Pseudomonas aeruginosa Elastase Disables ... - ATS Journals

Pseudomonas aeruginosa Elastase Disables Proteinase-Activated Receptor 2 in Respiratory Epithelial Cells Sophie Dulon, Dominique Leduc, Graeme S. Cottrell, Jacques D’Alayer, Kristina K. Hansen, Nigel W. Bunnett, Morley D. Hollenberg, Dominique Pidard*, and Michel Chignard* Unite´ de De´fense Inneé et Inflammation/Inserm Equipe 336, and Plate-Forme d’Analyse et de Microse´quençage des Proteínes, Institut Pasteur, Paris, France; Departments of Surgery and Physiology, University of California, San Francisco, California; and Department of Pharmacology and Therapeutics and Department of Medicine, University of Calgary, Calgary, Alberta, Canada

Pseudomonas aeruginosa, a major lung pathogen in cystic fibrosis (CF) patients, secretes an elastolytic metalloproteinase (EPa) contributing to bacterial pathogenicity. Proteinase-activated receptor 2 (PAR2), implicated in the pulmonary innate defense, is activated by the cleavage of its extracellular N-terminal domain, unmasking a new N-terminal sequence starting with SLIGKV, which binds intramolecularly and activates PAR2. We show that EPa cleaves the N-terminal domain of PAR2 from the cell surface without triggering receptor endocytosis as trypsin does. As evaluated by measurements of cytosolic calcium as well as prostaglandin E2 and interleukin-8 production, this cleavage does not activate PAR2, but rather disarms the receptor for subsequent activation by trypsin, but not by the synthetic receptor-activating peptide, SLIGKV-NH2. Proteolysis by EPa of synthetic peptides representing the N-terminal cleavage/activation sequences of either human or rat PAR2 indicates that cleavages resulting from EPa activity would not produce receptor-activating tethered ligands, but would disarm PAR2 in regard to any further activating proteolysis by activating proteinases. Our data indicate that a pathogen-derived proteinase like EPa can potentially silence the function of PAR2 in the respiratory tract, thereby altering the host innate defense mechanisms and respiratory functions, and thus contributing to pathogenesis in the setting of a disease like CF. Keywords: inflammation; infection; lung; cystic fibrosis; protease

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacillus, which acts as an opportunistic pathogen when there is a local or a general immunodeficiency in the host, as in patients with burns, acute ulcerative keratitis, or cystic fibrosis (CF) (1). In patients with CF, chronic colonization of the lung by P. aeruginosa currently accounts for the majority of the morbidity and mortality seen in this disease (1). Several virulence factors contribute to the pathogenicity of P. aeruginosa, including (1 ) surface structures such as pili, the flagella, and the mucoid glycocalyx, involved in the bacterial attachment to mucosal surfaces and in the resistance against phagocytosis and immunolysis;

(Received in original form August 26, 2004 and in final form February 2, 2005) This work was supported by a fellowship from “Vaincre la Mucoviscidose”, Paris, France (S.D.), by the Center National de la Recherche Scientifique (D.P.), by NIH grants DK43207 and DK57480 (N.W.B.), and by a term grant and a Group grant on Proteinases and Inflammation from the Canadian Institutes of Health Resarch (M.D.H.). The data have been presented in part at the World Congress on Inflammation, Vancouver, Canada, August 2–6, 2003. *These authors share senior authorship. Correspondence and requests for reprints should be addressed to Dr. Michel Chignard, Unite´ de De´fense Inneé et Inflammation, Institut National de la Sante´ et de la Recherche Me´dicale E336, Institut Pasteur, 25, rue du Dr. Roux, F-75724 Paris Cedex 15, France. E-mail: [email protected] Am J Respir Cell Mol Biol Vol 32. pp 411–419, 2005 Originally Published in Press as DOI: 10.1165/rcmb.2004-0274OC on February 10, 2005 Internet address: www.atsjournals.org

and (2) several enzymes, the most cytotoxic being exotoxin A (1, 2). Among the other enzymes is the elastolytic metalloproteinase LasB (EC 3.4.24.26), also known as pseudolysin or P. aeruginosa elastase (3), hereafter designated as EPa. Even though not all clinical isolates of P. aeruginosa produce EPa (4), for those that do, the proteinase is an important candidate in delineating the pathogenic status of the microorganism (2, 5, 6). Pathogenic proteolytic mechanisms include damage to tissues through hydrolysis of matrix components, as well as of different components of the immune system including the inactivation of complement factors and of IgG and IgA (5, 7). Moreover, in vitro exposure of human leukocytes to EPa results in the inhibition of cell chemotaxis, phagocytosis, and microbicidal activities (5, 7). EPa can also alter the protective physical barrier provided by the respiratory epithelial lining, as it cleaves the two tight junction proteins ZO-1 and ZO-2, resulting in an increase of the epithelial permeability (2). Proteinase-activated receptors (PARs) comprise a unique family of G protein–coupled, seven-transmembrane domain receptors, which are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser)-proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand which binds intramolecularly to activate the receptor (8, 9). The first PAR to be characterized was the thrombin receptor, or PAR1, and since then, three others have been identified, and named PAR2, PAR3 and PAR4 (8, 9). All PARs, except PAR3, can be selectively activated by short synthetic peptides that correspond to their tethered ligand receptor (8, 9). Thus, trypsin, the archetypal proteolytic agonist of PAR2, cleaves the extracellular N-terminal domain of human PAR2 at SKGR36↓37SLIGKV (where ↓ designates the cleavage site), unmasking the N-terminal intramolecular tethered ligand SLIGKV, while the synthetic peptide corresponding to this sequence (SLIGKV-NH2) activates PAR2 without the need for receptor cleavage (10, 11). PAR2 is highly expressed in the gastrointestinal tract (12), where it may be exposed to physiologic concentrations of pancreatic trypsin sufficient to activate the receptor, suggesting a possible function in this system (13). PAR2 is also expressed in tissues like prostate, kidney, liver, heart, vascular endothelium, skin, pancreas, and lung (12). In regard to the latter organ, several studies have shown that a functional PAR2 is expressed in the respiratory epithelium (12, 14, 15) as well as in many human respiratory epithelial cell lines (11, 16–21). Activation of PAR2 has been observed to cause a variety of responses, including, in the case of respiratory epithelial cells, the triggering of the secretion of prostanoids (16), cytokines (16, 19, 21), and metalloproteinases (20), as well as a mitogenic response in primary human cells in culture such as vascular endothelial cells, lung fibroblasts, and airway smooth muscle cells (8, 22). In vivo, activation of PAR2 leads to effects such as modulation of bronchomotor activity in guinea pigs (23),

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neutrophil extravasation and edema in rats (24), and eosinophil infiltration in mice (25). These observations are supported further by studies showing that PAR2-deficient mice have (1 ) a delayed onset of leukocyte rolling on an activated endothelium (26), and (2 ) a reduced eosinophil infiltration during allergic inflammation of the airways (25). However, other data also obtained in vivo suggest by contrast an anti-inflammatory role for PAR2 in some pathophysiologic situations, in keeping with its protection against histamine-induced bronchoconstriction (14), and LPS-induced neutrophil recruitment in the airways (27). Except for the intestine, it is of note that under normal physiologic conditions, PAR2-expressing organs or tissues are not known to be exposed to trypsin, and thus the tissue-localized physiologic activator(s) of PAR2 remain essentially unknown (8, 9, 15, 28). In the lung as in many tissues, one potential source of PAR2-activating proteinase is the mast cell. Upon degranulation, human mast cells release tryptase that has been reported to activate PAR2 in isolated cell preparations (8, 9, 28). However, a role for tryptase in activating PAR2 in vivo is open to question (8, 28). Other trypsin-like enzymes such as the membrane-type serine protease 1, or matriptase (29), the human airway trypsin-like protease, or HAT (30), or trypsin IV (31) are expressed in the lung and can activate PAR2, but a role for these locally expressed proteinases in PAR2 activation in vivo remains to be established. One potential source of PAR2-regulating enzymes is represented by the bacterial pathogens that can secrete proteinases, particularly at mucosal surfaces such as in the airways. For instance, recent studies reported that major dust mite antigens that are also Ser-proteinases (Der p3 and Der p9) or cysteine(Cys)proteinases (Der p1) can trigger signal transduction in a lungderived epithelial cell line in part by activating PAR2 (17, 19). Similarly, an arginine-specific Cys-proteinase produced by Porphyromonas gingivalis has been shown to activate PAR2 in an oral epithelial cell line (32). In the present study, we show that EPa, rather than activating PAR2, releases the receptor’s N-terminal tethered ligand sequence from the cell surface, thereby disarming the receptor and rendering it refractory to trypsin activation. As a consequence, the activation of respiratory epithelial cells via PAR2 would be suppressed by EPa. This inhibitory activity of a pathogenic enzyme, that could abrogate important pathways for intrinsic defense and functions of the lungs, may thus represent a novel aspect of the pathogenic mechanisms for P. aeruginosa.

MATERIALS AND METHODS Reagents A549 cells (ATCC CCL-185) are type II alveolar epithelial cells from a human adenocarcinoma, whereas 16HBE14o- cells, hereafter designated 16HBE, are human SV40-transformed bronchial epithelial cells and are a gift from Dr. D. C. Gruenert (University of Vermont, Colchester, VT) (33). A stable human PAR2–expressing cell line, KNRK-PAR2, was prepared essentially as previously described using a pcDNA3.1 (⫹) expression vector and the Kirsten murine sarcoma virus–transformed rat kidney (KNRK) cell line (11). The expression and functionality of PAR2 in these cells have been previously documented (11, 18). The PAR2 antiserum B5 was raised in rabbits against a synthetic 20-mer peptide encompassing the rat PAR2 sequence Gly30-Pro45 (30GPNSKGR↓ SLIGRLDTP45YGGC, hereafter designated as rP20, where ↓ designates the trypsin cleavage site, with the sequence YGGC added for derivatization), and cross-reacts with both rat and human PAR2 (13). The antiFlag mouse IgG2b mAb M1 raised against the Flag tag placed at the N-terminus of the PAR2 extracellular N-terminal domain (34), and the rabbit polyclonal Ab raised against the hemagglutinin (HA) tag placed at the PAR2 intracellular C-terminal end (34), were obtained from Sigma Chemical Co. (St Louis, MO). The mouse IgG2a mAb SAM11

raised against aa 37–50 of human PAR2 (35) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For fluorescence-activated cell sorter (FACS) analysis, the fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit Ab was obtained from Rockland (Gilbertsville, PA), the Texas Red–labeled goat anti-mouse Ab was obtained from Caltag Laboratories (Burlingame, CA), and the FITC-labeled goat anti-mouse Ab, as well as nonspecific IgG isotypes, were obtained from Dako A/S (Glostrup, Denmark). For confocal immunofluorescence microscopy experiments, the secondary Ab, either a FITC-labeled goat anti-mouse or a Texas Red–labeled goat anti-rabbit, were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Dulbecco’s modified Eagle’s medium (DMEM), F12/DMEM, and F12-K media (all from Gibco BRL, Paisley, Scotland, UK) were used for cultures of KNRK-PAR2, 16HBE, and A549 cells, respectively. L-glutamine, penicillin, streptomycin, fungizone, hygromycin B, Hanks’ balanced salt solution (HBSS), and trypsin-EDTA solutions were obtained from Gibco BRL. Heat-inactivated fetal calf serum (FCS) was from HyClone Laboratories (Logan, UT). FURA2/AM came from Calbiochem (La Jolla, CA). Purified elastase from P. aeruginosa (⬎ 90% purity by SDS-PAGE) was obtained from Elastin Products Co. (Owensville, MS) and was stored at ⫺20 ⬚C as a 0.5 mg/ml (ⵑ 15 ␮M) stock solution. It was found to possess a specific activity of 260 ⫾ 20 units/mg (mean ⫾ SEM, n ⫽ 3) using the substrate aminobenzoylAla-Gly-Leu-Ala-p-nitrobenzamide (Bachem, Torrence, CA), and a molecular mass of 33 kD was used to calculate the molar concentration of EPa (3). Purified EPa was alternatively obtained from Calbiochem, and this particular preparation was found to produce effects on PAR2, which were identical to those observed with the enzyme from Elastin Products Co. (data not shown). Bovine serum albumin (BSA) was from Euromedex (Strasbourg, France). Trypsin type XI from bovine pancreas, soybean trypsin inhibitor type I-S (SBTI), elastin-Congo red, and the EPa inhibitor, N-(␣-rhamnopyranosyloxyhydroxyphosphinyl)Leu-Trp (phosphoramidon, PA) (3) were from Sigma Chemical Co. Trypsin was prepared as a 10-␮M stock solution in 0.9% (wt/vol) NaCl and stored at ⫺20 ⬚C. The human PAR2-activating peptide SLIGKVNH2, corresponding to the tethered ligand exposed after trypsin cleavage of the receptor, and the reversed peptide VKGILS-NH2, taken as an inactive control peptide (11), were synthesized by Neosystem Laboratories (Strasbourg, France). A synthetic 20-mer, N-acylated, and C-amidated peptide corresponding to the human PAR2 sequence, Lys34Thr54, within the N-terminal receptor domain (Ac-KGR↓SLIGKV DGTSHVTGKGVT-NH2, hereafter designated as hP20; the arrow [↓] denotes the cleavage/activation site in the PAR2 N-terminal sequence targeted by trypsin) (10, 11) was prepared by MilleGen (Toulouse, France). The comparable 20-mer rat peptide, rP20, GPNSKGR↓SLIGRLDTPYGGC was synthesized by the Peptide Services Core at the University of Calgary Faculty of Medicine. All peptides were ⬎ 98% purity by HPLC and mass spectrometry analysis, and were taken up as stock solutions in deionized water or in 25 mM HEPES buffer, pH 7.4, in the range 1.6 to 37 mM, and stored at ⫺20 ⬚C.

Cell Cultures A549 and 16HBE cells were grown in their respective medium supplemented with 10% (vol/vol) FCS, 0.3 mg/ml L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 ␮g/ml fungizone. KNRK-PAR2 cells were grown in DMEM containing hygromycin B (0.15 mg/ml) supplemented with 10% FCS and penicillin, streptomycin, and fungizone as above. For routine passage, KNRK-PAR-2 cells were detached from culture flasks using the Versene medium (126 mM NaCl, 5 mM KCl, 1 mM EDTA, 50 mM Hepes, pH 7.4), whereas trypsin-EDTA was used for the A549 and 16HBE cells. All cell lines were cultured at 37 ⬚C in a 5% CO2, water-saturated atmosphere.

P. aeruginosa–Conditioned Medium The P. aeruginosa PAO1 reference strain was plated onto Luria broth agar and incubated at 37 ⬚C for 18 h. Then, one colony of the overnight culture was cultured in 10 ml of Luria broth at 37 ⬚C for 24 h under stirring on a shaker. Stationary phase culture was thus obtained and bacterial supernatant containing EPa and other secreted virulence factors, was separated from bacterial pellet by centrifugation at 10,000 ⫻ g for 5 min. Supernatant was further filtered on 0.22-␮m pore filters, and immediateley stored in aliquots at ⫺80 ⬚C. The elastolytic activity

Dulon, Leduc, Cottrell, et al.: Pseudomonas aeruginosa Elastase Disarms PAR-2

present in this P. aeruginosa–conditioned medium (Pa-CM) was measured using elastin-Congo red as a substrate and increasing concentrations of purified EPa for calibration, exactly as previously described (36) except that incubation of various dilutions of Pa-CM or of EPa with the substrate was for 1 h. Under the conditions of this assay, the elastolytic activity in Pa-CM amounted to 350 nM of purified EPa, and was inhibited by ⭓ 80% by the EPa-specific inhibitor PA.

FACS Analysis Adherent KNRK-PAR2, 16HBE, and A549 cells were detached from culture flasks using the Versene medium. Cells were pelleted by centrifugation and resuspended in HBSS-BSA 0.25% (wt/vol) at a final concentration of 1.5 ⫻ 106 cells/ml for KNRK-PAR2 and 3 ⫻ 106 cells/ml for 16HBE and A549 cells. Before immunostaining, KNRK-PAR2 cells were left untreated for 10 min at 37 ⬚C or exposed under the same conditions to various dilutions of Pa-CM in the absence or in the presence of 2 ␮M PA, to 7.5 nM to 75 nM EPa, or to 75 nM of EPa in the presence of 2 ␮M PA, whereas 16HBE and A549 cells were untreated or exposed to either 75 nM EPa or 50 nM trypsin for 10 min at 37 ⬚C. Proteolytic activities of proteinases were then inhibited with 3 ␮M SBTI for trypsin or 2 ␮M PA for EPa. After one wash, cell immunolabeling was achieved using either the PAR2 rabbit B5 antiserum or the rabbit pre-immune serum both at a 1:500 dilution, as well as the anti-Flag M1 or the SAM11 mAbs or mouse IgG isotypes, for a 1-h incubation at 4 ⬚C. After one wash, immunofluorescence staining was performed with a FITC-conjugated goat anti-rabbit Ab (20 ␮g/ml) for B5, a Texas Red–labeled goat anti-mouse Ab (50 ␮g/ml) for M1, or a FITC-conjugated goat anti-mouse Ab (25 ␮g/ml) for SAM11, all incubated at 4 ⬚C for 1 h. Then, cells were washed, resuspended in a medium containing 0.1 mg/ml propidium iodide (Sigma), except for Texas Red–labeled cells, and fluorescence emission was recorded using a FACScan cytometer (Becton Dickinson, Franklin Lakes, NJ). Propidium iodide–positive dead cells were excluded from analysis, which was performed using the CellQuest software (v3.3; Becton Dickinson), and binding of the B5 antiserum Ig, or of SAM11 and M1 IgG to their respective specific epitopes on PAR2 at the surface of viable cells was expressed as the geometric mean of fluorescence intensity, to which the background binding measured with the rabbit pre-immune serum or with IgG isotypes was substracted.

Immunolocalization of PAR2 KNRK-PAR2 cells were plated on coverslips 2 d before the start of the experiment. Cells were washed three times with DMEM containing 0.1% (wt/vol) BSA and then incubated with or without enzymes as appropriate for 60 min. Cells were then fixed with 4% (wt/vol) paraformaldehyde in 100 mM PBS, pH 7.4, for 20 min at 4 ⬚C, washed three times for 5 min with PBS supplemented with 1% (vol/vol) normal goat serum, 1 mM CaCl2 and 0.1% saponin, before incubation with the primary Abs (i.e., the mouse anti-Flag M1) at 1:1,000 and the rabbit anti-HA at 1:200, overnight at 4 ⬚C. Cells were again washed before addition of secondary Abs (FITC-labeled goat anti-mouse and Texas Red–labeled goat anti-rabbit both at 1:200) for 2 h at room temperature. After washing, cells were again fixed before washing with PBS and mounting the coverslips with Vectashield (Burlingame, CA). Cells were observed using a BioRad MRC 1,000 laser scanning confocal microscope (BioRad, Hercules, CA), a Zeiss Axiovert ⫻100 microscope, and a Zeiss ⫻100 Plan-Apochromat oil immersion objective (Carl Zeiss Inc., Thornwood, NY).

Measurement of Intracellular Ca2ⴙ Mobilization Adherent KNRK-PAR2, 16HBE, and A549 cells were detached from culture flasks using the Versene medium. After centrifugation, pulmonary epithelial cells were resuspended in their respective medium at a final concentration of 3 ⫻ 106 cells/ml, whereas KNRK-PAR2 cells were resuspended at a final concentration of 1.5 ⫻ 106 cells/ml. Cells were incubated with the calcium probe FURA 2/AM (5 ␮M for respiratory epithelial cells and 1 ␮M for KNRK-PAR2) for 30 min at room temperature. Cells were then washed twice in the assay buffer HBSS-BSA 0.25% (wt/vol) and resuspended at the same final concentration in the assay buffer containing 1 mM CaCl2 and 1 mM MgCl2. Aliquots (1 ml) of cell suspensions were equilibrated at 37 ⬚C for 3 min under stirring in a cuvette placed in a spectrofluorimeter. Cells were then exposed to

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75 nM EPa during 3 min, proteolytic activity was inhibited with 2 ␮M PA, and after 90 s, 50 nM trypsin or 100 ␮M of the activating peptide SLIGKV-NH2 were added and the changes in fluorescence were continously recorded for time periods of 1–4 min. As control, cells were incubated for 90 s in the spectrofluorimeter cuvette with 2 ␮M PA only before trypsin or SLIGKV-NH2 activation. In some sets of experiments, PAR2-activating agents (trypsin or peptide) were replaced by 1 ␮M bradykinin or 100 ␮M control reversed peptide, or KNRK-PAR2 cells were exposed to 2 ␮M PA before 75 nM EPa, before adding 50 nM trypsin or 100 ␮M of the peptide SLIGKV-NH2 after 3 min. Fluorescence was measured at wavelengths of 340 nm excitation and 510 nm emission. Cytosolic calcium ion concentrations were calculated as described previously (37), and data are expressed as the maximal variation of intracellular concentration of calcium elicited by the agonist over the basal concentration (⌬[Ca2⫹]i).

Measurement of Prostaglandin E2 and Interleukin-8 A549 cells were seeded at 1 ⫻ 106 cells/well in 24-well plates (Falcon, Becton Dickinson Labware), cultured at 37 ⬚C with 5% CO2 in complete medium until confluence, and then starved of FCS for 24 h. After one wash of the cell monolayers, they were exposed to 10 nM EPa in FCSfree culture medium or to medium alone for 5 min, then 2 ␮M PA was added to all wells and media were aspirated. Cells were next incubated with 50 nM trypsin, 200 ␮M SLIGKV-NH2 or VKGILS-NH2, or with FCS-free medium alone. SBTI (3 ␮M) was added in all wells after 1 h. For wells containing peptides, media were aspirated and replaced by FCS-free culture medium. Cell culture media were finally recovered after 6 h of incubation under cell culture conditions, centrifuged, and supernatants were processed for the measurements of prostaglandin (PG)E2 and of interleukin (IL)-8 production by ELISA (R&D System, Abingdon, UK).

PAR2 Peptide Fragmentation by Proteinases The hP20 peptide was adjusted to 500 ␮M in PBS and incubated at 37 ⬚C in a 20-␮l volume with purified proteinases, either trypsin or EPa, with a peptide/enzyme molar ratio varying from 100:1 to 10,000:1, and for 2–20 min. Controls consisted of the proteinase-free peptide or of proteinase dilutions alone incubated at 37 ⬚C for the longest periods of time in each given experiment. Enzymatic activity was stopped by acidifying the reaction mixture, and samples were processed either for direct peptide mass analysis using surface-enhanced laser desorption ionization–time of flight (SELDI-TOF) mass spectrometry (see below), or for HPLC separation of the cleavage products followed by N-terminal microsequencing. For the latter, samples of the hP20 peptide and its fragments generated at chosen time points during exposure to EPa (hP20/enzyme molar ratio, 10,000:1) were separated by HPLC onto a DEAE column (Vydac 218TP52, 250 mm, 2.1 mm diameter; Grace Vydac, Hesperia, CA) eluted with a 0–45% acetonitrile, 0.1% trifluoroacetic acid (TFA) gradient. Samples were collected and frozen until sequencing. Separated proteolytic products to be further analyzed were identified by their mass using SELDI-TOF mass analysis, and N-terminally microsequenced on an Applied Biosystems 473A sequencer. The rP20 peptide was subjected to proteolysis under slightly different conditions, to facilitate subsequent HPLC separation of the resulting proteolysis products, followed by mass determination using matrixassisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. GPNSKGRSLIGRLDTPYGGC (100 ␮M) was incubated at room temperature for 20 min with 750 nM EPa (peptide/ enzyme molar ratio, 135:1) in a final volume of 100 ␮l hydrolysis buffer (50 mM Tris, 10 mM CaCl2, 200 mM NaCl, pH 7.4), at which point the entire solution was injected onto a C4 HPLC column. The EPagenerated peptides were separated using a 0–45% acetonitrile, 0.1% TFA gradient over 30 min. The effluent fractions were monitored for absorbance at 215 nm and peak tubes were collected, freeze-dried, and subjected to MALDI-TOF mass analysis.

Mass Spectrometry Analysis of EPa Proteolysis Peptide Products For SELDI-TOF analysis, hP20 peptide or control samples (5 ␮l) were loaded on eight-spot, acetonitrile-preactivated hydrophobic surface H4 ProteinChip arrays (Ciphergen Biosystems, Fremont, CA), and airdried by evaporation. After five washes with 5 ␮l of deionized water,

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0.8 ␮l of matrix diluted one-twentieth (␣-cyano-4-hydroxy cinnamic acid saturated in 50% acetonitrile/0.1% TFA and diluted in the same solution) were applied on each spot and allowed to air-dry. Mass spectrometry analysis was performed in a PBS-II mass reader (Ciphergen Biosystems). Spectra were collected using an average 80 nitrogen laser shots with a 30-mJ laser intensity and a detector sensitivity of 6. Spectrum analysis was performed using the ProteinChip software version 3.1 (Ciphergen Biosystems), and internal mass calibration was performed by spotting a mixture of three peptides (Arg8-vasopressin, average mass 1,084.2 D; somastostatin, average mass 1,637.9 D; insulin B chain, average mass 3,495.94 D). Identification of the truncated peptides was conducted by using the PeptideMass analysis tool on the ExPASy Molecular Biology server (www.expasy.org), in regard to the initial hP20 sequence and based on the measured average masses. MALDI-TOF masses for the rP20 fragments isolated by HPLC were used to assign unequivocal peptide sequences by deconvolution of the masses, knowing the sequence of the enzyme substrate from which the fragments were generated.

Statistics Results are expressed as mean ⫾ SEM for the indicated number of independently performed experiments. Statistical significance between the different values was analyzed by using the nonparametric Wilcoxon’s test with a threshold of P ⭐ 0.05.

RESULTS Effect of P. aeruginosa Elastase on PAR2 Expression

First of all, the susceptibility of the extracellular N-terminal domain of PAR2 to cleavage by EPa was evaluated by exposing KNRK-PAR2 cells to decreasing dilutions of an extracellular milieu obtained from a stationary phase culture of P. aeruginosa (Pa-CM) known to contain large amounts of EPa (38), in our case 350 nM. Results of FACS analysis using the B5 antiserum, which maps an epitope located both upstream and downstream of the cleavage/activation site of PAR2 (13), showed a progressive disappearance of the targeted epitope up to ⬇ 90% with the 1/10 dilution as compared with untreated control cells, and this decrease was markedly inhibited by the preincubation of Pa-CM with PA, a specific inhibitor of EPa (Figure 1A). With this evidence that P. aeruginosa releases a material with EPa activity and which affects PAR2, cells were then exposed to increasing concentrations of purified EPa for 10 min in place of the Pa-CM. Results of FACS analysis using the B5 antiserum showed a progressive disappearance of the epitope in a concentration-dependent manner, up to ⬇ 75% for 75 nM EPa as compared with untreated control cells (Figure 1B). This concentration of EPa was then chosen for the subsequent experiments. The decrease of the B5 epitope display was attributable to the proteolytic activity of EPa as KNRK-PAR2 cells exposed to 75 nM EPa enzymatically blocked by PA, expressed the epitope as much as untreated control cells (Figure 1B). Results obtained with the anti-Flag M1 mAb showed a disappearance of its N-terminal epitope to a similar extent (85 ⫾ 6%, n ⫽ 3) on EPa-treated cells. Finally, 16HBE and A549 human respiratory cells were exposed to 75 nM EPa for 10 min to check wether the expression of the same epitope was similarly decreased for cells naturally expressing PAR2. As a positive control, cells were also exposed to 50 nM trypsin for 10 min, as this concentration of the archetypal PAR2-activating proteinase induces a near maximal (⬎ 90%) activation of respiratory epithelial cells in terms of cytosolic calcium increase, as well as a maximal reduction of the expression of the epitope targeted by the B5 antiserum (18). A significant reduction in the display of this epitope was observed after exposure of cells to 75 nM EPa, which amounted 72 ⫾ 15% and 84 ⫾ 11% for A549 and 16HBE cells, respectively, whereas trypsin induced a ⬇ 50% reduction in epitope display (Figure 1C).

Figure 1. Reduced expression of the PAR2 cleavage/activation domain in cells exposed to P. aeruginosa–conditioned medium or to purified EPa. (A and B ) KNRK-PAR2 cells (1.5 ⫻ 106 cells/ml) were incubated for 10 min at 37 ⬚C under stirring without (control) or with decreasing dilutions of Pa-CM (A ), or increasing concentrations of EPa from 7.5– 75 nM (B ). Alternatively, KNRK-PAR2 cells were incubated with Pa-CM dilutions (A ) or with 75 nM EPa (B ) previously incubated with 2 ␮M of PA (PA/Pa-CM and PA/EPa, respectively. (C ) 16HBE and A549 cells (3 ⫻ 106 cells/ml) were incubated without (control) or with 50 nM trypsin or 75 nM EPa for 10 min at 37 ⬚C under stirring before enzymatic activities were blocked with either 3 ␮M SBTI for trypsin or 2 ␮M PA for EPa. All cells were then incubated with the B5 antiserum or nonimmune rabbit serum at a 1:500 dilution, then with an FITC-coupled goat antirabbit Ab at 20 ␮g/ml. Binding of Abs was analyzed by FACS as described in MATERIALS AND METHODS. Each point or histogram represents the mean ⫾ SEM of three experiments, with the fluorescence of proteinasetreated cells expressed as the percentage of that of untreated control cells.

Effect of P. aeruginosa Elastase on PAR2 Cellular Localization

Based on the antigenic characteristics of the B5 and M1 Abs used for FACS analysis (13, 34), the disappearance of PAR2 N-terminal epitopes expression at the surface of KNRK-PAR2, 16HBE and A549 cells exposed to EPa, can be caused by (1 ) cleavage of the receptor at the cleavage/activation site Arg36Ser37, followed by endocytosis of the receptor as a consequence of its activation as it occurs with trypsin (18); or (2 ) cleavage(s) downstream of the activation site, as previously shown for PAR1 and PAR2 exposed to the leukocyte Ser-proteinases elastase, cathepsin G and/or proteinase 3 (18, 37, 39). We then repeated the FACS analysis using the SAM11 mAb, which epitope maps the sequence Ser37-Gly50 of human PAR2 (35), and which expression is not affected by the activating cleavage at Arg36-Ser37 per se, but by the endocytosis of the receptors that follows activation as reported with an Ab (PAR-2C) raised against an almost identical epitope mapping the sequence Ser37-Val53 of human PAR2 (35). Here again, in parallel with EPa, we challenged the cells with trypsin as a positive control. We also used SLIGKVNH2, the specific peptide agonist of PAR2, which activates the receptor without the need for a proteolysis of the cleavage/ activation site, and had thus the advantage to clearly dissociate the disappearance of the SAM11-recognized epitope due to proteolysis from that due to internalization of PAR2. Interestingly, with the SAM11 mAb, trypsin, SLIGKV-NH2 and EPa induced a quite comparable decrease of the cell labeling intensity, up to 58 ⫾ 10%, 56 ⫾ 5%, and 74 ⫾ 4%, respectively, as compared with untreated KNRK-PAR2 cells (Figure 2). We inferred that at least for SLIGKV-NH2 and trypsin, the disappearance of the epitope was due to the endocytosis of PAR2 after its activation. To block the internalization process, experiments were reproduced with KNRK-PAR2 cells depleted in cytosolic ATP by incubation for 30 min at room temperature with a cocktail of

Dulon, Leduc, Cottrell, et al.: Pseudomonas aeruginosa Elastase Disarms PAR-2

metabolic blockers (50 mM 2-deoxy-D-glucose, 0.05% [wt/vol] sodium azide, and 10 mM glucono-␦-lactone) (40) before exposure to trypsin, SLIGKV-NH2, or EPa. Under these conditions, it was observed that the labeling by SAM11 was fully maintained upon exposure to SLIGKV-NH2, and minimally diminished after trypsin treatment (Figure 2). By contrast, EPa decreased SAM11 epitope reactivity to a similar extent whether or not internalization was prevented (Figure 2). To confirm these results, we used confocal microscopy to examine the structure and cellular localization of PAR2 upon EPa treatment of KNRK-PAR2 cells. These cells express PAR2 with an extracellular N-terminal Flag epitope that is upstream from the site for trypsin cleavage and activation, and an intracellular C-terminal HA.11 epitope (Figure 3A). In untreated cells, Flag and HA.11 epitopes were colocalized at the plasma membrane and in intracellular locations (Figure 3B) that we have previously identified as the Golgi apparatus (34). Exposure to trypsin 10 nM for 60 min caused a loss of surface Flag immunoreactivity, which is indicative of receptor cleavage and removal of the Flag epitope. Immunoreactive HA.11 was detected in endosomes that did not contain immunoreactive Flag. Thus, these endosomes contain cleaved and activated PAR2, indicative of normal activation and endocytosis of the receptor (34). Exposure to EPa 10 nM for 60 min also removed the Flag epitope from the cell surface. However, in contrast to cells exposed to trypsin, the HA.11 epitope was still detected at the plasma membrane. These results suggest that EPa cleaves PAR2 to remove the extracellular Flag epitope, but that this cleavage does not activate the receptor and does not induce endocytosis.

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even totally suppressed, for KNRK-PAR2, 16HBE, and A549 cells, respectively, compared with the calcium signal caused by trypsin in untreated cells (Figure 4B). In parallel experiments, KNRK-PAR2, 16HBE, and A549 cells pretreated with 75 nM EPa and subsequently challenged with the activating peptide SLIGKV-NH2 (100 ␮M) instead of trypsin, showed a cytosolic calcium signal which was equivalent to that observed for untreated control cells (Figure 4B). Increasing the EPa concentrations up to 150 nM, or decreasing the peptide concentration down to 10 ␮M left the signal generated by SLIGKV-NH2 unchanged by EPa pretreatment (data not shown). It is of note that (1 ) the inhibitory effect of EPa on trypsin-induced cell activation was totally abrogated by blocking EPa activity with PA, and (2 ) when KNRK-PAR2 cells pretreated with 75 nM EPa were challenged with 1 ␮M bradykinin in place of trypsin, cytosolic calcium mobilization induced by bradykinin remained unaffected compared with that observed in untreated cells (data not illustrated). These data indicate that the inhibitory effect of EPa is very likely of proteolytic nature, and is restricted to PAR2. It has been recently demonstrated that activation of PAR2 expressed by human respiratory epithelial cells induces the release of various mediators, including the functionally important prostanoid PGE2 and chemokine IL-8 (16). We thus sought to determine if EPa could inhibit the release of PGE2 and of IL-8 normally induced by PAR2 activators. A549 cell monolayers

Effect of P. aeruginosa Elastase on PAR2-Mediated Cell Activation

From these data, we hypothesized that EPa cleaves PAR2 within or below the epitope recognized by the SAM11 mAb resulting in the removal of the tethered ligand without receptor activation. To test this hypothesis, we examined the effect of EPa on PAR2mediated cell functions. KNRK-PAR2, 16HBE, and A549 cells loaded with FURA-2 were challenged with 75 nM EPa. No elevation of intracellular calcium concentration was observed, indicating an absence of PAR2 activation. The lack of effect of EPa contrasted with the robust transient rise in cytosolic calcium induced by either trypsin (50 nM) or SLIGKV-NH2 (100 ␮M) (Figure 4A). When the same concentration of trypsin was applied to cells 4.5 min after their exposure to EPa, the trypsin-induced calcium signal was reduced by 74 ⫾ 11% and 88 ⫾ 3%, and

Figure 2. Proteolytic downexpression of the PAR2 extracellular N-terminal domain in KNRK-PAR2 cells exposed to EPa. KNRK-PAR2 cells (1.5 ⫻ 106 cells/ml), either untreated or depleted in cytosolic ATP as detailed in the text, were incubated without (control) or with 50 nM trypsin, 100 ␮M SLIGKV-NH2, or 75 nM EPa for 10 min at 37 ⬚C under stirring before enzymatic activities were blocked with either 3 ␮M SBTI for trypsin or 2 ␮M PA for EPa. Cells were then incubated with the SAM11 mAb or nonimmune IgG2a at 5 ␮g/ml, followed by an FITC-coupled goat anti-mouse Ab at 25 ␮g/ml. Binding of Ab were analyzed by FACS as described in MATERIALS AND METHODS. Histograms represent the means ⫾ SEM of three experiments, with the fluorescence of peptide- or proteinase-treated cells expressed as the percentage of that of non–proteinase-treated control cells.

Figure 3. Absence of endocytosis of cleaved PAR2 in KNRK-PAR2 cells exposed to EPa. (A ) Human PAR2 construct showing extracellular Flag epitope, intracellular HA.11 epitope, and trypsin cleavage site that exposes the tethered ligand and activates the receptor. (B ) Localization of immunoreactive Flag and HA.11 epitopes in untreated cells and after exposure to trypsin or EPa (10 nM) for 60 min. The same cells are shown in each given row. Note that Flag and HA.11 colocalize at the plasma membrane and in intracellular locations in untreated cells (arrows). Trypsin removes the Flag and induces endocytosis of HA.11 into endosomes that do not contain Flag and which thus contain cleaved and activated PAR2 (arrows). EPa removes the Flag but HA.11 remains at the cell surface, indicating that the receptor is cleaved and not activated (arrows). Scale bar ⫽ 10 ␮m.

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were exposed to 10 nM EPa for 5 min, and the proteolytic activity was then blocked by the addition of 2 ␮M PA. We chose a low concentration of enzyme and a short incubation time because cell monolayers were injured by higher EPa concentrations and longer incubation periods. Cells were then incubated for 1 h with trypsin (50 nM), the activating peptide SLIGKV-NH2, or the control reverse peptide (each at 200 ␮M), and culture media were harvested after 6 h, as detailed in Materials and Methods. In preliminary time course studies, it was observed that the concentrations of PGE2 and of IL-8 measured in the culture medium up to 24 h after activation with trypsin plateaued between 6 and 24 h (data not shown). It is of note that in the experiments dealing with the effects of EPa, the PAR2 agonists were incubated with cells for 1 h only after exposure to the bacterial proteinase or to a control medium, whereas production of PGE2 and of IL-8 by such activated cells was let to proceed for 6 h. The aim of this particular experimental setup was to minimize the activation of newly synthesized functional PAR2 receptors that are to be expressed at the cell surface after the EPa treatment, as it was observed after challenge with trypsin (34, 35). When activation thus proceeded for only 1 h, there was already a significant increase of PGE2 and of IL-8 production by trypsin- or SLIGKV-NH2–activated A549 cells. From a basal PGE2 production of 650 ⫾ 170 pg/ml (n ⫽ 6) by resting cells, the fold increase was of 2.01 ⫾ 0.47 and 3.10 ⫾ 0.31 upon activation by SLIGKV-NH2 and trypsin, respectively. Similarly, from a basal IL-8 production of 403 ⫾ 41 pg/ml (n ⫽ 6), SLIGKVNH2 and trypsin activation resulted in 2.43 ⫾ 0.08- and 2.60 ⫾ 0.69-fold increases, respectively (Figure 5). As expected, the reverse peptide VKGILS-NH2 was unable to trigger a significant increase in PGE2 and in IL-8 production (data not shown), as was also the case for exposure to EPa alone (Figure 5). When cells were pretreated with EPa before trypsin activation, the increase in PGE2 and in IL-8 concentrations recovered in the culture media was significantly reduced by around 50% and 55%, respectively (Figure 5). In contrast, PGE2 and IL-8 concentrations recovered from EPa-pretreated cells stimulated with SLIGKV-NH2 were not reduced as compared with those recov-

Figure 4. Impairement of PAR2-induced intracellular activation in cells exposed to EPa. Cells (1.5 ⫻ 106 cells/ml for KNRK-PAR2 cells and 3 ⫻ 106 cells/ml for 16HBE and A549) were loaded with FURA 2/AM and challenged with either EPa, trypsin, or SLIGKV-NH2. Variations of fluorescence reflecting changes of cytosolic Ca2⫹ concentrations were recorded over a total period of 10 min. (A ) KNRK-PAR2 cells challenged with EPa (75 nM), trypsin (50 nM), or SLIGKV-NH2 (100 ␮M). The results presented are representative of three different experiments displaying similar results. (B ) KNRK-PAR2, 16HBE and A549 cells challenged for 3 min with 75 nM EPa, then blocked with 2 ␮M PA, and trypsin (50 nM) or SLIGKV-NH2 (100 ␮M) added 90 s later. Histograms represent the means ⫾ SEM of three experiments with values expressed as the percentage of ⌬[Ca2⫹]i measured in trypsin- or SLIGKV-NH2–induced control cells not exposed to EPa.

ered from non pretreated cells (Figure 5). Thus, the inhibition of trypsin-induced, PAR2-dependent cell activation resulting from exposure to EPa appears likely to be due to a specific disarming cleavage of the PAR2 extracellular N-terminal domain, while the intramolecular SLIGKV-NH2 binding site remains unaffected. Fragmentation by P. aeruginosa Elastase of an N-Terminal PAR2 Peptide Mimic

The identification of the potential cleavage site(s) for EPa within the suspected proteinase-sensitive N-terminal domain of PAR2 was achieved by analyzing the peptide fragments released by EPa from two synthetic peptide substrates representing the N-terminal cleavage/activation sequences of human and rat PAR2. The synthetic peptide, hP20 (Lys34-Thr54) represents the sequence of human PAR2 containing the activating cleavage site Arg36-Ser37, the sequence recognized by the B5 Ab as well as the SAM11 epitope Ser37-Gly50. The rat PAR2 peptide rP20 presents to the enzyme a slightly different sequence, but an identical motif at the trypsin cleavage/activation site. Results for the analysis of the hP20 peptide cleavage products are summarized in Table 1, and are as follows; exposure of hP20 to trypsin, taken as the reference Ser-proteinase activating PAR2, in a substrate/enzyme molar ratio ⭓ 1,000:1 resulted, as expected, in a rapid (1–10 min) cleavage after the Arg residue corresponding to Arg36 in human PAR2 (10, 11). When the substrate/enzyme molar ratio was ⬍ 1,000:1, another cleavage site was found at a peptide bond corresponding to Lys51-Gly52 in human PAR2, as previously observed (39). A minor cleavage of PAR2 by trypsin at this latter peptide bond can actually explain the slight reduction in display of the SAM11 epitope which is observed even though endocytosis of trypsin-activated PAR2 has been blocked (Figure 2). Exposure of hP20 to EPa in an substrate/enzyme molar ratio ⭓ 5,000:1 resulted in early (2–10 min) cleavages generating peptide fragments with average mass values of 1,939.2 and 1,667.7 (Table 1), indicating that cleavages were after a Gly or after a Ser residue corresponding to the human PAR2 peptide

Figure 5. Inhibition of PAR2-induced production of PGE2 and of IL-8 by respiratory epithelial cells exposed to EPa. A549 cell monolayers were cultured with FCS-free medium for 24 h, then exposed to 10 nM EPa for 5 min or left untreated before 2 ␮M PA was added. Media were replaced by FCS-free medium alone (NS, nonstimulated), or containing 50 nM trypsin (⫹Tryp.) or 200 ␮M SLIGKV-NH2 (⫹SLIGKV). After 1 h, 3 ␮M SBTI was added, except in SLIGKV-NH2–treated cells, for which media were replaced with FCS-free culture medium. All culture media were then recovered after 6 h and processed for the ELISA measurement of PGE2 and of IL-8 production. Histograms represent the means ⫾ SEM of three to six experiments with values expressed as the fold increase in PGE2 and of Il-8 production over basal values measured in non EPatreated and unstimulated cells. *P ⬍ 0.05.

Dulon, Leduc, Cottrell, et al.: Pseudomonas aeruginosa Elastase Disarms PAR-2 TABLE 1. SELDI-TOF MASS SPECTROMETRY ANALYSIS OF PROTEINASE-INDUCED FRAGMENTATION OF THE PAR2-RELATED hP20 PEPTIDE Enzymatic Treatment* None Trypsin EPa

Mass (D)†

Peptides‡

2138.2 1755.0 1497.7 1939.2 1667.7 1469.0

KGRSLIGKVDGTSHVTGKGVT SLIGKVDGTSHVTGKGVT SLIGKVDGTSHVTGK KGRSLIGKVDGTSHVTGKG LIGKVDGTSHVTGKGVT LIGKVDGTSHVTGKG

* Peptide (500 ␮M) was exposed for 2–20 min at 37 ⬚C to proteinases in peptide/ enzyme molar ratios varying from 100–10,000, or to a control proteinase-free medium. † Values for the average masses oberved in an experiment representative of four to five. ‡ Truncated peptides were deduced from the masses fitted to the amino acid composition of the original hP20 peptide using the ExPASy PeptideMass software.

bonds Gly52-Val53 and Ser37-Leu38, respectively. For longer exposures (5–20 min) or for higher enzyme concentrations, combination of the two cleavages led to a 15-mer peptide with an average mass value of 1,469.0 (Table 1). With a mass accuracy of 0.05%, two different truncated sequences could fit with this latter mass. Then, the N-terminus of this peptide as well as of the peptide with an average mass value of 1,667.7 (see Table 1), was sequenced after HPLC separation of the fragments derived from the hP20 peptide exposed to EPa. For both peptides, the N-terminal sequence started with LI(G), confirming a unique cleavage after the serine at the fourth position in hP20. In keeping with the cleavage of the hP20 peptide at the Ser37-Leu38 bond, EPa hydrolysis of the rat rP20 peptide (GPNSKGRSLIGRLDTPYGGC) yielded as major products isolated by HPLC, peptides with masses that corresponded uniquely to GPNSKGRS ([M⫹H]⫹, 802.33) and the counterpart LIGRLDTPYGGC ([M⫹H]⫹, 1,264.65). Thus, the data obtained for the hydrolysis of the two peptides confirmed cleavage by EPa at the Ser37-Leu38 bond of human PAR2 and at the Ser38-Leu39 bond of rat PAR2, which would yield a tethered ligand sequence, LIGKV…for human and LIGRL… for rat, incapable of activating the corresponding receptor (see Discussion). Cleavage at the Gly52-Val53 bond of the tethered ligand, confirmed by the sequence analysis of the hP20 hydrolysis products, would release from the N-terminal sequence of the receptor, the epitopes detected by both the SAM11 and B5 Abs.

DISCUSSION Although the precise (patho)physiologic role for PAR2 in the lung has yet to be established with certainty, current evidence suggests that this proteolytically activated receptor is an important component of the intrinsic innate defense and functions of the lung (9, 15, 28). However, the endogenous or exogenous (such as pathogen-associated) proteinases that can modulate PAR2 activity have yet to be identified with certainty. Nonetheless, a number of endogenous candidates that in principle could regulate lung PAR2 activity (activation or inactivation) have been singled out, ranging from the neutrophil-derived Serproteinases cathepsin G and elastase (18) and the mast cell tryptase (8, 28) to the epithelial, membrane-tethered trypsinlike Ser-proteinases (29, 30) and extrapancreatic trypsin IV (31). Importantly, pathogen-derived proteinases, like those produced by allergenic dust mites (17, 19), have also been found capable of activating lung PAR2. In this regard, we were particularly interested to determine if the P. aeruginosa–derived metallopro-

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teinase, EPa, known to contribute to the virulence of this pathogen (2, 5, 6), might also be able to modulate PAR2 activity. The main finding of our study was that instead of activating PAR2, EPa was able to disarm the receptor, thereby preventing its subsequent activation by trypsin, but not by the PAR2activating peptide, SLIGKV-NH2. The ability of EPa to block the subsequent activation of cell surface PAR2 went hand in hand with its ability to remove the PAR2 epitopes recognized by the B5 and SAM11 Abs from the cell surface, without triggering receptor internalization and mobilization of intracellular pools. The epitopes recognized by these two Abs are situated either in the cleavage/activation sequence of the receptor (33SKGR↓ SLIGKVDGT45) or immediately downstream the cleavage/activation site (37SLIGKVDGTSHVTG50), respectively. In a previous study using the B5 antiserum, it has been found that epitopes both before and after the trypsin cleavage site (Arg36-Ser37) can be recognized independently (41). Thus, the loss of the B5 epitope would imply cleavage beyond Thr45 in the above sequence; this result also applies to the SAM11 Ab. This implication is completely supported by the analysis of the peptide cleavage products released by EPa from the synthetic human-derived PAR2 peptide hP20: 34KGRS↑LIGKVDGTSHVTGKG↑VT54, as indicated by the upward vertical arrows. We thus conclude that the disarming of PAR2 by EPa involves the release of the entire receptor-activating sequence from the cell surface, a process that would abrogate trypsin action, but not that of the receptoractivating peptide, SLIGKV-NH2. In addition, the cleavage of the receptor sequence at the Ser37-Leu38 bond, found in both the rat and human PAR2 sequences that we examined, would unmask a sequence, LIG…, which would be predicted to be inactive as a tethered ligand, according to our previous data (42). It is of note that one of the cleavage sites by EPa occurs at a Xaa-Leu peptide bond, where Xaa is Ser in PAR2, which has been described as one preferred cleavable bond for the thermolysin family of bacterial Zn2⫹-dependent metalloproteinases (3). Thus, cleavage of PAR2 by EPa at either or both of the sites identified by our analysis of the peptide proteolysis products would render the receptor inactive to further proteolytic activation. As for many other virulence factors secreted by P. aeruginosa, EPa (LasB) expression is upregulated in response to bacterial density increases, mainly through induction of the lasR quorum sensing system (4). Transcription and translation of the lasB gene actually results in a large, inactive preproenzyme molecule with a mass of ⵑ 55 kD. As it is translocated from the bacterial cytoplasm into the extracellular milieu through the periplasm, the protein is converted into a still inactive proenzyme, then finally into an active exoproteinase with a mass of ⵑ 33 kD (43). It is of note that the disarming activity of purified EPa appears at enzyme concentrations between 10 and 20 nM (see Figures 1 and 5), to be compared with the lowest active concentration present in the diluted Pa-CM, i.e. 7 nM for the 1/50 dilution. Given the persistence of P. aeruginosa in the lungs of individuals with CF, it is reasonable to assume that the enzyme could achieve a level that would be sufficient to disarm PAR2 activity toward its physiologic proteinase activators. Indeed, use of a specific ELISA to measure EPa concentrations in the sputum of patients with CF has revealed quantities as high as 3 to ⬎ 100 ␮g enzyme/mg sputum (44). In summary, our data support the conclusion that EPa disarms PAR2 through proteolysis of the extracellular N-terminal domain downstream from the trypsin cleavage/activation site. If the role of PAR2 in the lung is to contribute to the control of the innate response of this organ to invading organisms like P. aeruginosa and to the preservation of respiratory functions through production of mediators such as IL-8 and PGE2 (16,

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45), its crippling by EPa, and possibly by a combination of EPa and neutrophil proteinases (18) in an infectious/inflammatory context, could represent a novel mechanism that can account for some of the pathogenic activities of the microorganism by altering the respiratory defense mechanisms and functions in patients with CF, and thus participating in the deleterious evolution of the disease. This speculation would have to be properly evaluated in the future by looking at the clearance of P. aeruginosa and at the modulation of the innate immune response in PAR2 knockout versus wild-type mice. Conflict of Interest Statement : S.D. has no declared conflicts of interest; D.L. has no declared conflicts of interest; G.S.C has no declared conflicts of interest; J.D. has no declared conflicts of interest; K.K.H. has no declared conflicts of interest; N.W.B. consulted for Pfizer ($5,000 per year) from 2000–2004 and R. W. Johnson ($10,000 per year) from 1998–2004 and has been funded by AstraZeneca to lecture (2003) and receives grants from Pfizer ($100,000 per year) and AstraZeneca ($50,000 per year) from 2003–2005; M.D.H. has no declared conflicts of interest; D.P. has no declared conflicts of interest; and M.C. has no declared conflicts of interest. Acknowledgments : The authors thank Dr. Anne Dubouix (Laboratoire d’Hygie`ne ˆ pital de Rangueil, Toulouse, France) for the preparation of the et Bacte´riologie, Ho P. aeruginosa–conditioned medium, and Tyler Vanderputten and Denis McMaster (Peptide Services Core, University of Calgary, Calgary, Alberta, Canada) for assistance with peptide synthesis.

References 1. Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000;2:1051–1060. 2. Azghani AO. Pseudomonas aeruginosa and epithelial permeability: role of virulence factors elastase and exotoxin A. Am J Respir Cell Mol Biol 1996;15:132–140. 3. Morihara K. Pseudolysin and other pathogen endopeptidases of thermolysin family. Methods Enzymol 1995;248:242–253. 4. Cabrol S, Olliver A, Pier GB, Andremont A, Ruimy R. Transcription of quorum-sensing system genes in clinical and environmental isolates of Pseudomonas aeruginosa. J Bacteriol 2003;185:7222–7230. 5. Maeda H, Yamamoto T. Pathogenic mechanisms induced by microbial proteases in microbial infections. Biol Chem Hoppe Seyler 1996;377: 217–226. 6. Yanagihara K, Tomono K, Kaneko Y, Miyazaki Y, Tsukamoto K, Hirakata Y, Mukae H, Kadota J, Murata I, Kohno S. Role of elastase in a mouse model of chronic respiratory Pseudomonas aeruginosa infection that mimics diffuse panbronchiolitis. J Med Microbiol 2003; 52:531–535. 7. Buret A, Cripps AW. The immunoevasive activities of Pseudomonas aeruginosa: relevance for cystic fibrosis. Am Rev Respir Dis 1993; 148:793–805. 8. Hollenberg MD, Compton SJ. International Union of Pharmacology. XXVIII. Proteinase-activated receptors. Pharmacol Rev 2002;54:203– 217. 9. Ossovskaya VS, Bunnett NW. Protease-activated receptors: contribution to physiology and disease. Physiol Rev 2003;84:579–621. 10. Nystedt S, Emilsson K, Larsson AK, Strombeck B, Sundelin J. Molecular cloning and functional expression of the gene encoding the human proteinase-activated receptor 2. Eur J Biochem 1995;232:84–89. 11. Bo¨hm SK, Kong W, Bromme D, Smeekens SP, Anderson DC, Connolly A, Kahn M, Nelken NA, Coughlin SR, Payan DG, et al. Molecular cloning, expression and potential functions of the human proteinaseactivated receptor-2. Biochem J 1996;314:1009–1016. 12. D’Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P, Darrow AL, Santulli RJ, Brass LF, Andrade-Gordon P. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 1998;46:157–164. 13. Kong W, McConalogue K, Khitin LM, Hollenberg MD, Payan DG, Bo¨hm SK, Bunnett NW. Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2. Proc Natl Acad Sci USA 1997;94:8884–8889. 14. Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, Henry PJ, Carr MJ, Hamilton JR, Moffatt JD. A protective role for protease-activated receptors in the airways. Nature 1999;398:156–160. 15. Lan RS, Stewart GA, Henry PJ. Role of protease-activated receptors in airway function: a target for therapeutic intervention? Pharmacol Ther 2002;95:239–257.

16. Asokananthan N, Graham PT, Fink J, Knight DA, Bakker AJ, McWilliam AS, Thompson PJ, Stewart GA. Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells. J Immunol 2002;168:3577–3585. 17. Asokananthan N, Graham PT, Stewart DJ, Bakker AJ, Eidne KA, Thompson PJ, Stewart GA. House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. J Immunol 2002;169:4572–4578. 18. Dulon S, Cande´ C, Bunnett NW, Hollenberg MD, Chignard M, Pidard D. Proteinase-activated receptor-2 and human lung epithelial cells: disarming by neutrophil serine proteinases. Am J Respir Cell Mol Biol 2003;28:339–346. 19. Sun G, Stacey MA, Schmidt M, Mori L, Mattoli S. Interaction of mite allergens Der p3 and Der p9 with protease-activated receptor-2 expressed by lung epithelial cells. J Immunol 2001;167:1014–1021. 20. Vliagoftis H, Schwingshackl A, Milne CD, Duszyk M, Hollenberg MD, Wallace JL, Befus AD, Moqbel R. Proteinase-activated receptor-2mediated matrix metalloproteinase-9 release from airway epithelial cells. J Allergy Clin Immunol 2000;106:537–545. 21. Vliagoftis H, Befus AD, Hollenberg MD, Moqbel R. Airway epithelial cells release eosinophil survival-promoting factors (GM-CSF) after stimulation of proteinase-activated receptor 2. J Allergy Clin Immunol 2001;107:679–685. 22. Berger P, Perng DW, Thabrew H, Compton SJ, Cairns JA, McEuen AR, Marthan R, Tunon De Lara JM, Walls AF. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol 2001;91:1372–1379. 23. Ricciardolo FL, Steinhoff M, Amadesi S, Guerrini R, Tognetto M, Trevisani M, Creminon C, Bertrand C, Bunnett NW, Fabbri LM, et al. Presence and bronchomotor activity of protease-activated receptor-2 in guinea pig airways. Am J Respir Crit Care Med 2000;161:1672–1680. 24. Vergnolle N. Proteinase-activated receptor-2-activating peptides induce leukocyte rolling, adhesion, and extravasation in vivo. J Immunol 1999;163:5064–5069. 25. Schmidlin F, Amadesi S, Dabbagh K, Lewis DE, Knott P, Bunnett NW, Gater PR, Geppetti P, Bertrand C, Stevens ME. Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. J Immunol 2002;169:5315–5321. 26. Lindner JR, Kahn ML, Coughlin SR, Sambrano GR, Schauble E, Bernstein D, Foy D, Hafezi-Moghadam A, Ley K. Delayed onset of inflammation in protease-activated receptor-2-deficient mice. J Immunol 2000;165:6504–6510. 27. Moffatt JD, Jeffrey KL, Cocks TM. Protease-activated receptor-2 activating peptide SLIGRL inhibits bacterial lipopolysaccharide-induced recruitment of polymorphonuclear leukocytes into the airways of mice. Am J Respir Cell Mol Biol 2002;26:680–684. 28. Cocks TM, Moffatt JD. Protease-activated receptor-2 (PAR2) in the airways. Pulm Pharmacol Ther 2001;14:183–191. 29. Takeuchi T, Harris JL, Huang W, Yan KW, Coughlin SR, Craik CS. Cellular localization of membrane-type serine protease 1 and identification of protease-activated receptor-2 and single-chain urokinase-type plasminogen activator as substrates. J Biol Chem 2000;275:26333– 26342. 30. Miki M, Nakamura Y, Takahashi A, Nakaya Y, Eguchi H, Masegi T, Yoneda K, Yasuoka S, Sone S. Effect of human airway trypsin-like protease on intracellular free Ca2⫹ concentration in human bronchial epithelial cells. J Med Invest 2003;50:95–107. 31. Cottrell GS, Amadesi S, Grady EF, Bunnett NW. Trypsin IV, a novel agonist of protease-activated receptors 2 and 4. J Biol Chem 2004;279: 13532–13539. 32. Lourbakos A, Potempa J, Travis J, D’Andrea MR, Andrade-Gordon P, Santulli R, Mackie EJ, Pike RN. Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion. Infect Immun 2001;69:5121–5130. 33. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 1994;10:38–47. 34. Bo¨hm SK, Khitin LM, Grady EF, Aponte G, Payan DG, Bunnett NW. Mechanisms of desensitization and resensitization of proteinaseactivated receptor-2. J Biol Chem 1996;271:22003–22016.

Dulon, Leduc, Cottrell, et al.: Pseudomonas aeruginosa Elastase Disarms PAR-2 35. Molino M, Woolkalis MJ, Reavey-Cantwell J, Pratico D, AndradeGordon P, Barnathan ES, Brass LF. Endothelial cell thrombin receptors and PAR-2: two protease-activated receptors located in a single cellular environment. J Biol Chem 1997;272:11133–11141. 36. Caballero AR, Moreau JM, Engel LS, Marquart ME, Hill JM, O’Callaghan RJ. Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases. Anal Biochem 2001; 290:330–337. 37. Renesto P, Si-Tahar M, Moniatte M, Balloy V, Van Dorsselaer A, Pidard D, Chignard M. Specific inhibition of thrombin-induced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor. Blood 1997;89:1944–1953. 38. Do¨ring G, Obernesser HJ, Botzenhart K, Flehmig B, Hoiby N, Hofmann A. Proteases of Pseudomonas aeruginosa in patients with cystic fibrosis. J Infect Dis 1983;147:744–750. 39. Loew D, Perrault C, Morales M, Moog S, Ravanat C, Schuhler S, Arcone R, Pietropaolo C, Cazenave JP, van Dorsselaer A, et al. Proteolysis

40.

41.

42.

43. 44.

45.

419 of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry. Biochemistry 2000;39:10812–10822. Verhoeven AJ, Mommersteeg ME, Akkerman JW. Balanced contribution of glycolytic and adenylate pool in supply of metabolic energy in platelets. J Biol Chem 1985;260:2621–2624. Al-Ani B, Hollenberg MD. Selective tryptic cleavage at the tethered ligand site of the amino terminal domain of proteinase-activated receptor-2 in intact cells. J Pharmacol Exp Ther 2003;304:1120–1128. Hollenberg MD, Saifeddine M, al-Ani B. Proteinase-activated receptor-2 in rat aorta: structural requirements for agonist activity of receptoractivating peptides. Mol Pharmacol 1996;49:229–233. Galloway DR. Pseudomonas aeruginosa elastase and elastolysis revisited: recent developments. Mol Microbiol 1991;5:2315–2321. Azghani AO, Bedinghaus T, Klein R. Detection of elastase from Pseudomonas aeruginosa in sputum and its potential role in epithelial cell permeability. Lung 2000;178:181–189. Vancheri C, Mastruzzo C, Sortino MA, Crimi N. The lung as a privileged site for the beneficial actions of PGE2. Trends Immunol 2004;25:40–46.