Thymic stromal lymphopoietin (TSLP) is a novel cytokine that triggers dendritic cell-mediated T helper (Th)-2 inflammatory responses. Previous studies have demonstrated that human airway smooth muscle cells (HASMC) play a critical role in initiating or perpetuating airway inflammation by producing chemokines and cytokines. In this study, we first evaluated the expression of TSLP in primary HASMC and investigated how proinflammatory cytokines (TNF-α and IL-1β) and Th-2 cytokines (IL-4, IL-9) regulate TSLP production from HASMC. TSLP mRNA and protein were assessed by real-time RT-PCR, ELISA, and immunofluorescence from primary HASMC cultures. Primary HASMC express constitutive level of TSLP. Incubation of HASMC with IL-1 or TNF-α resulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1β and TNF-α has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase, showed a significant decrease in TSLP release on IL-1β and TNF-α treatment. Furthermore, TSLP immunoreactivity was present in ASM bundle from chronic obstructive pulmonary disease (COPD) and to lesser degree in normal subjects. Taken together, our data provide the first evidence of IL-1β- and TNF-α-induced TSLP expression in HASMC via (p38, p42/p44) MAPK signaling pathways. Our results raise the possibility that HASMC may play a role in COPD airway inflammation via TSLP-dependent pathway.
- mitogen-activated kinase
chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide (12). COPD is characterized by chronic inflammation in the airway lumen along with increased numbers of neutrophils, macrophages, CD8+ T cells, or mast cells in the airway walls and alveolar compartments (19). This complex disease state consists of emphysema, small airway disease, and chronic bronchitis with air flow obstruction (52). Proinflammatory cytokines may be the driving force behind the disease process (47).
Recently, interest has arisen because of the association of thymic stromal lymphopoietin (TSLP) with airway diseases (27, 58). TSLP is a novel IL-7-like cytokine, originally cloned from a murine thymic stromal cell line (13). In humans, TSLP gene is located on chromosome 5q22.1 next to the atopic cytokine cluster on 5q31 (39). The TSLP receptor (TSLPR) is a heterodimeric complex, consisting of the IL-7R-α chain and a common γ-like receptor chain (TSLPR-γ; Refs. 34, 36). More recently, experimental animal models show that TSLP expression was increased in the lungs of mice with antigen-induced asthma, whereas TSLPR-deficient mice had considerably attenuated disease (2). Lung-specific expression of a TSLP transgene induced airway inflammation and hyperreactivity characterized by T helper type 2 (Th-2) cytokines (57). A more recent study showed by in situ hybridization that TSLP expression was increased in asthmatic airways and correlated with both the expression of Th-2-attracting chemokines and with disease severity (56). These findings suggest that TSLP is an important factor necessary and sufficient for the initiation of airway inflammation.
As a critical airway cell, human airway smooth muscle cells (HASMC) are involved in the pathogenesis of airway diseases because these cells contribute to airway hyperresponsiveness and airway obstruction. In addition to their proliferative and contractile properties, studies have shown that cultured HASMC may express chemokines and cytokines, thereby acting as effector cells in initiating or perpetuating airway inflammation (16–18, 26, 40, 41, 49). With this background, we hypothesize that HASMC express TSLP and play a critical role in the pathogenesis of airway diseases. In this report, we first show that HASMC express TSLP in vitro and in vivo; IL-1β, TNF-α, or the combination upregulate the expression of TSLP in HASMC. p38 and p42/p44 ERK MAPK, but not phosphatidylinositol 3-kinase (PI3K), are essential for IL-1β- and TNF-α-mediated release of TSLP by HASMC.
MATERIALS AND METHODS
Recombinant human TNF-α and IL-1β, sheep polyclonal anti-human TSLP antibody (Ab), biotinylated sheep polyclonal anti-human TSLP, and recombinant human TSLP were purchased from R&D Systems. Sheep IgG control were from Sigma-Aldrich (Oakville, Ontario, Canada). Donkey anti-sheep IgG F(ab′)2 Alexa Fluor 488 and ProLong anti-fade were obtained from Molecular Probes (Eugene, OR). Donkey serum and normal human serum were from Cedarlane (Toronto, Ontario, Canada). FBS was from HyClone (Logan, UT). DMEM, Ham's F-12, trypsin-EDTA, antibiotics (penicillin, streptomycin), dNTP, SuperScript reverse transcriptase, and Taq polymerase were from Invitrogen Life Technologies (Grand Island, NY). The p38 MAPK inhibitor, SB-203580 [4-(4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5-(4′-pyridyl)-1H-imidazole], the p42/p44 ERK inhibitor, U-0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene], and the PI3K inhibitor, wortmannin, were purchased from Calbiochem (Mississauga, Ontario, Canada). Unless stated otherwise, all other reagents were obtained from Sigma-Aldrich.
Preparation of bronchial HASMC.
Bronchial HASMC were obtained from macroscopically healthy segments of the main bronchus after lung resection from surgical patients in accordance with procedures approved by the Human Research Ethics Board of the University of Manitoba, Winnipeg, Manitoba, Canada. Briefly, the muscle layer from each bronchial segment was dissected free from adventitia and submucosa under a binocular dissection microscope and then minced, and cells were dissociated enzymatically (600 U/ml collagenase I, 10 U/ml elastase, 2 U/ml Nagarse protease) for up to 60 min. Cells were seeded at a density of 8,000 cells per cm2 and grown at 37°C in DMEM supplemented with 10% FBS, sodium pyruvate (1 mM), l-glutamine (2 mM), nonessential amino acid mixture (1:100), gentamicin A (50 μg/ml), and amphotericin B (1.5 μg/ml). Media were replaced every 2 days, and confluent cultures were passaged and reseeded using a split ratio of 1:4. At confluence, primary HASMC exhibited spindle morphology and a hill-and-valley pattern that is characteristic of smooth muscle in culture. Moreover, using cultures up to passage 5, over 90% of the 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 (31). The growth rate of the HASMC from all lung resection donors was similar to what has been reported previously for HASMC cultures from healthy human transplant donors (31). In all experiments, cells were used at passages 2-5.
Confluent HASMC (passages 2-5) were growth-arrested by FBS deprivation for 48 h in Ham's F-12 medium containing 5 μg/ml human recombinant insulin, 5 μg/ml human transferrin, 5 ng/ml selenium, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). Cells were then stimulated in fresh FBS-free medium containing graded concentration (0.1, 1, 10, and 100 ng/ml) of human TNF-α, IL-1β, TNF-α plus IL-1β, or medium alone. In some experiments, cells were pretreated for 1 h with U-0126 (10 μM), SB-203580 (10 μM), or wortmannin (100 nM) before stimulation for 24 and 48 h with TNF-α (10 ng/ml), IL-1β (10 ng/ml), or both at 10 ng/ml. Supernatants were collected at 24 and 48 h, centrifuged at 1,200 rpm for 7 min at 4°C to remove cellular debris, and stored at −80°C until analysis by ELISA.
ELISA analysis of TSLP protein release in cell supernatants.
Immunoreactive TSLP within the supernatants was quantified using ELISA with matched Abs according to basic laboratory protocol provided by the manufacturer (R&D Systems, Minneapolis, MN). TSLP protein was quantified in reference to serial dilutions of recombinant standards falling within the linear part of the standard curve for each specific TSLP measured. The sensitivity limit of TSLP assay is 7.8 pg/ml. Each data point represents readings from a minimum of four independent assays performed in duplicate.
RNA isolation and RT-PCR.
Confluent HASMC (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-1β (10 ng/ml), TNF-α (10 ng/ml), or vehicle (medium alone) for 2, 6, and 20 h. Cells were harvested, and total cellular RNA was extracted using TRIzol method (Invitrogen Life Technologies, Gaithersburg, MD). The RNA concentration and purity were assessed with optical density measurements (8). Reverse transcription was performed by using 2 μg of total RNA in a first-strand cDNA synthesis reaction with SuperScript reverse transcriptase as recommended by the supplier (Invitrogen Life Technologies). PCR was performed by adding 2 μl of the reverse transcription product into 25 μl of total volume reaction containing 1× buffer, 200 μmol of each dNTP, 20 pmol of each oligonucleotide primer, and 0.2 unit of AmpliTaq polymerase. Oligonucleotide primers were synthesized on the basis of the entire coding region of the human TSLP (GenBank accession no. NM_033035) as follows: forward primer, 5′-TATGAGTGGGACCAAAAGTACCG-3′; and reverse primer, 5′-GGGATTGAAGGTTAGGCTCTGG-3′. Primers for housekeeping gene glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are forward primer 5′-AGCAATGCCTCCTGCACCACCAAC-3′ and reverse primer 5′-CCGGAGGGGCCATCCACAGTCT-3′. The PCR (TSLP, 35 cycles; GAPDH, 25 cycles) was conducted in a thermal cycler (Mastercycler, Eppendorf). Each cycle included denaturation (94°C, 1 min), annealing (TSLP, 62°C, 1 min; GAPDH, 55°C, 1 min), and extension (72°C, 1 min 30 s). The initial denaturation period was 5 min, and the final extension was 10 min. The size of the amplified TSLP and GAPDH fragment is 97 bp and 137 bp, respectively. GAPDH was amplified as internal control. Amplified products were analyzed by DNA gel electrophoresis in 2% agarose and visualized by ethidium bromide staining under ultraviolet illumination. The specificity of the amplified band was confirmed by sequencing (data not shown). The TSLP level was quantified by scanning densitometry and corrected for GAPDH in the same sample.
Real-time RT-PCR analysis.
Total cellular RNA extraction and reverse transcription was performed as described above. TSLP standards were prepared using PCR-amplified cDNA from IL-1β-stimulated peripheral blood mononuclear cells. PCR products were isolated from 2% wt/vol 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 real-time quantitative PCR, each reaction contained the following: 1× LightCycler DNA Master SYBR Green I (Roche), 25 mM MgCl2, 0.5 μM each primer, 0.07 μM TaqStart Ab (Clontech), and 1 and 0.5 μl (1:20 and 1:40 dilution) of cDNA matrix, in a final volume of 20 μl. 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, and 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 TSLP. 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 TSLP gene in stimulated cells was calculated first as the copy number ratio of TSLP per copy of GAPDH and then expressed as normalized values of fold increase over the value obtained with unstimulated control cells.
Immunofluorescence and confocal laser scanning microscopy.
Serum-fed HASMC grown on eight-well glass slides (Nalge Nunc, Naperville, IL) were cultured up to semiconfluence. Slides were fixed with 4% paraformaldehyde, air-dried, and stored at −20°C until use. Briefly, after treatment with universal blocking solution for 30 min (Dakocytomation, Carpinteria, CA), slides were incubated with purified sheep anti-human TSLP Ab or matched control Ig at a final dilution of 10 μg/ml overnight at 4°C and washed twice with Tris-buffered saline (TBS) followed by incubation for 2 h at room temperature with donkey anti-sheep IgG F(ab′)2 Alexa Fluor 488 (1:100 dilution). Slides were extensively washed with TBS and counterstained with nuclear stain PI for 10 min (Sigma). After washing with TBS, the slides were mounted with ProLong antifade. Samples were photographed on Olympus AX-70 microscope with a Photometrics PXL cooled CCD camera and Image-Pro Plus software (Carsen Group).
Immunohistochemistry was performed using tissue sections prepared from segments of the main bronchus after lung resection from surgical patients in accordance with procedures approved by the Human Research Ethics Board of the University of Manitoba, Winnipeg, Canada. Ten patients defined as COPD patients according to American Thoracic Society criteria and five normal controls were used for this study (Table 1). Deparaffinized sections were rehydrated in a series of graded concentrations of alcohol to water and then incubated for 30 min in 0.05% saponin in distilled H2O at room temperature. Sections were washed and then incubated with blocking solution (10% human normal serum, 10% donkey serum in TBS) for 30 min at room temperature. Sheep anti-human TSLP Ab or control Ig (both at 10 μg/ml) were added, and sections were incubated overnight at 4°C followed by donkey anti-sheep IgG F(ab′)2 Alexa Fluor 488 (1:100 dilution). Slides were then processed as described above.
Data were obtained from experiments performed in triplicate and repeated at least three times, and results are expressed as means ± SD. Statistical significance was determined using Mann-Whitney U test, and P values <0.05 were considered statistically significant.
IL-1β and TNF-α induces TSLP mRNA expression in HASMC.
We first determined whether HASMC express TSLP at the mRNA level on proinflammatory cytokine stimulation. RNA preparation from cytokine-stimulated serum-deprived HASMC was first analyzed by RT-PCR. As shown in Fig. 1A, HASMC depict constitutive TSLP mRNA that is enhanced significantly on IL-1β or TNF-α stimulation at 2, 6, and 20 h. Apart from IL-4, which mediates an increase of TSLP mRNA at 6 h, no significant effect on HASMC TSLP mRNA expression was detected following IL-9, IL-17, or IFN-γ cytokine (Fig. 1A). GAPDH products were of similar intensity between all samples, suggesting equality of the RNA preparations (Fig. 1A). The effect of IL-1β and TNF was then confirmed using quantitative real-time RT-PCR. As shown in Fig. 1B, HASMC treated with IL-1β or TNF-α showed an increase of TSLP mRNA level that reaches a maximum at 6 h for both cytokines. At 6 h, IL-1β and TNF-α induced 2.9 ± 0.2- and 3.9 ± 0.4-fold increase of TSLP mRNA level, respectively, compared with unstimulated cells. It is noteworthy that in contrast to IL-1β, TNF-α induction of TSLP mRNA expression was sustained up to 20 h.
TNF-α and IL-1β induce TSLP protein release from HASMC.
We then investigated whether HASMC released TSLP on IL-1β or TNF-α stimulation. Serum-deprived HASMC were stimulated with graded concentrations of IL-1β, TNF-α (0.1, 1, 10, 100 ng/ml), or medium alone for 24 and 48 h. Stimulation with TNF-α induced the release of TSLP in a dose-dependent manner at 24 and 48 h (Fig. 2A). At both 24- and 48-h time points, a statistically significant increase in TSLP release from HASMC occurred with 0.1, 1, 10, and 100 ng/ml IL-1β or TNF-α (P < 0.05 and P < 0.01, respectively; Fig. 2, A and B). No significant TSLP release could be detected in Th-2 (IL-4, IL-9) or Th-1 (IFN-γ) cytokine-stimulated HASMC (P > 0.05, data not shown).
We next investigated the combined effect of TNF-α and IL-1β stimulation on the induction of TSLP release from HASMC. As shown in Fig. 3, TNF-α or IL-1β alone (10 ng/ml) induced a significant increase in TSLP release compared with unstimulated HASMC (P < 0.05) at 24 h. Coincubation with IL-1β and TNF-α (both at 10 ng/ml) has an additive effect on TSLP release from HASMC (P < 0.05). Taken together, these results suggest that TSLP expression in primary HASMC is induced by IL-1β and TNF-α.
TSLP in cultured primary HASMC and bronchial HASMC from COPD subjects.
To further investigate the protein expression of TSLP by HASMC, immunofluorescence staining was performed with purified sheep anti-human TSLP Ab. A specific staining was clearly observed in confluent serum-fed primary HASMC (Fig. 4A). We then examined the expression of TSLP in bronchial HASMC within biopsies of 10 COPD patients and 5 normal donors. Figure 4C shows representative data of a specific signal in smooth muscle bundle from a COPD subject. TSLP staining was also detected in infiltrated inflammatory cells as well as in epithelial cells (data not shown). However, very weak or no staining was detectable in the smooth muscle area of a normal subject (Fig. 4E). A quantitative analysis of the TSLP staining in ASM area showed a 11.5-fold increase in COPD patients compared with normal donors (gray intensity per μm2 = 78.4 ± 24.1 in COPD vs. 6.8 ± 2.2 in normal subjects; Table 2). Substitution of the first Ab with matched IgG control eliminated the positive signal, demonstrating the specificity of the analysis (Fig. 4, B and D). Taken together, these results demonstrated that HASMC express TSLP both in vitro and in vivo.
IL-1β and TNF-α induce TSLP release from HASMC via MAPK (p38 and p42/p44 ERK) pathways.
To characterize the signaling pathways involved in IL-1β- and TNF-α-mediated TSLP release from HASMC, we performed experiments using SB-203580 and U-0126, specific and potent inhibitors of p38 and p42/p44 ERK MAPK. We also investigated the effects of wortmannin, a PI3K inhibitor. Treatment of HASMC with SB-203580 or U-0126 before stimulation with IL-1β, TNF-α, or both caused a significant inhibition of TSLP at 24 h (Fig. 5). In contrast, inhibition of PI3K with wortmannin had little or no effect on IL-1β-, TNF-α-, or combination-induced TSLP release by HASMC (Fig. 5). These results indicate that p38 and p42/p44 ERK MAPK, but not PI3K, are essential for IL-1β- and TNF-α-mediated release of TSLP by HASMC.
In this study, we investigated whether primary HASMC express TSLP in vitro and in vivo in COPD patients. Our data clearly show that HASMC can express both TSLP mRNA and protein on IL-1β or TNF-α stimulation. Pharmacological inhibitors of MAPK pathways (ERK1/2 and p38 MAPK but not PI3K) significantly suppressed TSLP release induced by TNF-α, IL-β, or the combination. These findings document that TSLP expression by bronchial smooth muscle is mediated at least via MAPK pathways. To ascertain whether TSLP is expressed by HASMC in vivo, we evaluated TSLP expression in bronchial biopsies of COPD and normal subjects. The expression of TSLP was present in ASM bundle from COPD and to lesser extent in normal subjects. This result is the first evidence of TSLP expression by HASMC in COPD and suggests that TSLP may play a role in the pathogenesis of airway inflammation.
TSLP, a four helix-bundle cytokine, was originally cloned from a mouse thymic stromal cell line (13). Human TSLP was found to be expressed by epithelial cells in peripheral mucosal-associated lymphoid tissue, where it activates myeloid dendritic cells to induce homeostatic proliferation of naïve and memory CD4+ T cells in the periphery (42, 55). Human TSLP expression was increased in epithelial cells of inflamed tonsils and keratinocytes of atopic dermatitis, and its expression is associated with Langerhans cell migration and activation (50). In our study, we found that in contrast to proinflammatory cytokines TNF-α and IL-1β, neither Th-1 (IFN-γ) nor Th-2 (IL-4, IL-9) cytokines induced TSLP from HASMC, suggesting that TSLP pathway is an upstream event in the cascade leading to airway inflammation. However, TSLP can synergize with IL-1β- and TNF-α-mediated Th-2 cytokine production as demonstrated by Allakhverdi et al. (1) on human mast cells.
COPD is characterized by irreversible airway flow limitation and chronic inflammation of the airways and parenchyma. In COPD patients, increased number of neutrophils and macrophages and their respective proteolytic activity have been associated with disease pathogenesis (21). Besides neutrophils and macrophages, CD8+ and CD4+ T cell and B cell lymphocytes are also increased in COPD, particularly in severe cases (20). Hogg et al. (21) assessed the inflammation in small airways of surgically resected lung tissue from patients with stage 0–4 COPD according to the severity classification of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) (11). They found increased numbers of infiltrating CD4+ and CD8+ lymphocytes and B cells within the small airways. Furthermore, a marked increase in lymphoid follicles in the small airways was observed in patients with GOLD stage 3–4 COPD compared with stage 0–2 (21). More recently, van der Strate et al. (53) showed that lymphoid follicles consisting of B cells and follicular dendritic cells with adjacent T cells were found in both the parenchyma and in bronchial wall of COPD patients with emphysema and in mouse model for cigarette smoke-induced emphysema. Ongoing somatic mutations were observed in 75% of B cell follicles indicating oligoclonal, antigen-specific proliferation (53). Since our present study showed the expression of TSLP within the airway by ASM cells, it is tempting to speculate that local TSLP may affect B cell proliferation and recruitment from the lymph node. In agreement with this hypothesis, TSLP has been shown to induce B and T cell lineage development (27). Furthermore, Astrakhan and coworkers (4), by using doxycycline-inducible, keratin 5-driven transgene-encoding TSLP, have recently demonstrated that local expression of TSLP caused a substantial increase in bone marrow B cell lymphopoiesis, resulting in premature exodus of immature cells to the periphery and an increase in antibody-secreting cells. The authors of this study concluded that even very low expression of TSLP potently promotes altered B cell development (4).
In COPD, although many studies have reported a predominance of Th-1 cytokines, there are a number of studies showing Th-2-biased response in the airways, especially in subjects who fulfill the criteria for diagnosis of chronic bronchitis (29, 35). Furthermore, tissue eosinophilia in bronchial biopsy specimens of nonasthmatic patients with chronic bronchitis have been reported (37, 44, 45). Although the mechanism behind this Th-2-mediated response remains poorly understood, many reports point out to cytotoxic CD8+ T cells in the expression of Th-2 cytokines (23, 54). A recent study by Barczyk et al. (6) has demonstrated an increase of IL-4 expression by CD8+ T cells in the lungs of patients with COPD. Interestingly, there is mounting evidence that CD8+ T cells are also capable of secreting Th-2 cytokines (10, 46) and contribute to eosinophilia. Moreover, human TSLP primes dendritic cells to induce naïve CD8+ T cell activation and differentiation into IL-5-producing cells (14). In light of these studies and our findings, it is tempting to speculate that TSLP expression by HASMC may influence immune regulation by interacting with and influencing local immune cells in COPD patients.
The central role of TNF-α and IL-1β in lung inflammation is not only supported by animal models, but also has been implicated in COPD patients (15). Patients with COPD have elevated numbers of neutrophils in the lungs, increased activation of neutrophils in peripheral blood, and increased TNF-α and IL-1β expression (12). In vivo, IL-1β and TNF-α may affect airway function by inducing cellular infiltrate, mucus hyperplasia, airway wall thickening and fibrosis, and enlargement of distal air spaces (9, 22, 24, 28). In vitro assays have demonstrated IL-1β and TNF-α enhance ASM contractility to acetylcholine and other contractile agonists through an increased mobilization of intracellular calcium (3). In this regard, our data demonstrate that TNF-α or IL-1β induced the release of TSLP from HASMC, an effect that was enhanced when both cytokines were combined, suggesting another pathway by which these proinflammatory cytokines can affect airway structural cells. Our data confirm a recent study by Lee and Ziegler (25) demonstrating an inducible TSLP mRNA expression with TNF-α and IL-1β stimulation of airway epithelial cells. However, in contrast to airway epithelial cells (25), stimulation of HASMC with LPS (Toll-like receptor 4 agonist) and cytosine phosphate guanine (CpG) (Toll-like receptor 9 agonist) (5, 48, 51) did not induce TSLP release (data not shown), suggesting perhaps a different signaling pathway in HASMC compared with epithelial cells.
The MAPK family is fundamental in mediating numerous changes in cell function such as cytokine expression, proliferation, and apoptosis (38, 43). On activation by upstream regulators, the MAPK 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 (43). In this study, pharmacological inhibitors of MAPK (p38 and ERK1/2) abolished IL-1β- and TNF-α-induced TSLP production from HASMC. This study suggests that TNF-α- and IL-1β-mediated TSLP expression involves MAPK pathways. In line with our data, TSLP promoter sequence contains an activator protein-1 binding site that seems to play a role in TSLP expression in human bronchial epithelial cells (25). Our data showed no substantial effect of PI3K inhibitor (wortmannin) on IL-1β- or TNF-α-induced TSLP release, suggesting that PI3K is not involved in TNF-α- or IL-1β-mediating TSLP expression in ASM cells.
In conclusion, our work provides the first evidence that HASMC express TSLP in vitro and in vivo. Proinflammatory cytokines TNF-α and IL-1β induce TSLP expression in HASMC via MAPK (p38 and ERK1/2). Furthermore, our study highlights the potential of TSLP in regulating immune response in COPD.
A. S. Gounni is supported by a Canadian Institutes of Health Research (CIHR) New Investigator Award. This work was supported by a CIHR operating Grant to A. S. Gounni.
We acknowledge Drs. Arash Shoja Saffar and R. Gosens for critical review of the manuscript and Stephane Dragon for help during the preparation of this manuscript.
Present address of K. Zhang: Dept. of Urology, Daping Hospital, Third Military Medical University, Chongqing 400042, P.R. China.
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