Proteolytic Regulation of the Receptor/CD87 on Monocytic Cells by and G

This information is current as Nathalie Beaufort, Dominique Leduc, Jean-Claude of September 25, 2021. Rousselle, Viktor Magdolen, Thomas Luther, Abdelkader Namane, Michel Chignard and Dominique Pidard J Immunol 2004; 172:540-549; ; doi: 10.4049/jimmunol.172.1.540

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by and Cathepsin G1

Nathalie Beaufort,* Dominique Leduc,* Jean-Claude Rousselle,† Viktor Magdolen,‡ Thomas Luther,§ Abdelkader Namane,† Michel Chignard,* and Dominique Pidard2*

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immuno- blotting and flow immunocytometry that the human neutrophil -proteinases elastase and cleave CD87 within Downloaded from the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spec- trometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each and suggests the existence of a mechanism regulating the CD87(D1-D2)-asso- ciated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind http://www.jimmunol.org/ urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells. The Journal of Immunology, 2004, 172: 540–549.

he urokinase-type receptor (CD87) It is also expressed to high levels in many types of tumor cells and is a highly glycosylated, single-chain polypeptide linked strongly contributes to their invasiveness and metastatic potential (4, T to the plasma membrane through a GPI anchor. It consists 5). Indeed, CD87 is a multiligand receptor that operates as a key of three structurally homologous domains of ϳ90 amino acids, element in (patho)physiological processes involving cell migration named D1, D2, and D3 from the N terminus to the C terminus of

and tissue remodeling, particularly during inflammation and cancer by guest on September 25, 2021 the molecule, each containing a conserved arrangement of disul- metastasis. Thus, CD87 binds with high affinity the Ser-proteinase fide bonds and separated by short interdomain linker sequences (1, urokinase-type plasminogen activator (uPA) which, once bound, cat- 2). CD87 is present on the surface of many cell types, including alyzes the conversion of plasminogen into , the latter partic- 3 monocytes/macrophages, polymorphonuclear neutrophils (PMNs), ipating in turn to the activation of various matrix metalloproteinases vascular endothelial and smooth muscle cells, and epithelial cells, and (MMPs), thus conferring to cells expressing CD87 a high potential for is overexpressed upon cell exposure to inflammatory mediators (3, 4). pericellular proteolysis (2, 4, 6). CD87 also binds the extracellular matrix adhesive protein vitronectin, thus greatly contributing to the adherence of leukocytic cells (7). High-affinity binding of both uPA *Unite«deDe«fense Inne«e et Inflammation, Institut National de la Sante«etdelaRe- cherche Me«dicale, Equipe 336, Institut Pasteur, Paris, France; †Plateforme de Pro- and vitronectin to CD87 requires the presence of D1 (2, 7). Yet, CD87 te«omique, Institut Pasteur, Paris, France; ‡Klinische Forschergruppe der Frauenklinik participates in cell adherence and migration through two other routes: der Technischen Universita¬t Mu¬nchen, Klinikum rechts der Isar, Mu¬nchen, Germany; and ¤Medizinisches Fachlabor Bautzen, Bautzen, Germany 1) a physical association and functional interaction with various in- ␣ ␤ Received for publication April 16, 2003. Accepted for publication October 15, 2003. tegrins, including the leukocyte M 2/CD11b-CD18 (8Ð10), and 2) an intrinsic chemotactic activity associated to the D1-D2 linker se- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance quence (11). In accordance with these properties demonstrated in with 18 U.S.C. Section 1734 solely to indicate this fact. vitro, CD87-deficient mice show an impaired recruitment of leuko- 1 This work was supported by the Centre National de la Recherche Scientifique (to cytes in response to local infections, resulting in increased mortality D.P.), by a fellowship from Ministe`re de la Recherche (to N.B.), and Institut Pasteur, Paris, France (to J.-C.R. and A.N.), and by Kapitel Klinische Forschung des Klini- (12Ð14). kums rechts der Isar, Mu¬nchen, Germany (to V.M.). It was presented in part at the CD87 is highly susceptible to endoproteolysis within the D1-D2 World Congress on Inflammation, Edinburgh, Scotland, September 22Ð26, 2001. linker sequence, which contains cleavage sites for proteinases in- 2 Address correspondence and reprint requests to Dr. Dominique Pidard, Unite«de volved in the inflammatory response, such as uPA itself, plasmin, De«fense Inne«e et Inflammation, Institut National de la Sante«et de la Recherche Me«di- cale Equipe 336, Institut Pasteur, 25 rue du Dr. Roux, F-75724 Paris cedex 15, France. and various MMPs (2, 15Ð19), and endoproteolytic processing of E-mail address: [email protected] CD87 with removal of D1 is a likely pathway for controlling peri- 3 Abbreviations used in this paper: PMN, polymorphonuclear neutrophil; uPA, uroki- cellular proteolysis as well as cell adherence and migration (20, nase-type plasminogen activator; MMP, matrix metalloproteinase; PI-PL, phosphati- 21). Along with these experimental observations, a soluble D1 dylinositol-specific phospholipase; EL, elastase; CG, cathepsin G; rhCD87[His]6, polyhistidine-tagged recombinant human CD87; ␣CT, ␣-; PR3, protein- domain can be detected in urine, likely originating from the vas- ␣ ␣ ase 3; 1ACT, 1-antichymotrypsin; AAPV-CMK, N-methoxysuccinyl-Ala-Ala-Pro- cular compartment (22, 23). However, soluble forms of full-length Val chloromethylketone; PMN-CM, PMN-conditioned milieu; NEM, N-ethylmale- imide; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; and truncated (D2D3) CD87 are also found in both blood and urine sD1, soluble D1; m, membrane. and are markedly increased during infections, inflammation, and

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 541

cancer (23Ð28). Mechanistic explanations for the release of such Cells species remain scarce, except for the hydrolysis of the GPI moiety U937 cell culture, differentiation, and membrane biotinylation. The by phosphatidylinositol-specific phospholipases C or D (PI-PLC or U937 human promonocytic cell line (42) (CRL-1593.2; American Type PI-PLD) (29Ð31). Although a juxtamembrane proteolytic cleavage Culture Collection, Manassas, VA) was grown as recommended by the supplier, in the presence of 100 U/ml penicillin, 100 ␮g/ml streptomycin, of CD87 has also been commonly suggested to occur, the protein- and 0.25 ␮g/ml Fungizone (Life Technologies). Before all experiments ase(s) involved in this process are still unidentified. reported hereafter, U937 cells at 0.5 ϫ 106 cells/ml were cultured for PMNs are major effector cells during innate host defense, and 48Ð72 h in the presence of 15 nM PMA (Sigma-Aldrich) to differentiate their primary recruitment and accumulation into injured tissues cells into monocytes/macrophages (3, 43), with Ͼ70% of cells becoming adherent to plastic and showing approximately 5-, 10-, and 90-fold in- appears to determine the subsequent mobilization of other blood ␣ creases in the expression of the membrane Ags CD14, CD11b/ M, and leukocytes (32, 33). Activated PMNs can locally release the con- CD87, respectively, as judged by FACS analysis (data not shown). Adher- tent of their numerous granules, including the primary/azurophilic ent U937 cells were detached by incubation (15 min, 25¡C) with HBSS granules containing Ser-proteinases such as elastase (EL) and ca- containing 1 mM EDTA, followed by neutralization with 1 mM of both CaCl and MgCl and two washings at 25¡C in HBSS with centrifugations thepsin G (CG), which have been shown to accumulate at inflam- 2 2 at 530 ϫ g for 15 min. When needed, biotinylation of cell surface mole- matory foci (33Ð35). These Ser-proteinases play a crucial role in cules was performed essentially as previously reported (44), using sulfo- extracellular proteolytic processes affecting not only matrix compo- succinimidyl-6-(biotinamido)hexanoate (EZ-Link Sulfo-NHS-LC-biotin; 7 nents, but also inflammatory effectors such as cytokines or cell mem- Pierce) at 1 mg/ml in suspensions of 2.5 ϫ 10 cells/ml in PBS (pH 8.0) containing 1 mM of both CaCl and MgCl and incubation for 30 min at brane receptors, and thus they may have immunomodulatory func- 2 2 25¡C. Cells were then diluted with PBS containing 50 mM glycine to tions in many (patho)physiological situations (33Ð36). In this context, quench unreacted biotin and were washed twice in HBSS as above. Either Downloaded from one striking feature is that they can participate to the chemotaxis of biotinylated or nonbiotinylated cells were finally resuspended in HBSS at PMNs and mononuclear cells both in vitro and in vivo (32, 35). 1Ð2.5 ϫ 106 cells/ml. The present study provides new insights into the proteolytic reg- Isolation of human blood monocytes. Monocytes were isolated from pe- ulation of leukocyte CD87 by demonstrating that the two major ripheral blood obtained from human volunteers with their informed consent (Etablissement Franc¸ais du Sang, Paris, France), based on the sequential PMN Ser-proteinases EL and CG cooperate to generate membrane aggregation and sheep RBC rosetting of PBMCs, resulting in a cell pop- and/or soluble fragments of potential biological importance and to ulation consisting of at least 85% monocytes, as previously described (45).

6 http://www.jimmunol.org/ reduce the capacity of inflammatory cells to initiate a uPA-depen- Monocyte suspensions were adjusted to 1 ϫ 10 cells/ml in HBSS. dent pericellular proteolytic cascade. Isolation and degranulation of human blood PMNs. PMNs were isolated from peripheral blood obtained from human volunteers with their informed consent (Etablissement Franc¸ais du Sang), as previously described (46). Isolated cells (Ͼ95% PMNs) were resuspended at 5 ϫ 106 cells/ml in Materials and Methods HBSS supplemented with 1.3 mM CaCl2 and 1 mM MgCl2. Degranulation ␮ Reagents was obtained by exposing cells to 5 g/ml cytochalasin B (Sigma-Aldrich) for 5 min under mild stirring, then to 0.5 ␮M fMLP (Sigma-Aldrich) for Recombinant human CD87 corresponding to the sequence Leu1-Arg281, another 5 min (47), and then cell suspensions were rapidly cooled to 4¡C. lacking the GPI anchor and fused to a C-terminal polyhistidine tag Proteinase-enriched, PMN-conditioned milieus (PMN-CMs) were obtained

by pelletting cells at 1700 ϫ g for 10 min at 4¡C and then clearing the by guest on September 25, 2021 (rhCD87[His]6), was from R&D Systems (Minneapolis, MN). Human leu- kocyte EL and CG were purified as previously described (37) from human supernatants from cell debris by centrifugation at 18,000 ϫ g for 15 min at Ϫ bone marrow PMNs. FITC-conjugated, high-m.w. human uPA was from 4¡C. PMN-CMs were stored at 80¡C. For preparation of control milieus, American Diagnostica (Greenwich, CT). -free bovine pancreatic cytochalasin B was omitted during incubation (47). Enzymatic activities of ␣-chymotrypsin (␣CT; 57 U/mg) and PI-PLC from Bacillus cereus (5100 EL and CG were measured in PMN-derived milieus through hydrolysis of U/mg) were from Sigma-Aldrich (St. Louis, MO). EL- and CG-free human specific synthetic substrates as previously described (46), using serial di- leukocyte (PR3), purified from purulent human sputum, was lutions of each purified enzyme to derive calibration curves. obtained from Elastin Products (Owensville, MO). Recombinant eglin C, a potent natural polypeptide for leukocyte Ser-proteinases Exposure of recombinant CD87 or CD87-expressing cells to (38), was provided by Dr. H. P. Schnebli (Novartis, Basel, Switzerland). proteinases ␣ ␣ Human 1-antichymotrypsin ( 1ACT), a -type plasma inhibitor spe- ␮ Exposure to purified proteinases. rhCD87[His]6 adjusted to 1 g/ml (20 cific for CG (34, 35), was from Calbiochem (Merck Eurolab, Darmstadt, nM) in 50 mM Tris, 100 mM NaCl (pH 7.4) either was mixed with varying Germany), whereas N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethylke- concentrations of purified EL or CG or was left untreated as control. After tone (AAPV-CMK), a synthetic inhibitor specific for EL (39), was from incubation for 5Ð30 min at 37¡C, enzymatic activities were neutralized by Sigma-Aldrich. Two synthetic peptides mapping the human CD87 se- ␮ ␮ 81 95 272 283 addition of 50 g/ml (6 M) eglin C, and samples were immediately sol- quences Ser -Cys within the D1-D2 linker domain and Asn -Gly at ubilized for 5 min at 100¡C in the presence of 2% (w/v) SDS and 5 mM Ͼ the C terminus of D3, respectively (1, 30), were prepared with 95% purity N-ethylmaleimide (SDS/NEM). by Eurogentec (Seraing, Belgium): the 15-mer Ser-Gly-Arg-Ala-Val-Thr-Tyr- Suspensions of biotinylated or unbiotinylated U937 cells or human Ser-Arg-Ser-Arg-Tyr-Leu-Glu-Cys (SGRAVTYSRSRYLEC, hereafter desig- blood monocytes in HBSS received 1 mM of both CaCl2 and MgCl2, and nated as the D1-D2 peptide) and the 12-mer Asn-His-Pro-Asp-Leu-Asp-Val- then varying concentrations of purified leukocyte proteinases were added Gln-Tyr-Arg-Ser-Gly (NHPDLDVQYRSG, hereafter designated as the D3 and incubation continued at 37¡C for 5Ð30 min, before were peptide). blocked with 6 ␮M eglin C. In some experiments, additional suspensions Abs used in this study were as follows: mouse mAbs 3931 and 3932, were incubated with 0.25Ð5 U/ml PI-PLC for 30 min under similar con- both IgG1 previously referred to as IIIF10 and IID7 (40) and recognizing ditions. A fraction of each suspension was kept at 4¡C for FACS analysis. sequences 52Ð60 of the D1 domain and 125Ð132 of the D2 Cells in the remaining suspensions were pelletted by centrifugation at domain of CD87, respectively, and mouse mAb 3936, an IgG2a originally 530 ϫ g for 15 min at 4¡C and then were solubilized (see Immunoblot described as clone F13/B10 against the Mo3f Ag (29, 41), reacting with an analysis of recombinant or cellular CD87). Cell supernatants were collected undetermined epitope in human CD87, all from American Diagnostica; and further centrifuged at 18,000 ϫ g for 30 min at 4¡C to eliminate cell debris nonspecific mAb of the IgG1 and IgG2a subclasses (Ancell, Bayport, MN); before being solubilized with SDS/NEM. His-probe (H-15), a rabbit polyclonal Ab against the [His]6 peptide tag (Santa Exposure to activated PMN-conditioned medium. rhCD87[His] was ad- Ј 6 Cruz Biotechnology, Santa Cruz, CA); the FITC-conjugated F(ab )2 goat Ab justed at 1 ␮g/ml in PMN-derived milieus prepared as described above, against mouse IgG (DAKO, Glostrup, Denmark); and HRP-conjugated Abs whereas U937 cells or human blood monocytes were resuspended at 1 ϫ against mouse or rabbit IgG (ImmunoPure; Pierce, Rockford, IL). 106/ml in these milieus after the last washing centrifugation. In both cases, Cell culture reagents were from Life Technologies (Paisley, U.K.), ex- incubations immediately started for 5Ð30 min at 37¡C and then enzymatic cept for heat-inactivated FCS from HyClone Laboratories (Logan, UT), activities were blocked by addition of 6 ␮M eglin C. An additional cell whereas chemicals for cell solubilization, SDS-PAGE, and protein transfer suspension made in HBSS containing Ca2ϩ and Mg2ϩ was incubated under

were from Sigma-Aldrich or Bio-Rad (Hercules, CA). similar conditions with 0.5 U/ml PI-PLC. Samples containing rhCD87[His]6 542 PROTEOLYSIS OF CD87/uPAR BY LEUKOCYTE PROTEINASES

and both cells and supernatants were solubilized as described above, whereas each given experiment. Enzymatic activity was stopped by acidifying the a fraction of each cell suspension was processed for FACS analysis. In some reaction mixture. Samples were immediately lyophilized, before being pro- experiments, PMN-CMs received proteinase inhibitors, i.e., 6 ␮M eglin C, 1 cessed for mass analysis. ␮ ␣ ␮ M 1ACT, or 25 M AAPV-CMK, 5 min before starting the incubation with rhCD87[His]6 or cells. Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry (MS) analysis Immunoblot analysis of recombinant or cellular CD87 Lyophilized samples were diluted in 10 ␮l of 0.1% (v/v) trifluoroacetic Cell solubilization and isolation of biotinylated proteins. After a final ␮ centrifugation, cells were resuspended at 2 ϫ 107 cells/ml and solubilized acid and desalted using -C18 ZipTip (Millipore) according to the proce- dure recommended by the manufacturer. Peptides were eluted directly on at 4¡C for 30 min in a radioimmunoprecipitation assay medium (pH 7.5) ␮ ␣ containing 1 mM PMSF, 10 ␮M aprotinin, 5 mM benzamidine, 100 ␮M the MALDI sample plate with 1 l of a 6-fold dilution of -cyano-4- leupeptin, 1.5 ␮M soybean , and 5 mM NEM. Insoluble hydroxy-cinnamic acid in acetonitrile/water/trifluoroacetic acid 70/30/ material was removed by an 18,000 ϫ g centrifugation at 4¡C for 30 min, 0.1% (v/v) (all chemicals from Sigma-Aldrich). After air drying of the and soluble proteins were stored at Ϫ80¡C. Protein concentrations were samples, MALDI-MS measurements were taken on a Voyager-DE STR measured using the BCA Protein Assay (Pierce). For SDS-PAGE and im- spectrometer (Applied Biosystems, Framingham, MA) equipped with a munoblot analysis, proteins were further solubilized with SDS/NEM. nitrogen laser (337 nm). Mass spectra were acquired in the reflector and Biotinylated membrane proteins were separated from nonbiotinylated delayed extraction modes using an acceleration voltage of 20 kV, a grid proteins by incubating 50Ð100 ␮g of solubilized total proteins with 40 ␮l voltage of 76%, and a delay extraction of 150 ns. Each mass spectrum was of a half-diluted slurry of NeutrAvidin-agarose beads (Pierce) for1hat an average of 250Ð500 laser shots. Masses were measured using close ␮ external calibration with a mixture of four peptides (des-Arg 1-bradykinin, 4¡Cinafinal volume adjusted to 250 l. After four washings in 10 mM ϩ ϩ ϩ ϩ Tris, 3 mM EDTA, 150 mM NaCl, 1% (v/v) Triton X-100 (pH 6.8), bio- monoisotopic mass [M H] : 904.4681; angiotensin 1, [M H] : 1296.6853; neurotensin, [MϩH]ϩ: 1672.9175; adrenocorticotropic hor- tinylated proteins were eluted by solubilization in 0.1 ml of the same me- ϩ

ϩ Downloaded from dium without Triton X-100 and containing SDS/NEM and heating for 5 mone fragment 18Ð39, [M H] : 2465.1989). Identification of the trun- min at 100¡C. cated peptides was conducted with GPMAW software, version 6.0 (Lighthouse Data, Odense, Denmark), in regard to the D1-D2 and D3 SDS-PAGE and immunoblot analysis. rhCD87[His]6 (15 ng) or cell pro- ␮ sequences. Search was performed using monoisotopic masses with a teins (10 g) were separated by SDS-PAGE as previously described (36), mass tolerance of 0.1%. with calibration using the Kaleidoscope Prestained Standards (Bio-Rad; Mr range, 7,600Ð216,000) for Mr determination. When required, disulfide Expression of data bonds were reduced before electrophoresis by adding 5% (v/v) 2-ME and

heating for 5 min at 100¡C. After SDS-PAGE, proteins were transferred to Results are expressed as mean Ϯ SEM for the indicated number of exper- http://www.jimmunol.org/ polyvinylidene fluoride membranes (Immobilon-P; Millipore, Bedford, iments or measures performed independently. MA). Membranes were first incubated for1hinTBST (pH 7.6) containing 0.5% (w/v) dried skimmed milk, and then were incubated for 1 h with the Results primary Ab in the same medium, washed in TBST, and finally incubated Recombinant human CD87 is cleaved within the D1-D2 linker for 1 h with the HRP-coupled secondary Ab. After washings, HRP activity was developed using the ECLϩ reagent kit (Amersham Biosciences, Little sequence by purified EL or CG and at its C terminus by CG Chalfont, U.K.), and the corresponding luminescence was revealed by ex- The proteolytic activity of EL and CG on the urokinase receptor posure of membranes to Kodak X-Omat AR films (Eastman Kodak, Roch- ester, NY). Quantification of luminescence was occasionally performed on was first evaluated on the polyhistidine-tagged, recombinant hu- membranes using an electronically cooled LAS-1000 Plus charge-coupled man CD87. After exposure to enzymes, quantitated immunoblot- by guest on September 25, 2021 device camera system and the Image Gauge 3.4 software (Fuji Photo Film, ting analysis indicated that rhCD87[His]6 was similarly altered by Tokyo, Japan). either one of the proteinases at low concentrations, i.e., 50Ð100 FACS analysis nM (Fig. 1, A and B). While the amount of full-length, three- Ϯ ϭ domain rhCD87[His]6 with Mr 54,200 3,400 (n 8) decreased, After exposure to enzymes, U937 cells or human blood monocytes were Ϯ distributed at 105 cells per well in a conical-bottom, 96-well microplate two molecular species were produced, one with Mr 38,100 (Nunc, Roskilde, Denmark). After centrifugation, cells were immunola- 2,000 only recognized by the anti-D2 mAb 3932 and the shortest Ϯ beled for1hat4¡C in 0.1 ml of HBSS containing 0.25% (w/v) BSA, 1 mM with Mr 15,700 2,600 only recognized by the anti-D1 mAb ␮ of both CaCl2 and MgCl2 (HBSS-BSA), and the primary Ab at 5 g/ml. 3931. Based on mass and antigenic reactivity, these fragments cor- After one washing in HBSS-BSA, immunofluorescence staining was per- respond to the D2D3 and D1 domains, respectively (40, 49). Re- formed by incubation at 4¡Cfor1hin0.1mlofHBSS-BSA containing a Ј ␮ lease of D1 from rhCD87[His]6 was observed whether intramo- secondary FITC-conjugated goat anti-mouse F(ab )2 Ab at 25 g/ml. Fi- nally, cells were washed once. lecular disulfide bonds were reduced or intact, indicating that For uPA binding assays, we used FITC-coupled uPA in a one-step pro- cleavage occurred within the D1-D2 linker sequence. EL at con- tocol adapted from a previously described procedure (48). After the initial centrations higher than 0.25 ␮M induced a further degradation of centrifugation in the microplate, cells were resuspended in 0.1 ml of Ј D2D3 and even more efficiently of D1, whereas a further proteol- HBSS-BSA containing 1.5Ð50 nM of either FITC-uPA or FITC-F(ab )2 as a control fluoresceinated protein or a mixture of FITC-uPA and a 20-fold ysis of only D2D3 was observed with CG, with D1 accumulating molar excess of unlabeled uPA, and then were incubated for1hat4¡C up to 1 ␮M of enzyme (Fig. 1, A and B). None of the Abs could before being washed three times. detect any D1- or D2D3-derived subfragments, indicating a rapid Cells were finally resuspended in HBSS-BSA containing 0.1 mg/ml pro- and extensive degradation of these domains in the presence of high pidium iodide (Sigma-Aldrich), and fluorescence emission was recorded using a FACScan cytometer (BD Biosciences, Franklin Lakes, NJ). Pro- enzyme concentrations. A limited amount of D2D3-like, truncated pidium iodide-positive dead cells were excluded from analysis, which was rhCD87[His]6 species could be observed in the initial untreated performed using CellQuest 3.3 software (BD Biosciences). Quantitative material (Fig. 1, A and C, lane 1), likely resulting from coexpres- binding of specific IgG or of uPA to the surface of viable cells was ex- sion with the full-length protein in the transfected cells, as ob- pressed as the geometric mean of fluorescence intensity histograms, after Ј served in many cell types expressing CD87 (15, 22, 50, 51) (see background binding provided by nonspecific IgG isotypes or FITC-F(ab )2 had been subtracted. next section). In addition, we examined whether EL or CG was active on the D1-D2 and D3 peptide fragmentation by proteinases C-terminal region of CD87. For this, proteinase-treated

Peptides reconstituted at 2 mg/ml (1.15Ð1.45 mM) in sterile deionized rhCD87[His]6 was assayed by immunoblotting for the presence of water were adjusted to 65 ␮M in HBSS and were incubated at 37¡Cina the C-terminal tag epitope. As shown in Fig. 1C, intact nontreated 10-␮l volume with purified proteinases, ␣CT, EL, CG, or a combination of the two latter, in a peptide/enzyme molar ratio varying from 10:1 to 1000:1 rhCD87[His]6 (lane 1) produced signals of similar intensities and for 1Ð30 min. Controls consisted of proteinase-free peptides or pro- whether reacted with the anti-D2 mAb (upper panel) or the anti- ␮ teinase dilutions alone incubated at 37¡C for the longest periods of time in [His]6 Ab (lower panel). Exposure to 0.5 MEL(lane 3) resulted, The Journal of Immunology 543

FIGURE 1. Cleavage of the D1-D2 linker sequence within recombinant CD87 by purified leukocyte EL or CG and of its C terminus by CG. A, ␮ ϳ rhCD87[His]6 (1 g/ml, 20 nM) was exposed at 37¡C to 0.05, 0.1, 0.25, 0.5, or 1 ␮M CG for 30 min or EL for 15 min (lanes 2Ð6 and 7Ð11, respectively) or was left untreated for 30 min (lane 1). Proteins (15 ng) were then separated by SDS-PAGE on 10% (upper panel) or 15% (lower panel) polyacrylamide gels under reducing conditions and finally were im- munoblotted using the anti-D2 mAb 3932 at 0.05 ␮g/ml (upper panel)or Downloaded from the anti-D1 mAb 3931 at 1 ␮g/ml (lower panel). Luminescent signals FIGURE 2. Modifications of membrane expression of CD87 in U937 associated with each molecular species were recorded with a charge-cou- cells or human blood monocytes exposed to purified EL or CG. Cells were pled device camera for quantification. Shown are portions of the resulting exposed at 37¡C to various concentrations of EL or CG for 15 or 30 min, digitized images corresponding to the location of the relevant Ags. B, respectively, or were incubated in the absence of proteinases, and mem- Quantitative evaluation of signals associated with each molecular species brane CD87 expression was then analyzed by FACS as described in Ma- terials and Methods. Anti-CD87 mAb 3931 (anti-D1), 3932 (anti-D2), or resulting from the exposure of rhCD87[His]6 to increasing concentrations of EL (upper graph)orCG(lower graph), expressed as the percentage of 3936 was used at 5 ␮g of IgG/ml. Results are expressed as the percentage http://www.jimmunol.org/ the maximal signal obtained for one species within a given experiment. of the mean fluorescence intensity value obtained for cells incubated in the Each data point is the mean Ϯ SEM of three independent experiments. C, absence of proteinase and are illustrated as means Ϯ SEM of five inde- ␮ pendent experiments. rhCD87[His]6 was exposed to 0.5 MCGorEL(lanes 2 and 3) or was left untreated (lane 1) for 30 min, except for EL (15 min), and then proteins were separated by SDS-PAGE on 10% acrylamide gels under reducing conditions. Immunoblotting used either the anti-D2 mAb (upper panel)or gressively decreased from 0.25 to 1 ␮M of enzyme, with an ϳ80% the anti-[His]6 His-probe Ab to label the C-terminal polyhistidine tag maximal reduction (Fig. 2, upper right panel). Time course anal- (lower panel) at 0.05 and 0.1 ␮g/ml, respectively. Portions of the films ysis with 0.5 ␮M CG showed that a 30-min exposure to the en- corresponding to the location of the relevant Ags, representative of three zyme was required for maximal effects (data not shown). Exposure by guest on September 25, 2021 different analyses, are depicted. of U937 cells to either enzyme did not increase the number of propidium iodide-positive dead cells. The modifications in CD87 expression resulting from cell exposure to the leukocyte Ser-pro- as expected, in a total conversion of the full-length molecule into teinases were compared with those produced by bacterial PI-PLC, D2D3, which still showed an equal reactivity with the anti-D2 and which hydrolyze the GPI structure between the inositolphosphate ring anti-[His]6 probes. The same concentration of CG (lane 2) resulted and the diacylglycerol moiety (53). Experiments not illustrated indi- in a partial conversion of rhCD87[His]6 into D2D3, but in this case cated that exposure of cells to 0.5 U/ml PI-PLC for 15 min resulted in both full-length and truncated species lost reactivity with the anti- maximal effects on GPI-anchored membrane proteins such as CD14

[His]6 Ab. CG, but not EL, thus appears to efficiently cleave CD87 and CD87. Under these conditions, PI-PLC maximally reduced at its C terminus. CD87-associated epitope display by 44 Ϯ 5%, 47 Ϯ 2%, and 40 Ϯ 10% (n ϭ 3) when using mAb 3931, 3932, or 3936, respectively, in EL cleaves membrane CD87 on monocytic cells preferentially agreement with previous observations (29, 30). within the D1-D2 linker sequence, whereas CG preferentially The evaluation of a leukocyte proteinase-dependent regulation cleaves a juxtamembrane sequence of CD87, as observed on the differentiated U937 cell line, was FACS analysis. The differentiated U937 cell line was used as a extended to primary mononuclear phagocytes. For this, we used model of CD87-expressing monocyte/macrophage-type cells (3, preparations of PBMCs highly enriched in monocytes, which ex- 43, 52). The proteolytic activity of purified EL and CG on mem- press CD87 (54). As judged by FACS analysis using the anti-D1 brane CD87 was first explored by FACS analysis, using the al- and anti-D2 mAbs (Fig. 2, lower panels), exposure of blood mono- ready mentioned anti-D1 mAb 3931 and anti-D2 mAb 3932 as cytes to EL up to 1 ␮M for 15 min resulted in an ϳ50% reduction well as mAb 3936, which recognizes a still unknown conforma- in display of the D1 domain, whereas display of the D2 domain tional epitope (41, 48). Exposure of cells to EL for 15 min (Fig. 2, was unaffected. Conversely, exposure to CG in the same range of upper left panel) resulted in a progressive decrease in binding of concentrations for 30 min led to a reduction in display of both the the anti-D1 mAb from 0.1 to 0.5 ␮M of enzyme, with an ϳ75% D1 and D2 domains, to ϳ50% of that observed on control un- maximal reduction. By contrast, binding of the anti-D2 mAb and treated cells. Thus, the susceptibility of membrane CD87 ex- of mAb 3936 did not markedly decrease up to 1 ␮M of enzyme. pressed on human blood monocytes to specific modifications by Time course analysis indicated that binding of 3931 was already EL or CG appears to be similar to that described using differen- reduced by ϳ65% after 5 min of exposure to 0.2 ␮M EL, whereas tiated U937 cells. binding of 3936 remained unchanged over a 30-min period (data Immunoblotting analysis. Although CD87 is easily detected in not shown). Exposure of cells to CG for 30 min had clearly dif- whole cell extracts by immunoblotting using domain-specific ferent consequences because binding of all three mAbs was pro- mAbs (17, 40), accurate evaluation of its proteolytic modifications 544 PROTEOLYSIS OF CD87/uPAR BY LEUKOCYTE PROTEINASES on plasma membranes was hampered by the presence of an intra- dicated that although exposure to EL resulted in the release of only cellular pool (44, 55) inaccessible to proteinases. This problem trace amounts of soluble D2D3 (sD2D3), exposure to CG induced was solved by cell surface biotinylation, which allows for specific the accumulation of both sD1D2D3 and sD2D3, with Mr values of analyzing of membrane proteins. As illustrated in Fig. 3A, when 63,300 Ϯ 1,600 and 50,700 Ϯ 2,000 (n ϭ 6), respectively (Fig. 3B, biotinylated membrane proteins obtained from control, proteinase- upper panel). Similar features were observed when supernatants untreated U937 cells are analyzed by immunoblot using the anti- were analyzed under nonreducing conditions (data not shown). Im- D2 mAb 3932 (lane 1), membrane CD87 appears as a major upper munoblotting with the anti-D1 mAb revealed a rather heteroge- Ϯ ϭ ϳ species with Mr 65,100 1,400 (n 7), which is also reactive neous material with a mean Mr of 17,500, corresponding to sol- with the anti-D1 mAb 3931 (data not shown) and corresponds to uble D1 (sD1) in the supernatants from EL-treated cells (Fig. 3B, the membrane full-length (mD1D2D3) CD87, whereas a minor lower panel), which disappeared with higher enzyme concentra- Ϯ lower species with Mr 49,700 2,000 reactive only with the anti- tions (data not shown). Supernatants derived from CG-treated cells D2 mAb represents a truncated, two-domain (mD2D3) form, as also showed the presence of sD1 species with similar Mr, but in previously observed (15, 22). It must be noted that a high level of lower amounts compared with EL-derived samples. of membrane CD87 results in a poorly focused and The two enzymes thus exert contrasted effects on membrane rather heterogeneous appearance of the various molecular species CD87, with EL proteolytically active mostly on the D1-D2 linker run in SDS-PAGE (55). sequence and within D1, and to a much lesser degree on the C- Immunoblotting analysis of membrane CD87 from biotinylated terminal part of the molecule, whereas CG is essentially active U937 cells exposed to EL or CG completed the data obtained in within this latter juxtamembrane domain of the receptor, with a

FACS analysis. Thus, EL induced, up to 0.5 ␮M, a proteolytic weaker activity on the D1-D2 region. Downloaded from transition from the mD1D2D3 to the truncated mD2D3 species (Fig. 3A, lanes 2 and 3), which accumulated at the cell surface and EL and CG secreted from PMNs cooperate for the generation of remained barely affected by the enzyme up to 3 ␮M (data not a major soluble D2D3 species shown). By contrast, exposure of cells to CG from 0.25 to 1 ␮M Evaluation of combined effects of EL and CG on CD87 structure (Fig. 3A, lanes 4Ð6) resulted in the disappearance of both the full- was performed by preparing conditioned milieus from degranu- length and truncated CD87 species. No fragments with Mr lower lated human blood PMNs, which comprise essentially EL and CG http://www.jimmunol.org/ than that of mD2D3 could be detected, and similar results were as proteinases (34). In agreement with previous data (36, 46), such obtained when considering proteins with intact disulfide bonds PMN-CMs contained concentrations of active EL approximately (data not shown). In parallel, analysis of the cell supernatants in- twice those of CG, respectively 0.39 Ϯ 0.07 ␮M and 0.20 Ϯ 0.05 ␮M(n ϭ 6). Activation of PMNs with fMLP in the absence of cytochalasin B provided control milieus with no detectable EL or CG activities. As illustrated in the upper and middle panels of Fig.

4, complete cleavage of rhCD87[His]6 occurred within 5 min of incubation in the proteinase-rich milieu (lane 2), generating a ma- Ϯ ϭ by guest on September 25, 2021 jor D2D3 form with Mr 38,700 1,000 (n 2), which remained

FIGURE 3. Cleavage and release of membrane CD87 in U937 cell sus- pensions exposed to purified EL or CG. U937 cells were surface-biotiny- lated, exposed to 0.1 or 0.5 ␮MEL(lanes 2 and 3) or to 0.25, 0.5, or 1 ␮M CG (lanes 4Ð6) as indicated in the legend to Fig. 2, or incubated in the absence of proteinase for 30 min (lane 1) before separation from the su- pernatants and solubilization of both fractions. A, Biotinylated membrane FIGURE 4. Cooperative cleavage of recombinant CD87 by EL and CG 5 ␮ proteins extracted from 10 cells were separated by SDS-PAGE on a 10% secreted by PMNs. rhCD87[His]6 (1 g/ml) was exposed at 37¡C for the acrylamide gel under reducing conditions. Immunoblotting was performed indicated periods of time to a proteinase-enriched PMN-CM (lane 2) pre- with the anti-D2 mAb as described in the legend to Fig. 1. B, Cell supernatants pared as indicated in Materials and Methods (the PMN-CMs used in this were solubilized, and volumes corresponding to 105 cells in the initial suspen- experiment contained 0.66 ␮M EL and 0.33 ␮M CG). Incubations in the sions were submitted to SDS-PAGE on 12% (upper panel)or15%(lower latter were also performed in the presence of Ser-proteinase inhibitors: 6 ␮ ␮ ␮ ␣ panel) acrylamide gels under reducing conditions and then were immunoblot- M eglin C (lane 1), 25 M AAPV-CMK (lane 3), or 1 M 1ACT (lane 4). ted with the anti-D2 (upper panel) or anti-D1 mAb (lower panel) as described Proteins were then separated by SDS-PAGE on 10% (upper and lower panels) in the legend to Fig. 1. Portions of the films corresponding to the location of or 15% (middle panel) polyacrylamide gels under reducing conditions, and the relevant Ags, representative of three independent experiments, are illus- immunoblotting was performed with the anti-D2 (upper panel) or anti-D1 trated. The asterisk in B, upper panel, indicates the position of a double band mAb (lower panel) or with the anti-[His]6 Ab (middle panel), as indicated in corresponding to cytokeratins that sometimes contaminate samples and are the legend to Fig. 1. Portions of the films corresponding to the location of the detected by Abs after reducing SDS-PAGE (56). relevant Ags, representative of three different analyses, are depicted. The Journal of Immunology 545 stable up to 30 min of incubation, and a free D1, which was further left panel, lane 2), but not with the anti-D1 mAb (data not shown), proteolyzed and became undetectable by 30 min. Blocking of leu- thus corresponding to sD2D3. Immunoblotting with the anti-D1 kocyte Ser-proteinase activities with eglin C totally abolished the mAb showed no detection of free D1 (Fig. 5A, lower left panel). conversion of rhCD87[His]6 into D2D3 (lane 1). The EL-specific Incubation in control PMN-derived milieus resulted in barely de- chloromethylketone inhibitor AAPV-CMK, which completely in- tectable material, except for traces of an sD1 species (lane 1). The hibited EL activity in PMN-CMs, drastically reduced formation of release of sD2D3 was inhibited when eglin C was present during D2D3 and degradation of free D1 (lane 3). By contrast, specific the incubation with PMN-CMs with, again, only small amounts of ␣ inhibition of CG up to 85% with 1ACT had limited effect on the sD1 being detected (lane 3). When EL was inhibited by AAPV- conversion of the full-length CD87 into D2D3 and on degradation CMK (lane 4), cell supernatants displayed both sD1D2D3, as con- of D1 (lane 4). Immunoblotting with the anti-[His]6 Ab (Fig. 4, firmed by its reactivity with the anti-D1 (data not shown), as well lower panel) showed a loss of the C-terminal tag upon as the anti-D2 mAb and sD2D3, with higher amounts of sD1 being Ϯ ϭ incubation with PMN-CMs (lane 2, 30-min incubation). This pro- clearly detectable with Mr 14,900 1,500 (n 3). By contrast, ␣ teolytic event was impaired by the presence of eglin C (lane 1) and when CG was inhibited with 1ACT (lane 5), only minute was still observed when EL was specifically blocked (lane 3), but amounts of sD2D3 could be detected, and no sD1. Importantly, was considerably reduced when CG was inhibited (lane 4). In all similar results were obtained when cell supernatants were analyzed cases, incubation in the control PMN-derived milieu had no effect under nonreducing conditions (Fig. 5A, upper right panel). To fur- on the structure of CD87 (data not shown). ther assess the specific nature of the CD87 fragment released upon We then tested the activity of PMN-CMs on cell membrane exposure of cells to PMN-derived proteinases, U937 cells were

CD87 (mCD87). Exposure of U937 cells to this milieu for 30 min exposed in parallel to 0.5 U/ml PI-PLC for 30 min, which resulted Downloaded from at 37¡C followed by FACS analysis indicated that binding of in the release of ϳ50% of the membrane-associated CD87 (see mAbs 3931, 3932, and 3936 was reduced to 13 Ϯ 6%, 46 Ϯ 5%, previous section). Immunoblot analysis of cell supernatants, and 63 Ϯ 6% (n ϭ 4), respectively, whereas exposure to the con- whether reduced or nonreduced, using the anti-D2 mAb indicated trol milieus had no effect on epitope display. The decrease in mAb that PI-PLC releases species reflecting the initial membrane-asso- binding could be detected as early as 5 min after cell exposure to ciated CD87, i.e., mostly sD1D2D3 and only trace amounts of

PMN-CMs (data not shown). Immunoblotting analysis of proteins sD2D3 (Fig. 5B, lanes 2), as opposed to the marked and only sD2D3 http://www.jimmunol.org/ in the cell fraction confirmed that, by 30 min of incubation in observed in supernatants of PMN-CM-treated cells (lanes 3). PMN-CMs, mCD87 had been extensively proteolyzed (data not Total inhibition of the proteolytic processing of CD87 by an shown). More interestingly, analysis of cell supernatants (Fig. 5A) inhibitor specific for EL and CG, i.e., eglin C (38), suggested that Ϯ ϭ showed that a major species with Mr 52,000 2,100 (n 3) had the proteolytic activity directed at CD87 in PMN-CMs was re- been released, which was reactive with the anti-D2 mAb (upper stricted to these two proteinases. However, PR3 is yet another Ser-proteinase stored in and secretable by PMNs upon degranula- tion, together with EL and CG (34, 57). Thus, we evaluated the potential activity of PR3 on CD87 by exposing U937 cells to pu- rified human PR3 at concentrations up to 1 ␮M for up to 45 min by guest on September 25, 2021 at 37¡C. Subsequent FACS analysis of PR3-treated cells showed no decreased display of CD87-associated epitopes recognized by mAb 3931, 3932, or 3936 (data not shown), thus excluding that PR3 participates in the proteolytic processing of CD87 exposed to PMN-CM. Thus, the natural combination of EL and CG results in a cleav-

age of rhCD87[His]6 as well as of mCD87 corresponding to the additive effects of each purified enzyme used separately at a con- centration similar to that measured in PMN-CMs (Figs. 1 and 3), i.e., a rapid release and further proteolysis of D1 essentially due to EL and an equally rapid cleavage at the C terminus of D3 mostly due to CG. Cooperation of the two proteinases acting on mono- cytic cells ultimately results in the release of a major, quite stable D2D3 species. FIGURE 5. Cooperative cleavage of membrane CD87 on U937 cells Determination of cleavage sites for EL and CG in CD87-related and release of truncated D2D3 species by EL and CG secreted from PMNs. peptides A, Nonbiotinylated U937 cells were exposed for 30 min at 37¡Ctothe control (lane 1) or proteinase-enriched (lanes 2Ð5) PMN-CMs used in Fig. Cleavage sites for EL and/or CG within identified proteinase-sen- 4. Incubations in fractions of the proteinase-enriched PMN-CMs were also sitive domains in CD87 were approached through MALDI-MS performed in the presence of Ser-proteinase inhibitors: 6 ␮M eglin C (lane analysis of the proteolytic fragmentation of two synthetic peptides ␮ ␮ ␣ 3), 25 M AAPV-CMK (lane 4), or 1 M 1ACT (lane 5). Cell super- mimicking the D1-D2 linker sequence, as well as the D3 C-termi- natants were then isolated from cells and their proteins were separated by nal domain ahead of the last cysteine residue engaged in disulfide SDS-PAGE as described in the legend to Fig. 3B, under reducing (left bonding (1, 2), respectively. For each MALDI-MS analysis of pep- panels) or nonreducing (right panel) conditions. Immunoblotting was per- tides submitted or not to the action of the two selected proteinases, formed using the anti-D2 (upper panels) or anti-D1 mAb (lower panel)as peptide identifications were achieved through the use of GPMAW described in the legend to Fig. 1. B, Nonbiotinylated U937 cells were exposed for 30 min at 37¡C to the control (lane 1) or proteinase-enriched software. Results are summarized in Table I and are as follows for ␣ PMN-CMs (lane 3) or to 0.5 U/ml PI-PLC (lane 2), and cell supernatants the D1-D2 peptide: 1) exposure to CT, taken as a reference Ser- were immunoblotted with the anti-D2 mAb as described in the legend. proteinase active on CD87, resulted, as expected from previous Portions of films corresponding to the location of the relevant Ags, repre- data (2, 18), in a single cleavage after a Tyr residue corresponding sentative of three independent experiments, are shown. to Tyr87 in human CD87; 2) exposure to EL resulted in a rapid 546 PROTEOLYSIS OF CD87/uPAR BY LEUKOCYTE PROTEINASES

Table I. MALDI-MS analysis of proteinase-induced fragmentation of CD87-related peptides D1-D2 and D3

Enzymatic Treatmenta [MϩH]ϩb Peptidesc

D1ÐD2 peptide None 1788.8780 SGRAVTYSRSRYLEC ␣CT 1012.4828 SRSRYLEC 795.3890 SGRAVTY EL 1276.6240 TYSRSRYLEC CG 1444.7610 SGRAVTYSRSRY 1012.5367 SRSRYLEC 795.4322 SGRAVTY ELϩCG 1276.6002 TYSRSRYLEC 932.4584 TYSRSRY FIGURE 6. Reduction of the capacity of U937 cells exposed to purified NHPDLDVQYRSG D3 peptide None 1441.6937 EL or CG, or to their combination, to bind uPA. U937 cells were exposed EL 851.4078 NHPDLDV to various concentrations of purified EL or CG or to the proteinase-en- CG 1298.6305 NHPDLDVQYR 1142.5208 NHPDLDVQY riched PMN-CMs or were incubated under control conditions as described (HPDLDVQYR) in the legends to Figs. 2 and 5, respectively. Cells were then incubated for 1hat4¡C with 50 nM FITC-uPA or with mAb 3931 (anti-D1) or 3936, a ␮ Peptides (65 M) were exposed for 1Ð30 min at 37¡C to proteinases in peptide/ both at 5 ␮g IgG/ml, and binding was assessed by FACS analysis as de-

enzyme molar ratios varying from 10 to 1000 or to a control proteinase-free medium. Downloaded from b Values for the [MϩH]ϩ monoisotopic ions observed in an experiment repre- scribed in Materials and Methods. Specific mean fluorescence intensity sentative of four for the D1ÐD2 peptide and two for the D3 peptide. was expressed as the percentage of the value obtained for cells incubated c Truncated peptides were deduced from the masses fitted to the amino acid com- in the absence of proteinase or in the control PMN-derived milieu. Results position of the original D1ÐD2 and D3 peptides using GPMAW software. are shown as means Ϯ SEM of three independent experiments.

85 cleavage (within 1 min) after a Val residue corresponding to Val , PMN-CMs exhibited a similar reduction of binding of FITC-uPA http://www.jimmunol.org/ thus confirming a previous observation (2); 3) exposure to CG (Fig. 6), whereas a control PMN-derived milieu had no effect (data resulted, within 15 min, in cleavages after Tyr residues corre- not shown). For comparison, incubation of cells with 0.5 U/ml sponding to Tyr87,asfor␣CT, and to Tyr92; and 4) exposure to a PI-PLC for 30 min reduced the binding capacity to 26 Ϯ 2% of the combination of EL and CG rapidly produced the EL-derived fragment control value (n ϭ 2). In the case of EL and PMN-CM, reduction corresponding to cleavage after Val85, with an [MϩH]ϩ value of in the binding of FITC-uPA appeared to be fairly well related to 1276.6448, which was then converted, through cleavage by CG after that of the anti-D1 mAb 3931 (Fig. 6). It must be noted that the Tyr92, into the heptapeptide TYSRSRY, which remained detectable conformational, yet unknown epitope recognized by mAb 3936, up to 30 min of proteolysis. which interferes with uPA binding and has been extensively used Exposure of the D3 peptide to CG resulted in its complete cleav- in immunohistochemistry studies (41, 48), appears to be unrelated by guest on September 25, 2021 age, within a few minutes, after residues corresponding to Arg281 to expression of domain D1. and to Tyr280 (or less likely Asn272) in human CD87 (1, 2). By contrast, only longer exposure to EL (30 min) resulted in some Discussion cleavage after the Val residue corresponding to Val278. Combina- Data reported here establish that the PMN-derived Ser-proteinases tion of the two proteinases for 30 min resulted in a mixture of the EL and CG cooperate for the proteolytic regulation of CD87 on three cleavages. monocytic cells. On one hand, EL is highly efficient at removing domain D1 while leaving most of the D2D3 truncated receptor Binding of uPA to monocytic cells is drastically reduced by EL associated with the membrane. In contrast, CG also has the capac- and CG ity to release D1, but actually appears more efficient in the prote- Membrane CD87 is a major for uPA, which the N- olysis of the CD87 C terminus, resulting in the release of both terminal domain can interact with several discrete sequences lo- full-length and truncated CD87 species. As depicted in Fig. 7, cated within all three domains of CD87 (2, 58). Because EL and combination of the two Ser-proteinases resulting from degranula- CG have the capacity to proteolytically affect some of these do- tion of PMNs ultimately leads to the accumulation of soluble mains, we next investigated how these proteinases could affect the D2D3, whereas D1 is not detected due to further proteolysis by interactions of uPA with its receptor, using a FITC-coupled uPA EL. The D1-D2 linker sequence covers residues Cys76-Cys95 in binding assay (48). When U937 cells were exposed at 4¡Ctoin- human CD87 (1, 2, 18), and cleavage sites for various proteinases creasing concentrations of FITC-uPA, FACS analysis indicated have been shown to include the peptide bonds after Arg83 and that a near saturating binding was observed for a ligand concen- Arg89 for uPA and potentially for plasmin, Val85 for EL, Thr86 for tration of 50 nM. Specificity of the interaction was further estab- various MMPs, and Tyr87 for ␣CT (2, 15, 16, 18). CG releases a ϳ lished through the observations that fluorescence associated with free D1 domain with Mr 16,000, indistinguishable from that gen- undifferentiated, i.e., CD87-negative, cells was reduced to that of erated by other proteinases (Ref. 59 and data not shown) and, in the nonspecific binding seen with an irrelevant FITC-conjugated agreement with its chymotrypsin-like Ser-proteinase activity (34, protein, whereas binding performed on differentiated cells in the 35), we have identified likely cleavage sites for CG within the presence of a 20-fold molar excess of unlabeled uPA resulted in D1-D2 linker sequence as peptide bonds after Tyr87 and/or Tyr92, similar nonspecific fluorescence. while confirming that EL can efficiently cleave after Val85.Re- Exposure of cells to EL for 15 min or to CG for 30 min resulted garding the juxtamembrane cleavage site(s), the release by CG of in an enzyme concentration-dependent reduction of their capacity full-length and truncated CD87 species from the cell surface sug- to bind FITC-uPA, with EL appearing slightly more efficient than gests that cleavage of D3 must occur below the last cysteine res- CG, but in both cases maximal inhibition reached Ͼ95% (Fig. 6). idue (Cys271) engaged in disulfide bonding (2), and indeed we Similarly, cells incubated for 30 min in the proteinase-enriched determined potential cleavage sites as the peptide bonds after The Journal of Immunology 547

which may prove to be particularly operative in situations of in- tense PMN recruitment and accumulation at inflammatory foci. Support to this assumption can be found in the observation 1) that the levels of soluble CD87 in inflammatory exudates taken from septic patients appear to correlate with the local accumulation of PMN (25), and 2) that free leukocyte Ser-proteinase activities in the range or even exceeding the concentrations of purified en- zymes used in the present study can be measured in biological fluids under certain pathological situations, such as those observed in the airspaces of patients with or cystic fibrosis (35, 62, 63). Removal of at least D1 from membrane CD87 expressed on inflammatory cells such as monocytes/macrophages should have FIGURE 7. Schematic representation of the combined effects of the major impacts on cell functions. As shown in the present study, neutrophil Ser-proteinases on CD87 structure and functions. A,Atanin- this drastically reduces the capacity of cells to bind uPA (Fig. 7), flammatory site, activated neutrophils can release amounts of the Ser-pro- teinases EL and CG sufficient to attack specific peptide bonds within both thus confirming that D1 indeed controls the high-affinity confor- the D1-D2 linker sequence (mostly EL) and the C-terminal part of D3 mation of CD87 for this ligand, although the binding site for uPA (mostly CG). B, Rapid release of D1 results in the abrogation of uPA is composite and implicates sequences in all three domains (2, 58). binding to the cell membrane and in the appearance of a membrane-bound Decreased uPA binding very likely results in a reduced pericellular Downloaded from truncated D2D3 species. C, Further proteolytic processes result in degra- generation of plasmin (2, 4) and, consequently, a reduced activa- dation of D1 and release of D2D3 from the cell membrane. A chemotactic tion of several MMPs (6). However, removal of D1 should also F motif ( ) can be exposed at the new N terminus of D2D3 or even can be profoundly perturb cell adherence and migration 1) by impairing released as a short peptide. the endocytosis of CD87 and its recycling to the leading edge of migrating cells, which depends on the formation of a trimolecular complex among plasminogen activator inhibitor-1, uPA, and http://www.jimmunol.org/ Tyr280 and Arg281, although not excluding a less likely cleavage CD87 (20), 2) by abrogating the high-affinity binding of CD87 to after Asn272. This assumption is supported by the fact that we were the extracellular matrix protein vitronectin (7), and 3) by disrupt- unable to detect any material containing the polyhistidine tag when ing the physical and functional interactions with integrins, partic- analyzing CG-treated rhCD87[His] by SDS-PAGE using Tris- 6 ularly with the leukocyte major integrin ␣ ␤ (8Ð10). Regarding tricine gels, indicating that the peptide bearing the tag had a mo- M 2 this latter point, it has been shown in several models that CD87- lecular mass lower than 1500 Da, the limit of separation of such deficient mice present a drastically reduced ␤ integrin-dependent gels (data not shown). Although it appears to be much less efficient, 2 recruitment of PMNs and monocytes at sites of experimentally EL may also cleave the C-terminal domain of CD87 after Val278,

induced infection and inflammation (12, 13). Limited proteolytic by guest on September 25, 2021 which could explain a very slight release of soluble D2D3 species truncation of CD87 by leukocyte Ser-proteinases could well be a from cells (Figs. 3B, lanes 2 and 3; and Fig. 5A, lane 5). negative feedback mechanism down-regulating ␣ ␤ -mediated The novel molecular mechanism that has been described allow- M 2 cell functions (8, 9). However, an excessive reduction of the ca- ing two leukocyte proteinases to complement each other to reduce cell surface expression of D1 and to generate membrane-bound pacity of inflammatory cells to migrate on and adhere to fibrin- and and soluble D2D3 species sheds some new light on previous ob- vitronectin-rich provisional inflammatory extracellular matrices and servations and has potential major (patho)physiological implica- to support pericellular proteolysis could be detrimental for the elim- tions. Thus, the presence of soluble forms of CD87, mostly full- ination of such matrices and for proper tissue healing (21, 64, 65). length or D2D3 species and more scarcely D1, is a common Finally, generation of membrane or soluble D2D3 species could feature of body fluids such as blood plasma or urine obtained from also be of a major importance in changing the cell phenotype. normal subjects (22, 23), and concentrations of these products are Indeed, CD87 is endowed with a chemotactic and promigratory elevated in several pathologies such as solid tumors (5, 24, 26), activity which can be, for a part, ascribed to the proteolytically bone marrow malignancies (23), and more particularly local or driven expression of a new N-terminal sequence after cleavage of systemic infectious and inflammatory disorders (25, 27, 28). Ac- the D1-D2 linker domain (11). This sequence must contain the 88 92 cordingly, cells in culture can release soluble CD87, as well as D1 active motif Ser -Arg-Ser-Arg-Tyr (SRSRY) and is chemotac- and D2D3 fragments (22, 60, 61), and shedding of CD87 is in- tic for monocytic cells in a nanomolar range of concentrations creased upon cell activation (19, 28, 61), including exposure of while remaining inactive in the full-length molecule (11, 66). CD87-expressing endothelial cells to isolated leukocytes (60). Al- D2D3 species are expressed on the plasma membrane of various though release of free D1 has been ascribed to the presence of cell types, and this expression is increased after endoproteolysis of active uPA, plasmin, and/or various MMPs in the cell environment the full-length CD87 (15, 17, 19, 50), as shown in this study with (15, 19), release of full-length or truncated CD87, which must monocytic cells exposed to leukocyte Ser-proteinases. Thus, this implicate juxtamembrane hydrolysis, has received little explana- process could provide cells with a cis-ortrans-acting, membrane- tion yet, except for the hydrolysis of the GPI moiety by bacterial bound chemokine-like structure (11, 20, 21). Maybe more impor- PI-PLC or host PI-PLD activities (29Ð31). Proteolysis of the C- tantly, the combined proteolysis of membrane CD87 by EL after terminal region of CD87 has been suggested to be a potential Val85 and by CG acting preferentially at the juxtamembrane D3 mechanism (19, 60, 61), although none of the proteinases consid- domain may release soluble D2D3 molecules bearing the SRSRY ered so far has been shown to cleave membrane CD87 outside the motif, whereas an even further possibility is that cleavage by CG D1-D2 linker sequence (16, 17, 19). We provide here the first after Tyr92, secondary to that after Val85 by EL, will release the demonstration of a secretable Ser-proteinase, i.e., CG, that exerts heptapeptide TYSRSRY (Fig. 7). Such proteolytic processings of its enzymatic activity essentially toward the CD87 C terminus. Our CD87 would produce molecules that might behave as true, fully observation offers an important alternative for the release of CD87, diffusible chemotactic agents, and thus would participate in the 548 PROTEOLYSIS OF CD87/uPAR BY LEUKOCYTE PROTEINASES orchestrated recruitment of leukocytes to an inflammatory site (14, 26. Stephens, R. W., A. N. Pedersen, H. J. Nielsen, M. J. A. Hamers, G. H¿yer- 32), a hypothesis currently under investigation. Hansen, E. R¿nne, E. Dybkjær, K. Dan¿, and N. Bru¬nner. 1997. ELISA deter- mination of soluble urokinase receptor in blood from healthy donors and cancer patients. Clin. Chem. 43:1868. Acknowledgments 27. Slot, O., N. Bru¬nner, H. Locht, P. Oxholm, and R. W. Stephens. 1999. Soluble We thank Viviane Balloy for assistance in the preparation of purified EL urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis. Ann. and CG, Dr. Anne-France Petit-Bertron for kindly providing human blood Rheum. Dis. 58:488. monocyte suspensions, and Drs. Agustin Valenzuela-Fernandez and Thi- 28. Florquin, S., J. G. van den Berg, D. P. Olszyna, N. Claessen, S. M. Opal, erry Planchenault for help in quantification of immunoblots. J. J. Weening, and T. van der Poll. 2001. Release of urokinase plasminogen activator receptor during urosepis and endotoxemia. Kidney Int. 59:2054. References 29. Mizukami, I. F., S. D. Vinjamuri, R. D. Trochelman, and R. F. Todd. 1990. A structural characterization of the Mo3 activation antigen expressed on the plasma 1. Roldan, A. L., M. V. Cubellis, M. T. Masucci, N. Behrendt, L. R. Lund, K. Dan¿, membrane of human mononuclear phagocytes. 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