Soluble ST2 Blocks Interleukin-33 Signaling in Allergic Airway Inflammation

Abstract

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively.
A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type
2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production
of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects
of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble
ST2 directly bound to IL-33 and suppressed activation of NF-κB in EL-4 cells stably expressing ST2L, suggesting that the complex
of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production
of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator
of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates
the biological activity of IL-33 in allergic airway inflammation.

Full text

Soluble ST2 Blocks Interleukin-33 Signaling in Allergic
Airway Inflammation
*
□
S
Received for publication, June 14, 2007, and in revised form, June 29, 2007 Published, JBC Papers in Press, July 10, 2007, DOI 10.1074/jbc.M704916200
Hiroko Hayakawa
‡
, Morisada Hayakawa
‡1
, Akihiro Kume
§
, and Shin-ichi Tominaga
‡
From the
‡
Department of Biochemistry and
§
Division of Genetic Therapeutics, Jichi Medical University, 3311-1 Yakushiji,
Shimotsuke-shi, Tochigi 329-0498, Japan
The ST2 gene produces a soluble secreted form and a trans-
membrane form, referred to as soluble ST2 and ST2L, respec-
tively. A recent study has reported that interleukin (IL)-33 is a
specific ligand of ST2L and induces production of T helper type
2 (Th2) cytokines. Although soluble ST2 is highly produced in
sera of asthmatic patients and plays a critical role for production
of Th2 cytokines, the function of soluble ST2 in relation to IL-33
signaling remains unclear. Here we show antagonistic effects of
soluble ST2 on IL-33 signaling using a murine thymoma EL-4
cells stably expressing ST2L and a murine model of asthma. Sol-
uble ST2 directly bound to IL-33 and suppressed activation of
NF-
␬
B in EL-4 cells stably expressing ST2L, suggesting that the
complex of soluble ST2 and IL-33 fails to bind to ST2L. In a
murine model of asthma, pretreatment with soluble ST2
reduced production of IL-4, IL-5, and IL-13 from IL-33-stimu-
lated splenocytes. These results indicate that soluble ST2 acts as
a negative regulator of Th2 cytokine production by the IL-33
signaling. Our study provides a molecular mechanism wherein
soluble ST2 modulates the biological activity of IL-33 in allergic
airway inflammation.
The interleukin (IL)-1
2
receptor family plays important roles
in inflammatory and immunological responses. The ST2 gene is
a member of the IL-1 receptor family, producing a soluble
secreted form and a transmembrane form, soluble ST2 and
ST2L, respectively (1–3). These proteins are generated by alter-
native splicing of pre-mRNA. The structure of ST2L is similar
to that of IL-1 receptor type I (IL-1RI), consisting of three extra-
cellular immunoglobulin domains and an intracellular Toll-
interleukin-1 receptor domain. Although the extracellular
domain is common to soluble ST2 and ST2L, soluble ST2 lacks
the transmembrane and intracellular Toll-interleukin-1 recep-
tor domains. The ST2 gene is expressed in several cells includ-
ing fibroblasts and mast cells (1, 4). In particular, ST2L is pref-
erentially expressed in murine and human Th2 cells and can be
utilized as a specific marker of Th2 cells in in vitro experiments
(5–8). Therefore, the function of ST2L has been suggested to
correlate with Th2 cell-mediated immunological responses.
However, ST2L has been an orphan receptor ever since it was
first reported (5). Late in 2005, IL-33, a newly discovered mem-
ber of the IL-1 cytokine family, was finally reported as a specific
ligand for ST2L (9).
The IL-33 gene, also described as a nuclear factor expressed
in high endothelial venules (NF-HEV) (10), codes a 31-kDa pro-
tein that does not contain a signal sequence for secretion, sim-
ilar to the IL-1
␣
, IL-1
␤
, and IL-18 genes (11, 12). Previous study
has demonstrated the processing and function of the IL-33 pro-
tein (9). The precursor 31-kDa protein (pre-IL-33) was cleaved
by caspase-1 into a mature 18-kDa protein (IL-33) in in vitro
experiments using a recombinant protein. Functional analysis
has shown that IL-33 bound to murine mast cells expressing
ST2L and stimulated the intracellular signaling pathway, lead-
ing to the activation of NF-
␬
B and mitogen-activated protein
kinases. In addition, the production of Th2 cytokines and
severe pathological changes in mucosal organs were induced by
administration of IL-33 to mice. Previous studies before the
discovery of IL-33 had already shown that ST2L is associated
with the production of Th2 cytokines. Levels of Th2 cytokines
were decreased in asthmatic mice by administration of the anti-
body that blocks ST2L and in a pulmonary granuloma model
using mice lacking the ST2 gene (7, 13). Therefore, these results
suggest that IL-33 signaling via ST2L plays important roles in
Th2 cell-mediated immunological responses including the pro-
duction of Th2 cytokines.
On the other hand, previous studies in human patients and
animal models have shown that the level of soluble ST2 in sera
was elevated in asthmatic disease (14, 15). Therefore, it has
been suggested that soluble ST2 may also play a critical role in
Th2 cell-mediated diseases. In fact, administration of a recom-
binant soluble ST2-Fc fusion protein or a soluble ST2 expres-
sion vector to asthmatic mice effectively attenuated inflamma-
tory responses and production of Th2 cytokines (7, 15). These
results of therapeutic experiments indicate that soluble ST2
negatively regulates the Th2 cell-mediated immunological
responses, in opposition to ST2L. However, the molecular
mechanism of negative regulation by soluble ST2 remains
unclear. In addition, it has not been addressed whether soluble
ST2 is associated with IL-33 signaling.
In this study, using a murine thymoma cell line, EL-4, stably
expressing ST2L and a murine model of asthma, we demon-
strated that soluble ST2 had a negative function in IL-33 signal-
* This work was supported by a Grant-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology of Japan. The costs of publication
of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1 and S2.
1
To whom correspondence should be addressed. Tel.: 81-285-58-7324; Fax:
81-285-44-2158; E-mail: morisada@jichi.ac.jp.
2
The abbreviations used are: IL, interleukin; Th, T helper; IFN-
␥
, interferon-
␥
;
OVA, ovalbumin; NF-
␬
B, nuclear factor-
␬
B; I
␬
B, inhibitor of NF-
␬
B; PBS,
phosphate-buffered saline; FITC, fluorescein isothiocyanate; RPE, R-phyco-
erythrin; EMSA, electrophoretic mobility shift assay; ELISA, enzyme-linked
immunosorbent assay; SAL, saline.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 36, pp. 26369 –26380, September 7, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
SEPTEMBER 7, 2007 •VOLUME 282• NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 26369
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ing. Binding and functional analyses showed that soluble ST2
inhibited the binding of IL-33 to ST2L-positive cells and that
the activation of NF-
␬
B and the production of Th2 cytokines in
the IL-33 signaling were suppressed in the presence of soluble
ST2. Our data suggest that soluble ST2 negatively modulates
the production of Th2 cytokines through IL-33 signaling in
allergic airway inflammation.
EXPERIMENTAL PROCEDURES
Animals—Male and female BALB/c mice, 7– 8 weeks of age,
were purchased from Japan SLC, Inc. (Shizuoka, Japan). All of
the mice were housed in an animal research facility of the Jichi
Medical University under pathogen-free conditions. All of the
experimental procedures were approved by the Animal
Research Ethics Board of Jichi Medical University.
Sensitization and Aeroallergen Challenge—The mice were
sensitized by intraperitoneal injection with 100
␮
g of ovalbu-
min (OVA) (Sigma-Aldrich) and 20 mg of aluminum potassium
sulfate (Sigma-Aldrich) in saline or 20 mg of aluminum potas-
sium sulfate alone in saline on days 0 and 7. On days 14 and 15,
the mice were challenged twice daily at intervals of 4 h with 1%
(w/v) OVA in saline or saline alone for 30 min using an ultra-
sonic nebulizer (Omron Corp., Tokyo, Japan) (15). The mice
were sacrificed 3– 48 h after the last aeroallergen challenge, and
sera and tissues were obtained for further analyses. Briefly,
blood was drawn from the caudal vena cava and left for 30 min,
and then the serum was separated by centrifugation. The sera
were stored at ⫺80 °C until assay.
Cell Culture—Human embryonic kidney HEK293T cells
were cultured in Dulbecco’s modified Eagle’s medium (Sigma-
Aldrich) supplemented with 10% fetal bovine serum (Thermo
Electron, Melbourne, Australia). Murine thymoma EL-4 cells
were cultured in RPMI 1640 medium (Sigma-Aldrich) supple-
mented with 5% fetal bovine serum (Sigma-Aldrich) and 50
␮
M
2-mercaptoethanol (RPMI 1640 growth medium).
Reverse Transcription-PCR Analysis—Total RNAs were iso-
lated from murine tissues using TRI reagent (Sigma-Aldrich).
The total RNA was treated with RNase-free DNase I; then first-
strand cDNA was synthesized as described previously (16). PCR
amplification was performed using 0.5 units of AmpliTaq Gold
DNA polymerase (Applied Biosystems, Foster, CA), 0.5
␮
M
each of the forward and reverse primers, and the first-strand
cDNAs derived from 0.25
␮
g of DNase I-treated RNA. After
cDNAs were treated at 94 °C for 10 min, PCR was carried out
for 25 (
␤
-actin), 28 (IL-33), or 33 (ST2 and ST2L) cycles at 94 °C
for 1 min, 60 °C for 1 min, and 72 °C for 1.5 min, followed by
treatment at 72 °C for 10 min. The nucleotide sequences of
primers used were as follows: ST2, forward 5⬘-TGGCATGAT-
AAGGCACACCATAAGGCT-3⬘ and reverse 5⬘-GTTAGTG-
TCTCTCTCCCTCCCATGC-3⬘; ST2L, forward 5⬘-TGCGTA-
CATCATTTACCCTCGGGTC-3⬘ and reverse 5⬘-TCTTGTG-
CCACAAGAGTGAAGTAGG-3⬘; IL-33, forward 5⬘-ATGA-
GACCTAGAATGAAGTATTCCA-3⬘ and reverse 5⬘-TTA-
GATTTTCGAGAGCTTAAACATA-3⬘; and
␤
-actin, forward
5⬘-ATCTACGAGGGCTATGCTCT-3⬘ and reverse 5⬘-TACT-
CCTGCTTGCTGATCCA-3⬘. Seven microliters of PCR prod-
ucts were developed by electrophoresis on 2% agarose gels, and
then the gels were stained with ethidium bromide. The inten-
sity of DNA bands was quantified using the public domain NIH
Image program (developed at the United States National Insti-
tutes of Health). The size of PCR products was as follows: ST2
(754 bp), ST2L (739 bp), IL-33 (801 bp), and
␤
-actin (576 bp).
Construction of Plasmids—Constructions of pET-21-mIL-33
and pET-21-mIL-1
␤
proceeded as follows. The coding regions
of mature IL-33 and IL-1
␤
proteins were obtained from cDNA
derived from spleens of BALB/c mice by PCR amplification.
The nucleotide sequences of primers containing an EcoRI or an
XhoI site were as follows: IL-33, forward 5⬘-GAATTCACAT-
TGAGCATCCAAGGAAC-3⬘ and reverse 5⬘-CTCGAGGAT-
TTTCGAGAGCTTAAACA-3⬘; IL-1
␤
, forward 5⬘-GAATTC-
GTTCCCATTAGACAGCTGCA-3⬘ and reverse 5⬘-CTCGA-
GGGAAGACACAGATTCCATGG-3⬘. PCR products were
digested with EcoRI and XhoI and then ligated into an EcoRI/
XhoI-digested pET-21a (⫹) vector (Novagen, Madison, WI).
Expression vectors of murine soluble ST2 were constructed
using pEF6-V5-His (Invitrogen) and pEF6-FLAG-His. Con-
struction of pEF6-FLAG-His proceeded as follows. A fragment
containing FLAG and His tags (FLAG-His) was created by
annealing a sense oligonucleotide (5⬘-GCGGCCGCTGACTA-
CAAGGATGACGATGACAAGCGTACCGGTC-3⬘) and an
antisense oligonucleotide (5⬘-GTTTAAACTCAATGGTGAT-
GGTGATGATGACCGGTACGCTTGT-3⬘), and the annealed
fragment was elongated and subcloned into a pCR2.1 TOPO
(Invitrogen). A NotI/PmeI-digested FLAG-His fragment was
ligated into a NotI/PmeI-digested pEF6-V5-His. Murine ST2
(mST2) cDNA was amplified from pEF-BOS-mST2 (17) by
PCR using a forward primer containing a KpnI site (5⬘-GG-
TACCATGATTGACAGACAGAGAAT-3⬘) and a reverse
primer containing a NotI site (5⬘-GCGGCCGCAGCAATGT-
GTGAGGGACACT-3⬘). A KpnI/NotI-digested PCR product
was ligated into a KpnI/NotI-digested pEF6-V5-His and pEF6-
FLAG-His to obtain final products pEF6-mST2-V5-His and
pEF6-mST2-FLAG-His, respectively. Constructions of pEF6-
mST2L-FLAG and pEF6-mIL-1RI-FLAG proceeded as follows.
Murine ST2L (mST2L) cDNA was amplified from pEF-BOS-
mST2L (5) by PCR using a forward primer containing a BamHI
site (5⬘-GGATCCATGATTGACAGACAGAGA-3⬘) and a
reverse primer containing a NdeI site (5⬘-CATATGAAAGT-
GTTTCAGGTCTAA-3⬘). The PCR product was subcloned
into a pCR2.1 TOPO (pCR2.1-mST2L). A FLAG fragment con-
taining a stop codon was created by annealing FLAG-s (5⬘-TA-
TGGATTACAAGGATGACGACGATAAGTAGA-3⬘) and
FLAG-as (5⬘-CTAGTCTACTTATCGTCGTCATCCTTGT-
AATCCA-3⬘), and the annealed fragment was ligated into a
NdeI/SpeI-digested pCR2.1-mST2L. Finally, the mST2L-
FLAG fragment was digested with BamHI and SpeI and ligated
into a BamHI/SpeI-digested pEF6/V5-His. Murine IL-1RI
(mIL-1RI) cDNA was obtained from cDNA derived from EL-4
cells by PCR amplification using primers containing a KpnI or a
NdeI site (forward 5⬘-GGTACCATGGAGAATATGAAAGT-
GCTA-3⬘ and reverse 5⬘-CATATGGCCGAGTGGTAAGTG-
TGTTGC-3⬘). Murine ST2L cDNA in pEF6-mST2L-FLAG was
replaced with mIL-1RI cDNA, using KpnI and NdeI digestion.
All of the constructs were confirmed to be correct by DNA
sequencing analysis.
Suppression of IL-33 Signaling by Soluble ST2
26370 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 36 •SEPTEMBER 7, 2007
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Purification of Recombinant IL-33 and IL-1
␤
Proteins—Re-
combinant murine IL-33 and IL-1
␤
proteins containing a T7
tag at the N terminus and a His tag at the C terminus (rIL-33
and rIL-1
␤
) were produced in bacteria. BL-21 Codon-Plus
(DE3)-RIL (Stratagene, La Jolla, CA) was transformed with
pET-21-mIL-33 or pET-21-mIL-1
␤
. The bacteria were cul-
tured at 37 °C until the A
600
reached 0.6; then expression of
recombinant protein was induced by the addition of isopropyl
␤
-D-thiogalactopyranoside to a final concentration of 1 mM.
Three hours after culture at 25 °C, the bacteria were harvested,
and the pellets were resuspended in lysis buffer (50 m
M
Na
2
HPO
4
, 300 mM NaCl, 10 mM imidazole) containing 1 mg/ml
lysozyme. After sonication, the soluble cytoplasmic fraction
was isolated by centrifugation. The fraction was loaded onto a
nickel-nitrilotriacetic acid-agarose (Qiagen) column. The pro-
teins were eluted with elution buffer (50 m
M Na
2
HPO
4
, 300 mM
NaCl, 250 mM imidazole). After dialysis against T7 tag binding
buffer (4.3 m
M Na
2
HPO
4
, 1.5 mM KH
2
PO
4
, 2.7 mM KCl, 137 mM
NaCl, 0.1% (v/v) Tween 20, pH 7.3), the proteins were purified
using a T7 tag affinity purification kit (Novagen). The proteins
were eluted from the column with 0.1
M citric acid (pH 2.2) and
neutralized with 2
M Tris base (pH 10.4). After desalting and
concentrating the protein using Centricon YM-3 (Millipore,
Bedford, MA), the purified proteins were dialyzed against PBS
(8 m
M Na
2
HPO
4
, 1.5 mM KH
2
PO
4
, 2.7 mM KCl, 137 mM NaCl).
The protein concentration was determined by the Bradford
method using protein assay dye reagent (Bio-Rad) with calibra-
tion using bovine serum albumin (Sigma-Aldrich). The protein
purity was evaluated using a silver staining kit (Daiichi Pure
Chemicals, Tokyo, Japan).
Purification of Recombinant Soluble ST2 Proteins—Recom-
binant soluble ST2 proteins containing V5-His or FLAG-His
tags at the C terminus (ST2-V5 and ST2-FLAG) were purified
as described previously (18). Briefly, HEK293T cells were tran-
siently transfected with pEF6-mST2-V5-His or pEF6-mST2-
FLAG-His using the calcium-phosphate method. Sixteen
hours after transfection, the cells were cultured in serum-
free Dulbecco’s modified Eagle’s medium for 48 h. The
secreted recombinant proteins in the culture supernatant
were purified by affinity chromatography using nickel-ni-
trilotriacetic acid-agarose (Qiagen). The proteins were
eluted with 50 m
M sodium phosphate buffer (pH 8.0) con-
taining 300 m
M NaCl and 250 mM imidazole. Desalting and
concentrating the proteins were performed using Centricon
YM-30 (Millipore). Finally, the purified proteins were dia-
lyzed against PBS. The method for measuring the protein
concentration is described under “Measurement of Soluble
ST2 and Cytokines. ” Deglycosylation with N-glycosidase F
(Roche Applied Science) was performed as described previ-
ously (17). The protein purity was evaluated using a silver
staining kit.
Establishment of Stable Cell Lines—Empty vector (pEF6-V5-
His) and expression vectors (pEF6-mST2L-FLAG and pEF6-
mIL-1RI-FLAG) were linearized with FspI. EL-4 cells (1 ⫻ 10
7
cells) were mixed with 50
␮
g of linearized plasmid DNA in
serum-free RPMI 1640 medium, and the mixtures were left for
10 min on ice. Electroporation was carried out using a Gene
Pulser (Bio-Rad) at 270 V and 960 microfarads, and then the
cells were left on ice for 10 min. The transfected cells were
returned to the RPMI 1640 growth medium and were incubated
at 37 °C in 5% CO
2
. Forty-eight hours after transfection, the
transfected cells were selected with blasticidin (Invitrogen).
Stable clones were cultured in RPMI 1640 growth medium con-
taining 6
␮
g/ml blasticidin.
Flow Cytometry—Splenocytes (1 ⫻ 10
6
cells) and stably
transfected EL-4 cells (5 ⫻ 10
5
cells) were used for flow cyto
-
metric analysis. Preparation of splenocytes proceeded as fol-
lows. The spleen was homogenized into a single-cell suspension
in PBS by filtration through nylon mesh (70
␮
m). After deple-
tion of erythrocytes by osmotic lysis, the splenocytes were
washed with PBS and resuspended in PBS containing 5% fetal
bovine serum. Subsequently, anti-mouse CD16/CD32 antibody
(BD Biosciences PharMingen, San Diego, CA) was mixed with
the splenocytes to block the Fc receptor for 5 min on ice. Bind-
ing analysis of IL-33 and IL-1
␤
on the cell surface was per-
formed as follows. The cells were mixed with 100 or 500 ng of
rIL-33 or rIL-1
␤
for1honice, followed by staining with bio-
tinylated anti-T7 tag antibody (Novagen) for 1 h on ice. Then
the cells were stained with R-phycoerythrin (RPE)-conju-
gated streptavidin (DakoCytomation, Glostrup, Denmark)
for 30 min on ice. In case of binding analysis in the presence
of soluble ST2, 1
␮
g of ST2-V5 was added to the cells at 1 h
before, at 1 h after, or at the same time as the addition of
rIL-33 or rIL-1
␤
. Binding of ST2-V5 was detected with flu-
orescein isothiocyanate (FITC)-conjugated anti-V5 anti-
body (Invitrogen) for1honice. ST2L or IL-1RI was stained
with FITC-conjugated anti-mouse T1/ST2 antibody (MD
Biosciences, Zu¨rich, Switzerland), RPE-conjugated anti-
mouse IL-1RI antibody (BD Biosciences PharMingen), or
each isotype control antibody for1honice. After the stained
cells were washed twice with PBS containing 5% fetal bovine
serum, the cells were resuspended in PBS and filtered through
nylon mesh (35
␮
m). Analysis was performed on Becton Dick-
inson LSR using Cell Quest software (BD Biosciences).
Immunoprecipitation—Five hundred ng of ST2-V5 was
mixed with 2
␮
g of rIL-33 or rIL-1
␤
in 500
␮
l of RIPA buffer (50
m
M Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40,
0.5% (w/v) deoxycholate, 0.1% (w/v) SDS), and the mixture was
agitated overnight at 4 °C. The protein complexes were immu-
noprecipitated with 40
␮
l of 50% (v/v) slurry of anti-T7 tag
antibody-conjugated agarose (Novagen). After the protein-
bound agarose was washed three times with RIPA buffer, bind-
ing protein complexes were eluted with 0.1
M citric acid (pH
2.2) and neutralized with the addition of 2
M Tris (pH 10.4). The
eluted proteins were subjected to Western blotting.
Western Blotting—The protein samples were separated by
electrophoresis on SDS-polyacrylamide gels. The proteins were
transferred to a polyvinylidene difluoride membrane (Milli-
pore) and were probed with mouse monoclonal anti-T7 tag
(Novagen), rabbit polyclonal anti-IL-33 (Adipogen Inc., Seoul,
South Korea), goat polyclonal anti-IL-1
␤
(Santa Cruz Biotech-
nology, Santa Cruz, CA), mouse monoclonal anti-V5 (Invitro-
gen), mouse monoclonal anti-His (C Term) (Invitrogen), rabbit
polyclonal anti-I
␬
B
␣
(Santa Cruz), or mouse monoclonal anti-
glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz) anti-
body as the primary antibody. The proteins were detected with
Suppression of IL-33 Signaling by Soluble ST2
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horseradish peroxidase-conjugated goat anti-mouse Ig (Bio-
Rad), horseradish peroxidase-conjugated horse anti-goat Ig
(Vector, CA), or horseradish peroxidase-conjugated donkey
anti-rabbit Ig (Amersham Biosciences) as the secondary anti-
body. The proteins were visualized using Immobilon Western
detection reagents (Millipore), and the membranes were
exposed to x-ray films (RX-U; Fuji Photo Film Co., Tokyo,
Japan).
Preparation of Cytoplasmic and Nuclear Extracts—Prepara-
tion of cytoplasmic and nuclear extracts from stably transfected
EL-4 cells was performed as described previously (16). Briefly,
the cells were harvested and rinsed with PBS. The cell pellets
were resuspended in 5 volumes of a buffer solution (10 m
M
Hepes-KOH, pH 8.0, 10 mM KCl, 1.5 mM MgCl
2
,1mM dithio
-
threitol). After addition of Nonidet P-40 to a final concentra-
tion of 0.1% (v/v), cytoplasmic extracts were separated by cen-
trifugation. The nuclei were resupended in 2.5 volumes of a
buffer solution (20 m
M Hepes-KOH, pH 8.0, 420 m M KCl, 1.5
m
M MgCl
2
, 0.2 mM EDTA, 25% (v/v) glycerol, 1 mM dithiothre
-
itol, 0.5 m
M phenylmethylsulfonyl fluoride) and mixed by agi-
tation. The debris was removed by centrifugation, and the
supernatants were dialyzed against a buffer solution (20 m
M
Hepes, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol,
1m
M dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride).
After centrifugation, the supernatants were harvested as
nuclear extracts. The protein concentration was determined by
the Bradford method. Cytoplasmic and nuclear extracts were
stored at ⫺80 °C until assay.
Electrophoretic Mobility Shift Assay (EMSA)—EMSA was
performed as described previously, with some modifications
(16, 19). Binding reactions were performed at 30 °C for 30 min
in a total volume of 15
␮
l containing nuclear extracts (5
␮
gof
protein) and 20,000 cpm of
32
P-labeled oligonucleotide probe.
Supershift assay was carried out using antibodies against p50,
p52, p65, and c-Rel (Santa Cruz). The protein-DNA complexes
were separated on 4% nondenaturing polyacrylamide gels at
100 V in 0.5 ⫻ TGE (50 m
M Tris-HCl, pH 8.0, 380 mM glycine,
2m
M EDTA) at 4 °C. The dried gels were exposed to Imaging
plates (Fuji Photo Film Co.) and analyzed using Typhoon 9410
(Amersham Biosciences). The oligonucleotide probe contain-
ing an NF-
␬
B binding site was created by annealing NF-
␬
B-s
(5⬘-AGTTGAGGGGACTTTCCCAGGC-3⬘) and NF-
␬
B-as
(5⬘-AGCCTGGGAAAGTCCCCTCAAC-3⬘).
Luciferase Assay—The luciferase assay was performed as
described previously (16). EL-4 cells stably expressing ST2L or
IL-1RI (1 ⫻ 10
7
cells) were transfected with plasmid DNAs (40
␮
g of firefly luciferase reporter plasmid, pNF-
␬
B-Luc, contain-
ing an NF-
␬
B binding site in the promoter, and 4
␮
gofthe
Renilla luciferase reporter plasmid pRL-TK (Promega, Madi-
son, WI)) by electroporation. Twenty-four hours after transfec-
tion, the cells were washed and cultured in serum-free medium
for 15 h. The transfected cells were treated with 500 ng/ml of
ST2-V5 for 3 h, followed by stimulation with 10 ng/ml rIL-33 or
rIL-1
␤
for 24 h. Then the cells were harvested and subjected to
luciferase assay using a dual luciferase reporter assay system
(Promega). Luciferase activity was measured by a luminometer
(Lumat LB9507; Berthold Technologies, Bad Wildbad, Ger-
many). The firefly luciferase activity was normalized against the
respective Renilla luciferase activity.
Stimulation of Splenocytes—Splenocytes were prepared as
described under “Flow Cytometry” and resuspended in RPMI
1640 growth medium. Splenocytes (4 ⫻ 10
7
cells/well) were
stimulated with 200
␮
g/ml OVA for 48 h in 6-well plate. The
OVA-stimulated cells were washed with PBS and resuspended
with serum-free RPMI 1640 medium at 2.5 ⫻ 10
6
cells/well in
48-well plate. After incubation for 15 h, ST2-FLAG was added
to a final concentration of 500 ng/ml for 3 h. Then the cells were
stimulated with 10 ng/ml rIL-33 or rIL-1
␤
for 48 h. The culture
supernatant was harvested and stored at ⫺80 °C until assay.
Measurement of Soluble ST2 and Cytokines—The concentra-
tions of soluble ST2 in sera and purified recombinant proteins
were measured by a sandwich enzyme-linked immunosorbent
assay (ELISA) as described previously (15). The concentrations
of IL-4, IL-5, IL-13, and IFN-
␥
in culture supernatants were
measured using ELISA kits (BIOSOURCE International Inc.,
Camarillo, CA).
Statistical Analysis—The data are represented as the
means ⫾ S.E. The data were analyzed by the Turkey-Kramer
test. A value of p ⬍ 0.05 was considered to be significant.
RESULTS
Specific Binding of IL-33 to ST2L-positive Cells—To study the
binding and function of IL-33, we developed systems for the
expression and purification of mature IL-33 as a recombinant
protein (designated as rIL-33) (Fig. 1A). In this study, recombi-
nant mature IL-1
␤
(designated as rIL-1
␤
) was also used for
control experiments, because IL-1
␤
has binding activity for
IL-1RI, but not for ST2L. Recombinant IL-33 and IL-1
␤
con-
taining T7 and His tags were expressed in bacteria and sub-
jected to affinity purification. The purities of rIL-33 and rIL-1
␤
proteins were examined by silver staining and Western blotting
(Fig. 1A, a and b). Although rIL-33 was purified as a single band,
the purification product of rIL-1
␤
contained a cleaved product.
Next, to establish a clear analysis system for the binding of
IL-33, we generated cell lines stably expressing ST2L or IL-1RI
using EL-4 cells (Fig. 1B). Although ST2L was hardly detected
in cells stably transfected with an empty vector (EV/EL-4) or an
IL-1RI expression vector (IL-1RI/EL-4), remarkable expression
of ST2L was observed in ST2L expression vector-transfected
cells (ST2L/EL-4). In addition to a constitutive expression of
IL-1RI in EL-4 cells, IL-1RI was further expressed in IL-1RI/
EL-4 cells. We examined the binding activity of IL-33 using
these stably transfected cell lines (Fig. 1C). The ST2L/EL-4 cells
clearly shifted according to the increasing concentration of
rIL-33 (Fig. 1C, middle panel). Conversely, EV/EL-4 and
IL-1RI/EL-4 cells showed little change corresponding to the
expression of ST2L. These results demonstrate that IL-33 spe-
cifically binds to ST2L.
Inhibition of IL-33 Binding Activity by Soluble ST2—The
amino acid sequence of soluble ST2 except for 9 amino acids in
the C terminus is the same as that of the extracellular domain of
ST2L (1, 3). Therefore, it seemed possible that soluble ST2
might also bind to IL-33. To investigate this possibility, we gen-
erated recombinant soluble ST2 containing either V5 or FLAG
and His tags in the C terminus (designated as ST2-V5 or ST2-
Suppression of IL-33 Signaling by Soluble ST2
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FLAG). Recombinant soluble ST2, expressed in HEK293T cells
and secreted into the culture supernatant, was affinity-purified.
The purity was confirmed by silver staining (Fig. 2A). SDS-
PAGE analysis showed that purified recombinant soluble ST2
was detected as a single broad band of 55– 65 kDa because of
N-linked glycosylation. After deglycosylation with N-glycosi-
dase F, the molecular mass shifted to 37 kDa, corresponding to
an unmodified form. We tested whether soluble ST2 directly
interacted with IL-33 in vitro (Fig. 2B). ST2-V5 was mixed with
either rIL-33 or rIL-1
␤
, and protein complexes were immuno-
precipitated with anti-T7 tag antibody-conjugated agarose and
then eluted from agarose. The eluates were analyzed by West-
ern blotting using anti-V5 and anti-T7 tag antibodies. Analysis
of input was performed using the
anti-His antibody because both
recombinant proteins were His-
tagged. These experiments clearly
revealed that ST2-V5 specifically
bound to rIL-33 but not to rIL-1
␤
(Fig. 2B, compare lanes 3–5).
To further study the interaction
between soluble ST2 and IL-33, we
analyzed the binding of rIL-33 to
ST2L/EL-4 cells or rIL-1
␤
to
IL-1RI/EL-4 cells in the presence of
ST2-V5 (Fig. 2C). Stably transfected
EL-4 cells were either left untreated
or treated with ST2-V5 and then
added with either rIL-33 or rIL-1
␤
.
ST2-V5 inhibited the binding of
rIL-33 to ST2L/EL-4 cells (Fig. 2C,
upper panel). In contrast, rIL-1
␤
binding activity for IL-1RI/EL-4
cells was not affected in the pres-
ence of ST2-V5 (Fig. 2C, lower
panel). Next, we examined whether
the additive order of soluble ST2
influences the binding activity of
IL-33 (Fig. 2D). ST2-V5 was added
to ST2L/EL-4 cells before, after, or
at the same time as the addition of
rIL-33. When rIL-33 was added
prior to ST2-V5, the binding of
IL-33 was hardly influenced by the
treatment with ST2-V5 (Fig. 2D,
compare panels c and d). On the
other hand, the binding of IL-33 was
inhibited by the treatment with
ST2-V5 at the same time or before
the addition of rIL-33 (Fig. 2D, com-
pare panels c, e, and f). In addition,
the binding of ST2-V5 to ST2L/
EL-4 cells was not observed regard-
less of the binding of IL-33 in this
system. These results indicate that
soluble ST2 specifically binds to free
IL-33 and inhibits the binding activ-
ity of IL-33 for ST2L and that the
ST2/IL-33 complex cannot bind to ST2L-positive cells.
Suppression of IL-33-induced NF-
␬
B Activation by Soluble
ST2—To study the activation of NF-
␬
B by IL-33 signaling,
we examined the DNA binding activity of NF-
␬
B by EMSA and
the degradation of I
␬
B
␣
by Western blotting (Fig. 3A and sup-
plemental Fig. S1). Nuclear and cytoplasmic extracts were pre-
pared from rIL-33- or rIL-1
␤
-stimulated EL-4 cells. EMSA
clearly showed that intracellular responses in the IL-33 and
IL-1
␤
signalings were consistent with the expression of ST2L
and IL-1RI, respectively. Stimulation with rIL-33 specifically
induced the DNA binding activity of NF-
␬
B (Fig. 3A, panel a,
lane 6) and the degradation of I
␬
B
␣
(Fig. 3A, panel b, lane 5)in
ST2L-EL-4 cells. On the other hand, stimulation with rIL-1
␤
FIGURE 1. Binding analysis of IL-33 to ST2L-positive cells. A, analysis of purified rIL-33 and rIL-1
␤
.
Purified proteins (100 ng) were separated on SDS-12.5% polyacrylamide gels, followed by silver staining
(panel a) and Western blotting (WB) with anti-T7 tag (
␣
T7), anti-mouse IL-33 (
␣
IL-33), and anti-mouse IL-1
␤
(
␣
IL-1
␤
) antibodies (panel b). White and black arrowheads indicate rIL-33 and rIL-1
␤
, respectively. An
asterisk indicates a cleaved product of rIL-1
␤
. Protein size is indicated in kDa at the left. B, expression
analysis of ST2L and IL-1RI in EL-4 cells. Stably transfected EL-4 cells were generated by introduction of an
empty vector, an ST2L expression vector, or an IL-1RI expression vector (EV, ST2L,orIL-1RI). Stably trans-
fected EL-4 cells (5 ⫻ 10
5
cells) were stained with FITC-conjugated anti-mouse T1/ST2 antibody (magenta
line), RPE-conjugated anti-mouse IL-1RI antibody (green line), or each isotype control antibody (purple
line). The gray-filled histogram shows unstained cells. C, binding analysis of rIL-33 to EL-4 cells. Stably
transfected EL-4 cells (5 ⫻ 10
5
cells) were mixed with 100 ng (blue line) or 500 ng (red line) of rIL-33 for 1 h.
Binding of rIL-33 was detected with biotinylated anti-T7 tag antibody and RPE-conjugated streptavidin.
The gray-filled histogram shows unstained cells. B and C, the data of clone numbers 1-1-A-3 (EV/EL-4 cells),
1-2-G-12 (ST2L/EL-4 cells), and 3-2-A-8 (IL-1RI/EL-4 cells) are represented in each figure.
Suppression of IL-33 Signaling by Soluble ST2
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induced the activation of NF-
␬
B (Fig. 3A, panel a, lanes 4, 7, and
10) and the degradation of I
␬
B
␣
(Fig. 3A, panel b, lanes 3, 6, and
9) in all cell lines, using constitutively expressed IL-1RI. Next,
we investigated components of acti-
vated NF-
␬
B using specific antibod-
ies against NF-
␬
B. Supershift assay
showed that the p50 and p65 sub-
units were contained in the IL-33-
induced DNA/NF-
␬
B complex (Fig.
3B, lanes 3 and 5), as well as in the
IL-1
␤
-induced DNA/NF-
␬
B com-
plex (Fig. 3B, lanes 8 and 10).
To further investigate the effect
of soluble ST2 on IL-33 signaling,
we examined the DNA binding
activity of NF-
␬
B and the degrada-
tion of I
␬
B
␣
in the presence of solu-
ble ST2 (Fig. 3C and supplemental
Fig. S2). Stably transfected EL-4
cells were either left untreated or
treated with ST2-V5 and then were
left unstimulated or stimulated with
rIL-33 or rIL-1
␤
. In IL-33-stimu-
lated ST2L/EL-4 cells, the DNA/
NF-
␬
B complex was gradually
decreased as the concentration of
ST2-V5 increased (Fig. 3C, panel a,
lanes 5–7). In contrast, pretreat-
ment with ST2-V5 led to the repres-
sion of I
␬
B
␣
degradation (Fig. 3C,
panel b, lanes 4–6). On the other
hand, ST2-V5 did not affect the
NF-
␬
B activation and I
␬
B
␣
degra-
dation in the IL-1
␤
signaling (Fig.
3C, panel a, lanes 11–13, and panel
b, lanes 10 –12). In addition, we
examined NF-
␬
B-dependent lucif-
erase activity in IL-33- or IL-1
␤
-
stimulated EL-4 cells (Fig. 3D).
Stimulation with IL-33 effectively
induced NF-
␬
B-dependent lucif-
erase activity in ST2L/EL-4 cells.
The IL-33-induced luciferase
activity also slightly increased in
IL-1RI/EL-4 cells coincident with
low expression levels of ST2L.
Furthermore, pretreatment with
ST2-V5 reduced the IL-33-in-
duced luciferase activities in both
cell lines. On the other hand,
IL-1
␤
-induced luciferase activities
in both cell lines were not affected
by the addition of ST2-V5. These
results demonstrate that soluble
ST2 specifically suppresses the
activation of NF-
␬
B by IL-33 sig-
naling via ST2L.
Expression of Soluble ST2 and
ST2L in a Murine Model of Asthma—Previous studies have
shown that expressions of soluble ST2 and ST2L increased in
asthma. To investigate the expressions of soluble ST2 and ST2L
FIGURE 2. Binding analysis of soluble ST2 to IL-33. A, analysis of purified recombinant soluble ST2. Purified
ST2-V5 and ST2-FLAG (100 ng) were left untreated or treated with N-glycosidase F. The proteins were separated
on SDS-10% polyacrylamide gel, followed by silver staining. Glycosylated and deglycosylated proteins are
indicated by black and gray arrowheads, respectively. N-Glycosidase F (PNGase F) is indicated by a white arrow-
head. B, analysis of interaction between ST2-V5 and rIL-33, or rIL-1
␤
. ST2-V5 (500 ng) was mixed with rIL-33 or
rIL-1
␤
(2
␮
g) in RIPA buffer. The protein complexes were immunoprecipitated with anti-T7 tag antibody-
conjugated agarose (IP:
␣
T7). The proteins were eluted with 0.1 M citric acid (pH 2.2) and neutralized with 2 M Tris
(pH 10.4), followed by Western blotting (WB) with anti-V5 (
␣
V5) and anti-T7 tag (
␣
T7) antibodies. Input was analyzed
by Western blotting with anti-His antibody (
␣
His) using 1/20 volumes of reaction mixture. Single and double asterisks
indicate heavy and light chains of immunoglobulin, respectively. A and B, protein size is indicated in kDa at the left.
C, effect of ST2-V5 on binding activity of rIL-33 or rIL-1
␤
. Stably transfected EL-4 cells (5 ⫻ 10
5
cells) were either left
untreated or treated with ST2-V5 (1
␮
g) for 1 h, and then rIL-33 or rIL-1
␤
(100 ng) was admixed for 1 h. Binding of
rIL-33 or rIL-1
␤
was detected with biotinylated anti-T7 tag antibody and RPE-conjugated streptavidin. Upper panel,
binding of rIL-33 to ST2L/EL-4 cells (clone, 1-2-G-12). Lower panel, binding of rIL-1
␤
to IL-1RI/EL-4 cells (clone, 3-2-A-
8). Blue- and orange-lined histograms represent cells untreated or treated with ST2-V5, respectively. The gray-filled
histogram shows unstained cells. D, effect of difference of ST2-V5-additive order on IL-33 binding activity. Binding of
ST2-V5 was detected with FITC-conjugated anti-V5 antibody. The additive order of proteins was as follows: panel a,
no addition (⫺/ ⫺);panel b, ST2-V5 for 1 h alone (⫺/ST2); panel c, rIL-33 for 1 h alone (⫺/IL-33); panel d, rIL-33 for 1 h
prior to ST2-V5 for another 1 h (IL-33/ST2); panel e, ST2-V5 and rIL-33 at the same time for 1 h (ST2 ⫹ IL-33); panel f,
ST2-V5 for 1 h prior to rIL-33 for another 1 h (ST2/IL-33).
Suppression of IL-33 Signaling by Soluble ST2
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FIGURE 3. Suppression of IL-33-induced NF-
␬
B activation by soluble ST2. A, analysis of intracellular responses in the IL-33 and IL-1
␤
signalings. Stably
transfected EL-4 cells (2 ⫻ 10
7
cells) were either left unstimulated or stimulated with rIL-33 or rIL-1
␤
(10 ng/ml) for 30 min, followed by preparation of
cytoplasmic and nuclear extracts. Panel a, EMSA using nuclear extracts with a
32
P-labeled oligonucleotide probe containing an NF-
␬
B-binding site. The
DNA-protein complexes were separated on a 4% nondenaturing polyacrylamide gel. Panel b, detection of I
␬
B
␣
and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) in cytoplasmic extracts. Cytoplasmic extracts were separated on SDS-12.5% polyacrylamide gels, followed by Western blotting
with anti-I
␬
B
␣
and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. B, supershift assay using anti-NF-
␬
B antibodies. Nuclear extracts were
kept on ice for 1 h without antibody (lanes 2 and 7) or with a series of anti-NF-
␬
B antibodies (lanes 3– 6 and 8–11), and then the
32
P-labeled oligonu
-
cleotide probe was admixed. The mixture was kept at 30 °C for 30 min and then subjected to EMSA. C, suppression of DNA binding activity of NF-
␬
Bby
the addition of ST2-V5. Stably transfected EL-4 cells were either left untreated or treated with ST2-V5 (10 and 100 ng) for 3 h and then left unstimulated
or stimulated with rIL-33 or rIL-1
␤
for 30 min. After stimulation, cytoplasmic and nuclear extracts were prepared. Panel a, DNA binding activity of NF-
␬
B
was analyzed by EMSA. Panel b, degradation of I
␬
B
␣
was analyzed by Western blotting. Lanes 1 of A (panel a), B, and C (panel a) contained the
32
P-labeled
oligonucleotide probe alone. The DNA/NF-
␬
B and supershifted complexes are indicated by white, gray (supershifted by
␣
p50), and black (supershifted
by
␣
p65) arrowheads, respectively. D, transcriptional activity of rIL-33-or rIL-1
␤
-induced NF-
␬
B. Stably transfected EL-4 cells (1 ⫻ 10
7
cells) were
transiently transfected with pNF-
␬
B-Luc (40
␮
g) and pRL-TK (4
␮
g). The transfected cells were either left untreated or treated with ST2-V5 (500 ng/ml)
for 3 h and then either left unstimulated or stimulated with rIL-33 or rIL-1
␤
(10 ng/ml) for 24 h. The cells were harvested and subjected to luciferase assay.
Firefly luciferase activity was normalized with Renilla luciferase activity, and the luciferase activity of the untreated and unstimulated cells was given a
reference value of 1. The data are shown as the means ⫾ S.E. from four independent experiments. **, p ⬍ 0.01, IL-33 alone versus ST2 plus IL-33). The data
of clone numbers 1-1-A-3 (EV/EL-4 cells), 1-2-G-12 (ST2L/EL-4 cells), and 3-2-A-8 (IL-1RI/EL-4 cells) are represented in each panel.
Suppression of IL-33 Signaling by Soluble ST2
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proteins in vivo, we utilized a murine model of asthma caused
by OVA. BALB/c mice were sensitized with saline (SAL) or
OVA and then challenged with an aerosol of SAL or OVA.
Twenty-four hours after the last aeroallergen challenge, the
concentration of soluble ST2 in sera was measured by sandwich
ELISA, and the expression of ST2L in splenocytes was detected
by flow cytometry. The level of soluble ST2 was predominantly
elevated in OVA-sensitized and -challenged (OVA/OVA) mice
(Fig. 4A). In contrast, the production of soluble ST2 in other
groups was low and showed no significant difference. However,
soluble ST2 was below the detectable level in bronchoalveolar
lavage fluids of any groups in our sandwich ELISA system (data
not shown). Moreover, the expression of ST2L in splenocytes
was apparently induced in OVA/OVA mice. On the other hand,
the expression of ST2L in SAL/OVA mice was as low as that in
untreated mice (Fig. 4B and data not shown). These results
indicate that the expressions of soluble ST2 and ST2L were
specifically induced in asthmatic mice by the combination of
sensitization and challenge with OVA.
In addition, we also examined the protein expression of IL-33
in asthmatic mice. So far, a detection system such as ELISA for
secreted murine IL-33 has not been developed. Therefore, we
tried to detect IL-33 protein by Western blotting using com-
mercially available anti-mouse IL-33 antibody. Cellular extracts
were prepared from the thymus of asthmatic mice because the
expression of IL-33 mRNA was highly induced in the thymus
(Fig. 5A). However, we could detect neither the precursor nor
the mature IL-33 protein in this experiment.
Expression of ST2, ST2L, and IL-33 mRNAs after the OVA
Challenge—To investigate the expressions of the ST2 and IL-33
genes in various tissues of OVA/OVA mice, we performed a
reverse transcription-PCR analysis (Fig. 5). Expression of the
ST2 gene was induced in the thymus, lung, lymph node, spleen,
and ovary after the last OVA challenge. However, the expres-
sion in the brain, heart, liver, kidney, and skeletal muscle was
low or absent, as was the case for the
testis (Fig. 5A and data not shown).
Although the expression of the ST2
gene in the stomach was detected,
the level of expression was not
altered before and after the OVA
challenge (data not shown). Inter-
estingly, biphasic expression of the
ST2 gene was observed in the thy-
mus, lung, lymph node, and spleen.
The expression of the ST2 gene was
increased at 3 h, dropped at 6 h, and
then increased again until 12 h or
24 h (Fig. 5). In addition, the expres-
sion profile was different in female
and male mice. Expression of the
ST2 gene was gradually induced in
the ovary after the OVA challenge,
but not in the testis. In the expres-
sion of the IL-33 gene, pronounced
biphasic expression was observed in
the thymus and lung (Fig. 5). IL-33
mRNA was expressed in the lymph
node, ovary, and testis; however, the expression was hardly
observed in the spleen. These results indicate that the expres-
sion of the ST2 gene is induced in immunological response-
associated tissues after the OVA challenge and that the expres-
sion of the IL-33 gene is also induced in several tissues of
asthmatic mice.
Suppression of the Production of Th2 Cytokines from IL-33-
stimulated Splenocytes—A previous study showed that IL-33
induces the production of Th2 cytokines (9). To study the
effects of soluble ST2 on the biological activity of IL-33, we
analyzed the production of Th2 cytokines from splenocytes of
asthmatic mice. We first examined whether rIL-33 binds to
splenocytes using flow cytometry (Fig. 6A). Splenocytes were
prepared from SAL/OVA and OVA/OVA mice at 24 h after the
last OVA challenge. Recombinant IL-33 apparently bound to
ST2L-positive splenocytes prepared from OVA/OVA mice.
Next, we analyzed the production of Th1 and Th2 cytokines.
Fig. 6B shows a scheme for the stimulation of splenocytes. The
splenocytes were stimulated with OVA for activation of lym-
phocytes. The OVA-stimulated splenocytes were either
untreated or treated with ST2-FLAG for 3 h. Subsequently, the
splenocytes were left unstimulated or stimulated with rIL-33
for 48 h, followed by harvest of culture supernatants. Stimula-
tion with rIL-33 specifically induced the productions of IL-4,
IL-5, and IL-13 from splenocytes of OVA/OVA mice, whereas
the production of these cytokines was reduced by pretreatment
with ST2-FLAG (Fig. 6C). On the other hand, the production of
IFN-
␥
was increased according to the reduction of Th2 cyto-
kine production in splenocytes of OVA/OVA mice. Although
the production of IFN-
␥
from splenocytes of SAL/OVA mice
was also induced by the addition of IL-33 alone or ST2-FLAG
plus IL-33, the reasons are presently unclear. Where the spleno-
cytes of OVA/OVA mice were unstimulated with OVA, IL-33-
induced production of Th2 cytokines was low (data not shown).
These results suggest that IL-33 induces the production of Th2
FIGURE 4. Expression of soluble ST2 and ST2L in a murine model of asthma. A, level of soluble ST2 in sera
after aeroallergen challenge. The mice were sensitized with saline and challenged with OVA (SAL/OVA), sensi-
tized and challenged with OVA (OVA/OVA), sensitized and challenged with saline (SAL/SAL), or sensitized with
OVA and challenged with saline (OVA/SAL). The sera were obtained at 24 h after the last aeroallergen challenge.
The concentration of soluble ST2 was measured by sandwich ELISA. The data are shown as the means ⫾ S.E.
(n ⫽ 10 mice/group; **, p ⬍ 0.01, OVA/OVA versus either SAL/OVA, SAL/SAL, or OVA/SAL). B, expression analysis
of ST2L in splenocytes. Splenocytes were prepared from SAL/OVA and OVA/OVA mice at 24 h after the last OVA
challenge. Splenocytes (1 ⫻ 10
6
cells) were stained with FITC-conjugated anti-mouse T1/ST2 antibody (solid
line) and then analyzed by flow cytometry. The thin-lined histogram shows unstained cells. Percentages of
ST2L-positive splenocytes are shown as the means ⫾ S.E. (n ⫽ 8 mice in SAL/OVA, n ⫽ 9 mice in OVA/OVA; *, p ⬍
0.05, OVA/OVA versus SAL/OVA).
Suppression of IL-33 Signaling by Soluble ST2
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cytokines from activated splenocytes via ST2L. Taken together,
soluble ST2 suppresses the IL-33-induced production of Th2
cytokines.
DISCUSSION
This study has examined the regulation of IL-33 signaling by
the soluble secreted form of the ST2 gene products (soluble
ST2). We found that soluble ST2 has antagonistic effects on
IL-33 signaling in allergic airway inflammation.
IL-33 is a member of the IL-1 cytokine family; the intracellu-
lar pathway of IL-33 signaling is similar to that of IL-1 signaling
(9). We have studied IL-33 signaling using EL-4 cells stably
expressing ST2L or IL-1RI (Figs. 1 and 3). IL-33 specifically
bound to ST2L/EL-4 cells, but not to IL-1RI/EL-4 cells. The
binding of IL-33 to ST2L induced the degradation of I
␬
B
␣
and
subsequent activation of DNA binding activity of NF-
␬
B. IL-33-
induced DNA/NF-
␬
B complex contained the p50 and p65 sub-
units. Furthermore, we found that soluble ST2 also bound to
FIGURE 5. Expression of ST2, ST2L, and IL-33 mRNAs after the last OVA challenge. A, reverse transcription-PCR analysis of expression of ST2, ST2L, and IL-33
mRNAs in tissues of asthmatic mice. DNase I-treated total RNAs were prepared from tissues of untreated mice (control; lanes C) and OVA/OVA mice at the
indicated time shown above each lane (3– 48 h) after the last OVA challenge and then were subjected to reverse transcription-PCR analysis.
␤
-Actin was
detected as an internal control. PCR products were separated on 2% agarose gels. The data show one of three independent experiments. B, kinetic analysis of
expression of ST2, ST2L, and IL-33 mRNAs. Densitometric analysis was performed using the public domain NIH Image program. Expression of ST2, ST2L, and
IL-33 mRNAs was normalized with that of
␤
-actin mRNA, and expression of control mice was given a reference value of 1. The data are shown as arbitrary units
(means ⫾ S.E.) from three independent experiments. Nd, no data for low level of gene expression.
Suppression of IL-33 Signaling by Soluble ST2
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IL-33 and that pretreatment with soluble ST2 suppressed the
IL-33-induced NF-
␬
B activation in ST2L/EL-4 cells (Figs. 2 and
3). Thus, our results indicate that the binding activity of IL-33
for ST2L was inhibited by the formation of an ST2/IL-33 com-
plex, leading to the suppression of IL-33 signaling.
Several studies have shown that soluble forms of cytokine
receptors function as positive or negative regulators in the
expression of cytokines and growth factors. The soluble form of
IL-1 receptor type II (sIL-1RII), IL-4 receptor
␣
-chain (sIL-
4R
␣
), and IL-13 receptor
␣
-chain 2 (sIL-13R
␣
2) have antago-
nistic effects on IL-1, IL-4, and IL-13 signalings, respectively
(20–22). In contrast, the soluble IL-6 receptor
␣
-chain (sIL-
6R
␣
) has agonistic effects on IL-6 signaling and modulates the
expression of chemokines (23, 24). Soluble forms of cytokine
receptors are generated by several mechanisms including alter-
native splicing of pre-mRNA and proteolytic cleavage of recep-
tors (25). In the case of the ST2 protein, soluble ST2 is gener-
ated by alternative splicing of pre-mRNA, and its amino acid
sequence is mostly consistent with
that of the extracellular domain of
ST2L (1, 3). Therefore, it was a rea-
sonable result that soluble ST2 pos-
sessed binding activity for IL-33.
Besides soluble ST2 and ST2L,
ST2V in humans and ST2LV in
chickens have been reported as var-
iant forms of the ST2 gene products
(26–28). These variants might also
be related to the regulation of IL-33
signaling because of containing the
extracellular domain.
In patients and model mice of
allergic asthma, the level of soluble
ST2 was elevated in sera (14, 15),
and the number of CD4-positive T
cells expressing ST2L was increased
in the lung and lymph nodes (29).
On the other hand, the expression
of IL-33 in allergic asthma is not
investigated yet. In this study, we
demonstrated that the expression of
IL-33 mRNA was induced in several
tissues after the OVA challenge in a
murine model of asthma (Fig. 5). In
addition, IL-33 bound to ST2L-pos-
itive splenocytes of asthmatic mice
and induced the productions of
IL-4, IL-5, and IL-13 (Fig. 6).
Although we could not clarify a level
and a time course for protein
expression of IL-33, these results
suggest that IL-33 expresses and
functions as a cytokine in allergic
asthma. Furthermore, we showed a
detailed expression profile of the
ST2 gene in asthmatic mice. The
ST2 gene was predominantly
expressed in the thymus, lung,
lymph nodes, and spleen (Fig. 5). The expression of the ST2
gene in these tissues was regulated by the distal promoter (data
not shown), which functions in Th2 cell-mediated immunolog-
ical responses (16, 30). The biphasic expression pattern of ST2
mRNA corresponds to the production pattern of soluble ST2 in
sera after the OVA challenge (15), suggesting that these tissues
might be sources of soluble ST2. In addition, the level of soluble
ST2 in sera was dramatically elevated in comparison with that
of ST2L on the cell surfaces of splenocytes after the OVA chal-
lenge (Fig. 4). This drastic increase of the soluble ST2 concen-
tration may contribute to the suppression of IL-33 signaling in
vivo. In fact, pretreatment with soluble ST2 was effective for the
suppression of IL-4, IL-5, and IL-13 productions from IL-33-
stimulated splenocytes in asthmatic mice (Fig. 6). This negative
effect on Th2 cytokine production was consistent with the
results of therapeutic experiments using a recombinant soluble
ST2-Fc protein or a soluble ST2 expression vector (7, 15). Thus,
our results strongly support the paradigm that soluble ST2 neg-
FIGURE 6. Suppression of Th2 cytokine production from IL-33-stimulated splenocytes of asthmatic mice.
A, binding analysis of rIL-33 to splenocytes by flow cytometry. Splenocytes were prepared from SAL/OVA and
OVA/OVA mice at 24 h after the last OVA challenge. The splenocytes (1 ⫻ 10
6
cells) were mixed with rIL-33 (1
␮
g) for 1 h. The splenocytes were stained with biotinylated anti-T7 tag antibody, RPE-conjugated streptavidin,
and FITC-conjugated anti-mouse T1/ST2 antibody. Percentages of IL-33-bound ST2L-positive splenocytes are
shown as the means ⫾ S.E. (n ⫽ 8 mice in SAL/OVA, n ⫽ 9 mice in OVA/OVA; *, p ⬍ 0.05, OVA/OVA versus
SAL/OVA). B, schematic diagram for stimulation of splenocytes. The splenocytes were prepared from SAL/OVA
and OVA/OVA miceat 24 h after the last OVA challenge. Stimulation of splenocytes was performed asdescribed
under “Experimental Procedures.” After the culture supernatants were harvested, the concentrations of IL-4,
IL-5, IL-13, and IFN-
␥
were measured by ELISA. C, levels of Th1 and Th2 cytokines in culture supernatants. White
and black bars indicate data obtained from splenocytes of SAL/OVA and OVA/OVA mice, respectively. The data
are shown as the means ⫾ S.E. (n ⫽ 8 mice in SAL/OVA, n ⫽ 9 mice in OVA/OVA; **, p ⬍ 0.01, IL-33 alone versus
ST2 plus IL-33 in splenocytes of OVA/OVA mice). ND, not detected.
Suppression of IL-33 Signaling by Soluble ST2
26378 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 36 •SEPTEMBER 7, 2007
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atively regulates the production of Th2 cytokines in allergic
airway inflammation.
Serum levels of soluble ST2 have been found to be elevated in
various diseases such as rheumatoid arthritis, systemic lupus
erythematosus, and idiopathic pulmonary fibrosis (31, 32), as
well as asthma. In addition, soluble ST2 suppresses the produc-
tion of inflammatory cytokines by stimulation with lipopolysac-
charides in murine macrophages and THP-1 cells, derived from
human monocytic leukemia (18, 33). Therefore, soluble ST2
may participate in the regulation of inflammatory cytokines
besides Th2 cytokines.
NF-
␬
B is a key regulator in the IL-33 signaling pathway,
although it remains unclear how NF-
␬
B regulates the expres-
sion of the Th2 cytokine genes. Previous studies using animal
models have provided evidence that NF-
␬
B plays an essential
role in Th2 cell-mediated immunological responses. The p50-
deficient (p50
⫺/⫺
) and c-Rel-deficient (c-Rel
⫺/⫺
) mice do not
develop allergic airway inflammation (34, 35). Furthermore,
p50 is required for the expression of transcription factor
GATA-3, which regulates the expression of Th2 cytokine genes
(36). Transgenic mice expressing dominant negative GATA-3
exhibit the inhibition of allergic inflammation (37). Hereafter,
analysis of the downstream regulation of NF-
␬
B is required to
advance understanding of IL-33 signaling. In addition, the
processing mechanism of the IL-33 protein has not been eluci-
dated in vivo. A recent study reported that pre-IL-33 also func-
tioned as a transcriptional repressor in nucleus besides acting as
a cytokine (38). Therefore, the regulation for secretion of native
IL-33 protein should be studied for better understanding of the
biological and pathological functions of IL-33.
In conclusion, we showed the biological function of soluble
ST2 in vitro and in vivo. We demonstrated that soluble ST2
suppresses the activation of NF-
␬
B and the production of
Th2 cytokines in IL-33 signaling, suggesting that this sup-
pression leads to attenuation of allergic inflammatory
responses in asthma. Furthermore, our findings may serve to
advance knowledge in relation to the biological functions of
IL-33 and therapeutic effects of soluble ST2 in allergic air-
way inflammation.
Acknowledgments—We thank Dr. K. Oshikawa and T. Ikahata for
technical advice. We are also grateful to R. Izawa and H. Ohto-Ozaki
for technical assistance.
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Suppression of IL-33 Signaling by Soluble ST2
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1
SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure. Suppression of IL-33-induced NF-κB activation by soluble ST2. A,
DNA-binding activity of NF-κB in the IL-33 and IL-1β signalings. B, Suppression of DNA-binding
activity of NF-κB by the addition of ST2-V5. a, DNA-binding activity of NF-κB in ST2L/EL-4
cells. b, DNA-binding activity of NF-κB in IL-1RI/EL-4 cells. A and B, Stimulation of the cells
and EMSA were performed as described in "Experimental Procedures". Clone numbers of stable
cell lines are indicated above lanes: EV/EL-4 (2-1-C-11 and 3-1-B-9), ST2L/EL-4 (1-1-G-12 and 3-2-
G-7), and IL-1RI/EL-4 (1-2-B-12 and 2-2-B-7). White arrowheads indicate bands of the DNA/NF-
κB complex.

Akihiro Kume and Shin-ichi Tominaga
Hiroko Hayakawa, Morisada Hayakawa,
Signaling in Allergic Airway Inflammation
Soluble ST2 Blocks Interleukin-33
Mechanisms of Signal Transduction:
doi: 10.1074/jbc.M704916200 originally published online July 10, 2007
2007, 282:26369-26380.J. Biol. Chem.
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