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*
Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom; and
Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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expression. Furthermore, using BALB/c
mice deficient in the expression of IL-4, IL-13, or both IL-13 and
IL-4, we demonstrate that IL-13-deficient mice are resistant to
infection and that there is an additive effect of deleting both IL-4
and IL-13. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
or Th2 cells (Th2-producing IL-4, IL-13, IL-5, and IL-10) during
experimental cutaneous leishmaniasis determines the outcome of
infection (1). In resistant mouse strains (such as
C57BL/6, CBA, and C3H/He), infection induces the onset of an
IL-12-dependent parasite-specific Th1 response characterized by the
enhanced expression of IFN- and results in the healing of the
infection. In contrast, susceptible BALB/c mice develop a
parasite-specific Th2 response characterized by the enhanced expression
of IL-4, progressive lesion development, and eventual death.
Following infection with L. major, the relative balance of
IL-4, IL-12, and IFN- has been proposed to be of primary importance
in determining the outcome of the disease. Although a role for IL-4 in
inducing susceptibility to infection has been demonstrated using mAb
treatment and transgenic expression (2, 3), recent reports
using IL-4-deficient mice have presented conflicting evidence for the
importance of IL-4 in the disease process. Noben-Trauth et al.
(4) reported that IL-4-deficient mice, on a BALB/c
background, remained susceptible to L. major, whereas both
Kopf et al. (5) and Mohrs et al. (6) showed
that the same mouse strain was resistant. Recently, IL-4R
-deficient
animals were reported to be more resistant to L. major than
IL-4-deficient mice (6, 7), highlighting the existence of
further contributory factors promoting susceptibility to infection.
Indeed, these and other reports have implied a role for IL-13 in
regulating responses to L. major infection
(6, 7, 8) although none have formally demonstrated a role for
IL-13.
Genetic mapping studies have identified that the IL-4 gene
lies within a genomic region associated with susceptibility in mouse
(9, 10, 11). Significantly, this region also includes the
IL-13 gene, and it is noteworthy that the IL-13 protein can
also utilize the IL-4R (12). Furthermore, recent
reports have highlighted the complex and integrated roles for IL-13 and
IL-4 in the development of Th2 cell responses (13, 14).
To assess directly the contribution of IL-13 to the progression of L. major infection, we have examined the responses of IL-13-transgenic mice and mice deficient for either IL-13 alone or doubly deficient for both IL-4 and IL-13. Our results indicate that IL-13 is an important component for susceptibility to L. major.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-13-transgenic and littermate control mice were as described previously (15) and had been backcrossed onto C57BL/6 mice for six generations. IL-13-transgenic mice were crossed with IL-4T-/- (16), which had been backcrossed for 10 generations onto C57BL/6. IL-13-deficient (17) and IL-4/13-deficient (14) mice were backcrossed for at least four generations with BALB/c. Wild-type littermates of the cytokine-deficient mice were also backcrossed with BALB/c and are referred to as crossbalb. IL-4-deficient (4) mice were generated on a BALB/c background. The mice were bred and maintained in the facility at the Medical Research Council Laboratory of Molecular Biology.
Parasites and Leishmania Ag
L. major (LV39) was maintained as described (18). Soluble Leishmania Ag (SLA)3 was prepared by rapidly freeze thawing a pellet of 5.6 x 109 L. major promastigotes. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL) according to the manufacturer’s instructions.
Infection
Eight- to 12-wk-old female mice were infected with two million metacyclic promastigotes in the hind footpad. Footpad thickness was determined by weekly measurement of the infected footpad with a Vernier caliper and subtracting the thickness of the contralateral uninfected footpad. Following infection, parasite numbers were determined in popliteal lymph nodes as described (19).
Preparation of cells
Infected mice were sacrificed and the popliteal lymph nodes and
blood were collected. Single-cell suspensions (4 x
106 cells/ml) were prepared from pooled popliteal
lymph nodes and cultured in RPMI 1640 with the following additions. LPS
stimulations were performed for 24 h in the presence of 3 µg/ml
LPS. Anti-CD3
Ab (0.25 µg/ml of clone 2C11; Becton Dickinson,
Mountain View, CA) stimulations were performed for 48 h.
Ag-specific responses were analyzed using 2.5 µg/ml SLA for 5 days.
Serum was collected and tested for parasite-specific Ab content.
ELISA assays
ELISAs were conducted upon Maxisorb 96-well immunoplates (Nunc, Naperville, IL) using standard Becton Dickinson ELISA protocols as described previously. Parasite-specific ELISAs were performed by coating 96-well plates with SLA at 2.5 µg/ml; bound Ig of diluted serum samples was detected using biotinylated monoclonal anti-Ig isotype detection Abs (Becton Dickinson). Cytokine ELISAs were as described elsewhere (13).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-13-Tg (15) and control C57BL/6 mice were infected
in the rear footpad with L. major LV39 promastigotes
(9, 18). Following infection, footpad inflammation was
monitored as an indicator of disease progression (9, 18).
As expected, C57BL/6 mice displayed an initial period of footpad
swelling lasting 
35 days, but thereafter the swelling decreased to
near normal by 91 days (Fig. 1
). In
contrast, the footpads of IL-13-transgenic mice continued to increase
in size before finally reaching a plateau by 49 days, and these animals
were unable to resolve the parasite infection even by 91 days
postinfection (Fig. 1). At this time, there was severe inflammation
along the length of the limb combined with ulceration and marked
deterioration of the integrity of the foot.
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Because IL-4 has been identified as a major factor in the
generation of susceptibility to L. major infection, we
determined the ability of IL-13 to promote susceptibility in the
absence of IL-4. The IL-13-transgenic mice were bred with
IL-4-deficient mice on a C57BL/6 background
(IL-4-/-(B6)IL13Tg) and infected with L.
major alongside IL-4-deficient mice
(IL-4-/-(B6)) and wild-type controls. By day 60
postinfection, the footpads of both IL-4-/-(B6)
and wild-type mice had returned to normal (Fig. 2). However, despite the absence of IL-4,
the IL-4-/-(B6)IL13Tg mice failed to control
the parasite infection (Fig. 2). Furthermore, the parasite burden was
1000-fold higher in IL-13-transgenic mice at 70 days postinfection than
in controls [104–105
parasites/1 x 106 transgenic lymph node
cells vs 10–102 parasites/1 x
106 wild-type lymph node cells
(19)]. Thus, the ability of IL-13 to induce
susceptibility to infection is not dependent on IL-4.
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Th1-like responses have been shown to promote healing of infection
(20, 21, 22). Therefore, we examined whether the
overexpression of IL-13 had perturbed the generation of a Th1 cytokine
response to L. major. The cytokine production from popliteal
lymph node cells derived from infected mice was analyzed. IL-12
production was reduced in cell cultures from transgenic mice as
compared with those from C57BL/6 controls (Fig. 3A). This decrease was
mirrored by substantially reduced levels of Ag-specific IFN-
production by the IL-13-transgenic mice (Fig. 3B).
Significantly, cultures from IL-13 transgenics produced elevated levels
of IL-5 as compared with wild-type (Fig. 3C). These data
demonstrate that the Th1-like cytokine response in the IL-13-transgenic
mice is suppressed.
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Because overexpression of IL-13 promoted susceptibility to L.
major infection, we addressed whether ablation of IL-13
expression would confer resistance on normally susceptible BALB/c mice.
IL-13-deficient (IL-13-/-) and doubly
IL-4/13-deficient
(IL-4-/-IL-13-/-) mice
backcrossed onto a BALB/c genetic background (13, 14) were
infected with L. major LV39. In addition, IL-4-deficient
(IL-4-/-) mice generated on a BALB/c background
(4) were also infected, and footpad swelling was measured
at 7-day intervals. BALB/c mice and backcrossed (crossbalb) control
mice failed to control parasite infection, and, by 35 days
postinfection, these animals were sacrificed due to the extent of
footpad inflammation and ulceration (Fig. 4). Importantly, the
IL-13-/- mice were able to control infection
and by day 56 had normal footpads (Fig. 4). Similarly, IL-4-deficient
mice were also found to be resistant to infection (Fig. 4). In both
cases, the severity of infection of IL-4-deficient or IL-13-deficient
mice was similar to that of C57BL/6 mice (Fig. 4). Interestingly, the
double IL-4/13-deficient animals had the least severe footpad swellings
of any group and resolved the infection more quickly (Fig. 4).
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A). For
example, IL-13-/- mice had elevated IL-4 levels
in comparison to C57BL/6 but were nevertheless able to control parasite
infection. Similarly, there was no significant correlation between
IFN- expression and resistance to infection, although there were
slightly enhanced levels of IFN- production in both
IL-4-/- and
IL-4-/-IL-13-/- mice
(Fig. 5A). IL-12 expression levels also failed to correlate
with a resistant phenotype (Fig. 5C). In addition, there was
no correlation between Ab response and resistance to infection in the
various cytokine-deficient mice (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The healing response to L. major is characterized by the
development of a Th1 response with elevated levels of IFN- and
IL-12. Th1 responses are dependent on the early production of IL-12 by
macrophages and dendritic cells (23, 24). This pulse of
IL-12 leads to enhanced expression of IFN- by T cells, which in turn
maintains the Th1 phenotype. Consequently, suppression or ablation of
IFN- and IL-12 can inhibit parasite clearance and result in the
development of susceptibility to infection. Neutralization of IL-12
induces a susceptible phenotype (25, 26), whereas
treatment of susceptible mice with exogenous IL-12 allows them to heal
their lesions (27, 28). Similarly, ablation of IFN-
using Ab treatment (20) or gene targeting (21, 22) causes susceptibility in normally resistant mouse strains.
Since IL-13 has been shown to inhibit the production of IL-12 by
macrophages and to inhibit L. major parasite killing in
vitro (29, 30), it is probable that by overexpressing or
ablating IL-13, the levels of IL-12 production will be modified. Thus,
overexpression of IL-13 in transgenic animals may be acting to prevent
the onset of a curative Th1 response following infection. Indeed, the
levels of both IL-12 and IFN- were considerably reduced in the
infected transgenic animals, whereas the background cytokine milieu was
characterized by the presence of Th2 cytokines such as IL-5. These data
are in keeping with previous studies which have demonstrated that IL-13
plays an important positive role in the onset and magnitude of Th2-like
responses to gastrointestinal helminth infection and synchronous
pulmonary granuloma production (13, 14). Equally, IL-13
may be acting directly on the macrophage by inhibiting parasite killing
mechanisms. Thus, these alternative pathways are not mutually exclusive
and would represent an integrated immune response to L.
major infection. In addition, it is also possible that in the case
of the IL-13-transgenic mice, the observed decrease in IL-12 production
may be due to the increased parasite load suppressing normal macrophage
function.
Genetic mapping and functional studies have implicated a number of loci
that may influence susceptibility to L. major infection. One
such susceptibility locus has been mapped to the gene cluster on mouse
chromosome 11 containing both IL-4 and IL-13 (10, 11).
Although previous investigations have linked IL-4 with susceptibility,
we now demonstrate conclusively that both IL-4 and IL-13 are factors
that can induce susceptibility to L. major infection and
that IL-13 can mediate this role even in the absence of IL-4
expression. Furthermore, our studies also help clarify the inconclusive
results obtained using mouse strains with gene disruptions in IL-4-
and/or IL-13-signaling pathways, including IL-4R-deficient mice
(6, 7), STAT6, and BCL6-deficient mice
(8).
Reports examining the responses of IL-4-deficient mice to L. major have produced conflicting results. Noben-Trauth et al. (4) initially reported that IL-4-deficient mice generated on a BALB/c background remained susceptible to L. major (LV39). However, Mohrs et al. (6), and our results shown above, found that the same IL-4-deficient BALB/c mice were resistant to L. major (LV39). It is difficult to identify the reason for these conflicting results although it is well documented that the virulence of L. major differs markedly between strains. Therefore, one explanation for these differing results might be substrain differences of LV39 between laboratories.
Our experiments illustrate a novel and previously unreported role for IL-13 in the induction of susceptibility to L. major infection. Importantly, our results also demonstrate that IL-13 acts independently of IL-4 and thus identifies IL-13 as a major component of the immune response to L. major. In addition, the finding that IL-4/13-deficient animals have enhanced resistance to infection reinforces the notion that these cytokines act in combination to produce robust Th2 responses and inhibit Th1 differentiation and parasite killing.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew N. J. McKenzie, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, U.K. E-mail address:
3 Abbreviation used in this paper: SLA, soluble Leishmania Ag.
Received for publication October 1, 1999. Accepted for publication November 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major. J. Immunol. 143:266.[Abstract]
-deficient mice infected with Leishmania major. J. Exp. Med. 179:1367. receptor are susceptible to infection with Leishmania major but mount a polarised T helper cell 1-type CD4+ T cell response. J. Exp. Med. 181:961.This article has been cited by other articles:
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) mice or C57BL/6 (
)
littermate controls were infected in the rear hind footpad with 2
x 106 promastigotes, and their footpad swelling was
measured. Data are representative of four experiments with five to
eight animals per group. Data represent means plus SD.
), and C57BL/6 (
) and IL-13-transgenic (
) mice, stimulated, and
analyzed using ELISA. A, IL-12 levels following
stimulation with LPS (3 µg/ml) for 24 h. B,
IFN-
) mice were infected in the rear
footpad with 2 x 106 promastigotes, and their footpad
swelling was measured. Student’s t test was used to
compare the difference in footpad depth between IL-4/13-deficient and
C57BL/6 mice; *, p values < 0.005. Data are
representative of two experiments with 6–15 animals per group. Data
represent means plus SD.
