Natural Soluble Interleukin-15Rα Is Generated by Cleavage That Involves the Tumor Necrosis Factor-α-converting Enzyme (TACE/ADAM17)
2004; Elsevier BV; Volume: 279; Issue: 39 Linguagem: Inglês
10.1074/jbc.m404125200
ISSN1083-351X
AutoresVadim Budagian, Elena Bulanova, Zane Orinska, Andreas Ludwig, Stefan Rose‐John, Paul Säftig, Ernest C. Borden, Silvia Bulfone‐Paus,
Tópico(s)T-cell and B-cell Immunology
ResumoThis study shows that the high affinity α-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Rα (sIL-15Rα) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Rα is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-α-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Rα, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Rα. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Rα shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Rα, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Rα generation in mice. The existence of natural sIL-15Rα offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention. This study shows that the high affinity α-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Rα (sIL-15Rα) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Rα is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-α-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Rα, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Rα. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Rα shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Rα, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Rα generation in mice. The existence of natural sIL-15Rα offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention. Natural soluble interleukin-15Rα is generated by cleavage that involves the tumor necrosis factor-α-converting enzyme (TACE/ADAM17).Journal of Biological ChemistryVol. 286Issue 11PreviewVOLUME 279 (2004) PAGES 40368–40375 Full-Text PDF Open Access Many cytokine receptors are naturally expressed in both membrane-linked and soluble forms (1Fernandez-Bortran R. Exp. Opin. Investig. Drugs. 2000; 9: 497-514Crossref PubMed Scopus (49) Google Scholar, 2Heaney M.L. Golde D.W. J. Leukocyte Biol. 1998; 6: 135-146Crossref Scopus (145) Google Scholar, 3Jones S.A. Rose-John S. Biochim. Biophys. Acta. 2002; 1592: 251-263Crossref PubMed Scopus (226) Google Scholar, 4Müllberg J. Althoff K. Jostock T. Rose-John S. Eur. Cytokine. Netw. 2000; 11: 27-38PubMed Google Scholar). A number of soluble proteins corresponding to the extracellular portions of transmembrane receptors and adhesion molecules have been identified in biological fluids (1Fernandez-Bortran R. Exp. Opin. Investig. Drugs. 2000; 9: 497-514Crossref PubMed Scopus (49) Google Scholar, 2Heaney M.L. Golde D.W. J. Leukocyte Biol. 1998; 6: 135-146Crossref Scopus (145) Google Scholar). The progress in this field balanced the concept of free ligands and bound receptors by demonstrating a widespread existence of soluble receptors and membrane-anchored ligands. Soluble receptors commonly consist of the extracellular portions or ectodomains of membrane-bound forms and thereby retain the ability to bind ligand. They typically function as natural antagonists, carrier molecules, or chaperones to protect their ligands from proteolytic degradation and increase the half-life and in some cases act as biological agonists (1Fernandez-Bortran R. Exp. Opin. Investig. Drugs. 2000; 9: 497-514Crossref PubMed Scopus (49) Google Scholar, 2Heaney M.L. Golde D.W. J. Leukocyte Biol. 1998; 6: 135-146Crossref Scopus (145) Google Scholar, 3Jones S.A. Rose-John S. Biochim. Biophys. Acta. 2002; 1592: 251-263Crossref PubMed Scopus (226) Google Scholar). Two major mechanisms account for the physiological or pathological generation of soluble receptors. These include alternative mRNA splicing that gives rise to a secreted polypeptide lacking a transmembrane and/or other regions or proteolytic cleavage of membrane-bound receptor proteins from the cell surface by proteases, a process also known as receptor shedding (1Fernandez-Bortran R. Exp. Opin. Investig. Drugs. 2000; 9: 497-514Crossref PubMed Scopus (49) Google Scholar, 2Heaney M.L. Golde D.W. J. Leukocyte Biol. 1998; 6: 135-146Crossref Scopus (145) Google Scholar, 3Jones S.A. Rose-John S. Biochim. Biophys. Acta. 2002; 1592: 251-263Crossref PubMed Scopus (226) Google Scholar). Both membrane-bound and soluble proteases can mediate ectodomain shedding (5Becherer J.D. Blobel C.P. Curr. Top. Dev. Biol. 2003; 54: 101-123Crossref PubMed Google Scholar, 6Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (562) Google Scholar). Members of the protease superfamily, including the matrix metalloproteinases (MPs) 1The abbreviations used are: MP, matrix metalloproteinases; ADAM, a disintegrin and metalloproteinase; TACE, TNFα-converting enzyme; TNFα, tumor necrosis factor α; IL, interleukin; IL-15Rα, IL-15 receptor α; sIL-15Rα, soluble IL-15Rα; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; FP, fusion protein; ELISA, enzyme-linked immunosorbent assay; MEF, mouse embryonic fibroblast; LPS, lipopolysaccharide; CTLL, cytotoxic T cell line. , membrane-tethered matrix MPs, and zinc-dependent ADAM (a disintegrin and metalloproteinase) family MPs have been shown to be responsible for the cleavage of the majority of shed proteins (6Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (562) Google Scholar). Among these, members of the ADAM family are particularly important (6Hooper N.M. Karran E.H. Turner A.J. Biochem. J. 1997; 321: 265-279Crossref PubMed Scopus (562) Google Scholar, 7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1387) Google Scholar, 8Black R.A. Int. J. Biochem. Cell Biol. 2002; 34: 1-5Crossref PubMed Scopus (299) Google Scholar). ADAM17 or TNFα-converting enzyme (TACE) has surfaced recently as a central mammalian ectodomain sheddase (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1387) Google Scholar, 8Black R.A. Int. J. Biochem. Cell Biol. 2002; 34: 1-5Crossref PubMed Scopus (299) Google Scholar). TACE plays a critical role in the ectodomain shedding of many soluble proteins, including TNFα (9Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2762) Google Scholar), TNFRI and II (10Dri P. Gasparini C. Menegazzi R. Cramer R. Alberi L. Presani G. Garbisa S. Patriarca P. J. Immunol. 2000; 165: 2165-2172Crossref PubMed Scopus (73) Google Scholar), l-selectin (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1387) Google Scholar), IL-6R (11Althoff K. Reddy P. Voltz N. Rose-John S. Müllberg J. Eur. J. Biochem. 2000; 267: 2624-2631Crossref PubMed Scopus (151) Google Scholar), CD30 (12Hansen H.P. Dietrich S. Kisseleva T. Mokros T. Mentlein R. Lange H.H. Murphy G. Lemke H. J. Immunol. 2000; 165: 6703-6709Crossref PubMed Scopus (88) Google Scholar), CD40 (13Contin C. Pitard V. Itai T. Nagata S. Moreau J.F. Dechanet-Merville J. J. Biol. Chem. 2003; 278: 32801-32809Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), growth hormone receptor (14Zhang Y. Jiang J. Black R.A. Baumann G. Frank S.J. Endocrinology. 2000; 141: 4342-4348Crossref PubMed Scopus (120) Google Scholar), erythropoietin receptor (15Westphal G. Braun K. Debus J. Clin. Exp. Med. 2002; 2: 45-52Crossref PubMed Scopus (18) Google Scholar), transforming growth factor β (16Cui X. Shimizu I. Lu G. J. Hepatol. 2003; 39: 731-737Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and others. Of more than 30 other members of the ADAM family known up to date, ADAM10/Kuzbanian shares significant sequence homology with TACE. Both TACE and ADAM10 have been implicated in the shedding of the amyloid precursor protein (17Slack B.E. Ma L.K. Seah C.C. Biochem. J. 2001; 357: 787-794Crossref PubMed Scopus (129) Google Scholar), cellular prion protein (18Vincent B. Paitel E. Saftig P. Frobert Y. Hartmann D. De Strooper B. Grassi J. Lopez-Perez E. Checler F. J. Biol. Chem. 2001; 276: 37743-37746Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), IL-6 receptor after cellular cholesterol depletion (19Matthews V. Schuster B. Schütze S. Bussmeyer I. Ludwig A. Hundhausen C. Sadowski T. Hartmann D. Kallen K.-J. Rose-John S. J. Biol. Chem. 2003; 278: 38829-38839Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar), and TNFα (20Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In addition, ADAM10/Kuzbanian is involved in cleavage of Notch (20Blobel C.P. Cell. 1997; 90: 589-592Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar), Notch ligand Delta (21Six E. Ndiaye D. Laabi Y. Brou C. Gupta-Rossi N. Israel A. Logeat F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7638-7643Crossref PubMed Scopus (221) Google Scholar), and fractalkine (22Hundhausen C. Misztela D. Berkhout T.A. Broadway N. Saftig P. Reiss K. Hartmann D. Fahrenholz F. Postina R. Matthews V. Kallen K.J. Rose-John S. Ludwig A. Blood. 2003; 102: 1186-1195Crossref PubMed Scopus (571) Google Scholar). The IL-15 receptor α (IL-15Rα) chain is a specific high affinity receptor that constitutes together with the IL-2 receptor β (IL-2Rβ) and the IL-2 receptor γ (IL-2Rγ/γc) subunits a trimeric receptor for IL-15 (23Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D.N. Morris S.W. Parki L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 24Giri J.G. Kumaki S. Ahdieh M. Friend D.J. Loomis A. Shanebek K. DuBose R. Cosman D. Park L.S. Anderson D.M. EMBO J. 1995; 14: 3464-3663Crossref Scopus (570) Google Scholar). IL-15Rα is structurally related to the IL-2Rα chain and alone is capable of high affinity binding of IL-15 (Kd ∼ 10–11m) (23Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D.N. Morris S.W. Parki L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Both IL-15Rα and IL-15 are expressed by a variety of tissues and cell types, including monocytes/macrophages, keratinocytes, fibroblasts, nerve, muscle, and epithelial cells (25Musso T. Calosso L. Zucca M. Millesimo M. Ravarino D. Giovarelli M. Malavasi F. Negro Ponzi A. Paus R. Bulfone-Paus S. Blood. 1999; 93: 3531-3539Crossref PubMed Google Scholar, 26Quinn L.S. Anderson B.G. Drivdahl R.H. Alvarez B. Argiles J.M. Exp. Cell Res. 2002; 280: 55-63Crossref PubMed Scopus (160) Google Scholar, 27Bulfone-Paus S. Bulanova E. Pohl T. Budagian V. Dürkop H. Rückert R. Kunzendorf U. Paus R. Krause H. FASEB J. 1999; 13: 1575-1585Crossref PubMed Scopus (139) Google Scholar, 28Satoh J. Kurohara K. Yukitake M. Kuroda Y. J. Neurol. Sci. 1998; 155: 170-177Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 29Shinozaki M. Hirahashi J. Lebedeva T. Liew F.Y. Salant D.J. Maron R. Kelley V.R. J. Clin. Investig. 2002; 109: 951-960Crossref PubMed Scopus (79) Google Scholar). Several different isoforms of human and murine IL-15Rα as a result of alternative splicing of the IL-15Rα gene were recently described (23Anderson D.M. Kumaki S. Ahdieh M. Bertles J. Tometsko M. Loomis A. Giri J. Copeland N.G. Gilbert D.J. Jenkins N.A. Valentine V. Shapiro D.N. Morris S.W. Parki L.S. Cosman D. J. Biol. Chem. 1995; 270: 29862-29869Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 30Dubois S. Magrangeas F. Lehours P. Raher S. Bernard J. Boisteau O. Leroy S. Minvielle S. Godard A. Jacques Y. J. Biol. Chem. 1999; 274: 26978-26984Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 31Bulanova E. Budagian V. Orinska Z. Krause H. Paus R. Bulfone-Paus S. J. Immunol. 2003; 170: 5045-5055Crossref PubMed Scopus (46) Google Scholar). Recombinant soluble IL-15Rα prevents collagen-mediated arthritis (32Ruchatz H. Leung B.P. Wei X.Q. McInnes I.B. Liew F.Y. J. Immunol. 1998; 160: 5654-5660PubMed Google Scholar), inhibits short-term carrageenan-induced inflammation (33Wei X. Orchardson M. Gracie J.A. Leung B.P. Gao B. Guan H. Niedbala W. Paterson G.K. McInnes I.B. Liew F.Y. J. Immunol. 2001; 167: 277-282Crossref PubMed Scopus (86) Google Scholar), and enhances cardiac allograft survival (34Smith S.G. Bolton E.M. Ruchatz H. Wei X. Liew F.Y. Bradley J.A. J. Immunol. 2000; 165: 3444-3450Crossref PubMed Scopus (79) Google Scholar). Although recombinant soluble IL-15Rα has been used for several in vivo studies (32Ruchatz H. Leung B.P. Wei X.Q. McInnes I.B. Liew F.Y. J. Immunol. 1998; 160: 5654-5660PubMed Google Scholar, 33Wei X. Orchardson M. Gracie J.A. Leung B.P. Gao B. Guan H. Niedbala W. Paterson G.K. McInnes I.B. Liew F.Y. J. Immunol. 2001; 167: 277-282Crossref PubMed Scopus (86) Google Scholar, 34Smith S.G. Bolton E.M. Ruchatz H. Wei X. Liew F.Y. Bradley J.A. J. Immunol. 2000; 165: 3444-3450Crossref PubMed Scopus (79) Google Scholar), no data concerning the existence of natural sIL-15Rα were reported thus far. In this study, we demonstrated the presence of natural soluble IL-15Rα (sIL-15Rα) in mouse serum. Furthermore, murine fibroblasts constitutively release sIL-15Rα into the culture medium, and this process is further stimulated by PMA. We provide several lines of evidence that constitutive and PMA-inducible IL-15Rα cleavage involves the activity of TACE by using specific MP inhibitors, by overexpression of the enzyme, and by using TACE-deficient fibroblasts. Cytokines, Antibodies, Inhibitors, Recombinant Proteins, and Vectors—Recombinant human IL-15, IL-2, and TNFα were purchased from TEBU (London, UK). Lipopolysaccharide (LPS), cycloheximide, phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). Antibodies against IL-15Rα (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against ADAM17 and ADAM10 were from Chemicon (Hofheim, Germany), and biotinylated goat-anti-mouse IL-15Rα was from R&D Systems (Wiesbaden, Germany). Rabbit anti-goat horseradish peroxidase conjugates (Amersham Biosciences) were used as secondary antibodies. IL-15-IgG2b and IL-2-IgG2b fusion proteins (FPs) were produced as described previously (35Bulfone-Paus S. Ungureanu D. Pohl T. Lindner G. Paus R. Rückert R. Krause H. Kunzeldorf U. Nat. Med. 1997; 3: 1124-1128Crossref PubMed Scopus (292) Google Scholar). Recombinant sIL-15Rα was produced as described previously (32Ruchatz H. Leung B.P. Wei X.Q. McInnes I.B. Liew F.Y. J. Immunol. 1998; 160: 5654-5660PubMed Google Scholar). In brief, the His6-tagged recombinant protein was expressed in Escherichia coli (strain BL21), extracted from bacteria under denaturing conditions and purified using a nickel agarose purification system (Qiagen, Dorking, UK) according to the manufacturer's recommendations. The purity of recombinant sIL-15Rα was analyzed by SDS-PAGE and Western blotting using specific anti-IL-15Rα antibodies. Purified recombinant sIL-15Rα inhibited IL-15-but not IL-2-mediated proliferation of CTLL cells (data not shown). A broad-spectrum hydroxamic acid-based MP inhibitor batimastate (BB94) was purchased from GlaxoSmithKline (Harlow, UK). GW280264X (a potent inhibitor of TACE and ADAM10 metalloproteinases) and GI254023X (an inhibitor of ADAM10) were described previously (22Hundhausen C. Misztela D. Berkhout T.A. Broadway N. Saftig P. Reiss K. Hartmann D. Fahrenholz F. Postina R. Matthews V. Kallen K.J. Rose-John S. Ludwig A. Blood. 2003; 102: 1186-1195Crossref PubMed Scopus (571) Google Scholar). Murine IL-15Rα was cloned into pcDNA3.1 expression vector (Invitrogen) as described previously (27Bulfone-Paus S. Bulanova E. Pohl T. Budagian V. Dürkop H. Rückert R. Kunzendorf U. Paus R. Krause H. FASEB J. 1999; 13: 1575-1585Crossref PubMed Scopus (139) Google Scholar). Murine TACE and human ADAM10 cDNA were cloned into pDC304 vector. Mice—Female mice of the inbred strains Balb/c, CH3, and C57BL/6 were obtained from Charles River Laboratories (Sulzfeld, Germany). IL-15Rα–/–mice were bred in Research Center Borstel under specific pathogen-free conditions. Mice were bled from the tail vein, and sera were analyzed by ELISA and Western blotting. Cell Culture, Stimulation, and Transfection Conditions—Ras-Myc-retrovirus-immortalized TACE–/–and simian virus large T-antigenimmortalized ADAM10–/–mouse embryonic fibroblasts (MEFs) and respective wild type cells were generated and characterized as described elsewhere (7Peschon J.J. Slack J.L. Reddy P. Stocking K.L. Sunnarborg S.W. Lee D.C. Russell W.E. Castner B.J. Johnson R.S. Fitzner J.N. Boyce R.W. Nelson N. Kozlosky C.J. Wolfson M.F. Rauch C.T. Cerretti D.P. Paxton R.J. March C.J. Black R.A. Science. 1998; 282: 1281-1284Crossref PubMed Scopus (1387) Google Scholar, 36Hartmann D. De Strooper B. Serneels L. Craessaerts K. Herreman A. Annaert W. Umans L. Lubke T. Lena Illert A. von Figura K. Saftig P. Hum. Mol. Genet. 2002; 11: 2615-2624Crossref PubMed Google Scholar). Murine fibrosarcoma L929 (ECACC), its TNFα-resistant derivative L929R, and primate fibroblast COS-7 (American Type Culture Collection) cell lines were maintained in RPMI 1640, and MEFs were cultured in Dulbecco's modified Eagle's medium. Culture medium was supplemented with 10% fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were stimulated with PMA (200 ng/ml) in fetal calf serum-free medium. For transient transfection, cells were seeded at 5 × 105/well in 6-well plates. COS-7 cells were transfected using the GenePORTER 2 transfection kit (Gene Therapy Systems, San Diego, CA), and MEFs were transfected using LipofectAMINE 2000 (Invitrogen). Transfection efficiency was confirmed by Western blotting using specific antibodies. Conditioned medium was collected 48 h after transfection, concentrated 10-fold using 10-kDa cutoff filtration units (Vivaspin; Vivascience, Hannover, Germany), and analyzed by immunoprecipitation and Western blotting for the presence of sIL-15Rα. ELISA for sIL-15Rα—A 96-well plate (Greiner, Hamburg, Germany) was coated overnight at 4 °C with 1 μg/ml of IL-15-IgG2b FP, which served as a capture protein for sIL-15Rα. Wells were blocked with 3% BSA in PBS for 2 h. Samples (50 μl/well) were added to the plate and incubated overnight. Serial dilutions of murine recombinant sIL-15Rα were used for standardization. Bound sIL-15Rα was detected using biotinylated anti-mouse IL-15Rα antibodies followed by incubation with streptavidin-peroxidase. Chromogenic substrate (R&D Systems) was used for visualization, and reaction was stopped after 20 min of incubation by addition of1NH2SO4. Optical density was determined at 450 nm using ELISA reader (Dynatech, Denkendorf, Germany). The detection limit of ELISA was 20 pg/ml of recombinant sIL-15Rα. The specificity of newly developed sIL-15Rα ELISA was convincingly demonstrated by the complete absence of detectable sIL-15Rα using IL-2-IgG2b FP as a coating reagent. Immunoprecipitation and Western Blotting—Nonidet P-40 (0.5% final concentration) and mixture of protease inhibitors were added to supernatants and immunoprecipitation with anti-IL-15Rα antibodies was performed for 2 h at 4 °C. Immunocomplexes were captured on protein G-agarose. To analyze glycosylation, after immunoprecipitation, the samples were treated with 250 mU of N-glycosidase F (Roche) for 3 h at 37 °C according to the manufacturer's instructions. Samples were resuspended in SDS-PAGE sample buffer (62.5 mm Tris-HCL, pH 8.0, 1% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.01% bromphenol blue), boiled for 5 min, and analyzed on 10% SDS-PAGE. The resolved proteins were transferred onto nitrocellulose (Bio-Rad). Blots were blocked for 1 h in phosphate-buffered saline containing 0.05% Tween 20 (phosphate-buffered saline-T) and 3% BSA (Sigma). After incubations with primary and secondary antibodies and washing with phosphate-buffered saline/Tween 20, visualization of specific proteins was carried out by an enhanced chemiluminescence (ECL) method using ECL Western blotting detection reagents (Amersham Biosciences) according to the manufacturer's instructions. Cellular extracts were prepared and analyzed by Western blotting as described elsewhere (31Bulanova E. Budagian V. Orinska Z. Krause H. Paus R. Bulfone-Paus S. J. Immunol. 2003; 170: 5045-5055Crossref PubMed Scopus (46) Google Scholar). Reverse Transcription-PCR—RNA was extracted from cells using TRIzol reagent (Invitrogen). cDNA was synthesized from 5 μg of total RNA using random oligonucleotides as primers and SuperScriptII kit (Invitrogen). cDNA was amplified by standard PCR procedure as described previously (31Bulanova E. Budagian V. Orinska Z. Krause H. Paus R. Bulfone-Paus S. J. Immunol. 2003; 170: 5045-5055Crossref PubMed Scopus (46) Google Scholar). The following primers were used: murine TACE: sense, 5′-GCGGCGTCTCCTCATCCT-3′; antisense, 5′-TTATATTCTGCCCCATCTGTGTTG-3′; and β-actin: sense, 5′-GTGGGGCGCCCCAGGCACCA-3′; antisense, 5′-CTCCTTAATGTCACGCACGATTTC-3′. All primers were purchased from Metabion (Planegg-Martinsried, Germany). Amplification of β-actin message was used to normalize the amount of cDNA. A mock PCR (without cDNA) was included to exclude contamination in all experiments. CTLL Assay—Serial 2-fold dilutions of the supernatant were added to microtiter plates containing 1 × 104 CTLL-16 cells in a final volume of 200 μl. The cells were incubated for 20 h at 37 °C and pulse-labeled for another 4 h with 0.5 μCi of [3H]thymidine (5 Ci/mmol). Thymidine incorporation was quantified by liquid scintillation counting (PerkinElmer Life and Analytical Sciences). Standards of IL-15, IL-2, and sIL-15Rα were included in each experiment. Flow Cytometric Analysis—Adherent cells were harvested from culture plates using acutase (PAA Laboratories, Coelbe, Germany). IL-15Rα expression was evaluated by incubation of cells with IL-15-IgG2b FP as described previously (31Bulanova E. Budagian V. Orinska Z. Krause H. Paus R. Bulfone-Paus S. J. Immunol. 2003; 170: 5045-5055Crossref PubMed Scopus (46) Google Scholar), and analyzed by flow cytometry using FACScalibur (BD Biosciences) and CELLQuest software. Negative controls consisted of isotype-matched, nonspecific antibodies (BD Phar-Mingen). The fluorescence signal of the labeled cells was calculated as median fluorescence intensity of the cell population. Data Analysis—All experiments were performed in at least three independent assays that yielded highly comparable results. Data are summarized as mean ± S.D. Statistical analysis of the results was performed by Student's t test for unpaired samples. A p value of < 0.05 was considered as statistically significant. Murine Fibroblasts Release Soluble IL-15Rα—Murine L929 fibrosarcoma cells abundantly express IL-15Rα mRNA and protein (27Bulfone-Paus S. Bulanova E. Pohl T. Budagian V. Dürkop H. Rückert R. Kunzendorf U. Paus R. Krause H. FASEB J. 1999; 13: 1575-1585Crossref PubMed Scopus (139) Google Scholar). Membrane-bound IL-15Rα could be detected on the cell surface of these fibroblasts by flow cytometry and confocal microscopy (data not shown). To investigate whether these cells release sIL-15Rα into the culture medium, conditioned media were collected from L929 fibroblasts after 3–5 days of culture and analyzed by CTLL assay for the ability to block specifically IL-15-mediated proliferation of CTLL cells. In parallel, the stimulation with IL-2 was assessed, which served here as a control. In fact, IL-15-but not IL-2-induced proliferation of CTLL cells was strongly inhibited by the addition of the conditioned medium from L929 fibroblasts (Fig. 1A). Reportedly, a variety of proteins may be released from the cell surface after stimulation with PMA, a potent activator of protein kinase C, by triggering a metalloproteinase-dependent ectodomain sheddase machinery (37Hwang C. Gatanaga M. Granger G.A. Gatanaga T. J. Immunol. 1993; 151: 5631-5638PubMed Google Scholar, 38Arribas J. Massague J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (133) Google Scholar). This pathway is defined as inducible shedding and seems to be highly conserved among multiple cell types. Thus, we investigated whether PMA treatment could enhance the release of sIL-15Rα from murine fibroblasts. To this end, L929 cells were treated with PMA for different time intervals, or left untreated. Interestingly, these experiments revealed that PMA had the ability to up-regulate the amount of membrane IL-15Rα within the first hour of stimulation as demonstrated by IL-15-IgG2b FP staining of the membrane-bound IL-15Rα and flow cytometry analysis (Fig. 1B). However, the increase in membrane-linked IL-15Rα was transitory and dropped substantially within the second hour of PMA treatment. After 2 h, L929 fibroblasts clearly had less membrane-bound IL-15Rα, resulting in 52% reduction of median fluorescence intensity compared with the untreated control (Fig. 1B). Given that down-modulation of IL-15Rα from the cell surface may result from its release into the culture medium, cell-free supernatants from PMA-stimulated and untreated cells were assayed for the presence of the soluble α chain by ELISA. Indeed, PMA treatment induced the release of the soluble form to the culture medium from L929 cells, increasing the amount of sIL-15Rα 3-fold (Fig. 1C). These results were further corroborated by Western blotting analysis of the culture medium. Conditioned media from these cells were harvested after 2 h of culture, and sIL-15Rα was immunoprecipitated using specific antibodies. As illustrated in Fig. 1D, immunoprecipitation and Western blotting experiments confirmed that sIL-15Rα is present in the cell supernatants as a single band protein with molecular mass of about 30 kDa. Re-appearance of the membrane-bound IL-15Rα was detected after 24 h of PMA removal (data not shown). It has been reported that certain ligands could induce release of respective receptors to the culture medium. Such liganddriven release of soluble form of the cognate receptor has been demonstrated for IL-4 and TNFα (10Dri P. Gasparini C. Menegazzi R. Cramer R. Alberi L. Presani G. Garbisa S. Patriarca P. J. Immunol. 2000; 165: 2165-2172Crossref PubMed Scopus (73) Google Scholar, 39Chilton P.M. Fernandez-Botran R. J. Immunol. 1993; 151: 5907-5917PubMed Google Scholar). Thus, the ability of IL-15 to induce sIL-15Rα release was tested. Parallel with IL-15, we also tested for the ability to induce release of sIL-15Rα several other agents, including TNFα and LPS. In L929 cells, TNFα can reportedly induce apoptosis, necrosis, or even both at once (27Bulfone-Paus S. Bulanova E. Pohl T. Budagian V. Dürkop H. Rückert R. Kunzendorf U. Paus R. Krause H. FASEB J. 1999; 13: 1575-1585Crossref PubMed Scopus (139) Google Scholar, 40Humphreys D.T. Wilson M.R. Cytokine. 1999; 11: 773-782Crossref PubMed Scopus (45) Google Scholar), whereas LPS treatment triggers expression of chemokines by these cells. 2E. Bulanova and S. Bulfone-Paus, unpublished observations. L929 cells were treated with IL-15, TNFα, or LPS for different time intervals, and shedding of the IL-15Rα chain was evaluated by FACS and ELISA. It is interesting that only TNFα was able to induce shedding of IL-15Rα in L929 cells to the levels comparable with PMA treatment, leading to
Referência(s)