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Inhibition of interleukin-33 signaling attenuates the severity of experimental arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 3, March 2009, pp 738–749
DOI 10.1002/art.24305
© 2009, American College of Rheumatology
Inhibition of Interleukin-33 Signaling Attenuates the
Severity of Experimental Arthritis
Gaby Palmer,1 Dominique Talabot-Ayer,1 Céline Lamacchia,1 Dean Toy,2
Christian A. Seemayer,3 Sébastien Viatte,1 Axel Finckh,1 Dirk E. Smith,2 and Cem Gabay1
Objective. Interleukin-33 (IL-33; or, IL-1F11) was
recently identified as the ligand of the IL-1 family
receptor T1/ST2. The aim of this study was to examine
IL-33 production in human and mouse joints and to
investigate the role of IL-33 and T1/ST2 in experimental
arthritis.
Methods. IL-33 expression was examined in human synovial tissue, rheumatoid arthritis (RA) synovial
fibroblasts, and arthritic mouse joints. Mice with
collagen-induced arthritis (CIA) were treated with
blocking anti-ST2 antibody or control antibody beginning at the onset of disease. Arthritis severity was
assessed by clinical and histologic scoring. Draining
lymph node (LN) cell responses were examined ex vivo,
and joint messenger RNA (mRNA) was used for expression profiling.
Results. IL-33 was highly expressed in human RA
synovium. In cultured synovial fibroblasts, IL-33 expression was strongly induced by IL-1␤ and/or tumor
necrosis factor ␣. Furthermore, IL-33 mRNA was detected in the joints of mice with CIA and increased
during the early phase of the disease. Administration of
a blocking anti-ST2 antibody at the onset of disease
attenuated the severity of CIA and reduced joint destruction. Anti-ST2 antibody treatment was associated
with a marked decrease in interferon-␥ production as
well as with a more limited reduction in IL-17 production by ex vivo–stimulated draining LN cells. Finally,
RANKL mRNA levels in the joint were reduced by
anti-ST2 treatment.
Conclusion. IL-33 is produced locally in inflamed
joints, and neutralization of IL-33 signaling has a
therapeutic effect on the course of arthritis. These
observations suggest that locally produced IL-33 may
contribute to the pathogenesis of joint inflammation
and destruction.
Interleukin-33 (IL-33; or, IL-1F11) was recently
identified as a ligand for the orphan IL-1 family receptor
T1/ST2. IL-33 is produced as a 30-kd propeptide and,
like IL-1␤ and IL-18, is cleaved by caspase 1 to generate
mature 18-kd IL-33, at least in vitro (1). Mature IL-33
has been reported to mediate its biologic effects via
T1/ST2 binding by activating NF-␬B and MAP kinases
(1). T1/ST2-dependent IL-33 responses resemble classic
IL-1–like signaling, and several recent studies identified
the IL-1 receptor accessory protein as the coreceptor
involved in IL-33 signaling (2–4).
A number of studies have established T1/ST2
(also referred to as ST2L) as a selective marker of both
murine and human Th2 lymphocytes (for review, see ref.
5). In addition, T1/ST2 is highly expressed on mast cells
(6). Several recent reports describe a stimulatory effect
of IL-33 on cytokine and chemokine secretion in mast
cells (3,7–10), suggesting that, in addition to promoting
Th2 responses, IL-33 exhibits proinflammatory potential
by inducing the production of a number of inflammatory
mediators in mast cells. T1/ST2 also exists as a soluble
isoform (sST2) obtained by differential messenger RNA
(mRNA) processing. Soluble ST2 is identical with the
extracellular region of the long T1/ST2 isoform except
Dr. Palmer’s work was supported by a grant from the de
Reuter Foundation. Dr. Gabay’s work was supported by the Swiss
National Science Foundation (grant 3200-107592/1).
1
Gaby Palmer, PhD, Dominique Talabot-Ayer, MS, Céline
Lamacchia, MS, Sébastien Viatte, MD, PhD, Axel Finckh, MD, MS,
Cem Gabay, MD: University Hospital of Geneva, and University of
Geneva School of Medicine, Geneva, Switzerland; 2Dean Toy, MS,
Dirk E. Smith, MS: Amgen, Inc., Seattle, Washington; 3Christian A.
Seemayer, MD: University Hospital of Geneva, Geneva, Switzerland.
Mr. Toy and Mr. Smith own stock or stock options in Amgen,
Inc. Dr. Gabay has received consulting fees, speaking fees, and/or
honoraria from Roche, Novartis, Wyeth, Merck, Bristol-Myers Squibb,
Serono, and Abbott (less than $10,000 each).
Address correspondence and reprint requests to Cem Gabay,
MD, Division of Rheumatology, University Hospital of Geneva, 26
Avenue Beau-Séjour, 1211 Geneva 14, Switzerland. E-mail: cem.
gabay@hcuge.ch.
Submitted for publication April 24, 2008; accepted in revised
form November 7, 2008.
738
ROLE OF IL-33 IN ARTHRITIS
for 9 additional amino acids, and it was recently demonstrated to act as an antagonistic decoy receptor for
IL-33 (11). Serum concentrations of sST2 are elevated in
patients with various inflammatory disorders (5). Increased levels of sST2 have also been observed in the
synovial fluid of patients with rheumatoid arthritis (RA)
as compared with patients with osteoarthritis (12).
Several studies have described a role of T1/ST2
and sST2 in regulating inflammatory responses (13–16).
Interestingly, mast cells have been recognized as important mediators of the pathogenesis of arthritis (17,18),
suggesting that IL-33–mediated mast cell activation
might play a role in joint inflammation. The injection of
an anti-T1/ST2 antibody had previously been reported to
exacerbate collagen-induced arthritis (CIA), but this
result was suggested to be due to complementdependent Th2 clone lysis rather than to an effect of the
antibody on T1/ST2 signaling (19). Indeed, another
study indicated that administration of sST2 decreased
CIA (20). At the time, the mechanism proposed to
explain this effect was direct inhibition of macrophage
activation by sST2 via a putative sST2 receptor expressed at the macrophage surface. However, the identification of IL-33 as the bona fide ligand for T1/ST2
suggests neutralization of IL-33 by sST2 (11) as an
alternative explanation.
Expression of IL-33 mRNA has previously been
detected by in situ hybridization in endothelial cells
(ECs) in human RA synovium, suggesting that ECs
constitute a major cellular source of IL-33 in inflamed
synovial tissue (21). Interestingly, proIL-33 had previously been described in high endothelial venule (HEV)
ECs as a nuclear protein and was thus called nuclear
factor from HEVs (22). Like proIL-1␣ (23), nuclear
proIL-33 may exert unique biologic activities independent of caspase 1 cleavage and cell surface receptor
binding (21,22).
In the present study, we further examined IL-33
production in human and mouse joints and investigated
the role of IL-33 and T1/ST2 in experimental arthritis.
The results described herein demonstrate expression of
the IL-33 protein in human RA synovium. Furthermore,
we show for the first time that, in addition to ECs,
synovial fibroblasts produce IL-33 and that its expression is increased by proinflammatory stimuli. Consistent
with these findings, IL-33 is produced locally in inflamed
mouse joints. Finally, the administration of antibodies
that block T1/ST2 signaling attenuated the severity of
CIA, thus suggesting that IL-33 plays a pathogenic role
in arthritis.
739
MATERIALS AND METHODS
Mice. Male DBA/1 mice were obtained from Elevage
Janvier (Le Genest St. Isle, France). BALB/c mice were
supplied by The Jackson Laboratory (Bar Harbor, ME). The
mice were housed under conventional conditions. Animal
experiments were approved by the Geneva (Switzerland) Veterinarian Office or by the Amgen (Seattle, WA) Animal Care
and Use Committee.
Human synovial tissue. Formalin-fixed paraffinembedded tissue samples were obtained from an Amgen tissue
collection and from the Department of Clinical Pathology,
University Hospital of Geneva. RA synovial biopsy samples
were obtained from a 72-year-old woman undergoing joint
replacement surgery and from a 47-year-old man undergoing
knee arthroscopy. Normal control synovial tissue was obtained
at autopsy from a 52-year-old woman who died of an unrelated
illness.
Immunohistochemistry. Formalin-fixed paraffinembedded tissue samples were stained for IL-33 protein
expression using an immunoperoxidase protocol. Briefly, sections were deparaffinized, rehydrated, and heated in a steamer
with antigen recovery buffer (DIVA buffer; BioCare Medical,
Concord, CA) for 60 minutes. Slides were then blocked and
incubated with a polyclonal goat anti-human IL-33 antibody
(AF3625; R&D Systems, Minneapolis, MN). Subsequently,
slides were washed, blocked for endogenous peroxidase activity, incubated with secondary antibody–peroxidase complexes
(ABC Elite; Vector, Burlingame, CA), and developed with
diaminobenzidine. In additional experiments, staining was
performed using a monoclonal anti-human IL-33 antibody
(IL33305B; Alexis, Lausen, Switzerland), which yielded an
identical staining pattern (data not shown). To characterize
IL-33–positive cells, double immunostaining was performed
with anti-CD34 and anti–D2-40 antibodies (M7165 and
M3619, respectively; Dako, Baar, Switzerland) to stain vascular
and lymphatic ECs or with an anti-CD68 antibody (M0814;
Dako) to identify cells of the myeloid lineage and developed
using the EnVision G兩2 alkaline phosphatase system with
permanent red (Dako). To assess staining specificity, negative
controls were performed using unrelated isotype-matched
control antibodies.
Culture of human synovial fibroblasts. Cell culture
reagents were obtained from Invitrogen Life Technologies
(Basel, Switzerland). Synovium was obtained from RA patients
undergoing knee or hip joint replacement. Synovial fibroblasts
were isolated by collagenase digestion, cultured as described
previously (24), and used between passages 3 and 13.
Reverse transcriptase–polymerase chain reaction
(RT-PCR). Human synovial fibroblasts were grown to confluence in 6-well plates and stimulated with IL-1␤ (1 ng/ml) and
tumor necrosis factor ␣ (TNF␣) (10 ng/ml) for 6 hours. Cells
were solubilized in TRIzol reagent (Gibco Life Technologies,
Basel, Switzerland), and RNA was isolated according to the
manufacturer’s instructions. Total RNA (2 ␮g) was digested by
DNase I and reverse-transcribed, and IL-33 levels were examined by quantitative PCR using the iCycler iQ Real-Time PCR
Detection System, iQ SYBR Green Supermix (both from
Bio-Rad, Philadelphia, PA), and the following primers: 5⬘-GGTGTTACTGAGTTACTATGAG-3⬘ (forward) and 5⬘-GGAGCTCCACAGAGTGTTCCTTG-3⬘ (reverse) (GenBank
740
accession no. AY905581). IL-33 expression was normalized to
expression of the housekeeping gene ␤-glucuronidase (␤-glu)
using the following human ␤-glu primers: 5⬘-CCTCTGATGTTCACTGAAG-3⬘ (forward) and 5⬘-CTCTCGTCGGTGACTG-3⬘ (reverse) (GenBank accession no. NM_000181.1).
The annealing temperature was 60°C. IL-33 mRNA expression
was corrected for ␤-glu mRNA levels, and the IL-33:␤-glu
ratios were normalized to the maximal value observed in each
experiment. Non–reverse-transcribed RNA samples and water
were included as negative controls. The identity of the amplified products was confirmed by sequencing.
Western blotting. Human synovial fibroblasts were
grown to confluence in 6-well plates and stimulated with IL-1␤
(1 ng/ml) and TNF␣ (10 ng/ml) for 24 hours before lysis in 500
␮l Laemmli buffer. For each sample, 40 ␮l of total cell lysate
was fractionated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis and transferred to a Porablot membrane
(Macherey-Nagel, Düren, Germany). The membrane was
blocked in phosphate buffered saline (PBS), 0.05% Triton
X-100, and 5% bovine serum albumin (BSA), and immunoblotting was performed with a biotinylated Nessy-1 monoclonal
anti-human IL-33 antibody (1:1,000 dilution; Alexis) and visualized by enhanced chemiluminescence (Amersham Pharmacia
Biotech, Dübendorf, Switzerland) using streptavidin–
horseradish peroxidase (1:20,000 dilution; R&D Systems,
Abingdon, UK).
Immunofluorescence. Human synovial fibroblasts were
plated on chamber slides and stimulated with IL-1␤ (1 ng/ml)
and TNF␣ (10 ng/ml) for 48 hours. Cells were fixed for 10
minutes (with PBS, 4% paraformaldehyde, and RT), permeabilized for 10 minutes (with PBS and 0.1% Triton X-100), and
blocked for 1 hour (with PBS, 1% BSA, and 10% normal goat
serum) before staining with the Nessy-1 antibody (1:100 dilution) and detection with a phycoerythrin-labeled anti-mouse
IgG1 secondary antibody. To assess staining specificity, negative controls were performed in the absence of the primary
antibody. Slides were mounted using a mounting medium
containing 4⬘,6-diamidino-2-phenylindole (Vector).
Assessment of binding and in vitro blocking activity of
monoclonal anti-ST2 antibody. A rat monoclonal anti-murine
ST2 antibody directed against the extracellular domain of
recombinant murine ST2 was produced by Amgen. P815 cells
were cultured in Dulbecco’s modified Eagle’s medium with
10% fetal bovine serum (FBS) and penicillin/streptomycin. To
investigate anti-ST2 antibody binding specificity, P815 cells
were stained in PBS and 1% FBS with biotinylated anti-ST2
antibody (10 ␮g/ml) in the presence or absence of unlabeled
anti-ST2 antibody (10-fold excess). To evaluate the blocking
activity of the anti-ST2 antibody, P815 cells were stimulated
with recombinant murine IL-33 (10 ng/ml; R&D Systems) for
72 hours before IL-6 production was assessed by enzymelinked immunosorbent assay (ELISA; R&D Systems). The
monoclonal anti-ST2 antibody or an isotype-matched control
antibody was added to the cells at the indicated concentrations
30 minutes before stimulation with IL-33.
Assessment of in vivo blocking activity of monoclonal
anti-ST2 antibody. Female BALB/c mice (age ⬃4 months)
were injected intraperitoneally (IP) with 250 ␮g of monoclonal
anti-ST2 antibody, mouse sST2-Fc (produced at Amgen), or
control rat IgG (5 mice/group). Two hours later, mice were
challenged intranasally with 200 ng of recombinant murine
PALMER ET AL
IL-33 or mouse serum albumin (MSA) as a control protein.
Mice were killed 24 hours later, and IL-5 levels in the
bronchoalveolar lavage (BAL) fluid were assessed by ELISA
(R&D Systems).
Induction and assessment of CIA. CIA was induced in
male DBA/1 mice as described elsewhere (25), except that for
the booster injection on day 21, 100 ␮g type II collagen (CII)
in PBS was given IP. Monoclonal rat IgG1 anti-murine ST2
antibody or isotype-matched control antibody M139 (rat IgG1
anti–keyhole limpet hemocyanin; Amgen) was injected (150
␮g/mouse IP) every 2–3 days starting on day 21. From this time
point onward, arthritis severity in each paw was assessed by
clinical scoring on a 3-point scale (25). Mice were killed on day
42 after the first immunization.
RNase protection assay and RNA expression profiling
of arthritic mouse joints. Ankle joints from mice with CIA
were homogenized in TRIzol reagent for extraction of total
RNA. IL-33 and GAPDH mRNA expression was analyzed by
RNase protection assay. A probe for murine IL-33 corresponding to bp 1–264 of the IMAGE3593927 expressed sequence tag
complementary DNA (cDNA) clone (GenBank accession no.
BC003847) was cloned into Bluescript SK⫹ (Stratagene, La
Jolla, CA). The probe for murine GAPDH has been described
previously (26). Linearized probes were transcribed with T7
RNA polymerase (Promega, Madison, WI) to synthesize 32Plabeled riboprobes complementary to IL-33 and GAPDH
mRNA. The riboprobes were purified, and 2 ⫻ 105 counts per
minute of each probe was hybridized to 10 ␮g of total RNA in
80% formamide, 40 mM PIPES (pH 6.7), 400 mM NaCl, and 1
mM EDTA before digestion with RNases A and T1. The
RNase-protected probes were resolved on denaturing polyacrylamide gels, imaged by autoradiography, and quantified by
phosphorimaging (Cyclone Storage Phosphor System;
PerkinElmer, Zaventem, Belgium). IL-33 levels were normalized to GAPDH expression.
Quantitative PCR profiling of cDNA synthesized from
joint tissue mRNA was performed using a custom low-density
array and standard TaqMan reagents (Applied Biosystems,
Foster City, CA). Expression of 62 genes (see Supplementary
Table 1, available on the Arthritis & Rheumatism web site at
http://www3.interscience.wiley.com/journal/76509746/home)
was measured simultaneously, and expression levels were
normalized to murine hypoxanthine guanine phosphoribosyltransferase.
Histologic grading of arthritis. Knee joints were fixed
in 10% buffered formalin, decalcified in 15% EDTA, dehydrated, and embedded in paraffin. Sagittal sections (5 ␮m)
were stained with hematoxylin and eosin or toluidine blue and
graded by a pathologist (CAS) in a blinded manner. Cartilage
erosion, joint destruction, and inflammation including “pannus” formation were scored on a scale of 0–4 (0 ⫽ normal, 1 ⫽
minimal, 2 ⫽ moderate, 3 ⫽ severe, and 4 ⫽ very severe) (27).
The amount of polymorphonuclear neutrophils was scored on
a scale of 0–4 (0 ⫽ no neutrophils present and 4 ⫽ maximal
neutrophil infiltration).
T cell proliferation assay. Draining lymph node (LN)
cells were harvested and seeded at 4 ⫻ 105/well in 96-well
plates in 200 ␮l RPMI 1640 medium containing 100 IU/ml of
penicillin, 100 ␮ g/ml of streptomycin, 5 ⫻ 10 ⫺5 M
␤-mercaptoethanol, and 1% heat-inactivated mouse serum.
Cells were incubated for 72 hours without or with 100 ␮g/ml
ROLE OF IL-33 IN ARTHRITIS
741
Figure 1. Analysis of interleukin-33 (IL-33) expression in human synovium. A and B, Expression of the IL-33 protein was assessed by
immunohistochemistry in human rheumatoid arthritis (RA) (A) and normal (B) synovium. IL-33 staining (brown) was detected in endothelial cells
(EC) both in inflamed and in normal synovium. In addition, in RA synovium, IL-33 immunoreactivity was observed in high endothelial venule (HEV)
ECs in lymphoid aggregates (asterisks), in synovial lining cells (LC), in cells morphologically consistent with fibroblasts (solid arrowheads), and in
cells resembling mononuclear inflammatory cells (open arrowheads) in the sublining. C, IL-33–expressing cells were further characterized by double
staining. Left, Human RA synovium was stained for IL-33 (brown) and for vascular and lymphatic EC markers (red). Some examples of
double-positive IL-33–expressing ECs (solid arrowheads) and of nonendothelial IL-33–positive cells (open arrowheads) are indicated. Right,
Human RA synovium was stained for IL-33 (brown) and for CD68 to label inflammatory cells (red). Some examples of double-positive
IL-33–expressing inflammatory cells (solid arrowheads), CD68-negative IL-33–expressing cells (open arrowheads), and IL-33–negative inflammatory cells (asterisks) are indicated. Staining specificity was assessed using isotype-matched antibodies as negative controls. Original magnifications
are indicated.
742
PALMER ET AL
CII or 5 ␮g/ml of concanavalin A (Con A; Amersham Pharmacia Biotech). During the final 18 hours of incubation,
3
H-thymidine was added at 1 ␮Ci/well.
Determination of cytokine and antibody production.
Draining LN cells were harvested at the time mice were killed
and cultured for 72 hours without or with 100 ␮g/ml of CII or
5 ␮g/ml of Con A. Culture supernatants were harvested, and
levels of interferon-␥ (IFN␥), IL-17, TNF␣, and IL-10 were
quantified by ELISA using DuoSet ELISA Development
Systems (R&D Systems). The detection limits were 16 pg/ml
for IL-17 and TNF␣ and 31 pg/ml for IFN␥ and IL-10. Serum
levels of anti-CII total IgG, IgG2a, and IgG1 were measured as
described previously (25).
Statistical analysis. Significance of differences was
calculated by one-way analysis of variance with Fisher’s protected least significant difference test or Dunnett’s multiple
comparison test, as indicated. To assess differences in the
longitudinal evolution of arthritis outcomes, we used generalized linear mixed models for repeated measures (28). The tests
were conducted at a 2-sided alpha level of 0.05 and performed
with Stata version 9.2 for Windows (StataCorp, College Station, TX). A difference between experimental groups was
considered significant when the P value was less than 0.05.
RESULTS
IL-33 is expressed in human synovial tissue, in
cultured human RA fibroblasts, and in arthritic mouse
joints. Expression of the IL-33 protein was assessed by
immunohistochemistry in human RA and normal synovium (Figures 1A and B). IL-33 staining was detected in
ECs both in inflamed and in normal synovial tissue.
However, the RA sample contained an increased number of blood vessels displaying strong IL-33 expression in
EC nuclei. The RA sample also showed an increased
number of cells with immunoreactive nuclei and cytoplasm that were morphologically consistent with fibroblasts, as well as immunoreactive cells resembling mononuclear inflammatory cells. Consistently, double staining
of RA synovial sections for IL-33 and EC markers
clearly demonstrated the presence of other IL-33–
expressing cells in addition to ECs (Figure 1C). Furthermore, double staining for IL-33 and CD68 identified a
subset of CD68-positive inflammatory cells as an additional source of IL-33 (Figure 1C). Finally, synovial
lining cells in the RA sample also showed a light
cytoplasmic staining, but it was only slightly darker than
the isotype control and was therefore equivocal.
Human RA synovial fibroblasts constitutively expressed low levels of IL-33 mRNA and protein. IL-33
mRNA and protein expression were strongly increased
after treatment of the cells with IL-1␤ and/or TNF␣
(Figures 2A and B). In cell lysates, predominantly
Figure 2. Interleukin-33 (IL-33) expression in human synovial fibroblasts and arthritic mouse joints. A, IL-33 mRNA levels in
synovial fibroblasts that were left unstimulated (open bar) or
stimulated for 6 hours with 1 ng/ml IL-1␤ (gray bar), 10 ng/ml tumor
necrosis factor ␣ (TNF␣; hatched bar), or IL-1␤ and TNF␣ (solid
bar) were analyzed by real-time reverse transcriptase–polymerase
chain reaction. Values are the mean and SEM of data obtained
using 3 independent cultures. ⴱ ⫽ P ⬍ 0.05 versus unstimulated
synovial fibroblasts. B, Synovial fibroblasts were left unstimulated
or were stimulated with IL-1␤ and TNF␣ for 24 hours before IL-33
protein expression in cell lysates was analyzed by Western blotting.
Lane 1, Recombinant mature human IL-33; lane 2, unstimulated
synovial fibroblast lysate; lane 3, IL-1␤/TNF␣–stimulated synovial
fibroblast lysate. C, Localization of IL-33 in unstimulated (left) or
IL-1␤/TNF␣–stimulated (right) synovial fibroblasts was analyzed by
immunostaining. Phycoerythrin-labeled anti–IL-33 staining (red)
was detected mainly in cell nuclei, as visualized with 4⬘,6-diamidino2-phenylindole (blue) (original magnification ⫻ 200). D, IL-33
mRNA expression in the paws of naive and arthritic mice during
early (days 1–8) or late (days 14–23) collagen-induced arthritis
(CIA) was quantified in 1 paw from a naive mouse, 4 unaffected
paws from arthritic mice, 5 arthritic paws from mice with early CIA,
and 5 arthritic paws from mice with late CIA. Values are the mean
and SEM. ⴱ ⫽ P ⬍ 0.05 versus unaffected paws and versus arthritic
paws of mice with late CIA, by one-way analysis of variance with
Fisher’s protected least significant difference test. GUSB ⫽ ␤-glucuronidase; AU ⫽ arbitrary units.
ROLE OF IL-33 IN ARTHRITIS
unprocessed 30-kd proIL-33 was detected by Western
blotting (Figure 2B). Immunocytochemistry showed that
this proIL-33 localized predominantly in the cell nucleus
(Figure 2C). As a negative control, no staining was
detected in IL-1␤– and TNF␣-stimulated synovial fibroblasts in the absence of the anti–IL-33 antibody (data
not shown). In contrast to our findings in synovial
fibroblasts, we could not detect IL-33 expression in
primary human articular chondrocytes (data not shown).
We also investigated whether human resident
joint cells express the T1/ST2 receptor, which would
make them responsive to IL-33. Human synovial fibroblasts expressed mRNA encoding short sST2, but not
the long signaling T1/ST2 isoform (data not shown).
Consistently, treatment with recombinant mature IL-33
enhanced neither IL-6 secretion nor IL-33 mRNA expression in synovial fibroblasts (data not shown). Similarly, we could detect neither T1/ST2 expression nor
responsiveness to IL-33 in human articular chondrocytes.
We next used an RNase protection assay to
investigate the expression of IL-33 mRNA in mice with
CIA. IL-33 expression was increased during the early,
inflammatory phase of CIA (Figure 2D).
A blocking antibody against the T1/ST2 receptor
decreases the severity of CIA. To investigate the role of
IL-33 and T1/ST2 in experimental arthritis, we tested a
potential therapeutic effect of a blocking anti-ST2 antibody during CIA. The anti-ST2 antibody was generated
by immunizing rats with the extracellular domain of
recombinant mouse ST2. The resulting monoclonal antibody recognizes recombinant ST2 protein in platebased assays (data not shown), as well as cell surface ST2
as assessed by flow cytometry of ST2-positive P815 cells
(Figure 3A). The potency of this blocking anti-ST2
antibody was first assessed in vitro by examining its
ability to inhibit IL-6 secretion induced by IL-33 in P815
mastocytoma cells (Figure 3B). In this assay, anti-ST2
effectively inhibited the biologic activity of IL-33, with
an estimated 50% inhibition concentration of 470 pM.
The blocking ability of the anti-ST2 antibody was comparable with that of a well-described monoclonal antiST2 antibody DJ8 (data not shown) (29).
Next, we evaluated the ability of anti-ST2 to
inhibit the biologic effects of recombinant mature IL-33
in vivo. Mice were treated IP with anti-ST2, sST2-Fc, or
control rat IgG prior to pulmonary challenge with IL-33
or MSA (Figure 3C). Intranasal instillation of IL-33 led
to increased IL-5 levels in BAL fluid, and treatment of
mice with anti-ST2 antibody or sST2-Fc efficiently in-
743
Figure 3. Characterization of a blocking monoclonal anti-ST2 antibody. A, Binding of the anti-ST2 antibody to cell surface ST2 as
assessed by flow cytometry on P815 cells in the presence or absence of
unlabeled anti-ST2 antibody. An unrelated isotype-matched antibody
was used as a negative control. B, Potency of the anti-ST2 antibody in
vitro. P815 cells were stimulated with 10 ng/ml recombinant mature
interleukin-33 (IL-33) for 72 hours before IL-6 production was assessed in culture supernatants. Monoclonal anti-ST2 antibody (Ab) or
an isotype-matched control antibody was added to the cells at the
indicated final concentrations 30 minutes prior to stimulation with
IL-33. Experiments were performed in triplicate for each condition;
results are representative of 3 independent experiments. C, Efficacy of
the anti-ST2 antibody in vivo. IL-5 levels were assessed in bronchoalveolar lavage fluid obtained from mice 24 hours after intranasal
instillation of recombinant mature IL-33 (200 ng/mouse). BALB/c
mice (n ⫽ 5 per group) were injected intraperitoneally (250 ␮g/mouse)
with an isotype-matched control antibody 2 hours before intranasal
instillation of mouse serum albumin (MSA) or with an isotypematched control antibody, the monoclonal anti-ST2 antibody, or
soluble ST2 (sST2)–Fc 2 hours before intranasal instillation of IL-33.
ⴱⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus isotype control plus IL-33, by
one-way analysis of variance with Dunnett’s multiple comparison test.
IC50 ⫽ 50% inhibition concentration.
hibited the effect of IL-33 in this experimental system.
Taken together, these results demonstrate that the
744
PALMER ET AL
Figure 4. Treatment with anti-ST2 antibody decreases the severity of collagen-induced arthritis. A, Incidence of arthritis in anti-ST2 antibody–
treated mice (n ⫽ 10) and isotype-matched control antibody–treated mice (n ⫽ 10). The onset of arthritis was significantly retarded in anti-ST2
antibody–treated mice (P ⬍ 0.05 by longitudinal model for binomial data). B, Clinical severity scores in anti-ST2 antibody–treated mice and control
antibody–treated mice. Values are the mean and SEM. Anti-ST2 antibody treatment significantly decreased the evolution of severity scores (P ⫽
0.01 by mixed model for repeated measures) and disease severity at the end of the experiment (ⴱ ⫽ P ⬍ 0.05 versus control antibody treatment).
C, Number of affected paws in anti-ST2–treated mice and control antibody–treated mice. Values are the mean and SEM. The number of affected
paws tended to be lower in anti-ST2 antibody–treated mice (P ⫽ 0.11 by longitudinal model for ordinal data). D, Severe inflammation, pannus
formation, cartilage infiltration, and joint destruction in an isotype-matched control antibody–treated mouse, but not in an anti-ST2 antibody–
treated mouse (hematoxylin and eosin stained; original magnifications are indicated). E, Histologic scores for inflammation, cartilage erosion, and
neutrophil infiltration. Scores in anti-ST2 antibody–treated mice (n ⫽ 10) (solid bars) and control antibody–treated mice (n ⫽ 10) (open bars) are
the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus control antibody–treated mice, by one-way analysis of variance with Fisher’s protected least significant
difference test.
ROLE OF IL-33 IN ARTHRITIS
monoclonal anti-ST2 antibody is an effective inhibitor of
IL-33 activity in vitro and in vivo.
We then tested the effect of the anti-ST2 antibody in CIA. Treatment of mice with blocking anti-ST2
antibody, initiated at the onset of arthritis, slightly
delayed the incidence of the disease (Figure 4A). In
addition, the evolution of clinical severity scores was
significantly reduced in anti-ST2 antibody–treated mice
(P ⫽ 0.01) (Figure 4B), and the clinical severity at the
end of the experiment was significantly lower in anti-ST2
antibody–treated mice compared with control-treated
mice. The number of affected paws also tended to be
lower in the anti-ST2 antibody–treated group, although
the difference was not statistically significant (P ⫽ 0.11)
(Figure 4C). Histologic scoring of the joints at the end of
the experiment confirmed decreased severity of arthritis
and indicated in particular decreased inflammation and
cartilage erosion in anti-ST2 antibody–treated mice
compared with isotype control–treated mice (Figures 4D
and E). Neutrophil infiltration also tended to be reduced, although the reduction was not statistically
significant.
A blocking antibody against the T1/ST2 receptor
decreases T cell IFN␥ production during CIA. The in
vitro proliferative response of draining LN cells to CII
was slightly, but significantly, reduced in cells isolated
from anti-ST2 antibody–treated mice compared with
isotype-matched control antibody–treated mice (Figure
5A). Interestingly, despite this rather modest effect on
cell proliferation, IFN␥ production was almost completely abrogated in draining LN cells isolated from
anti-ST2 antibody–treated mice (Figure 5B). IL-17,
TNF␣, and IL-10 production by draining LN cells was
only slightly reduced in cells isolated from anti-ST2
antibody–treated mice. The effect of anti-ST2 treatment
on IFN␥ production was antigen specific, since Con
A–induced IFN␥ production was not impaired in draining LN cells isolated from anti-ST2 antibody–treated
mice (mean ⫾ SEM IFN␥ concentration 11.02 ⫾ 0.62
ng/ml versus 15.87 ⫾ 0.30 ng/ml in cells from isotypematched control antibody–treated mice versus those
from anti-ST2 antibody–treated mice, respectively; P ⬍
0.05). Furthermore, this inhibitory effect could not be
reproduced when the anti-ST2 antibody (50 ␮g/ml) was
added in vitro during restimulation with antigen to
draining LN cells isolated from immunized mice that
had not been treated with the anti-ST2 antibody in vivo
(data not shown). Finally, anticollagen IgG titers (total
IgG, IgG2a, and IgG1) were not different in anti-ST2
antibody–treated mice and control antibody–treated
mice (data not shown).
745
Figure 5. Anti-ST2 antibody treatment decreases interferon-␥ (IFN␥)
production by draining lymph node (LN) cells. A, Proliferation of
draining LN cells isolated from anti-ST2 antibody–treated mice (n ⫽
10) and isotype-matched control antibody–treated mice (n ⫽ 10). Cells
were stimulated in vitro with 100 ␮g/ml type II collagen (CII) or were
left unstimulated. Values are the mean and SEM. Proliferation was
increased by stimulation with CII (ⴱ ⫽ P ⬍ 0.05 versus unstimulated
cultures). The proliferative response to CII was slightly reduced in cells
from anti-ST2 antibody–treated mice (& ⫽ P ⬍ 0.05 versus cells from
control antibody–treated mice with similar stimulation). B, IFN␥,
interleukin-17 (IL-17), tumor necrosis factor ␣ (TNF␣), and IL-10 production by draining LN cells isolated from anti-ST2 antibody–treated
mice (n ⫽ 10) and control antibody–treated mice (n ⫽ 10). Cells were
stimulated in vitro with 100 ␮g/ml CII or were left unstimulated.
Values are the mean and SEM. Cytokine production was increased by
stimulation with CII (ⴱ ⫽ P ⬍ 0.05 versus unstimulated cultures). The
IFN␥ response to CII was markedly inhibited in cells from anti-ST2
antibody–treated mice. IL-17, IL-10, and TNF␣ responses were also
slightly reduced (& ⫽ P ⬍ 0.05 versus cells from control antibody–
treated mice with similar stimulation, by one-way analysis of variance
with Fisher’s protected least significant difference test).
A blocking antibody against the T1/ST2 IL-33
receptor decreases joint RANKL mRNA expression during CIA. We analyzed the expression of 62 different
transcripts (see Supplementary Table 1, available on the
746
PALMER ET AL
Figure 6. Treatment with anti-ST2 antibody decreases joint RANKL mRNA expression. Expression of 62 different inflammation-related gene
transcripts was assessed in the ankle joints of anti-ST2 antibody–treated mice (n ⫽ 10) and isotype-matched control antibody–treated mice (n ⫽ 10),
as described in Materials and Methods. Expression of RANKL, tumor necrosis factor ␣ (TNF␣), matrix metalloproteinase 9 (MMP-9), and inducible
nitric oxide synthase (iNOS) was reduced in anti-ST2–treated animals. Each symbol represents a single mouse. Horizontal lines show the mean.
RANKL mRNA expression was significantly reduced in anti-ST2 antibody–treated mice. Differences in expression of TNF␣, MMP-9, and iNOS did
not reach statistical significance. All P values were assessed by one-way analysis of variance with Dunnett’s multiple comparison test.
Arthritis & Rheumatism web site at http://www3.
interscience.wiley.com/journal/76509746/home) by
quantitative PCR of the total RNA isolated from ankle
joints of anti-ST2 antibody–treated mice and isotype
control antibody–treated mice. Among the transcripts
examined, we observed a significant decrease in expression of RANKL as well as a trend toward decreased
expression of inflammation-related genes, such as those
for TNF␣, matrix metalloproteinase 9 (MMP-9), and
inducible nitric oxide synthase (iNOS), although the
differences between the groups did not reach statistical
significance (Figure 6).
DISCUSSION
We report that IL-33, a novel member of the IL-1
family of cytokines, is expressed in human RA synovium.
Consistent with previous observations, ECs appear to be
a major source of IL-33 in the inflamed synovium (21).
Interestingly, we also observed expression of nuclear
IL-33 in ECs in normal synovium, suggesting constitutive IL-33 production by this cell type. Moreover, IL-33
staining indicated that cells resembling synovial fibroblasts, as well as a subset of CD68-positive mononuclear
inflammatory cells, constitute additional cellular sources
of IL-33 in the rheumatoid synovium. Consistently, we
observed that cultured synovial fibroblasts produced
IL-33 and that its expression was increased in response
to IL-1␤ and/or TNF␣, 2 common proinflammatory
mediators present in the rheumatoid synovial tissue and
fluid. IL-33 mRNA was also detected in the joints of
mice with CIA during the early phase of the inflammatory process. The kinetics of IL-33 mRNA production
are similar to those found previously for other cytokines
showing that, for example, IL-1␤ levels peak during the
ROLE OF IL-33 IN ARTHRITIS
early inflammatory phase of the disease, while expression of antiinflammatory cytokines predominates later in
the disease process (30).
The administration of a monoclonal antibody
that block ST2 signaling, which was initiated at the time
of arthritis onset, attenuated the severity of arthritis and
the signs of structural damage. In addition, anti-ST2
antibody treatment was associated with a marked decrease in IFN␥ production as well as with a significant,
but more limited, reduction in IL-17 production by ex
vivo–stimulated draining LN cells. Finally, anti-ST2
treatment also decreased the expression of RANKL
mRNA in the joints. These findings indicate that IL-33 is
produced locally in inflamed joints and that neutralization of IL-33 signaling has a therapeutic effect on the
course of arthritis.
Overall, these results are consistent with those of
a previous study demonstrating a protective effect of
sST2 in CIA (20). Although the previous study explored
a different hypothesis and did not address the role of
IL-33 specifically, in retrospect, the biologic effects of
sST2 could be attributed to IL-33 neutralization. The
authors found that administration of an sST2-Fc fusion
protein at disease onset significantly attenuated the
severity of arthritis. In addition, cellular infiltration,
synovial hyperplasia, and joint erosion were markedly
reduced in the joints of sST2-Fc–treated mice compared
with controls. Similar to our findings, treatment with
sST2-Fc also modulated the immune response, decreasing spleen cell IFN␥, TNF␣, IL-6, and IL-12 production
upon restimulation with CII in vitro. Finally, sST2
treatment was reported to down-regulate serum levels of
IL-6, IL-12, and TNF␣. In the present experiment, we
tried to assess serum levels of IFN␥ and IL-17 at the
time that mice were killed; however, the levels of both
cytokines were below the detection limits of the ELISAs.
Taken together, the results of these 2 studies strongly
suggest a role for IL-33 in the pathogenesis of arthritis.
ST2 is known as a selective marker of Th2
lymphocytes (5), and studies using anti-ST2 antibodies
or sST2-Fc demonstrated an important role for this
receptor in Th2 responses, although there were conflicting results regarding the contribution of this receptor to
Th2 effector functions in ST2-deficient mice (31–35).
Systemic administration of recombinant IL-33 to mice
induced prototypical Th2 responses, with increased levels of IL-4, IL-5, and IL-13, enhanced IgE and IgA
production, and pathologic changes in the lungs and
digestive tract (1). However, our findings of a marked
decrease in IFN␥ production by draining LN cells from
747
anti-ST2 antibody–treated mice, as well as the decreased
production of IFN␥ and TNF␣ by ex vivo–stimulated
spleen cells reported after administration of ST2-Fc in
mice with CIA, suggest that IL-33 is also involved in the
development of Th1 responses (20). In addition, IL-33
was recently reported to promote the production of
IFN␥ by natural killer (NK) cells in a model of parasitic
infection in SCID mice (36). Furthermore, in human
Th2, NK, and invariant NK T cells, IL-33 stimulated the
production of IFN␥ in an antigen-dependent and
-independent manner (37).
Taken together, these results indicate that IL-33
can induce both Th1 and Th2 responses according to the
stimulation, the cell type, and the cytokine environment
involved. These findings are reminiscent of previous
observations concerning IL-18, a cytokine originally
described as a potent inducer of IFN␥ by Th1 cells,
which is also able to stimulate Th2 responses depending
on the system examined (38).
IL-17 production by draining LN cells was also
somewhat reduced in anti-ST2–treated mice, suggesting
that, like other IL-1 cytokine family members, IL-33 can
enhance Th17 responses (39–41). However, it is unknown whether IL-33 is able to stimulate the production
of IL-17 directly or whether IL-33 acts indirectly through
the induction of cytokines such as IL-1 and IL-6. Interestingly, in separate studies we have observed that
systemic administration of IL-33 leads to increases in
both IL-1 and IL-6 levels in C57BL/6 and BALB/c mice
(Toy D, Smith DE: unpublished observations).
RANKL mRNA expression was reduced in the
joints of anti-ST2–treated mice, suggesting that anti-ST2
treatment affects local as well as systemic responses and
may beneficially affect RANK-mediated bone erosions.
In addition, expression of several inflammation-related
genes, such as TNF␣, MMP-9, and iNOS, also tended to
be lower in anti-ST2–treated mice.
Like other members of the IL-1 family, such as
IL-1␣, IL-1␤, and IL-18, IL-33 is expressed as a propeptide and lacks a typical leader sequence for protein
secretion. Our results indicate that, like ECs, synovial
fibroblasts express predominantly the pro form of IL-33
and that it is primarily detected in cell nuclei (21,22).
Potential nuclear functions of IL-33 in these cells are as
yet unclear. ProIL-1␣ is also mainly detected in cell
nuclei, where it has been shown to exert several activities
independent of receptor binding, such as regulation of
cell proliferation, senescence, motility, apoptosis, and
inflammatory responses (23,42–44). IL-33 may thus exert different biologic functions both inside and outside
the cells, as previously shown for proIL-1␣ or for high
748
PALMER ET AL
mobility group box chromosomal protein 1 (45). The
ability of an antireceptor antibody to ameliorate signs of
disease suggests that at least some of the inflammation is
driven by extracellular IL-33.
In conclusion, we report that IL-33, a novel
member of the IL-1 family of cytokines, is expressed in
human RA synovium and synovial fibroblasts and in the
joints of mice with CIA. Administration of a blocking
anti-ST2 antibody attenuated the severity of CIA and
was associated with a marked decrease in IFN␥ production by draining LN cells. Our findings indicate that
IL-33 is produced locally in inflamed joints and that
neutralization of IL-33 signaling has a therapeutic effect
on the course of arthritis.
ACKNOWLEDGMENTS
We would like to thank Philippe Henchoz and David
Swart for their expert technical assistance, Jim Rottman and
Kim Merrimam for human immunohistochemistry data, and
Thomas McKee and Patricia Gindre for their help with double
immunostainings.
AUTHOR CONTRIBUTIONS
Dr. Gabay had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Palmer, Gabay.
Acquisition of data. Palmer, Talabot-Ayer, Lamacchia, Toy, Seemayer,
Viatte.
Analysis and interpretation of data. Palmer, Talabot-Ayer, Lamacchia, Toy, Seemayer, Viatte, Finckh, Smith, Gabay.
Manuscript preparation. Palmer, Finckh, Smith, Gabay.
Statistical analysis. Palmer, Toy, Finckh.
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