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Leukotriene C₄ Induces TGF-₁ Production in Airway Epithelium via p38 Kinase Pathway

Department of Chest Medicine, and Division of Chest Surgery, Taipei Veterans General Hospital; School of Medicine, and School of Medical Technology and Engineering, National Yang-Ming University, Taipei; and Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County, Taiwan

Correspondence and requests for reprints should be addressed to Diahn-Warng Perng, M.D., Ph.D., Department of Chest Medicine, Taipei Veterans General Hospital, 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan. E-mail: dwperng{at}vghtpe.gov.tw

	Abstract

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Cysteinyl leukotrienes (CysLTs) play an important role in the pathogenesis of airway remodeling. We investigated the interaction between epithelium and CysLTC₄, and the contribution of this interaction to airway fibrosis. Human airway epithelial cells were grown on air–liquid interface culture inserts. CysLTC₄ was employed to stimulate the cells. Conditioned medium following CysLTC₄ stimulation was coincubated with human lung fibroblasts. Our results have demonstrated that CysLTC₄ stimulates airway epithelial cells, through a p38 mitogen-activated protein kinase (MAPK) activation mechanism, to produce transforming growth factor ₁ (TGF-₁), which results in fibroblast proliferation. The selective p38 MAPK inhibitor S203580 successfully inhibits p38 MAPK phosphorylation and subsequent TGF-₁ production. CysLT₁ receptor antagonist montelukast and corticosteroid inhibit TGF-₁ production at the mRNA and protein levels. When treated with LTC₄, the conditioned medium from epithelial cells enhances fibroblast proliferation, this mitogenic effect being attributed to TGF-₁ and LTC₄ remaining in the culture medium. In addition, LTC₄ itself acts as a potential growth factor for lung fibroblasts. These data indicate that interactions between LTC₄ and airway epithelial cells may contribute to the pathogenesis of airway remodeling. Early intervention to stop these processes may be useful in preventing airway fibrosis in chronic allergic inflammation.

Key Words: airway epithelial cell • fibroblast • leukotriene C₄ • MAP kinase • TGF-₁

	Introduction

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Cysteinyl leukotrienes (CysLTs) are produced mainly by eosinophils and mast cells, both of which contribute greatly to the pathogenesis of asthma (1, 2). CysLTs play an important role in the airway remodeling seen in persistent asthma, which includes an increase of airway goblet cells, mucus, blood vessels, smooth muscle, myofibroblasts, and airway fibrosis (3). The role of CysLTs in airway remodeling has been studied in a mouse model of asthma characterized by smooth muscle cell hyperplasia and collagen deposition beneath the airway epithelial cell layers and lung interstitium (4). Leukotriene C₄ (LTC₄) can induce human skin fibroblast proliferation (5) as well as rat lung fibroblast proliferation and collagen synthesis (6). The airway epithelium is likely to be exposed to high levels of LTC₄ in chronic allergic airway inflammation (7–9), but the responses of the airway epithelium following this exposure remain unclear.

Studies performed in trachea and lung explants have demonstrated that airway epithelial damage may induce a fibrotic response (10, 11). Increasing evidence supports the theory that airway epithelial cells are the primary source of cytokines and growth factors release, which promotes fibroblast migration, proliferation, and myofibroblast differentiation (12). Through in situ hybridization and immunocytochemistry, airway epithelial cells have been shown to be the main site of synthesis of platelet-derived growth factor, transforming growth factor- (TGF-), and tumor necrosis factor-, all of which play a central role in the development of pulmonary fibrosis (13–17). TGF-₁ is a critical mediator of lung fibrosis in animal models; it is a strong extracellular matrix inducer and is chemotactic for fibroblasts and polymorphonuclear neutrophils (18, 19). Targeted overexpression of TGF-₁ leads to progressive fibrosis (20).

This study attempts to investigate the interaction between the airway epithelium and LTC₄, and the contribution of this interaction to airway remodeling. Human airway epithelial cells were grown on modified air–liquid interface culture inserts, and LTC₄ was then employed to stimulate the epithelial cells. The conditioned medium following LTC₄ stimulation was coincubated with human lung fibroblasts. We report that LTC₄ activates MAP kinase p38 and upregulates TGF-₁ gene expression, which subsequently induces TGF-₁ production by epithelial cells. The conditioned medium enhances fibroblast proliferation and the mitogenesis is then to a great extent blocked by anti–TGF-₁ antibodies. LTC₄ itself can also induce fibroblast proliferation. These results demonstrate that interactions between LTC₄ and airway epithelial cells may contribute greatly to the pathogenesis of airway remodeling.

	MATERIALS AND METHODS

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Modified Air–Liquid Interface Culture for Human Airway Epithelial Cells
Preparation of the modified air–liquid interface culture for human airway epithelial cells (HAECs) has been described in detail previously (21). Human bronchus, obtained from surgical lobectomy for lung cancer, was rinsed several times with Leibovitz's L-15 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). The tissue was cut into 1- to 2-mm² pieces, and three to four pieces of tissue were planted with the epithelium side facing down onto 6-well culture inserts (Becton Dickinson Labware, Franklin Lakes, NJ) with a membrane growth area of 4.2 cm² (pore size 0.4 µm) and coated with type IV collagen (50 µg/cm²). Two milliliters of medium containing antibiotics/antimycotic, human epidermoid growth factor (1 ng/ml), insulin (2.5 µg/ml), transferrin (2.5 µg/ml), hydrocortisone (1 µg/ml), glutamine (2 mM), and 0.1% fetal bovine serum (FBS) in RPMI 1640 and Medium 199 (vol/vol 1:1; Gibco BRL Life Technologies, Gaithersburg, MD) were added to the basal chamber, and 100 µl were added to the insert. The culture medium in the basal chamber was changed every 48–72 h, while no medium was added to the insert. The airway epithelial cells were grown on a porous membrane, on which they formed a continuous epithelial sheet with the basal aspect exposed to the medium and the apical surface exposed to air. Cells grown on the inserts were confluent after 7–10 d of incubation, and the tissue fragments were then transferred to fresh inserts to obtain new growth of epithelial cells. Cells were then dissociated using 0.02% trypsin–EDTA solution and seeded in 24-well culture inserts (growth area of membrane 0.3 cm², pore size 0.4 µm) coated with collagen, to determine the extent of TGF-₁ release after stimulation by various concentrations of LTC₄.

Human Lung Fibroblast Culture
Lung parenchyma, obtained from surgical lobectomy for lung cancer, was rinsed several times with Leibovitz's L-15 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (0.25 µg/ml). The tissue was cut into 1- to 2-mm² pieces, and three to four pieces of tissue were planted onto 6-well culture plates. The culture medium contained antibiotics/antimycotics, glutamine (2 mM), and 10% FBS in DMEM. Cells were detached from the plates by trypsinization and seeded onto 24-well culture plates for the proliferation studies.

LTC₄ Stimulation and TGF-₁ Release
Cells (100 µl) were seeded in 24-well culture inserts at a density of 1 x 10⁵ cells/ml and grown in culture medium (500 µl per basal chamber). At confluence, LTC₄ (Sigma, St. Louis, MO) was added to the apical compartment at varying concentrations. To suppress the effect of mediator release induced by LTC₄, cells were pretreated with dexamethasone (Sigma) and montelukast (leukotriene receptor₁ antagonist; Merck and Co., Inc., Rahway, NJ) for 2 h before changing to fresh medium and adding LTC₄ to the apical compartment. Supernatants were collected at regular intervals and stored at –80°C until assayed for mediators. Levels of TGF-₁ were assayed by ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).

Semiquantative Reverse Transcription–Polymerase Chain Reaction for TGF-₁ mRNA Expression
After removal of supernatants for mediator detection, total cellular RNA was isolated from cell monolayers using a High Pure RNA Isolation Kit (Roche Molecular Biochemicals, Mannheim, Germany). The RNA (1 µg) was reverse transcribed into cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI). An aliquot of cDNA was then subjected to 28 cycles of PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using a standard procedure: denaturing at 94°C for 2 min, hybridizing at 55°C for 30 s, and elongating at 72°C for 1.5 min. Another aliquot of cDNA was subjected to 35 cycles of PCR for TGF-₁ using a standard procedure: denaturing at 95°C for 2 min, annealing at 56°C for 40 s, and elongating at 72°C for 5 min. The TGF-₁–specific primer pair (Gibco BRL Life Technologies) amplified a 240-bp PCR product, composed of 5' primer GGGACTAT CCACCTGCAAGA and 3' primer CCTCCTTGGCGT AGTAGTCG. The constitutively expressed gene, GAPDH, was used as an internal control. The primers for GAPDH were 5' primer ATCAAGAA GGTGGTG AAGCAGG and 3' primer GCAACTGTGAGGAGG GGA GATT, generating a 385-bp PCR product. The respective amplified products were electrophoresed in a 2% agarose gel containing ethidium bromide (0.5 µg/ml) and viewed under an ultraviolet illuminator. The image was photographed, stored, and analyzed by a photodocumentation system using Photo-Capt software (ETS Vilber-Lourmat Inc., Marne LuVallee Cedex, France). Each band was quantified by calculating the ratio of target cDNA signal to the GAPDH control, and the mRNA expression was presented as a percentage of the GAPDH signal.

Real-Time Quantitative Polymerase Chain Reaction
The cDNAs were amplified and detected using an Applied Biosystems Prism 7,000 Sequence Detection System (Foster City, CA). cDNA (1 µl) was added to a reaction mixture (12.5 µl and 1.25 µl of Assays-on Demand Gene Expression Assay mixture containing TGF-₁ primers and double fluorescently labeled probes; Applied Biosystems), in a final volume of 25 µl. The conditions for thermal cycling were 50°C for 2 min, 10 min at 95°C, and then 40 cycles of 15 s at 95°C, and 1 min at 60°C. The cycle threshold value used to assess the quantity of target gene expression was determined by how much the fluorescence exceeded a preset limit. The amount of TGF-₁ RNA message in each sample was calculated on the basis of the relative standard curve generated with the RNA from epithelial cells. The data have been normalized to the expression of GAPDH.

Western Blot Analysis of MAP Kinases
Primary epithelial cells were exposed to LTC₄ in the presence or absence of inhibitors of MAP kinase activity. Pre-incubation of SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole), a selective p38 MAP kinase inhibitor (Cell Signaling Technology, Beverly, MA), with cells stimulated by LTC₄ was employed to prevent p38 MAP kinase activity. At the end of treatment, cells were lysed on ice in lysis buffer containing 50 mM Tris · HCl at pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, pepstatin A (1 µg/ml), aprotinin (0.2 U/ml), leupeptin (0.5 µg/ml), and 1 mM Na₃VO₄. The protein concentration was determined using a bicinchoninic acid protein assay (Pierce Chemicals, Rockford, IL) with bovine serum albumin as the standard. Equal amounts of total cell lysates (15 µg) were solubilized in a sample buffer by boiling for 10 min, fractionated on a 7.5% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The membrane was washed with 0.1% Tween 20 supplemented with Tris-buffered saline (TBS) and incubated in a blocking buffer (TBS containing 5% nonfat dry milk and 0.1% Tween 20). Anti–phospho-p46/54 (SAPK/JNK, Thr¹⁸³/Tyr¹⁸⁵), anti–phospho-p44/42 (Thr²⁰²/Tyr²⁰⁴) antibody, or anti–phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²) antibody (Cell Signaling Technology) in a 1:1,000 dilution was then applied at 4°C overnight, with gentle shaking. After washing three times with TBS, blots were incubated with a 1:2,000 dilution of a horseradish peroxidase–conjugated secondary antibody (Cell Signaling Technology) for 1 h. The protein bands were viewed using enhanced chemiluminescence (Amersham Pharmacia Biotech, Sunnyvale, CA) and autoradiography with Kodak X-ray film.

p38 MAP Kinase Activity Assay
The activity of p38 MAPK was measured using a p38 MAPK assay kit (Cell Signaling Technology) according to the manufacturer's instructions. Cell lysates (200 µg) were incubated overnight with 20 µl of immobilized phospho-p38 MAPK (Thr¹⁸⁰/Tyr¹⁸²) monoclonal antibody. Phospho-p38 MAPK was then immunoprecipitated from the lysate and used in a kinase reaction with ATP (100 µM) and ATF-2 fusion protein (2 µg). The products of the kinase reaction were separated on an SDS polyacrylamide gel (10%) and immunoblotted with a phospho–ATF-2 antibody (Thr71). Immunoreactive bands were detected as described above.

Fibroblast Proliferation
Fibroblasts (6 x 10⁴ cells/100 µl) were seeded to 24-well culture plates. Conditioned medium from the airway epithelial cell culture-supernatant with various treatments (24 h after stimulation) was added to the lung fibroblasts to determine the growth stimulatory activity. Cell viability was determined by light microscopy and dye exclusion with trypan blue. Cell numbers were measured by direct counting of the cells using a hemocytometer.

Statistics
Data were expressed as means ± SE. Statistical analysis for multiple comparisons was performed by ANOVA. Student's t test (for cytokine assay data) or the paired Student's t test (for the mRNA expression data) was employed. A difference of P < 0.05 was considered significant.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Effect of LTC₄ on TGF-₁ Release
The effect of LTC₄ on the generation of TGF-₁ from HAECs is shown in Figure 1. The concentrations of TGF-₁ from epithelial cells were not detectable when LTC₄ was lower than 10 ng/ml. The maximal concentration of TGF-₁ (80.6 ± 4.7 pg/ml) was detected in the presence of LTC₄ (100 ng/ml) at 24 h of incubation. The levels of TGF-₁ were undetectable at 3, 6, and 12 h of incubation. To suppress the LTC₄-induced TGF-₁ release, epithelial cells were treated with increasing concentrations of either dexamethasone (1–1,000 µM) or montelukast (1–1,000 nM). Dexamethasone or montelukast alone had no effect on the TGF-₁ release of unstimulated cells. Incubation of dexamethasone (100 µM) or montelukast (100 nM) with cells maximally stimulated with LTC₄ (100 ng/ml) significantly inhibited their production of TGF-₁(Figure 1).

View larger version (10K): [in this window] [in a new window]   Figure 1. Effect of LTC4 on TGF-1 generation by human airway epithelial cells. TGF-1 level is determined in supernatants of epithelial cells incubated for 24 h with buffer alone, varying concentrations of LTC4, as well as LTC4 (100 ng/ml) with montelukast (Mon, 100 nM) or dexamethasone (Dexa, 100 µM). Mean TGF-1 values (± SEM) are shown for four to six experiments performed in duplicate.

TGF-₁ mRNA Expression

View larger version (32K): [in this window] [in a new window]   Figure 2. TGF-1 mRNA expression after LTC4 stimulation. (A) RT-PCR analysis of expression of TGF-1 mRNA and the housekeeping gene GAPDH after incubation of epithelial cells for 3 h with varying concentrations of LTC4. (B) The effect of dexamethasone (100 µM) and montelukast (Mon, 100 nM) on the induction of TGF-1 mRNA. (C) Relative TGF-1 mRNA expression normalized to internal control GAPDH. Data are shown for three experiments performed in duplicate and expressed as percent of those of cells without LTC4 stimulation. *P < 0.05 compared with cells treated with buffer alone or with LTC4 plus dexamethasone or montelukast.

View larger version (69K): [in this window] [in a new window]   Figure 3. Effect of LTC4 on MAP kinase activation. Cells were treated with LTC4 (100 ng) for the times indicated and harvested for Western blotting. Immunodetection was performed using specific antibodies to phosphorylated p38, p44/42, and p46/54 (p38-p, p44/42-p and p46/54-p), or pan-p38 (p38), pan-p44/42 (p44/42), and pan-p46/54 (p46/54, SAPK/JNK). Blots presented are representative of three separate experiments.

View larger version (45K): [in this window] [in a new window]   Figure 4. Effect of SB203580 on LTC4-induced p38 MAP kinase activation and TGF-1 production. Cells were treated with LTC4 (100 ng/ml) in the presence or absence of SB203580 (10 µM and 20 µM, respectively) for 1 h, then harvested for Western blotting. Representative immunoblots for phosphorylated p38 and ATF-2 are shown (A). Supernatant for TGF-1 measurement was collected after 24 h of stimulation (B). Data are presented for four separate experiments.

View larger version (11K): [in this window] [in a new window]   Figure 5. Mitogenic effect on fibroblasts. Fibroblasts were cultured with conditioned medium from epithelial cells stimulated by buffer (CM-buffer), LTC4 1–1,000 ng/ml (CM-LTC4 1, 10, 100, and 1,000), LTC4 100 ng/ml after pretreatment with dexamethasone 100 µM (CM-LTC4+Dexa), montelukast 100 nM (CM-LTC4+Mon), or CM-buffer with additional LTC4 100 ng/ml. Data are shown for four separate experiments.

View larger version (13K): [in this window] [in a new window]   Figure 6. The effect of suppression of TGF-1 on fibroblast proliferation. Fibroblasts were incubated with conditioned medium from epithelial cells stimulated by buffer (CM buffer), LTC4 100 ng/ml (CM LTC4), or incubated with CM buffer with additional recombinant TGF-1 at a concentration of 1 to 1,000 pg/ml (CM buffer+TGF). Anti–TGF-1 antibodies (0.3 µg/ml), mouse IgG (0.3 µg/ml), or montelukast (100 nM) were added to conditioned medium (LTC4) to suppress the mitogenic effects of TGF-1 or LTC4. Data are shown for four separate experiments.

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

CysLT₁ receptor antagonist montelukast has been reported to significantly reduce the airway eosinophil infiltration, mucus plugging, smooth muscle hyperplasia, and subepithelial fibrosis in an animal model, suggesting that CysLTs may be important in the pathogenesis of airway remodeling. Instillation of TGF-₁ in mouse trachea has caused subepithelial collagen deposition in the airways. Increased TGF-₁ expression in bronchial mucosa is seen in individuals with asthma, and the levels correlate with the depth of basement membrane thickness and fibroblast numbers (22). In addition, the level of TGF-₁ in the bronchoalveolar lavage fluid is significantly increased 24 h after allergen challenge in atopic asthma (46.0 versus 21.5 pg/ml) (23). The above evidence and our results provide greater insight into the pathogenesis of airway remodeling in chronic allergic inflammation. In our preliminary results, TGF-₁ was detected only in the supernatant collected from the apical compartment after 3 h and 6 h of stimulation. After 24 h of stimulation, TGF-₁ could be detected in the both apical and basal compartments. TGF-₁ (25-kD protein) may diffuse into the basal compartment through the cell junction. This may explain why epithelium-secreted TGF-₁ can induce fibroblast proliferation. Being exposed to allergens, activated mast cells and eosinophils release CysLTs in the airways, which stimulate epithelial cells to produce TGF-₁. This fibrogenic mediator subsequently stimulates fibroblast proliferation and collagen deposition in the basement membrane. In the meantime, CysLTs are able to induce similar activities, which are related to airway fibrosis.

MAP kinases play important roles in signal transduction through a cascade of protein phosphorylation, which induces a variety of cellular responses such as apoptosis, proliferation, and cytokine production. The p38 kinase is activated in a variety of cell types in response to growth factors, lipopolysaccharides, and proinflammatory cytokines. Extracellular regulated kinase (ERK-1, p44) and ERK-2 (p42) are considered to be involved mostly in cell growth, differentiation, and development (24). Results from our study show that LTC₄ stimulates epithelial cells through the activation of MAP kinase p38, but not p44/42 or SAPK/JNK, to produce TGF-₁. So far, little is known about the intracellular signal transduction of CysLTs receptors, which have been recognized as G-protein–coupled receptors (25). In human monocytic leukemia cells, LTD₄ has been shown to increase intracellular calcium concentration and activated MAP kinase (26). However, the precise signaling cascade starting from the activation of CysLTs receptors, and leading to MAP kinase phosphorylation, gene transcription, and protein translation still needs further investigation.

In addition to increasing collagen deposition, TGF-₁ was also found to be linked to airway hyperresponsiveness (27). Dexamethasone successfully inhibited LTC₄-induced TGF-₁ production at the gene transcription and protein synthesis levels. A question arising from this finding is whether the long-term use of corticosteroids can affect airway remodeling in individuals with asthma. Sont and colleagues, using airway hyperresponsiveness in addition to lung function and symptoms as guides to long-term treatment, reported that inhaled corticosteroid can significantly reduce the thickness of subepithelial fibrosis in a 2-yr period (28). Our results may provide a reasonable scientific explanation for this clinical finding.

It has been reported that levels of LTB₄ and LTC₄ are greatly elevated in lung tissue obtained from patients with newly diagnosed idiopathic pulmonary fibrosis, as compared with levels measured in regions of nonfibrotic lung tissue obtained from control subjects (29). CysLT₁ receptor antagonist, montelukast, is able to reverse airway remodeling by ovalbumin sensitization/challenge (4). Further studies are required to prove whether the anti-fibrogenesis effect of CysLT₁ receptor blockade may be beneficial in terms of prevention and management of airway remodeling in chronic persistent asthma.

In conclusion, interaction of LTC₄ and epithelial cells may significantly contribute to the pathogenesis of airway remodeling. Early intervention to stop these processes may be useful in preventing airway fibrosis in chronic allergic inflammation.

	Footnotes

 
^* These authors have made equal contributions to this study.

This study was supported in part by National Science Council Research Grant NSC91-2314-B075-063 and Taipei Veterans General Hospital Grant VGH93-185.

Originally Published in Press as DOI: 10.1165/rcmb.2005-0068OC on September 22, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 15, 2005

Accepted in final form August 29, 2005

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