Advances in Brief Myeloma Cells Release Soluble Interleukin-6Ra in Relation to Disease Progression by Two Distinct Mechanisms: Alternative Splicing and Proteolytic Cleavage

Multiple myeloma (MM) is a plasma-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow. Interleukin (IL)-6 is an essential survival and growth factor for myeloma cells that exerts its activity through a cell surface receptor composed of an 80-kDa ligand binding molecule (IL-6Ra) and a 130-kDa signal-transducing molecule. Of major interest, the soluble form of the IL-6Ra (sIL-6Ra) is an agonistic molecule able to potentiate IL-6 activity and a strong prognostic factor in MM. In the present study, we demonstrate that purified myeloma cells from all of the patients with MM and human myeloma cell lines release sIL-6Ra. The level of sIL-6Ra release correlates with disease activity and is clearly up-regulated during tumoral expansion in vivo and immortalization in vitro. Of note, this sIL-6Ra release is strongly reduced (50%) by a hydroxamate-based metalloproteinase inhibitor underlying the importance of shedding in the production of sIL6Ra by myeloma cells. Using specific IL-6Ra primers flanking the transmembrane domain, we demonstrate by PCR the presence of two IL-6R mRNAs corresponding to the membrane IL-6Ra and to the sIL-6Ra generated through alternative splicing in myeloma cells. In conclusion, we show that: (a) native myeloma cells and human myeloma cell lines release sIL-6Ra by two distinct mechanisms: alternative splicing and proteolytic cleavage of the membrane IL-6Ra; and (b) the release of the sIL6Ra, which is an agonist of IL-6, correlates with disease progression, explaining in part its strong prognostic value in vivo. Introduction MM is a plasma-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow. IL-6 is an essential survival and growth factor for myeloma cells (1, 2). It exerts its activity through a cell surface receptor composed of an 80-kDa ligand binding molecule (IL-6Ra, CD126) and a 130-kDa signal-transducing molecule (gp130, CD130; Ref. 3). The soluble form of the IL-6Ra (i.e., sgp80, sIL-6Ra) is an agonistic molecule that has been shown to potentiate IL-6 activity both in vitro and in vivo (4–6). In MM, two important observations have been made concerning the sIL-6Ra. First, elevated serum sIL-6Ra levels have been found in MM patients in correlation with a poor prognosis (7). Moreover it has been shown that elevated serum sIL-6Ra levels are higher in MM than in monoclonal gammopathy of undetermined significance (MGUS), allowing to discriminate MM patients from MGUS patients (8). Second, sIL-6Ra increases the proliferative response of myeloma cells to IL-6 and again behaves as a potent agonist of IL-6 (9, 10). The sIL-6Ra seems to be generated through two nonexclusive mechanisms: (a) a proteolytic cleavage of the membrane IL-6Ra by a metalloproteinase (11, 12), and (b) the production of a sIL-6Ra form lacking the transmembrane domain (13, 14). In HMCLs, it has been shown that a metalloproteinase inhibitor significantly reduced the release of sIL-6Ra without abolishing it (12). Moreover, an mRNA encoding a soluble form of the IL-6Ra has been identified in the U266 HMCL (14). Therefore, we have investigated the capacity of native myeloma cells to release sIL-6Ra and the mechanism involved in the generation of sIL-6Ra. We demonstrate that myeloma cells themselves release sIL-6Ra by two distinct mechanisms, alternative splicing and proteolytic cleavage of the membrane IL-6Ra, and we determined the contribution of both mechanisms. Furthermore, we show that this release correlates with disease progression, explaining in part the strong prognostic value of serum sIL-6Ra in patients with MM. Materials and Methods mAbs and Reagents. The hydroxamate-based metalloproteinase inhibitors BB-3103 and BB-94 were kindly provided Received 3/5/99; revised 7/12/99; accepted 7/24/99. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant 9659 from Association pour la Recherche sur le Cancer and Programme Hospitalier de Recherche Clinique Grant BRD/ 97/8-K. 2 Both authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at U463, Institut de Biologie, 9, quai Moncousu 44093 Nantes cedex 01, France. Phone: 33-2-40-08-47-66; Fax: 33-2-40-08-47-78; E-mail: mamiot@nantes. inserm.fr. 4 The abbreviations used are: MM, multiple myeloma; IL, interleukin; HMCL, human myeloma cell line; PMA, phorbol 12-myristate 13acetate. 2693 Vol. 5, 2693–2697, October 1999 Clinical Cancer Research Research. on September 12, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from by British Biotech Pharmaceuticals Limited (Oxford, United Kingdom; Ref. 15). PMA was from Sigma Chemical Co. (St. Louis, MO). B-B4, an anti-CD138 mAb was a gift from Dr. J. Wijdenes (Diaclone Research, Besançon, France). Purified B-B4 was biotinylated as described previously (16). Patients. Thirty-five MM patients have been included in the current study. The diagnostic criteria for MM were those of the Southwest Oncology Group (17). Nineteen MM patients were previously untreated (diagnosis), and 16 patients had progressive disease (relapse). HMCLs. LP1, OPM-2, and NCI-H929 were purchased from DSM (Scheiwg, Germany). JJN3 and AMO-1 were kindly provided by Drs. B. Van Camp and J. Minowada, respectively. U266 and RPMI-8226 were obtained from the American Type Culture Collection. XG-1, XG-2, XG-6, SBN-1, and MDN were established previously in the laboratory. BCN was recently established in the laboratory from a patient with a secondary plasma cell leukemia. BCN displays a specific phenotype of myeloma cell line (i.e., CD138, CD38, CD19, and CD28). The XG-, SBN-1, MDN, and BCN cell lines were dependent on IL-6 for their growth and were cultured in the presence of 3 ng/ml recombinant IL-6. Purification of Myeloma Cells and Preparation of Conditioned Media. Mononuclear cells were isolated by FicollHypaque density centrifugation from bone marrow aspirates or peripheral blood from patients with MM or plasma cell leukemia. The percentage of plasma cells was then determined by morphology (May-Grunwald-Giemsa staining) and checked by flow cytometry using two-color staining with anti-CD38 and anti-B-B4 mAbs, as described (16). Only samples with a plasmocytosis superior to 8% were used for the subsequent purification. The purification of myeloma cells was performed using a MACS separator (Miltenyi Biotec, Germany). Cells were incubated with MACS microbeads coupled to CD138 before proceeding to magnetic separation on MS separation column (Miltenyi Biotec). The purity of myeloma cells was evaluated by standard morphology (MayGrunwald-Giemsa stained cytospins), and only cell populations with a purity above 99% were used for the preparation of conditioned media. B-B4 myeloma cells were seeded in 96-well plates at the concentration of 10 cells/ml, and media were harvested after 24 h. The cultures were done in RPMI 1640 with 2% FCS. In all cases, the labeling index of myeloma cells was ,3%, thus, the number of myeloma cells after 24 h of culture was not significantly increased. Determination of Soluble gp80 Production. The concentration of soluble gp80 in cell supernatants was determined using a sandwich ELISA (Pelikine Compact, Tebu, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, the Netherlands). The sensitivity of the test was 10 pg/ml. Assays were performed according to the manufacturer’s recommendations. RNA isolation and Reverse Transcription-PCR Amplification. Total cellular RNAs were prepared from 2.10 purified myeloma cells using Trizol (Life Technologies, Inc.). All RNAs were reverse transcribed with 400 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), according to the manufacturer’s protocol. PCR was performed in a thermal-cycler (PCR Express; Hybaid) for 35 cycles of denaturation at 94°C for 1 min, followed by annealing at 62°C for 1.5 min and extension at 72°C for 1 min. Oligonucleotides used for PCR of IL-6Ra were as described by Horiuchi et al. (13): IL-6R-A (Thr 313) 59-ACGCCTTGGACAGAATCCAG-39 and IL-6R-B (Pro 456) 59-TGGCTCGAGGTATTGTCAGA-39.