1Centre for Respiratory Research, University College London, United Kingdom; 2Pulmonary and Critical Care Medicine, Vanderbilt University; 3St. Thomas Hospital, Nashville, Tennessee; 4Asthma and Allergy Research Institute & Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, Australia; and 5Respiratory Division-Heart Institute (InCor), University of São Paulo, São Paulo, Brazil
Submitted 12 May 2004 ; accepted in final form 15 November 2004
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ABSTRACT |
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proteinase-activated receptor; pleura
Proteinase-activated receptors (PARs) belong to a family of novel G protein-coupled, seven-transmembrane receptors (9). To date, four types of PARs have been described. PAR1, PAR3, and PAR4 (but not PAR2) are thrombin receptors (16, 17, 38), whereas trypsin, tryptase, and coagulation factor Xa have been shown to activate PAR2 (22). PAR2 is believed to be distinct from the other PARs, and evidence to date suggests that it has a regulatory role in inflammation (11, 14, 36), especially in epithelial cells e.g., airway epithelial cells (3) and keratinocytes (15). The visceral and parietal pleurae are lined by a monolayer of mesothelial cells, which are cells of mesenchymal origin with epithelial characteristics. However, whether PAR2 is present on mesothelial cells and has functional significance in the pleura has not been studied.
The results of the present study show that PAR2 is present in human pleural tissues, in both normal and pathological states, and plays a role in pleural inflammatory responses. We demonstrate that activation of PAR2 on mesothelial cells, by its known physiological activators as well as by a specific activating peptide, elicits chemokine production in vitro. In vivo, activation of PAR2 on pleural mesothelial cells results in chemokine release and neutrophil recruitment into the pleural cavity.
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METHODS |
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Immunohistochemistry
To establish whether PAR2 is present in the pleura, parietal (n = 19) and visceral (n = 11) pleural biopsy samples from patients who underwent thoracoscopy or thoracotomy were collected. Thin (5 µm) sections were immunostained using a mouse monoclonal anti-PAR2-antibody, SAM II (kindly provided by Prof. Lawrence Brass, University of Pennsylvania) (32). A modification of the avidin-biotin-peroxidase complex method was used as previously described (18). Sections were fixed by immersion in ice-cold acetone for 10 min and then rinsed in phosphate-buffered saline (PBS, pH 7.6). Endogenous peroxidase activity was quenched by the addition of 0.5% (vol/vol) hydrogen peroxide for 5 min. The sections were incubated with 10% (vol/vol) normal swine serum to reduce background staining, followed by incubation with the primary antibody at a dilution of 1:50 for monoclonal and 1:100 for polyclonal antibodies for 1 h at room temperature. The sections were incubated with biotin-conjugated rabbit anti-mouse IgG (Dako 1:200) for 45 min. After repeated washes in 1 M Tris-buffered saline (pH 7.4), sections were incubated with peroxidase-conjugated streptavidin for 45 min. Immunostaining was visualized by the addition of 3,3-diaminobenzidine (Sigma, St. Louis, MO) and hydrogen peroxide and counterstained with Gills hematoxylin. Negative control experiments were performed using isotype-matched immunoglobulins.
The diagnoses from hospital records were independently verified by a qualified pulmonologist (Y. C. G. Lee). Pleural biopsies were collected from patients who had abnormal pleurae (7 postcoronary artery bypass graft pleuritis, 2 empyema, and 1 fibrous tumor of the pleura) and from 11 patients undergoing lobectomy (for lung carcinoma) in whom normal visceral pleurae (histologically confirmed) were obtained at sites distant from the tumor.
To quantify the amount of PAR2 in the pleura, a semiquantitative system was used as described by Detre et al. (10). For each sample, six random fields were counted by an experienced scorer (D. A. Knight) blinded to the clinical data. The intensity of staining was graded from 0 to 3 (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong) under x10 magnification. The percentage of stain-positive cells was also counted at x40 magnification and semiquantitatively graded from 1 to 6 (1 = <5%, 2 = 519%, 3 = 2039%, 4 = 4059%, 5 = 6079%, and 6 = 80100%). The total score was the product of the intensity and the quantity scores.
Reagents
SLIGRL-NH2, a known specific PAR2-activating peptide, and a control peptide, LSIGRL-NH2 [previously shown not to activate PAR2 (3)], were obtained from United Biotech Research (Seattle, WA) for the in vivo and in vitro studies. The peptides were confirmed to be of >95% purity by mass spectrometry and HPLC chromatograph. The peptides, tryptase (Calbiochem, La Jolla, CA), trypsin (Sigma), and coagulation factor Xa (Calbiochem) were diluted in serum-free Dulbecco's modified Eagle's medium (DMEM, Sigma) for cell culture experiments. The peptides were diluted with sterile PBS for the in vivo experiments.
In Vitro Experiments
Murine mesothelial cell harvesting and culture. Pleural and peritoneal mesothelial cells were harvested from C57BL/6 wild-type mice. Pleural mesothelial cells were harvested as we have previously described (23, 27). In brief, mice were euthanized with CO2, and the abdomen opened to expose the diaphragm. Under direct vision, Hanks' balanced salt solution (Life Technologies, Grand Island, NY) was injected into the pleural cavity from beneath the diaphragm and aspirated after 2 min. Then, 1.0 ml of 0.25% trypsin-EDTA (Life Technologies) was injected into the pleural space in the same way and left in situ for 10 min during which the mice were rotated. The solution was then aspirated and transported in DMEM with fetal bovine serum (FBS, Life Technologies). Peritoneal and omental mesothelial cells were also harvested and incubated in 0.25% trypsin-EDTA at 37°C for 30 min as described previously (13).
Cells were centrifuged at 300 g for 5 min, resuspended in DMEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine, and cultured in 75-cm2 flasks at 37°C and 5% CO2. The medium was changed the following day to remove nonadherent cells and thereafter every 35 days. Cells between passages 2 and 4 were used in the experiments.
Mesothelial cell cytokine production by PAR2-activating peptides. To study the in vitro cytokine response to PAR2 activation, we treated murine mesothelial cells with SLIGRL-NH2 or LSIGRL-NH2 in log doses (10, 100, and 1000 µM) or with culture media alone. Cells were plated in 48-well plates, and the medium was changed to serum-free DMEM immediately before the study. Supernatants were collected at the end of 4 or 24 h and stored at 70°C until assay. Cells in each well were then lysed with 500 µl of a lysis agent containing 0.5% SDS. Protein levels of the lysate were determined by a BCA protein assay (Pierce Chemical, Rockford, IL) with an incubation time of 2 h at room temperature. All cytokine measurements were normalized to the corresponding protein concentration to ensure that differences were not due to variation in cell number in individual wells. The study was repeated for a total of seven or eight wells for each agent at each time point. The results of the first and the repeat study were highly consistent, and the combined results are presented.
Mesothelial cell cytokine production by PAR2 physiological activators. To establish the effect of known physiological activators of PAR2 on MIP-2 production by mesothelial cells, we repeated the above experiments using log concentrations of tryptase (1100 nM), trypsin (101,000 nM), and coagulation factor Xa (101,000 nM) for 24 h. The media and cell lysate were collected as described above.
Inhibition of PAR2-induced cytokine production from mesothelial cell. To study the effects of steroids on PAR2-induced MIP-2 production, we cultured murine mesothelial cells with dexamethasone (1071011 M) and stimulated them with 100 µM SLIGRL-NH2. Cells cultured with media alone, and with the highest dose of dexamethasone without the activating peptide, were used as controls. The medium was collected after 24 h and assayed for cytokines, as described above.
In Vivo Experiments
Wild-type C57BL/6 male mice were used. The mice were sedated with isoflurane (Baxter, Deerfield, IL) followed by an intraperitoneal injection of 35 mg/kg of ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, IA) and 5 mg/kg of xylazine hydrochloride (Fermenta, Kansas City, MO). The left chest was shaved, and the skin was sterilized with alcohol. Reagents were delivered intrapleurally by a single injection via a 1-ml syringe with an ultrafine (28 G) needle from underneath the left lateral ribs.
To validate the method of injection, mice were given an intrapleural injection of 0.21.0 ml of 0.9% saline with trypan blue dye. In all mice, expansion of the chest cavity and increased respiratory rates were observed immediately on intrapleural injection, confirming accurate intrapleural delivery. Respiration usually returned to baseline on recovery from sedation. The mice were killed within 30 min. The injected solution was accurately delivered into the pleural space with even distribution of the dye throughout the visceral and parietal surfaces of left and right pleural spaces. In eight mice injected with 1.0 ml of volume, 80100% of the injected solution was recovered at 30 min.
In the 4-h study, 16 mice were injected intrapleurally with either 10 mg/kg of SLIGRL-NH2, 10 mg/kg of LSIGRL-NH2, or PBS in 0.75 ml. The same experiment was also performed for 24 h. The only variation was that a higher injection volume of 1.0 ml was used for the 24-h study. In the 4-h study, three mice died: one at the induction of anesthesia and two within 15 min of intrapleural injection. The mice were killed after 4 or 24 h by CO2 inhalation. The abdominal cavity was opened to expose the diaphragm. For the 4-h experiment, residual pleural fluid was aspirated from beneath the diaphragm under direct vision. For the 24-h experiment, none of the mice had residual pleural fluids. A pleural lavage was then performed with 1.0 ml of PBS. The lavage fluid was recovered after 5 min and placed on ice.
Total and differential cell counts were measured in the pleural fluids. The pleural fluids and the lavage fluids were centrifuged at 1,000 g for 10 min. The cell pellets were resuspended in 1.0 ml of 1% bovine serum albumin in sterile physiological saline for counting in a hemacytometer. We obtained differential cell counts by mounting 50,000 cells onto a cytocentrifuge slide and staining them with a modified Wright's stain (Diff-Quik; Baxter, Miami, FL), as we have previously described for bronchoalveolar lavage fluids (1921). The neutrophil counts in 20 random high-power fields on each slide were recorded by an experienced reader (M. A. Koay), who was blinded to the treatment groups. The supernatant was stored at 70°C until cytokine measurement. At the time of death, blood was also collected from the inferior vena cava, centrifuged at 1,000 g for 10 min, and stored at 70°C until assayed.
Measurements of Cytokines
Macrophage inflammatory protein (MIP)-2, TNF-, and vascular endothelial growth factor (VEGF) concentrations were determined with enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN).
Statistical Analysis
Data were analyzed with a SigmaStat version 2.03 program (San Rafael, CA).
All data are expressed as means ± SE. The differences among groups were compared by one-way analysis of variance (ANOVA) or one-way ANOVA on ranks. Multiple comparisons (post hoc test) were performed using Tukey's or Dunn's test. Student's t-test and Mann-Whitney ranked-sum test were used to compare differences between two treatment groups (for parametric and nonparametric data respectively). Pearson's correlation was used to measure linear regression. P < 0.05 was considered significant.
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RESULTS |
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By immunohistochemistry, PAR2 was found to be strongly present in all the parietal and visceral pleural samples examined. The staining illustrated high density of PAR2 present in the mesothelial cells of the pleura (Fig. 1, top). PAR2 was also seen on the endothelium of the small vessels present in the biopsies (Fig. 1, bottom left). A similar pattern of PAR2 expression was also observed in pleural tissues from rabbits and C57BL/6 mice (not shown).
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In Vitro Experiments
The addition of the PAR2-activating peptide elicited a definite dose-dependent increase in MIP-2 [the murine homolog of human interleukin 8 (IL-8)] release compared with the addition of the control peptide or medium controls (Fig. 2A). The PAR2-activating peptide at 10 and 100 µM stimulated a twofold (P < 0.05) and a 40-fold (P = 0.001) increase in MIP-2 release respectively over medium controls. The response was maintained even at a peptide concentration of 1,000 µM, which stimulated a 1,330-fold increase in MIP-2 release compared with medium controls (P < 0.001). The PAR2-activating peptide stimulated significantly higher MIP-2 production compared with the control peptide at 10, 100, and 1,000 µM by twofold (P < 0.05), 32-fold (P < 0.01), and 75-fold (P < 0.01), respectively.
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Similar to its effect on MIP-2 production, the PAR2-activating peptide stimulated a significant dose-dependent increase in mesothelial cell TNF- production compared with the control peptide and the medium controls (Fig. 2B). The PAR2-mediated release of MIP-2 was inhibited by dexamethasone in a dose-dependent fashion (Fig. 3).
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To further confirm the proinflammatory role of PAR2 activation seen in the in vitro experiments, we injected mice intrapleurally with PAR2-activating peptides. In the 4-h experiment, the volume of pleural effusion recovered was very similar among the three groups: PAR2-activating peptide group 588 ± 99 µl, control peptide group 620 ± 25 µl, and PBS group 612 ± 43 µl (P = 0.92).
The pleural fluid MIP-2 levels in mice injected with PAR2-activating peptide (2,710 ± 165 pg/ml) were significantly higher than those injected with the control peptide (880 ± 357 pg/ml) or with PBS (85 ± 46 pg/ml) by 3- and 30-fold, respectively (P < 0.001 for both, Fig. 5A). Similarly, pleural fluid TNF- levels were significantly higher in mice given PAR2-activating peptide than those given the control peptide or PBS (43.2 ± 6.4 vs. 7.0 ± 3.1 vs. 0.0 ± 0.0 pg/ml, respectively, P < 0.001 for both; Fig. 5B). MIP-2 and the TNF-
levels were strongly correlated both in the pleural fluids (r = 0.92, P < 0.00001) and in the serum (r = 0.76, P = 0.002). Although there were marked differences of the cytokine levels in the pleural fluid, no significant differences were found in the serum levels of MIP-2 and TNF-
among the three groups (data not shown).
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The total number of nucleated cells in the pleural fluid was higher in the PAR2-activating peptide group (442 ± 98 x 104) than in the control peptide (179 ± 49 x 104) or the PBS control groups (223 ± 75 x 104) (P = 0.07). The predominant cell type in the pleural fluids (of all mice) was the mesothelial cell. When the inflammatory cells were counted, the number of neutrophils in the pleural fluids was significantly higher (131 ± 86 in 20 high-power fields) in the mice treated with PAR2 peptide than in those given the control peptide (3 ± 1) and those given PBS (5 ± 1) (P < 0.05, Fig. 6).
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When the same experiment was repeated at 24 h, there was no residual pleural fluid in any of the mice. As a result, pleural lavages were performed. The MIP-2 levels in the lavage fluid samples were low with no significant differences in the lavage fluid MIP-2 levels among the three groups (activating peptide group, 3.7 ± 1.0; reverse peptide group, 1.3 ± 0.5; PBS group, 8.1 ± 3.3 pg/ml; P = NS).
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DISCUSSION |
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Chemokine production and neutrophil influx underlie the pleural inflammatory process common to most pleural diseases (24), but the mechanism of mesothelial cell production of key chemokines has seldom been investigated. The pleural cavity is susceptible to attacks from pathogens or particulates (e.g., asbestos fibers) (8), either via direct invasion from a subpleural pulmonary focus or from hematogenous spread. Mounting a prompt inflammatory response to remove invading microbes is essential to prevent pleural space infection, such as empyema, which carries significant morbidity and mortality. However, in normal circumstances, relatively few leukocytes are present in the pleural space (33). Effective immunological defense in the pleural cavity relies on the appropriate production of potent chemokines by mesothelial cells (2, 26). IL-8 (or MIP-2 in mice) holds a pivotal role in the recruitment of neutrophils, monocytes, and lymphocytes to the pleural cavity (5). Conversely, antagonizing IL-8 activity can significantly impair recruitment of inflammatory cells to the pleural space (7) and may contribute to increased severity and lethality of pleural infections.
The mechanisms regulating mesothelial cell release of IL-8 (or MIP-2) must therefore be effective and efficient. This study shows that PAR2, a seven-transmembrane receptor, is present in high levels in the human pleura. Stimulation of PAR2 on mesothelial cells induced the prompt production of key inflammatory cytokines, and the direct stimulation of PAR2 in vivo resulted in significant neutrophil recruitment to the pleural space.
The role of PAR2 in inflammation has been investigated in different tissues and contrasting effects have been reported. Moffatt et al. (31) showed that activation of PAR2 inhibits leukocyte recruitment in murine airways in vivo following endotoxin challenges. Fiorucci et al. (12) also reported anti-inflammatory effects of PAR2 on lymphocytes from colonic lamina propria and a protective effect in mice against colitis. In contrast, PAR2 stimulation has been shown to induce inflammatory cytokine release in many cell types, including airway epithelial cells (3, 4), macrophages (34), eosinophils (30, 37), keratinocytes (15), and microvascular endothelial cells (35), and can potently stimulate the release of proinflammatory neurogenic peptides from neurons (36). Additionally, PAR2-deficient mice display delayed responses to inflammatory challenges following trauma (28).
Our data raise further questions that require exploration. To date, the most well-characterized naturally occurring activators of PAR2 include trypsin, tryptase, and coagulation factor Xa (22). However, it is widely speculated that more PAR2 agonists exist but are yet uncovered, as PAR2 has been localized to tissues where the known activators (e.g., trypsin or tryptase) are not normally present. It is therefore important to determine the activators of PAR2 in the human pleura, especially in common disease states. The downstream pathways activated by PAR2 stimulation are poorly understood. In our study, dexamethasone inhibited PAR2-mediated MIP-2 release, but the precise point in the downstream pathway where this inhibitory effect is occurring is currently unclear. As a family, PARs are G protein-coupled receptors, but the detailed intracellular mechanisms that lead to inflammatory cytokine production in mesothelial cells remains to be explored. In our study, PAR2 was highly expressed in all the human (visceral and parietal pleurae) samples with no significant differences in mesothelial PAR2 expression between patients with or without pleural diseases. Hence, PAR2-mediated inflammatory effects in pleural diseases are unlikely to be controlled by the receptor expression.
The PAR2 agonist peptide used in our study, SLIGRL-NH2, is the most commonly used peptide in published studies (3, 12, 28, 31). It should be noted, however, that SLIGRL-NH2, like many other PAR-activating peptides, may have additional non-PAR2-mediated effects, which may be tissue specific. Using arteries taken from PAR2 knockout mice, McGuire et al. (29) found that SLIGRL-NH2 was significantly more specific than another agonist peptide, tc-LIGRLO-NH2, which induced marked biological activities in knockout tissues. We believe it is highly likely that the MIP-2 release in our experiments was PAR2 mediated, since the dose-dependent stimulatory effect of the specific peptide, SLIGRL-NH2, was reproduced with known physiological PAR2 activators with very different substrate specificities (i.e., tryptase, trypsin, and coagulation factor Xa) and since the control peptide was ineffective in MIP-2 stimulation. Future studies with the use of PAR2/ mesothelial cells may help confirm the specificity of the effect induced by PAR2-activating peptides.
The response to PAR2-activating peptide was dose dependent from 10 µM onward, with significant effects obtained relative to the control peptide. We extended our treatment doses to an unusually high dose of 1,000 µM and found that the mesothelial cells continued to respond with a striking 1,330-fold increase in MIP-2 production over medium controls. This suggests that PAR2 activation can potentially induce massive release of potent chemokines in the pleural cavity.
The scrambled peptide used, LSIGRL-NH2, is one of the most commonly employed control peptides and has been shown to be virtually inactive on PAR2. In the present study, there was no increase in MIP-2 production at 10 and 100 µM, and, in our previous studies, LSIGRL-NH2 did not induce inflammatory cytokine production by epithelial cells at concentrations up to 400 µM (3). In the present study, LSIGRL-NH2 stimulated a mild inflammatory response over medium control at the highest dose (1,000 µM) tested. However, at that concentration, the MIP-2 production induced by LSIGRL-NH2 was only 1.3% (or 75-fold lower than) that by the activating peptide (SLIGRL-NH2). The small induction by LSIGRL-NH2 at 1,000 µM may be explained on the basis of cross-reactivity between the peptides at this very high concentration due to their close structural similarity.
In summary, this study is the first to demonstrate the presence of PAR2 in the pleural mesothelium and to demonstrate that the activation of this receptor is capable of eliciting significant release of inflammatory chemokines in vitro and neutrophil recruitment to the pleural cavity in vivo. Elucidation of the mechanisms of PAR2 function, and its activators in pleural diseases, has important clinical significance. On one hand, augmentation of PAR2-mediated pleural inflammation may help eradicate infective agents or other unwanted material in the pleural space. Equally, regulating unwanted or excessive pleural inflammation can be of therapeutic value, for instance in pleuritis secondary to connective tissue disorders or drug reactions. Excessive acute inflammation can produce severe pain, and if persistent, inflammation can result in the development of pleural fibrosis, in the form of adhesions or fibrothorax (25). Strategies to antagonize PAR2 action may thus have potential clinical benefits. Finally, the pleural mesothelium shares significant similarities with the peritoneal and pericardial mesothelia, and our results may have relevance to peritoneal and pericardial diseases.
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GRANTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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