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Abstract |
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Abstract
Introduction
Materials and Methods
Results
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Acute and chronic interstitial lung diseases are accompanied by evidence of inflammation and vascular injury. Thrombin activity in bronchoalveolar lavage fluid from such conditions is often increased, as well as
interleukin (IL)-8. We observed that conditioned medium from lung fibroblasts exposed to thrombin has
chemotactic activity for polymorphonuclear cells, and that this activity can be abolished by antibody to IL-8.
We report that thrombin stimulates expression of IL-8 in human lung fibroblasts on both the messenger RNA and protein levels in a time- and dose-dependent manner. Stimulation of IL-8 expression by thrombin is inhibited by specific thrombin inhibitors. Synthetic thrombin receptor agonist peptide-14 mimics thrombin's stimulation of IL-8 expression in a dose-dependent manner consistent with the idea that upregulation of IL-8 by thrombin in human lung fibroblasts requires cleavage of proteolytically activated receptor-I. We demonstrate further that thrombin-induced IL-8 synthesis is regulated by protein kinase (PK) C. PKC-
may be involved in the upregulation of lung fibroblast IL-8 by thrombin because stimulation of
lung fibroblasts with thrombin caused significant upregulation of PKC- and because PKC- antisense
oligonucleotides inhibited the accumulation of PKC- protein and IL-8 protein. Our data suggest that the
PKC- isoform increase observed after thrombin stimulation is required for thrombin-induced IL-8 formation by human lung fibroblasts.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Thrombin, a multifunctional serine protease generated at
sites of vascular injury, has central functions in thrombosis
and hemostasis but also promotes a wide range of cellular
responses (1, 2). Thrombin mediates a variety of inflammatory and tissue-repair responses associated with vascular injury (3). Current concepts regarding pathogenesis
of chronic fibrotic lung diseases involve microvascular injury with fibroproliferative repair culminating in pulmonary fibrosis (6, 7). Vascular injury, which occurs early in
fibrotic lung disorders, is characterized by disruption of
the endothelium and damage to the intima of the blood
vessel (3, 8, 9). Thrombin is one of the first factors to appear after vascular injury and can persist at sites of injury
when released from fibrin clots undergoing degradation by
plasmin (10). Thrombin is chemotactic for monocytes and
is mitogenic for lymphocytes and mesenchymal cells (11-
13). It enhances production of platelet-derived growth factor (PDGF), a potent mitogen for smooth-muscle cells and
fibroblasts (12, 14, 15), and latent transforming growth factor (TGF)-
1 (TGF-1) in smooth-muscle cells (16). Recently it has been found that thrombin also induces extracellular matrix proteins such as fibronectin in epithelial cells and in fibroblasts (17) and procollagen in smooth-muscle cells (10) and endothelial cells (18). Thus, thrombin may have profibrogenic activity as well.
Thrombin's participation in the pulmonary fibroproliferative process is poorly understood. Thrombin alone is
mitogenic and chemotactic for lung fibroblasts (5, 14, 19).
Previously, we reported that the mitogenic effect of
thrombin on human lung fibroblasts is mediated mainly
via the upregulation of the PDGF 
-receptor and its
ligand, PDGF-AA (14). Additionally, thrombin stimulates
lung fibroblasts to secrete other mediators involved in pulmonary fibrosis, such as the proinflammatory cytokine interleukin (IL)-8 (20). Factors such as PDGF (14), TGF-1
(16), IL-6 (21), IL-8 (20), fibronectin (17), and collagen
(10, 18), each of which appears to be stimulated by thrombin, and thrombin itself, are elevated in bronchoalveolar
lavage (BAL) fluid of patients with interstitial lung disease
such as idiopathic pulmonary fibrosis and scleroderma
lung disease (14, 20, 22). This suggests that together
they may function in pulmonary fibrosis and that thrombin, by inducing these factors, may initiate some of the
events during pulmonary fibrosis.
Cellular responses induced by thrombin are due in most
cases to activation of the proteolytically activated receptor-I (PAR-I) (26). In the proteolytic pathway -thrombin
cleaves its receptor's N-terminal extension site to create a
new N-terminus that functions as a tethered ligand and activates the receptor (26). A synthetic 14-amino acid thrombin receptor agonist peptide (SFLLRNPNDKYEPF) known
as TRAP-14, corresponding to the N-terminal portion of
the receptor, induces most of cellular responses characteristic of native thrombin, e.g., platelet aggregation, increased endothelial cell calcium concentrations, activation
of phospholipase C, neutrophil adhesion, and induction of
procollagen, IL-6, and TGF-1 (10, 16, 26, 27, 28).
Signal transduction mechanisms underlying the various
cellular responses to thrombin have been found to be dependent on the protein kinase (PK) C pathway (15, 29).
The mechanism whereby thrombin induces IL-8 production is not well understood. The present study was undertaken to investigate the mechanism of thrombin-induced IL-8 synthesis in human lung fibroblasts, including whether
this induction is mediated by PAR-I and whether it is regulated by PKC signaling. Our data suggest that thrombin is
a potent inducer of IL-8 in human lung fibroblasts, that
this induction requires proteolytic cleavage of the thrombin receptor PAR-I, and that such induction is regulated
by PKC- signaling.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
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Discussion
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Reagents
PKC activators were phorbol 12-myristate 13-acetate (PMA)
(30 ng/ml) (Sigma Chemical Co., St. Louis, MO), 12-deoxyphorbol 13-phenyl acetate (dPP) (5 nM) (Alexis Biochemicals Corp., San Diego, CA); inhibitors were PMA (20 ng/
ml) and calphostin C (100 nM) (Calbiochem, La Jolla, CA).
Phosphorothioate-modified PKC oligonucleotides were
synthesized in the Biochemistry and Molecular Biology facility at the Medical University of South Carolina (Charleston, SC): antisense oligonucleotide for PKC- (5'-CGGGAAAACGTCAGCCAT-3') and sense oligonucleotide
(5'-ATGGCTGACGTTTTCCCG-3') as a control. Lipofectin (10 µg/ml) (GIBCO BRL, Grand Island, NY), leukotriene (LT) B4 (100 nM) (Sigma), -thrombin (0.0 to 10 U/ml), catalytically inactivated diisopropylfluorophosphate
(DIP)--thrombin (500 ng/ml), and -thrombin (500 ng/
ml) were made from -thrombin as described previously
(2). D-Phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone
(PPACK) (5 nM) and hirudin (1 µM) were obtained from Calbiochem.
Cell Culture
Normal human adult lung fibroblast cell line CCD 34Lu was obtained from American Type Culture Collection (ATCC; Rockville, MD). Monolayer cultures were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO BRL) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 µg/ml gentamicin sulfate, and 5 µg/ml amphotericin B at 37°C in 10% CO2.
Synthesis of Peptide
TRAP-14 was synthesized employing the tBOC method of Merrifield (30) on an automated peptide synthesizer (Applied Biosystems, Foster City, CA). The peptide was purified to greater than 95% homogeneity by high-performance liquid chromatography. The correct sequence was confirmed by automated gas phase sequencing on a Proton 2090E sequencer.
RNA Preparation and Northern Blot Analysis
Total RNA was extracted using an acid guanidinium thiocyanate-phenol chloroform method (31) from human lung fibroblasts stimulated with various concentrations (1 to 10 U/ml) of thrombin and with specific thrombin inhibitors PPACK (5 nM), hirudin (1 µM), and thrombin (10 U/ml) for 4 h. Total RNA was also extracted from the cells stimulated with thrombin (5 U/ml) for various time intervals (0 to 48 h). RNA (10 µg) was subjected to electrophoresis on 1% agarose formaldehyde gel and blotted to nylon filters (S&S Nytran, Schleicher and Schuell, Keene, NH). The filters were crosslinked by ultraviolet irradiation using a Stratalinker (Stratagene, La Jolla, CA) and prehybridized for 2 h at 65°C. They were then hybridized at 42°C for 24 h in hybridization buffer (50% deionized formamide, 20% dextran sulfate, 1% sodium dodecyl sulfate [SDS], 1 M NaCl, and herring sperm DNA, 0.1 mg/ml). Blots were then washed twice in 2× saline sodium citrate (SSC) (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS for 5 min at room temperature and twice in 0.1× SSC, 0.1% SDS for 15 min at 42°C. The following probes were used: human IL-8, 550-base pair (bp) EcoRI fragment kindly obtained from Dr. M. Bong (Washington University, St. Louis, MO); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 437-bp BamHI, EcoRI fragment. Probes were labeled with a random-primed DNA labeling kit. IL-8 messenger RNA (mRNA) level was expressed as a ratio of expression in stimulated cells to expression in unstimulated cells. Relative intensity of the signals with each probe was quantified using a PhosphorImager (Molecular Dynamics, Sunnyville, CA).
Determination of IL-8 by Enzyme-Linked Immunosorbent Assay
The concentration of IL-8 from conditioned media and
cell extracts (prepared as for Western blot analysis, described later) of human lung fibroblasts was determined
by means of a two-site enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies (Quantikine
Assay; R&D Systems, Minneapolis, MN). Cells were stimulated in serum-free DMEM with various concentrations
(0.1 to 10 U/ml) of thrombin or DIP--thrombin (500 ng/ml),
or -thrombin (500 ng/ml), or TRAP-14 (1-200 µM). The
effect of 5 nM PPACK or 1 µM hirudin on stimulation of
IL-8 expression by thrombin was determined. The amount
of culture medium from fibroblasts was adjusted to cell
count in each experiment. In time-course experiments the amount of culture medium from fibroblasts was adjusted
to protein level of cell extracts, and equal amounts of protein from cell extracts were applied for ELISA tests. Absorbance was determined using an ELISA microplate
reader (Anthos 2001; Anthos Labtec Instruments, Frederick, MD) at 450 nm wavelength. Concentrations of IL-8
(pg/ml) were calculated from a standard curve.
Neutrophil Migration Assay
Whole blood was collected into syringes containing ethylenediaminetetraacetic acid (4 mM final concentration), then diluted with an equal part of phosphate-buffered saline and layered on a Percoll gradient with a density of 1.082. The gradient was centrifuged at 1,200 × g for 20 min. The upper layer of mononuclear cells was removed, and the lower layer (erythrocytes, granulocytes) was subjected to ammonium chloride lysis buffer to remove erythrocytes. The remaining cells, 90 to 95% neutrophils, were resuspended in DMEM and added to the insert of a cell migration chamber (Biocoat control inserts; Becton Dickinson Labware, Bedford, MA). Lung fibroblasts were grown to confluency and then treated with thrombin (5 U/ml) for 24 h. Part of the conditioned media from fibroblasts stimulated with thrombin was incubated with monoclonal anti- IL-8 antibody (2.5 µg/ml) (R&D Systems) for 2 h at 37°C. Recombinant IL-8 (100 nM) (R&D Systems), thrombin (5 U/ml), and collected medium from fibroblasts stimulated with thrombin (5 U/ml) were added to the lower well of the migration system to serve as chemoattractant. Neutrophils (2.5 × 105/well) were applied to each insert and after 4 h incubation, cells that had migrated into the lower chamber were counted with a Coulter counter. Chemotaxis to LTB4 (100 nM) was used as a positive control for each experiment. Neutrophils were obtained from four different donors and assays were performed in duplicate.
Determination of the Effect of PKC Activators and Inhibitors on Thrombin-Induced IL-8 Production by Lung Fibroblasts
To activate PKC, lung fibroblasts (34Lu) were pretreated with PMA (30 ng/ml) for 15 min, or dPP (5 nM) for 1 h. Then thrombin (5 U/ml) was added for 24 h. To inhibit PKC isoforms, cells were pretreated with PMA (20 ng/ml) for 3 d or with calphostin C (100 nM) for 40 min before thrombin treatment. After 24 h of thrombin treatment, conditioned medium was collected and the concentration of IL-8 protein in the medium was determined by ELISA.
Western Blot Analysis of PKC Isoforms
Western blot analysis was performed as described elsewhere (14), with some modifications. Briefly, soluble cytoplasmic proteins from normal lung fibroblasts (34Lu;
ATCC) stimulated with thrombin (5 U/ml) for 5, 10, 20, and 60 min and 24 h were prepared in Laemmli buffer
(30). Equal amounts of protein (determined by Bio-Rad protein assay) were applied to each well and electrophoresed in 6% SDS-polyacrylamide gel for 2 h at 20 mA. The
gel was electroblotted onto nitrocellulose filters and incubated for 60 min in Tris-buffered saline (100 mM NaCl,
50 mM Tris HCl [pH 7.4], and 0.01% sodium azide) containing 5% nonfat powdered milk. Then filters were incubated with monoclonal antibody against PKC- and other
PKC isoforms (, 
, 
, 
, and 
) (dilutions 1:1,000 and
1:5,000, respectively) (Transduction Lab, Lexington, KY)
overnight. After washing, filters were incubated with rabbit antimouse immunoglobulin G antibody conjugated
with peroxidase (1:2,500 dilution) (Calbiochem) for 1 h,
washed, then developed using the enhanced chemiluminescence system (ECL System; Amersham Corporation,
Arlington Heights, IL).
PKC Oligonucleotide Treatment of Cells
Antisense oligonucleotides for PKC- and appropriate
sense oligonucleotides (control) were used at final concentrations of 0.8 to 2 µM. Oligonucleotides were dissolved in
culture medium, sterilized by filtration through 0.2-µm
cellulose acetate filters, and added to cell cultures of human lung fibroblasts (34Lu; ATCC) at the indicated concentrations. Cells were pretreated with lipofectin (10 µg/ml)
(to increase permeability of the cells) and oligonucleotides (0.8 to 2 µM) for 4 h, then medium was changed and incubations were performed for 2 d with oligonucleotides (0.8 to 2 µM) only and were treated with thrombin (5 U/ml)
for an additional 24 h. Cells were then used for protein determination of PKC- and PKC- by Western blot analysis and conditioned media were used for IL-8 determination by ELISA.
Statistical Analysis
IL-8 levels were expressed as means ± standard deviation (SD) of results obtained from three individual experiments performed in duplicate or triplicate. Statistical analysis was performed using GraphPad InStat software, version 2.03.
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Abstract
Introduction
Materials and Methods
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Discussion
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Thrombin Increases IL-8 mRNA Expression in Human Lung Fibroblasts
Human lung fibroblasts stimulated with thrombin at concentrations ranging from 1 to 10 U/ml showed a dose- dependent increase in IL-8 mRNA levels with maximal expression at 10 U/ml of thrombin. Thrombin's effect on IL-8 synthesis was blocked by the specific thrombin inhibitors PPACK (5 nM) and hirudin (1 µM) (Figure 1). Quantitative analysis of IL-8 mRNA expression after stimulation with 10 U/ml of thrombin at time points from 0.5 to 24 h revealed an early induction starting between 1 and 2 h with maximal upregulation by 6 h, suggesting a direct induction of IL-8 mRNA by thrombin. After 24 h, IL-8 mRNA levels returned to baseline (Figure 2).
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Thrombin Increases IL-8 Protein Production by Human Lung Fibroblasts
Human lung fibroblasts stimulated with thrombin at concentrations ranging from 0.1 to 10 U/ml showed a dose- dependent increase in IL-8 protein in the cell culture supernatant (Figure 3). Maximal IL-8 protein concentration was noted after exposure to 10 U/ml of thrombin and was 10-fold higher than control levels. The specific thrombin inhibitors PPACK (5 nM) and hirudin (1 µM) inhibited thrombin- induced IL-8 secretion by approximately 70 and 60%, respectively (Figure 3). We obtained similar results with primary cultures of lung fibroblasts (Ludwicka-Bradley and colleagues, unpublished observations), but because primary cultures are not easily available all experiments presented here were performed with an ATCC cell line of lung fibroblasts.
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We also compared the levels of IL-8 protein in the culture medium and the cell layer. Thrombin (5 U/ml) treatment increased IL-8 protein expression in the cell layer. Maximum cell extract levels were detected at 6 h, yet elevated expression was still detectable at 24 h. Levels of IL-8 in the culture medium increased throughout the 24-h incubation (Figure 4), reflecting secretion of IL-8 by lung fibroblasts. Much higher levels of IL-8 in the medium than in the cell layer observed were due to constantly synthesized IL-8 and rapidly secreted into the medium.
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Chemotactic Activities of Conditioned Media from Lung Fibroblasts Stimulated with Thrombin
Conditioned medium from human lung fibroblasts exposed to thrombin (5 U/ml) had chemotactic activity for polymorphonuclear cells (neutrophils). This activity was abolished by monoclonal antibody to IL-8, indicating that the chemotactic activity was due to IL-8 secreted by the fibroblasts in response to thrombin (Table 1). The chemotactic activity of the IL-8 induced by thrombin was comparable to the chemotactic activity of recombinant IL-8 (100 nM) and of LTB4 (100 nM), a known neutrophil chemoattractant. These data suggest that lung fibroblast secretion of IL-8 may contribute significantly to the inflammatory events in fibroproliferative conditions.
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TRAP-14 Stimulates IL-8 Secretion by Human Lung Fibroblasts
Next we sought to establish whether thrombin's effect on
fibroblast IL-8 secretion is mediated via proteolytic cleavage of PAR-I. Because it has been shown that the synthetic TRAP-14 mimics thrombin's stimulation of a number of cellular responses, we determined the expression of
IL-8 protein by human lung fibroblasts stimulated with
various concentrations of TRAP-14. We found that thrombin receptor agonist increases IL-8 secretion in a dose-
dependent manner at a maximum level at 200 µM TRAP-14 (Figure 5). To confirm these data we used two other
thrombins: DIP--thrombin with disrupted catalytic activity, and -thrombin with catalytic activity but disrupted
binding site. DIP--thrombin did not induce IL-8 production in human lung fibroblasts, whereas -thrombin had an
upregulatory effect that was 15-fold smaller than -thrombin (Ludwicka-Bradley and associates, unpublished observations). Together these data show that thrombin's proteolytic pathway is involved in IL-8 secretion from human
lung fibroblasts stimulated with thrombin.
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Effect of PKC Activators and Inhibitors on Thrombin-Induced IL-8 Synthesis
Because many thrombin-mediated cellular responses are regulated by the PKC pathway, we examined whether the PKC pathway is involved in thrombin-induced IL-8 formation by human lung fibroblasts. Testing various PKC activators and inhibitors, we found that activation of lung fibroblasts either with the potent PKC activator PMA, or dPP, another specific activator of PKC, led to a significant increase in IL-8 synthesis induced by thrombin (Figure 6A). On the other hand, PKC depletion by pretreatment of cells with PMA for 3 d or inactivation with calphostin C, a specific PKC inhibitor, abolished thrombin-induced IL-8 synthesis (Figure 6B).
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Involvement of PKC- in Thrombin-Induced
IL-8 Formation
We observed that thrombin upregulates PKC- after 10 min stimulation in lung fibroblasts when compared with
nontreated cells and that the level remains elevated after
24 h of thrombin treatment (Figure 7). Therefore, to test
the idea that PKC- is an intermediate in the induction of
IL-8 by thrombin, we treated cells with antisense oligonucleotides and sense (control) for PKC-. Western blot
analysis showed that the accumulation of this isoform at
the protein level is blocked by antisense oligonucleotide
(Figure 8A). Sense oligonucleotide for PKC- did not affect PKC- protein level (Ludwicka-Bradley and coworkers, unpublished observations). Antisense oligonucleotide for PKC- did not affect other isoforms such as PKC-
(Figure 8B), confirming that downregulation of PKC-
protein level was specific. Moreover, antisense oligonucleotide for PKC-, but not sense oligonucleotide, inhibited
thrombin-induced IL-8 formation by 50% (Figure 9). These
data suggest that PKC- is one intermediate in the induction
of IL-8 expression by thrombin in human lung fibroblasts.
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The importance of thrombin in mediating nonthrombotic responses is not fully understood. As a multifunctional cytokine regulating proliferation of many cells, thrombin stimulates expression of other cytokines and also activates the respective cytokine receptor (1, 14, 21, 32). Thrombin appears after vascular injury (33) characteristic of early events of interstitial lung diseases and induces proinflammatory cytokines, e.g., IL-6 in endothelial cells and fibroblasts (27) and IL-8 in endothelial cells (32). Previously we reported that thrombin activity is significantly increased in BAL fluid from scleroderma (SSc) patients with pulmonary fibrosis (14), who also have a significant increase in IL-8 levels (23). These observations have been confirmed by others (24, 25). The importance of IL-8 in the pathogenesis of a variety of acute and chronic lung diseases is well established (34), but the mechanism of its induction is not well understood. In the present study we investigated the effect of thrombin on IL-8 expression in human lung fibroblasts. We found that thrombin induces IL-8 mRNA expression as well as IL-8 protein synthesis. Both effects are dose- and time-dependent, and we conclude that thrombin is a potent inducer of IL-8 in human lung fibroblasts. Observations that thrombin-induced IL-8 production by these cells is inhibited by the specific thrombin inhibitors PPACK and hirudin confirm this conclusion.
IL-8 may play a key role in the pathogenesis of pulmonary fibrosis by recruiting and activating neutrophils within the local microenvironment. In patients with adult respiratory distress syndrome in whom thrombin and its byproducts are elevated, IL-8 is increased both systemically and in BAL fluid, where the concentration of IL-8 correlates with mortality (34, 35). Inhibition of IL-8 is protective against acute lung injury in certain animal models of lung disease (36, 37), further implicating IL-8 in the pathogenesis of lung fibrosis. Further, anti-IL-8 antibodies significantly ameliorate tissue damage associated with reperfusion injury and chemical pneumonitis (37, 38).
IL-8's effects on polymorphonuclear cells (PMNs) are well described and include chemoattraction, upregulation of adhesion molecules, enhancement of transendothelial migration, and stimulation of respiratory burst (35, 39). Chemotaxis is an important functional response to IL-8 and is a key event in the recruitment of neutrophils in inflammation. In idiopathic pulmonary fibrosis and in SSc lung disease, IL-8 present in epithelial lining fluid may cause the influx of neutrophils. In such conditions, levels of IL-8 in BAL fluid are correlated with fibrosing alveolitis and a greater degree of lung injury (25). We have shown that conditioned medium from human lung fibroblasts stimulated with thrombin has chemotactic activity for PMNs and that this activity is abolished by anti-IL-8 antibody (Table 1). The chemotactic activity of the IL-8 induced by thrombin was comparable to the activity of 100 nM of the neutrophil chemoattractant LTB4. Therefore, our results suggest that lung fibroblasts stimulated with thrombin may contribute to pulmonary inflammation by releasing IL-8 at levels capable of promoting neutrophil migration into lungs.
Another objective of our study was to investigate whether the response of human lung fibroblasts to thrombin is mediated by the proteolytically activated thrombin receptor PAR-I. A significant role for the thrombin receptor in vascular pathology has recently been postulated (40). The thrombin receptor is expressed at low levels in normal arteries but is upregulated after vascular injury (40). High expression of thrombin receptor is observed in rheumatoid synovial tissue and atherosclerotic lesions (40, 41) and has been found to contribute to the progression of vascular lesions (33). Upregulated thrombin receptor has also been found in glomerulonephritis (42). Another fact of great importance is that the thrombin receptor is tissue-specific. Studies in knockout mice lacking PAR-I have shown that PAR-I plays only a minor role in platelet activation (43), while all known thrombin responses in fibroblasts were ablated (44, 45).
To investigate whether thrombin-induced IL-8 formation requires proteolytic activation of the thrombin receptor, we stimulated human lung fibroblasts with the synthetic TRAP-14, which has been shown to induce a number
of cellular responses characteristic of native thrombin (46-
52). We found that TRAP-14 stimulates production of IL-8
protein by human lung fibroblasts, albeit to a somewhat
lesser extent than does native -thrombin. To confirm these results we employed DIP--thrombin, which has no
enzymatic activity but still can bind receptor via anion
binding exosite, and -thrombin, which is enzymatically
active but has the binding site disrupted. DIP--thrombin
did not induce IL-8 whereas -thrombin had an upregulatory
effect, although that was lower than -thrombin. These
data suggest that thrombin-induced IL-8 in lung fibroblasts is mediated by thrombin's proteolytic mechanism of
receptor activation, a feature consistent with the known
properties of the seven-transmembrane domain receptor
for thrombin.
Several cellular responses to thrombin are mediated via
PKC signal transduction (53, 54). We demonstrated that
thrombin-induced IL-8 production by lung fibroblasts is
also regulated by a PKC-dependent pathway. Thrombin's
induction of IL-8 was significantly suppressed by the specific PKC inhibitor calphostin C and enhanced by PMA or
the specific PKC activator dPP. Among various PKC isoforms, PKC- is well expressed in human lung fibroblasts.
We found that thrombin upregulates PKC- protein levels
in normal lung fibroblasts. PKC- antisense oligonucleotide
downregulated but did not completely abolish thrombin-induced IL-8 formation, and therefore this may suggest involvement of other PKC isoforms in thrombin's induction of IL-8.
Our results provide a compelling link between thrombin and the proinflammatory cytokine IL-8 and provide additional support for the notion that thrombin may have important physiologic consequences in inflammatory diseases of the lung. These studies suggest that lung fibroblasts, by secreting IL-8, may contribute to the neutrophilic alveolitis characteristic of interstitial lung diseases such as idiopathic pulmonary fibrosis and SSc lung disease. Future therapeutic strategies may be derived from knowledge of the regulatory mechanisms whereby thrombin upregulates cytokine expression.
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Footnotes |
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Abbreviations: analysis of variance, ANOVA; bronchoalveolar lavage, BAL; diisopropyl fluorophosphate, DIP; Dulbecco's modified Eagle's medium, DMEM; 12-deoxyphorbol 13-phenyl acetate, dPP; enzyme-linked immunosorbent assay, ELISA; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; interleukin, IL; leukotriene, LT; messenger RNA, mRNA; proteolytically activated receptor-I, PAR-I; platelet-derived growth factor, PDGF; protein kinase, PK; phorbol 12-myristate 13-acetate, PMA; polymorphonuclear cells, PMNs; D-phenylalanyl-L-prolyl-L-arginyl-chloromethylketone, PPACK; standard deviation, SD; sodium dodecyl sulfate, SDS; serum-free medium, SFM; scleroderma, SSc; saline sodium citrate, SSC; transforming growth factor, TGF; 14-amino acid thrombin receptor agonist peptide-14, TRAP-14.
(Received in original form December 10, 1998 and in revised form August 18, 1999).
Acknowldgments:Acknowledgments: This work was supported in part by grants from the RGK Foundation, the United Scleroderma Foundation, the Medical University of South Carolina's Environmental Biosciences Program, and a Clinical Research Center Grant from the National Institutes of Health (RR1070-1). The authors thank Maria Trojanowska for critical reading of the manuscript and Vicki Kivett and Ann Donaldson for manuscript and figure preparation.
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References
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