TNFα and IFNγ inversely modulate expression of the IL

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 20, 2006). doi:10.1152/ajplung.00301.2005
TNF and IFN inversely modulate expression of the IL-17E receptor in Airway
Smooth Muscle Cells
Stéphane Lajoie-Kadoch*, Philippe Joubert*, Séverine Létuvé*. Andrew J. Halayko†,
James G. Martin*, Abdellilah Soussi-Gounni‡, and Qutayba Hamid*
*Meakins-Christie Laboratories, McGill University, Montréal, Québec, Canada.
Departments of †Physiology and ‡Immunology University of Manitoba, Winnipeg,
Manitoba, Canada.
Running title: Expression and Modulation of IL-17BR by Airway Smooth Muscle
Cells.
Correspondence address:
Qutayba Hamid
Meakins-Christie Laboratories
McGill University
3626 St-Urbain Street
Montreal, Canada, H2X 2P2
Phone: (514) 398-3864 x00143
Fax: (514) 398-7483
[email protected]
Copyright © 2006 by the American Physiological Society.
Abstract
The interleukin-17B receptor (IL-17BR) is expressed in a variety of tissues and is
upregulated under inflammatory conditions. This receptor binds both its cognate
ligand IL-17B and IL-17E/IL-25, a novel cytokine known to promote Th2 responses.
The present study illustrates that airway smooth muscle cells express IL-17BR in vitro
and that its expression is upregulated by TNFE and downregulated by IFNF. Our data
indicates that TNFE upregulates IL-17BR mainly through NF-kB as assessed with the
IKK2 inhibitor, AS602868. In addition, both IFN-gamma and dexamethasone are able
to antagonize a TNFE-induced IL-17BR increase in mRNA expression. The MEK
inhibitor, U0126, totally reversed the inhibition observed with IFNF, suggesting the
involvement of the ERK pathway in this effect. In addition, upon stimulation with IL17E, ASMC increase their expression of extracellular matrix components, namely
pro-collagen E-1 and lumican mRNA. Furthermore, immunohistochemical analysis of
biopsies from asthmatic subjects reveals that this receptor is abundant in smooth
muscle layers. This is the first report showing IL-17BR receptor in structural cells of
the airways. Our results suggest a potential pro-remodelling effect of IL-17E on
airway smooth muscle cells through the induction of extracellular matrix and that its
receptor is upregulated by proinflammatory conditions.
Keywords: human, stromal cells, cytokine receptors, cytokines, extracellular matrix.
Introduction
Interest in the interleukin (IL)-17 family has been stimulated by the publication of
data suggesting that its founding member, IL-17, may contribute to the pathogenesis
of conditions such as multiple sclerosis, arthritis and asthma (8, 38, 40, 42, 65, 67,
73). The expression of this cytokine is significantly upregulated in the airways of
subjects with moderate-to-severe asthma (8). IL-17 is proinflammatory in nature and
is produced primarily by activated memory CD4+ and CD8+ T lymphocytes (14, 56,
77), and does not appear to be particularly associated with either the Th1 or Th2
subset of these cells (26). IL-17 is especially potent at synergizing with IL-1J or
TNFE to induce production of cytokines such as IL-6, IL-8 and GM-CSF (2, 19, 70).
Among the six members of the IL-17 family, the homodimer IL-17E, also known as
IL-25, is unique in that it is able to elicit protypical Th2 responses such as peripheral
and lung eosinophilia, increased serum IgE, increased respiratory tract mucus
production, as well as the induction of IL-4, IL-5 and IL-13 gene expression (13, 24,
32, 48). Recent in vitro experiments have shown that IL-17E is produced by Th2
cells, mast cells and macrophages (13, 25, 31). IL-17E binds to the interleukin-17B
receptor (IL-17BR) also known as IL-17Rh1 (34) and Evi27 (68), and is expressed in
a variety of human tissues including the lung. IL-17E activates NF- B (34, 74) and
stimulates IL-8 production on binding to IL-17BR in two human renal carcinoma cell
lines, TK-10 and 293 (34). It also induces p38 and JNK phosphorylation in
eosinophils (74).
Airway smooth muscle cells (ASMC) are responsible for the airway narrowing of
asthma. However their role is not limited to this action, rather, they appear to play an
active role in the pathogenesis of this condition through various mechanisms
including airway remodelling (24, 49, 61). ASMC have been shown to express
receptors for a variety of cytokines and chemokines, including IL-17 receptor (53),
and respond to their cognate ligands by releasing other proinflammatory mediators
and extracellular matrix (ECM) proteins. One of the characteristic features of
remodelling is the increased deposition of ECM, in particular collagen I, III, V, and
proteoglycans (27, 28). We hypothesized that ASMC harboured IL-17BR and that
proinflammatory cytokines could modulate its expression. We also assessed the
potential of IL-17E to induce expression of ECM components. Here, we provide data
showing the presence of the IL-17E receptor, IL-17BR, in ASMC in vivo and in vitro,
as well as its modulation by TNFE and IFNF. We also show that there was increased
expression of pro-collagen 1 and lumican mRNA increased in these cells in response
to IL-17E. Taken together, these data suggest the involvement of IL-17E and its
receptor in the initiation and/or perpetuation of airway remodelling in asthma.
Materials and Methods
Materials
Smooth muscle Growth medium was purchased from Cambrex (East Rutherford, NJ,
USA). LabTek chamber slides were bought from Nalgene Nunc (Naperville, IL,
USA). Human recombinant cytokines, mouse monoclonal anti-IL-17BR and isotype
antibodies were obtained from R&D Systems (Minneapolis, MN, USA). QuantiTect
SYBR Green PCR kit, RNeasy mini and micro kit extraction columns, HiSpeed Maxi
plasmid kit and the QIAquick PCR purification kit were purchased from Qiagen
(Mississauga, ON, Canada). Trypan Blue dye, Superscript II, Taq Platinum
polymerase, PCR primers, 100 bp DNA ladder and restriction enzymes were obtained
from Invitrogen (Burlington, ON, Canada). Bradford assay reagent and Western blot
precision plus molecular weight standards were purchased from Bio-Rad
(Mississauga, ON, Canada). Donkey anti-mouse HRP-conjugated secondary antibody,
oligo(dT)12-18 primer, RNAguard, Hybond-P polyvinylidene fluoride (PVDF)
membrane, ECL Plus, and X-ray films were purchased from Amersham Pharmacia
Biotech (Baie d'Urfé, QC, Canada). The LightCycler real-time PCR, the minicomplete protease inhibitor cocktail tablets were bought from Roche Diagnostics
(Laval, QC, Canada). pGEM-T was purchased from Promega (Madison, WI, USA).
Pharmacological inhibitors (U0126, SB203580, and SP600125, resuspended in
DMSO), dexamethasone 21-phosphate disodium salt (resuspended in water) and Fast
Red were purchased from Sigma-Aldrich (Oakville, ON, Canada). The specific IKK2
inhibitor, AS602868, was a generous gift from Dr. Ian M. Adcock (National Heart
and Lung Institute, Imperial College London). Alexa Fluor 555 labelled goat antimouse IgG was obtained from Molecular Probes (Eugene, OR). Universal Blocking
Solution and mouse alkaline phosphatase anti-alkaline phosphatase (APAAP) were
bought from Dako, (Mississauga, ON, Canada), Gill modified hematoxylin was from
Surgipath (Winnipeg, MB, Canada). The BX51 Olympus epifluorescence microscope
attached to a CoolSNAP-Pro color digital camera was purchased from Carsen Group
(Markham, ON, Canada). The Cap-Sure Macro LCM Caps and the PixCell II System
were obtained from Arcturus (Mountain View, CA, USA).
Preparation of human bronchial ASMC
Primary human airway smooth muscle cells (ASMC) were obtained from main
bronchial airway segments (0.5–1.0 cm diameter) in pathologically uninvolved
segments of resected lung specimens using isolation methods described previously
(17, 46). Cells were then seeded at a density of 105 cells/cm2 and grown at 37°C in
Smooth Muscle Growth medium. At confluence, primary human ASMC exhibited
spindle morphology and a hill-and-valley pattern characteristic of smooth muscle in
culture. In cultures up to passage 5, over 90% of the cells at confluence retained
smooth muscle-specific E-actin, SM22, and calponin protein expression, and were
able to mobilize intracellular Ca2+ in response to acetylcholine. Growth rate
(determined by cell number) of ASMC from all lung resection donors was similar to
that reported previously for ASMC cultures from healthy human transplant donors.
Morphologically, the ASMC from lung resection donors and from healthy human
transplant donors were indistinguishable. Cell viability was always above 95% as
assessed by Trypan Blue dye exclusion.
Human bronchial tissue.
Human bronchial biopsy tissue samples were obtained from the Montreal Chest
Institute following approval of the protocol by an institutional Ethics Committee.
Immunofluorescence on primary ASMC
ASMC were grown to 50-60% confluence in LabTek chamber slides. The slides were
briefly rinsed in PBS, then fixed in 4 % paraformaldehyde solution for 20 min at room
temperature and washed again in PBS before drying. Fixed slides were stored at -80ºC
until use. When processed, the slides were first incubated with a solution of 50 µM
NH4Cl for 30 min, followed by blocking in 20% normal goat serum. Slides were then
exposed overnight to a solution of mouse anti-human IL-17BR (5 µg/ml), or isotypematched control antibody. Slides were extensively washed and incubated with a 1/750
dilution of Alexa Fluor 555 labelled goat anti-mouse IgG. Cell nuclei were visualized
by staining with Hoechst 33342 (2 µg/ml) and slides were examined with a BX51
Olympus epifluorescence microscope attached to a CoolSNAP-Pro color digital
camera.
RNA extraction and cDNA preparation
Total RNA was isolated from (i) cultured ASMC and (ii) ASMC bundles from human
airway biopsies (by laser capture microdissection) using RNeasy mini and micro kit
extraction columns, respectively. cDNA was generated in a 30 µl reaction, using 500
ng total RNA as template, oligo(dT)12-18 primer and Superscript II Reverse
Transcriptase, in the presence of RNAguard.
Preparation of standards for Real-time PCR
Quantification of the housekeeping gene, ribosomal protein S9 (S9), IL-17BR, procollagen
I, versican and lumican cDNA was achieved by constructing standard
curves using serial dilutions of purified cDNA. Briefly, conventional PCR was
employed to amplify the genes of interest from unstimulated ASMC cDNA, using
Platinum Taq polymerase and oligonucleotide primers. The primers were designed to
span at least one intron and, in the case of IL-17BR, to include the region encoding
the transmembrane domain. Primer sequences are as follows: S9-sense 5’TGCTGACGCTTGATGAGAAG-3’;
S9-antisense
5’-
CGCAGAGAGAAGTCGATGTG-3’;
IL-17BR-sense
5’-
AACAGGCGTCCCTTTCCCTCTGGA-3’;
IL-17BR-antisense
5’-
TTCTTGATCCTTTCGTGCCTCCAC-3’
collagen1-sense:
5’-
CTGGTCCCCAAGGCTTCCAAGG-3’;
collagen1-antisense:
5’-
CTTCACCCTTAGCACCAACAGC-3’;
versican-sense:
5’-
GAATTGGAGACCCAACCAGCC-3’;
versican-antisense:
5’-
GGTATAGCCCATCTTCCATTTCC-3’;
CCTGGTTGAGCTGGATCTGT-3’;
lumican-sense:
5’-
lumican-antisense:
5’TGGTTTCTGAGATGCGATTG-3’. All PCR amplicons were purified using the
QIAquick PCR purification kit and cloned in pGEM-T. Plasmids were sequenced to
confirm identity and integrity. Large-scale preparations of each construct were
purified using the HiSpeed Plasmid Maxi Kit. 10 µg of each construct was then
digested with Pst I and Sac II and the resulting fragments were gel-purified as
previously described. A 10-fold dilution series ranging from 10-1 to 10-10 ng/µl of each
amplicon was then prepared in Tris-HCl pH 8.0.
Real-time PCR
Relative quantification of S9, IL-17BR, pro-collagen
I, lumican and versican was
performed using the LightCycler. PCR reactions were performed in a 20 µl volume
containing 7 µl water, 1 µl cDNA, 0.3 µM of each primer, and 10 µl of 2X QuantiTect
SYBR Green PCR Master Mix. Specificity of the amplified products was assessed by
melting curve analysis and gel electrophoresis.
Western blotting
Cells were rinsed in ice-cold PBS and incubated in lysis buffer (150 mM NaCl; 10
mM Tris-HCl pH 7.4; 1 mM EDTA; 1 mM EGTA; 1% Triton X-100; 0.5% NP40,
100 mM sodium fluoride; 10 mM sodium pyrophosphate; 2 mM sodium
orthovanadate) containing a mini-complete protease inhibitor cocktail tablet for 30
minutes on ice. Extracts were clarified at 14,000 x g at 4VC for 20 minutes and protein
concentration was determined using the Bradford assay. 30 µg of protein was then
resolved by one-dimensional 10% SDS-PAGE and transferred to Hybond-P
polyvinylidene difluoride (PVDF) membranes. Blocking of membranes was carried
out using 5% BSA in TBST (0.05% Tween-20, 10 mM TBS, pH 7.5) for 2 h at room
temperature. Blots were incubated overnight at 4VC with either 0.5 µg/ml of mouse
monoclonal anti-IL-17BR antibody or with anti-IL-17BR preincubated with a 10
molar excess of rhIL-17BR/Fc chimera for 2 h at room temperature. After washing,
membranes were treated with HRP-conjugated anti-mouse secondary antibody for 1 h
at room temperature. After washing, signal was developed using the ECL Plus kit and
detected by exposure to X-ray film.
Immunohistochemistry
Immunohistochemistry was performed on sections of major airways from 6 asthmatic
subjects. Briefly, paraffin-embedded sections were deparaffinized and heat-induced
antigen retrieval was performed using 10 mM citrate buffer. Slides were then
extensively washed in PBS, blocked for 30 min in Universal Blocking Solution and
incubated overnight at 4ºC with 25 ug/ml murine anti-human IL-17BR, or IgG2B
isotype control, antibodies. Sections were subsequently washed twice in TBS prior to
45 minute incubation at room temperature with rabbit anti-mouse Ig followed by
mouse alkaline phosphatase anti-alkaline phosphatase (APAAP). The reaction was
developed using Fast Red. Tissue was then counterstained with Gill modified
hematoxylin and examined by light microscopy. Images were acquired with the BX51
microscope.
Laser capture microdissection of ASMC bundles.
Laser-capture microdissection (LCM) was performed as described elsewhere (18).
Briefly, slides were completely air-dried and desiccated to prevent the activation of
endogenous RNAse in the tissues. LCM of smooth muscle bundles was performed
using the PixCell II System. The interval between staining of slides and completion of
microdissection was 1 to 2 hours. After samples were captured on Cap-Sure Macro
LCM Caps, cells were lysed with RLT lysis buffer (RNeasy kit), and samples were
stored at W80°C until RNA extraction.
Statistical analysis
Data were obtained from at least three independent experiments. Results were
expressed as mean ± SD and analyzed by ANOVA or by unpaired two-tailed Student
T test. Values of p<0.05 were considered significant.
Results
IL-17BR is expressed by cultured primary human ASMC.
In order to determine whether human cultured ASMC constitutively expressed IL17BR, RNA and protein lysates obtained from unstimulated cells from different
donors were analyzed by RT-PCR, Western blot and immunofluorescence. The RTPCR data illustrated that IL-17BR was constitutively expressed in all our human
ASMC sources as shown in Fig. 1A. Western blot analysis from the same ASMC
demonstrated basal expression of IL-17BR (Fig. 1A). No signal was detected when
the membrane was incubated with anti-IL-17BR antibody plus rhIL-17BR,
confirming
the
specificity
of
the
55
kDa
band
observed
(Fig.
1A).
Immunofluorescence also revealed receptor protein expression by primary cultured
ASMC (Fig. 1C and D). Compared to isotype control, specific fluorescent staining
was observed on the cell membrane, as well as in the cytoplasm.
IL-17BR mRNA and protein expression are modulated in a reciprocal manner by
TNF+ and IFN, in ASMC.
We wanted to investigate whether IL-17BR expression could be regulated by TNFE
and IFNF. Real-time PCR and Western blot were used to evaluate receptor
modulation by these cytokines. TNFE upregulated IL-17BR in ASMC (Fig 2A). A
time course of the effect of TNFE shows a rapid peak expression for both mRNA and
protein after 4 h (Fig. 2A). Furthermore, we observed a concentration-related effect of
TNFE on IL-17BR message and protein levels after 4 h stimulation (Fig. 2B). Levels
of IL-17BR appeared to reach a plateau at 10 ng/ml TNFE at the mRNA level.
In contrast, treatment of cells with IFNF resulted in a decrease in the expression of IL17BR mRNA and protein (Fig. 2B). IL-17BR mRNA showed an early tendency for
downregulation by IFNF, however, the maximal inhibitory effects for mRNA and
protein were reached after 12 h. IFNF also inhibited the receptor’s expression at the
mRNA and protein levels in a concentration-related manner (Fig. 2B) up until 10
ng/ml, the 100 ng/ml concentration did not further inhibit the receptor.
Th2 cytokines do not affect IL-17BR expression in ASMC.
We wished to assess whether IL-17BR expression was sensitive to the prototypical
Th2 cytokines IL-4 and IL-13. These cytokines had no noticeable effect on the
receptor in ASMC after 4 or 24 h of stimulation (Fig. 3). In the same conditions the
Th1 cytokine IFNF induced a significant inhibition of IL-17BR mRNA.
Effect of pharmacological inhibitors on IL-17BR expression
As previously illustrated (Fig. 2 and 3), TNF induced a robust increase in IL-17BR
mRNA, and although upregulation of IL-17BR mRNA by TNFE was strongest at 4 h,
the receptor remained induced after 24 h. This time point was used to study the effects
of TNFE plus, either IFNF or dexamethasone. Addition of increasing concentrations
of IFNF to TNFE-treated cells abrogated IL-17BR expression in a concentrationrelated fashion (Fig 4A). The addition of the synthetic corticosteroid dexamethasone
to TNFE-stimulated ASMC caused an even greater reduction in expression of IL17BR message (Fig 4B). Inhibition of IKK2 with AS602868 produced a marked
inhibition of Il-17BR expression with a similar potency to that of dexamethasone (Fig
4C). The JNK inhibitor SP600125 caused a partial decrease in IL-17BR message (Fig
4C). The inhibitor of ERK phosphorylation, U0126, appeared to produce some
inhibition of IL-17BR expression, although this effect was only significant at the
concentration of 1 µM. The p38 phosphorylation inhibitor SB203580 showed no
effect (Fig 4C). Of note, the fold increase values observed with TNFE+DMSO
compared to DMSO were less than those observed with TNFE alone as compared to
medium (Fig 2A). This may be related to an effect of DMSO on ASMC.
The inhibitory effect of IFNF on IL-17BR expression was reversed by U0126 in a
concentration-related manner (Fig. 4D). Treatment with SB203580 and SP600125,
had no significant effect on this parameter (Fig. 4D).
IL-17E stimulates ECM mRNA expression in ASMC.
Cells were stimulated with either 100 ng/ml IL-17E or equivalent in vehicle (4 mM
HCl/ 0.1 % BSA) for 4, 24 and 48 h and analyzed for their expression of ECM
components mRNA: pro-collagen
I, lumican and versican. There was a trend
towards increased ECM mRNA in IL-17E-treated cells. Statistically significant data
points showed that pro-collagen EI was on average 1.6 fold upregulated after 24 h and
lumican was 1.2 fold increased after 4 h (Fig. 5) when compared to vehicle-treated
cells at their respective time points. Pro-collagen EI appeared to remain elevated after
48 h of stimulation (p = 0.054).
IL-17BR is expressed in vivo.
We investigated IL-17BR expression in vivo, using immunohistochemistry on airway
biopsies obtained from 6 different asthmatic donors. Positive staining was evident in
the smooth muscle layers (Fig. 6B) demonstrating the relative abundance of this
receptor in the airways. No staining was observed using isotype control antibody (Fig.
6A). We confirmed in vivo expression of IL-17BR by laser capture microdissection of
ASMC bundles from suitable biopsies (n=3). cDNA obtained from these
microdissected tissues were analyzed by real-time PCR and showed that the IL-17BR
141 bp amplicon was present in laser-captured ASMC bundles (lanes 1-3). Cultured
ASMC were used as a positive control (Fig. 6C). Non-specific bands observed below
100 bp correspond to primer-dimers (lanes 1 and – ctrl)
Discussion
We report for the first time the expression and modulation of the IL-17E receptor in a
primary structural cell type. We show here that IL-17BR is expressed in vitro by
primary ASMC and in vivo in airway biopsies with predominant staining in smooth
muscle layers. Previous reports show that dendritic cells, leukocytes and some
structural cell lines express this receptor (16, 34, 68). Interestingly, a functional IL17R has recently been described on airway smooth muscle cells (53).
IL-17BR and IL-17R share partial amino acid homology but bind different IL-17
family members, IL-17BR binds IL-17E and to a lesser extent IL-17B but not IL-17,
or IL-17C (34). To date, there has been one report describing the modulation of IL17BR (16). The authors found that IL-4, -10, and -13 upregulated IL-17BR mRNA in
human dendritic cells, although the report of baseline levels of the receptor is unclear
(16). In ASMC, IL-4 and IL-13 did not have any effect on the constitutive expression
of IL-17BR. However, our data shows that IL-17BR message and protein were readily
upregulated by TNFE, an effect observed for IL-17R in human colonic myofibroblasts
(2). Preliminary bioinformatic analysis of the IL-17BR promoter region revealed
several NF- B binding sites (57) which could account for the TNFE-induced
upregulation of the IL-17BR transcript we observed. TNFE is a well known inducer of
NF- B in ASMC (1). Consequently, we evaluated the effect of AS602868, a specific
inhibitor of the I B kinase 2 (IKK2), important for TNFE-mediated NF- B activation
(15, 43). As expected, inhibition of NF-kB led to a marked reduction of TNFEinduced IL-17BR mRNA. IL-17BR expression is also corticosteroid sensitive, as
demonstrated by the potent inhibition of TNF -induced IL-17BR by dexamethasone.
Besides activating the I B kinase (IKK) pathway, TNFE can turn on the main MAPK
signalling cascades including, p42/44, p38 and c-Jun N-terminal kinase (JNK) (37,
79). The effect of inhibiting JNK was less drastic than with either dexamethasone or
AS602868, SP600125 reduced TNFE-induced IL-17BR levels to baseline.
Taken together, our data show NF-kB and to a lesser extent JNK play a role in the
induction of IL-17BR by TNFE. Most likely, NF- B causes a direct transactivation of
the IL-17BR promoter.
IFNF reduced the constitutive expression of IL-17BR, a finding also observed in
dendritic cells (16). Furthermore, the increase in IL-17BR transcript induced by TNFE
could be antagonized at the mRNA level by the addition of IFNF. The mechanism by
which IFNF downregulates IL-17BR is unknown; IFNF has been shown to target gene
expression at different levels. IFNF downregulates IL-4-induced IgE and IL-4R as
well as collagen I expression by mechanisms involving transcriptional suppression,
and posttranscriptional inhibition mediated by increased mRNA degradation (51, 63,
71). The IL-17BR promoter sequence displays two potential IFNF response elements
(IgRE) that bind Y-Box-1, previously shown to be responsible for IFNF-mediated
collagen I E2 downregulation (20, 21). Furthermore, the 3’-untranslated region of the
mouse and human IL-17BR transcript contain 3 and 2 Shaw-Kamen motifs (58) (5’AUUUA-3’), respectively. This pentamer motif was previously shown to be present
in several immediate early genes, such as cytokines, cytokine receptors, and
oncogenes, and confers instability to mRNAs. Indeed, we have preliminary data
suggesting that IFNF promotes IL-17BR mRNA decay when added to actinomycin Dtreated ASMC. Collectively, these observations hint at the possible downregulation of
IL-17BR by IFNF via possible IgRE elements present in its promoter and/or through
an increase in mRNA decay.
Besides the traditional Jak/STAT pathway involved in IFNF signalling, this cytokine
has been shown to activate ERK 1/2 and p38 in some cell types, such as cardiac
myocytes, macrophages, glioma cells, human bronchial epithelial cells and human
vascular smooth muscle cells (9, 33, 47, 60, 62). IFNF can also induce ERK 1/2, and
JNK phosphorylation in ASMC (data not shown). It has also been reported that IFNF
can induce, through JAK2, the activation of Raf-1, an upstream kinase involved in
ERK phosphorylation (76). Moreover, the MEK inhibitor U0126 can abrogate IFNFinduced gene expression in the RAW murine macrophage cell line (22). Accordingly,
preincubation of ASMC with the MAPK inhibitor U0126 totally reversed the IFNFmediated IL-17BR downregulation. However p38 or JNK phosphorylation inhibition
had no significant effect. This suggests that IFNF exerts its inhibition at least through
the MEK-ERK pathway.
Shi et al (59) found that IL-17BR mRNA was significantly upregulated in a murine
model of intestinal inflammation, a condition characterized by elevated TNF levels
in humans (50). In asthma, TNFE is increased in the airways and is linked to disease
severity. No real consensus exists in the literature about IFNF expression in asthma (5,
7, 10, 35, 44, 54, 55, 66, 75), since published data shows that levels of this cytokine
are either increased, decreased or unchanged in asthma and upregulated in subjects
treated with corticosteroids (4, 5, 66, 75). Airways exposed to increased TNFE and
decreased IFNF concentrations could thus favour IL-17BR expression in vivo by
smooth muscle. This phenomenon could modulate their sensitivity to IL-17E released
by airway-infiltrated Th2 cells and/or mast cells. Accordingly, mice that were
administered either recombinant IL-17E or an adenoviral IL-17E construct, developed
a robust eosinophilic infiltration of the airways and stimulated the release of the Th2
cytokines IL-4, -5, and -13 (13, 24, 32). IFNF inhibits Th2 cells and Th2 cytokine
release as well as eosinophilia in sensitized mice after exposure to allergen and could
therefore antagonize the effects of IL-17E. Indeed, one of the positive effects of
increasing IFNF in asthmatic patients, through allergen immunotherapy (12), could be
the reduction of IL-17BR expression on ASMC and other cell types and the
subsequent reduction of IL-17E bioactivity in vivo.
One of the hallmarks of remodelling is the increased deposition of ECM proteins in
the airways (23, 29, 72). We wanted to investigate whether IL-17BR was involved in
airway remodelling. We evaluated ASMC response to IL-17E in producing
extracellular matrix (ECM) components. Reports have shown that cultured ASMC
can produce matrix proteins such as collagen I, versican, and decorin (30, 52). In
addition, IL-17 has been associated with an increased deposition of type I collagen in
the airways of moderate to severe asthmatics, but it is thought that IL-17 could induce
collagen I deposition indirectly through the induction of the profibrotic cytokines IL-6
and IL-11 (45, 69) which have been shown to enhance collagen and tissue inhibitor of
metalloprotease (TIMP)-1 production (11, 39, 41, 64). IL-17E has also been shown to
induce IL-6 production in eosinophils (74) and human cartilage (6).
Primary ASMC cultured in the presence of IL-17E showed increased mRNA
expression for lumican, and especially collagen E1 (60% increase approximately). For
comparison, TGF- 1, considered a positive inducer of collagen EI. TGF- 1 produced
a 150% increase in collagen EI mRNA (data not shown). In contrast, IFNF, a known
inhibitor of collagen EI synthesis led to a 40-50% decrease in pro-collagen E1 mRNA
(data not shown). Similar values were found in the literature regarding collagen EI
mRNA modulation by TGF- 1 and IFNF in fibroblasts and hepatic stellate cells (3,
36, 78). This initial finding implies that IL-17E could participate with other Th2 and
profibrotic cytokines and contribute to airway remodelling observed in asthmatics.
Altogether, our results provide initial insight into the in vitro and in vivo expression as
well as the in vitro regulation of the IL-17E receptor and a potential mechanism of
action of this novel cytokine on ASMC. Further work is necessary to better
understand the mechanisms that lead to the modulation of IL-17BR in ASMC by
TNFE and IFNF respectively. Moreover, it would be of considerable interest to assess
if IL-17E and IL-17BR are overexpressed in asthma, an area of ongoing research in
our laboratory.
Acknowledgements:
We would like to thank Andrea K. Mogas for her expert technical assistance.
Grants:
Funding: Canadian Institutes of Health Research (CIHR)
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FIGURE 1. IL-17BR is expressed in cultured airway smooth muscle cells. IL-17BR
mRNA (A) and protein (B) are constitutively expressed by cultured ASMC from
different donors (n=4). Specificity of the 55 kDa band in the western blot was
assessed using ASMC protein extract from donor 1 incubated with anti-IL-17BR
preincubated with rhIL-17BR. Detection of IL-17BR in cultured ASMC was
performed by immunofluorescence using an Alexa Fluor 555-labelled antibody. (C)
200X magnification of the staining obtained with the isotype control or (D) anti-IL17BR antibody.
FIGURE 2. Time and concentration related effect of TNFE and IFNF on IL-17BR
mRNA and protein expression in ASMC. Cells were starved for 24 h before treatment
with cytokines, cDNA and protein samples were analyzed by real-time PCR (bar
graphs) and Western blot (insets). A) Time and B) concentration-related expression of
IL-17BR mRNA and protein in ASMC in response to TNFE. C) Time and D)
concentration-related, expression of IL-17BR mRNA and protein in ASMC after
treatment with IFNF. RNA was extracted at stated time and concentration and reversetranscribed. cDNA was analyzed by real-time PCR, and data are represented as the
mean of 3 to 5 independent experiments ± SD (*p<0.05). Western blots shown are
representative of 3 independent experiments.
FIGURE 3. Effect of Th2-type cytokines on IL-17BR mRNA expression in ASMC.
Cells were starved for 24 h and treated with 10 ng/ml IL-4, IL-13, or IFNF. RNA was
extracted at 4 and 24 h and reverse-transcribed. cDNA was analyzed for IL-17BR
levels by real-time PCR. Data represent the mean of 3 to 6 independent experiments ±
SD (*, p<0.05;**p <0.01).
FIGURE 4. Effect of various inhibitors on IL-17BR expression. ASMC were starved
for 24 h prior to stimulation and then treated with cytokines. Cells were stimulated 24
h with TNFE (10 ng/ml) and increasing concentrations of A) IFNF or B)
dexamethasone. C) ASMC were stimulated 4 h with TNFE (10 ng/ml) and increasing
concentrations of MAPK (U0126, SB203580 and SP600125) or IKK2 (AS =
AS602868) inhibitors. D) ASMC were stimulated 24 h with IFNF (10 ng/ml) and
increasing concentrations of MAPK inhibitors. Effect of the solvent (DMSO, 0.2%)
was tested for the equivalent volume used in the highest inhibitor concentration (20
µM). RNA was extracted after 4 or 24 h. RNA samples were reverse-transcribed and
cDNA was analyzed for IL-17BR levels. Data are expressed as the mean of 3 to 4
independent experiments ± SD (*p<0.05; **p<0.01; # =significantly different from
media or DMSO, †= significantly different from TNFE or IFNF alone).
FIGURE 5. Effect of IL-17E on the expression of ECM mRNA in cultured ASMC.
Cells were starved for 24 h and treated with 100 ng/ml IL-17E or its vehicle (4 mM
HCl/0.1% BSA) and RNA was extracted at 4, 24 and 48 h. RNA samples were
reverse-transcribed and cDNA was analyzed for pro-collagen
I, lumican and
versican levels. Real time PCR data represents the mean of 4-8 independent
experiments ± SD. (* p <0.05; **p<0.01).
FIGURE 6. Expression of IL-17BR in vivo. IL-17BR is expressed at the protein level
as assessed by immunohistochemistry. Representative staining of a human airway
biopsy showing positive immunoreactivity in smooth muscle layers. 200X
magnification of an airway biopsy treated with the isotype control (A) or the anti-IL17BR (B) antibody followed by APAAP detection. Expression of IL-17BR mRNA in
ASMC bundles obtained by laser capture microdissection (lanes 1-3). Cultured
ASMC were used as a positive control (C).