of September 13, 2015.
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Human Lung Tissue Cells
IL-33 Mediates Inflammatory Responses in
Saito and Akio Matsuda
Futamura, Noriko Hashimoto, Kenji Matsumoto, Hirohisa
Akiko Yagami, Kanami Orihara, Hideaki Morita, Kyoko
2010; 185:5743-5750; Prepublished online 6
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The Journal of Immunology
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The Journal of Immunology
IL-33 Mediates Inflammatory Responses in Human Lung
Akiko Yagami,*,†,1Kanami Orihara,*,1Hideaki Morita,*,‡Kyoko Futamura,*,x
Noriko Hashimoto,* Kenji Matsumoto,* Hirohisa Saito,* and Akio Matsuda*
IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2
cells and mastcells. Althoughpolymorphic variation inST2 is strongly associatedwith asthma, it is currently unclear whether IL-33
acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33–responsive cells among
primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or
smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by
fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells signifi-
cantly reduced the IL-33–dependent upregulation of IL-8, suggesting that IL-33–mediated responses in these cells occur via the
ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and
epithelial cells. The IL-33–mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid
treatment. In contrast, the effect of corticosteroid treatment on the IL-33–mediated responses of endothelial cells was only partial.
IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required
for the IL-33–mediated responses of endothelial cells, whereas ERK was required for IL-33–mediated IL-8 production
by epithelial cells. Taken together, these findings suggest that IL-33–mediated inflammatory responses of lung tissue cells may
be involved in the chronic allergic inflammation of the asthmatic airway.
numerous studies established that the ST2 receptor is a selective
marker on both murine and human Th2 cells (2). Recent studies
have demonstrated that ST2 is also expressed on mast cells (3, 4),
eosinophils (5, 6), and basophils (7), but not on Th1 cells or neu-
trophils. IL-33 potently drives the production of proinflam-
matory Th2-associated cytokines, including IL-4, IL-5, and IL-13,
by in vitro polarized Th2 cells (1), mast cells (3, 4, 8), and
basophils (9). These hematopoietic cells also produce other in-
flammatory cytokines and chemokines, including IL-6 and IL-8,
via IL-33 stimulation (3, 4, 6, 8, 9). More recently, Allakhverdi
et al. (10) demonstrated that circulating CD34+hematopoietic
progenitor cells expressed ST2 and responded to IL-33 by rapidly
The Journal of Immunology, 2010, 185: 5743–5750.
nterleukin-33 is a newly identified member of the IL-1 family
that is a ligand for the orphan IL-1 family receptor ST2 (also
called IL1RL1, DER4, Fit-1, or T1) (1). Over the past decade,
releasing high levels of Th2-associated cytokines. Furthermore,
IL-33 not only drives the production of cytokines/chemokines by
various hematopoietic cells but also directly activates eosinophils
(5, 6), basophils (7), and dendritic cells (11). These activities
suggest potential roles for IL-33 in Th2-associated immune
responses, and thus IL-33 is thought to be closely associated with
allergic inflammatory diseases, including asthma.
Indeed, a very recent article reported increased IL-33 levels in
the bronchoalveolar lavage fluid from subjects with moderate
asthma compared with that in mild asthmatics and controls without
asthma (12). The same group also reported that bronchial epi-
thelium (12) and airway smooth muscle cells (13) from asthmatics
expressed elevated levels of IL-33 compared with that in healthy
controls. Furthermore, a recent genome-wide association study
showed that a single-nucleotide polymorphism in ST2/IL1RL1
was most strongly associated with asthma in a collection of 10
different populations (14). A single-nucleotide polymorphism in
IL-33 that showed a suggestive association with the circulating
eosinophil count was also significantly associated with atopic
asthma (14). These findings further support the pathophysiological
relevance of the IL-33/ST2 pathway to asthma.
Lung tissue cells as well as a number of inflammatory cells are
known to participate in airway inflammatory responses and play
important roles in the pathogenesis of asthma. Chronic inflam-
mation in the lung leads to persistent structural alterations in the
airway wall (i.e., airway remodeling), which is thought to cause
irreversible airflow obstruction and exacerbation of asthma (15).
Airway remodeling consists of several structural alterations, such
as goblet cell hyperplasia, subepithelial fibrosis, smooth muscle
cell hypertrophy/hyperplasia, and angiogenesis in the lung (15).
However, it is currently unclear whether IL-33, a pro-Th2
cytokine, acts directly on lung tissue cells at sites where airway
remodeling occurs. We therefore designed this study to identify
IL-33–responsive cells among human lung tissue cells and found
*Department of Allergy and Immunology, National Research Institute for Child
Health and Development;
of Medicine, Toyoake; andxDepartment of Dermatology, Nagoya University, School
of Medicine, Nagoya, Japan
‡Department of Pediatrics, Keio University, School of
†Department of Dermatology, Fujita Health University, School
1A.Y. and K.O. contributed equally to this work.
Received for publication November 30, 2009. Accepted for publication September 8,
This work was supported in part by National Institute of Biomedical Innovation
Grants ID05-24 and ID05-41 and Japan Health Science Foundation Grant KH51046.
Address correspondence and reprint requests to Dr. Akio Matsuda, Department of
Allergy and Immunology, National Research Institute for Child Health and Develop-
ment, 2-10-1 Okura, Setagaya-ku, 157-8535, Tokyo, Japan. E-mail address: amatsuda@
Abbreviations used in this paper: BSMC, bronchial smooth muscle cell; FP, flutica-
sone propionate; HCAEC, human coronary artery endothelial cell; HMVEC-LBl,
human microvascular endothelial cells from lung blood vessels; NHBE, normal
human bronchial epithelial cell; NHLF, normal human lung fibroblast; siRNA, small
interference RNA; sST2, soluble ST2; ST2L, membrane-bound ST2.
by guest on September 13, 2015
that IL-33 acts directly on pulmonary microvascular endothelial
cells and epithelial cells, but not on smooth muscle cells or
fibroblasts, via the ST2 receptor. More importantly, we found that
Th2 cytokines, such as IL-4 and IL-13, significantly enhanced ST2
expression and function in both endothelial and epithelial cells.
These findings suggest that IL-33–mediated inflammatory re-
sponses in lung tissue cells may be crucially involved in the
chronic allergic inflammation of the asthmatic airway.
Materials and Methods
Recombinant human IL-33 was purchased from PeproTech (Rocky
Hill, NJ). Recombinant human ST2-Fc chimera was purchased from R&D
Systems (Minneapolis, MN). PD98059 and SB202190 were purchased
from Calbiochem (La Jolla, CA). Fluticasone propionate was purchased
from Sigma (St. Louis, MO).
Primary human cell culture, treatment, and transfection
Normal human bronchial epithelial cells (NHBEs), normal human lung
fibroblasts (NHLFs), bronchial smooth muscle cells (BSMCs), human
microvascular endothelial cells from lung blood vessels (HMVEC-LBl),
neonatal normal human epidermal keratinocytes, normal human dermal
fibroblasts, normal HUVECs, and normal human coronary artery endothe-
lial cells (HCAECs) were purchased from Lonza (Walkersville, MD) and
maintained exactly as recommended by the manufacturer. NHBEs were
All the experiments described in this study were performed using sec-
ond- or third-passage cells in 70–80% confluent monolayers unless other-
All the cells were treated with different concentrations of IL-33 for up
to 24 h or with 10 ng/ml IL-4 for up to 48 h. In some experiments, NHBEs
and HMVEC-LBl were treated with different concentrations of PD98059
or SB202190 for 30 min prior to stimulation with IL-33 (Fig. 7).
Both the SAGM BulletKit and EGM-2MV BulletKit (Lonza), which are
optimized for use with NHBEs and HMVEC-LBl, respectively, contain
hydrocortisone. Therefore,experimentsexamining the effects offluticasone
propionate (Fig. 5) were performed after hydrocortisone deprivation for 24
h, as previously described (16). All other experiments described in this
study were performed using a complete medium suited for each type of cell
NHBEs and HMVEC-LBl were seeded at 5 3 104cells/well in 12-
well culture plates and cultured until the cells reached 50–60% conflu-
ence. Then, the cells were transfected with small interference RNA
(siRNA) against ST2 (No. SI00114618; Qiagen, Valencia, CA), STAT6
(No. SI02662905; Qiagen), or nontargeting control siRNA (No. 1027281;
Qiagen) at 5 nM (NHBEs) or 10 nM (HMVEC-LBl) using HiPerFect
transfection reagent (Qiagen) in accordance with the manufacturer’s
instructions. The transfected cells were further grown for 48 h and then
stimulated with the indicated cytokine(s).
Quantitative real-time PCR
Total RNA extraction, cDNA synthesis, and quantitative real-time PCR
were performed as previously described (16, 17). Primer sets for six genes
were synthesized at Fasmac (Kanagawa, Japan): ST2L (sense, 59-CTGTC-
TGGCCCTGAATTTGC-39; antisense, 59-AGCAGAGTGGCCTCAATC-
CA-39), sST2 (sense, 59- CTGTCTGGCCCTGAATTTGC-39; antisense,
59-TGGAACCACACTCCATTCTGC-39), IL-8 (sense, 59-GTCTGCTAG-
CCAGGATCCACAA-39; antisense, 59-GAGAAACCAAGGCACAGTGG-
AA-39), IL-6 (sense, 59- CAATAACCACCCCTgACCCA-39; antisense, 59-
GCGCAGAATGAGATGAGTTGTC-39), STAT6 (sense, 59-TCTGACCG-
GCTGATCATTGG-39; antisense, 59-CCAATCTCTGAGTCGCTGAAGC-
39), and b-actin (sense, CCCAGCCATGTACGTTGCTAT-39; antisense,
59-TCACCGGAGTCCATCACGAT-39). To determine the exact copy num-
bers of the target genes, quantified concentrations of the purified PCR
products of ST2L, soluble ST2 (sST2), IL-8, IL-6, STAT6, and b-actin
were serially diluted and used as standards in each experiment. Aliquots of
cDNA equivalent to 5 ng of the total RNA samples were used for each real-
time PCR. The mRNA expression levels were normalized to the b-actin
level in each sample.
The concentrations of the sST2, IL-8, IL-6, and MCP-1 proteins in cell-free
supernatants were measured with specific ELISA kits (R&D Systems) in
accordance with the manufacturer’s instructions.
Cells were seeded into 6-well plates at 1 3 105cells/well and cultured until
subconfluent (2 or 3 d). The cells were then treated for the indicated time
periods with 10 ng/ml IL-4 (for ST2 blotting, see Fig. 3D) or 10 ng/ml
IL-33 (for phospho-MAPK blotting, see Fig. 6). Whole-cell lysates were
extracted with 200 ml NuPAGE sample buffer (Invitrogen, Carlsbad, CA)
containing 5% 2-ME and lysed by sonication. Equal amounts of whole-cell
lysates were separated by SDS-PAGE (5–15% Ready Gels J; Bio-Rad,
Hercules, CA) gel electrophoresis and transferred to nitrocellulose mem-
branes (iBlot Gel Transfer Stacks, mini; Invitrogen). Immunoblotting was
performed using the following Abs: clone 97203, mouse mAb for ST2/IL-
1R4 (R&D Systems); clone D13.14.4E, rabbit mAb for phospho-p44/42
MAPK (Erk1/2) (Thr202/Tyr204); rabbit polyclonal Ab for phospho-p38
MAPK (Thr180/Tyr182) (Cell Signaling Technology, Danvers, MA); and
clone AC-15, mouse mAb for b-actin (Sigma), in accordance with the
All data are presented as the mean 6 SD. Differences between groups were
analyzed using ANOVA with Bonferroni’s post hoc test and were con-
sidered to be significant when p , 0.05.
Preferential expression of ST2 among lung tissue cells
The ST2 gene encodes, by alternative splicing, both membrane-
bound ST2L, which is a receptor for IL-33, and sST2, which is
ST2 mRNA in lung tissue cells and other human primary cells. We
found that both ST2L and sST2 were preferentially expressed in
microvascular endothelial cells (HMVEC-LBl) and airway epi-
thelial cells (NHBEs), but not in lung fibroblasts (NHLFs), smooth
muscle cells (BSMCs), epidermal keratinocytes (neonatal normal
human epidermal keratinocytes), or normal human dermal fibro-
blasts (Fig. 1A, open bars). ST2 mRNA expression was also ob-
served in other human endothelial cells, such as umbilical vein
endothelial cells (HUVECs) and coronary artery endothelial cells
(HCAECs), suggesting that ST2 is characteristically expressed in
human vascular endothelial cells. We further confirmed that the
secreted sST2 level in the culture supernatant of each type of cell
IL-33–mediated inflammatory responses in lung tissue cells
Because we had elucidated the cell type distribution pattern of
ST2L expression, we next examined the biological significance
of ST2 expression in lung tissue cells. We examined the ability of
IL-33 to induce the production of various cytokines/chemokines
by those cells. Consequently, we found that there was good cor-
respondence between the ST2L mRNA distribution and IL-33
responsiveness. More specifically, neither NHLFs nor BSMCs,
which did not express ST2L mRNA, responded to IL-33 (Fig. 1B,
yellow and green bars). In NHBEs, IL-33 induced IL-8 pro-
duction, detected in the supernatants of 24-h cultures in a dose-
dependent manner (Fig. 1B, upper graph, blue bars). However,
IL-33 induced neither IL-6 nor MCP-1 production by NHBEs. Of
note, HMVEC-LBl showed dose-dependent, enhanced production
of IL-6 and MCP-1 in addition to IL-8 in response to treatment
with IL-33 for 24 h (Fig. 1B, red bars). Thus, IL-33 induced
stronger responses in HMVEC-LBl than in NHBEs in accordance
with the levels of ST2 expression in each type of cell. Although
we looked for production of other cytokines/chemokines, includ-
ing IL-4, IL-5, IL-10, IL-12, IL-13, TNF-a, IL-1b, and IP-10,
none were found in either HMVEC-LBl or NHBEs.
IL-33 mediates inflammatory responses via the ST2 receptor in
lung tissue cells
To elucidate the role of ST2 in IL-33–mediated inflammatory
responses in lung tissue cells, we depleted ST2 mRNA by using
5744IL-33 ACTIONS IN LUNG TISSUE CELLS
by guest on September 13, 2015
siRNA specific for ST2 (No. SI00114618; Qiagen), designed
to target a site within the sequence shared by ST2L and sST2.
NHBEs (Fig. 2A) and HMVEC-LBl (Fig. 2B) were transfected
with siRNA against nontargeting control siRNA or ST2 and then
stimulated with IL-33 for 6 h. Control experiments demonstrated
that both ST2L and sST2 mRNA were significantly suppressed
by the ST2 siRNA compared with the levels of ST2L and sST2
transcripts, respectively, in nontargeting control siRNA-trans-
fected cells. Induction of IL-8 (NHBEs, HMVEC-LBl) and IL-6
(HMVEC-LBl) by IL-33 was significantly inhibited by the trans-
fection of ST2 siRNA, suggesting that IL-33–mediated responses
in these cells occur via an ST2-dependent pathway. Furthermore,
we found that IL-33–mediated responses in HMVEC-LBl were
IL-33 specific because they were almost completely suppressed
by simultaneous treatment with IL-33 and recombinant ST2-Fc
chimera (Fig. 2C).
Th2 cytokines enhance the expression and function of ST2 in
lung tissue cells
IL-33 is a potent inducer of Th2 immunity, and we thus examined
the effects of Th2 cytokines such as IL-4 on the expression and
function of ST2 in lung tissue cells. As shown in Fig. 3A, both
ST2L mRNA and sST2 mRNA were significantly upregulated by
10 ng/ml IL-4 treatment in a time-dependent manner. Importan-
tly, this IL-4–mediated upregulation of the ST2 genes was ob-
served in IL-33–responsive cells such as NHBEs and HMVEC-
LBl but not in the IL-33–unresponsive cells such as NHLFs and
BSMCs. We further confirmed that the sST2 protein levels accu-
mulated in the culture supernatants of NHBEs and HMVEC-LBl
in response to IL-4 treatment correlated well with their respective
sST2 mRNA levels (Fig. 3B, left graph).
IL-13 is another Th2 cytokine that plays a prominent role in
the pathogenesis of allergic inflammation. IL-13 and IL-4 share
many functional properties, stemming from the fact that they
share the a subunit of the IL-4R. In fact, we found that IL-13
also induced sST2 production by HMVEC-LBl (Fig. 3B, right
graph). IL-4 or IL-13 stimulation of cells leads to activation of
multiple signaling pathways via IL-4R a, one of which involves
a transcription factor, STAT6. Therefore, to examine the role of
STAT6 on IL-4–induced expression of ST2, we depleted STAT6
mRNA by using siRNA for STAT6 (No. SI02662905; Qiagen).
The siRNA for STAT6 or nontargeting control siRNA was trans-
fected into HMVEC-LBl. The transfected cells were further
cultured for 48 h and then stimulated with 10 ng/ml IL-4 for
24 h. The efficiency of STAT6 mRNA depletion was more than
70% compared with the level of STAT6 transcripts in control
siRNA-transfected cells, which was confirmed by real-time
PCR (Fig. 3C, left graph). Transfection of STAT6 siRNA sig-
nificantly reduced the IL-4–dependent upregulation of both
ST2L mRNA and sST2 mRNA (Fig. 3C, right two graphs),
suggesting that STAT6 is required for IL-4–enhanced expression
of ST2 genes.
To confirm the IL-4–enhanced expression of ST2 at the protein
level, whole-cell lysates from IL-4–stimulated HMVEC-LBl and
NHBEs were subjected to SDS-PAGE followed by immunoblot-
ting with an anti-ST2 Ab or an anti–b-actin Ab as a loading
control. We found that IL-4 significantly enhanced ST2L protein
in the whole-cell lysates of both HMVEC-LBl and NHBEs (Fig.
3D), in parallel with upregulation of ST2L mRNA in these cells
(Fig. 3A, upper graph).
We next examined whether IL-33–mediated responses of lung
tissue cells were further enhanced by IL-4 pretreatment. NHBEs
and HMVEC-LBl were pretreated with 10 ng/ml IL-4 for 48 h
and then stimulated with 10 ng/ml IL-33 for the indicated pe-
riods. IL-4–pretreated cells showed significantly enhanced IL-33–
mediated responses, including the induction of IL-8 and IL-6
mRNA (Fig. 4). Thus, Th2 cytokines significantly enhanced ST2
expression and function in both lung endothelial and epithelial
Effects of corticosteroid on IL-33–mediated responses in
epithelial and microvascular endothelial cells
Currently, inhaled corticosteroids are a first-line therapy and
known to be one of the most effective therapies available for
asthma (19). Therefore, we next examined the effect of cortico-
steroid on the responses of both NHBEs and HMVEC-LBl to
IL-33. Fluticasone propionate (FP) treatment showed significant
attenuation of IL-33–mediated IL-8 production by NHBEs even
at a low FP concentration (1 nM) (reduction to 28% of the pro-
duction in the absence of corticosteroid), and the production was
almost completely suppressed by 100 nM FP treatment (Fig. 5A).
In contrast, FP treatment showed only partial attenuation of IL-
33–mediated IL-6, IL-8, and MCP-1 production by HMVEC-LBl
(reduction to 80, 63, and 74% of the respective production in
the absence of corticosteroid) even at a high concentration of FP
(100 nM) (Fig. 5B). The higher levels of IL-8 production by
NHBEs compared with the results observed in Fig. 1B may be
due to the hydrocortisone deprivation before IL-33 stimulation.
Because IL-33–mediated IL-8 production by NHBEs was sen-
sitively inhibited by corticosteroid treatment, we presume that
human primary cells. Total RNA was isolated from various human pri-
mary cells in growth phase, and the levels of mRNA for ST2L and sST2
were measured by quantitative real-time PCR (open bars). Concentrations
of secreted sST2 protein in the culture supernatants were quantified by
ELISA (solid bars). B, IL-33–mediated cytokine-chemokine production by
lung tissue cells. NHBEs (blue), NHLFs (yellow), BSMCs (green), and
HMVEC-LBl (red) were treated with the indicated concentrations of IL-33
for 24 h. Protein concentrations in the culture supernatants are shown. Data
are shown as the mean 6 SD of triplicate samples and are representative of
experiments using at least two different lots from individual donors of
NHBEs, NHLFs, BSMCs, and HMVEC-LBl.
A, Expression of mRNA for ST2L and sST2 in cultured
The Journal of Immunology5745
by guest on September 13, 2015
IL-33 robustly enhanced IL-8 production in the absence of cor-
IL-33–induced phosphorylation of MAPK in epithelial and
microvascular endothelial cells
We next sought to evaluate the signaling pathways involved in the
IL-33 responses in both NHBEs and HMVEC-LBl. Although the
signaling pathways activated by IL-33 remain poorly understood, it
was reported that IL-33–mediated IL-8 production by human mast
cells is mediated by a signaling pathway involving p38 MAPK (8).
Therefore, we investigated whether IL-33 induces phosphorylation
of MAPK, including ERK and p38, in NHBEs and HMVEC-LBl.
In HMVEC-LBl, transient phosphorylation of both ERK and p38
was observed after 5 to 15 min treatment with IL-33 (Fig. 6). In
contrast, in NHBEs, phosphorylation of ERK was observed for up
to 60 min of treatment with IL-33, whereas constitutive phos-
phorylation of p38 was unaffected.
Effects of ERK and p38 MAPK inhibitors on IL-33–mediated
responses in microvascular endothelial cells and epithelial
To verify which MAPK was involved in the IL-33–mediated
responses in HMVEC-LBl, the cells were treated with various
concentrations of ERK inhibitor PD98059 or p38 inhibitor
SB202190 for 30 min prior to treatment with IL-33. IL-33–
mediated productions of IL-8, IL-6, and MCP-1 were dramatically
and dose-dependently reduced by the addition of p38 inhibitor
SB202190 but not by ERK inhibitor PD98059 (Fig. 7A). These
results clearly indicate that p38 MAPK is required for IL-33–
mediated responses in HMVEC-LBl.
In contrast, IL-33–mediated production of IL-8 by NHBEs was
significantly reduced by the addition of ERK inhibitor PD98059,
but not by p38 inhibitor SB202190 (Fig. 7B). This indicates that,
conversely from HMVEC-LBl, ERK is required for IL-33–medi-
ated IL-8 production by NHBEs.
In this study, we found that IL-33, a pro-Th2 cytokine, acts directly
on pulmonary microvascular endothelial cells and epithelial cells
and mediates inflammatory responses.
First, our investigation of lung tissue cells found that both ST2L
and sST2 were preferentially expressed in microvascular endo-
thelial cells (HMVEC-LBl) and airway epithelial cells (NHBEs)
but not in either lung fibroblasts (NHLFs) or smooth muscle cells
(BSMCs) (Fig. 1A). Furthermore, there was good correspondence
between the ST2 distribution (Fig. 1A) and IL-33 responsiveness
(Fig. 1B) among these lung tissue cells. This suggests that IL-33
mediates its actions via the ST2 receptor on both HMVEC-LBl
and NHBEs. Indeed, depletion of ST2 mRNA significantly re-
duced the IL-33–mediated responses of these cells (Fig. 2).
Neither NHLFs nor BSMCs showed any expression of ST2
(Fig. 1A) or responsiveness to IL-33 (Fig. 1B), suggesting that
LBl (B) were transfected with siRNA against ST2 (solid bars) or nontargeting control siRNA (open bars) at 5 nM (NHBEs) or 10 nM (HMVEC-LBl).
The transfected cells were further cultured for 48 h and then stimulated with 10 ng/ml IL-33 for 6 h. The levels of mRNA for ST2L, sST2, IL-8, and IL-6
were determined by real-time PCR. Data are shown as the mean 6 SD of triplicate samples and are representative of two individual experiments. pp ,
0.05; ppp , 0.01 compared with nontargeting control siRNA. C, IL-33–mediated responses in cultured HMVEC-LBl were IL-33 specific. HMVEC-LBl
were stimulated in the presence of 10 ng/ml IL-33 and 10 mg/ml neutralizing ST2-Fc chimera for 24 h. Protein concentrations in the culture supernatants
are shown. Data are shown as the mean 6 SD of triplicate samples and are representative of two individual experiments. ppp , 0.01 compared with
10 ng/ml IL-33.
IL-33 mediates inflammatory responses via the ST2 receptor on HMVEC-LBl and NHBEs. A and B, Cultured NHBEs (A) and HMVEC-
5746 IL-33 ACTIONS IN LUNG TISSUE CELLS
by guest on September 13, 2015
IL-33 does not act directly on these cells in the asthmatic airway.
Of note, ST2 was preferentially expressed in vascular endothelial
cells, including HUVECs and HCAECs (Fig. 1A). These obser-
vations are consistent with recent reports of sST2 secretion by
human venous and arterial endothelial cells (20, 21).
IL-33 drives production of Th2-associated cytokines, including
Unlike in those hematopoietic cells, IL-33–mediated cytokine-
chemokine production by the lung tissue cells was rather limited
(Fig. 1B), and we found no production of Th2-associated cytokines
(data not shown). It was recently reported that the ST2/IL-33
pathway is necessary not only for the development of an allergic
inflammatory response but also for its maintenance (22). Thus, the
actions of IL-33 on lung tissue cells may not contribute to the
development of allergicinflammation butrather to the maintenance
of chronic inflammation. It should be noted that Th2 cytokines,
such as IL-4, significantly enhanced ST2 expression (Fig. 3) and
function (Fig. 4) in both lung endothelial and epithelial cells.
These findings are important when considering chronic inflamma-
tion in the lung and suggest that allergic individuals may be more
susceptible to IL-33–mediated inflammatory responses of lung
tissue cells than nonallergic individuals. Aoki et al. (21) recently
reported that IL-33 stimulated secretion of IL-6 and IL-8 by
HUVECs. Notably, they showed that ST2 gene expression in
HUVECs was growth-dependent and was downregulated when
the cells were differentiated to form vascular structures on an ex-
tracellular membrane matrix in vitro, whereas vascular endothelial
growth factor gene expression was not downregulated. These
results suggest that blood vessels normally would not respond to
IL-33. In contrast, Th2-inflamed lung blood vessels and/or epi-
thelium seem to be potential targets for the actions of IL-33.
Although the results of this study were limited to in vitro
experiments, several reportsby others have shed lighton the invivo
roles of IL-33 by exogenous administration of recombinant IL-33
to mice (1, 23–25) or by transgenic overexpression of IL-33 in
mice (26). Those studies have independently provided evidence
that excessive expression of IL-33 in vivo might lead to an in-
crease in the number of inflammatory cells in the airway via re-
lease of endogenous Th2 cytokines and chemokines. Notably,
Zhiguang et al. (26) showed that pulmonary inflammation with
infiltration of inflammatory cells was observed around the blood
vessels in the airway of IL-33–transgenic mice, supporting our
conclusion from this study that pulmonary endothelial cells can
be direct targets of IL-33. Furthermore, both administration and
periods. A, The levels of mRNA for ST2L and sST2 are shown. B, The accumulated sST2 protein levels after 10 ng/ml IL-4 treatment (left graph) or 10
ng/ml IL-13 treatment (right graph) for the indicated periods are shown. ppp , 0.01 compared with no cytokine treatment (0 h). C, STAT6 is required for
IL-4–enhanced expression of ST2 in HMVEC-LBl. Cultured HMVEC-LBl were transfected with siRNA against STAT6 (solid bars) or nontargeting control
siRNA (open bars). The transfected cells were further cultured for 48 h and then stimulated with 10 ng/ml IL-4 for 24 h. The levels of mRNA for STAT6,
ST2L, and sST2 were determined by real-time PCR. pp , 0.05; ppp , 0.01 compared with nontargeting control siRNA. D, Whole-cell lysates from IL-4–
stimulated HMVEC-LBl and NHBEs were harvested, and ST2L and b-actin were analyzed by Western blotting. The fold induction of ST2L protein was
determined by densitometry and normalized to the respective b-actin level (lowergraph). Data are shown as the mean 6 SD of triplicate samples (A–C) and
are representative of three (A, B) or two (C, D) individual experiments.
Effects of Th2 cytokines on the expression of ST2 in cultured lung tissue cells. Cells were treated with 10 ng/ml IL-4 for the indicated
The Journal of Immunology 5747
by guest on September 13, 2015
transgenic overexpression of IL-33 in mice led to increased
numbers of neutrophils as well as eosinophils in the airway
(23, 25, 26). Neutrophils are not regarded as direct target cells
of IL-33 because they have few ST2 receptors on their surface.
Therefore, we surmise that IL-33 can promote neutrophil in-
filtration in the airway through IL-33–induced release of neutro-
phil chemoattractants, including IL-8 family members, by lung
Today, inhaled corticosteroids are a first-line anti-inflammatory
treatment and known to be one of the most effective therapies
available for asthma (19). Indeed, FP treatment showed significant
attenuation of IL-33–mediated IL-8 production by NHBEs even at
a low FP concentration (1 nM), and that production was almost
completely abrogated by 100 nM FP treatment (Fig. 5A), sug-
gesting that corticosteroids are capable of effectively reducing
IL-33–mediated airway epithelial inflammation. In contrast, FP
treatment was only partially effective against IL-33’s actions on
microvascular endothelial cells (Fig. 5B), which were found to
be the main IL-33–targeted cells among the lung tissue cells
(Fig. 1B). We recently showed that corticosteroid treatment was
also only weakly effective on TNF-a–mediated microvascular
inflammation, including chemokine production (16). In addition,
corticosteroid enhanced TNF-a–mediated leukocyte adhesion
to pulmonary microvascular endothelial cells via upregulation
of cell-surface expression of ICAM-1 and VCAM-1 (16). Taken
together, those various findings suggest that the poor effect of
corticosteroid on TNF-a/IL-33–mediated inflammatory responses
is not only a specific feature of airway microvessels but is also
crucially involved in the refractoriness seen in the asthmatics.
Notably, Pre ´fontaine et al. (13) recently demonstrated that dexa-
methasone fails to abolish TNF-a–induced IL-33 upregulation in
primary human airway smooth muscle cells, further suggesting
a contribution of IL-33 as well as TNF-a to the refractory phe-
notype of certain asthmatics treated with corticosteroids.
Persistent chronic inflammation in the lung leads to structural
alterations in the airway wall (i.e., airway remodeling), which is
thought to cause irreversible airflow obstruction and exacerbation
of asthma (15). Recent compelling evidence has demonstrated that
pretreatment of NHBEs and HMVEC-LBl. Cultured NHBEs (A) or
HMVEC-LBl (B) were treated with 10 ng/ml IL-4 for 48 h, and then the
cells were washed twice with HBSS and replaced with fresh medium
containing 10 ng/ml IL-33 for the indicated periods. The levels of mRNA
for IL-8 and IL-6 were determined by real-time PCR. Data are shown as
the mean 6 SD of triplicate samples and are representative of three in-
IL-33–mediated responses were further enhanced by IL-4
NHBEs and HMVEC-LBl. Cultured NHBEs (A) and HMVEC-LBl (B)
were simultaneously treated with IL-33 and FP for 24 h at the indicated
concentrations. Concentrations of IL-8, IL-6, and MCP-1 in the culture
supernatants are shown. Data are shown as the mean 6 SD of triplicate
samples and are representative of at least three individual experiments.
pp , 0.05; ppp , 0.01 compared with 10 ng/ml IL-33.
Effects of corticosteroid on IL-33–mediated responses in
HMVEC-LBl and NHBEs. Whole-cell lysates were examined at the in-
dicated time points after stimulation with 10 ng/ml IL-33 for the expres-
sion of phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), phospho-p38
MAPK (Thr180/Tyr182), and b-actin (as a loading control).
IL-33–induced phosphorylation of MAPK in cultured
5748IL-33 ACTIONS IN LUNG TISSUE CELLS
by guest on September 13, 2015
airway hypervascularity in severe asthma, an element of airway
remodeling resulting from accelerated angiogenesis, responds
poorly to corticosteroid treatment and is clinically involved in
reduced lung function (27–29). We previously showed that auto-
crine CXCR2 chemokines, such as IL-8, are indispensable for
lung angiogenesis in a corticosteroid-insensitive manner (16, 17,
30). As shown in our current study, IL-33 can induce IL-8 pro-
duction by pulmonary endothelial and epithelial cells (Fig. 1B),
suggesting that IL-33 is involved in lung angiogenesis and the
resultant airway hypervascularity. As a matter of fact, Choi et al.
(31) recently demonstrated that IL-33 promotes angiogenesis and
vascular permeability by stimulating endothelial NO production
via the ST2 receptor.
As shown in Fig. 1B, both IL-6 and MCP-1, which were also
secreted by IL-33–stimulated pulmonary microvascular endo-
thelial cells, are known to be critically involved in allergic in-
flammation (32–34). Therefore, these proinflammatory mediators
originating from IL-33–stimulated pulmonary microvessels may
also play roles in the maintenance of chronic allergic inflam-
mation of the asthmatic airway.
Although IL-33/ST2 signaling pathways remain poorly un-
derstood, it could be expected that the signaling molecules are
similar to those for other IL-1 family cytokines. Indeed, it was
reported that IL-33, as well as IL-1b, can enhance MAPK (ERK
and p38) phosphorylation in both murine (1) and human (8) mast
cells. We confirmed that IL-33 can activate MAPK (ERK and
p38) phosphorylation in HMVEC-LBl (Fig. 6). Moreover, as was
reported for IL-33–induced IL-8 production by human mast cells
(8), IL-33–induced production of each of IL-8, IL-6, and MCP-1
by HMVEC-LBl was dramatically and dose-dependently reduced
by treatment with a p38 inhibitor, SB202190, but not with an
ERK inhibitor, PD98059 (Fig. 7A). This suggests that the IL-33–
mediated signaling pathway in human microvascular endothelial
cells is similar to that in human mast cells. In contrast, ERK,
but not p38, is required for IL-33–mediated IL-8 production by
NHBEs (Fig. 7B). Because ST2L mRNA remained at a lower level
in NHBEs than in HMVEC-LBl (Fig. 1A), we initially considered
that NHBEs respond only partially to IL-33 and produce only IL-8
(not IL-6 or MCP-1) (Fig. 1B) simply due to a smaller number of
ST2 protein molecules on the surface of NHBEs compared with
that of HMVEC-LBl. However, as described above, we found
a distinct difference between these cells in their requirements for
MAPK in the IL-33–mediated signaling pathway. These mecha-
nistic differences between HMVEC-LBl and NHBEs in their
IL-33–mediated signaling pathways should be further elucidated.
Nevertheless, these observations suggest that, contrary to our ini-
tial expectation, IL-33–mediated responses in NHBEs use signal
transduction pathways that are distinct from the pathways in
HMVEC-LBl and human mast cells.
The main sources of IL-33 involved in the pathogenesis of
asthma remain controversial. IL-33 was originally identified as
NF-high endothelial venules, which is an NF preferentially
expressed in high endothelial venules (35). The same group also
reported that endothelial cells constitute a major source of IL-33
mRNA in chronically inflamed tissues from patients with rheu-
matoid arthritis and Crohn’s disease (36). Furthermore, they
showed abundant nuclear expression of IL-33 in endothelial cells
from both large and small blood vessels in most normal human
tissues (37), suggesting that endothelial cells constitute major
sources of IL-33 in vivo. Indeed, we also confirmed expression of
IL-33 mRNA and protein in whole-cell lysates, but not culture
supernatants, of HMVEC-LBl by real-time PCR and ELISA, re-
spectively (data not shown). Although further studies are clearly
needed, we speculate that endogenous IL-33 released from in-
flamed and/or injured blood vessels acts on neighboring vessels
as an endogenous “danger signal” (37), leading to chronic in-
flammatory responses. Notably, release of the IL-33 “danger sig-
nal” by damaged/injured endothelial cells has recently been dem-
onstrated (38), lending further support to the endogenous “danger
In conclusion, IL-33, a pro-Th2 cytokine, acts directly on pul-
monary microvascular endothelial cells and epithelial cells (among
lung tissue cells), which express its ST2 receptor. Importantly,
Th2 cytokines significantly enhanced ST2 expression and function
in both endothelial and epithelial cells. Furthermore, the responses
of those cells, especially microvascular endothelial cells, to IL-33
are almost refractory to corticosteroid treatment, and we thus an-
ticipate that IL-33 and/or its receptor, ST2, may be able to be
exploited as a novel target for development of curative drugs for
inhibitor (PD98059) and p38 MAPK in-
hibitor (SB202190) on IL-33–mediated
responses in HMVEC-LBl and NHBEs.
Cultured HMVEC-LBl (A) and NHBEs
(B) were treated with the indicated con-
centrations of PD98059 or SB202190 for
30 min prior to stimulation with 10 ng/ml
IL-33 for 24 h. Data are shown as the
mean 6 SD of triplicate samples and are
representative of three individual experi-
ments. pp , 0.05; ppp , 0.01 compared
with 10 ng/ml IL-33.
Divergent effects of ERK
The Journal of Immunology5749
by guest on September 13, 2015
Acknowledgments Download full-text
We thank Shuhei Fukuda of the Department of Allergy and Immunology,
The authors have no financial conflicts of interest.
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