This information is current as
of June 17, 2017.
IL-17A Induces Eotaxin-1/CC Chemokine
Ligand 11 Expression in Human Airway
Smooth Muscle Cells: Role of MAPK (Erk1/2,
JNK, and p38) Pathways
Muhammad Shahidur Rahman, Akira Yamasaki, Jie Yang,
Lianyu Shan, Andrew J. Halayko and Abdelilah Soussi
Gounni
References
Subscription
Permissions
Email Alerts
This article cites 62 articles, 21 of which you can access for free at:
http://www.jimmunol.org/content/177/6/4064.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
J Immunol 2006; 177:4064-4071; ;
doi: 10.4049/jimmunol.177.6.4064
http://www.jimmunol.org/content/177/6/4064
The Journal of Immunology
IL-17A Induces Eotaxin-1/CC Chemokine Ligand 11
Expression in Human Airway Smooth Muscle Cells:
Role of MAPK (Erk1/2, JNK, and p38) Pathways1
Muhammad Shahidur Rahman,* Akira Yamasaki,*† Jie Yang,* Lianyu Shan,*
Andrew J. Halayko,†‡ and Abdelilah Soussi Gounni2*
A
irway smooth muscle (ASM)3 cells are key determinants
of asthma due to their ability to contract in response to
inflammatory cell products (1, 2). Due to intrinsic phenotype plasticity, airway myocytes exhibit a capacity for multifunctional behavior and contribute to local inflammation and fibrosis (3). Emerging evidence suggests that ASM cells can
contribute directly to the pathogenesis of asthma by expressing
adhesion and costimulatory molecules, and by secreting multiple
proinflammatory cytokines and chemokines (4, 5).
Eotaxin-1/CCL11 is a CC chemokine that was first identified as
an important chemoattractant for eosinophils in lungs from Agsensitized and -challenged guinea pigs (6). Increased eotaxin-1/
CCL11 mRNA expression has been found both in bronchial biopsies and in cells from bronchoalveolar lavage of mild human
asthmatics (7–9). Moreover, measured concentrations of this chemokine appear to correlate well with the severity of the disease
*Department of Immunology, †Department of Physiology, and ‡Section of Respiratory Diseases, University of Manitoba, Winnipeg, Manitoba, Canada
Received for publication October 18, 2005. Accepted for publication June 29, 2006.
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.
1
This work was supported by a grant from the Canadian Institutes of Health Research
(CIHR) (to A.S.G.). M.S.R. was supported by a fellowship from CIHR National
Training Program in Allergy and Asthma (NTPAA). A.Y. was supported by a fellowship from CIHR-NTPAA and Manitoba Institute of Child Health. A.S.G. is supported by a CIHR New Investigator Award.
2
Address correspondence and reprint requests to Dr. Abdelilah Soussi Gounni, Department of Immunology, 606 Basic Medical Sciences Building, Faculty of Medicine,
730 William Avenue, Winnipeg, Manitoba, Canada R3E 0W3. E-mail address:
[email protected]
3
Abbreviation used in this paper: ASM, airway smooth muscle.
Copyright © 2006 by The American Association of Immunologists, Inc.
(10). Other studies have shown that eotaxin-1/CCL11 neutralization in mice substantially reduced eosinophil recruitment after Ag
challenge, thus indicating an important functional role in promoting airway inflammation in asthma (11, 12). Studies using asthmatic airways and isolated primary cells indicate ASM cells are a
significant source for eotaxin-1/CCL11 production both in vivo (4)
and in vitro (13, 14).
Human IL-17A is the founding member of an emerging cytokine family, with pleiotropic biological activities and is released by
activated CD4⫹ T cells (15). Exaggerated levels of IL-17A are
observed in chronic inflammatory disorders (16, 17), suggesting a
potential role of this cytokine in the initiation or maintenance of
inflammatory responses. Although IL-17A has been shown to be
present in higher levels in respiratory secretions from asthmatics
(18) and increased concentrations of IL-17A correlate with airway
hyperresponsiveness (19), the mechanism by which this cytokine
may influence allergic disease processes remains unknown.
In the current study, we show for the first time that IL-17A alone
can induce eotaxin-1/CCL11 mRNA expression and protein release
from ASM cells. This effect of IL-17A alone, or in combination with
IL-1␤, is dependent on de novo protein and mRNA synthesis. Transfection studies using eotaxin-1/CCL11 promoter luciferase construct
showed that IL-17A induced eotaxin-1/CCL11 transcription in ASM
cells. IL-17A signaling via MAPK (p38, p42/p44 ERK, and JNK),
and probably STAT-3, appeared to be required for the induction of
eotaxin-1/CCL11 synthesis and release in ASM cells.
Materials and Methods
Reagents
Recombinant human IL-17A, IL-1␤, IL-4, neutralizing mouse anti-human
IL-17A mAb, mouse anti-human p38 ␣ mAb, mouse anti-human ERK1/2
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with
airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine
(eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells
with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing
anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was
abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter
construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional
level. IL-17A also enhanced significantly IL-1␤-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM
cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a
significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of
MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the
first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11
production. The Journal of Immunology, 2006, 177: 4064 – 4071.
The Journal of Immunology
mAb, affinity-purified rabbit anti-phopsho-p38 MAPK (T180/Y182), and
affinity-purified rabbit anti-phospho-ERK1/2 (T202/Y204) were purchased
from R&D Systems. Rabbit anti-total and phospho-specific SAPK/JNK
(T183/Y185) Abs was purchased from Cell Signaling. Mouse IgG1 antiphospho-tyrosine-specific STAT-5, STAT-6 were purchased from BD Biosciences. Mouse mAb anti-phospho-tyrosine STAT-3 (Y705), affinity-purified rabbit anti-total STAT-3, STAT-5, and STAT-6 were from Santa
Cruz. Mouse IgG isotype control (clone MOPC21), actinomycin D, cycloheximide, and p-nitrophenyl phosphate disodium substrate were from
Sigma-Aldrich. The p38 MAPK inhibitor, SB203580 (4-[4-fluorophenyl]5-[4-pyridyl]-1H-imidazole); the p42/p44 ERK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenyl-thio]butadiene); the JNK inhibitor
II, SP600125 (anthrax(1–9-c-d)pyrazol-6(2H)-one; 1,9-pyrazoloanthrone);
the JAK inhibitor I (2-(1–1-dimethylethyl)-9-fluoro-3,6 dihydro-7Hbenz[H]-immidaz[4,5]isoquinlin-7-one); and the PI3K inhibitor (wortmannin) were purchased from Calbiochem. All cell culture media (DMEM and
F-12), antibiotics (penicillin, streptomycin), and cell culture reagents were
obtained from Invitrogen Life Technologies, and FBS was from HyClone
Laboratories. The anti-human smooth muscle actin Ab was obtained from
DakoCytomation. Alkaline phosphatase-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories.
Isolation and culture of human ASM cells
Cell culture and ELISA analysis of chemokine release in cell
supernatants
Semiconfluent ASM cells (75%; passages 2–5) were growth arrested for
48 h in FBS-free Ham’s F-12 containing 5 ␮g/ml recombinant human
insulin, 5 ␮g/ml human transferrin, 5 ng/ml selenium, and antibiotics (100
U/ml penicillin and 100 ␮g/ml streptomycin). Cells were then cultured in
fresh, serum-free Ham’s F-12 with graded concentrations (0.1, 1, 10, and
100 ng/ml) of recombinant human IL-17A, IL-1␤ (0.1, 1, 10, 100 ng/ml),
combination or medium alone. Supernatants were then harvested at 24
and/or 48 h. For neutralizing experiments, mouse anti-human IL-17A neutralizing mAb or mouse IgG control was added for 2 h to serum-deprived
ASM cells before stimulation with IL-17A for 24 h. In some experiments,
cells were pretreated for 1 h with actinomycin D (1 ␮g/ml), cycloheximide
(5 ␮g/ml), SB203580 (10 ␮M), U 0126 (10 ␮M), SP600125 (40 nM) or
wortmannin (100 nM), JAK inhibitor (15 nM) before stimulation for 24
and 48 h with IL-17A (10 ng/ml), IL-1␤ (10 ng/ml), or both at 10 ng/ml.
Control cells were cultured in the presence of an equivalent amount of
DMSO. Supernatants collected in different experiments were centrifuged at
1200 rpm for 7 min at 4°C to remove cellular debris, and stored at ⫺80°C
until analysis was conducted by ELISA. Immunoreactive eotaxin-1/CCL11
was measured by ELISA as described previously (21). Immunoreactive
IL-6 and IL-8/CXCL8 were quantified by ELISA using matched Abs from
Pierce Endogen (Biosource International) according to basic laboratory
protocols. The sensitivity limit for both IL-6 and IL-8/CXCL8 was 10 and
7.8 pg/ml for eotaxin-1/CCL11. Each data point represents readings from
a minimum of four independent assays performed in triplicate.
RNA isolation and real-time RT-PCR analysis
Confluent human ASM cells (passages 2–5) were growth arrested for 48 h
in serum-free medium as described above. Cells were then stimulated in
fresh FBS-free medium containing human recombinant IL-17A, IL-1␤,
IL-17A plus IL-1␤ (both at 10 ng/ml), or vehicle (medium alone) for 6 h.
In some experiments, cells were pretreated for 1 h with actinomycin D (1
␮g/ml) before stimulation with cytokines. Cells were harvested and total
cellular RNA was extracted using the guanidinium isothiocyanate method
(22). Reverse transcription was performed using 1 ␮g of total RNA in a
first-strand cDNA synthesis in a 20-␮l reaction with SuperScript reverse
transcriptase as recommended by the supplier (Invitrogen Life Technolo-
gies). Relative levels of eotaxin-1/CCL11 mRNA were analyzed using
quantitative real-time PCR analysis by LightCycler (Roche). Primers for
housekeeping gene GAPDH, and standard controls were developed in our
laboratory. The forward and reverse specific primer sequences used, the
size of the amplified fragment, and the annealing temperature for GAPDH
are as follows: 5⬘-AGCAATGCCTCCTGCACCACCAAC-3⬘ and 5⬘CCGGAGGGGCCATCCACAGTCT-3⬘, 137 bp, 59°C. DNA standards
were prepared from PCR using cDNA from cells stimulated by IL-1␤. PCR
products were isolated from 0.5% w/v agarose gel using QIAEX II Agarose
Gel Extraction kit (Qiagen). The amount of extracted DNA was quantified
by spectrophotometry and expressed as copy number. A serial dilution was
used to generate each standard curve. For eotaxin-1/CCL11, supplied
primer set and standard were used (Roche). For real-time quantitative PCR,
each reaction contained the following: 1⫻ LightCycler-DNA master
SYBRGreen I (Roche), 25 mM MgCl2, 0.5 ␮M of each primer, 0.07 ␮M
TaqStart Ab (Clontech), and 1 and 0.5 ␮l (1/20 and 1/40 dilution) of cDNA
matrix, in a final volume of 20 ␮l. After 10 min of denaturation at 95°C,
the reactions were cycled 40 times for 5 s at 95°C, 10 s at the annealing
temperature, 7 s at 72°C for GAPDH and 35 times for 10 s at 95°C, 10 s
at the annealing temperature, and 16 s at 72°C for eotaxin-1/CCL11, respectively. Product specificity was determined by melting curve analysis,
and by visualization of PCR products on agarose gels. Calculation of the
relative amount of each cDNA species was performed according to standard protocols. Briefly, the amplification of eotaxin-1/CCL11 gene in stimulated cells was calculated first as the copy number ratio of eotaxin-1/
CCL11 per copy of GAPDH, and then expressed as normalized values of
fold increase over the value obtained with unstimulated control cells.
Assessment of ERKp42/p44, p38 MAP, JNK, and STAT-3, -5, -6
phosphorylation
Nearly confluent ASM cells were growth arrested by FBS deprivation for
48 h as described above. Cells were then stimulated in fresh FBS-free
medium with IL-17A (10 ng/ml), or medium alone. At selected time points,
the cells were washed once with cold PBS, and total proteins were extracted with lysis buffer (1% Nonidet P-40, PMSF, 2 mM sodium vanadate,
0.1% sodium deoxycolate, and protease inhibitor mixture (Roche)). Harvested lysates were centrifuged for 10 min at 4°C to pellet cellular debris.
The supernatants were removed and stored at ⫺70°C. Protein lysate (10
␮g) was loaded on 10% SDS-PAGE, followed by transfer to nitrocellulose
membranes (Invitrogen Life Technologies). The blots were then blocked
with 5% nonfat dry milk in TBS/0.1% Tween (TBST) for 1 h at room
temperature, and then incubated overnight at 4°C with Abs specific for
phosphorylated ERK1/2 (T202/Y204), p38 MAPK (T180/Y182), JNK
(T183/Y185), STAT-3 (Y705), STAT-5 (Y694), and STAT-6 (Y641). After washing with TBST, the blots were incubated with goat anti-mouse or
goat anti-rabbit HRP-conjugated secondary Abs and bands were revealed
with ECL reagents (Amersham Biosciences). After stripping, total antiERK, p38 MAPK, JNK, STAT-3, STAT-5, STAT-6, and ␤-actin were
used as loading control.
Luciferase reporter constructs and cell transfection
Eotaxin1/CCL11 promoter fragment was amplified from human cell
genomic DNA using PCR with specific primers (23). Briefly, a 2.2-kb
amplified fragment was inserted into pGL3-Basic vector (Promega) yielding the reporter construct pGL3-EO2. Constructs used for transient transfection were purified by cesium chloride density gradients. ASM cells (4 ⫻
104) were seeded into 24-well culture plates in fresh complete DMEM.
After 24 h at 70% confluency, cells were transfected with wild-type plasmid pGL3-EO2 eotaxin-1/CCL11 promoter luciferase construct (23). Transient transfection of ASM cells was performed using ExGen 500 in vitro
transfection reagent (Fermentas) according to the manufacturer’s instructions. In each well, 0.8 ␮g of eotaxin-1/CCL11 promoter-luciferase DNA
and 0.2 ␮g of Renilla luciferase reporter vector-pRL-TK (Promega) were
cotransfected for 24 h. Then, cells were washed and stimulated with IL17A (10 ng/ml), IL-1␤ (10 ng/ml), or both at 10 ng/ml each. IL-4 (100
ng/ml) was used as positive control. After 12 h of cytokine stimulation,
cells were washed twice with PBS and cell lysates were collected with 100
␮l of reporter lysis buffer (Promega). The luciferase activity was measured
by the Dual-Luciferase Assay System kit (Promega) using a luminometer
(model LB9501; Berthold Lumat). Briefly, 20 ␮l of cell lysate was mixed
with 100 ␮l of Luciferase Assay Reagent II (Promega) and firefly luciferase activity was first recorded. Then, 100 ␮l of Stop-and-Glo Reagent
(Promega) was added, and Renilla luciferase activity was measured. All
values are normalized to Renilla luciferase activity and expressed relative
to the control transfected nonstimulated cells.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Human bronchial smooth muscle cells were obtained from macroscopically
healthy segments of second to fourth generation lobar or main bronchus of
patients undergoing surgery for lung carcinoma in accordance with the
procedures approved by the Ethics Committee of the University of Manitoba (Winnipeg, Canada). Informed consent for ASM harvesting was obtained from all patients. Primary ASM cells were isolated from explants as
described previously (20). Cells were cultured in DMEM supplemented
with 10% FBS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ␮g/ml) at 37°C with 5% CO2. At confluence, primary human
ASM cells exhibited spindle morphology and a hill-and-valley pattern that
is characteristic of smooth muscle in culture. Moreover, ASM cells at confluence retain smooth muscle-specific actin, SM22, and calponin protein
expression, and mobilize intracellular Ca2⫹ in response to acetylcholine, a
physiologically relevant contractile agonist (20).
4065
4066
MECHANISMS OF IL-17A-MEDIATED CCL11 EXPRESSION IN ASM CELLS
Statistical analysis
Results are expressed as means ⫾ SD. Differences between the groups
were analyzed using Kruskal-Wallis with Dunne test. Differences between
pairs were assessed by Mann-Whitney U test. Values of p ⬍ 0.05 were
considered statistically significant.
Results
rIL-17A induces eotaxin-1/CCL11 protein release from ASM
cells
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
To investigate whether IL-17A influences eotaxin-1/CCL11 release from ASM cells, serum-deprived ASM cells were stimulated
with a wide range of IL-17A concentrations (0.1–100 ng/ml).
Stimulation with IL-17A induced the release of eotaxin-1/CCL11
in a dose-dependent manner with a maximum induction at 48 h
(Fig. 1A). At both 24- and 48-h time points, a statistically significant increase in eotaxin-1/CCL11 release from ASM cells occurred with 1, 10, and 100 ng/ml IL-17A ( p ⬍ 0.05; Fig. 1A).
Furthermore, analysis of the same supernatants revealed a normal
level of IL-6 (Fig. 1B) and CXCL-8/IL-8 (data not shown) as we
reported previously (24). Although at 100 ng/ml IL-17A seems to
induce more eotaxin-1/CCL11 release (Fig 1A), analysis of data
from five ASM cell lines showed no statistical significance in
eotaxin-1/CCL11 release of cells stimulated with 10 or 100 ng/ml
(fold increase, 2.5 ⫾ 0.7 vs 3.3 ⫾ 1.2, respectively, at 48 h; p ⫽
0.67).
Coincubation with an anti-IL-17A neutralizing mAb prevented
IL-17A-mediated eotaxin-1/CCL11 release ( p ⬍ 0.01) (Fig. 1C).
In contrast, mouse IgG isotype control, had no effect on IL-17Ainduced eotaxin-1/CCL11 release by ASM cells. Taken together,
these results suggest that eotaxin-1/CCL11 release in primary
ASM cells is selectively mediated by IL-17A.
IL-17A enhances IL-1␤-mediated eotaxin-1/CCL11 expression in
ASM cells by de novo mRNA and protein synthesis
Because cells are exposed to multiple proinflammatory cytokines
in the microenvironment of inflammatory diseases, we next investigated the combined effect of IL-17A and IL-1␤ stimulation on the
induction of eotaxin-1/CCL11 release from ASM cells. As shown
in Fig. 2A, IL-17A or IL-1␤ alone (0.1, 1, 10, and 100 ng/ml)
induced a significant increase in eotaxin-1/CCL11 release compared with unstimulated ASM cells ( p ⬍ 0.01). Addition of IL17A (1 ng or 100 ng/ml) to a suboptimal dose of IL-1␤ (0.1 ng/ml)
induced a significant increase of eotaxin-1/CCL11 release in ASM
cells ( p ⬍ 0.05; Fig. 2A). Interestingly, IL-17A at 1, 10, or 100
ng/ml combined with IL-1␤ at 1 or 10 ng/ml, but not at 100 ng/ml,
resulted in enhanced eotaxin-1/CCl-11 release from ASM cells
( p ⬍ 0.05; Fig 2A).
Previous studies have demonstrated that eotaxin-1/CCL11 expression is controlled at many points, particularly at the transcriptional and posttranscriptional levels (25). Thus, we next tested
whether blocking mRNA and protein synthesis affected eotaxin1/CCL11 production induced by IL-17A alone or in combination
with IL-1␤ in ASM cells. Cells were pretreated with actinomycin
D followed by stimulation with IL-17A, IL-1␤, or both for 48 h.
As observed in Fig. 2B, actinomycin D and cycloheximide abrogated eotaxin-1/CCL11 release from ASM cells treated with IL17A, IL-1␤, or both ( p ⬍ 0.05). The inhibition of eotaxin-1/
CCL11 mRNA expression by actinomycin D was also confirmed
by quantitative real-time RT-PCR. ASM cells treated for 6 h with
IL-17A, IL-1␤, or both induced 1.8-, 9.4-, and 13.8-fold increase
of eotaxin-1/CCL11 mRNA level, respectively, compared with unstimulated cells (Fig. 2C). Interestingly, this effect was significantly inhibited with actinomycin D treatment ( p ⬍ 0.05; Fig. 2C).
FIGURE 1. Time course and concentration response of IL-17A-induced
eotaxin-1/CCL11 (A) and IL-6 (B) release from ASM cells. Growth-arrested ASM cells were left unstimulated (medium alone) or treated with
indicated concentrations of IL-17A for 24 and 48 h. Each point represents
mean ⫾ SD of triplicate values from one experiment and is representative
of five experiments done with cells from five different donors, cell passages
3–5. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.001 compared with unstimulated ASM cells.
C, Effects of a monoclonal anti-IL-17A neutralizing Ab on release of
eotaxin-1/CCL11 from ASM cells, as determined by ELISA. Growth-arrested cells were left unstimulated (medium alone) or treated with IL-17A
for 48 h with or without pretreatment with anti-IL-17A Ab or isotype
control IgG at indicated doses for 2 h. Each point represents mean ⫾ SD
of triplicate values from one experiment and is representative of five experiments done with cells from five different donors, cell passages 3– 4. ⴱ,
p ⬍ 0.01 compared with IL-17A-stimulated ASM cells.
Similar results were observed at 24 h (data not shown). Actinomycin D and cycloheximide had no effect on eotaxin-1/CCL11
mRNA expression in unstimulated cells (data not shown).
The Journal of Immunology
4067
IL-1␤ (mean value of fold increase compared with baseline: 4.3
and 11.9, respectively; Fig. 3). As expected from previous studies
(25), IL-4 proved to be a positive control for activation of the
eotaxin-1/CCL11 promoter (Fig. 3). Furthermore, eoatxin-1/
CCL11 promoter activity was further increased when ASM cells
were stimulated with a combination of IL-1␤ and IL-17A (Fig. 3).
Collectively, these data suggest that IL-17A induces eotaxin-1/
CCL11 via at least a transcriptional mechanism in ASM cells.
IL-17A induces eotaxin-1/CCL11 release from ASM cells via
MAPK (p38, JNK, and ERK1/2)
IL-17A mediates rapid phosphorylation of MAPK p38, JNK, and
ERK1/2 in ASM cells
FIGURE 2. A, IL-17A enhances IL-1␤-mediated eotaxin-1/CCL11 release from ASM cells. Growth-arrested cells were left unstimulated (medium alone) or treated with IL-17A, IL-1␤, or with the combination at
different concentration for 48 h. Each point represents mean ⫾ SD of
triplicate values of one experiment of three performed on cells from three
different donors, cell passages 3–5. ⴱⴱ, p ⬍ 0.05 compared with medium
alone; ⴱ, p ⬍ 0.026; and ␺, p ⬎ 0.05 compared with IL-1␤-stimulated cells.
B, Effect of actinomycin D and cycloheximide on IL-17A-induced eotaxin1/CCL11 release from ASM cells. Growth-arrested cells were left unstimulated (medium alone) or treated with IL-17A, IL-1␤, or with the combination for 48 h with or without pretreatment with actinomycin D or
cycloheximide for 1 h. C, Actinomycin D abrogates IL-17A-mediated
eotaxin-1/CCL11 mRNA expression in ASM cells. Growth-arrested cells
were left unstimulated (medium alone) or treated with IL-17A, IL-1␤, or
with the combination for 6 h, and mRNA was harvested. Real-time RTPCR was then performed as described in Materials and Methods. Data in
B and C represent the mean ⫾ SD of triplicate values from three independent experiments. ⴱ, p ⬍ 0.05 compared with IL-17A-, IL-1␤-, and IL17A- plus IL-1␤-stimulated cells, respectively, in the absence of drugs.
IL-17A induces eotaxin-1/CCL11 promoter activity
To further investigate mechanisms by which IL-17A mediated
eotaxin-1/CCL11 transcription, ASM cells were transiently transfected with a luciferase reporter construct driven by a 2.2-kb
eotaxin-1/CCL11 proximal promoter (25). Primary human ASM
cells transfected with CCL11 promoter construct showed a significant increase in luciferase activity in response to IL-17A and
To further confirm the involvement of MAPK in IL-17A-mediated
eotaxin-1/CCL11 expression in ASM cells, we performed Western
blot analysis using specific Abs for the phosphorylated regulatory
sites on both p38, JNK, and ERK1/2 MAPK. IL-17A induced rapid
and marked phosphorylation of p38, ERK1/2, and JNK (Fig. 5).
Phosphorylation reached a maximum 5–10 min after IL-17A exposure, and then gradually declined to near baseline levels over the
subsequent 120 min. Taken together, our data demonstrate that
IL-17A-mediated eotaxin-1/CCL11 expression in ASM cells involves the MAPK pathways (p38, ERK1/2, and JNK).
FIGURE 3. IL-17A enhanced IL-1␤-mediated eotaxin-1/CCL11 promoter activity in ASM cells. Human ASM cells were transfected with a
luciferase reporter construct driven by the 2.2-kb eotaxin-1/CCL11 proximal promoter. Twenty-four hours posttransfection, cells were stimulated
with cytokines and further incubated for 12 h as described in Materials and
Methods. Data represent one experiment of four done with cells from four
different donors. Data were normalized according to the Renilla luciferase
activity.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Previous studies suggest that IL-17A-induced signaling may differ
between divergent resident lung cell populations (26). To characterize the signaling pathways involved in IL-17A-mediated
eotaxin-1/CCL11 release from ASM cells, we first performed experiments using SB203580, U0126, or SP600125 specific and potent inhibitors of MAPK p38, p42/p44 ERK, and JNK, respectively. We also investigated the effects of wortmannin, a specific
PI3K inhibitor. Treatment of ASM cells with SB203580, U0126,
or SP600125 before IL-17A stimulation caused a significant and
substantial inhibition of eotaxin-1/CCL11 release ( p ⬍ 0.05; Fig.
4A). In contrast, inhibition of PI3K with wortmannin had little or
no effect on IL-17A-induced eotaxin-1/CCL11 release by human
airway myocytes (Fig. 4B). ASM cell pretreatment with U0126,
SB203580, SP600125, or wortmannin profoundly reduced
eotaxin-1/CCL11 release induced by IL-1␤ or IL-17A in combination with IL-1␤ suggesting that signaling via MAPK, JNK, and
PI3K pathways are required for the induction of eotaxin-1/CCL11
by IL-1␤ ( p ⬍ 0.01; Fig. 4, A and B). These results indicate that
p38 MAPK, JNK, and p42/p44 ERK, but not PI3K, are essential
for IL-17A-mediated release of eotaxin-1/CCL11 by human ASM
cells.
4068
MECHANISMS OF IL-17A-MEDIATED CCL11 EXPRESSION IN ASM CELLS
inhibitor (JAK inhibitor I). Treatment of ASM cells with a JAK
inhibitor I before IL-17A stimulation caused a significant and substantial inhibition of eotaxin-1/CCL11 release ( p ⬍ 0.001; Fig.
6B). This result indicates that STAT pathway may participate in
IL-17A-mediated release of eotaxin-1/CCL11 by human ASM
cells.
FIGURE 4. Effects of p38 MAPK, p42/p44 ERK, JNK (A), and PI3K
inhibitors (B) on IL-17A-mediated eotaxin-1/CCL11 release from ASM
cells. Growth-arrested cells were left unstimulated (medium alone) or
treated with IL-17A, IL-1␤, or with the combination for 48 h with or
without pretreatment for 1 h with inhibitors of p38 MAPK (SB203580),
p42/p44 ERK (U0126), JNK (SP600125), and PI3K (wortmannin). Each
point represents mean ⫾ SD of triplicate values from three experiments
done with cells from three different donors, cell passages 4 –5. Results are
expressed as fold increase over control values (medium alone). ⴱ, p ⬍ 0.05
compared with IL-17A-, IL-1␤-, or IL-17A-plus-IL-1␤-stimulated cells,
respectively, in the absence of the inhibitors.
IL-17A causes rapid phosphorylation of STAT-3 but not STAT-6
or STAT-5
Eotaxin-1/CCL11 expression has been shown to be dependent on
STAT-6 activation in various inflammatory and structural cells
including ASM cells (25, 27). To determine STAT-6 activation in
response to IL-17A in ASM cells, total cell protein was probed
with an Ab specific for tyrosine-phosphorylated STAT-6 and total
STAT-6. As shown in Fig. 6, IL-17A stimulation did not induce
STAT-6 tyrosine phosphorylation over a 2-h time period in ASM
cells.
A previous study in a monocytic cell line has shown that IL-17A
receptor signaling can activate STAT-3 and STAT-5 (28). We then
determined the tyrosine phosphorylation of both STAT-3 and
STAT-5 in IL-17A-stimulated ASM cells. IL-17A induced 2- and
1.8-fold increases of STAT-3 phosphorylation in ASM cells at 5
and 10 min, respectively, reaching baseline levels at 20 min. However, IL-17A did not induce noticeable STAT-5 phosphorylation in
ASM cells (normalized to total STAT-5). These data suggest that
IL-17A may induce eotaxin-1/CCL11 release via STAT-3.
To further characterize the involvement of STAT-3 signaling
pathways in IL-17A-mediated eotaxin-1/CCL11 release from
ASM cells, we performed experiments using an universal JAK
Discussion
The current study was designed to explore the role of IL-17A and
the signaling pathways by which it mediated eotaxin-1/CCL11 expression in ASM cells. Our data demonstrate for the first time that
IL-17A, a cytokine involved primarily in inducing neutrophilic
inflammatory responses, can induce the synthesis and release of
eotaxin-1/CCL11 in human primary ASM cells. Furthermore, the
induced expression of eoatxin-1/CCL11 by IL-17A in human
ASM cells depended on de novo mRNA and protein synthesis,
suggesting transcriptional and posttranscriptional regulatory mechanisms. Promoter luciferase reporter assay revealed that IL-17A
significantly induced eotaxin-1/CCL11 promoter activity in ASM
cells. Inhibitors of MAPK (p38, JNK, and ERK1/2), but not PI3K,
prevented IL-17A-induced eotaxin-1/CCL11 production from
ASM cells, and IL-17A induced rapid phosphorylation of MAPK
(p38, JNK, ERK1/2). Finally, phosphorylation of STAT-3 but not
STAT-6 or STAT-5 occurred upon IL-17A stimulation of ASM
cells. This study suggests that IL-17A mediates eoatxin-1/CCL11
expression at least at a transcriptional level and involves MAPK
pathways but not PI3K or STAT-6. The results of this study not
only have functional implications for ASM cell-mediated inflammation, but also provide a novel mechanism by which IL-17A may
contribute to the development of airway diseases such as asthma.
IL-17A is a homodimeric protein, with pleiotropic biological
activities, that can be released by activated CD4⫹ T cells (15, 29,
30). IL-17A has been shown to be capable of stimulating the production of various cytokines such as IL-6, IL-8, and GM-CSF from
diverse cell types, such as fibroblasts, keratinocytes, and renal epithelial cells (17, 31–33). A potential role of IL-17A in the initiation or maintenance of inflammatory responses is suggested by
elevated IL-17A expression in mononuclear cells from patients
with multiple sclerosis (16), rheumatoid arthritis (34), or systemic
lupus erythematosus (35). Furthermore, studies using a murine
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. IL-17A induces p38 MAPK, JNK, and ERK-1/2 phosphorylation in human ASM cells. Growth-arrested ASM cells were stimulated
with IL-17A or medium for 5, 10, 20, 30, 60, and 120 min. Reactions were
stopped and cell lysates were immunoblotted with phosphospecific antip38, JNK, ERK1/2 Abs and detected by ECL as described in Materials and
Methods. Total p38, JNK, and ERK1/2 protein was determined by reprobing the blot with anti-p38, JNK, and ERK1/2 specific Abs. The results
represent one of similar results from four independent experiments.
The Journal of Immunology
4069
FIGURE 6. A, IL-17A mediates STAT-3 but not STAT-5 or STAT-6 phosphorylation. Cells were stimulated as described in Fig. 5. Total cell lysates
were analyzed with anti-phospho-tyrosine STAT-3, STAT-5, or STAT-6 Abs. The same blot was stripped and reprobed with anti-STAT-3, STAT-5, or
STAT-6. The results represent one of similar results from five independent experiments. B, JAK inhibitor (JAK inhibitor I) abrogates IL-17A-mediated
eotaxin-1/CCL11 release from ASM cells. Growth-arrested cells were left unstimulated (medium alone) or treated with IL-17A (10 ng/ml) for 24 h with
or without pretreatment for 1 h with JAK inhibitor. Each point represents mean ⫾ SD of triplicate values and the results represent one of three independent
experiments. ⴱ, p ⬍ 0.03 compared with IL-17A-stimulated cells in the absence of the inhibitors.
has been shown to inhibit neutrophil recruitment in a mouse model
of endoxemia (46) and down-regulate CXC chemokines particularly IL-8/CXCL8 in human dermal microvascular endothelial
cells (47). In light of prior evidence and present data, it is tempting
to speculate that IL-17A-mediated eotaxin-1/CCL11 release may
down-regulate exaggerated neutrophilic inflammation by suppressing CXC chemokine release during acute inflammation, hence creating a negative feedback to establish tissue homeostasis. Interestingly, it is worth to mention that many prototypic proinflammatory
cytokines such as TNF-␣ exhibit both immunosuppressive and immunoregulatory roles depending on the microenvironment and cellular targets (48). Further studies in vivo using animal models are
needed to address this possibility.
The MAPK family is fundamental in mediating numerous
changes in cell function such as cytokine expression, proliferation,
and apoptosis (49, 50). The p38 MAPK, JNK, and ERK play a
central role in these cell responses. Upon activation by upstream
regulators, the MAP kinases translocate to the cell nucleus where
they transform the action of nuclear transcription factors and kinases, which in turn cause changes in cell function such as release
of cytokines (50). Using pharmacological inhibitors of MAPK and
investigating the kinetics of phosphorylation of p38, ERK1/2, and
JNK, we demonstrate that IL-17A-induced eotaxin-1/CCL11 release in ASM cells involves MAPK p38, JNK, and p42/p44 ERK.
These results are in agreement with previous studies in human
bronchial epithelial cells, colonic myofibroblasts and chondrocytes
(32, 51–54). Because the IL-17RA cytoplasmic domain encompasses ⬎500 aa but lacks identifiable signaling motifs within this
region, it is plausible that numerous signaling pathways can be
activated by this receptor such as PI3K and STATs. Our data
showed no substantial effect of PI3K inhibitor (wortmannin) on
IL-17A-induced eotaxin-1/CCL11 release, suggesting that PI3K is
not involved in IL-17A signaling in ASM cells, as observed in
human bronchial epithelial cells (51). Furthermore, because IL-4R
signaling involves PI3K (55), our results may also imply that IL17A does not mediate its effect through IL-4 release and autocrine
mechanism. Interestingly, we found that PI3K signal transduction
is involved in IL-1␤-induced eotaxin-1/CCL11 release from ASM.
This difference in signaling pathways may explain the difference in
patterns of eotaxin-1/CCL11 release observed with IL-17A or
IL-1␤ stimulation. The potential synergy between IL-17A and
IL-1␤ at the intracellular level is currently under investigation.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
model have clearly demonstrated the role of IL-17A network in
inducing neutrophil recruitment to inflammatory sites (36). In the
current study, we show additional effects mediated by IL-17A.
This cytokine induced eotaxin-1/CCL11 in ASM cells, and in combination with IL-1␤, exceeding the maximal induction of eotaxin1/CCL11 mRNA and protein caused by either cytokine alone. Our
data are in agreement with previous studies where IL-17A alone,
or in combination with proinflammatory cytokines, enhanced chemokine and cytokine expression (37). Similar to what we recently
showed for chronic obstructive pulmonary disease patients (24),
IL-17RA protein expression was detected in vivo within ASM area
of an allergic bronchopulmonary aspergillosis patient airway (data
not shown). This preliminary result suggests that ASM cells of
allergic patients may be one of the cellular targets for IL-17A,
contributing with IL-1␤ to eotaxin-1/CCL11 expression in the airways. Taken together, these findings suggest that IL-17A might
play a general proinflammatory role through a combined effect
with other cytokines such as IL-1␤.
Eotaxin-1/CCL11 is a chemokine belonging to the CC family,
and was first isolated from lung lavage fluid of a guinea pig model
of allergic disease (6). It has been shown to be a potent chemoattractant for eosinophils both in vitro and in vivo (11, 12, 38 – 40).
Increased production of eotaxin-1/CCL11 has been associated with
allergic diseases such as asthma (7–10, 41), allergic rhinitis (42),
and atopic dermatitis (43). Previous studies have shown that Th2
cytokines, particularly IL-4 can induce eotaxin-1/CCL11 release in
many structural cells including ASM cells (13, 14). Previously, it
has been demonstrated that human ASM cells express eotaxin-1/
CCL11 following TNF-␣ and/or IL-1␤ stimulation (4). The
eotaxin-1/CCL11 produced and secreted by ASM cells may then
amplify the chemokine signal generated by infiltrating inflammatory cells in the airway, thereby augmenting the recruitment of
eosinophils, basophils, and Th2 lymphocytes to the airways. The
accumulation of these inflammatory cells may subsequently contribute to the development of airway hyperresponsiveness, local
inflammation, and tissue injury through the release of granular
enzymes and other cytokines (44).
Our current study shows that IL-17A-dependent activation of
ASM cells can also induce eotaxin-1/CCL11, thus revealing a new
pathway for induction of eotaxin-1/CCL11 within the airways. Because an earlier study in primary bronchial epithelial cells did not
report similar findings (45), the effect of IL-17A may be cell specific. Besides its role in attracting eosinophils, eotaxin-1/CCL11
4070
MECHANISMS OF IL-17A-MEDIATED CCL11 EXPRESSION IN ASM CELLS
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Acknowledgments
We thank Dr. Jutta Horejs-Hoeck (Institute for Chemistry and Biochemistry, Salzburg, Austria) for the gift of CCL-11 promoter construct.
Disclosures
The authors have no financial conflict of interest.
22.
23.
24.
References
1. Armour, C., P. Johnson, S. Anticevich, A. Ammit, K. McKay, M. Hughes, and
J. Black. 1997. Mediators on human airway smooth muscle. Clin. Exp. Pharmacol. Physiol. 24: 269 –272.
2. Hakonarson, H., C. Carter, C. Kim, and M. M. Grunstein. 1999. Altered expression and action of the low-affinity IgE receptor Fc␧RII (CD23) in asthmatic
airway smooth muscle. J. Allergy Clin. Immunol. 104: 575–584.
3. Halayko, A. J., and Y. Amrani. 2003. Mechanisms of inflammation-mediated
airway smooth muscle plasticity and airways remodeling in asthma. Respir.
Physiol. Neurobiol. 137: 209 –222.
4. Ghaffar, O., Q. Hamid, P. M. Renzi, Z. Allakhverdi, S. Molet, J. C. Hogg,
S. A. Shore, A. D. Luster, and B. Lamkhioued. 1999. Constitutive and cytokinestimulated expression of eotaxin by human airway smooth muscle cells.
Am. J. Respir. Crit. Care Med. 159: 1933–1942.
5. Hirst, S. J., M. P. Hallsworth, Q. Peng, and T. H. Lee. 2002. Selective induction
of eotaxin release by interleukin-13 or interleukin-4 in human airway smooth
muscle cells is synergistic with interleukin-1␤ and is mediated by the interleukin-4 receptor ␣-chain. Am. J. Respir. Crit. Care Med. 165: 1161–1171.
6. Jose, P. J., D. A. Griffiths-Johnson, P. D. Collins, D. T. Walsh, R. Moqbel,
N. F. Totty, O. Truong, J. J. Hsuan, and T. J. Williams. 1994. Eotaxin: a potent
eosinophil chemoattractant cytokine detected in a guinea pig model of allergic
airways inflammation. J. Exp. Med. 179: 881– 887.
7. Ying, S., Q. Meng, K. Zeibecoglou, D. S. Robinson, A. Macfarlane, M. Humbert,
and A. B. Kay. 1999. Eosinophil chemotactic chemokines (eotaxin, eotaxin-2,
RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C
chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic
(Intrinsic) asthmatics. J. Immunol. 163: 6321– 6329.
8. Lamkhioued, B., P. M. Renzi, S. Abi-Younes, E. A. Garcia-Zepada,
Z. Allakhverdi, O. Ghaffar, M. D. Rothenberg, A. D. Luster, and Q. Hamid. 1997.
Increased expression of eotaxin in bronchoalveolar lavage and airways of asth-
25.
26.
27.
28.
29.
30.
31.
32.
33.
matics contributes to the chemotaxis of eosinophils to the site of inflammation.
J. Immunol. 159: 4593– 4601.
Brown, J. R., J. Kleimberg, M. Marini, G. Sun, A. Bellini, and S. Mattoli. 1998.
Kinetics of eotaxin expression and its relationship to eosinophil accumulation and
activation in bronchial biopsies and bronchoalveolar lavage (BAL) of asthmatic
patients after allergen inhalation. Clin. Exp. Immunol. 114: 137–146.
Nakamura, H., S. T. Weiss, E. Israel, A. D. Luster, J. M. Drazen, and C. M. Lilly.
1999. Eotaxin and impaired lung function in asthma. Am. J. Respir. Crit. Care
Med. 160: 1952–1956.
Teixeira, M. M., T. N. Wells, N. W. Lukacs, A. E. Proudfoot, S. L. Kunkel,
T. J. Williams, and P. G. Hellewell. 1997. Chemokine-induced eosinophil recruitment: evidence of a role for endogenous eotaxin in an in vivo allergy model
in mouse skin. J. Clin. Invest. 100: 1657–1666.
Gonzalo, J. A., C. M. Lloyd, D. Wen, J. P. Albar, T. N. Wells, A. Proudfoot,
A. C. Martinez, M. Dorf, T. Bjerke, A. J. Coyle, and J. C. Gutierrez-Ramos. 1998.
The coordinated action of CC chemokines in the lung orchestrates allergic inflammation and airway hyperresponsiveness. J. Exp. Med. 188: 157–167.
Laporte, J. C., P. E. Moore, S. Baraldo, M. H. Jouvin, T. L. Church,
I. N. Schwartzman, R. A. Panettieri, Jr., J. P. Kinet, and S. A. Shore. 2001. Direct
effects of interleukin-13 on signaling pathways for physiological responses in
cultured human airway smooth muscle cells. Am. J. Respir. Crit. Care Med. 164:
141–148.
Moore, P. E., T. L. Church, D. D. Chism, R. A. Panettieri, Jr., and S. A. Shore.
2002. IL-13 and IL-4 cause eotaxin release in human airway smooth muscle cells:
a role for ERK. Am. J. Physiol. 282: L847–L853.
Yao, Z., S. L. Painter, W. C. Fanslow, D. Ulrich, B. M. Macduff, M. K. Spriggs,
and R. J. Armitage. 1995. Human IL-17: a novel cytokine derived from T cells.
J. Immunol. 155: 5483–5486.
Matusevicius, D., P. Kivisakk, B. He, N. Kostulas, V. Ozenci, S. Fredrikson, and
H. Link. 1999. Interleukin-17 mRNA expression in blood and CSF mononuclear
cells is augmented in multiple sclerosis. Mult. Scler. 5: 101–104.
Albanesi, C., C. Scarponi, A. Cavani, M. Federici, F. Nasorri, and G. Girolomoni.
2000. Interleukin-17 is produced by both Th1 and Th2 lymphocytes, and modulates interferon-␥- and interleukin-4-induced activation of human keratinocytes.
J. Invest. Dermatol. 115: 81– 87.
Molet, S., Q. Hamid, F. Davoine, E. Nutku, R. Taha, N. Page, R. Olivenstein,
J. Elias, and J. Chakir. 2001. IL-17 is increased in asthmatic airways and induces
human bronchial fibroblasts to produce cytokines. J. Allergy Clin. Immunol. 108:
430 – 438.
Barczyk, A., W. Pierzchala, and E. Sozanska. 2003. Interleukin-17 in sputum
correlates with airway hyperresponsiveness to methacholine. Respir. Med. 97:
726 –733.
Hamann, K. J., J. E. Vieira, A. J. Halayko, D. Dorscheid, S. R. White,
S. M. Forsythe, B. Camoretti-Mercado, K. F. Rabe, and J. Solway. 2000. Fas
cross-linking induces apoptosis in human airway smooth muscle cells.
Am. J. Physiol. 278: L618 –L624.
Gounni, A. S., Q. Hamid, S. M. Rahman, J. Hoeck, J. Yang, and L. Shan. 2004.
IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle
cells. J. Immunol. 173: 2771–2779.
Chirgwin, J. M., A. E. Przybyla, R. J. MacDonald, and W. J. Rutter. 1979.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294 –5299.
Hoeck, J., and M. Woisetschlager. 2001. Activation of eotaxin-3/CCLl26 gene
expression in human dermal fibroblasts is mediated by STAT6. J. Immunol. 167:
3216 –3222.
Rahman, M. S., J. Yang, L. Y. Shan, H. Unruh, X. Yang, A. J. Halayko, and
A. S. Gounni. 2005. IL-17R activation of human airway smooth muscle cells
induces CXCL-8 production via a transcriptional-dependent mechanism. Clin.
Immunol. 115: 268 –276.
Hoeck, J., and M. Woisetschlager. 2001. STAT6 mediates eotaxin-1 expression
in IL-4 or TNF-␣-induced fibroblasts. J. Immunol. 166: 4507– 4515.
Kolls, J. K., and A. Linden. 2004. Interleukin-17 family members and inflammation. Immunity 21: 467– 476.
Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, and W. E. Paul. 1999. The
IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol.
17: 701–738.
Subramaniam, S. V., R. S. Cooper, and S. E. Adunyah. 1999. Evidence for the
involvement of JAK/STAT pathway in the signaling mechanism of interleukin17. Biochem. Biophys. Res. Commun. 262: 14 –19.
Fossiez, F., O. Djossou, P. Chomarat, L. Flores-Romo, S. Ait-Yahia, C. Maat,
J. J. Pin, P. Garrone, E. Garcia, S. Saeland, et al. 1996. T cell interleukin-17
induces stromal cells to produce proinflammatory and hematopoietic cytokines.
J. Exp. Med. 183: 2593–2603.
Rouvier, E., M. F. Luciani, M. G. Mattei, F. Denizot, and P. Golstein. 1993.
CTLA-8, cloned from an activated T cell, bearing AU-rich messenger RNA instability sequences, and homologous to a herpesvirus saimiri gene. J. Immunol.
150: 5445–5456.
Van Kooten, C., J. G. Boonstra, M. E. Paape, F. Fossiez, J. Banchereau,
S. Lebecque, J. A. Bruijn, J. W. De Fijter, L. A. Van Es, and M. R. Daha. 1998.
Interleukin-17 activates human renal epithelial cells in vitro and is expressed
during renal allograft rejection. J. Am. Soc. Nephrol. 9: 1526 –1534.
Attur, M. G., R. N. Patel, S. B. Abramson, and A. R. Amin. 1997. Interleukin-17
up-regulation of nitric oxide production in human osteoarthritis cartilage. Arthritis Rheum. 40: 1050 –1053.
Cai, X. Y., C. P. Gommoll, Jr., L. Justice, S. K. Narula, and J. S. Fine. 1998.
Regulation of granulocyte colony-stimulating factor gene expression by interleukin-17. Immunol. Lett. 62: 51–58.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
STAT-6 plays a pivotal role in IL-4-mediated eotaxin-1/CCL11
expression (27). In our study, a significant increase in STAT-3 but
not STAT-6 or STAT-5 phosphorylation was detected following
IL-17A stimulation, which may suggest that IL-17A used a different pathway than IL-4. These findings are in agreement with
oncostatin-M-induced eotaxin-1/CCL11 expression in human
ASM cells and mouse fibroblasts (56, 57). Transcriptional activation of cytokine-responsive genes requires coordinated cooperation between STATs and other sequence-specific transcription factors that recruit transcriptional coregulators and components of the
basal transcription machinery to the promoter. Promoter of
eotaxin-1/CCL11 contains response elements for NF-␬B in addition to STAT-6 binding sites (25). Preliminary EMSA data using
nuclear protein from ASM cells of two subjects revealed that IL17A induced STAT-6 and NF-␬B binding to eotaxin-1/CCL11
promoter region (our unpublished data). Although IL-17A did not
have any effect on STAT-6 phosphorylation, it is likely that IL17A may affect STAT-6 cooperation with other transcription factors such as NF-␬B or C/EBP (58 – 60), recruitment of transcriptional coactivators at the promoter region (61), or posttranslational
modifications of STAT-6 by serine threonine kinases (62). A study
on ASM cells isolated from STAT-6 knockout mice and transfected ASM cells with dominant-negative STAT-6 will be useful
to address those possibilities.
In conclusion, this study is the first demonstration of the capacity for IL-17A to induce eotaxin-1/CCL11 expression by ASM
cells. Induction occurs at least through a transcriptional mechanism and involves signaling pathways that include MAPK (p38,
JNK, and p42/p44 ERK) and probably STAT-3. Our findings support the concept that IL-17A might act as an amplifier of airway
eosinophilic inflammation by enhancing eotaxin-1/CCL11 expression. Our results also provide a better understanding of IL-17AASM cell interactions and their role in airway inflammatory
responses.
The Journal of Immunology
49. Puddicombe, S. M., and D. E. Davies. 2000. The role of MAP kinases in intracellular signal transduction in bronchial epithelium. Clin. Exp. Allergy 30: 7–11.
50. Robinson, M. J., and M. H. Cobb. 1997. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 9: 180 –186.
51. Laan, M., J. Lotvall, K. F. Chung, and A. Linden. 2001. IL-17-induced cytokine
release in human bronchial epithelial cells in vitro: role of mitogen-activated
protein (MAP) kinases. Br. J. Pharmacol. 133: 200 –206.
52. Hata, K., A. Andoh, M. Shimada, S. Fujino, S. Bamba, Y. Araki, T. Okuno,
Y. Fujiyama, and T. Bamba. 2002. IL-17 stimulates inflammatory responses via
NF-␬B and MAP kinase pathways in human colonic myofibroblasts.
Am. J. Physiol. 282: G1035–G1044.
53. Shalom-Barak, T., J. Quach, and M. Lotz. 1998. Interleukin-17-induced gene
expression in articular chondrocytes is associated with activation of mitogenactivated protein kinases and NF-␬B. J. Biol. Chem. 273: 27467–27473.
54. Martel-Pelletier, J., F. Mineau, D. Jovanovic, J. A. Di Battista, and J. P. Pelletier.
1999. Mitogen-activated protein kinase and nuclear factor ␬B together regulate
interleukin-17-induced nitric oxide production in human osteoarthritic chondrocytes: possible role of transactivating factor mitogen-activated protein kinaseactivated proten kinase (MAPKAPK). Arthritis Rheum. 42: 2399 –2409.
55. Zamorano, J., and A. D. Keegan. 1998. Regulation of apoptosis by tyrosinecontaining domains of IL-4R␣: Y497 and Y713, but not the STAT6-docking
tyrosines, signal protection from apoptosis. J. Immunol. 161: 859 – 867.
56. Faffe, D. S., L. Flynt, M. Mellema, P. E. Moore, E. S. Silverman,
V. Subramaniam, M. R. Jones, J. P. Mizgerd, T. Whitehead, A. Imrich, et al.
2005. Oncostatin M causes eotaxin-1 release from airway smooth muscle: synergy with IL-4 and IL-13. J. Allergy Clin. Immunol. 115: 514 –520.
57. Langdon, C., C. Kerr, L. Tong, and C. D. Richards. 2003. Oncostatin M regulates
eotaxin expression in fibroblasts and eosinophilic inflammation in C57BL/6 mice.
J. Immunol. 170: 548 –555.
58. Shen, C. H., and J. Stavnezer. 1998. Interaction of stat6 and NF-␬B: direct association and synergistic activation of interleukin-4-induced transcription. Mol.
Cell Biol. 18: 3395–3404.
59. Stutz, A. M., J. Hoeck, F. Natt, B. Cuenoud, and M. Woisetschlager. 2001.
Inhibition of interleukin-4- and CD40-induced IgE germline gene promoter activity by 2⬘-aminoethoxy-modified triplex-forming oligonucleotides. J. Biol.
Chem. 276: 11759 –11765.
60. Mikita, T., M. Kurama, and U. Schindler. 1998. Synergistic activation of the
germline epsilon promoter mediated by Stat6 and C/EBP ␤. J. Immunol. 161:
1822–1828.
61. Yang, J., S. Aittomaki, M. Pesu, K. Carter, J. Saarinen, N. Kalkkinen, E. Kieff,
and O. Silvennoinen. 2002. Identification of p100 as a coactivator for STAT6 that
bridges STAT6 with RNA polymerase II. EMBO J. 21: 4950 – 4958.
62. Pesu, M., K. Takaluoma, S. Aittomaki, A. Lagerstedt, K. Saksela, P. E. Kovanen,
and O. Silvennoinen. 2000. Interleukin-4-induced transcriptional activation by
stat6 involves multiple serine/threonine kinase pathways and serine phosphorylation of stat6. Blood 95: 494 –502.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
34. Ziolkowska, M., A. Koc, G. Luszczykiewicz, K. Ksiezopolska-Pietrzak,
E. Klimczak, H. Chwalinska-Sadowska, and W. Maslinski. 2000. High levels of
IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via
cyclosporin A-sensitive mechanism. J. Immunol. 164: 2832–2838.
35. Wong, C. K., C. Y. Ho, E. K. Li, and C. W. Lam. 2000. Elevation of proinflammatory cytokine (IL-18, IL-17, IL-12) and Th2 cytokine (IL-4) concentrations in
patients with systemic lupus erythematosus. Lupus 9: 589 –593.
36. Hellings, P. W., A. Kasran, Z. Liu, P. Vandekerckhove, A. Wuyts, L. Overbergh,
C. Mathieu, and J. L. Ceuppens. 2003. Interleukin-17 orchestrates the granulocyte
influx into airways after allergen inhalation in a mouse model of allergic asthma.
Am. J. Respir. Cell Mol. Biol. 28: 42–50.
37. Kehlen, A., K. Thiele, D. Riemann, N. Rainov, and J. Langner. 1999. Interleukin-17 stimulates the expression of I␬B ␣ mRNA and the secretion of IL-6 and
IL-8 in glioblastoma cell lines. J. Neuroimmunol. 101: 1– 6.
38. Garcia-Zepeda, E. A., M. E. Rothenberg, R. T. Ownbey, J. Celestin, P. Leder, and
A. D. Luster. 1996. Human eotaxin is a specific chemoattractant for eosinophil
cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2:
449 – 456.
39. Rothenberg, M. E., R. Ownbey, P. D. Mehlhop, P. M. Loiselle, M. van de Rijn,
J. V. Bonventre, H. C. Oettgen, P. Leder, and A. D. Luster. 1996. Eotaxin triggers
eosinophil-selective chemotaxis and calcium flux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Mol. Med.
2: 334 –348.
40. Zimmermann, N., G. K. Hershey, P. S. Foster, and M. E. Rothenberg. 2003.
Chemokines in asthma: cooperative interaction between chemokines and IL-13.
J. Allergy Clin. Immunol. 111: 227–243.
41. Griffiths-Johnson, D. A., P. D. Collins, P. J. Jose, and T. J. Williams. 1997.
Animal models of asthma: role of chemokines. Methods Enzymol. 288: 241–266.
42. Greiff, L., H. Petersen, E. Mattsson, M. Andersson, J. S. Erjefalt, M. Linden,
C. Svensson, and C. G. Persson. 2001. Mucosal output of eotaxin in allergic
rhinitis and its attenuation by topical glucocorticosteroid treatment. Clin. Exp.
Allergy 31: 1321–1327.
43. Yawalkar, N., M. Uguccioni, J. Scharer, J. Braunwalder, S. Karlen, B. Dewald,
L. R. Braathen, and M. Baggiolini. 1999. Enhanced expression of eotaxin and
CCR3 in atopic dermatitis. J. Invest. Dermatol. 113: 43– 48.
44. Busse, W. W., S. Banks-Schlegel, and S. E. Wenzel. 2000. Pathophysiology of
severe asthma. J. Allergy Clin. Immunol. 106: 1033–1042.
45. Kawaguchi, M., F. Kokubu, H. Kuga, S. Matsukura, H. Hoshino, K. Ieki, T. Imai,
M. Adachi, and S. K. Huang. 2001. Modulation of bronchial epithelial cells by
IL-17. J. Allergy Clin. Immunol. 108: 804 – 809.
46. Cheng, S. S., N. W. Lukacs, and S. L. Kunkel. 2002. Eotaxin/CCL11 is a negative
regulator of neutrophil recruitment in a murine model of endotoxemia. Exp. Mol.
Pathol. 73: 1– 8.
47. Cheng, S. S., N. W. Lukacs, and S. L. Kunkel. 2002. Eotaxin/CCL11 suppresses
IL-8/CXCL8 secretion from human dermal microvascular endothelial cells. J. Immunol. 168: 2887–2894.
48. O’Shea, J. J., A. Ma, and P. Lipsky. 2002. Cytokines and autoimmunity. Nat. Rev.
Immunol. 2: 37– 45.
4071