Haematologica 2003 - Supplements
Share Embed Flag
Download ePaper

Haematologica 2003 - Supplements

Haematologica 2003 - Supplements Haematologica 2003 - Supplements

supplements.haematologica.org
from supplements.haematologica.org More from this publisher
13.11.2014 Views

110 IL-7 IS INDUCED BY IL-6 IN HUMAN MYELOMA CELLS: ROLE IN MULTIPLE MYELOMA. Nicola Giuliani, Simona Colla, Giovanni Roti, Sabrina Bonomini, Magda Hojden, Gabriella Sammarelli, Francesca Morandi, Regis Bataille, Vittorio Rizzoli Chair of Hematology, University of Parma, Italy, INSERM U463, Nantes, France. It has been recently shown that IL-7 stimulates IL-6 secretion by bone marrow (BM) stromal cells that express IL-7 receptor (IL- 7R) as well as long lived plasma cell progenitors. On the basis of this evidence the production of IL-7 and its potential role in multiple myeloma (MM) has been investigated in this study. First we tested IL-7/IL-7R expression in human myeloma cell lines (HMCL) and in purified CD138+ MM cells obtained at the diagnosis. We found that RPMI-8226, U266, OPM-2, XG-6 and fresh MM cells obtained from 12 patients were positive for IL-7 mRNA. On the other hand IL-7R mRNA was not expressed in any HMCL tested while the EBV positive cell line ARH-77 was positive for IL-7R. Using an ELISA assay IL-7 was detected in the supernatant of HMCL, in contrast IL-7 was undetectable in conditioned medium of mononuclear cells or normal CD19+ B cells or B leukemic cell line REH. IL-7 levels were significantly up-regulated when RPMI-8226, U266, XG-6 were cultured in presence of IL-6 (20-50 ng/ml) while IL-6 did not induce IL-7 production in normal B cells and REH as well as in EBV positive cells and in B cells obtained from patients with acute lymphoblastic leukemia, previously evaluated for CD126 expression by flow cytometry. A stimulatory effect of IL-7 on IL-7R positive cells ARH-77 proliferation was found (+12%±1;p

integrin in IGF-1-triggered MM cell adhesion and migration. A functional blocking antibody against β1 integrin and a RGD peptide, as well as cytochalasin D (cyt D: a cytoskeleton inhibitory agent), significantly reduced IGF-1-induced cell adhesion. Immunoprecipitation studies demonstrated that IGF-1 rapidly and transiently induces association of IGF-1R and 1 integrin, which is associated with phosphorylation of IGF-1R, IRS-1, and p85/PI3K. IGF-1 also triggers phosphorylation of AKT and ERK. We next demonstrated that IGF-1R and 1 integrin comigrate to cholesterol-rich microdomains on plasma membrane (membrane rafts) following IGF-1 stimulation, using western blotting for the Triton-insoluble and Triton-soluble fraction of the cells and dual immunofluorescence staining. Using methyl-β-cyclodextrin to disrupt membrane raft structure, we further show that β1 integrin-mediated IGF-1-stimulated adhesion requires intact lipid rafts. In addition, IGF-1 (100 ng/ml and 40 ng/ml for MM.1S and OPM6, respectively) triggers polymerization of F-actin, indicating focal adhesion formation, and immunoprecipitation experiments confirm that IGF-1 induces interaction of β1 integrin with cytoskeletal and signaling proteins localized at focal adhesions, including focal adhesion kinase (p125FAK), α-actinin, and paxillin. Pretreatment of cyt D abrogates activation of p125FAK and paxillin induced by IGF-1. Importantly, IGF-1-induced MM cell adhesion to FN is achieved only when 1 integrin and PI3K/AKT are activated. In a 96-well transmigration assay, IGF-1 induces a 2-3 (MM.1S) and 3-4-fold (OPM6) increase in migration. Conversely, IR3 and a blocking anti-β1 integrin mAb, as well as Cyt D, abrogate MM cell transmigration. Interestingly, IGF-1 induces MM cell migration independent of de no vo protein synthesis. Finally, primary CD138+ MM cells isolated from 3 patients were also responsive to IGF-1-induced adhesion on FN-coated plates and BM stromal cells. Together, these studies demonstrate that IGF-1 induces MM cell adhesion and migration, suggesting a role of IGF-1 in the trafficking and localization of these cells in the BM microenvironment. Morover, these results identify a functional cooperation between IGF-1R and β1 integrin in MM homing, supporting blockade of IGF-1/IGF1R system as a novel treatment strategy of MM. 113 HHV-8 viral IL-6 homologue is as active as human IL-6 on human myeloma cells Andreas Günther1, Frank Bakker1, Wolfgang Baum1, Renate Burger1, Frank Neipel2 and Martin Gramatzki1 1Department of Medicine III / Div. of Hematology/Oncology and 2Institute for Clinical and Molecular Virology, University of Erlangen, Germany Kaposi-Sarcoma-associated Virus/ Human-Herpesvirus-8 (KSHV/HHV-8) is associated with at least three human lymphoproliferative disorders: primary effusion lymphomas (PEL), Castleman’s disease (CD) and plasmablastic lymphoma. However, the role of HHV-8 in multiple myeloma (MM) is still controversial. Some groups detected HHV-8-DNA in bone marrow samples of MM patients whereas other groups failed to confirm these data. An important issue is the potential functional role of HHV-8 which encodes for several viral cytokines. Most interest was given to the viral homologue of IL-6 (vIL-6) since human IL-6 (huIL-6) is not only an essential growth and survival factor for malignant plasma cells but plays an important role in PEL, CD and plasmablastic lymphoma. We could show that prokaryotically expressed vIL-6 (pro-v-IL-6) has biological activity on human myeloma cells in vitro (Burger et al., Blood 1998). However, the vIL-6 concentrations needed for a maximum stimulatory growth effect on the strictly IL-6 dependent human myeloma cell line INA-6 were dramatically higher (4000 ng/ml) than those needed for recombinant huIL-6 (1.25 ng/ml) This requirement for very high amounts of vIL-6 argued against a causative role of vIL-6 in human diseases. However, this study was performed with prokaryotically expressed recombinant vIL-6 which could be less active than vIL-6 expressed in human cells. Now, we could overcome these limitations. First we used conditioned medium (CM) of the HHV-8 infected PEL cell line BCBL-1, containing only small amounts of hu IL-6 (45 pg/ml). The addition of 50% CM was able to induce a proliferative response of the INA-6 plasma cell line which would have required 1 ng/ml of recombinant huIL-6. Anti-gp130 antibody (Ab) or the combination of anti-IL-6 receptor and anti-gp130 Abs, but not anti-IL-6R Ab alone, were effective in inhibiting BCBL-1 CM activity on INA-6. These results are similar to those obtained with vIL-6 and indicate that the growth induction of BCBL-1 CM is mainly caused by vIL-6. In a second approach we used a baculovirus vector expression system in eukaryotic cells. Eukaryotically expressed vIL-6 (eu-v-IL-6) had a significantly higher activity than pro-v-IL-6 and induced a maximum stimulatory growth response in INA-6 cells at the same concentration as huIL-6 (1.25 ng/ml). Similar to the results published previously for pro-vIL-6 and obtained for BCBL-1 CM. The growth effects induced by eu-v-IL-6 could be blocked by anti-gp 130 Ab or a combination of anti-IL-6R and anti-gp130 Abs, but not anti-IL-6R Ab alone. Thus, our data indicate that the HHV-8 viral homologue of IL-6 is as active on human cells as hu IL-6. HHV-8 vIL-6 may therefore act as a substitute for the human cytokine in multiple myeloma and HHV-8 associated diseases and could play a causative role in their development. 114 Macrophage inflammatory protein 1-alpha (MIP-1α) triggers migration and signaling cascades mediating survival and proliferation in multiple myeloma cells Suzanne Lentzsch, Margarete Gries, Martin Janz, Ralf Bargou, Bernd Dorken, and Markus Y. Mapara. Humboldt University of Berlin, Charite Campus Buch, Robert- Roessle-Klinik, 13125 Berlin, Germany, Tel.: +49-30-94171370, FAX:+49-30-94171209, e-mail: lentzsch@rrk-berlin.de Recently, several studies have suggested that MIP-1 contributes to the pathophysiology of multiple myeloma (MM). Thus, it has been demonstrated that MIP-1α is an osteoclast stimulatory factor and that MIP-1α neutralizing antibody (ab) or antisense inhibit bone destruction and reduce tumor load in a SCID-mice model of MM. Furthermore, MM patients have significantly higher bone marrow plasma levels of MIP-1α than healthy controls. The current study was designed to determine the direct effects of MIP-1α on MM cells. We found expression of very high levels of MIP-1α and its receptor CCR5 in MM cell lines and patientderived primary cells. Furthermore, we were able to demonstrate that MIP-1α significantly increased proliferation of different MM cell lines (MM1.S, H929, OPM-2, INA-6) and patient-derived primary MM cells. MIP-1α specific migration could be observed in a dose-dependent fashion up to 21 fold compared to control. In MM cell line and patient MM cells, MIP-1α induces phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) as well as AKT and its downstream target FKHR. STAT3 was not activated by MIP-1α. In addition, inhibition of AKT activation by the PI3-Kinase (PI3-K) inhibitors wortmannin S138

110<br />

IL-7 IS INDUCED BY IL-6 IN HUMAN MYELOMA CELLS:<br />

ROLE IN MULTIPLE MYELOMA.<br />

Nicola Giuliani, Simona Colla, Giovanni Roti, Sabrina<br />

Bonomini, Magda Hojden, Gabriella Sammarelli, Francesca<br />

Morandi, Regis Bataille, Vittorio Rizzoli<br />

Chair of Hematology, University of Parma, Italy, INSERM U463,<br />

Nantes, France.<br />

It has been recently shown that IL-7 stimulates IL-6 secretion by<br />

bone marrow (BM) stromal cells that express IL-7 receptor (IL-<br />

7R) as well as long lived plasma cell progenitors. On the basis of<br />

this evidence the production of IL-7 and its potential role in<br />

multiple myeloma (MM) has been investigated in this study.<br />

First we tested IL-7/IL-7R expression in human myeloma cell<br />

lines (HMCL) and in purified CD138+ MM cells obtained at the<br />

diagnosis. We found that RPMI-8226, U266, OPM-2, XG-6 and<br />

fresh MM cells obtained from 12 patients were positive for IL-7<br />

mRNA. On the other hand IL-7R mRNA was not expressed in<br />

any HMCL tested while the EBV positive cell line ARH-77 was<br />

positive for IL-7R. Using an ELISA assay IL-7 was detected in<br />

the supernatant of HMCL, in contrast IL-7 was undetectable in<br />

conditioned medium of mononuclear cells or normal CD19+ B<br />

cells or B leukemic cell line REH. IL-7 levels were significantly<br />

up-regulated when RPMI-8226, U266, XG-6 were cultured in<br />

presence of IL-6 (20-50 ng/ml) while IL-6 did not induce IL-7<br />

production in normal B cells and REH as well as in EBV positive<br />

cells and in B cells obtained from patients with acute<br />

lymphoblastic leukemia, previously evaluated for CD126<br />

expression by flow cytometry.<br />

A stimulatory effect of IL-7 on IL-7R positive cells ARH-77<br />

proliferation was found (+12%±1;p

logo

products

Resources

Company

Terms of service
Privacy policy
Cookie policy
Cookie settings
Imprint
Change language
Made with love in Switzerland
© 2024 Yumpu.com all rights reserved

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!