Page Articles 1 of 34 in PresS. Am J Physiol Lung Cell Mol Physiol (May 18, 2007). doi:10.1152/ajplung.00045.2007 1
Constitutive and Inducible Thymic Stromal Lymphopoietin Expression in Human Airway Smooth Muscle cells: Role in COPD Keqin Zhang1, LianyuShan1, Muhammad Sahidu Rahman1, Helmut Unruh2, Andrew Halayko3, Abdelilah Soussi Gounni*1,
1
Department of Immunology, 2Section of Thoracic Surgery, 3Department of Physiology,
University of Manitoba, Winnipeg, Manitoba, Canada
Running head: Human Airway Smooth Muscle cells express TSLP
* Address for correspondence: Dr. Abdelilah Soussi Gounni Department of Immunology, Faculty of Medicine University of Manitoba, Rm 606 Basic Medical Sciences Building 730 William Avenue, Winnipeg, MB Canada, R3E 0W3 Phone: 1-204-975-7750 Fax: 1-204-789-3921 Email:
[email protected]
Footnote: ASG is supported by a CIHR New Investigator Award. This work was supported by a grant from the Canadian Institutes of Health Research (CIHR) to ASG.
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Copyright © 2007 by the American Physiological Society.
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Abstract 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 pro-inflammatory cytokines (TNF- and IL-1 ) 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 , TNFresulted in a significant increase of TSLP mRNA and protein release from HASMC. Furthermore, combination of IL-1 and TNF- has an additive effect on TSLP release by HASMC. Primary HASMC pretreated with inhibitors of p38 or p42/p44 ERK MAPK, but not PI3K, showed a significant decrease in TSLP release upon IL-1 and TNF
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-1 and TNF- -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.
Key words: TSLP, signaling, airway smooth muscle cells, inflammation, COPD
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Abbreviations:
HASMC: human airway smooth muscle cells TSLP:
thymic stromal lymphopoeitin
TNF: tumor necrosis factor TSLPR: thymic stromal lymphopoeitin receptor TLR: toll like receptor DC: dendritic cells
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Introduction 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). Pro-inflammatory cytokines may be the driving force behind the disease process (47).
Recently interest has arisen because of the association of 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 TLSP receptor (TLSPR) is heterodimeric, consisting of the IL-7R– chain and a common receptor–like chain (TSLPR- ) (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 (Th2) 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 Th2-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.
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As a critical airway cell, 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-1 , TNF
or the combination up-regulate the expression of TSLP in
HASMC. p38 and p42/p44 ERK MAPK, but not PI3K, are essential for IL-1 TNF
and
-mediated release of TSLP by HASMC.
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Materials and Methods Reagents Recombinant human TNF-
and IL-1 , sheep polyclonal anti-human TSLP Ab,
biotinylated sheep polyclonal anti-human TSLP and recombinant human TSLP were purchased from R&D Systems (Minneapolis, MN). Sheep IgG control were from Sigma-Aldrich (Oakville, Ontario, Canada). Donkey anti-Sheep IgG F(ab')2 AlexaFluor 488, ProLong® anti-fade were obtained from Molecular Probes (Eugene, Oregon). Donkey serum and normal human serum were from Cedarlane (Toronto, Ontario). FBS was from HyClone Laboratories (Logan, UT). DMEM, Ham’s F12, trypsin-EDTA, antibiotics (penicillin, streptomycin), dNTP, SuperScript reverse transcriptase, and Taq polymerase were from Invitrogen Life Technologies (Grand Island, NY). The p38 MAPK inhibitor, SB203580 (4-[4-fluorophenyl]-5-[4-pyridyl] 1Himidazole), the p42/p44ERK inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenyl-thio]butadiene), and the PI3K inhibitor (wortmannin), were purchased from Calbiochem (Mississauga, Ontario). 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, Canada. Briefly, the muscle layer from each bronchial segment was dissected free from adventitia and submucosa under a binocular dissection microscope, then was minced, and cells were dissociated enzymatically (600 U/ml collagenase I, 10 U/ml elastase, 2 U/ml 6
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Nagarse protease) for up to 60 min. Cells were seeded at a density of 8000 cells/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 was replaced every 2 days, and confluent cultures were passaged and reseeded using a split ratio of 1:4. When necessary, cells were stored at liquid nitrogen in 10% DMSO/90% FBS. 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 to 5.
Cell stimulation
Confluent HASMC (passage 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- , IL-1 , TNF- plus IL-1 , or medium alone. In some experiments, cells were pretreated for 1 h with U0126 (10µM), SB203580 (10µM) or Wortmannin (100 nM) before stimulation for 24 and 48 h with
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TNF- (10ng/ml), IL-1 (10 ng/ml), or both at 10 ng/ml. Supernatants were collected at 24 and 48 h, centrifuged at 1200 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 manufactory (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 7.8pg/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 48h in serum-free medium as described above. Cells were then stimulated in fresh FBS-free medium containing human recombinant IL-1
(10 ng/ml), TNF
(10 ng/ml), or vehicle (medium alone) for 2 h, 6 h
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 1x buffer, 200 µmol of each dNTPs, 20 pmol of each 8
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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
5'-TATGAGTGGGACCAAAAGTACCG-3'; 5'-GGGATTGAAGGTTAGGCTCTGG-3'.
follows: and
Primers
forward reverse
for
primer, primer,
housekeeping
gene
glyceraldhyde-3-phosphate dehydrogenase (GAPDH) are as follows: 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, Germany). Each cycle including denaturation (94°C, 1 min), annealing (TSLP: 62°C, 1 min, GAPDH: 55°C, 1min), 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 137bp, 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-1 stimulated PBMC. PCR products were isolated from 2 % w/v agarose gel using QIAEX II Agarose Gel 9
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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: 1x LightCycler-DNA master SYBRGreen I (Roche), 25 mM MgCl2, 0.5 µM of 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, 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, respectively. Product specificity was determined by melting curve analysis, and by visualization of PCR products on agarose gels. Calculation of the relative amount of each cDNA species was performed according to standard protocols. Briefly, the amplification of 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 8 well glass slides (Naig Nunc, Napierville, IL) were cultured up to semi confluence. Slides were fixed with 4% paraformaldehyde, air-dried, and stored at −20ዊC until use. Briefly, after treatment with universal blocking solution for 30 min (DakoCytomation, Carpenteria, CA), slides were incubated with purified sheep anti-human TSLP Ab or matched control Ig at a final dilution of 10µg/ml overnight at 4°C, washed twice with Tris-Buffered Saline (TBS) followed by incubation for 2 h at room temperature with donkey anti-sheep IgG F(ab )2 AlexaFluor® 488 (1: 100 dilution). Slides 10
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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 AX70 microscope with a Photometrics PXL cooled CCD Camera and Image-Pro® Plus Software (Carsen Group Inc, Ontario)
Immunohistochemistry
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 the American Thoracic Society Criteria and five normal controls were used for this study (Table I). Deparaffinized sections were rehydrated in a series of graded concentrations of alcohol to water, then incubated sections for 30 minutes in 0.05% saponin in distilled H2O at room temperature. Washed sections with TBS, then incubated sections 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
Statistical analysis Data were obtained from experiments performed in triplicate and repeated at least three times, and results are expressed as mean ± SD. Statistical significance was determined using Mann-Whitney U test, and p values
