171:115.8 CD4+ T cells in BALB/c mice infected with Leishmania major instructsTh2 cell development and susceptibility to infection. Immunity 6:541.[Medline]
during infection of mice with Plasmodium chabaudi chabaudi. Immunobiology 179:353.[Medline]
-deficient mice in chronic leishmaniasis reveal a protective role for IL-13 receptor signaling. J. Immunol. 162:7302.This article has been cited by other articles:
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