1 Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, The Royal Free and University College Medical School, The Rayne Institute, London WC1E 6JJ; 3 Respiratory Diseases Unit, Glaxo Wellcome Research and Development Limited, Medicines Research Centre, Stevenage SG1 2NY, United Kingdom; and 2 Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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Mast cells play a potentially important role in fibroproliferative diseases, releasing mediators including tryptase that are capable of stimulating fibroblast proliferation and procollagen synthesis. The mechanism by which tryptase stimulates fibroblast proliferation is unclear, although recent studies suggest it can activate protease-activated receptor (PAR)-2. We therefore investigated the role of PAR-2 in tryptase-induced proliferation of human fetal lung and adult lung parenchymal and airway fibroblasts and, for comparative purposes, adult dermal fibroblasts. Tryptase (0.7-70 mU/ml) induced concentration-dependent increases in proliferation of all fibroblasts studied. Antipain, bis(5-amidino-2-benzimidazolyl)methane, and benzamidine inhibited tryptase-induced fibroblast proliferation, demonstrating that proteolytic activity is required for the proliferative effects of tryptase. RT-PCR demonstrated the presence of PAR-2 mRNA, and immunohistochemical staining localized PAR-2 to the cell surface of lung fibroblasts. In addition, specific PAR-2 activating peptides, SLIGKV and SLIGRL, mimicked the proliferative effects of tryptase. In contrast, human dermal fibroblasts only weakly stained with the PAR-2 antibody, PAR-2 mRNA was almost undetectable, and fibroblasts did not respond to PAR-2 activating peptides. These results suggest that tryptase induces lung, but not dermal, fibroblast proliferation via activation of PAR-2 and are consistent with the hypothesis that the release of tryptase from activated mast cells may play an important role in the fibroproliferative response observed in asthma, chronic obstructive pulmonary disease, and patients with pulmonary fibrosis.
asthma; airway remodeling; interstitial fibrosis; serine proteinase
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INTRODUCTION |
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MANY DISEASES OF THE LUNG are associated with a fibroproliferative response, with increased numbers of fibroblasts/myofibroblasts and excessive deposition of extracellular matrix proteins. These include interstitial lung diseases such as cryptogenic fibrosing alveolitis, sarcoidosis, bronchopulmonary dysplasia, farmer's lung, bronchiolitis obliterans organizing pneumonia, pulmonary fibrosis associated with systemic sclerosis, and rheumatoid arthritis (38) as well as diseases such as asthma and chronic obstructive pulmonary disease where there is an increase in fibroblast number and extracellular matrix deposited around the airways (7, 15, 53). However, the mechanism by which fibroblast proliferation and excessive extracellular matrix deposition occurs is unclear.
Lung tissue from patients with these diseases has been shown to contain increased numbers of mast cells (13, 24, 30, 39, 49, 50, 52), and these are often found in close apposition to fibroblasts (28, 34). The increase in mediators derived from mast cells, such as histamine and tryptase, in bronchoalveolar lavage fluid from these patients (1, 11, 13, 21, 39, 63, 66) and their state of degranulation (34) suggest that they are in a state of activation. In addition, bronchoalveolar lavage fluid from asthmatic patients contains an increased number of mast cells (60, 65), and the number of mast cells in bronchial biopsies has been related to the degree of subepithelial thickening within the airway wall of asthmatic patients (19, 20, 22). Together, these data suggest that the mediators released from mast cells may play an important role in stimulating fibroblast proliferation and collagen synthesis.
One mediator that is released in a high concentration from degranulating mast cells is tryptase. It is a 134-kDa serine protease stored in the granule as an active tetrameric enzyme bound to heparin (48). Tryptase is a potent stimulant of fibroblast, epithelial, and smooth muscle cell proliferation (8-10, 27) and is capable of stimulating synthesis of type I collagen by human fibroblasts (9, 26). The mechanisms by which tryptase exerts these cellular effects is unclear, although recent evidence suggests that tryptase may activate one member of the protease-activated receptor (PAR) family, PAR-2 (18).
PAR-2 is a member of the PAR family that includes PAR-1 (62), PAR-3 (32), and PAR-4 (33, 67). Activation of these receptors requires the cleavage of the extracellular NH2-terminal domain, revealing a new peptide sequence (tethered ligand) that is able to bind to sites within the second extracellular loop of the seven-transmembrane receptor (36). It has been suggested that trypsin (5, 6, 44, 45) and, more recently, tryptase (14, 25, 40, 42) may activate PAR-2. In addition, PAR-2 can be selectively activated by the peptides serine-leucine-isoleucine-glycine-lysine-valine (SLIGKV) and serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL), which correspond to the first six amino acids of the new NH2-terminal domain exposed after cleavage of human and rat PAR-2, respectively (5, 6, 35, 43). Tryptase-induced calcium mobilization in various cells has been shown to be mediated via PAR-2 (14, 42, 57). The role of PAR-2 in cell proliferation is controversial, with activation of PAR-2 in human vascular endothelial cells and keratinocytes causing proliferative and antiproliferative effects, respectively (17, 41). The role of PAR-2 in tryptase-induced fibroblast proliferation is currently unknown, although it has been suggested that dermal and arterial fibroblasts do not express PAR-2 mRNA (3, 56, 57).
In this study, we investigated the role of PAR-2 in mediating the effects of tryptase on lung fibroblast proliferation. To address this, we examined the expression and localization of PAR-2 in human lung fibroblasts with RT-PCR and immunohistochemical staining. In addition, we compared the mitogenic effects of tryptase with those of trypsin and the specific PAR-2 activating peptides.
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METHODS |
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Materials. Dulbecco's modified Eagle's medium (DMEM), glutamine, penicillin-streptomycin, Fungizone, and trypsin-EDTA solutions were all obtained from Life Technologies (Paisley, UK); newborn calf serum (NCS) was obtained from Imperial Laboratories (Andover, UK); phosphate-buffered saline (PBS) was from Oxoid (Basingstoke, UK); antipain, benzamidine, BSA, holo-transferrin, methylene blue, and cell dissociation solution were obtained from Sigma (Poole, UK); human fetal lung fibroblasts (HFL1) were purchased from American Type Culture Collection (Manassas, VA); and human mast cell tryptase was purchased from Bioprocessing (Scarborough, ME). Tryptase activity is expressed in milliunits and was defined as the amount of enzyme that gave a change of 1 absorbance unit/min at 410 nm with N-benzyl-DL-arginine p-nitroanilide (pNA) as the substrate. The batches of tryptase used in these studies had specific activities ranging from 5,275 to 6,089 mU/mg of total protein. Rat PAR-2 activating peptide SLIGRL was purchased from Neosystems Laboratoire (Strasbourg, France). Human PAR-2 activating peptide SLIGKV and the control peptides leucine-serine-isoleucine-glycine-lysine-valine (LSIGKV) and leucine-serine-isoleucine-glycine-arginine-leucine (LSIGRL) were kindly synthesized by Dr. Robert Mecham (Washington University School of Medicine, St. Louis, MO). All peptides were in the free carboxylate form. The synthetic peptides were prepared by conventional solid-phase synthesis on an Applied Biosystems model 431A synthesizer with FastMoc chemistry. The sequence and purity of the peptides were confirmed by electrospray mass spectrometry. All synthetic peptides were soluble in the tissue culture medium at the concentrations tested. Bis(5-amidino-2-benzimidazolyl)methane (BABIM) was a kind gift from Dr. David Andrews (Glaxo Wellcome Research and Development, Stevenage, UK); rabbit anti-rat PAR-2 serum (B5) was raised in rabbits against a peptide that spans the rat PAR-2 trypsin cleavage/activation site: 30GPNSKGR(trypsin cleavage site)SLIGRLDT46P-TGGC coupled to keyhole limpet hemocyanin (YGGC for coupling). The use of the B5 antibody for immunohistochemical studies has been previously described (14, 35). Goat anti-rabbit IgG conjugated to FITC was obtained from DAKO (High Wycombe, UK). TRIzol Reagent and Superscript Kit were obtained from Life Technologies; agarose, ethidium bromide, and mineral oil were obtained from Sigma.
Tissue explant. Fibroblast cell lines were established from
lung tissue obtained from patients undergoing resection of localized lung tumors. For parenchymal lung fibroblasts, tissue was carefully cut
into pieces of <1 mm3 that were placed into
10-cm-diameter petri dishes (Corning Costar, Cambridge, MA) with 2 ml
of DMEM containing penicillin (100 U/ml), streptomycin (100 µg/ml),
Fungizone (amphotericin B; 2.5 µg/ml), and 10% NCS and incubated at
37°C in a humidified atmosphere of air containing 10%
CO2. When the tissue had firmly attached to the plastic
surface, a further 8 ml of culture medium were slowly added to the
petri dish and incubated as before. The medium was changed every 7 days. For airway fibroblasts, the airways were carefully dissected free
of parenchymal connective tissue, and <1-mm3 pieces of
tissue were cultured as for the parenchymal tissue. Samples of skin
obtained from patients undergoing breast reduction surgery were
dissected free of fatty tissue, and 1-mm3 pieces were
placed, dermal side down, into petri dishes containing 2 ml of DMEM as
described above and cultured. After ~3 wk of culture, fibroblasts
were seen growing out from the explanted tissue. These were cultured
for at least a further 2 wk until confluent islands of cells were
observed. The cultures were then passaged with a split ratio of 1:2
into 75-cm2 flasks (Falcon, Marathon, London, UK)
containing culture medium without Fungizone. Thereafter, confluent
cultures were passaged with a split ratio of 1:4. Experiments were
performed with cells between passages 2 and 8. Human
fetal lung fibroblasts (HFL1) were grown to confluence in DMEM
containing penicillin (100 U/ml), streptomycin (100 µg/ml), and 5%
NCS in 75-cm2 flasks. For the experiments, cells were used
between passages 15 and 23. Fibroblast lines were
characterized immunohistochemically to confirm their purity. Staining
with antibodies to cytokeratin, von Willebrand factor, and desmin was
negative, indicating that the cultures did not contain significant
numbers of epithelial or mesothelial cells, endothelial cells, or
smooth muscle cells. Greater than 95% of the cells stained positively
for vimentin, and between 20 and 30% of the cells were also positive
for -smooth muscle actin, confirming the
fibroblast/myofibroblast phenotype of the cells lines. All cells
were tested for mycoplasma infection.
Cell proliferation. In pilot studies, tryptase-induced cell
proliferation was assessed in subconfluent and confluent cultures. In
subconfluent cultures, tryptase at concentrations 3.5 mU/ml caused a small mitogenic response; however, at concentrations > 3.5 mU/ml, tryptase caused cell detachment (data not shown). In studies
with confluent cultures, cell detachment was not seen until
concentrations > 80 mU/ml were used. In parallel control plates, cell
numbers were assessed at the time of agonist addition and at the end of
the incubation 48 h later. There was an ~10% decrease in cell number
(76,328 ± 3,841 cells at time of addition compared with 67,109 ± 5,258 cells after 48-h incubation); however, this was not significant
(P = 0.18). Therefore, all subsequent studies were performed in
confluent cultures with the following protocol. Cells were trypsinized
[trypsin (0.05% wt/vol)-EDTA (0.02%
wt/vol)] and seeded onto 96-well microtiter plates (Nunc, Life Technologies) at 104 cells/well in 100 µl of DMEM
containing penicillin, streptomycin, and NCS for 5 days as described in
Tissue explant. When the cells were visually
confluent, the medium was replaced with 100 µl of serum-free medium
(incubation medium) containing BSA (1 mg/ml) and holo-transferrin (1 µg/ml) and incubated for a further 24 h. The medium was replaced with
100 µl of fresh incubation medium containing growth factors
(tryptase, 0.7-70 mU/ml; trypsin, 1-20 nM; activating
peptides SLIGKV and SLIGRL, 0.01-1 mM; control peptides LSIGKV and
LSIGRL, 1 mM; and NCS, 5%, or tryptase, 17 mU/ml) that had been
preincubated with various protease inhibitors (antipain, benzamidine,
or BABIM at concentrations of 1-100 µM) for 60 min at 37°C.
Cells were then incubated for a further 48 h.
Cell number was assessed by the use of a spectrophotometric assay previously described (47). Briefly, the medium was removed, and the plates were washed in PBS. The cells were fixed in Formalin-saline (10% vol/vol Formalin in 0.15 M NaCl) for at least 30 min. The Formalin-saline was removed, and the plates were blotted dry. The cells were stained with 100 µl/well of methylene blue (1% wt/vol in 0.01 M borate buffer) for 30 min. The excess dye was removed by washing in 0.01 M borate buffer (pH 8.5) with a plate washer (Denley Instruments Cellwash, Life Science International, Basingstoke, UK). The bound dye was then eluted from the cells by the addition of 100 µl of acidified alcohol (0.01 M HCl-ethanol, 1:1 vol/vol), and the absorbance was measured at 650 nm with a microplate spectrophotometer (Titretek Multiscan MCC/340 MK II, Flow Laboratories, Rickmansworth, UK). Results are expressed as a percent change in the mean absorbance compared with that in cells exposed to incubation medium alone. Changes in fibroblast cell number were confirmed by direct cell counting with a hemacytometer (British Drug House/Merck, Lutterworth, UK).
PAR-2 localization. Confluent cultures of fibroblasts were treated with nonenzymatic cell dissociation fluid to obtain single-cell suspensions. Cells were seeded onto eight-well chamber slides (Lab-Tek Permanox, Nunc, Life Technologies) at a density of 104 cells/well in 400 µl of DMEM containing 5% NCS. The cells were incubated at 37°C in a humidified atmosphere of air containing 10% CO2 for 48 h. The cells were fixed with 4% paraformaldehyde for 3 min at room temperature. The fixed cells were washed with PBS and treated with normal swine serum for 20 min followed by three 5-min washes with PBS. The cells were incubated with rabbit anti-rat PAR-2 antiserum (1:1,000 dilution) for 16 h at 4°C. Normal rabbit serum (1:1,000) or rabbit IgG (20 ng/ml) was used instead of the primary antiserum as a negative control. The cells were thoroughly washed and then incubated with a secondary swine anti-rabbit IgG conjugated to FITC for 60 min at room temperature. After three 5-min washes with PBS, the chambers were removed, and coverslips were mounted with an antifade glycerol-PBS-based mountant (AF1, Citiflour Products, Canterbury, UK). The cells were visualized by confocal microscopy (Leica TCS NT, Leica UK, Milton Keynes, UK). Digital images were obtained with a Polaroid Digital Palette HR6000 (Leica UK), and photomicrographs were obtained with Polaroid Presentation Chrome 35-mm (ASA100) film.
RT-PCR for PAR-2 in lung and dermal fibroblasts. Human fetal lung, adult airway, and adult dermal fibroblasts were grown to confluence as described in Tissue explant. Total RNA was extracted from the cells with TRIzol Reagent (GIBCO BRL, Life Technologies) according to the manufacturer's instructions. First-strand cDNA was synthesized by reverse transcription of the RNA with the Superscript Preamplification System for First Strand cDNA Synthesis (GIBCO BRL, Life Technologies) according to the manufacturer's instructions. PCR amplification was performed with 2.5 U of Taq DNA polymerase on 5 µg of cDNA with the following oligonucleotide primers for human PAR-2: forward, 5'-GTTGATGGCACATCCCACGTC-3', and reverse, 5'-GTACAGGGCATAGACATGGC-3'. The reaction was allowed to proceed for 35 cycles at 94°C for 30 s, 50°C for 1 min, and 72°C for 1 min. The quality of the PCR product was checked by 1.5% agarose gel electrophoresis at 90 V for 45 min and visualized by ethidium bromide. Its identity was confirmed by sequencing with AmpliTaq DNA polymerase FS (Perkin-Elmer) fluorescently labeled dye-terminator chemistry and the use of an Applied Biosystems 377 PRISM automated sequencer. The signal obtained in the PCR amplification of PAR-2 cDNA was compared with the product generated from a glyceraldehyde-3-phosphate dehydrogenase PCR control with the following primer pair: forward, 5'-ACCACAGTCCATGCCATCAC-3', and reverse, 5'-TCCACCACCCTGTTGCTGTA-3', giving an expected product size of 452 bp.
Assay for inhibition of tryptase activity. Tryptase activity and its inhibition by serine protease inhibitors were assayed with the synthetic peptide substrate tosyl-Gly-Pro-Arg pNA. Stock solutions of substrate and inhibitors were dissolved in DMSO. Twenty microliters of tryptase (17 mU/ml final concentration) were added to the reaction mixture containing 50 µl of 10 mM Tris and 120 mM NaCl at pH 7.8, 20 µl of substrate (stock solution, 2 mM), and 10 µl of antipain, BABIM, or benzamidine to give a final concentration of 100 µM. The reaction volume was 100 µl, with a final DMSO concentration of 1.8%. Absorbance was measured at 405 nm with a 630/650-nm reference filter. The results are expressed as a percent inhibition of tryptase-induced cleavage of the synthetic peptide.
Statistics. In cell proliferation studies, means ± SE were calculated. The variation between data sets was tested with ANOVA, and the significance was tested with unpaired t-tests, with a Bonferroni modification for multicomparison of data (23). Differences were considered significant when P was <0.05.
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RESULTS |
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Effect of tryptase on fibroblast proliferation. Figure
1 shows the effect of tryptase on human
fetal lung fibroblast proliferation. Tryptase stimulated proliferation
of fibroblasts in a concentration-dependent manner. Proliferation was
induced at concentrations of 0.7 mU/ml and above, with a maximal
stimulation of ~50% obtained at a concentration of 35 mU/ml. The
effect of tryptase was reproducible; in eight separate experiments,
17.6 mU/ml of tryptase stimulated fibroblast proliferation by 38 ± 2%. The effect of tryptase on fibroblast proliferation was confirmed
by direct cell counting. Values were generally higher than for the
spectrophotometric assay. Tryptase at concentrations of 3.5 and 17.6 mU/ml increased cell numbers above those seen in parallel control
plates by ~25 and 70%, respectively (control, 124,200 ± 12,800 cells; 3.5 mU/ml, 156,700 ± 32,500 cells; 17.6 mU/ml, 210,000 ± 11,100 cells).
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Tryptase also caused concentration-dependent increases in the
proliferation of fibroblasts derived from adult human lung parenchyma and airways over a similar concentration range to that seen with fetal
lung fibroblasts (Fig. 2, A and
B, respectively). Tryptase stimulated proliferation of adult
parenchymal fibroblasts to an extent similar to that observed in fetal
lung fibroblasts. The airway fibroblasts appeared to be more
responsive, with values approximately double those observed for
parenchymal cells stimulated with the same concentrations of tryptase.
Tryptase also caused reproducible, concentration-dependent increases in
human dermal fibroblast proliferation over a similar concentration
range (0.7-17.6 mU/ml) to that seen with the lung fibroblasts
(Fig. 2C).
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Effect of proteolytic inhibitors on tryptase-induced fibroblast
proliferation. The effect of proteolytic inhibitors on
tryptase-induced fibroblast proliferation is shown in Fig.
3. The nonselective serine protease
inhibitor antipain and the relative selective tryptase inhibitors BABIM
and benzamidine caused concentration-dependent inhibition of
tryptase-induced human fetal lung fibroblast proliferation (Fig. 3). At
the highest concentration used (100 µM), antipain, BABIM, and
benzamidine inhibited the response to tryptase by 68 ± 17.7, 100 ± 6.8, and 70 ± 5.8%, respectively. The addition of the inhibitors alone to the cells had no effect on basal cell proliferation (Fig. 3).
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Tryptase-induced cleavage of the synthetic peptide substrate tosyl-Gly-Pro-Arg pNA was inhibited by all three serine protease inhibitors tested. Antipain and BABIM at a concentration of 100 µM caused inhibition of 87.8 ± 3.3 and 93.4 ± 2.5%, respectively, similar to their degree of inhibition of tryptase-induced fibroblast proliferation. However, benzamidine (100 µM) appeared less effective in the biochemical assay, inhibiting tryptase-induced cleavage by 24 ± 13.0%.
Effect of trypsin on fibroblast proliferation. Trypsin, a known
activator of PAR-2, also induced concentration-dependent increases in
lung fibroblast proliferation. Proliferation was induced at concentrations of 5 nM and above, with a maximal achievable stimulation at 20 nM (Fig. 4). At higher
concentrations, the cells detached from the plate. Trypsin also induced
proliferation of dermal fibroblasts but to a lesser extent (Fig.
4).
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Effect of PAR-2 activating peptides on fibroblast
proliferation. The PAR-2 activating peptides corresponding to the
human (SLIGKV) and rat (SLIGRL) sequences also caused increases in
fetal lung fibroblast proliferation (Fig.
5). At a concentration of 1 mM, SLIGKV and
SLIGRL caused stimulation of 22 ± 2 and 37 ± 1.5%, respectively,
above medium control values (P < 0.01). This was confirmed by
direct cell counts with SLIGKV (1 mM) and SLIGRL (1 mM), which
increased cell number by ~25 and 50%, respectively (control,
55,200 ± 4,900 cells; SLIGKV, 67,800 ± 900 cells; SLIGRL, 82,800 ± 5,600 cells). Coincubation with the peptidase inhibitor phosphoramidon (10 µM), bestatin (10 µM), or thiorphan (10 µM) did not increase the activity of either peptide agonist (data not
shown). Neither of the control peptide sequences at concentrations similar to those used with the activating peptides affected fibroblast proliferation (1 mM LSIGKV, 1 ± 1%; 1 mM LSIGRL, 3 ± 1%).
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Both SLIGKV and SLIGRL caused a concentration-dependent stimulation of
adult parenchymal and airway fibroblast proliferation. Stimulation was
seen at concentrations of 10 µM and above. Figure 6 shows the effects of the activating and
control peptides at a concentration of 1 mM. As shown for human fetal
lung fibroblasts, SLIGRL generally caused a greater degree of
stimulation than SLIGKV in both adult parenchymal and airway
fibroblasts. The control peptides did not affect fibroblast
proliferation (Fig. 6). In contrast to the lung fibroblasts, both the
human and rat PAR-2 activating peptides failed to stimulate human
dermal fibroblast proliferation (Fig. 6).
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Immunolocalization of PAR-2. Figure
7 shows the localization of PAR-2 in human
fetal lung and adult parenchymal and airway fibroblasts after fixation
for 3 min at room temperature. PAR-2 was clearly localized to the cell
surface as demonstrated by a greater intensity of staining around the
perimeter of the cells in the confocal images (Fig. 7,
A-C). Weak staining was also observed in two
different human adult dermal fibroblast cell lines (Fig. 7D).
Fluorescent staining was absent in control preparations with normal
rabbit serum (1:1,000 dilution) or rabbit IgG (20 ng/ml; data not
shown).
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Detection of PAR-2 mRNA by RT-PCR. RT-PCR with primers targeted
to human PAR-2 amplified a product corresponding to the predicted size
(813 bp) and sequence, indicating the presence of PAR-2 mRNA in human
fetal lung and adult airway fibroblasts. A similar PCR product was also
detected in human dermal fibroblasts but at much lower intensity
relative to the signal observed in the lung and airway fibroblasts.
RT-PCR with primers to glyceraldehyde-3-phosphate dehydrogenase gave a
product of similar intensity in all cell types studied (Fig.
8). Similar results were obtained with
another airway and dermal fibroblast line (data not shown).
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DISCUSSION |
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In this study, we have shown that human mast cell tryptase causes proliferation of fibroblasts derived from fetal and adult human lung parenchyma and adult airways. Tryptase-induced fibroblast proliferation was inhibited by proteolytic inhibitors, demonstrating that the effect was dependent on its catalytic activity. In addition, as far as we are aware, we have demonstrated for the first time the presence of PAR-2 on the surface of lung fibroblasts and that PAR-2 activating peptides corresponding to either the human or rat sequence mimicked the proliferative effect of tryptase. These data suggest that tryptase induces lung fibroblast proliferation via cleavage and activation of PAR-2 located on the fibroblast cell surface. In contrast, although tryptase stimulated proliferation of human adult dermal fibroblasts, the PAR-2 activating peptides had no effect, suggesting that in this cell line tryptase was acting via other mechanisms.
Tryptase has previously been shown to be a potent mitogen for fibroblasts derived from the lung (9, 27, 54) and dermis (51). The concentrations of tryptase at which activity was observed in the present study were comparable to those previously reported, expressed in either units of enzymatic activity or molar concentration. The present study confirms previous data and extends these observations to show that tryptase can stimulate proliferation of fibroblasts derived from both the lung parenchyma and airway.
Tryptase-induced increases in proliferation were inhibited by the serine protease inhibitors antipain, BABIM, and benzamidine at concentrations that inhibit its enzyme activity and that have been used in previous studies to inhibit the effects of tryptase (9, 10, 12, 59, 64). Together, these data demonstrate that the tryptase-induced fibroblast proliferation is dependent on the catalytic activity of the enzyme.
The evidence that tryptase-induced proliferation requires its proteolytic activity suggests that a member of the PAR family may be involved in the response (18, 32, 45, 62, 67). It has recently been shown that tryptase induces phosphoinositol hydrolysis and calcium mobilization in endothelial cells, colonic myocytes, and keratinocytes (14, 42, 57) and that these effects are mimicked by the PAR-2 activating peptides (2, 31, 37, 41, 43, 46, 55, 57, 58). In this study, we demonstrated that PAR-2 activating peptides also mimic the effects of tryptase on lung fibroblast proliferation. In contrast, the control peptides, in which the first two amino acids were reversed (LSIGKV and LSIGRL), were without activity, demonstrating the specificity of the responses to the activating peptides. Because PAR-2 activating peptides do not cross-react with other known PARs (4, 29, 36), this suggests that the proliferative effects of tryptase on lung fibroblasts may be mediated via PAR-2.
Further evidence for the presence of PAR-2 on the cell surface of fibroblasts was obtained by immunohistochemical staining. We used a rabbit anti-rat PAR-2 antiserum, which has previously been used to demonstrate PAR-2 on the cell surface of both rat enterocytes and colonic myocytes and a rat kidney epithelial cell line transfected with human cDNA for PAR-2 (14, 35). We have demonstrated the presence of PAR-2 on the cell surface of both human fetal and adult parenchymal and airway fibroblasts. As far as we are aware, this is the first time that the B5 antiserum has been used to identify PAR-2 on human cells, although its cross-reactivity has previously been confirmed by the expression of human PAR-2 in rat cells (35). This result was not surprising given the similarity of this region of the receptor sequence between human and rodent PAR-2 (6, 44, 45, 55) and given that an antibody to a similar region of the human PAR-2 sequence also cross-reacts with rat PAR-2 (14, 35). In addition, we have demonstrated the expression of PAR-2 mRNA by RT-PCR in human fetal lung and adult airway fibroblasts. Further evidence demonstrating the presence of PAR-2 in the human lung has been provided by Northern analysis (6, 44) and immunolocalization (16), although in this study, PAR-2 was only identified within the epithelial layer and bronchial smooth muscle. Thus the evidence suggests that PAR-2 is present in the human lung, and we have shown in this study with RT-PCR, immunohistochemistry, and the use of PAR-2 activating peptides that PAR-2 is present on human lung fibroblasts and may be activated by tryptase to induce fibroblast proliferation.
In contrast to our observations in human lung fibroblasts, previous studies (16, 56) have suggested that human dermal fibroblasts do not express PAR-2 mRNA. From our data with human dermal fibroblasts, we observed very weak mRNA expression and immunostaining for PAR-2. In addition, we were unable to obtain responses to the PAR-2 activating peptides, suggesting there may be insufficient receptors to elicit a proliferative response. Furthermore, the response to trypsin, another known activator of PAR-2, was limited. Interestingly, we obtained responses to tryptase that were similar to those seen on lung fibroblasts. This result suggests that tryptase may cause its proliferative effects either 1) via a subtype of PAR-2 that is not activated by the PAR-2 activating peptides; 2) via a novel protease-activated receptor; 3) via PAR-4, which can be activated equally well by trypsin and thrombin (33, 67); or 4) via a non-PAR mechanism. Furthermore, a recent study (61) suggests the presence of a non-PAR-2, trypsin-sensitive receptor in the rat jejunum, providing further evidence for the existence of multiple PAR-2-like receptors. If it can be confirmed that there are subtypes of PAR-2 or multiple tryptase-sensitive PARs, this would have important implications for the pharmacological regulation of tryptase-mediated cellular effects. The discovery of distinct PAR-2 subtypes would raise the possibility of developing tissue-specific PAR-2 inhibitors.
In summary, we have confirmed that tryptase is mitogenic for both fetal and adult lung fibroblasts derived from either the parenchyma or airway. Proteolytic cleavage is required for the proliferative activity of tryptase. We also demonstrated the expression and localization of PAR-2 to the cell surface of the lung fibroblasts and that activation of these receptors with PAR-2 activating peptides induced proliferation. These data are consistent with the hypothesis that tryptase-induced lung fibroblast proliferation occurs via the activation of PAR-2 and that release of tryptase from activated mast cells may play an important role in fibroblast proliferation and the extracellular matrix protein deposition observed in the airways of patients with asthma and chronic obstructive pulmonary disease and in the lungs of patients with pulmonary fibrosis.
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ACKNOWLEDGEMENTS |
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This work was funded by Glaxo Wellcome Research and Development Limited.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. J. McAnulty, Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, The Royal Free and University College Medical School, The Rayne Institute, 5 University St., London WC1E 6JJ, UK (E-mail: r.mcanulty{at}ucl.ac.uk).
Received 19 March 1999; accepted in final form 25 August 1999.
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