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Am J Physiol Lung Cell Mol Physiol 290: L1238-L1246, 2006. First published January 20, 2006; doi:10.1152/ajplung.00301.2005
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TNF-{alpha} and IFN-{gamma} inversely modulate expression of the IL-17E receptor in airway smooth muscle cells

Stéphane Lajoie-Kadoch,1 Philippe Joubert,1 Séverine Létuvé,1 Andrew J. Halayko,2 James G. Martin,1 Abdellilah Soussi-Gounni,3 and Qutayba Hamid1

1Meakins-Christie Laboratories, McGill University, Montréal, Québec; and Departments of 2Physiology and 3Immunology, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 13 July 2005 ; accepted in final form 13 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 shows that airway smooth muscle cells express IL-17BR in vitro and that its expression is upregulated by TNF-{alpha} and downregulated by IFN-{gamma}. Our data indicate that TNF-{alpha} upregulates IL-17BR mainly through nuclear factor-{kappa}B as assessed with the I{kappa}B kinase 2 inhibitor AS-602868. In addition, both IFN-{gamma} and dexamethasone are able to antagonize a TNF-{alpha}-induced IL-17BR increase in mRNA expression. The mitogen-activated protein kinase kinase inhibitor U0126 totally reversed the inhibition observed with IFN-{gamma}, suggesting the involvement of the extracellular signal-regulated kinase pathway in this effect. In addition, on stimulation with IL-17E, airway smooth muscle cells increase their expression of ECM components, namely procollagen-{alpha}I 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 proremodeling effect of IL-17E on airway smooth muscle cells through the induction of ECM and that its receptor is upregulated by proinflammatory conditions.

human; stromal cells; cytokine receptors; cytokines; extracellular matrix; tumor necrosis factor-{alpha}; interferon-{gamma}; interleukin-17E

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, 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 T helper (Th) 1 or Th2 subset of these cells (26). IL-17 is especially potent at synergizing with IL-1beta or TNF-{alpha} to induce production of cytokines such as IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (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 IL-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 nuclear factor (NF)-{kappa}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 c-Jun NH2-terminal kinase (JNK) phosphorylation in eosinophils (74).

Airway smooth muscle cells (ASMCs) 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). ASMCs 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 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 harbored 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 TNF-{alpha} and IFN-{gamma}. We also show that there was increased expression of procollagen-{alpha}I, and that 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 remodeling in asthma.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Smooth muscle growth medium was purchased from Cambrex (East Rutherford, NJ). LabTek chamber slides were bought from Nalgene Nunc (Naperville, IL). Human recombinant cytokines, mouse monoclonal anti-IL-17BR, and isotype antibodies were obtained from R&D Systems (Minneapolis, MN). 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 horseradish peroxidase-conjugated secondary antibody, oligo(dT)12-18 primer, RNAguard, Hybond-P polyvinylidene fluoride membrane, ECL Plus, and X-ray films were purchased from Amersham Pharmacia Biotech (Baie d’Urfé, QC, Canada). The LightCycler real-time PCR and the mini-complete protease inhibitor cocktail tablets were bought from Roche Diagnostics (Laval, QC, Canada). pGEM-T was purchased from Promega (Madison, WI). Pharmacological inhibitors (U0126, SB-203580, and SP-600125, resuspended in DMSO), dexamethasone 21-phosphate disodium salt (resuspended in water), and fast red were purchased from Sigma-Aldrich (Oakville, ON, Canada). The specific I{kappa}B kinase (IKK) 2 inhibitor, AS-602868, was a generous gift from Dr. Ian M. Adcock (National Heart and Lung Institute, Imperial College London, London, UK). Alexa Fluor 555-labeled goat anti-mouse IgG was obtained from Molecular Probes (Eugene, OR). Universal blocking solution and mouse alkaline phosphatase anti-alkaline phosphatase were bought from Dako (Mississauga, ON, Canada), and 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 laser-capture microdissection (LCM) caps and the PixCell II system were obtained from Arcturus (Mountain View, CA).

Preparation of human bronchial ASMCs. Primary human ASMCs were obtained from main bronchial airway segments (0.5- to 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 ASMCs 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 {alpha}-actin, SM22, and calponin protein expression, and were able to mobilize intracellular Ca2+ in response to acetylcholine. Growth rate (determined by cell number) of ASMCs from all lung resection donors was similar to that reported previously for ASMC cultures from healthy human transplant donors. Morphologically, the ASMCs 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 ASMCs. ASMCs 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 –80°C 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 isotype-matched control antibody. Slides were extensively washed and incubated with a 1/750 dilution of Alexa Fluor 555-labeled 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 1) cultured ASMCs and 2) ASMC bundles from human airway biopsies (by LCM) 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-{alpha}I, and 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'; collagen-1 sense 5'-CTGGTCCCCAAGGCTTCCAAGG-3'; collagen-1 antisense 5'-CTTCACCCTTAGCACCAACAGC-3'; versican sense 5'-GAATTGGAGACCCAACCAGCC-3'; versican antisense 5'-GGTATAGCCCATCTTCCATTTCC-3'; lumican sense 5'-CCTGGTTGAGCTGGATCTGT-3'; 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. Ten micrograms of each construct were then digested with PstI and SacII, 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, procollagen-{alpha}1, lumican, and versican was performed using the LightCycler. PCR reactions were performed in a 20-µl volume containing 7 µl of water, 1 µl of cDNA, 0.3 µM of each primer, and 10 µl of x2 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% Nonidet P-40, 100 mM sodium fluoride; 10 mM sodium pyrophosphate; 2 mM sodium orthovanadate) containing a mini-complete protease inhibitor cocktail tablet for 30 min on ice. Extracts were clarified at 14,000 g at 4°C for 20 min, and protein concentration was determined using the Bradford assay. Thirty micrograms of protein were then resolved by one-dimensional 10% SDS-PAGE and transferred to Hybond-P polyvinylidene fluoride membranes. Blocking of membranes was carried out using 5% BSA in TBS-Tween (0.05% Tween 20, 10 mM TBS, pH 7.5) for 2 h at room temperature. Blots were incubated overnight at 4°C 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 horseradish peroxidase-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 six 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 4°C with 25 µg/ml murine anti-human IL-17BR or IgG2B isotype control antibodies. Sections were subsequently washed twice in TBS before 45-min incubation at room temperature with rabbit anti-mouse Ig followed by mouse alkaline phosphatase anti-alkaline phosphatase. 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.

LCM of ASMC bundles. 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 1–2 h. After samples were captured on Cap-Sure macro LCM caps, cells were lysed with RLT lysis buffer (RNeasy kit), and samples were stored at –80°C until RNA extraction.

Statistical analysis. Data were obtained from at least three independent experiments. Results were expressed as means ± SD and analyzed by ANOVA or by unpaired two-tailed Student’s t-test. Values of P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
IL-17BR is expressed by cultured primary human ASMCs. To determine whether human cultured ASMCs constitutively expressed IL-17BR, RNA and protein lysates obtained from unstimulated cells from different donors were analyzed by RT-PCR, Western blot, and immunofluorescence. The RT-PCR 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 ASMCs (Fig. 1, C and D). Compared with isotype control, specific fluorescent staining was observed on the cell membrane, as well as in the cytoplasm.


Figure 1
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  		Fig. 1. IL-17B receptor (IL-17BR) is expressed in cultured airway smooth muscle cells (ASMCs). IL-17BR mRNA (A) and protein (B) are constitutively expressed by cultured ASMCs 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 recombinant human IL-17BR. Detection of IL-17BR in cultured ASMCs was performed by immunofluorescence using an Alexa Fluor 555-labeled antibody. C and D: x200 magnification of the staining obtained with the isotype control (C) or anti-IL-17BR antibody (D).

 
IL-17BR mRNA and protein expression are modulated in a reciprocal manner by TNF-{alpha} and IFN-{gamma} in ASMCs. We wanted to investigate whether IL-17BR expression could be regulated by TNF-{alpha} and IFN-{gamma}. Real-time PCR and Western blot were used to evaluate receptor modulation by these cytokines. TNF-{alpha} upregulated IL-17BR in ASMCs (Fig. 2A). A time course of the effect of TNF-{alpha} shows a rapid peak expression for both mRNA and protein after 4 h (Fig. 2A). Furthermore, we observed a concentration-related effect of TNF-{alpha} on IL-17BR message and protein levels after 4 h of stimulation (Fig. 2B). Levels of IL-17BR appeared to reach a plateau at 10 ng/ml TNF-{alpha} at the mRNA level.


Figure 2
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  		Fig. 2. Time- and concentration-related effect of TNF-{alpha} and IFN-{gamma} on IL-17BR mRNA and protein expression in ASMCs. Cells were starved for 24 h before treatment with cytokines, and cDNA and protein samples were analyzed by real-time PCR (bar graphs) and Western blot (insets). Time (A) and concentration-related (B) expression of IL-17BR mRNA and protein in ASMCs in response to TNF-{alpha}. Time (C) and concentration-related (D) expression of IL-17BR mRNA and protein in ASMCs after treatment with IFN-{gamma}. RNA was extracted at stated time and concentration and reverse transcribed. cDNA was analyzed by real-time PCR, and data are means ± SD of 3–5 independent experiments. Western blots are representative of 3 independent experiments. *P < 0.05; P < 0.01.

 
In contrast, treatment of cells with IFN-{gamma} resulted in a decrease in the expression of IL-17BR mRNA and protein (Fig. 2B). IL-17BR mRNA showed an early tendency for downregulation by IFN-{gamma}; however, the maximal inhibitory effects for mRNA and protein were reached after 12 h. IFN-{gamma} also inhibited the receptor’s expression at the mRNA and protein levels in a concentration-related manner (Fig. 2B). The concentration-related effect is noticeable from concentrations of IFN-{gamma} ranging from 0 to 10 ng/ml. Increasing the concentration to 100 ng/ml did not further inhibit the receptor’s expression.

Th2 cytokines do not affect IL-17BR expression in ASMCs. 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 ASMCs after 4 or 24 h of stimulation (Fig. 3). In the same conditions, the Th1 cytokine IFN-{gamma} induced a significant inhibition of IL-17BR mRNA.


Figure 3
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  		Fig. 3. Effect of T helper (Th) 2-type cytokines on IL-17BR mRNA expression in ASMCs. Cells were starved for 24 h and treated with 10 ng/ml IL-4, IL-13, or IFN-{gamma}. RNA was extracted at 4 and 24 h and reverse transcribed. cDNA was analyzed for IL-17BR levels by real-time PCR. Data are means ± SD of 3–6 independent experiments. *P < 0.05; **P < 0.01.

 
Effect of pharmacological inhibitors on IL-17BR expression. As previously illustrated (Figs. 2 and 3), TNF-{alpha} induced a robust increase in IL-17BR mRNA, and although upregulation of IL-17BR mRNA by TNF-{alpha} was strongest at 4 h, the receptor remained induced after 24 h. This time point was used to study the effects of TNF-{alpha} plus either IFN-{gamma} or dexamethasone. Addition of increasing concentrations of IFN-{gamma} to TNF-{alpha}-treated cells abrogated IL-17BR expression in a concentration-related fashion (Fig. 4A). The addition of the synthetic corticosteroid dexamethasone to TNF-{alpha}-stimulated ASMCs caused an even greater reduction in expression of IL-17BR message (Fig. 4B). Inhibition of IKK2 with AS-602868 produced a marked inhibition of IL-17BR expression with a similar potency to that of dexamethasone (Fig. 4C). The JNK inhibitor SP-600125 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 SB-203580 showed no effect (Fig. 4C). Of note, the fold increase values observed with TNF-{alpha} + DMSO compared with DMSO were less than those observed with TNF-{alpha} alone compared with medium (Fig. 2A). This may be related to an effect of DMSO on ASMCs.


Figure 4
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  		Fig. 4. Effect of various inhibitors on IL-17BR expression. ASMCs were starved for 24 h before stimulation and then treated with cytokines. Cells were stimulated 24 h with TNF-{alpha} (10 ng/ml) and increasing concentrations of IFN-{gamma} (A) or dexamethasone (B). C: ASMCs were stimulated 4 h with TNF-{alpha} (10 ng/ml) and increasing concentrations of MAPK (U0126, SB-203580, and SP-600125) or IKK2 [AS-602868 (AS)] inhibitors. D: ASMCs were stimulated 24 h with IFN-{gamma} (10 ng/ml) and increasing concentrations of MAPK inhibitors. Effect of the solvent (0.2% DMSO) 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 means ± SD of 3–4 independent experiments. *P < 0.05; **P < 0.01. #Significantly different from media or DMSO. {dagger}Significantly different from TNF-{alpha} or IFN-{gamma} alone.

 
The inhibitory effect of IFN-{gamma} on IL-17BR expression was reversed by U0126 in a concentration-related manner (Fig. 4D). Treatment with SB-203580 and SP-600125 had no significant effect on this parameter (Fig. 4D).

IL-17E stimulates ECM mRNA expression in ASMCs. 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: procollagen-{alpha}I, lumican, and versican. There was a trend toward increased ECM mRNA in IL-17E-treated cells. Statistically significant data points showed that procollagen-{alpha}I was on average 1.6-fold upregulated after 24 h, and lumican was 1.2-fold increased after 4 h (Fig. 5) compared with vehicle-treated cells at their respective time points. Procollagen-{alpha}I appeared to remain elevated after 48 h of stimulation (P = 0.054).


Figure 5
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  		Fig. 5. Effect of IL-17E on the expression of ECM mRNA in cultured ASMCs. 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 procollagen {alpha}I, lumican, and versican levels. Real-time PCR data are means ± SD of 4–8 independent experiments. *P < 0.05; **P < 0.01.

 
IL-17BR is expressed in vivo. We investigated IL-17BR expression in vivo, using immunohistochemistry on airway biopsies obtained from six 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 LCM 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 ASMCs were used as a positive control (Fig. 6C). Nonspecific bands observed below 100 bp correspond to primer-dimers (lane 1 and control).


Figure 6
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  		Fig. 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. Shown is x200 magnification of an airway biopsy treated with the isotype control (A) or the anti-IL-17BR (B) antibody followed by alkaline phosphatase anti-alkaline phosphatase detection. Expression of IL-17BR mRNA in ASMC bundles obtained by laser capture microdissection (lanes 1–3). C: cultured ASMCs were used as a positive control.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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 ASMCs 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 IL-17R has recently been described on ASMCs (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 IL-17BR (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 ASMCs, IL-4 and IL-13 did not have any effect on the constitutive expression of IL-17BR. However, our data show that IL-17BR message and protein were readily upregulated by TNF-{alpha}, an effect observed for IL-17R in human colonic myofibroblasts (2). Preliminary bioinformatic analysis of the IL-17BR promoter region revealed several NF-{kappa}B binding sites (57), which could account for the TNF-{alpha}-induced upregulation of the IL-17BR transcript we observed. TNF-{alpha} is a well- known inducer of NF-{kappa}B in ASMCs (1). Consequently, we evaluated the effect of AS-602868, a specific inhibitor of the IKK2, important for TNF-{alpha}-mediated NF-{kappa}B activation (15, 43). As expected, inhibition of NF-{kappa}B led to a marked reduction of TNF-{alpha}-induced IL-17BR mRNA. IL-17BR expression is also corticosteroid sensitive, as demonstrated by the potent inhibition of TNF-{alpha}-induced IL-17BR by dexamethasone. Besides activating the IKK pathway, TNF-{alpha} can turn on the main MAPK signaling cascades, including p42/44, p38, and JNK (37, 79). The effect of inhibiting JNK was less drastic than with either dexamethasone or AS-602868: SP-600125 reduced TNF{alpha}-induced IL-17BR levels to baseline.

Taken together, our data show NF-{kappa}B and to a lesser extent JNK play a role in the induction of IL-17BR by TNF-{alpha}. Most likely, NF-{kappa}B causes a direct transactivation of the IL-17BR promoter.

IFN-{gamma} reduced the constitutive expression of IL-17BR, a finding also observed in dendritic cells (16). Furthermore, the increase in IL-17BR transcript induced by TNF-{alpha} could be antagonized at the mRNA level by the addition of IFN-{gamma}. The mechanism by which IFN-{gamma} downregulates IL-17BR is unknown; IFN-{gamma} has been shown to target gene expression at different levels. IFN-{gamma} 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 IFN-{gamma} response elements that bind Y-Box-1, previously shown to be responsible for IFN-{gamma}-mediated collagen I {alpha}2 downregulation (20, 21). Furthermore, the 3'-untranslated region of the mouse and human IL-17BR transcript contains 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 IFN-{gamma} promotes IL-17BR mRNA decay when added to actinomycin D-treated ASMCs. Collectively, these observations hint at the possible downregulation of IL-17BR by IFN-{gamma} via possible IFN-{gamma} response elements present in its promoter and/or through an increase in mRNA decay.

Other than the traditional JAK-STAT pathway involved in IFN-{gamma} signaling, this cytokine has been shown to activate ERK1/2 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). IFN-{gamma} can also induce ERK1/2 and JNK phosphorylation in ASMCs (data not shown). It has also been reported that IFN-{gamma} can induce, through JAK2, the activation of Raf-1, an upstream kinase involved in ERK phosphorylation (76). Moreover, the MEmitogen-activated protein kinase kinase inhibitor U0126 can abrogate IFN-{gamma}-induced gene expression in the RAW murine macrophage cell line (22). Accordingly, preincubation of ASMCs with the MAPK inhibitor U0126 totally reversed the IFN-{gamma}-mediated IL-17BR downregulation. However, p38 or JNK phosphorylation inhibition had no significant effect. This suggests that IFN-{gamma} exerts its inhibition at least through the MAPK kinase-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-{alpha} levels in humans (50). In asthma, TNF-{alpha} is increased in the airways and is linked to disease severity. No real consensus exists in the literature about IFN-{gamma} expression in asthma (5, 7, 10, 35, 44, 54, 55, 66, 75), since published data show 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 TNF-{alpha} and decreased IFN-{gamma} concentrations could thus favor 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). IFN-{gamma} 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 IFN-{gamma} in asthmatic patients through allergen immunotherapy (12) could be the reduction of IL-17BR expression on ASMCs and other cell types and the subsequent reduction of IL-17E bioactivity in vivo.

One of the hallmarks of remodeling 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 ECM components. Reports have shown that cultured ASMCs 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-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 ASMCs cultured in the presence of IL-17E showed increased mRNA expression for lumican and especially collagen-{alpha}I (an ~60% increase). For comparison, TGF-beta1, considered a positive inducer of collagen-{alpha}I, produced a 150% increase in collagen-{alpha}I mRNA (data not shown). In contrast, IFN-{gamma}, a known inhibitor of collagen-{alpha}I synthesis, led to a 40–50% decrease in procollagen-{alpha}I mRNA (data not shown). Similar values were found in the literature regarding collagen-{alpha}I mRNA modulation by TGF-beta1 and IFN-{gamma} 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 ASMCs. Further work is necessary to better understand the mechanisms that lead to the modulation of IL-17BR in ASMCs by TNF-{alpha} and IFN-{gamma}, respectively. Moreover, it would be of considerable interest to assess whether IL-17E and IL-17BR are overexpressed in asthma, an area of ongoing research in our laboratory.


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This study was funded by the Canadian Institutes of Health Research.


   ACKNOWLEDGMENTS
 
We thank Andrea K. Mogas for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Q. Hamid, Meakins-Christie Laboratories, McGill Univ., 3626 St-Urbain St., Montreal, Canada H2X 2P2 (e-mail: qutayba.hamid{at}mcgill.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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