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Published ahead of print on February 23, 2006, doi:10.1165/rcmb.2005-0362OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 35, pp. 133-141, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0362OC

Proteinase-Activated Receptor2 Agonists Upregulate Granulocyte Colony-Stimulating Factor, IL-8, and VCAM-1 Expression in Human Bronchial Fibroblasts

Rithwik Ramachandran, Alyn H. Morice and Steven J. Compton

Respiratory Medicine, Division of Academic Medicine, Post Graduate Medical Institute of the University of Hull in association with the Hull York Medical School, East Yorkshire, United Kingdom

Correspondence and requests for reprints should be addressed to Steven J Compton, Respiratory Medicine, Division of Academic Medicine, Post Graduate Medical Institute of the University of Hull in association with the Hull York Medical School, East Yorkshire, HU16 5JQ, United Kingdom. E-mail: S.J.Compton{at}Hull.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Proteinase-activated receptors (PARs) are a novel family of G-protein–coupled receptors. PAR2 has been implicated in inflammatory airways disease. Although fibroblasts are pathologically important in the airways, the proinflammatory role of PAR2 in these cells remains unknown. We assessed PAR expression and functionality in human primary bronchial fibroblasts (HPBFs) before assessing PAR2-mediated HPBF proliferation, cytokine production, and adhesion molecule expression. RT-PCR and flow cytometry demonstrated that HPBFs express hPAR1, hPAR2, and hPAR3, but not hPAR4. Intracellular calcium signaling in HPBFs in response to PAR agonists showed that only hPAR1 and hPAR2 were functional receptors. We used the MTT assay to assess HPBF proliferation. Of the PAR2 agonist proteinases or selective PAR2-activating peptides (PAR2-APs) tested, none stimulated HPBF proliferation, whereas thrombin was a HPBF growth factor. mRNA for IL-8 and granulocyte colony-stimulating factor (G-CSF) was upregulated after addition of SLIGKV-NH2 when assessed by RT-PCR. No significant increase in G-CSF or IL-8 protein was detected. Trypsin stimulated IL-8 and G-CSF release from HPBF in a time- and dose-dependent manner. Leupeptin and soya trypsin inhibitor abrogated trypsin-stimulated cytokine release, indicating a requirement for trypsin's proteolytic activity. Trypsin and SLIGKV-NH2 stimulated an increase in VCAM-1 expression at 12 h after treatment, which declined thereafter. PAR2-driven upregulation of VCAM-1 cell surface expression and the release of IL-8 and G-CSF from bronchial fibroblasts may be important in promoting neutrophilic airways inflammation.

Key Words: adhesion molecules • cytokines • diseases • inflammation • lung


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Proteinase-activated receptors (PARs) are a novel family of G-protein–coupled receptors that have been implicated in a number of pathologic processes, such as inflammation, pain, and tissue repair (1). Four members of this family have been identified: PAR1 (2, 3) and PAR3 (4), which are primarily thrombin-activated receptors; PAR2 (5), a trypsin- and tryptase-activated receptor; and PAR4 (6, 7), which responds to trypsin and thrombin. Activation of PARs typically involves the proteolytic cleavage of an N-terminal sequence, exposing a "tethered ligand," which initiates receptor signaling (1). All PARs (with the exception of PAR3) respond to extraneous treatment with short synthetic PAR-APs, possessing sequences corresponding to that of the proteolytically revealed ligand (1).

PAR2 has been reported to mediate proinflammatory responses in vivo and in vitro (8–12). For example, subplantar injection of the selective PAR2-AP, SLIGRL-NH2, or the more potent peptide trans-cinnamoyl-LIGRLO-NH2 induces significant edema in the rat hind paw (12). A study in PAR2 knockout mice and in mice overexpressing PAR2 has shown that PAR2 activation mediates the infiltration of eosinophils into the airways (13). In addition, PAR2 mediates the activation of inflammatory cells, such as eosinophils and neutrophils (10, 14). Further, in vitro studies have demonstrated that PAR2 activation on airway epithelial cells results in the expression and release of inflammatory cytokines (8, 14). The ability of human lung fibroblasts to release chemotactic cytokines, such as eotaxin and IL-8 (15, 16), has provided recent evidence that these cells may also play an important role in airways inflammation. PAR2 expression has been reported on fibroblasts of various origins (17, 18), and activation of PAR2 has been reported to stimulate lung and human airway fibroblast proliferation (17, 19). Despite evidence that fibroblasts play an important role in airways disease (20), the role of PAR2 in modulating proinflammatory responses in human primary bronchial fibroblasts (HPBFs) is unknown.

We investigated PAR expression and functionality on HPBF before assessing the ability of PAR2 to induce HPBF proliferation, cytokine release, and adhesion molecule expression.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Dulbecco's modified Eagle's medium, sodium pyruvate, antibiotic-antimycotic (penicillin G sodium, streptomycin sulfate, and amphotericin B), FBS, trypsin EDTA, and enzyme-free cell dissociation buffer were purchased from Gibco (Paisley, Renfrewshire, UK). Gel Extraction Kits, Rneasy RNA isolation kits, Hot Start Taq DNA Polymerase, and Omniscript RT-PCR kits were obtained from Qiagen (Crawley, UK). dNTPs and random primers were obtained from Invitrogen (Paisley, Renfrewshire, UK). Fibroblast growth medium kits (fibroblast basal medium, fibroblast growth supplements, and 50 µg gentamycin and 0.1 µg amphotericin B) were obtained from TCS Cell Works (Botolph Claydon, Buckinghamshire, UK). Mouse ExtrAvidin Peroxidase Staining Kit Extra-2, serum replacement media, N{alpha}-Benzoyl-DL-arginine 4-nitroanilide hydrochloride, leupeptin hydrochloride, soya trypsin inhibitor (STI), heparin agarose, Sephacryl S200, and anti-mouse FITC–conjugated secondary antibody were purchased from Sigma (Poole, Dorset, UK). Taq DNA polymerase was obtained from New England Biolabs (Hitchin, Hertfordshire, UK). Primers were purchased from Sigma-Genosys (Pampisford, Cambridgeshire, UK) and from MWG-biotech (Ebersberg, Germany). The human fibroblast–specific marker monoclonal antibody (Clone 5B5) was obtained from DAKO (Ely, Cambridgeshire, UK). Human IL-8 and G-CSF matched antibody pairs, human recombinant IL-8, human recombinant G-CSF, anti-human VCAM-1 and ICAM-1 monoclonal antibodies, and anti-human E-selectin fluorescein conjugated monoclonal antibody were purchased from R&D Systems (Abingdon, Oxon, UK). Cell titre AQueous one solution proliferation assay reagent was from Promega (Southampton, Hampshire, UK). PAR-APs were synthesized by the Peptide Synthesis Facility, University of Calgary (AB, Canada). All other chemicals and reagents were purchased from Sigma-Aldrich (Poole, Dorset, UK) unless otherwise stated.

Cell Culture
HPBF cultures were established as previously described (17) from normal conducting bronchial tissue explants obtained under informed consent from patients undergoing thoracotomy. Ethics approval was obtained from the Hull and East Riding local research ethics committee. Briefly, explants were dissected into pieces of < 1 mm3 and were placed into individual wells of a 24-well culture plate with Dulbecco's modified Eagle's medium containing 20% FBS, 1% sodium pyruvate, and 1% antibiotic/antimycotic. The medium was renewed every 3 d. After ~ 3 wk, isolated fibroblasts were observed, and by 4 wk, confluent fibroblast cultures were obtained. The cells were harvested by trypsinization and transferred to 25-cm2 flasks in 5 ml of fresh HPBF medium (fibroblast basal medium with fibroblast growth supplements and 50 µg gentamycin and 0.1 µg amphotericin B). Confluent cultures were passaged with a split ratio of 1:3. The purity of fibroblast cultures was consistently over 99% as established morphologically by their typical spindle shape and characteristic swirl formation when confluent and immunocytochemically using antibody Clone 5B5 (Dako) and an antivimentin antibody (Sigma). HPBF were used between passages 2–8 for all experiments.

Tryptase Extraction
Human lung tryptase was purified as described previously (21) and stored in 2M NaCl/20 mM MES buffer (pH 6.1) at –80°C. Human lung was obtained according to procedures approved by the University of Calgary, Faculty of Medicine ethics committee. One unit of tryptase activity was defined as the amount of tryptase required to hydrolyze 1 µM of benzoyl-DL-arginine 4-nitroanilide hydrochloride per min at 25°C. Tryptase purity was assessed by specific activity (> 2.5 mU/µg of protein) and SDS/PAGE on a 12% gel, wherein tryptase was identified by Western blot analysis using the tryptase-specific monoclonal antibody AA5 (provided by Dr. Andrew Walls, Respiratory Cell and Molecular Biology, University of Southampton, UK). The identity of the tryptase was confirmed by amino-acid-sequence analysis of protein recovered from Western blot transfer (Alberta Peptide Institute, University of Alberta, Edmonton, AB, Canada). Tryptase was used in the presence of heparin (2:1 heparin/tryptase molar ratio), and concentrations used in all experiments were calculated based on the molecular mass (134 kD) of the tryptase tetramer.

RT-PCR
HPBF were grown to confluence in 6-well plates as described above and placed in serum-free medium (fibroblast basal medium plus serum replacement media) for 48 h before mRNA was extracted using an RNeasy minikit according to the manufacturer's instructions. In separate experiments, HPBF cultured in 6-well plates were quiesced for 48 h before challenge with SLIGKV-NH2 (200 µM) or TNF-{alpha} (50 ng/ml) over a 48-h time course. The mRNA was quantified with a GeneQuant spectrophotometer (Amersham Biosciences, Buckinghamshire, UK), and cDNA was synthesized by reverse transcription of 2 µg of mRNA with an omniscript RT kit. The reverse transcription reaction was cycled at 24°C for 10 min, 37°C for 50 min, and 95°C for 10 min. PCR was performed for beta-actin on all cDNA samples to ensure successful quantification and reverse transcription. PCR amplification of HPBF cDNA for the PARs, a range of proinflammatory cytokines and the adhesion molecules E-selectin, VCAM-1, and ICAM-1, were performed on 1 µl of cDNA with the specific oligonucleotide primers (Table 1). Water blanks were included in all assays. PCR was also performed for PAR4 using human genomic DNA template to confirm the efficacy of the primer sets. PCR reactions for beta-actin, PAR1, PAR2, PAR3, E-selectin, ICAM-1, IL-1{alpha}, IL-1beta, IL-8, IL-18, G-CSF, GM-CSF, and RANTES were performed for 35 cycles at 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. VCAM-1, IL-6, and eotaxin PCR reactions involved 35 cycles at 94°C for 1 min, 59°C for 1 min, and 72°C for 1 min. PCR for IL-11 was performed for 35 cycles at 94°C for 1 min, 69°C for 1 min, and 72°C for 1 min. All the PCR reactions included an initial denaturation step at 94°C for 3 min and a final extension at 72°C for 10 min. PCR for PAR4 was performed using a touchdown PCR method with initial denaturation at 94°C for 3 min, followed by five cycles of 94°C, 70°C, and 72°C (each for 30 s) and then 35 cycles of 94°C, 55°C, and 72°C (each for 30 s), and a final extension at 72°C for 10 min. The PCR products were resolved by running the samples on a 1.3% agarose gel and visualized by ethidium bromide under UV light. The PCR products were purified using a gel purification kit (Qiagen) and sequenced by fluorescence-based automated cycling sequencing (Qiagen sequencing service, Hilden, Germany), and their identities were confirmed by comparison to published sequences.


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  		TABLE 1. PROTEINASE-ACTIVATED RECEPTOR–, CYTOKINE-, AND ADHESION MOLECULE–SPECIFIC OLIGONUCLEOTIDE SEQUENCES

 
Flow Cytometry
PAR Expression. HPBFs were grown to confluence in 6-well plates and placed in serum-free medium for 48 h. Cells were washed with ice-cold PBS, harvested with cold enzyme-free cell dissociation buffer, placed on ice for 5 min, centrifuged at 4°C, and resuspended in 200 µl of ice-cold PBS in the presence of PAR-specific primary antibodies or isotype-matched negative controls and incubated for 90 min on ice. Cells were again pelleted by centrifugation, resuspended in ice-cold PBS, and incubated for 30 min with FITC-conjugated secondary antibodies.

Adhesion Molecule Expression Analysis. Cell surface ICAM-1 expression was assessed in cells treated with SLIGKV-NH2 (200 µM) or TNF-{alpha} (100 ng/ml) for 0, 6, 12, 24, 48, and 72 h. VCAM-1 cell surface expression was assessed in cells treated with trypsin (25 nM), SLIGKV-NH2 (200 µM), or TNF-{alpha} (50 ng/ml) for 0, 6, 8, 10, 12, and 14 h. E-selectin expression was assessed in cells that had been treated with SLIGKV-NH2 (200 µM) or TNF-{alpha} (100 ng/ml) for 0, 0.5, 1, 2, 4, and 6 h. Cells were harvested and resuspended in ice-cold PBS with VCAM-1– or ICAM-1–specific primary antibodies for 90 min. After centrifugation, cells were placed on ice for 30 min with the appropriate FITC-conjugated secondary antibodies. HPBFs were assessed for E-selectin expression by incubating cells with an E-selectin–specific FITC-conjugated antibody in ice-cold PBS for 90 min. After a final PBS wash sequence, cells were pelleted by centrifugation, resuspended in 300 µl of cold PBS, and analyzed on a Becton Dickinson flow cytometer.

Intracellular Calcium Mobilization
Intracellular calcium mobilization in HPBFs was monitored as described previously (22). Briefly, HPBF were seeded onto 12-mm glass coverslips and grown to confluence in 2 ml of HPBF medium. Upon confluence, the medium was replaced with HPBF media containing 0.25 mM sulfinpyrazone and 7 µM Fluo-3 acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR) before incubation for 30 min at 37°C. The coverslips were briefly washed in calcium assay buffer (CAB) (150 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, 0.25 mM sulfinpyrazone [pH 7.4]) before mounting on a microincubation platform (Harvard Apparatus, Holliston, MA) with 500 µl of CAB on a Nikon Diaphot inverted microscope. Cells were stimulated with test agonists by replacing the CAB on the microincubation platform with agonists prediluted in CAB. Fluo-3 fluorescence was measured through a 20x objective using ImageMaster system software (Photon Technology International, London, ON, Canada) with excitation at 480 nm and emission recorded through a dichroic filter cube with the appropriate filter (Nikon) and an intensified CCD video camera (Photon Technology International, London, ON, Canada). At least 20 cells were analyzed for each coverslip tested.

Measurement of Cell Proliferation
Proliferation of HPBF was monitored using the CellTiter 96 AQueous One Solution proliferation assay (Promega). Briefly, cells were seeded into 96-well plates in HPBF medium at a density of 8,000 cells/well and allowed to adhere for 24 h before being placed in serum-free medium for 24 h. Cells were then treated for 0, 24, 48, 72, and 96 h with SLIGKV-NH2 (200 µM) in the presence or absence of amastatin (10 µM) or with trypsin (25 nM). In addition, mitogenic responses to the potent new aminopeptidase resistant PAR2 agonist peptide 2-Furoyl-LIGRLO-NH2 (50 µM) (23) were measured. As a positive control, cells in separate wells were placed in medium containing FBS (10%) or thrombin (5 nM) for 0, 24, 48, 72, and 96 h. In separate experiments, responses of HPBF to varying concentrations of SLIGKV-NH2 (0, 10, 20, 100, and 200 µM) or tryptase (0, 1, 3, 10, and 30 nM) were monitored. At the end of 96 h, the medium in all the wells was carefully replaced with 100 µl of fresh serum-free medium, and 20 µl of CellTiter 96 AQueous One Solution was added to each of the wells. The plates were incubated in the dark at 37°C in a humidified 5% CO2 atmosphere for 2 h before absorbance was measured at 490 nm using a Lambda scan spectral series (MWG-Biotech) 96-well plate reader. Cell numbers were calculated by comparing absorbance readings of treated cells with the absorbances of a standard curve of known cell numbers.

Investigation of Cytokine Release
HPBFs were grown to confluence in 6-well culture plates and quiesced for 48 h in serum-free medium. Quiescent HPBF were incubated with trypsin (50 nM) or SLIGKV-NH2 (200 µM) for 0, 3, 6, 24, and 48 h in serum-free medium. As a positive control, cells were treated with TNF-{alpha} (50 ng/ml) for 6 h. In separate experiments, quiescent HPBF were incubated with trypsin (0–100 nM) or SLIGKV-NH2 (0–200 µM) for 48 h. In inhibition studies, trypsin (20 nM) was preinbuated on ice for 1 h with leupeptin (20 µg/ml) or STI (100 µg/ml) before being added to the cells. Supernatants were collected and stored at –80°C. mRNA from SLIGKV-NH2 time-course treated cells was extracted and reverse transcribed as described previously. The SLIGKV-NH2 treated time course samples were screened by PCR with primers targeted to a range of proinflammatory cytokines (Table 1). Where PCR results indicated an upregulation of a specific cytokine, the appropriate ELISAs were performed on the culture supernatants obtained from the treated cells to assess the presence or upregulation of the specific cytokine protein levels. For costimulation experiments, quiescent HPBF in 24-well plates were costimulated with IL-1{alpha} (10 ng/ml) and SLIGKV-NH2 (200 µM) or TNF {alpha} (50 ng/ml) and SLIGKV-NH2 (200 µM) for 24 h. All supernatants were removed and stored at –80°C before ELISA analysis according to the manufacturers' protocols.

Detection of Proteolytic Activity in Culture Supernatants
Cell culture supernatants (100 µl) from HPBFs treated with SLIGKV-NH2 (0–200 µM) were transferred to separate wells of a 96-well plate under sterile conditions. To each well, 5 ng/ml of recombinant IL-8 or G-CSF was added, and the plate was incubated for 24 h at 37°C. The concentrations of IL-8 and G-CSF in the culture supernatants were analyzed by ELISA according to the manufacturers' protocols.

Statistics
Unless stated otherwise, data are expressed as the mean ± SEM. Data were analyzed by the paired two-tailed Student's t test, taking P < 0.05 as statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
PAR Expression and Function on HPBFs
RT-PCR of cDNA derived from confluent cultures of HPBF indicated significant mRNA expression for PAR1, PAR2, and PAR3 (Figure 1A). PAR4 mRNA expression was not detected. PCR of genomic DNA with our PAR4 primer set generated a product of the expected size (244 bp). DNA sequencing confirmed the identity of each PCR product (data not shown). HPBF cultured from at least six separate tissue samples displayed a similar PAR profile to that shown in Figure 1A. To identify whether PAR expression was altered when cells were quiesced compared with cells grown in full growth media, we performed RT-PCR on cDNA isolated from HPBFs that had been placed in serum-free media for 24 h. The PAR mRNA expression profile for quiescent HPBF displayed no observable difference when compared with that of HPBF in full growth media (data not shown).


Figure 1
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  		Figure 1. Analysis of PAR expression on HPBFs. (A) RT-PCR detection of PAR mRNA in HPBF. RNA was extracted, reverse transcribed, and amplified with specific PAR primers. Lane 1, 1 kb+ ladder; Lane 2, PAR1 (450 bp); Lane 3, PAR2 (550 bp); Lane 4, PAR3 (500 bp); Lane 5, PAR4; Lane 6, PAR4 in genomic DNA (200 bp); Lane 7, water blank. (B) FACS analysis of cell-surface PAR expression in HPBFs. Cells were stained with PAR-specific antibodies, and isotype-matched irrelevant antibodies were used as the negative controls. (i) PAR1; (ii) PAR2; (iii) PAR3; (iv) PAR4. Results are representative of six separate experiments performed with HPBFs cultured from six different subjects.

 
We determined whether PAR protein at the HPBF cell surface correlated with the data derived by RT-PCR. Flow cytometry data revealed, in accordance with the RT-PCR results, that HPBF expressed PAR1, PAR2, and PAR3 but not PAR4 protein on their cell surface when compared with the appropriate isotype control antibodies (Figure 1B).

Calcium imaging was used to determine PAR functionality at the HPBF cell surface (Figure 2). Addition of the selective PAR2-AP SLIGKV-NH2 (200 µM) triggered the typical robust but transient PAR2 calcium signal in the HPBFs (Figure 2A). Addition of trypsin after the application of SLIGKV-NH2 resulted in no observable response. However, addition of thrombin thereafter stimulated a robust calcium signal in these cells (Figure 2A). Addition of the selective PAR1-AP TFLLR-NH2 (200 µM) resulted in a robust calcium signal (Figure 2B). Thrombin had no observable effect after TFLLR-NH2 challenge, although the cells were responsive to the PAR2-AP SLIGKV-NH2 (Figure 2B). Finally, the selective PAR4-AP AYPGQV-NH2 (200 µM) had no observable effect on HPBFs (Figure 2C), although the addition of thrombin after the application of the PAR4-AP resulted in a robust calcium response.


Figure 2
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  		Figure 2. PAR calcium signaling and desensitization in HPBFs. HPBFs seeded on coverslips were loaded with fluo-3 (7 µM) and challenged with different agonists prediluted in calcium assay buffer. Responses were monitored by fluorescence spectrophotometry (excitation 480 nm, emission 530 nm). Arrows indicate when test agents were added to the cells. Fluorescence was measured in individual cells using an ICCD video camera and image master video microscopy acquisition program. At least 20 cells were analyzed for each coverslip of cells treated. Results are representative of six separate experiments using cells grown from six different subjects.

 
PAR2-Mediated HPBF Proliferation
The PAR2-AP SLIGKV-NH2 (200 µM) failed to promote any significant increase in cell number compared with untreated cells up to the 96 h tested (Figure 3A). Trypsin (25 nM) also failed to stimulate an increase in HPBF cell number at the time points tested (Figure 3A). Further experiments demonstrated that SLIGKV-NH2 concentrations of 10, 20, 100, and 200 µM also failed to stimulate a significant increase in HPBF number at the 96-h time point (data not shown). To ascertain whether the SLIGKV-NH2 peptide was being degraded due to aminopeptidase activity, the aminopeptidase inhibitor amastatin (10 µM) was added in combination with SLIGKV-NH2 to HPBF. The addition of amastatin in combination with SLIGKV-NH2 had a negligible effect on the ability of SLIGKV-NH2 to stimulate HPBF proliferation (data not shown). We next investigated proliferative responses to the highly potent aminopeptidase resistant and selective PAR2-AP, 2-Furoyl-LIGRLO-NH2 (50 µM) (23). 2-Furoyl-LIGRLO-NH2 failed to stimulate any significant increase in HPBF cell number over the 96-h test period (Figure 3A). The positive control, 10% FBS, stimulated significant increases in cell numbers at all the time points tested (Figure 3A). Thrombin, a PAR1 agonist, also triggered a significant increase in HPBF cell number (128 ± 8% and 141 ± 8% at 72 and 96 h, respectively) (Figure 3B).


Figure 3
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  		Figure 3. Measurement of PAR2-mediated HPBF proliferation. (A) Proliferative response of HPBFs to various PAR2 agonists. (B) Proliferative response of HPBFs to thrombin (5 nM). Proliferation was assessed using an MTS assay over a 96-h time course. FBS (10%) was used as the positive control. Before the addition of test agonists, all cells were placed in serum-free media for 24 h. Untreated (NT) cell number was recorded at the 96-h time point. Data are expressed as the mean percentage of control ± SEM from at least three different experiments performed in triplicate using cells derived from different subjects. Control untreated cells are at 100%. **P < 0.01; ***P < 0.001 versus untreated cells.

 
A previous report has shown that mast cell tryptase can stimulate the proliferation of primary parenchymal lung fibroblast cultures (17). We failed to observe any significant increase in HPBF number in response to tryptase concentrations of 1, 3, 10, and 30 nM at 96 h after stimulation, with cell numbers obtained being 109 ± 8%, 105 ± 7%, 111 ± 10%, 110 ± 6% percentage of untreated control cells (mean ± SEM from three separate experiments each performed in triplicate). FBS (10%) used as the positive control in the same experiments stimulated an increase of 182 ± 12% at the 96-h time point tested.

Effect of PAR2 Stimulation on Cytokine Production in HPBFs
Incubation of quiescent HPBF with the specific PAR2-AP SLIGKV-NH2 (200 µM) resulted in the specific upregulation of mRNA for IL-8 (Figure 4A) and G-CSF (Figure 4B). IL-8 mRNA levels were upregulated as early as 3 h after treatment and remained higher than baseline up to the 48-h time point tested. For G-CSF, mRNA levels were upregulated as early as 3 h after treatment and declined to near baseline levels by the 48-h time point. For beta-actin mRNA levels, no difference was observed in the treated samples compared with no treatment (Figure 4C). No increases in mRNA levels of IL-1{alpha}, IL-1beta, IL-6, IL-11, IL-18, GM-CSF, eotaxin, or RANTES were observed in response to SLIGKV-NH2 (200 µM) over the 48-h period tested (data not shown). TNF-{alpha} (50 ng/ml), the positive control, stimulated a clear upregulation of mRNA for all the cytokines tested at the 6-h time point (data not shown).


Figure 4
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  		Figure 4. RT-PCR analysis of cytokine expression after PAR2 activation in HPBFs. RNA from PAR2–activated HPBF was extracted, reverse transcribed, and amplified with specific primers for (A) IL-8, (B) G-CSF, and (C) beta-actin. Lane 1, no treatment (NT); Lane 2, TNF-{alpha} (50 ng/ml) 6-h treatment; Lanes 3–6, SLIGKV-NH2 (200 µM) treatment for 3, 6, 24, and 48 h, respectively. Images are representative of six different experiments performed using cells derived from six different subjects.

 
ELISAs were performed to assess whether SLIGKV-NH2–stimulated increases in mRNA levels of IL-8 and G-CSF in HPBF correlated with subsequent increases in soluble cytokine levels (untreated controls taken at 48 h) in the culture supernatants. Over a 48-h time course, we failed to detect any significant SLIGKV-NH2–stimulated increase in the levels of soluble IL-8 or G-CSF in culture supernatants using ELISA (Figures 5A and 5B, respectively). The positive control TNF-{alpha} stimulated a significant increase in soluble IL-8 (139 ± 22%) and G-CSF (129 ± 16%) levels at the 6-h time point (Figures 5A and 5B), with concentrations recorded in culture supernatants being 4.9 ± 0.8 ng/ml and 3.8 ± 0.6 ng/ml, respectively (mean ± SEM, n = 6). SLIGKV-NH2 (50–200 µM) failed to stimulate IL-8 or G-CSF release after 24 h incubation with HPBFs (Figures 5C and 5D). To assess whether soluble IL-8 and G-CSF were being degraded by potential proteinases in the culture supernatants, we spiked SLIGKV-NH2–treated (50–200 µM) culture supernatants with 5 ng of recombinant IL-8 or G-CSF before incubating the supernatants at 37°C for 24 h before performing ELISA. No significant reduction in IL-8 or G-CSF was observed in spiked SLIGKV-NH2 culture supernatants when compared with untreated controls (data not shown). Costimulation of HPBFs with SLIGKV-NH2 (2–200 µM) and IL-1{alpha} (10 ng/ml) or SLIGKV-NH2 (2–200 µM) and TNF-{alpha} (50 ng/ml) also failed to stimulate any observable increase in IL-8 or G-CSF release when compared with IL-1{alpha} or TNF-{alpha} alone (data not shown). Trypsin (50 nM) stimulated a marked increase in IL-8 release from HPBFs over a 48-h time course (Figure 5E) while stimulating a modest increase in G-CSF release over the same 48-h time course (Figure 5F). Trypsin stimulated a concentration-dependent increase in IL-8 and G-CSF release from 10–100 nM (Figures 5G and 5H, respectively). Preincubation of trypsin (20 nM) with STI (100 µg/ml) or leupeptin (20 µg/ml) inhibited trypsin-induced IL-8 and G-CSF release from HPBF (Figures 5G and 5H, respectively). STI (100 µg/ml) and leupeptin (20 µg/ml) alone did not stimulate IL-8 or G-CSF release from HPBF (Figures 5G and 5H, respectively).


Figure 5
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  		Figure 5. ELISA analysis of IL-8 and G-CSF release from HPBFs after PAR2 agonist challenge. (A) IL-8 and (B) G-CSF in culture supernatants from HPBFs treated with SLIGKV-NH2 over a 48-h time course. (C) IL-8 and (D) G-CSF in culture supernatants from HPBFs treated with 0–200 µM SLIGKV-NH2 for 24 h. (E) IL-8 and (F) G-CSF in culture supernatants from HPBFs treated with trypsin (50 nM) over a 48-h time course. (G) IL-8 and (H) G-CSF in culture supernatants from HPBFs treated with trypsin (0–100 nM) or trypsin (50 nM) preincubated with the proteinase inhibitors leupeptin (Leu, 20 µg/ml) or STI (100 µg/ml). TNF-{alpha} (50 ng/ml) was used as the positive control. Data are expressed as mean percentage of control ± SEM from two to six different experiments performed in triplicate using cells derived from different subjects. NT, no treatment. *P < 0.05; **P < 0.01; ***P < 0.001 versus NT. #P < 0.01 versus 20 nM trypsin-treated cells.

 
Effect of PAR2 Activation on HPBF Adhesion Molecule Expression
Increases in the cell surface expression of the adhesion molecules VCAM-1 and ICAM-1 were investigated in PAR2-stimulated HPBFs. RT-PCR displayed a clear increase in mRNA for VCAM-1 and ICAM-1 in response to TNF-{alpha} (50 ng/ml) (Figures 6B and 6D). TNF-{alpha}–induced upregulation of VCAM-1 mRNA was observed at 3 h after treatment, with levels peaking at the 6-h time point and remaining higher than untreated controls up to the 48-h time point tested (Figure 6B). TNF-{alpha}–induced ICAM-1 mRNA upregulation was transient, with a clear increase observed at 6 h after treatment (Figure 6D). SLIGKV-NH2 (200 µM) induced a clear increase in mRNA expression for VCAM-1 at the 3-h time point, which declined to baseline levels thereafter (Figure 6A). No increase in ICAM-1 mRNA was observed in response to SLIGKV-NH2 (200 µM) challenge (Figure 6C). We have further revealed that HPBFs do not express mRNA for E-selectin in control samples or in response to SLIGKV-NH2 or TNF-{alpha} stimuli (data not shown).


Figure 6
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  		Figure 6. RT-PCR analysis of VCAM-1 and ICAM-1 mRNA expression in HPBFs after PAR2 activation. (A) VCAM-1 in SLIGKV-NH2–treated HPBFs. Lane 1, 1 kb+ ladder; Lane 2, untreated (NT); Lanes 3–6, SLIGKV-NH2 (200 µM) treatment for 3, 6, 24, and 48 h, respectively; Lane 8, water blank. (B) VCAM-1 in TNF-{alpha}–treated HPBFs. Lane 1, 1 kb+ ladder; Lane 2, untreated (NT); Lanes 3–6, TNF-{alpha} (50 ng/ml) treatment for 3, 6, 24, and 48 h, respectively; Lane 8, water blank. (C) ICAM-1 in SLIGKV-NH2–treated HPBFs. Lane 1, 1 kb+ ladder; Lane 2, no treatment (NT); Lanes 3–6, SLIGKV-NH2 (200 µM) treatment for 3, 6, 24, and 48 h, respectively; Lane 8, water blank. (D) ICAM-1 in TNF-{alpha}–treated HPBFs. Lane 1, 1 kb+ ladder; Lane 2, no treatment (NT); Lanes 3–6, TNF-{alpha} (200 µM) treatment for 3, 6, 24, and 48 h, respectively. Images are representative of three different experiments performed using cells derived from different subjects.

 
Finally, we sought to determine whether PAR2 activation modulates HPBF cell surface VCAM-1 expression (Figure 7). SLIGKV-NH2 (200 µM)–stimulated HPBF VCAM-1 expression was apparent 8 h after stimulation (Figures 7A [solid bars] and 7Bvi), with maximum expression at 12 h (Figures 7A [solid bars] and 7Bviii). At the 14-h time point, VCAM-1 expression had begun to decline compared with the expression observed at 12 h (Figures 7A [solid bars] and 7Bx). Responses to the endogenous PAR2 agonist trypsin (25 nM) was also investigated. Upregulation of VCAM-1 in response to trypsin was observed at the 10-h and 12-h time points (Figures 7A [hatched bars] and 7Bxi–xv), which had declined to baseline by 14 h. No increases in cell surface levels of ICAM-1 were observed in response to SLIGKV-NH2 (data not shown), but TNF-{alpha} (100 ng/ml) induced an increase at the 12-h and 24-h time points (data not shown). No increase in cell surface expression of E-selectin was observed in response to SLIGKV-NH2 or TNF-{alpha} treatment for 0, 0.5, 1, 2, 4, and 6 h (data not shown).


Figure 7
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  		Figure 7. Flow cytometry analysis of VCAM-1 expression on HPBFs after PAR2 activation. (A) VCAM-1 levels in TNF-{alpha}–treated (50 ng/ml), SLIGKV-NH2–treated (200 µM), and trypsin-treated (25 nM) HPBFs. Data are expressed as percentage median fluorescence intensity ± SEM from three different flow cytometry experiments using cells from different subjects. No treatment (NT) control cells are at 100%. *P < 0.05 versus NT cells. (B) Representative trace of flow cytometry detection of VCAM-1 upregulation by trypsin-, SLIGKV-NH2–, and TNF-{alpha}–activated HPBFs. HPBFs were NT or stimulated with TNF-{alpha} (50 ng/ml), SLIGKV-NH2 (200 µM), or trypsin (25 nM) over a 14-h time course. Results are representative of three separate experiments using cells grown from three different subjects.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
We demonstrate that HPBFs express functional PAR1 and PAR2 and that activation of PAR2 with SLIGKV-NH2 results in the upregulation of IL-8 and G-CSF mRNA but not cytokine release. In contrast, trypsin stimulated significant IL-8 and G-CSF release from HPBFs. SLIGKV-NH2 and trypsin stimulated the upregulation of HPBF VCAM-1 cell surface expression. These findings provide evidence to suggest that PAR2 on HPBF may play a role in the recruitment, survival, and infiltration of neutrophils during an inflammatory response in the upper airways.

We initially sought to determine the PAR profile on HPBFs using a panel of techniques. Using RT-PCR, we show that HPBFs express mRNA for PAR1, PAR2, and PAR3 but not PAR4. This expression pattern remained unchanged even when the cells were quiesced in a serum-free media. In accord with the PAR profile observed with RT-PCR, the FACS data indicated that PAR1, PAR2, and PAR3, but not PAR4, were present at the cell surface of HPBFs. In contrast, the calcium signaling experiments demonstrated that HPBFs express functional PAR1 and PAR2 but not PAR3 or PAR4. Knowing that PAR3 cannot be activated with agonist peptides corresponding to the tethered ligand sequence (24), we used an indirect receptor desensitization approach for determining PAR3 signaling. Addition of thrombin, a PAR1, PAR3, and PAR4 agonist, after PAR1 desensitization with the specific PAR1-AP TFLLR-NH2 resulted in no observable calcium signal, suggesting that PAR3 in the HPBF does not signal through calcium. Our finding that PAR3 does not signal through calcium in HPBFs is in accord with a previous report on endothelial cells (25). Thus, HPBFs express functional PAR1 and PAR2, whereas PAR3 seems not to signal through calcium, and PAR4 is not detectable.

Because PAR2 agonists have previously been shown to stimulate fibroblast proliferation from various tissue sources (17, 19, 26, 27), we investigated whether PAR2 could trigger the proliferation of HPBFs. Our data consistently showed that a panel of PAR2 agonists failed to induce proliferation of HPBFs at the times points and concentrations tested. We confirmed by using amastatin, an aminopeptidase inhibitor, and through the use of an aminopeptidase-resistant PAR2-AP, 2-Furoyl-LIGRLO-NH2 (23), that this lack of proliferation observed was unlikely to be due to the degradation of the PAR2-AP. We further demonstrated that trypsin and tryptase were also incapable of stimulating HPBF proliferation. The origin of the fibroblasts may provide an explanation for our contrasting results to that reported by Akers and colleagues (17), whose lung fibroblasts were derived from airway and lung tissue in close proximity to the parenchyma. Other studies (26, 27) used established fibroblasts cell lines, and these may respond differently to primary cells. HPBFs could proliferate in our assay system because FBS and thrombin stimulated a significant proliferative response in these cells. Finally, our finding that thrombin could clearly stimulate HPBFs to proliferate suggests that PAR2 plays a minor role in the proliferation of fibroblasts derived from normal conducting bronchial tissue. We conclude that PAR2 activation alone fails to stimulate HPBF proliferation, whereas thrombin is a growth factor for HPBF.

Because PAR2 has been implicated in proinflammatory events (1), we investigated whether the PAR2 agonists trypsin and SLIGKV-NH2 could induce cytokine release from HPBFs. From the panel of cytokines tested, RT-PCR detected the upregulation of mRNA for IL-8 and G-CSF in HPBF in response to the selective PAR2-AP SLIGKV-NH2. The increases in mRNA for IL-8 and G-CSF seen in HPBFs were specific because we detected no changes in the levels of IL-1{alpha}, IL-1beta, IL-6, IL-11, IL-18, eotaxin, GM-CSF, or RANTES. However, we were unable to detect by ELISA any increases in the levels of secreted IL-8 or G-CSF protein in PAR2-AP–activated HPBF supernatants. Trypsin stimulated an increase in IL-8 and G-CSF release from HPBFs that was time and dose dependent. Further, we demonstrated that leupeptin and STI inhibited trypsin-stimulated cytokine release, indicating that this effect was also catalytic site dependent. The reason for this discrepancy in the ability of trypsin and SLIGKV-NH2 to trigger cytokine release raises the possibility that trypsin is activating other signaling pathways separate from those of PAR2, as has been reported for tryptase activation of airway smooth muscle cells (28). Recent reports have described the inability of PAR-APs to activate the full repertoire of PAR intracellular signaling pathways when compared with their respective endogenous proteinases (29, 30). This provides a plausible explanation for differences in trypsin- and SLIGKV-NH2–induced cytokine release. Further studies are required to unravel this interesting observation.

Finally, we determined whether PAR2 activation could modulate the expression of the adhesion molecules ICAM-1 and VCAM-1. Trypsin and the selective PAR2-AP SLIGKV-NH2 stimulated the upregulation of VCAM-1 with maximum expression observed at 12 h. These kinetics are consistent with those reported previously for VCAM-1 upregulation on fibroblasts in response to cytokine stimuli (31). A recent report has shown that PAR2 activation on neutrophils upregulates the expression of very late antigen-4, the neutrophilic ligand for VCAM-1 (14). Our finding that PAR2 upregulation of VCAM-1 in HPBFs provides a complementary mechanism that may facilitate neutrophil migration through an inflamed airway.

The combination of IL-8, G-CSF release, and the upregulation of VCAM-1 expression on HPBF in response to PAR2 agonists indicates that PAR2 may play a role in facilitating a neutrophilic specific response in the airways. In conclusion, our findings suggest that PAR2 on fibroblasts may play an important role in inflammatory airways disease.


   Acknowledgments
 
The authors thank Mike Cowan (Surgery Unit, Castle Hill Hospital, Cottingham, UK) for assistance in procuring bronchial tissue and Professor Morley D. Hollenberg (Department of Pharmacology, University of Calgary, Canada) for providing human lung tissue and for the use of facilities for the extraction of human lung tryptase.


   Footnotes
 
This work was supported by a University of Hull research committee Ph.D. studentship (R.R.).

Originally Published in Press as DOI: 10.1165/rcmb.2005-0362OC on February 23, 2006

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 September 22, 2005

Accepted in final form February 16, 2006


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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