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*
Schistosomiasis Immunology and Pathology Unit, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
Preclinical Research, Genetics Institute, Andover, MA 01810;
Genetics Institute, Cambridge, MA 02140; and
§
The Biomedical Research Institute, Rockville, MD 20852
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Fc fusion
protein, significantly reduced the size of pulmonary granulomas in
unsensitized as well as egg-sensitized mice. Blocking IL-13 also
significantly reduced total serum IgE levels. Interestingly, however,
IL-13 blockade did not affect the evolving egg-induced Th2-type
cytokine response. IL-4, IL-5, as well as IL-13 responses were
indistinguishable in control-Fc- and soluble IL-13R 2-Fc fusion
protein-treated animals. The smaller granulomas were also
phenotypically like the control Fc-treated mice, displaying a similar
eosinophil content. Additional studies in IL-4-deficient mice
demonstrated that IL-13 was produced, but at much lower levels than in
wild-type mice, while IL-4 expression was completely independent of
IL-13. Moreover, while granuloma formation was partially reduced in
IL-4-deficient mice, blocking IL-13 in these animals almost completely
abrogated granuloma development and the pulmonary eosinophilia, while
it simultaneously increased IFN-
production. Together, these data
demonstrate that IL-13 serves as an important mediator of Th2-mediated
inflammation and plays a role in eliciting IgE responses triggered by
schistosome eggs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Schistosoma mansoni-infected mice, egg-induced granuloma formation has been characterized as a CD4+ T cell-mediated delayed-type hypersensitivity reaction (14, 15) and is associated with a dominant Th2-type cytokine production profile (16, 17). Depletion of the Th2 cytokine IL-4, both in infected mice (18, 19) and in animals i.v. injected with schistosome eggs (20, 21), revealed an important role for the cytokine in driving the egg-induced Th2-mediated inflammatory response, as well as the accompanying rise in IgE Ab levels (18). Nevertheless, in these reports as well as in studies performed recently in IL-4-deficient mice, it has become clear that a reduced but significant Th2-type cytokine response can develop and that marked, albeit diminished, granulomatous inflammation is observed even in the absence of IL-4 (20, 21, 22, 23, 24). These findings suggest that the host response to schistosome eggs may not be totally dependent upon IL-4 and that other Th2-associated cytokines, such as IL-13, which is induced by schistosome eggs (25), might serve as important mediators in the generation of egg-induced granulomas.
IL-13 is produced by Th2 cells and is closely related to IL-4 in many activities (26, 27). The nucleotide sequence of murine IL-13 is 30% homologous with IL-4, but has limited homology with any other cytokine (26). Although IL-4 and IL-13 share many of the same biological activities, some studies indicate that there are indeed functional differences between the two cytokines (26). Similar to IL-4, IL-13 inhibits the production of inflammatory cytokines (28, 29) and up-regulates MHC class II and CD23 expression on monocytes/macrophages as well as on B cells (29). The cytokine also induces IgE class switch in human cells in vitro and triggers IgG and IgM synthesis (28, 30, 31, 32, 33). Most importantly, however, in contrast to IL-4, IL-13 does not affect resting or activated T cells (26). Because almost all of the above studies were performed using in vitro systems, little is known about the immunoregulatory properties of IL-13 in vivo and whether the cytokine plays any role in the establishment of Th2 responses or in regulating the effector functions typically attributed to Th2 cells.
The aim of this study was to assess the role of IL-13 in the
inflammatory response induced by S. mansoni eggs. We
employed a commonly used model of pulmonary granuloma formation in
which purified schistosome eggs are injected i.v. into the lungs of
unsensitized or egg-sensitized mice (21). IL-13 activity was blocked by
treating mice with soluble IL-13R 2-Fc fusion protein
(sIL-13R2-Fc),3 which
effectively blocks IL-13 binding to its receptor in vivo (34). In these
experiments, we focused on the contribution of IL-13 to the egg-induced
inflammatory response and to the generation of IgE Abs and tissue
eosinophilia. We also examined whether IL-13 influenced the local and
systemic cytokine balance by determining changes in cytokine mRNA
expression at the site of lesion formation. Our results demonstrate
that IL-13 plays a significant role in egg-induced granulomatous
inflammation, but suggest that the changes observed are not simply due
to alterations in the cytokine response. In addition, we show that
blocking IL-13 results in significantly reduced IgE titers. These
findings suggest that IL-13 acts as an important effector cytokine for
Th2-mediated inflammatory reactions in the lung.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to eight-week-old female C57BL/6 mice were purchased from the Division of Cancer Treatment, National Cancer Institute (Frederick, MD). IL-4-deficient mice were constructed by gene targeting in embryonic stem cells, as previously described (35), and were generously provided by Dr. Werner Müller (Institute for Genetics, University of Cologne, Germany). The mice were used at the twelfth generation backcross to C57BL/6.
Reagents
sIL-13R2-Fc.
A pED expression vector containing DNA encoding the murine
sIL-13R2-Fc extracellular domain, fused in frame to the hinge
CH2/CH3 regions of human IgG1, was transfected into CHO cells (36).
IL-13R2-Fc is the high affinity binding chain for IL-13 and is
closely related to the murine low affinity IL-13 binding subunit
IL-13R1 (34). Transfected CHO cells were grown in serum-free medium,
and culture supernatants that contained secreted sIL-13R2-Fc were
filtered through a Millipore Millidisk and concentrated 20-fold with a
Millipore Pellicon 30,000 m.w. cutoff membrane, then adsorbed to
recombinant protein A-Sepharose that had been equilibrated with
PBS, pH 7.4, washed with PBS, and eluted with 20 mM citrate, 0.2 M
NaCl, pH 3. The eluate was neutralized to pH 7.5 with an equal volume
of 0.2 M potassium phosphate, pH 8, and was formulated into PBS, pH
7.3, using an Amicon stirred cell with a YM30 membrane. The final
product was 95% pure by SDS-PAGE analysis and had a concentration of 3
mg/ml (determined using A280 and a theoretical extinction
coefficient of 1.93 ml/mg · cm). Endotoxin contamination was <2
EU/mg, as determined with the Cape Cod Associates limulus amebocyte
lysate assay (Woods Hole, MA). The in vitro ID50, as
determined by ability to neutralize 3 ng/ml of murine IL-13 in the B9
proliferation assay (37), was approximately 10 ng/ml. Human IgG
(control-Fc), which was used as a control for sIL-13R2-Fc, was
affinity purified by recombinant protein A-Sepharose
chromatography, as described for sIL-13R2-Fc, from a 10% solution
of human Ig that is commercially available for i.v. administration
(Miles, Elkhart, IN). The final preparation had an endotoxin
concentration of <0.4 EU/mg. Both reagents were provided by Genetics
Institute (Cambridge, MA).
Induction of pulmonary granulomas
The induction of synchronous egg-induced granulomas was performed as described previously (21). S. mansoni eggs were separated from the livers of infected mice (Biomedical Research Institute, Rockville, MD) and enriched for mature eggs. To induce primary pulmonary granulomas, mice were injected with 5000 eggs i.v. Secondary granulomas were induced in i.p. egg-sensitized mice (sensitized with 5000 eggs). Mice were sensitized 2 wk before the i.v. egg challenge. Animals were killed on days 3, 6, or 9 postchallenge.
At the time of sacrifice, the lungs were removed for histology and RNA extraction. The lymph nodes draining the lung and spleens were removed to prepare cell suspensions for in vitro culture. Serum was obtained for total IgE and Ag-specific Ab measurements.
Treatments
Animals were treated either with a human control-Fc or with the
sIL-13R2-Fc by i.p. injection in 0.5 ml PBS. In primary pulmonary
granuloma experiments, 200 µg/mouse/day was administered on days -1,
0, 2, 5, 8, and 11 postchallenge. There was no effect observed with the
control- Fc alone. In the secondary granuloma experiments, mice were
treated every other day during the 2-wk i.p. egg sensitization period,
only at the time of challenge, or throughout the period of
sensitization and challenge. Either 200, or in some experiments 400
µg/mouse/day was administered. The optimal concentration for in vivo
use was based on kinetic assays and on dose-response experiments in
mice. The 200 µg dose was chosen as the optimal dose, although 400
and 800 µg doses were equally effective at suppressing granuloma
formation. In additional experiments, egg-sensitized mice were treated
with mAbs to CD4 or CD8 to deplete CD4+/CD8+ T
cells at the time of i.v. egg challenge. Mice were injected i.p. with
0.5 mg GL113 (anti-ßGal control mAb), 0.5 mg GK1.5 (anti-CD4
mAb), or 0.25 mg clone 2.43 (anti-CD8 mAb) diluted in 0.5 ml PBS on
days -4, -1, and +3 post-i.v. egg challenge.
Histopathology
For measurement of granulomas, the left lung was inflated with Bouin-Hollande fixative and processed as previously described (18). The size of the pulmonary granulomas was determined in histologic sections stained by Wright’s Giemsa stain (Histopath of America, Clinton, MD). The diameters of each granuloma containing a single egg were measured with an ocular micrometer, and the volume of each granuloma was calculated assuming a spherical shape. Two diameters from each granuloma are used, using the longest and shortest diameter of each lesion for the measurement. An average of 30 granulomas per mouse was included in the analysis. All histologic examinations were scored by the same individual in a blinded fashion to control for consistency. The percentage of eosinophils and other cell types was evaluated in the same sections.
Isolation and purification of RNA
The right lung from each animal was placed in 1 ml of RNA-STAT 60 (Tel-Test, Friendswood, TX), frozen on dry ice, and kept at -70°C until use. Tissues were homogenized using a tissue polytron (Omni International, Waterbury, CT), and total RNA was extracted following the recommendations of the manufacturer. The RNA was resuspended in diethyl-pyrocarbonate-treated water and quantitated spectrophotometrically.
RT-PCR detection of cytokine mRNA
A RT-PCR procedure was used to determine relative quantities of
mRNA for IL-4, IL-5, IFN-, IL-1ß, TNF-, and
hypoxanthine-guanine phosphoribosyl transferase (HPRT). The cDNA was
obtained after reverse transcription of 1 µg of RNA, as described
(21). The primers and probes for all genes except CD4 and CD8 were
previously published (21, 25). The primers and probes used to amplify
CD4 and CD8 were as follows: CD4 upstream primer, 5'-TGT GCC GAG CCA
TCT CTC TTA GG-3'; CD4 downstream primer, 5'-GCA CTG AGA GTG TCA TGC
CGA AC-3'; CD4 probe, 5'-GTC TCT AAC CCC TTG ACA GAG-3'; CD8 upstream
primer, 5'-ATG CAG CCA TGG CTC TGG CTG G-3'; CD8 downstream primer,
5'-GCA TGT CAG GCC CTT CTG GGT C-3'; and CD8 probe, CTC CTG GAG TTC TTC
CAA AGG AG-3'. The PCR cycles used for each cytokine were as follows:
IL-4 (33), IL-5 (31), IFN- (29), IL-1ß (29), TNF- (38), CD4
(33), CD8 (33), and HPRT (23).
Analysis and quantification of PCR products
The amplified DNA was analyzed by electrophoresis, Southern blotting, and hybridization with nonradioactive cytokine-specific probes, as previously described (21). The PCR products were detected using a ECL detection system (Amersham). The chemoluminescent signals were quantified using a flat-bed scanner (Microtek model 600 ZS, Torrance, CA). Arbitrary densitometric units were calculated by dividing the cytokine or cell surface marker OD units by the individual HPRT OD units and multiplying the result by 100.
In vitro cultures
Lung-associated lymph node (LALN) cells (thoracic/mediastinal)
and spleens were extracted from the mice, and single cell suspensions
were prepared. RBC were lysed by osmotic treatment with ACK
lysing buffer (Biofluids, Rockville, MD). Cells were placed in RPMI
1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 U/ml
penicillin, 100 µg/ml streptomycin, 25 mM HEPES, 1 mM sodium
pyruvate, 0.1 mM nonessential amino acids, and 50 µM 2-ME at 37°C
in 5% CO2. Cells were plated in 24-well plates (3 x
106/ml) and stimulated with soluble egg Ag (SEA) at 20
µg/ml or with Con A at 5 µg/ml, and supernatants were collected
after 72 h to measure the levels of IL-4, IL-5, IL-13, and
IFN-. Additional SEA-stimulated cultures were also treated with 50
µg/ml of anti-CD4 mAb (GK1.5). Cultures treated with anti-CD4
mAb alone showed no change in cytokine expression when compared with
that observed in medium control cultures (data not shown). IL-5 and
IFN- were measured using specific sandwich ELISA (38). IL-13 levels
were measured using murine IL-13 ELISA kits (R&D Systems, Minneapolis,
MN). Cytokine levels were calculated from curves prepared with
recombinant cytokines. IL-4 was measured using the IL-4-sensitive cell
line CT.4S. Proliferation of these cells was quantified by
[3H]TdR incorporation, and the amount of cytokine was
determined by comparison with known amounts of rIL-4.
Serum IgE and Ab isotype analysis
Total IgE in serum was determined using a specific sandwich ELISA (PharMingen, San Diego, CA) and quantified according to a reference IgE standard (anti-trinitrophenol IgE; PharMingen). Plates were coated with anti-mouse IgE capture mAb from clone R35-72 in 0.1 M NaHCO3, pH 8.2, overnight at 4°C. The secondary mAb was a biotinylated anti-mouse IgE from clone R35-92. The streptavidin-peroxidase reagent was diluted 1/1000 in PBS/1% BSA. Isotype-specific Ab titers were evaluated by indirect ELISA. Immulon 4 plates were coated with 10 µg/ml of SEA (50 µl/well) diluted in PBS, and serum samples were analyzed using serial twofold dilutions. Second-step horseradish peroxidase-conjugated rabbit anti-mouse IgG, IgG1, IgG2a, and IgG2b Abs (Zymed Laboratories, San Francisco, CA) were employed at a 1/1000 dilution. The absorbance in the wells was read at 405 nm using a Vmax Kinetic Microplate Reader (Molecular Devices, Palo, Alto, CA) after adding 100 µl of ABTS:H2O2 one-step substrate.
Statistics
Statistical significance was determined by Student’s t test, and p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Initially, we determined to what extent IL-13 and IL-4 were induced during the initiation of an immune response to eggs (primary response) and in animals that had been sensitized to parasite eggs before experimental challenge (secondary response). The production of IL-4 and IL-13 both in vitro and in vivo was directly compared with changes in granuloma development. In these studies, naive or i.p. egg-sensitized mice were injected i.v. with eggs, and at 1, 3, 6, and 14 days, animals were sacrificed and the LALN cells were processed for cytokine analysis. Granuloma size was determined and lung tissues were also processed to determine the changes in IL-13 and IL-4 mRNA expression in the local milieu of granuloma formation.
LALN cells were cultured in vitro and restimulated with SEA, and 72-h
culture supernatants were analyzed for IL-13 and IL-4 by ELISA. As
shown in Fig. 1
, B and
C, Ag-specific IL-4 and IL-13 responses were first detected
in i.v. egg-challenged naive mice as early as day 3 following egg
embolization. Peak expression for both cytokines occurred on day 6 and
then declined markedly by day 14. Peak granuloma size (Fig. 1A) correlated with the peak in cytokine expression. The
cytokine response in egg-sensitized mice followed a similar kinetic
pattern, except that the overall levels of IL-4 and IL-13 were much
higher than in the naive mice, and the granulomas were larger,
confirming an anamnestic-type response in sensitized animals. Again,
the peak in the cytokine response corresponded with the peak in
granulomatous inflammation (Fig. 1, right panels).
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A). IL-4 levels peaked on
day 6 and were markedly decreased by day 14, while IL-13 mRNA remained
at similarly elevated levels on both days 6 and 14. In egg-sensitized
mice, peak induction IL-4 and IL-13 mRNA occurred at much earlier time
points than in the unsensitized animals (Fig. 2B). IL-4 and
IL-13 mRNA reached maximal levels as early as 1 day following egg
embolization, and while IL-13 remained at high levels at all time
points, IL-4 mRNA expression dropped rapidly after day 3 and almost
reached baseline levels between days 6 and 14. These data suggest that
IL-4 mRNA expression is more tightly regulated, while the induction of
IL-13 mRNA, at least at the site of granuloma formation, is more
sustained. Again, similar to the lymph node data, the peak in IL-13
mRNA expression closely correlated with the peak in granuloma size
(Fig. 1
A).
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IL-4 and IL-13 were both initially described as Th2 cytokines, yet
recent studies examining the cellular sources of IL-4 have suggested
that CD4+ T cells may not be the only source of these
important Th2-associated cytokines (6). Therefore, to begin to address
the cellular source of IL-13 in this model, we examined whether the
production of IL-13 was dependent on a CD4+ T cell
response. For these experiments, naive and egg-sensitized mice were
challenged with eggs, and LALN cells were isolated at various time
points and then restimulated in vitro with SEA, either in the presence
or absence of anti-CD4 mAbs. As shown in Fig. 1, B and
C, inclusion of the anti-CD4 mAb to SEA-stimulated
cultures markedly reduced the IL-4 and IL-13 response in both the
primary and secondary egg challenge models. Indeed, the marked
reduction in IL-4 and IL-13 production observed at the peak time point
(day 6) suggests that the expression of both cytokines is highly
dependent on CD4+ T cells.
To further examine the dependence of the IL-4/IL-13 response on
CD4+ T cells, additional experiments were performed in
which either CD4 or CD8+ T cells were depleted in vivo. For
these experiments, mice were sensitized with eggs i.p. and then 4 wk
later, at the time of secondary i.v. egg challenge, depleted of either
CD4 or CD8+ T cells. Changes in the expression of CD4 and
CD8 mRNA were used to determine the extent to which the cell
populations were depleted at the site of granuloma formation. As shown
in Fig. 3, there was a marked increase in
both CD4 and CD8 mRNA expression following egg embolization. As
expected, CD4-depleted mice showed a highly significant reduction in
CD4 mRNA and no change in their CD8 levels, while CD8-depleted mice
showed an opposite and equally significant reduction in CD8 mRNA
expression. Interestingly, however, only the CD4-depleted mice showed a
significant reduction in IL-4 and IL-13 mRNA expression, again
demonstrating that IL-4 and IL-13 mRNA expression in vivo is highly
dependent on CD4+ T cells (Fig. 3).
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2-Fc reduces granulomatous inflammation in naive and
egg-sensitized mice
To assess the role of IL-13 in the development and modulation of
granuloma formation, we treated mice with a soluble IL-13R fusion
protein to block the activity of the cytokine. The murine IL-13R2
capable of binding IL-13, but not IL-4, IL-2, IL-7, IL-9, or IL-15, was
fused with a human Fc and effectively blocks IL-13 activity both in
vitro and in vivo (34).
Different protocols were employed in these studies so that we could
address the contribution of IL-13 to the initiation of a Th2-mediated
inflammatory response, as well as determine whether IL-13 blockade
could affect established Th2 responses. As shown in Fig. 4A, sIL-13R2-Fc
significantly reduced primary pulmonary granuloma formation in
unsensitized mice. In additional experiments, mice were sensitized with
eggs i.p. to establish an egg-specific Th2 response, and then 2 to 3 wk
later, animals were challenged i.v. with eggs. Mice were treated with
sIL-13R2-Fc during both the period of sensitization and again at the
time of challenge (Fig. 4B, Expts. 1 and 2) or only at the
time of challenge (Fig. 4B, Expt. 3). With both protocols,
sensitized mice showed significant reductions in lesion formation. In a
final adjuvant-type protocol, we wanted to determine whether treatment
with sIL-13R2-Fc during the period of sensitization alone would
modify subsequent granulomatous responses. In this situation, IL-13
blockade had little or no effect (data not shown).
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Although in vitro studies suggested that IL-13 does not
directly influence Th2 cell development, it is possible that it may
indirectly influence CD4+ Th2 cell differentiation in vivo,
due to its role as a regulator of APC monokine secretion and
surface Ag expression (26, 28, 30). Therefore, to begin to address the
mechanism for the reduced granulomatous response, we analyzed the
effects of IL-13 blockade on related responses during Th2-dominated
reactions, including eosinophil differentiation and IgE production. In
addition, changes in the expression of Th1- and Th2-associated
cytokines were monitored both in vitro and in vivo after sIL-13R2-Fc
treatment.
Previous studies have established that alterations in granuloma
formation are often accompanied by significant changes in the
local and systemic cytokine response (25, 38, 39, 40). Therefore,
to examine whether IL-13 blockade influenced Th cell cytokine
production, LALN cells were isolated from control-Fc- and
sIL-13R2-Fc-treated mice at multiple time points following i.v. egg
challenge. Isolated lymphocytes were placed in culture and restimulated
with either SEA or mitogen (Con A), and the production of IFN-,
IL-4, IL-5, and IL-13 was examined in the 72-h supernatants. As shown
in Fig. 5, marked increases in IL-4,
IL-5, and IL-13 were observed in all SEA- and Con A-stimulated cultures
prepared from egg-challenged mice, and no major differences were
detected among the control-Fc- and sIL-13R2-Fc-treated animals at
any time point. There was only minor IFN- expression in response to
SEA, and the peak Con A-induced IFN- response detected at early time
points (day 3) was down-regulated significantly at days 6 and 9
postchallenge (Fig. 5). However, in two separate experiments in
unsensitized mice (primary response), treatment with sIL-13R2-Fc
induced a modest increase in SEA-specific IFN- production on day 14
postchallenge (data not shown). Nevertheless, this was not accompanied
by decreases in the expression of any Th2-type cytokines. Together
these data demonstrate that a highly polarized Th2-type cytokine
pattern develops in egg-challenged mice, and that IL-13 blockade fails
to significantly modify this response even in mice treated throughout
the period of sensitization and challenge. Identical results were
obtained with cultured spleen cells obtained from similarly treated
animals (data not shown).
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) known to play a role in
the granulomatous response (41, 42, 43). As shown in Fig. 6, marked increases in IL-4, IL-5,
IL-1ß, and TNF- mRNA were observed after i.v. egg challenge.
Nevertheless, there were no significant differences in cytokine mRNA
responses detected among the control-Fc- or sIL-13R2-Fc-treated
mice, and there was no evidence for an increase in IFN- mRNA
expression. We have also looked at earlier time points (days 3 and 6),
but again found no evidence for a significantly altered cytokine mRNA
response (data not shown).
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It is well known that Ab isotype switching in B cells is greatly
influenced by the cytokine milieu. In mice, high IgG1/IgE and low IgG2
Ab isotype titers associate with Th2 responses, while IgG2 and low
IgG1/IgE titers are observed in Th1-type cellular immune responses (44, 45). Tissue eosinophilia is similarly regulated by the Th2/Th1 cytokine
balance, with IL-5 positively and IFN- negatively regulating
eosinophil accumulation (46). Because IL-13 blockade down-regulated the
overall Th2-mediated inflammatory response, we wanted to determine
whether these related responses were similarly affected by IL-13
antagonism.
To address this issue, we compared the cellular composition of
granulomas in both control-Fc- and sIL-13R2-Fc-treated mice and
monitored their serum Ab response. While lesion size was decreased
significantly after IL-13 blockade, the cellular composition of the
granulomas did not vary significantly. In all experiments, both
control-Fc- and sIL-13R2-Fc-treated mice had granulomas that were
composed of between 50–70% eosinophils, with the remaining
cells in all groups being predominantly macrophages with moderate
numbers of lymphocytes. Although the overall composition of granulomas
did not change, the total numbers of eosinophils recruited to the lungs
must be decreased in proportion to the reduction in granuloma size.
There was also no obvious difference in the amount of collagen
deposition in or around the granulomas, as determined by microscopic
analysis of sections stained with picrosirius red, which stains
collagen specifically (data not shown).
Total serum IgE titers were reduced significantly as a result of IL-13
blockade (Fig. 7A). However,
the reduction in total IgE Ab expression was only observed in mice that
were treated with the sIL-13R2-Fc both at the time of sensitization
and again at challenge. There was no significant reduction in IgE
titers in mice that were treated with the sIL-13R2-Fc only at the
time of sensitization or during the i.v. challenge alone (data not
shown). Marked SEA-specific IgG1 and IgG2b Ab responses were also
induced by schistosome eggs; however, no significant differences in Ab
titers were detected among the two groups (Fig. 7B).
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While the role of IL-4 in regulating granuloma formation is still
incompletely understood (6), IL-4 clearly plays a key role in IgE
production (47), and most likely indirectly influences IL-5, and thus
eosinophil differentiation through its role as a differentiation factor
for CD4+ Th2 cells (1). Thus, IL-13 blockade could
indirectly affect granuloma formation and IgE production by
down-regulating IL-4 responses. It should be noted, however, that
sIL-13R2-Fc does not block IL-4 activity directly (34). To rule out
the possibility that sIL-13R2-Fc was playing an indirect role by
modulating IL-4 levels, LALN cells from egg-challenged control-Fc- and
sIL-13R2-Fc-treated mice were examined for IL-4 production after
restimulation with SEA or Con A. As was observed with the other
Th2-associated cytokines, continuous in vivo treatment with the
sIL-13R2-Fc had no significant effect on the capacity of LALN cells
to produce IL-4 at any time point (Fig. 5). Comparable findings were
also observed with spleen cells obtained from similarly treated animals
(data not shown).
Studies were also conducted in IL-4-deficient mice to examine whether
IL-13 production was modulated or controlled by IL-4. As shown in Fig. 8, Ag-specific IL-13 production was
decreased almost 10-fold in IL-4-deficient mice when compared with the
levels observed in WT egg-injected animals. A modest IL-13 response was
detected, however, in the IL-4-deficient animals, confirming that a
low, but significant, IL-13 response can develop in the absence of IL-4
(22, 23, 24).
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production
While previous studies have demonstrated an important role
for IL-4 in granuloma formation (20, 21), studies in IL-4-deficient
mice have shown that reduced, but significant, egg-induced inflammatory
responses persist in these animals (22, 23, 48). Because IL-13
production is maintained at reduced levels in these mice (Fig. 8), we
wanted to determine whether the low-grade inflammatory response was
influenced by this cytokine. In these experiments, IL-4-deficient mice
were sensitized and challenged i.v. with eggs. The knockout mice were
treated throughout the period of sensitization and again at challenge
with either control-Fc or sIL-13R2-Fc. Granuloma formation in both
groups was compared with the levels observed in sensitized WT animals.
While granuloma size was reduced in IL-4-deficient animals, lesion
formation was almost completely abrogated in sIL-13R2-Fc-treated
IL-4 knockout mice (Fig. 9A). Even more
striking was the complete absence of eosinophils in the small
inflammatory foci of doubly-deficient animals (Fig. 9B).
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,
IFN- production was partially up-regulated in control-Fc-treated
IL-4-deficient mice, while IL-5 production was almost completely
ablated. Interestingly, the sIL-13R2-Fc-treated IL-4-deficient mice
showed an even more striking increase in their SEA-specific IFN-
response, suggesting that IL-4 and IL-13 may act together to suppress
type 1 cytokine expression. The latter findings may at least in part
explain the marked reduction in granuloma formation and tissue
eosinophilia observed in the double IL-4/IL-13-deficient animals (Fig. 9A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-Fc to block IL-13 activity in vivo and then examined in
detail the effects on the evolving Th2-associated egg response.
Different protocols were explored so that we could determine whether
IL-13 blockade either would influence the development of a Th2-mediated
inflammatory response or could be used to modulate established
responses. In naive mice, granulomas peak in size at about 2 wk after
i.v. challenge with eggs (21). In these animals, treatment with the
sIL-13R2-Fc reduced peak granuloma volume by more than 50%. In
separate studies, mice were sensitized i.p. with eggs to establish a
polarized Th2 response and then challenged i.v. with eggs and treated
with the sIL-13R2-Fc at the time of challenge or during both the
period of sensitization and challenge. In these experiments, granuloma
size was routinely reduced between 28 and 50% (Fig. 4). Interestingly,
the magnitude of the reduction approached the levels observed in
IL-4-deficient animals (Fig. 9A). Most striking, however,
was the almost complete abrogation of granulomatous inflammation in
IL-4-deficient animals after IL-13 blockade. The collective results
from all of the blocking studies clearly show that IL-13 plays a role
in the egg-induced inflammatory response.
Schistosome granulomas are rich in eosinophils, and egg deposition is
accompanied by marked increases in serum IgE. Interestingly, previous
studies have established that IL-13 exhibits chemotactic activity for
human eosinophils and may also play a role in their survival by
stimulating the production of IL-3 and granulocyte-macrophage CSF (49).
IL-13 has also been shown to induce IgG4 and IgE synthesis, and directs
IgE isotype switching in human B cells (32). Thus, IL-13 may also
participate in other aspects of the egg-induced Th2 response. We
therefore wanted to determine whether the altered granulomatous
response in sIL-13R2-Fc-treated mice was accompanied by changes in
eosinophil accumulation or IgE production. Although granuloma size was
reduced in both naive and sensitized mice by IL-13 blockade, the
cellular phenotype of the lesions was not significantly different.
Nevertheless, the total number of eosinophils in the lungs was
decreased since the lesions in sIL-13R2-Fc-treated mice were
smaller.
Total serum IgE responses were also significantly reduced in
egg-injected sIL-13R2-Fc-treated mice. This finding, however, was
restricted to animals treated with IL-13R2-Fc both during
sensitization and at the time of challenge (Fig. 7A). There
was no effect on IgE levels in animals that had been treated with the
IL-13 antagonist only at the time of challenge (data not shown). This
was surprising since, in contrast to its effects on human Ab responses,
IL-13 is not believed to induce IgE production by mouse cells (26). It
is possible, however, that the reduced IgE levels are more a
consequence of the overall reduction in the host’s reaction to the
eggs rather than a direct effect of IL-13 on B cell Ig
production. This conclusion seems likely since mouse B cells are
believed to lack functional IL-13R (26). Nevertheless, a novel
IL-4-independent pathway for IgE switching in the mouse was described
(50) that might be influenced by IL-13, and recent studies in IL-13
transgenic animals have confirmed an IL-4-independent role for IL-13 in
regulating IgE responses (51).
To begin to address the mechanism behind the reduced granulomatous
response in sIL-13R2-Fc-treated mice, we first examined whether
IL-13 blockade altered the cytokine response. Modulation of granuloma
formation is often accompanied by changes in the Th1/Th2-type cytokine
response (52). Although in vitro studies established that IL-13 does
not directly affect CD4+ T cell differentiation as does
IL-4 (53), the cytokine could indirectly influence T cell
differentiation in vivo by affecting Ag presentation. IL-13 was
described initially as a potent down-regulatory cytokine for macrophage
function and, like IL-4 and IL-10 (54, 55), modulates cytokine
production (56, 57) as well as MHC class II expression in macrophages
(58). To determine whether there was any effect on Th1/Th2-type
cytokine production, we isolated LALN and spleen cells from
egg-injected/sIL-13R2-Fc-treated mice and examined IFN-
production as a marker of a Th1-type response, and IL-4, IL-5, and
IL-13 as markers of a Th2-type response. Although we observed modest
increases in IFN- production in sIL-13R2-Fc-treated primary
egg-challenged mice (data not shown), this finding was not observed in
any of the secondary challenge experiments. At no time point did we
detect any significant difference in cytokine production by cultured
lymphocytes from egg-sensitized mice, even after continuous treatment
with sIL-13R2-Fc (Fig. 5).
We also examined the cytokine mRNA response at the site of granuloma
formation and extended our analysis to include TNF- and IL-1ß, two
inflammatory cytokines induced in both infected (21) and egg-injected
mice (39). TNF- and IL-1ß are both believed to play a role in
granuloma formation (41, 42), and in vitro experiments have
demonstrated that IL-13 decreases their expression in LPS-activated
macrophages (56). Therefore, the effects of IL-13 could be focused more
on the regulation of inflammatory mediators than on the production of
Th2-associated cytokines. As shown in Fig. 6, however, we detected no
significant alteration in the cytokine mRNA responses after treatment
with sIL-13R2-Fc.
IL-4 has clearly been shown to be a key mediator of pulmonary granuloma
formation, and parasite-induced IgE responses are believed to be almost
entirely dependent upon IL-4 (18, 47, 59). Therefore, although IL-13
blockade does not directly inhibit IL-4 activity, it was possible that
even modest effects on IL-4 production could explain the reduction in
granuloma formation and IgE production in the sIL-13R2-Fc-treated
mice. To rule out the possibility that IL-13 blockade was indirectly
affecting IL-4 levels, we examined IL-4 production at the site of
granuloma formation (Fig. 6), as well as in the draining lymph nodes
and spleens of both control-Fc- and sIL-13R2-Fc-treated mice at
multiple time points (Fig. 6A). In all of our studies in WT
mice, however, we failed to detect any significant effect on the IL-4
response.
In contrast to these findings, however, IL-13 production was highly
dependent upon IL-4. SEA-specific IL-13 production was reduced almost
10-fold in egg-injected IL-4-deficient mice (Fig. 6). These findings
are thus consistent with the well-established role of IL-4 as the key
differentiation factor for Th2 cells (1). Nevertheless, significant
IL-13 was detected in the culture supernatants of LALN cells obtained
from IL-4-deficient mice. Previous studies have also demonstrated the
maintenance of low-level Th2 responses in IL-4-deficient animals
(20, 21, 22, 23, 24). Interestingly, we showed that this reduced IL-13 response was
in large part responsible for the granulomatous response of
IL-4-deficient mice. Moreover, the lesions in double-deficient mice,
unlike their single cytokine-deficient counterparts, were also
completely devoid of eosinophils. These findings further emphasize the
IL-4-independent effects of IL-13 in the granulomatous response and
suggest that IL-13 may act at the effector end of the Th2 response.
Previous studies addressing the role of IL-13 relied primarily on in
vitro studies to examine its role in immunoregulation (26). The
development of the sIL-13R2-Fc provided a novel approach for
studying the effects of IL-13 in vivo. The combined results from this
study demonstrate for the first time that IL-13 is an important factor
in Th2-mediated inflammation in the lung. The data showing that
Th1/Th2-type cytokine responses were not significantly affected by
IL-13 blockade suggest that IL-13 may in fact be a key effector
cytokine for granuloma formation. Thus, unlike IL-4, whose main role
may be as a differentiation factor for egg-induced Th2 responses, IL-13
may play more of a direct role in the inflammatory response. The
transient nature of IL-4 mRNA expression and the more sustained IL-13
response at the site of lesion formation might support such a
hypothesis (Fig. 2). Indeed, IL-13 has
been shown to regulate adhesion molecule expression on human
endothelial cells (60), and the expression of ICAM-1 in particular is
important to granuloma development (61). The fact that granuloma
formation and tissue eosinophilia were only partially reduced by IL-4
or IL-13 deficiencies, but were nearly completely ablated in
IL-4-deficient/sIL-13R2-Fc-treated mice, suggests that both
cytokines are equally important to the overall granulomatous response.
The observation that IFN- production was partially up-regulated in
IL-4-deficient mice, and even more so in double IL-4/IL-13-deficient
animals, might suggest an alternative hypothesis whereby IL-4 and IL-13
act together to suppress the antiinflammatory type 1 cytokine response
(Fig. 10). Regardless of the exact mechanism, these findings are
particularly important for schistosomiasis, since it is chronic
egg-associated pathology that leads to the development of severe
disease in infected humans. Moreover, these data may also extend to
other infectious diseases (62), asthma (63), or allergic-type reactions
(64), in which Th2 responses and the production of IL-13 are linked
with pathology.
|
Acknowledgments |
|---|
2-Fc, Pat Caspar
for performing the interleukin-4 bioassays, and Fred Lewis from the
Biomedical Research Institute for providing schistosome eggs. We also
grateful to Alan Sher, Karl Hoffmann, and Joe Sypek for their helpful
comments and suggestions. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Thomas A. Wynn, Immunobiology Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, 9000 Rockville Pike, Building 4/126, Bethesda, MD 20892-0425.
3 Abbreviations used in this paper: sIL-13R2-Fc, soluble IL-13 receptor 2-Fc fusion protein; HPRT, hypoxanthine-guanine phosphoribosyl transferase; LALN, lung-associated lymph node; SEA, soluble egg antigen; WT, wild type.
Received for publication May 21, 1998. Accepted for publication September 25, 1998.
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P. G. Fallon, E. J. Richardson, G. J. McKenzie, and A. N. J. McKenzie Schistosome Infection of Transgenic Mice Defines Distinct and Contrasting Pathogenic Roles for IL-4 and IL-13: IL-13 Is a Profibrotic Agent J. Immunol., March 1, 2000; 164(5): 2585 - 2591. [Abstract] [Full Text] [PDF]
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P. W. Mola, I. O. Farah, T. M. Kariuki, M. Nyindo, R. E. Blanton, and C. L. King Cytokine Control of the Granulomatous Response in Schistosoma mansoni-Infected Baboons: Role of Exposure and Treatment Infect. Immun., December 1, 1999; 67(12): 6565 - 6571. [Abstract] [Full Text] [PDF]
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D. Jankovic, M. C. Kullberg, N. Noben-Trauth, P. Caspar, J. M. Ward, A. W. Cheever, W. E. Paul, and A. Sher Schistosome-Infected IL-4 Receptor Knockout (KO) Mice, in Contrast to IL-4 KO Mice, Fail to Develop Granulomatous Pathology While Maintaining the Same Lymphokine Expression Profile J. Immunol., July 1, 1999; 163(1): 337 - 342. [Abstract] [Full Text] [PDF]
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N. Noben-Trauth, W. E. Paul, and D. L. Sacks IL-4- and IL-4 Receptor-Deficient BALB/c Mice Reveal Differences in Susceptibility to Leishmania major Parasite Substrains J. Immunol., May 15, 1999; 162(10): 6132 - 6140. [Abstract] [Full Text] [PDF]
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A. W. Cheever, R. W. Poindexter, and T. A. Wynn Egg Laying Is Delayed but Worm Fecundity Is Normal in SCID Mice Infected with Schistosoma japonicum and S. mansoni with or without Recombinant Tumor Necrosis Factor Alpha Treatment Infect. Immun., May 1, 1999; 67(5): 2201 - 2208. [Abstract] [Full Text] [PDF]
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 I.-C. HO, J.I. KIM, S.J. SZABO, and L.H. GLIMCHER Tissue-specific Regulation of Cytokine Gene Expression Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 573 - 584. [Abstract] [PDF]
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