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• W1966365320 abstract "gp130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XG1 hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells. gp130 transducing receptor is involved in the formation of high affinity receptors for the cytokines of the interleukin-6 (IL-6) family. Recruitment of gp130 by IL-6 associated to its receptor leads to the dimerization of the transducing component. In the present study we did characterize the B-S12 monoclonal antibody raised against gp130 and able to elicit IL-6 type biological activities. B-S12 antibody triggered strongly the proliferation of TF1 and XG1 hematopoietic cell lines and was able to increase the synthesis of acute phase proteins in HepG2 hepatoma cell line. B-S12 also behaved as a synergistic factor with granulocyte-macrophage colony-stimulating factor for both proliferation and differentiation of CD34-positive hematopoietic cell progenitors. By using a symmetric enzyme-linked immunosorbent assay, allowing the detection of dimeric forms of soluble gp130, we found that addition of B-S12 to gp130 led to its dimerization. Analysis of the tyrosine phosphorylation events in gp130 and Jak kinase family members revealed that B-S12 quickly induced the phosphorylation of gp130 in a neural derived cell line, and that Jak1 and Jak2 were also recruited. In conclusion, we show that gp130 cross-linking with the B-S12 monoclonal antibody was sufficient to generate functional IL-6 type responses in hematopoietic, neural, and hepatic cells. INTRODUCTIONgp130 transducing receptor is involved in the formation of high affinity receptors for the members of the IL-6 1The abbreviations used are: ILinterleukinLIFleukemia inhibitory factorOSMoncostatin MCNTFciliary neurotrophic factorCT-1cardiotrophin-1GM-CSFgranulocyte-macrophage colony-stimulating factorG-CSFgranulocyte colony-stimulating factorELISAenzyme-linked immunosorbent assay. family(1.Hibi M. Murakami M. Saito H.T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1090) Google Scholar). In addition to IL-6, this cytokine family is composed of interleukin-11 (IL-11)(2.Paul S.R. Bennett F. Calvetti J.A. Kelleher K. Wood C.R. O'Hara R.M. Leary A.C. Sibley B. Clark S.C. Williams D.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7512-7516Crossref PubMed Scopus (557) Google Scholar), leukemia inhibitory factor (LIF)(3.Gearing D.P. Gough N.M. King J.A. Hilton D.J. Nicola N.A. Simpson R.J. Nice E.C. Kelso A. Metcalf D. EMBO J. 1987; 6: 3995-4002Crossref PubMed Scopus (451) Google Scholar, 4.Moreau J.F. Donaldson D.D. Bennett F. Witek Giannotti J. Clark S.C. Wong G.G. Nature. 1988; 336: 690-692Crossref PubMed Scopus (235) Google Scholar), oncostatin M (OSM)(5.Zarling J.M. Shoyab M. Marquardt H. Hanson M.B. Lioubin M.N. Todaro J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9739-9743Crossref PubMed Scopus (364) Google Scholar), ciliary neurotrophic factor (CNTF)(6.Lin L.F. Mismer D. Lile J.D. Armes L.G. Butler E.T. Vannice J.L. Collins F. Science. 1989; 246: 1023-1025Crossref PubMed Scopus (356) Google Scholar), and cardiotrophin-1 (CT-1)(7.Pennica D. King K.L. Shaw K.J. Luis E. Rullamas J. Luoh S.-M. Darbonne W.C. Knutzon D.S. Yen R. Chien K.R. Baker J.B. Wood W.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1142-1146Crossref PubMed Scopus (497) Google Scholar). Many biological properties of the IL-6-related molecules are shared, such as activation of hepatocyte transcription(8.Baumann H. Ziegler S.F. Mosley B. Morella K.K. Pajovic S. Gearing D.P. J. Biol. Chem. 1993; 268: 8414-8417Abstract Full Text PDF PubMed Google Scholar), activation of neural proliferation and differentiation(9.Yamamori T. Fukada K. Aebersold R. Korsching S. Fann M.J. Patterson P.H. Science. 1989; 246: 1412-1416Crossref PubMed Scopus (500) Google Scholar), and regulation of hematopoiesis (10.Leary A.G. Wong G.G. Clark S.C. Smith A.G. Ogawa M. Blood. 1990; 75: 1960-1964Crossref PubMed Google Scholar, 11.Musashi M. Clark S.C. Sudo T. Urdal D.L. Ogawa M. Blood. 1991; 78: 1448-1451Crossref PubMed Google Scholar). Moreover LIF, CNTF, CT-1, and OSM display biological effects in the early stages of embryonic development, allowing the in vitro growth of embryonic stem cells in an undifferentiated state (12.Smith A.G. Heath J.K. Donaldson D.D. Wong G.G. Moreau J. Stahl M. Rogers D. Nature. 1988; 336: 688-690Crossref PubMed Scopus (1450) Google Scholar, 13.Conover J.C. Ip N.Y. Poueymirou W.T. Bates B. Goldfarb M.P. DeChiara T.M. Yancopoulos G.D. Development. 1993; 119: 559-565Crossref PubMed Google Scholar, 14.Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar). IL-6 and IL-11 are also important modulators of the immune response by regulating immunoglobulin secretion(15.Kishimoto T. Akira S. Taga T. Science. 1992; 258: 593-597Crossref PubMed Scopus (788) Google Scholar, 16.Yin T.G. Schendel P. Yang Y.C. J. Exp. Med. 1992; 175: 211-216Crossref PubMed Scopus (91) Google Scholar). Gene inactivation experiments in the mouse have confirmed the implication of these cytokines in vivo by showing that LIF was essential for embryo implantation (17.Stewart C. Kaspar P. Brunet L.J. Bhatt H. Gadi I. Kontgen F. Abbondanzo S.J. Nature. 1992; 359: 76-79Crossref PubMed Scopus (1752) Google Scholar) and that CNTF/CNTF receptor interaction was critical for motoneuron development(18.Masu Y. Wolf E. Holtmann B. Sendtner M. Brem G. Thoenen H. Nature. 1993; 365: 27-32Crossref PubMed Scopus (531) Google Scholar, 19.DeChiara T.M. Vejsada R. Poueymirou W.T. Acheson A. Suri C. Conover J.C. Friedman B. McClain J. Pan L. Stahl N. Ip N. Kato A. Yancopoulos G.D. Cell. 1995; 83: 313-322Abstract Full Text PDF PubMed Scopus (341) Google Scholar). In addition, mice lacking IL-6 displayed some deficiencies in bone remodeling, inflammatory response, and immunoglobulin secretion(20.Kopf M. Lamers M. Bluethman H. Köhler G. Nature. 1994; 376: 339-342Crossref Scopus (1499) Google Scholar, 21.Poli V. Balena R. Fattori E. Markatos A. Yamamoto M. Tanaka H. Ciliberto G. Rodan G.A. Costantini F. EMBO J. 1994; 13: 1189-1196Crossref PubMed Scopus (655) Google Scholar).The shared use of gp130 receptor in their multimeric receptor explained in part the redundancy of their biological properties. The recruitment of the gp130 transduction pathway by IL-6 also implicates an IL-6-binding protein known as IL-6 receptor(22.Yamasaki K. Taga T. Hirata H. Kawanishi Y. Seed B. Taniguchi T. Hirano T. Kishimoto T. Science. 1988; 241: 825-828Crossref PubMed Scopus (878) Google Scholar), which can be produced either as a transmembrane protein or as a truncated soluble product (23.Lust J.A. Donovan K.A. Kline M.P. Greipp P.R. Kyle R.A. Maihle N.J. Cytokine. 1992; 4: 96-101Crossref PubMed Scopus (294) Google Scholar). Both forms of IL-6 receptor can associate IL-6 to induce gp130 dimerization and downstream signaling events(1.Hibi M. Murakami M. Saito H.T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1090) Google Scholar, 24.Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar). More recently, an IL-11 binding chain was isolated and a similar activation process for gp130 is suspected(25.Hilton D.J. Hilton A.A. Raicevic A. Rakar S. Smith M.H. Gough N.M. Begley C.G. Metcalf D. Nicola N.A. Willson T.A. EMBO J. 1994; 13: 465-475Crossref Scopus (253) Google Scholar, 26.Chérel M. Sorel M. Lebeau B. Dubois S. Moreau J.F. Bataille R. Minvielle S. Jacques Y. Blood. 1995; 86: 2534-2540Crossref PubMed Google Scholar). Regarding to the receptors for LIF, OSM, and CT-1, gp130 will associate to a second transducing subunit referred to as gp190/LIF receptor(14.Pennica D. Shaw K.J. Swanson T.A. Moore M.W. Shelton D.L. Zioncheck K.A. Rosenthal A. Taga T. Paoni N.F. Wood W.I. J. Biol. Chem. 1995; 270: 10915-10922Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 27.Gearing D.P. Thut C.J. VandenBos T. Gimpel S.D. Delaney P.B. King J. Price V. Cosman D. Beckmann M.P. EMBO J. 1991; 10: 2839-2848Crossref PubMed Scopus (513) Google Scholar, 28.Gearing D.P. Comeau M.R. Friend D.J. Gimpel S.D. Thut C.J. McGourty J. Brasher K.K. King J.A. Gillis S. Mosley B. Ziegler S.F. Cosman D. Science. 1992; 255: 1434-1437Crossref PubMed Scopus (790) Google Scholar). For CNTF a third additional component or α CNTF receptor was identified and is required to reinforce the interaction of CNTF with the transducing receptor complex composed of gp130 and gp190/LIF receptor(29.Davis S. Aldrich T.H. Valenzuela D.M. Wong V.V. Furth M.E. Squinto S.P. Yancopoulos G.D. Science. 1991; 253: 59-63Crossref PubMed Scopus (534) Google Scholar, 30.Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (587) Google Scholar).Dimerization of the transducing subunits initiates intracellular signaling by activating members of a family of cytoplasmic receptor-associated tyrosine kinases, referred to as Jak (Janus kinase) (for review, see (31.Ihle J.N. McKerr I. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar)). Both gp130 and gp190/LIF receptor can associate Jak1, Jak2, and Tyk2(32.Stahl N. Boulton T.G. Farrugella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (840) Google Scholar). The information is next relayed by a family of transcription factors known as STATs (signal transducers and activators of transcription), which are activated in the cytoplasm before translocation to the nucleus(31.Ihle J.N. McKerr I. Trends Genet. 1995; 11: 69-74Abstract Full Text PDF PubMed Scopus (818) Google Scholar). IL-6-related cytokines will preferentially activate STAT1 and STAT3(33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 34.Lai C.F. Ripperger J. Morella K.K. Wang Y. Gearing D.P. Fey G. Baumann H. J. Biol. Chem. 1995; 270: 14847-14850Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 35.Lütticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar).Induction of gp130 dimerization or its heterodimerization with LIF receptor was well analyzed in the case of IL-6 and CNTF/LIF receptors, respectively(24.Murakami M. Hibi M. Nakagawa N. Nakagawa T. Yasukawa K. Yamanishi K. Taga T. Kishimoto T. Science. 1993; 260: 1808-1810Crossref PubMed Scopus (638) Google Scholar, 30.Davis S. Aldrich T.H. Stahl N. Pan L. Taga T. Kishimoto T. Ip N.Y. Yancopoulos G.D. Science. 1993; 260: 1805-1808Crossref PubMed Scopus (587) Google Scholar). Studies regarding to the downstream activation processes mediated through the gp130/gp130 or gp130/gp190 pathways were also performed by constructing chimeric receptors composed in their external parts of G-CSF receptor, epidermal growth factor, or TRKc and by the intracellular domains of gp130 or gp190(33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 36.Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Crossref PubMed Google Scholar). In the present study we have analyzed the agonistic properties of the B-S12 antibody directed against gp130, and we show that cross-linking of gp130 transducing subunit was sufficient to elicit IL-6 type responses.MATERIALS AND METHODSCells and ReagentsThe SK-N-MC neuroblastoma cell line (ATCC, Rockville, MD), and the HepG2 cell line (ATCC) were routinely grown in RPMI culture medium supplemented with 10% fetal calf serum. For the multifactor-dependent TF1 cell line (37.Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (713) Google Scholar) and the XG1 myeloma cell line(38.Klein B. Zhang X.G. Jourdan M. Content J. Houssiau F. Aarden L. Piechaczyk M. Bataille R. Blood. 1989; 73: 517-522Crossref PubMed Google Scholar), the culture medium was supplemented, respectively, with 1 ng/ml GM-CSF and 1 ng/ml IL-6. Purified human recombinant LIF (108 units/mg) produced in Chinese hamster ovary cell line, Escherichia coli recombinant GM-CSF (108 units/mg), and human soluble IL-6 receptor were kindly donated by Drs. K. Turner and M. Stahl (Genetics Institute, Boston, MA). IL-6 (107 units/mg) and OSM (106 units/mg) were purchased from Peprotech (Canton, MA). Soluble human gp130 was bought from R& Systems (Minneapolis, MN). Purified B-S12 monoclonal antibody (IgG1) obtained as described previously 2S. Chevalier, M. Fourcin, O. Robledo, J. Wijdenes, A. Pouplard-Barthelaix, and H. Gascan, submitted for publication. was kindly provided by Dr. John Wijdenes from Diaclone (Besançon, France). B-S12 and control IgG1 Fab fragments were generated by using immobilized papain from Pierce, following the manufacturer's instructions. Fab fragments were then separated from Fc fragments on a protein A column.BioassaysSerial dilutions of antibodies or cytokines were added to 15 × 103 TF1 or XG1 cells in triplicate in the assay(39.Fourcin M. Chevalier S. Lebrun J.J. Kelly P. Pouplard A. Wijdenes J. Gascan H. Eur. J. Immunol. 1994; 24: 277-280Crossref PubMed Scopus (50) Google Scholar). The cells were then incubated for 72 h before being pulsed with 0.5 μCi of [3H]Tdr for the last 4 h of the culture. HepG2 hepatoma cell line was plated in triplicate in 96-well plates at a concentration of 50 × 103 cells/well in 200 μl of RPMI medium containing 10% fetal calf serum, 10-6M dexamethasone, and serial dilutions of IL-6 or antibodies (40.Gauldie J.C. Richards C. Harnish D. Lansdorp P. Baumann H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7251-7255Crossref PubMed Scopus (1358) Google Scholar). After 48 h, the supernatants were harvested and the haptoglobin content determined by ELISA as described previously(41.Lecron J.C. Roblot P. Chevalier S. Morel F. Alderman E. Gombert J. Gascan H. Clin. Exp. Immunol. 1993; 92: 23-26Crossref PubMed Scopus (43) Google Scholar).2 Hematopoietic progenitor cells were purified from bone marrow from informed donors by using the CD34-positive cell selection system from Applied Immune Science (Menlo Park, CA) and following the manufacturer's instructions. Purity of the cells was controlled on a FACScan by using an anti-CD34 phycoerythrin-labeled antibody (Becton Dickinson, Mountain View, CA) and was routinely >97% CD34-positive cells. One hundred CD34-positive purified cells were seeded in 24-well plates in triplicate in Iscove's medium containing 20% fetal calf serum, 0.8% methylcellulose, 5 × 10-5M β-mercaptoethanol, and the indicated combination of cytokines or antibodies. After a 14-day culture period, the colony number was scored.Soluble gp130 Symmetric ELISADetection of the dimeric form of soluble gp130 was achieved by coating the ELISA plates with the B-T12 monoclonal antibody2 at a concentration of 10 μg/ml in 100 mM carbonate buffer, pH 8.6. After washing and a saturation step, 2 nM soluble gp130 supplemented with 2 nM IL-6 plus 2 nM soluble IL-6 receptor, or 6 nM B-S12 anti-gp130 antibody were added to the wells for an overnight incubation at 4°C. Biotinylated B-T12 mAb was used as tracer antibody at a final concentration of 1 μg/ml. After a 6-h incubation at 37°C, streptavidin peroxidase (Dako, Glostrup, Denmark) was added at a 1/20,000 dilution for an additional 1-h incubation step. ABTS was used as substrate and the reading performed at 405 nM.Tyrosine Phosphorylation AnalysisCells were stimulated with OSM or antibodies before being lysed in 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100 and proteinase inhibitors. After pelleting insoluble material and protein standardization, the supernatants were immunoprecipitated in the presence of B-T2 antibody raised against gp1302, anti-Jak1 antibody (Upstate Biotechnology, Inc. (UBI), Lake Placid, NY), or anti-Jak2 antibody (UBI). The complexes were then isolated with beads coupled to protein A, submitted to SDS-polyacrylamide gel electrophoresis, and transferred onto an Immobilon membrane (Millipore, Bedford, MA). The membranes were incubated in the presence of 4G10 anti-phosphotyrosine mAb, followed by an incubation with a goat anti-mouse immunoglobulin polyclonal antibody labeled with peroxidase (Tago, Camarillo, CA). The reaction was visualized on a x-ray film with ECL reagent (Amersham, Les Ulis, France) following the manufacturer's instructions.RESULTS AND DISCUSSIONB-S12 Antibody Triggered the Proliferative Response of Hematopoietic Cell Lines and Increased Protein Transcription in HepG2 Hepatoma Cell LineWe recently characterized a set of new monoclonal antibodies raised against the gp130 transducing protein.2 During the time course of the antibody screening, we did identify the B-S12 mAb, which, in contrast to most other identified antibodies, displayed agonistic properties in several bioassays. The stimulatory properties of the B-S12 antibody were first analyzed by using two human hematopoietic cell lines, XG1 and TF1, respectively, known to be sensitive to the cytokines activating the gp130 transduction pathway and both gp130 and gp190/LIF receptor systems(38.Klein B. Zhang X.G. Jourdan M. Content J. Houssiau F. Aarden L. Piechaczyk M. Bataille R. Blood. 1989; 73: 517-522Crossref PubMed Google Scholar, 39.Fourcin M. Chevalier S. Lebrun J.J. Kelly P. Pouplard A. Wijdenes J. Gascan H. Eur. J. Immunol. 1994; 24: 277-280Crossref PubMed Scopus (50) Google Scholar). B-S12 mAb was found to strongly trigger the proliferation of TF1 erythroleukemia cell line and XG1 multiple myeloma cell line, whereas a control IgG1 did not support the proliferation of these cells (Fig. 1, A and B). This result indicates that the B-S12 antibody can bypass the cytokine requirement to activate the gp130 pathway, and that recruitment of an α binding component was not necessary to generate a functional response. A series of reports had shown that the integrity of two proximal motifs in the intracellular part of gp130, and known as box 1 and 2, were sufficient to generate a proliferative response(33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 36.Baumann H. Symes A.J. Comeau M.R. Morella K.K. Wang Y. Friend D. Ziegler S.F. Fink J.S. Gearing D.P. Mol. Cell. Biol. 1994; 14: 138-146Crossref PubMed Google Scholar, 42.Murakami M. Narazaki M. Hibi M. Yawata H. Asukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar). On the other hand, it has been demonstrated that regulation of gene transcription in hepatoma or neuroblastoma cell lines was under the dependence of an additional gp130 motif called box 3, and implicating the recruitment of STAT3 shuttle transducing protein(33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 42.Murakami M. Narazaki M. Hibi M. Yawata H. Asukawa K. Hamaguchi M. Taga T. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11349-11353Crossref PubMed Scopus (486) Google Scholar). In line with these observations, we have tried to further characterize the functional properties of the B-S12 antibody in order to know, if in addition to its ability to stimulate cell proliferation, the antibody could also increase gene transcription. The experiments were performed in HepG2 hepatoma cell line where the induction of haptoglobin secretion was monitored. Fig. 1C shows that B-S12 antibody was able to up-regulate the induction of haptoglobin secretion, whereas an IgG1 control antibody was without effect. Nevertheless, B-S12 was about a thousandfold less potent than IL-6 used as positive control. This result shows that gp130 activation pathway by the B-S12 antibody led not only to a proliferative response, as observed in the hematopoietic cell lines, but also to an increase gene transcription in hepatic cells.B-S12 Antibody Induced Both Proliferation and Differentiation of CD34 Hematopoietic ProgenitorsSeveral IL-6-related cytokines were reported to display synergistic activities on both proliferation and differentiation processes of hematopoietic cells. To examine the potential role of the B-S12 anti-gp130 antibody on the expansion of hematopoietic cell progenitors, its effect was tested in combination with GM-CSF on purified human CD34-positive cells by using a methylcellulose clonal assay. B-S12 antibody alone, like the IgG1 isotype control did not induce colony formation (Table 1). Addition of 1 ng/ml GM-CSF to the culture stimulated the generation of 11 ± 1.4 colonies after a 2-week culture period. To assess the synergistic effect of IL-6 type cytokines in the culture system, LIF was preferred to IL-6 because this latest cytokine only displayed weak effects in human CD34 cell culture and the adjunction of soluble IL-6 receptor was usually required to observe a response(43.Sui X. Tsuji K. Tanaka R. Tajima S. Muraoka K. Ebihara Y. Ikebuchi K. Yasukawa K. Taga T. Kishimoto T. Nakahata T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2859-2863Crossref PubMed Scopus (138) Google Scholar). Addition of LIF to GM-CSF doubled the score of colonies in the cultures in agreement with the published studies(10.Leary A.G. Wong G.G. Clark S.C. Smith A.G. Ogawa M. Blood. 1990; 75: 1960-1964Crossref PubMed Google Scholar, 44.Verfaillie C. McGlave P. Blood. 1991; 77: 263-270Crossref PubMed Google Scholar). Similar results were obtained by introducing B-S12 antibody in combination with GM-CSF in the CD34-positive cell cultures. Moreover, the average size of the observed colonies generated in the presence of B-S12 and GM-CSF was twice larger than those detected in the presence of GM-CSF alone. 3In the present study, the number of granulocyte-macrophage colony-forming units, blast cell colonies, and mixed colonies has been cumulated; the phenotypic detail of the generated colonies will be presented in another study. These experiments show that B-S12 antibody, like most of the cytokines belonging to the IL-6 family, synergistically promoted potent expansion of human hematopoietic progenitor cells in vitro.Tabled 1B-S12 Antibody-induced Dimerization of the gp130 Transducing ProteinFab fragments were generated from the B-S12 antibody and tested in the proliferative TF1 assay to determine whether the observed agonistic properties of the antibody were dependent on the presence of the two functional sites expressed by the immunoglobulin. Fig. 2A shows that B-S12-derived Fab fragments failed to trigger the proliferation of the TF1 cell line. Cross-linking of the Fab fragments by using a second antibody recognizing the mouse immunoglobulins did not allow the restoration of a functional signal, indicating that a very precise configuration of the antibody was likely required to bring the proliferative information. In addition, coating of Fab fragments to a plastic dish did not restore the signal (data not shown). The lost of biological properties of the Fab fragments indicates that B-S12 might induce a dimerization of gp130 transducing protein to generate a biological response. To assess this hypothesis, induction of gp130 dimerization was analyzed in a symmetric ELISA where the same B-T12 anti-gp130 mAb was used as coating and tracer antibody. In these conditions the detection of an increased optical density value will be a reflection of the dimerization of the soluble form of gp130. As shown in Fig. 2B, addition of 2 nM soluble IL-6 receptor and IL-6 to 2 nM soluble gp130 conducted to an increased signal in the symmetric gp130 ELISA. This result sustains the notion that IL-6, IL-6 receptor and gp130 can reassociate together in solution to generate a trimeric, or an hexameric complex as reported before(45.Paonessa G. Graziani R. De Serio A. Savino R. Ciapponi L. Lahm A. Salvati A. Toniatti C. Ciliberto G. EMBO J. 1995; 14: 1942-1951Crossref PubMed Scopus (206) Google Scholar, 46.Ward L. Howlett G.J. Discolo G. Yasukawa K. Hammacher J. Moritz R. Simpson R.J. J. Biol. Chem. 1994; 269: 23286-23289Abstract Full Text PDF PubMed Google Scholar). In a similar manner introduction of the B-S12 antibody, instead of IL-6 and its soluble receptor, led to a dimerization of the soluble form of gp130 transducing protein. Interestingly B-S12 antibody failed to inhibit IL-6-driven proliferation of the TF1 cell line and did not interfere with the binding of radiolabeled IL-6 to its high affinity receptor (data not shown). These results indicate that the antibody and the cytokine recruited gp130 through different functional sites.Figure 2:B-S12 anti-gp130 antibody-induced dimerization of the receptor transducing component. In A, 15,000 TF1 cells were cultured in the presence 20 μg/ml B-S12 mAb (black bars), IgG1 control mAb (hatched bars), or 20 μg/ml Fab-derived fragments from B-S12 antibody and IgG1 isotype control, or a combination Fab-derived fragments (20 μg/ml) plus 20 μg/ml goat anti-mouse polyclonal antibody (Gam). After a 72-h culture period, a [3H]Tdr pulse was performed and the incorporated radioactivity determined by using a β counter. In B, soluble gp130-induced dimerization was measured in a symmetric ELISA using the same B-T12 anti-gp130 mAb as coating and tracer antibody. After coating and plate saturation, 2 nM soluble gp130, 2 nM IL-6, 2 nM soluble IL-6 receptor, or 6 nM B-S12 mAb were added to the wells as indicated for an overnight incubation step. Biotinylated B-T12 mAb was used as second antibody and peroxidase-labeled avidin used for the detection. Reading was carried out at 405 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)gp130 and Jak Family Members Were Tyrosine-phosphorylated in Response to B-S12 StimulationReceptor activation by the IL-6 family of cytokines results in the tyrosine phosphorylation of the transducing receptor subunits and downstream regulatory proteins(32.Stahl N. Boulton T.G. Farrugella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (840) Google Scholar, 33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar). Functional activation of gp130 transducer by B-S12 antibody was assessed by analyzing tyrosine phosphorylation of the transducing protein. The experiments were performed by using the SK-N-MC neuroblastoma cell line, since IL-6-related cytokines displayed many effects in neuronal tissue and, in addition, gp130 tyrosine phosphorylation was readily detectable in this cell line. Fig. 3A shows that the recruitment of gp130, and its tyrosine phosphorylation by the B-S12 antibody was dose-dependent. After a 5-min interaction with the agonistic antibody tyrosine phosphorylation of gp130 could already be detected (Fig. 3B). The observed signal peaked after a 20-min contact, but remained weaker than the one observed by incubating the SK-N-MC cell line in the presence of OSM. A control antibody added in the culture for 20 min did not activate gp130 tyrosine phosphorylation underlining the specificity of the detected signal. Jak1, Jak2, and Tyk2 kinases have been shown to be implicated in the activation of gp130 and gp190/LIF receptor(32.Stahl N. Boulton T.G. Farrugella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (840) Google Scholar, 33.Stahl N. Farrugella T.J. Boulton T.G. Zhong Z. Darnell J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (864) Google Scholar, 34.Lai C.F. Ripperger J. Morella K.K. Wang Y." @default.
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• W1966365320 title "gp130 Transducing Receptor Cross-linking Is Sufficient to Induce Interleukin-6 Type Responses" @default.
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