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Previous Article | Next Article 
Infection and Immunity, August 2007, p. 3848-3858, Vol. 75, No. 8
0019-9567/07/$08.00+0 doi:10.1128/IAI.00015-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Sophie de Bentzmann,3
Jean-Claude Rousselle,4
Abdelkader Namane,4
Michel Chignard,1,2 and
Dominique Pidard1,2*
INSERM, U874, Paris F-75015, France,1 Unité de Défense Innée et Inflammation, Institut Pasteur, Paris F-75015, France,2 CNRS, IBSM-UPR9027, Marseille F-13402, France,3 Plate-Forme de Protéomique, Institut Pasteur, Paris F-75015, France4
Received 4 January 2007/ Returned for modification 6 March 2007/ Accepted 2 May 2007
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The urokinase-type plasminogen activator (uPA) receptor (uPAR, also designated CD87 or Mo3 antigen) is a highly glycosylated glycosylphosphatidylinositol (GPI)-anchored cell membrane protein. It is composed of three homologous globular domains, named D1, D2, and D3, from the amino terminus to the carboxy terminus of the molecule, each containing a conserved arrangement of disulfide bonds and separated by short interdomain linker sequences (43). uPAR is ubiquitously expressed in human tissues, being present on the surface of many leukocyte subsets as well as on endothelial and epithelial cells (8, 12, 53). Its expression is increased upon the exposure of cells to a wide range of inflammatory mediators (12, 53). uPAR is a pleiotropic receptor that is pivotal in (patho)physiological processes involving cell migration and tissue remodeling, particularly during infection and inflammation. Thus, uPAR binds with high affinity to the serine (Ser) proteinase uPA, which, once bound, catalyzes the conversion of plasminogen into plasmin. The latter activates several matrix metalloproteinases (MMPs), thus conferring a high potential for pericellular proteolysis, ECM processing, and cell motility to cells expressing uPAR (8, 43). uPAR also participates in cell adherence and migration in other ways, including (i) its capacity to bind the ECM adhesive protein vitronectin (Vn) (44), (ii) a physical and functional interaction with various integrins (30), and (iii) an intrinsic chemotactic activity that is associated with a peptide motif present within the interdomain sequence linking domains D1 and D2 and which targets various types of inflammatory cells (39). In accordance with these properties, uPAR-deficient mice show an impaired recruitment and activation of leukocytes at sites of infection, resulting in impaired bacterial clearance and increased host mortality (19, 39), as demonstrated in a model of P. aeruginosa pulmonary infection (20). The use of uPAR-deficient animal models also indicates that uPAR may play a prominent role in tissue repair and healing after an inflammatory event (59).
uPAR is highly susceptible to endoproteolysis within the D1-D2 linker sequence and/or at the carboxy terminus of D3. These two regions contain cleavage sites for proteinases involved in the inflammatory response, such as uPA, plasmin, and various MMPs, as well as Ser proteinases secreted from activated leukocytes (4, 5, 23, 29). Since the D1 domain plays a crucial role in the interactions of uPAR with its various ligands, the endoproteolytic removal of D1 is thus a likely pathway for controlling pericellular proteolysis as well as cell adherence and migration (8, 40, 44). Indeed, soluble uPAR species (suPAR), which include the free D1 domain but also truncated D2D3 forms as well as full-length (D1D2D3) species, can be detected in the biological fluids of healthy individuals (40). Importantly, suPAR concentrations are markedly increased in infectious and inflammatory disorders and can be of prognostic value for a negative outcome, including in lung diseases (40).
Despite the prominent role played by uPAR in the host's capacity to mount adequate innate and adaptive immune responses to pathogens, particularly in the lungs (20, 39), to date, only a few studies have investigated a direct interaction between bacteria or bacterial secretable products and uPAR (6, 13, 26). In this context, the present study provides new insights into the proteolytic regulation of leukocytic and epithelial uPAR by demonstrating that the major P. aeruginosa proteinase, LasB, generates membrane and/or soluble truncated forms of this receptor and thus has an impact on the interactions between uPAR and its physiological ligands.
(Part of this work was presented at the 26th European Cystic Fibrosis Conference, Belfast, United Kingdom, 4 to 7 June 2003.)
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15 µM, based on a molecular mass of 33 kDa) and had a specific activity of 260 units/mg protein. The fungal thermolysin/LasB-specific inhibitor N-(
-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp (phosphoramidon [PA]) (27) and the LasB substrate elastin-Congo red were obtained from Sigma. The following two synthetic N-acylated and C-amidated peptides mapping the human uPAR sequences Ser81-Cys95 within the D1-D2 linker domain and Asn272-Gly283 at the carboxy terminus of D3, respectively (43), were prepared with >95% purity by Eurogentec (Seraing, Belgium): the 15-mer peptide SGRAVTYSRSRYLEC, hereafter designated the D1-D2 peptide, and the 12-mer peptide NHPDLDVQYRSG, hereafter designated the D3 peptide.
Antibodies used in this study were anti-uPAR mouse monoclonal antibodies (mAbs) 3931 (against the D1 domain) and 3932 (against the D2 domain) (both immunoglobulin G1 [IgG1] mAbs) and 3936, an IgG2a mAb reacting with an undetermined epitope in the D2D3 portion of human uPAR (4) (all from American Diagnostica); mouse IgG1 mAbs R2 and R4 directed against different epitopes located in the D3 domain (23, 33) and provided by Gunilla Høyer-Hansen (The Finsen Laboratory, Copenhagen, Denmark); mouse IgG2b mAb MY4 from Coulter Corp. (Miami, FL); mouse mAbs ICRF44 (IgG1) and IB4 (IgG2a), directed against epitopes on the CD11b/M and CD18/ß2 subunits of the Mß2 integrin, respectively, as well as nonspecific control mAbs of the IgG1, IgG2a, or IgG2b subclass from Ancell (Bayport, MN); His probe (H-15), a rabbit polyclonal antibody against the His6 peptide tag, from Santa Cruz Biotechnologies (Santa Cruz, CA); a rabbit polyclonal antibody directed against the mature, 33-kDa LasB protein obtained from a commercial source; an FITC-conjugated F(ab')2 goat antibody against mouse IgG from Dako (Glostrup, Denmark); and horseradish peroxidase (HRP)-conjugated antibodies against mouse IgG (ImmunoPure) from Pierce (Rockford, IL) or from Jackson Laboratories (West Grove, PA) (as were HRP-conjugated anti-rabbit IgG goat antibodies [AffiniPure]).
Cell culture reagents were all obtained from Gibco BRL (Paysley, Scotland), except for heat-inactivated fetal calf serum, which was obtained from HyClone (Logan, UT), whereas chemicals for cell solubilization, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and protein electrotransfer were obtained from Sigma or from Bio-Rad (Hercules, CA).
Cell cultures and membrane protein biotinylation. (i) Monocytic cells. The U937 human promonocytic cell line (50) (ATCC CRL-1593.2; American Type Culture Collection, Manassas, VA) was grown and differentiated into a monocyte/macrophage phenotype by exposure to phorbol 13-myristate 12-acetate (Sigma-Aldrich) (4). Differentiated adherent U937 cells were recovered, washed, and, when needed, surface biotinylated using sulfosuccinimidyl-6-(biotinamido)hexanoate (EZ-Link Sulfo-NHS-LC-biotin; Pierce, Rockford, IL) as previously reported (4). Cells were finally resuspended in Hanks' balanced salt solution (HBSS) at 1 x 106 cells/ml for further treatment (see below).
(ii) Respiratory epithelial cells. 16HBE14o– cells, hereafter designated 16HBE cells, are human simian virus 40-transfected bronchial epithelial cells (14) and were a gift from D. C. Gruenert (University of Vermont, Colchester, VT). Cells were cultured to confluence in 24-well plates (Techno Plastic Products, Trasadingen, Switzerland) as described previously (17), and when required, biotinylation of cell surface molecules was performed using EZ-Link Sulfo-NHS-LC-biotin (5). Cell monolayers were washed twice with HBSS before further treatment (see below).
(iii) P. aeruginosa CM.
The P. aeruginosa PAO1 reference strain producing LasB protease and strain PAO1lasB9
(referred to as PDO240), in which the lasB gene sequence had been deleted and replaced with a spectinomycin resistance DNA fragment (37), were grown overnight at 37°C in Luria broth (LB) up to stationary phase. Bacterium-free culture supernatants were obtained by a double-step centrifugation, including a first run at 6,000 x g for 10 min followed by a second one at 12,000 x g for 10 min. Supernatants were further filtered through 0.45-µm membranes to provide conditioned media (CM), hereafter designated PAO1-CM and PDO240-CM, respectively, which were immediately frozen in aliquots at –80°C when used for uPAR cleavage experiments (see below). A subfraction was submitted to tetra-acetic acid precipitation for protein content analysis (see below). The elastolytic activity present in PAO1-CM and in PDO240-CM was measured using elastin-Congo red as a substrate and increasing concentrations of purified LasB in the range of 12.5 to 100 nM for calibration, exactly as previously described (10) except that the incubation time for various dilutions of CM or of LasB with the substrate was 30 min.
Exposure of recombinant uPAR- or uPAR-expressing cells to LasB or to P. aeruginosa CM. (i) rhuPAR.
rhuPAR-His6 adjusted to 1 µg/ml (20 nM) in 50 mM Tris-100 mM NaCl (pH 7.4) was mixed either with purified LasB in the range of 15 to 300 nM (final concentrations; enzyme-to-substrate [E/S] molar ratio of 
1:1 to 15:1) or with a preformed mixture of LasB and 50 µM PA or was left without proteinase as a control, and incubation then proceeded for 5 to 30 min at 37°C. In some experiments, rhuPAR-His-6 was incubated in parallel with increasing dilutions of PAO1-CM or of PDO240-CM for 30 min or, as control, with the LB culture medium alone or diluted CM preincubated with 50 µM PA. At the end of the exposure of rhuPAR-His6 to the protease or to media, the enzymatic activity was neutralized by the addition of PA, and samples were immediately solubilized in the presence of 2% (wt/vol) SDS and 5 mM N-ethylmaleimide (SDS-NEM) by boiling at 100°C for 5 min in the presence of 5% (vol/vol) 2-mercaptoethanol for the reduction of disulfide bonds when required. In some experiments, part of the samples was kept unsolubilized and stored at –20°C until the interaction of rhuPAR-His6 with immobilized Vn was measured (see below).
(ii) U937 cell suspensions. Suspensions of U937 cells in HBSS received 1 mM of both CaCl2 and MgCl2 (HBSS-CaMg), LasB in the range of 37.5 to 150 nM was then added, and incubation continued at 37°C from 5 to 40 min before the enzyme was blocked with 50 µM PA. In some experiments, part of the suspensions was incubated in parallel with 0.5 U/ml PI-PLC. A fraction of each cell sample was kept at 4°C for fluorescence-activated cell sorting (FACS) analysis (see below). The remaining cells were pelleted by centrifugation at 530 x g for 15 min at 4°C and then solubilized as described below, whereas extracellular fluids were collected and further centrifuged at 18,000 x g for 30 min at 4°C to eliminate cell debris before being directly solubilized with SDS-NEM, as described above, or stored at –80°C.
(iii) 16HBE cell monolayers. 16HBE cell monolayers covered with 200 µl HBSS-CaMg received various amounts of LasB in the range of 37.5 to 600 nM, and incubation continued at 37°C from 15 to 60 min before the enzymatic activity was blocked with 50 µM PA. In parallel incubations, cell monolayers were exposed to 1 U/ml PI-PLC. Extracellular fluids were collected and kept as described above. 16HBE cell monolayers were washed twice with HBSS and then solubilized as described below.
SDS-PAGE and immunoblot analysis. (i) Cell solubilization and isolation of biotinylated proteins. U937 cells in suspension (2 x 107 cells/ml) and 16HBE cell monolayers were solubilized in the presence of a cocktail of protease inhibitors, the concentration of extracted soluble proteins was measured, and biotinylated membrane proteins were separated from nonbiotinylated proteins and finally solubilized in SDS-NEM exactly as previously described (4, 5).
(ii) SDS-PAGE and immunoblotting. rhuPAR-His6 (10 to 15 ng per lane), biotinylated cell membrane proteins (extracted from 5 µg of total U937 or 16HBE cell proteins per lane), or proteins present in the extracellular fluids (30 µl per lane, corresponding to the equivalent of 2.5 or 15 µg of total cell proteins for U937 or 16HBE cells, respectively) were separated by SDS-PAGE, as previously described (4, 5), with either intact or reduced disulfide bonds. Following SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore Corp., Bedford, MA), and uPAR-related protein species were probed by immunoblotting, as previously described (4, 5), by using a panel of domain-specific, anti-uPAR antibodies as detailed in Results and in the figure legends. The chemiluminescent reaction kit ECL+ (Amersham Biosciences, Little Chalfont, United Kingdom) was used to reveal immune complexes in immunoblotting analysis. Calibration of gels for measurement of relative molecular weight (Mr) was done using Broad Range or Kaleidoscope prestained standard proteins (Bio-Rad).
For analysis of protein content in P. aeruginosa culture CM, tetra-acetic acid-precipitated protein samples were suspended in SDS sample buffer and loaded onto a 12% polyacrylamide gel for SDS-PAGE. Proteins in polyacrylamide gels were either stained with Coomassie blue or transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) together with the Kaleidoscope prestained standard proteins. Immunoblot detection of the LasB protein was performed using a primary anti-LasB rabbit antibody at a 1:500 dilution and a HRP-conjugated anti-rabbit IgG goat antibody at a dilution of 1:5,000 in Tris-buffered saline containing 10% (wt/vol) skim milk and 0.1% (vol/vol) Tween 20. The presence of the protein was revealed using the Supersignal Pico chemiluminescence revelation kit (Pierce).
Determination of cleavage sites for LasB within uPAR. (i) Fragmentation of D1-D2 and D3 peptides by LasB and mass spectrometry analysis. Peptides reconstituted at 2 mg/ml in sterile deionized water were adjusted to 65 µM in HBSS and incubated at 37°C in a 10-µl volume with LasB (E/S ratio, 1:25 or 1:100) for 5 or 15 min. Controls consisted of proteinase-free peptides 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. Samples were immediately lyophilized before being processed for mass analysis using matrix-assisted laser desorption ionization-time of flight mass spectrometry on a Voyager-DE STR spectrometer (Applied Biosystems Inc., Framingham, MA) exactly as previously described (4). Identification of the truncated peptides was carried out with GPMAW software, version 6.0 (Lighthouse Data, Odense, Denmark), for the D1-D2 and D3 sequences. Searching was performed using monoisotopic masses with a mass tolerance of 0.1%.
(ii) Amino-terminal amino acid sequencing. Protein carrier-free rhuPAR-His6 was resuspended in HBSS at 100 µg/ml (2 µM) and was incubated overnight at 37°C with 10 or 100 nM of LasB (E/S ratio, 1:20 or 1:200) or left untreated as a control. Proteins were then solubilized and reduced as described above before SDS-PAGE separation and transfer onto polyvinylidene difluoride membranes. Protein bands corresponding to the D2D3 species (see Results for details) were visualized by amido black staining, excised, and subjected to five cycles of amino-terminal microsequencing using an Applied Biosystems ABI 494 protein sequencer.
FACS analysis. Following exposure to LasB, U937 cells were distributed at 105 cells per well in conical-bottom 96-well microplates (Nunc, Roskilde, Denmark). After centrifugation, cells were immunolabeled for 1 h at 4°C in 0.1 ml of HBSS-CaMg containing 0.25% (wt/vol) bovine serum albumin (BSA) (Euromedex, Strasbourg, France) (HBSS-CaMg-BSA) and the primary mAb at 5 µg/ml, washed, and then incubated with a secondary FITC-conjugated goat anti-mouse F(ab')2 antibody (4). The one-step FITC-labeled uPA binding assay was performed as detailed previously (4). Briefly, cells pelleted in microplates were resuspended in 0.1 ml of HBSS-CaMg-BSA containing 50 nM of either FITC-uPA or FITC-F(ab')2 as a control fluoresceinated protein and then incubated for 1 h at 4°C before being washed three times. Quantitative binding of FITC-labeled proteins to the surface of the selected viable cells (defined as those remaining negative for labeling with propidium iodide, performed immediately before fluorescence measurement) was measured using a FACScan cytometer coupled to CellQuest 3.3 software (Becton Dickinson, Franklin Lakes, NJ) and expressed as the geometric mean of fluorescence intensity histograms after background binding provided by nonspecific IgG isotypes or FITC-F(ab')2 had been subtracted.
Measurement of soluble extracellular uPAR. The concentration of suPAR in cell-free U937 or 16HBE extracellular fluids was determined by use of a specific quantitative enzyme-linked immunosorbent assay (ELISA) (Quantikine ELISA for human uPAR; R&D Systems) according to the manufacturer's instructions. The detection limit of the assay was 62.5 pg/ml suPAR.
Vn-uPAR interaction assay. The capacity of LasB-treated rhuPAR-His6 to bind Vn was assayed essentially according to a previously reported procedure (47). Briefly, Maxisorb 96-well plates (Nunc) were coated with 5 µg/ml purified Vn in a phosphate-buffered saline (PBS) solution (0.1 ml/well) overnight at 4°C and then saturated with 1% BSA in PBS (PBS-BSA) (0.2 ml/well) for 1 h at room temperature. Wells then received 5 nM uPA together with rhuPAR-His6 (final concentration, 10 nM), which had been previously exposed to either LasB in the range of 30 to 150 nM for 30 min at 37°C, before LasB was blocked with 50 µM PA, or 150 nM LasB premixed with 50 µM PA or left unexposed to the proteinase. All reactants were diluted in PBS-BSA (0.1 ml/well), and incubation continued for 1 h at 4°C. After washing in PBS containing 0.1% (vol/vol) Tween 20, wells were probed for bound rhuPAR-His6 using anti-D3 mAb R2 or R4 (2 µg/ml), followed by a HRP-conjugated rabbit anti-mouse Ig antibody (1/1,000 dilution). After washing, the immune complexes were quantified through a colorimetric assay for HRP activity using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (KPL, Gaithersburg, MD). Following acidification of the medium, the optical density (OD) in wells was measured at 405 nm in an ELISA plate reader. Vn-bound uPAR was calculated by subtracting the background OD measured in wells that did not receive rhuPAR-His6 and was expressed as the percentage of the binding measured with rhuPAR-His6 that was not treated with LasB.
Statistical analysis. Results are expressed as means ± standard errors of the means (SEM) for the indicated number of experiments. Statistical analysis was performed by use of the univariate Student's t test for paired samples implemented using StatView software, version 5.0 (Abacus Concepts, Berkeley, CA).
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64 kDa (mean value over four determinations), was found, as expected, to react with both mAb 3931 (anti-D1) and mAb 3932 (anti-D2) (Fig. 1, left and middle panels, respectively) as well as with mAb R2 (anti-D3) (data not shown). However, when exposed to LasB in the range of 15 to 150 nM for 30 min and then immunoblotted with mAb 3932, rhuPAR-His6 appeared to be converted, in a LasB concentration-dependent manner, into smaller molecular species of 43 kDa. These fragments did not react with the anti-D1 mAb 3931 (Fig. 1, compare left and middle panels) but were still reactive with mAb R2 (data not shown), thus corresponding to a truncated receptor composed of the D2 and D3 domains (D2D3) (4, 23). The conversion of rhuPAR-His6 into D2D3 was already detectable 5 min after exposure to 75 nM LasB and was complete at 30 min (data not shown and Fig. 1, middle, respectively). It should be noted that minute amounts of a D2D3-like species were present in the rhuPAR-His6 sample before exposure to LasB (Fig. 1, middle). These were accompanied by traces of the free D1 domain with a mass of 18.5 kDa, as judged by immunoblotting with mAb 3931 (Fig. 1, left). Interestingly, the efficient LasB-induced conversion of rhuPAR-His6 into the D2D3 species, which resisted further endoproteolysis up to 150 nM LasB (Fig. 1, middle), was not accompanied by an accumulation of free D1 in the reaction mixture. Indeed, this species was undetectable at the highest proteinase concentrations (Fig. 1, left), and no D1-derived subfragments could be detected by immunoblotting. Similar results were observed when intramolecular disulfide bonds were maintained for SDS-PAGE (data not shown). Altogether, these data demonstrated that LasB can efficiently cleave soluble uPAR within the sequence linking D1 to D2, while the released D1 domain is likely to be extensively degraded.
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Cleavage sites in the D1-D2 linker sequence were identified through amino-terminal sequencing of the truncated D2D3 species resulting from exposure of rhuPAR-His6 to LasB. These D2D3 molecules were found to contain a predominant species with the amino-terminal sequence YSRSR, while at the lowest E/S ratio (1:200), a less abundant species with an amino-terminal VTYSR sequence could also be detected (<10% of the total D2D3 material analyzed on an amino acid molar basis). These results thus established that cleavage sites for LasB in the amino-terminal part of uPAR are at the Ala84-Val85 and at the Thr86-Tyr87 peptide bonds (43). Because this approach was not applicable to the characterization of the cleavage sites at the carboxy terminus of D3, these were determined through matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the proteolytic fragmentation of a synthetic peptide, which mimicked the D3 carboxy-terminal sequence ahead of the last cysteine residue engaged in intradomain disulfide bonding (43). Exposure of this peptide (sequence COO-NHPDLDVQYRSG-NH2) (monoisotopic ion [M + H]+ value, 1,441.6466) to LasB for 5 to 15 min and at E/S ratios of 1:100 to 1:25 resulted in a marked but nevertheless incomplete hydrolysis, producing one single shorter detectable species with an [M + H]+ value of 979.4548 (values are representative of two determinations). This corresponded to peptide COO-NHPDLDVQ-NH2, thus demonstrating a single cleavage at the Gln-Tyr peptide bond, analogous to Gln279-Tyr280 in human uPAR (43). A similar approach applied to a synthetic peptide mimicking the D1-D2 linker sequence (COO-SGRAVTYSRSYLEC-NH2) confirmed the capacity of LasB to specifically cleave the Ala-Val and Thr-Tyr peptide bonds (data not shown).
LasB is the principal protease secreted by P. aeruginosa that can target uPAR.
The secretory proteome (secretome) of clinical strains of P. aeruginosa is known to contain a number of proteases other than LasB, including the elastolytic metalloprotease LasA and the Ser proteinase PrpL (or protease IV) (10, 49). In order to establish whether or not LasB was uniquely responsible for the cleavage of uPAR, we compared the proteolytic capacity of the secretome from the LasB-producing strain PAO1 to that of the LasB-deficient strain PDO240, designed on the PAO1 genetic background (37). One-dimensional SDS-PAGE analysis of proteins present in the bacterial culture CM coupled to specific immunoblotting confirmed that LasB is a major product in the secretome of strain PAO1, as previously reported (49), whereas it was undetectable in the secretome of strain PDO240 (Fig. 2A). Measurement of the proteolytic activity present in the CM, measured on elastin-Congo red, confirmed that PAO1-CM contained a marked activity, which was found to be approximately equivalent to that produced by 500 nM purified LasB (mean value of three determinations) and was inhibited to a mean of 85% by the LasB-specific inhibitor PA (27) and to 90% by 75 mM EDTA, as previously reported (10, 17). Conversely, barely detectable elastolytic activity was observed in PDO240-CM (
5% of that measured in PAO1-CM), in agreement with previous observations (37). When exposed to these milieus and then assayed for immunoblotting with anti-D2 mAb 3932, rhuPAR-His6 was found to be readily cleaved by PAO1-CM and progressively converted into the truncated D2D3 species for dilutions of the milieu ranging from 1/5 to 1/40, i.e., equivalent to 100 to 12.5 nM LasB. The pattern of cleavage was similar to that resulting from the exposure of rhuPAR-His6 to purified LasB in the same range of concentrations (Fig. 2B, top). Importantly, cleavage was totally blocked by the preincubation of PAO1-CM with the inhibitor PA, whereas it was undetectable when rhuPAR-His6 was exposed to PD0240-CM at the lowest dilution (1/5), compared to the control incubations made in LB culture medium alone or in the presence of PA (Fig. 2B, top). Moreover, immunoblotting with anti-D1 mAb 3931 showed that the freed D1 domain was degraded at the highest levels of protease activities (Fig. 2B, bottom).
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The proteolytic activity of LasB on membrane uPAR was first explored on U937 cells through flow immunocytometry. Exposure of cells to the proteinase in the range of 30 to 300 nM for 30 min resulted in a LasB concentration-dependent decrease in binding of anti-D1 mAb 3931 to a maximal reduction of epitope surface expression of 85% (Fig. 3A). By contrast, the binding of mAbs 3932 and 3936, both targeting D2D3, did not significantly decrease following cell exposure to LasB up to 300 nM. Time course analyses indicated that the binding of mAb 3931 was already reduced by 20% and 50% after 10 and 20 min of exposure to 75 nM LasB, respectively. Incubation of cells with LasB premixed with its inhibitor PA maintained the binding of mAb 3931 to levels observed in control cells, indicating that the impact of LasB on the expression of the D1 domain relied on the proteinase activity. No change in the binding capacity of U937 cells exposed to LasB could be noted when antibodies directed towards cell surface antigens such as CD11b, CD18, and CD14 were used, thus indicating a rather restricted activity of LasB on leukocytic membrane proteins (data not shown). Finally, the exposure of U937 cells to up to 300 nM LasB did not increase the number of propidium iodide-positive dead cells, indicating that LasB was not cytotoxic under our experimental conditions (data not shown).
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Similar effects of LasB on membrane uPAR were observed when 16HBE bronchial epithelial cell monolayers were exposed to the proteinase. As for the monocytic cells, SDS-PAGE/immunoblotting analysis of biotinylated 16HBE cell membrane proteins indicated that epithelial membrane uPAR occurred as two molecular species (5) (here well focused in SDS-polyacrylamide gels), i.e., the major D1D2D3 form with a molecular mass of 72 kDa (mean value over five determinations) and a minor truncated D2D3 species of 53 kDa (Fig. 3C). Exposure of cells to 150 nM LasB resulted in a marked conversion of full-length uPAR into the D2D3 species as early as 15 min, which was almost complete by 60 min (Fig. 3C, left). Increasing the concentration of LasB up to 600 nM over a 30-min period did not result in more extensive proteolysis of the truncated membrane uPAR (Fig. 3C, right).
LasB induces shedding of membrane uPAR.
Besides the removal of domain D1 from membrane-associated uPAR as described above, LasB also resulted in the shedding of soluble forms of the truncated receptor. Indeed, immunoblotting analysis of cell-free extracellular milieus using anti-D2 mAb 3932 revealed that the exposure of 16HBE epithelial cell monolayers to LasB in the range of 150 to 600 nM and from 15 to 60 min led to an increasing release of a soluble species showing a molecular mass of 45 kDa (Fig. 4A), which was unreactive with anti-D1 mAb 3931 (data not shown), thus corresponding to the truncated D2D3 form. The identity of this species was confirmed by comparison with those resulting from the exposure of cells to a bacterial PI-PLC. This phospholipase hydrolyzes the GPI structure between the inositol phosphate ring and the diacylglycerol moiety (43) and thus released full-length (molecular mass, 61 kDa) and truncated (molecular mass, 47 kDa) uPAR species in a ratio that mirrored that observed for membrane uPAR on normal 16HBE cells (compare Fig. 4A and 3C). Immunoblot analysis of the milieus taken from monocytic U937 cell suspensions exposed to LasB (Fig. 4B) similarly showed that part of the membrane D2D3 species generated by the bacterial proteinase was shed into the extracellular environment for enzyme concentrations of 
75 nM. It is important to note that free D1 domains were never detected in the extracellular milieus of LasB-treated cells using our immunoblotting approach, thus suggesting that this species was efficiently degraded by the proteinase, as has been noted with recombinant uPAR.
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Interaction of uPAR with uPA and Vn is abrogated following cleavage of the receptor by LasB. Membrane uPAR is a binding site for two ligands with major importance in pericellular proteolysis and cell adherence, i.e., the proteinase uPA and the adhesive protein Vn (8, 44). Amino-terminal domains of both ligands can interact with several discrete sequences located within all three domains of uPAR, although the presence of domain D1 is mandatory for high-affinity interactions of the ligands with the receptor (43, 44). Since LasB can proteolytically remove domain D1, we used an FITC-coupled uPA binding assay to ascertain that the proteinase can affect the interaction of uPA with the U937 cell surface. Exposure to LasB for 30 min indeed resulted in an enzyme concentration-dependent reduction of the cell capacity to bind FITC-uPA. This was closely related to the specific disappearance of domain D1 as judged through the decreased binding of anti-D1 mAb 3931, as opposed to the unchanged binding of anti-D2 mAb 3932 (Fig. 5A). Binding of uPA-FITC reached a maximal 65% reduction at the highest LasB concentration tested, i.e., 150 nM. As previously observed (4, 43), the exposure of U937 cells to 0.5 U/ml PI-PLC resulted in a similar decrease in the binding capacity of uPA (64% and 69% reduction by two determinations) via the partial shedding of full-length uPAR, as shown in Fig. 4.
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We have now established that the human uPAR is a target for LasB, which is able to efficiently cleave discrete peptide bonds in the D1-D2 linker sequence and to generate a truncated (D2D3) species. The exposure of soluble or cell-associated uPAR to the proteinase in the range of 15 to 75 nM and for periods of time as short as 5 to 15 min already results in a marked proteolysis of D1. As a bacterial Zn2+-dependent metalloproteinase of the thermolysin family, LasB cleaves at the amino group side of the amino acid located at the P'1 position in the sessile peptide bond, preferably a bulky apolar or hydrophobic amino acid, whereas the presence of Ala at the P1 position increases the rate of hydrolysis (38, 41). The cleavage sites identified within D1-D2, Ala84-Val85 and Thr86-Tyr87 (Fig. 6), are in agreement with the enzymatic properties of LasB, and indeed, hydrolysis at Xaa-Val and Xaa-Tyr peptide bonds has previously been reported for this proteinase (1, 17, 41). Interestingly, a truncated (D2D3) species corresponding to cleavage at the Thr86-Tyr87 position was found to be the most abundant species following the interaction of uPAR with LasB, and this position is also a major cleavage site identified within the D1-D2 linker region for some mammalian metalloproteinases of the MMP family known to shed domain D1 and to regulate uPAR-dependent cell functions (2, 29). Because soluble free D1 remains undetectable by our immunoblot assay, we propose that the removal of D1 by LasB is accompanied by a further, extensive degradation of this domain. A similar feature has been previously observed for some proteinases that are active on uPAR (2, 4, 5), indicating a marked susceptibility of this important functional domain to endoproteolysis.
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150 nM) and after 30 min, LasB was able to release a quantity of suPAR from the surface of respiratory epithelial cells equivalent to that shed by a bacterial phospholipase acting on the GPI moiety. In contrast, when applied to monocytic cells, LasB was markedly less efficient for shedding uPAR than was PI-PLC, which is known to release about 50% of membrane-associated uPAR (4), while LasB released about 5%. This infers that the accessibility of LasB to the juxtamembrane region of uPAR may vary depending on the cell type and/or membrane environment in which this receptor is expressed. In contrast to free D1, the D2D3 species generated by LasB resisted further degradation by the proteinase and accumulated on the cell surface and in the extracellular milieu, even at the highest LasB concentration used in our study (i.e., 600 nM). LasB is thus a new member, and the first of bacterial origin, of a group of proteinases, also including the polymorphonuclear leukocyte Ser proteinases neutrophil elastase and cathepsin G as well as plasmin, which are capable of cleaving both the D1-D2 linker and the D3 carboxy terminus of human uPAR and of releasing soluble D2D3 species (4, 5). In this respect, it remains to be determined whether LasB can generate soluble D2D3 molecules endowed with chemotactic and promigratory activities. Indeed, we show here that it cleaves the D1-D2 linker domain slightly upstream of the S88RSRY sequence, which has been identified as being the minimal chemotactic epitope (8, 39, 40) (Fig. 6).
Experimental and/or clinical evidence points to a role for LasB as a major modulator of host cellular responses during P. aeruginosa infections. As mentioned previously, LasB is able to alter the activity of many immune and inflammatory human cells, including leukocytes and epithelial cells, by proteolytically acting on membrane receptors (9, 17, 24, 56). It is noteworthy that the concentrations of LasB (in the range of 10 to 100 nM) as well as the duration of exposure to the proteinase (within 60 min) usually found to be effective for the cleavage of domains of different receptors are similar to those reported here for uPAR and appear to be lower than those required for the cleavage of extracellular immune effector proteins (1, 35, 51). LasB quantitatively represents one of the major proteins/proteases secreted in culture milieus by various strains of P. aeruginosa, including clinical isolates from CF patients (49) (Fig. 2A), and is the only one that targets uPAR, as established in the present study by showing that the secretome from a LasB-deficient strain does not produce cleavage of the receptor, in contrast to that of a LasB-producing strain. Furthermore, the mucopurulent respiratory liquid from such patients has been found to markedly stimulate the transcription of the lasB gene (57). Indeed, both transcripts of lasB (51) and the LasB protein (25, 54) can be detected in sputum obtained from infected CF patients. Specific quantitative immunodetection or measurement of the enzymatic activity indicates that LasB concentrations in sputum or bronchoalveolar lavage fluid are highly variable between patients, ranging from a few nmol/liter up to levels of 300 nM (25, 54). Because the protein material experimentally extracted from sputum as well as lavage fluid represents diluted airway fluids, one could thus expect the local concentrations of LasB in the immediate vicinity of host cells to be higher than the concentrations measured ex vivo and to be well within the range of those active on uPAR. Finally, it has been shown that high titers of antibodies are continuously produced against LasB in many chronically infected CF patients, suggesting a sustained secretion of the proteinase from the time of initial infection (16), while respiratory fluids obtained from such patients contain host protein degradation products, some of which may be specific markers for LasB activity in vivo (1, 9, 51). Taken together, these observations provide strong support for the assumption that LasB, among the numerous P. aeruginosa proteinases, may be an important virulence factor (7, 16), particularly by dampening the host immune responses through the alteration of membrane receptors. This may well apply to uPAR, since it has been shown that experimental P. aeruginosa corneal infections in mice are accompanied by fragmentation of the receptor, leading to the shedding of D1 and the appearance of truncated D2D3 species in the infected tissues (6). Along with this experimental observation, there is recent clinical evidence that suPAR measured in sputum from patients with various infectious/inflammatory lung diseases is found at the highest levels in a series of CF patients all infected with P. aeruginosa, with an approximately 10-fold increase in concentrations compared to those found in healthy subjects (58). Although the proteolytic system(s) responsible for the shedding of uPAR in these in vivo settings has not yet been characterized, our present in vitro demonstration that LasB can target uPAR suggests that it may well be a major participant in the proteolytic processing of the receptor, together with host proteolytic systems such as the leukocyte Ser proteinases and the uPA/plasmin/MMP cascade (4, 5, 23, 29).
We have confirmed in this study that the proteolysis of D1, as it results from LasB activity on uPAR, has a major negative impact on the capacity of cells to bind uPA. Such a cleavage of uPAR may constitute a survival advantage for the bacteria at various levels. First, the engagement of uPAR through its D1 domain with uPA and with membrane partners of the integrin family is a major molecular mechanism involved in recruitment and in bactericidal activities of inflammatory leukocytes that are triggered in response to various pathogens, including P. aeruginosa (19, 20, 39). The disruption of these molecular interactions due to the removal of D1 may participate, together with the proteolytic alteration of other immune effectors (1, 9, 32), in the inappropriate dampening of the early immune response seen during P. aeruginosa infections and may thus help the bacteria to escape rapid clearance and to colonize the respiratory tract (28, 55). Subsequently, as for many pathogenic bacteria (31), P. aeruginosa appears to be able to use the host uPA/plasmin/MMP cascade, besides its own proteinases, to facilitate its dissemination in tissues through matrilysis. This occurs through the binding of plasminogen on the bacterial surface and its activation by uPA (18) as well as through a bypass of the uPAR/uPA-dependent controlled activation of MMPs via the removal of D1 and the direct unbalanced activation of MMPs by LasB (15). Moreover, more unusual interactions between P. aeruginosa and uPA appear to exist, since in vitro, uPA was able to markedly increase bacterial growth under stringent culture conditions (low oxygen and low bacterial density) and to stimulate the production of bacterial proteinases (21, 22). By decreasing the uPAR-dependent uptake of uPA by host cells, cleavage of uPAR may thus increase uPA bioavailability to P. aeruginosa, with the consequent stimulation of both bacterial growth and dissemination.
Finally, cleavage of uPAR may well participate in the detrimental progression of lung tissues chronically infected with P. aeruginosa towards fibrosis, bronchiectasis, and emphysema (28, 52, 55). Indeed, the proteolysis of D1 by LasB expectedly abrogates the capacity of uPAR to bind, in a uPA-dependent manner, the inflammatory ECM component Vn (8, 44). The trimolecular interaction between uPAR, uPA, and Vn supports the migration and adherence of airway monocytes/macrophages to the Vn-rich provisional ECM and, subsequently, its remodeling and elimination during tissue healing (48), whereas the proliferation and migration of smooth muscle and epithelial cells within inflammatory ECM also depend partly on the engagement of uPAR with uPA and Vn and with membrane integrins (8, 44). A disruption of these molecular axes through of removal of D1 may thus have profound negative impacts on tissue repair and the return to homeostasis (59).
Although the biological consequences of the interactions between the P. aeruginosa proteinase LasB and the host receptor uPAR require further investigation, we hypothesize that the cleavage and shedding of uPAR could represent an important step during the complex interplay between the host and the pathogen, allowing the latter to more easily infect and colonize susceptible tissues, such as those of the respiratory tract.
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ACKNOWLEDGMENTS |
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We are indebted to Gunilla Høyer-Hansen (The Finsen Laboratory, Copenhagen, Denmark) for providing us with monoclonal antibodies R2 and R4, and we thank Jacques D'Alayer (Plate-Forme d'Analyze et de Microséquençage des Protéines, Institut Pasteur, Paris, France) for performing protein amino-terminal sequencing and Mary Osborne-Pellegrin for editing of the manuscript.
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FOOTNOTES |
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Published ahead of print on 21 May 2007.
Present address: Klinische Forschergruppe der Frauenklinik der Technische Universität München, München, Germany.
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