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MYELOMA BIOLOGY AND THERAPY
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MULTIPLE MYELOMA
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MYELOMA BIOLOGY AND THERAPY Present Status and Future Developments Norihiro Nishimoto, MD, Yoshihito Shima, MD, Kazuyuki Yoshizaki, MD, and Tadamitsu Kishimoto, MD
Multiple myeloma is a neoplastic disease characterized by the monoclonal expansion of lymphoplasmacytic cells in the bone marrow.13 The microenvironment of this site seems to be favorable for myeloma growth, probably because bone marrow stromal cells produce several cytokines including interleukin-6 (IL-6), which has been identified as the major growth factor of myeloma cell^.^*,^^ IL-6 is also responsible for the progressive bone resorption characteristic of myeloma, which causes the bone pain, fractures, osteoporosis, or hypercalcemia observed in myeloma patients. On the basis of these findings, new therapeutic strategies employing the blocking of IL-6 signal transduction have been designed for the treatment of this so far incurable disease. This review reports on the pathophysiologic roles of cytokines, specifically IL-6, as well as the new therapeutic approaches for multiple myeloma based on these roles. CYTOKINES INVOLVED IN THE EXPANSION OF MULTIPLE MYELOMA Expression of Cytokine Receptors
The expansion of poorly proliferating neoplastic tumors such as multiple myeloma is determined by the equilibrium between the prolifFrom the Department of Medicine III, Osaka University Medical School (NN, YS, TK) and the Department of Medical Science I, School of Health and Sport Sciences, Osaka University (KY), Osaka, Japan.
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eration and programmed death of ,tumor cells. Accordingly, the cytokines that augment the growth of myeloma cells are as important for the development of this disease as the factors that modulate the programmed cell death. To identify the cytokines involved in the development of multiple myeloma, immunofluorescence analysis using a panel of monoclonal antibodies against cytokine receptors was performed.” The majority of the tested myeloma cell lines and freshly isolated myeloma cells from examined patients expressed 80-kDa IL-6 receptor (IL6R), to which IL-6 can directly bind, and its signal transducing molecule, gp130, on their cell surface, although the growth of cells expressing IL6R did not always depend on IL-6. Interferon-y receptor (IFNyR) and Fas antigen/APO-1 were also expressed on most of the tested samples, whereas some of them expressed IL-1R type I, IL-2RP, IL-7R, granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), stem cell factor receptor (SCFR), membrane-bound stem cell factor (MBSCF), and tumor necrosis factor receptor (TNFR) type 11. IL-1R type 11, IL-2Ra, IL-4R, IL-8R, and TNFR type I were not detected at all. These findings suggest that IL-6, Fas, IFNy, IL-1, IL-2, IL-7, GM-CSF, SCF, and TNF at least have some biologic effect on myeloma cells. The expression of gp130 also suggests the involvement of gpl30-related cytokines such as leukemia inhibitory factor (LIF), oncostatin M(OM), IL-11, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1) in multiple myeloma. The response patterns to these cytokines, however, may differ from cell to cell because of the different expression of cytokine-specific binding receptors. IL-6 as a Growth Factor for Myeloma Cells
IL-6 was originally identified as a B-cell differentiation factor (BCDF/BSF-2) that induces final maturation of B cells into antibodyproducing cells.35Now it has been shown to be a pleiotropic cytokine with a wide range of biologic activities such as support of hematopoiesis, induction of acute phase reactions, regulation of immune response, and neural differentiation? One of the most interesting activities of IL-6 is to stimulate the growth of myeloma cells. Kawano et a142first reported that IL-6 is a possible autocrine growth factor for both myeloma cell line U266 and myeloma cells freshly explanted from patients. The evidence supporting this conclusion is threefold. First, recombinant IL-6 was found to induce in vitro growth of myeloma cells. Second, myeloma cells spontaneously produced IL-6 and expressed 1L-6 receptor. Third, in vitro growth of myeloma cells was specifically inhibited by anti-IL-6 antibody. In support of this hypothesis, Schwab et al” found evidence of the growth inhibition of the U266 myeloma cell line by IL-6 antisense oligonucleotides; this inhibition was reversed by addition of recombinant IL-6. Similar findings to support the autocrine growth mechanism mediated by IL-6 have been reported by several groups.29,33, 36, 39, 51, 6o
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On the other hand, Klein et a147have proposed a paracrine instead of an autocrine growth mechanism. They observed a spontaneous proliferation in short-term culture of myeloma cells freshly isolated, but not purified, from patients; this proliferation was also inhibited by neutralizing antibodies to IL-6. They demonstrated, however, that IL-6 mRNA was significantly expressed in bone marrow stromal cells but not in myeloma cells. Moreover, they described that myeloma cell line U266 and RPM18226 did not secrete IL-6 or express mRNA for IL-6; also, anti-IL-6 antibodies did not inhibit their proliferation, nor recombinant IL-6 stimulate it. Several reports have also described IL-6 as a paracrine growth factor 73, 76, 84 This view is specifically supported by the of myeloma cells.7, evidence that macrophages, which are the source of large amounts of IL-6 or recombinant IL-6, can generate the reproducible establishment of myeloma cell lines, which used to be extremely difficult to establish especially from bone marrow.", 84 Because IL-6 production from myeloma cells is much less than that from stromal cells and because myeloma cell lines with IL-6 autocrine growth mechanism are rare, the paracrine hypothesis has been sustained. Close cellular contact between myeloma cells and bone marrow stromal cells trigger marrow stromal cells to produce a large amount of IL-6, which supports the growth and final differentiation of malignant plasma cells.21,s2, Several findings indicate that IL-6 is a likely growth factor in vivo, too. The serum levels of IL-6 reflect the disease severity16, ", 7s* 79* and correlate with bone marrow plasmacytosis and serum levels of lactate dehydrogenase and p2 microglobulin, which are the known prognostic factors of multiple myeloma.75These levels also inversely correlate with h e m ~ g l o b i nIn .~~ addition, patients with high IL-6 levels were found to have more frequent osteolytic bone lesions." Finally, in vivo administration of murine anti-IL-6 monoclonal antibodies to patients with advanced myeloma resulted in a decrease of malignant plasma cells in the S-phase of the DNA cell cycle and an improvement of tumor-associated toxicities such as fever, bone pain, and ~achexia.'~, 48 These findings strongly suggest IL-6 is a major growth factor of myeloma cells both in vitro and in vivo. 583
gpl30-Related Cytokines also Stimulate Myeloma Cell Growth
Recent cytokine studies have revealed that several cytokines have several overlapping multiple biologic functions. This functional redundancy can be explained by the utilization of a common signal transducer. Because IL-6, IL-11, CNTF, LIF, OM, and CT-1 utilize gp130 as a common signal transducer, these cytokines were assumed to be able to augment the proliferation of myeloma cells. In fact, LIF and OM were found to be a growth factor for malignant plasma cells freshly isolated from patients.60Furthermore, Zhang et aP5 showed that IL-11 and CNTF, as
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well as LIF, OM, and IL-6, stimulated the proliferation of myeloma cell lines. The responsiveness of the myeloma cells to these cytokines seems to depend on whether the cell’s express the ligand-binding receptors. Because these cytokines exist in vivo, we need to consider them when devising a therapeutic strategy that utilizes blocking IL-6 signal transduction.
Other Growth Factors for Myeloma Cells
Since Mellstedt et alS5= first reported the efficacy of IFNa in 1979, it has been used as a therapeutic agent for multiple myeloma.l* 23, 63, 66, Most studies, but not all, have proved the overall response to IFNa or its ability, in combination with conventional chemotherapy, to prolong the plateau phase of remission; however, the precise mechanism by which IFNa exerts its therapeutic effect on myeloma has not been elucidated. An autocrine loop of IL-&mediated cell growth was disrupted by IFNa in an IL-6-dependent U266 myeloma cell line through the downregulation of IL-6R and g ~ 1 3 0On . ~ the ~ other hand, two reports state that IFNa augmented the growth of myeloma cell lines and that this augmentation depended on the presence of IL-6 in ~ i t r o .73~More~, over, Blade et all9 found that aggressive plasma cell leukemia developed during IFNa therapy. IFNy has been found to inhibit the growth of myeloma cell lines and freshly explanted myeloma cells.4o, 65 Portier et al,65as well as the present authors,6l reported that the mechanism of this growth inhibition by IFNy operated through the downregulation of IL-6R, although Jemberg-Wiklund et a140could not observe this modulation of IL-6R. Because Quesada et aP6 reported that in vivo administration of recombinant IFNy did not improve the disease activity, further study is required to clarify the exact mechanism of IFNy on the growth of myeloma cells. IL-3 acts synergystically with IL-6 to stimulate proliferation and differentiation of myeloma cells in vitro.18 A large number of malignant plasma cells could be obtained from peripheral blood mononucleax cells cultured in the presence of IL-3 and IL-6, suggesting the presence of myeloma precursors in blood. GM-CSF can accelerate myeloma cell growth through the enhancement of the cells’ response to IL-6, because neutralizing anti-IL-6 antibody abrogates the stimulatory effect of GMCSF and because GM-CSF does not augment IL-6 production from bone marrow cells.83IL-5 has also been reported as a growth factor independent of IL-6.7 Because IL-3, GM-CSF, and IL-5 use a common p signal they may act similarly on myeloma cells. The effect of IL-la and IL-1p on myeloma cell growth remains controversial. According to some investigators, IL-la or IL-1p could accelerate myeloma cell growth through the enhancement of IL-6 production,22,43 but this activity could not be confirmed by other^.^ IL-1 is, however, thought to be an activating factor for osteoclasts responsible 557
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for the osteolytic lesions or hypercalcemia typical of this disease. Therefore, IL-1 must be involved in the development of multiple myeloma. IL-10 is a growth factor for myeloma cells different than IL-6, because the stimulating activity of IL-10 is not affected by either anti-IL6 or anti-IL-6 receptor antib0dies.5~TNF has also been identified as a myeloma cell growth factor through an independent pathway of IL6.20,32 We have also observed a growth-stimulating effect of SCF on the myeloma cells that express c-kit (SCFR) on their surface (unpublished observation). The variety of response patterns of myeloma cells to the previously mentioned growth factors suggests the heterogeneity of myeloma cells. Regulation of Apoptosis in Myeloma Cells
Recently, the clonogenic cell in myeloma has been regarded as a memory B cell or a plasmablast that has already gone through the stage of somatic hypermutation and antigen selection.", 67 These cells result from the rescue of germinal center cells from apoptosis, and bone marrow provides a favorable microenvironment for this step. For the expansion of poorly proliferating myeloma cells, prevention of apoptosis is as important as the stimulation by growth factors. As mentioned earlier, a majority of myeloma cells expressed Fas antigen, which can mediate the apoptosis of various human cells including myeloid cells, as well as T and B lymphoblastoid cells. Fas antigen expressed on myeloma cells can mediate the signal for apoptosis of both freshly isolated myeloma cells and cell lines, although this effect may vary from patient to patient.72 Similar observations have been reported by others.%,81 Whether the cells undergo apoptosis in vitro in response to the signal mediated by the Fas antigen might be related to the relative strength of bcl-2, bcl-XL, and bcl-Xs expression. Another question is how the myeloma cells sensitive to anti-Fas monoclonal antibody in vitro can survive in vivo. Because IL-6 was found to prevent the Fas antigen-mediated apoptosisX as well as thakinduced by dexamethasone30in vitro, IL-6 may play an important role in the prevention of apoptosis in vivo in addition to its function as a growth factor. We also observed in myeloma patients a significant increase of a soluble form of the Fas antigen that may interfere with the interaction between Fas antigen and its ligand (unpublished observation). Cytokines Responsible for the Progressive Bone Resorption Characteristic of Myeloma
In normal individuals, there is a balance between the process of bone formation by osteoblasts and bone resorption by osteoclasts. Such bone remodeling is regulated by local factors, referred to as osteotropic cytukines, that are generated in the microenvironment of the remodeling
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unit. In 1974, Mundy et aP7 reported that the osteoclast stimulating factor produced by marrow cells is responsible for osteolytic bone lesion of multiple myeloma. Garrett et a127found bone-resorbing activity in the culture supernatant of a tumor cell line derived from a myeloma patient with osteolytic bone lesion and hypercalcemia, and they determined that this activity originated from a lymphotoxin (TNF-P). In the supernatant of freshly isolated myeloma compartments, a large amount of IL-1P and a lesser amount of IL-la were detected and found responsible for most of the osteoclast stimulating factor activity.25, Furthermore, IL-1 and TNF as well as parathyroid hormone were found to induce the local production of IL-6 in bone by osteoblasts.26,37, 53 IL-6 is also produced by osteoclasts and stimulates early osteoclast precursor formation from cells present in CFU-GM colonies.50Simultaneous treatment with IL-6 and soluble IL-6 receptors induced osteoclast precursor cells to authentic osteoclasts characterized by the presence of tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation in dentine slices.78These findings, taken together with the evidence that myeloma patients with high IL-6 levels had more frequent osteolytic bone lesions,@show that IL-6 may play a major role in generating osteolytic bone lesion and osteoporosis in multiple myeloma (Fig. 1). NEW THERAPEUTIC APPROACHES UTILIZING INTERFERENCE WITH IL-6 SIGNAL TRANSDUCTION
In comparison with conventional chemotherapy and radiation therapy, early myeloablative therapy supported by autologous stem cells appears effective4,6, lo, 11, 13, 14, 31, 38; however, problems such as residual myeloma cells detectable after autografting still remain.24Therefore, a new therapeutic strategy is still needed. On the basis of the evidence that IL-6 is a potent growth factor for myeloma cells, interference with IL-6 signal transduction may constitute a new therapeutic strategy for this disease. For this purpose, several therapeutic approaches are proposed: (1) inhibition of IL-6 production; (2) neutralization of IL-6; (3) blockade of IL-6 binding onto IL-6R; (4) blockade of IL-6/IL-6R compkx binding to gp130; (5) suppression of IL6R and/or gp130 expression; and (6) blockade of the intracytoplasmic signal from gp130. Klein et a148treated a patient with advanced plasma cell leukemia
Figure 1. Pathological significance of interleukin-6 (IL-6) in multiple myeloma. Cellular contact between myeloma cells and bone marrow stromal cells triggers the latter to produce a large amount of IL-6, which aids the proliferation and final differentiation of myeloma cells. IL-6 may also be responsible for osteolytic bone lesions by activating osteoclasts as well as for renal function disorders by stimulating mesangial cell proliferation.
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with neutralizing murine anti-human IL-6 monoclonal antibodies. In vivo administration of antibodies to the patient alleviated fever and improved the serum M-component, serum calcium, and C-reactive protein in association with a decrease in the number of malignant plasma cells in the S-phase of the DNA cell cycle. This therapeutic efficacy was transient, however, because human antibodies against murine anti-IL-6 monoclonal antibodies appeared. Another explanation for the loss of efficacy was that the concentration of anti-IL-6 monoclonal antibodies was insufficient to neutralize IL-6 that had increased after the treatment. In fact, myeloma cells explanted from the patient after recurrence showed the same IL-Mependent growth in vitro as before treatment. In addition, Bataille et all7 presented clinical data for 10 patients with advanced myeloma who were treated with murine anti-IL-6 monoclonal antibody. Marked inhibition of plasmablastic cell proliferation was observed in two patients, and an antiproliferative effect marked by a significant reduction of the labeling index of bone marrow myeloma cells was found in three patients. None of the patients studied, however, achieved remission or improved outcome as judged by standard clinical criteria. The loss of the efficacy of murine anti-IL-6 monoclonal antibodies in those patients is also likely to have been the result of an increase of in vivo production of IL-6 induced by this treatment, which is too high to be neutralized. It may also be due to the emergence of human antibodies against murine antibodies, especially in patients with treatments longer than 1 month. Although remission could not be achieved, the blocking of IL-6 signal transduction is anticipated to become a new therapeutic strategy for multiple myeloma. Murine antibodies are so highly immunogenic in humans as to limit their therapeutic value for human patients. To overcome this difficulty, murine antibodies have been engineered to look like human antibodies. Reshaped human I'M-1 (rhPM-1) is constructed by grafting the complementarity determining regions from the mouse anti-human IL-6R antibody I'M-1 onto human IgG to recreate a well-functioning antigenbinding site in a reshaped huplan antibody.69rhPM-1 looks very much like a human antibody and can therefore be expected to be a poor immunogen in humans and maintain a prolonged effect when administered in vivo. The inhibitory activity of rhPM-1 on myeloma cell growth in vitro is equivalent to that of both mouse and chimeric I'M-1. Moreover, rhPM-l was found to inhibit both the tumor formation of human plasmacytomas and M-protein synthesis in IL-6 transgenic, severely combined immunodeficient mice, which were inoculated with human myeloma cells (our unpublished results). On the basis of these findings, we administered rhPM-1 to patients with advanced multiple myeloma and observed improvement in tumor associated toxicities (unpublished observation). IL-6 receptor antagonists can be another device acting as a therapeutic agent to block the binding of IL-6 to ILdR as well as to anti-IL-6R antibodies. Factors that decrease production of IL-6 or expression of IL-6R and
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gp130 can be expected to be potential therapeutic agents for multiple myeloma. All-trans retinoic acid (ATRA) reportedly inhibits the growth of myeloma cell line U266 as a result of downregulation of IL-6R and subsequent inhibition of IL-&mediated autocrine We also demonstrated the growth inhibitory activity of ATRA on freshly explanted myeloma cells. This effect is due partly to heterologous or homologous downregulation of IL-6R and gp130 and to suppression of IL-6 production from myeloma as well as bone marrow stromal cells. Steroid therapy and interferon therapy are thought to exert their effect partly through a similar mechanism. A phase I1 clinical trial of ATRA for patients with advanced refractory myeloma was initiated59;however, the results appear to be discouraging because the patients developed severe hypercalcemia following administration of ATRA in association with an increase in serum IL-6 levels, which had previously been observed in ATRA treatment for acute promyelocytic l e ~ k e m i a Further .~ investigation will be required to determine whether this treatment is beneficial. Recent studies of the IL-6 signaling pathway have revealed that binding of IL-6 to IL-6R results in the homodimerization of gp130 and activation of the tyrosine kinase JAK2 and the consequent initiation of the Ras-development MAP kinase cascade. This cascade ultimately leads to the activation of the transcription factor, NF-IL6. In addition, JAK2 may directly tyrosine-phosphorylate the acute-phase response factor (APRF)/signal transductions and activators of transcription 3 (STAT 3), which then rapidly migrates to the nucleus and binds to the DNA. These transcription factors are essential for the expression of biologic activity of IL-6 as an acute-phase protein inducer.45,46 Although the signaling pathway for the growth-stimulatory activity on myeloma cells has not been elucidated yet, the antisense oligonucleotides for these molecules may be effective for blocking the growth signals for myeloma cells. Other therapeutic approaches have been initiated in combination with myeloablative therapy. Monoclonal antibodies specific to myeloma cells are clinically useful not only for characterization of myeloma cells but also for in vivo or in vitro purging of myeloma cells. Goldmacher et alZ8reported the development of a potent anti-CD38 immunotoxin capable of killing myeloma cells. A chimeric anti-CD38 antibody consisting of the Fab portion of the murine monoclonal antibody linked to an Fc molecule derived from human IgGl was constructed to mediate antibody-dependent cellular cytotoxicity, which can kill myeloma cells in vivo.R Finally, IL-6-Pseudomonas exotoxin derivatives could also be effective for ex vivo marrow purging for myeloma patients.49 CONCLUSION
Since the introduction of melphalan for the treatment of multiple myeloma, many therapeutic approaches have been attempted to enhance the remission and survival rates that can be achieved by combination
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chemotherapy with melphalan and predonisolone; however, the results do not seem to be satisfactory. Will it be possible to overcome this refractory disease in the near future? Recent advances in molecular biology have made it possible to identify the precursors for myeloma cells and elucidate the mechanisms of their proliferation and differentiation. In cytokine studies in particular, remarkable progress has been made in the understanding of the pathophysiology of multiple myeloma. We believe that an approach based on these pathophysiologic findings should provide us with a breakthrough to prevail over this so far incurable disease. ACKNOWLEDGMENT We thank Ms. A. Nobuhara for her outstanding secretarial assistance.
References 1. Ahre A, Bjorkholm M, Mellstedt H, et al: Human leukocyte interferon and intermittent high-dose melphalan-prednisone administration in the treatment of multiple myeloma: A randomized clinical trial from the Myeloma Group of Central Sweden. Cancer Treat Rep 68:1331, 1984 2. Akira S, Taga T, Kishimoto T Interleukin-6 in biology and medicine. Adv Immunol 54:1, 1993 3. Akiyama H, Nakamura N, Nagasaka S, et al: Hypercalcaemia due to all-trans retinoic acid [letter]. Lancet 339:308, 1992 4. Alexanian R, Dimopoulos M: The treatment of multiple myeloma. N Engl J Med 330:484, 1994 5. Alexanian R, Dimopoulos MA, Delasalle K, Barlogie B: Primary dexamethasone treatment of multiple myeloma. Blood 80887, 1992 6. Alexanian R, Dimopoulos MA, Hester J, et al: Early myeloablative therapy for multiple myeloma. Blood M4278, 1994 7. Anderson KC, Jones RM, Morimoto C, et al: Response patterns of purified myeloma cells to hematopoietic growth factors Blood 73:1915, 1989 8. Anderson KC, Barut BA, Ritz J, et al: Monoclonal antibody-purged autologous bone marrow transplantation therapy for multiple myeloma. Blood 77712, 1991 9. Anderson KC, Andersen J, Soiffer R, et al: Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 82:2568, 1993 10. Attal M, Harousseau JL, et al: A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. N Engl J Med 225:91, 1996 11. Attal M, Huguet F, Schlaifer D, et al: Intensive combined therapy for previously untreated aggressive myeloma. Blood 79:1130, 1992 12. Bakkus MH, Heirman C, Van-Riet I, et al: Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 80:2326, 1992 13. Barlogie 8, Epstein J, et al: Plasma Cell Myeloma-New Biological Insights and Advances in Therapy. Blood 73:865, 1989 14. Barlogie 8, Jagannath S, et al: Superiority of tendon autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood, 1997 15. Barut BA, Zon LI, Cochran MK, et a 1 Role of interleukin 6 in the growth of myelomaderived cell lines. Leuk Res 16:951, 1992 16. Bataille R, Jourdan M, Zhang XG, Klein B Serum levels of interleukin 6, a potent
MYELOMA BIOLOGY AND THERAPY
17. 18. 19. 20.
21.
22. 23.
24. 25. 26. 27. 28. 29. 30. 31.
32.
' 33. 34. 35. 36. 37. 38. 39.
169
myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias. J Clin Invest 849008, 1989 Bataille R, Barlogie 8, Lu ZY, et al: Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood 86:685, 1995 Bergui L, Schena M, Gaidano G, et al: Interleukin 3 and interleukin 6 synergistically promote the proliferation and differentiation of malignant plasma cell precursors in multiple myeloma. J Exp Med 170:613, 1989 Blade J, Lopez-Guillermo A, Tassies D, et al: Development of aggressive plasma cell leukaemia under interferon-alpha therapy [see comments]. Br J Haematol79:523,1991 Borset M, Waage A, Brekke OL, Helseth E: TNF and IL-6 are potent growth factors for OH-2, a novel human myeloma cell line. Eur J Haematol 53:31, 1994 Caligaris-Cappio F, Bergui L, Gregoretti MG, et al: Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 772688,1991 Carter A, Merchav S, Silvian-Draxler I, Tatarsky I: The role of interleukin-1 and tumour necrosis factor-alpha in human multiple myeloma. Br J Haematol 74424,1990 Cooper MR, Dear K, McIntyre OR, et al: A randomized clinical trial comparing melphalan/prednisone with or without interferon alfa-2b in newly diagnosed patients with multiple myeloma: A Cancer and Leukemia Group B study. J Clin Oncol 11:155, 1993 Corradini P, Voena C, Astolfi M, et a1 High-dose sequential chemoradiotherapy in multiple myeloma: Residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting. Blood 85:1596, 1995 Cozzolino F, Torcia M, Aldinucci D, et al: Production of interleukin-1 by bone marrow myeloma cells. Blood 74:380, 1989 Feyen JH, Elford P, Di-Padova FE, Trechsel U Interleukin-6 is produced by bone and modulated by parathyroid hormone. J Bone Miner Res 4:633, 1989 Garrett IR, Durie BG, Nedwin GE, et a1 Production of lymphotoxin, a bone-resorbing cytokine, by cultured human myeloma cells. N Engl J Med 317526, 1987 Goldmacher VS, Bourret LA, Levine BA, et al: Anti-CD38-blocked ricin: An immunotoxin for the treatment of multiple myeloma. Blood 84:3017, 1994 Goto H, Shimazaki C, Tatsumi T, et al: Establishment of a novel myeloma cell line KPMM2 carrying t(3;14)(qZl;q32), which proliferates specifically in response to interleukin-6 through an autocrine mechanism. Leukemia 9:711, 1995 Hardin J, MacLeod S, Grigorieva I, et al: Interleukin-6 prevents dexamethasoneinduced myeloma cell death. Blood 84:3063, 1994 Harousseau JL, Attal M, Divine M, et al: Autologous stem cell transplantation after first remission induction treatment in multiple myeloma: A report of the French Registry on autologous transplantation in multiple myeloma. Blood 85:3077, 1995 Hata H, Matsuzaki H, Takatsuki K: Autocrine growth by two cytokmes, interleukin6 and tumor necrosis factor alpha, in the myeloma cell line KHM-IA. Acta Haematol 83133, 1990 Hata H, Xiao H, Petrucci MT, et a1 Interleukin-6 gene expression in multiple myeloma: A characteristic of immature tumor cells. Blood 81:3357, 1993 Hata H, Matsuzaki H, Takeya M, et a1 Expression of Fas/Apo-1 (CD95)and apoptosis in tumor cells from patients with plasma cell disorders. Blood 86:1939, 1995 Hirano T, Yasukawa K, Harada H, et a1 Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 32473, 1986 Hitzler JK, Martinez-Valdez H, Bergsagel DB, et al: Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease. Blood 78:1996, 1991 Ishimi Y, Miyaura C, Jin CH, et a1 IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 145:3297, 1990 Jagannath S, Vesole DH, Glenn L, et al: Low-risk intensive therapy for r'nultiple myeloma with combined autologous bone marrow and blood stem cell support. Blood 80:1666, 1992 Jemberg H, Pettersson M, Kishimoto T, Nilsson K Heterogeneity in response to interleukin 6 (IL-6), expression of IL-6 and IL-6 receptor mRNA in a panel of
170
40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
54. 55. 55a. 56. 57. 58. 59. 60. 61. 62.
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established human multiple myeloma cell l i e s [published erratum appears in Leukemia 1991 June 5(6): following 5301 Leukemia 5955, 1991 Jemberg-Wiklund H, Pettersson M, Nilsson K: Recombinant interferon-gamma inhibits the growth of IL-Mependent human multiple myeloma cell lines in vitro. Eur J Haematol46:231, 1991 Jourdan M, Zhang XG, Portier M, et al: IFN-alpha induces autocrine production of IL-6 in myeloma cell l i e s . J Immunol 147:4402, 1991 Kawano M, Hirano T, Matsuda T, et al: Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 33283, 1988 Kawano M, Tanaka H, Ishikawa H, et a1 Interleukin-1 accelerates autocrine growth of myeloma cells through interleukin-6 in human myeloma. Blood 732145, 1989 Kawano M, Yamamoto I, Iwato K, et al: Interleukin-1 beta rather than lymphotoxin as the major bone resorbing activity in human multiple myeloma. Blood 73:1646,1989 Kishimoto T, Taga T, Akira S Cytokine signal transduction. Cell 76:253, 1994 Kishimoto T, Akira S, Narazaki M, Taga T Interleukin-6 family of cytokines and gp130. Blood 86:1243, 1995 Klein B, Zhang XG, Jourdan M, et al: Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 73517, 1989 Klein B, Wijdenes J, Zhang XG, et al: Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. Blood 78:1198, 1991 Kreitman RJ, Siegall CB, FitzGerald DJ, et a1 Interleukin-6 fused to a mutant form of Pseudornonas exotoxin kills malignant cells from patients with multiple myeloma. Blood 79:1775, 1992 Kurihara N, Bertolini D, Suda T, et al: IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release. J Immunol 1M4226, 1990 Levy Y, Tsapis A, Brouet J C Interleukin-6 antisense oligonucleotides inhibit the growth of human myeloma cell lines. J Clin Invest 88:696, 1991 Lokhorst HM, Lamme T, de-Smet M, et al: Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures. Blood 842269,1994 Lowik CW, van-der-Ruijm G, Bloys H, et al: Parathyroid hormone (PTH) and PTHlike protein (PLP) stimulate interleukin-6 production by osteogenic cells: A possible role of interleukin-6 in osteoclastogenesis. Biochem Biophys Res Commun 162:1546, 1989 Lu ZY, Zhang XG, Rodriguez C, et al: Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells. Blood 85:2521, 1995 Mandelli F, Awisati G, Amadori S, et al: Maintenance treatment with recombinant interferon alfa-2b in patients with multiple myeloma responding to conventional induction chemotherapy. N Engl J Med 322:1430,1990 Mellstedt H, Ahre A, Bjorkholm M, et al: Interferon therapy in myelomatosis. Lancet s 1945, 1979 Miyajima A, Kitamura T, Harada N, et a1 Cytokine receptors and signal transduction. Annu Rev Immunol10:295,1992 Mundy G R Evidence for the secretion of an osteoclast stimulating factor in myeloma. N Engl J Med 291:1041, 1974 Nachbaur DM, Herold M, Maneschg A, Huber H Serum levels of interleukin-6 in multiple myeloma and other hematological disorders: Correlation with disease activity and other prognostic parameters. Ann Hematol6254, 1991 Niesvizky R, Siege1 DS, Busquets X, et a1 Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma. Br J Haematol 89217, 1995 Nishimoto N, Ogata A, Shima Y, et al: Oncostatin M, leukemia inhibitory factor, and interleukin 6 induce the proliferation of human plasmacytoma cells via the common signal transducer, gp130. J Exp Med 179:1343, 1994 Ogata A, Nishimoto N, Shima Y, et al: Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction. Blood 843040,1994 Okuno Y, Takahashi T, Suzuki A, et al: Establishment and characterization of four
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myeloma cell lines which are responsive to interleukin-6 for their growth. Leukemia 5:585, 1991 63. Osterborg A, Bjorkholm M, Bjoreman M, et a1 Natural interferon-alpha in combination with melphalan/prednisone versus melphalan/prednisone in the treatment of multiple myeloma stages I1 and 111: A randomized study from the Myeloma Group of Central Sweden. Blood 81:1428, 1993 64 Pelliiemi TI',lrjala K, Mattila K, et al: Immunoreactive interleukin-6 and acute phase proteins as pro&ostic factors in multiple myeloma. Finnish Leukemia Group. Blood 85:765. 1995 65. Portier M, Zhang XG, Caron E, et a1 Gamma-interferon in multiple myeloma: Inhibition of interleukin-6 (IL-6)-dependent myeloma cell growth and downregulation of IL-6-receptor expression in vitro. Blood 81:3076, 1993 66. Quesada JR, Alexanian R, Hawkins M, et a 1 Treatment of multiple myeloma with recombinant alpha-interferon. Blood 67275, 1986 67. Ralph QM, Brisco MJ, Joshua DE, et al: Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 82202, 1993 68. Salmon SE, Durie BG, Young L, et al: Effects of cloned human leukocyte interferons in the human tumor stem cell assay. J Clin Oncol 1:217, 1983 69. Sat0 K, Tsuchiya M, Saldanha J, et al: Reshaping a human antibody to inhibit the interleukin Mependent tumor cell growth. Cancer Res 53851, 1993 70. Schwab G, Siegall CB, Aarden LA, et al: Characterization of an interleukin-&mediated autocrine growth loop in the human multiple myeloma cell line, U266. Blood 77587,1991 71. Schwabe M, Brini AT, Bosco MC, et al: Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-&dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains. J Clin Invest 94:2317, 1994 72. Shima Y, Nishimoto N, Ogata A, et al: Myeloma cells express Fas antigen/APO-1 ( 0 9 5 ) but o d y some are sensitive to anti-Fas antibody resulting in apoptosis. Blood 85757,1995 73. Shimizu S, Yoshioka R, Hirose Y, et al: Establishment of two interleukin 6 (B cell stimulatory factor 2/interferon beta 2)-dependent human bone marrow-derived myeloma cell lines. J Exp Med 169:339, 1989 74. Sidell N, Taga T, Hirano T, et a1 Retinoic acid-induced growth inhibition of a human myeloma cell line via down-regulation of IL-6 receptors. J Immunol 146:3809, 1991 75. Solary E, Guiguet M, Zeller V, et al: Radioimmunoassay for the measurement of serum IL-6 and its correlation with tumour cell mass parameters in multiple myeloma. Am J Hematol 39363, 1992 76. Sonneveld P, Schoester M, de-Leeuw K In vitro Ig-synthesis and proliferative activity in multiple myeloma are stimulated by different growth factors. Br J Haematol 79:589, 1991 77. Stevenson FK, Bell AJ, Cusack R, et al: Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD3S antibody. Blood 771071, 1991 78. Tamura T, Udagawa N, Takahashi N, et al: Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proc Natl Acad Sci USA 90:11924, 1993 79. Tienhaara A, Pulkki K, Mattila K, et al: Serum immunoreactive interleukin-6 and Creactive protein levels in patients with multiple myeloma at diagnosis. Br J Haematol 86:391, 1994 80. Uchiyama H, Barut BA, Mohrbacher AF, et al: Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion. Blood 823712, 1993 81. Westendorf JJ, Lammert JM, Jelinek D F Expression and function of Fas'(AP0-I/ CD95) in patient myeloma cells and myeloma cell lines. Blood 85:3566, 1995 82. Zhang XG, Klein 8, Bataille R Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma. Blood 74:11, 1989 83. Zhang XG, Bataille R, Jourdan M, et al: Granulocyte-macrophage colony-stimulating
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factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells [see comments]. Blood 76:2599, 1990 84. Zhang XG, Gaillard JP, Robillard N, et al: Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma. Blood 83:3654, 1994 85. Zhang XG, Gu JJ, Lu ZY, et a1 Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. J Exp Med 179:1337, 1994 Address reprint requests to Tadamitsu Kishimoto, MD Department of Medicine 111 Osaka University Medical School 2-2 Yamada-oka, Suita Osaka 565 Japan
0889-8588/97 $0.00
MULTIPLE MYELOMA
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MYELOMA BIOLOGY AND THERAPY Present Status and Future Developments Norihiro Nishimoto, MD, Yoshihito Shima, MD, Kazuyuki Yoshizaki, MD, and Tadamitsu Kishimoto, MD
Multiple myeloma is a neoplastic disease characterized by the monoclonal expansion of lymphoplasmacytic cells in the bone marrow.13 The microenvironment of this site seems to be favorable for myeloma growth, probably because bone marrow stromal cells produce several cytokines including interleukin-6 (IL-6), which has been identified as the major growth factor of myeloma cell^.^*,^^ IL-6 is also responsible for the progressive bone resorption characteristic of myeloma, which causes the bone pain, fractures, osteoporosis, or hypercalcemia observed in myeloma patients. On the basis of these findings, new therapeutic strategies employing the blocking of IL-6 signal transduction have been designed for the treatment of this so far incurable disease. This review reports on the pathophysiologic roles of cytokines, specifically IL-6, as well as the new therapeutic approaches for multiple myeloma based on these roles. CYTOKINES INVOLVED IN THE EXPANSION OF MULTIPLE MYELOMA Expression of Cytokine Receptors
The expansion of poorly proliferating neoplastic tumors such as multiple myeloma is determined by the equilibrium between the prolifFrom the Department of Medicine III, Osaka University Medical School (NN, YS, TK) and the Department of Medical Science I, School of Health and Sport Sciences, Osaka University (KY), Osaka, Japan.
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eration and programmed death of ,tumor cells. Accordingly, the cytokines that augment the growth of myeloma cells are as important for the development of this disease as the factors that modulate the programmed cell death. To identify the cytokines involved in the development of multiple myeloma, immunofluorescence analysis using a panel of monoclonal antibodies against cytokine receptors was performed.” The majority of the tested myeloma cell lines and freshly isolated myeloma cells from examined patients expressed 80-kDa IL-6 receptor (IL6R), to which IL-6 can directly bind, and its signal transducing molecule, gp130, on their cell surface, although the growth of cells expressing IL6R did not always depend on IL-6. Interferon-y receptor (IFNyR) and Fas antigen/APO-1 were also expressed on most of the tested samples, whereas some of them expressed IL-1R type I, IL-2RP, IL-7R, granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), stem cell factor receptor (SCFR), membrane-bound stem cell factor (MBSCF), and tumor necrosis factor receptor (TNFR) type 11. IL-1R type 11, IL-2Ra, IL-4R, IL-8R, and TNFR type I were not detected at all. These findings suggest that IL-6, Fas, IFNy, IL-1, IL-2, IL-7, GM-CSF, SCF, and TNF at least have some biologic effect on myeloma cells. The expression of gp130 also suggests the involvement of gpl30-related cytokines such as leukemia inhibitory factor (LIF), oncostatin M(OM), IL-11, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1) in multiple myeloma. The response patterns to these cytokines, however, may differ from cell to cell because of the different expression of cytokine-specific binding receptors. IL-6 as a Growth Factor for Myeloma Cells
IL-6 was originally identified as a B-cell differentiation factor (BCDF/BSF-2) that induces final maturation of B cells into antibodyproducing cells.35Now it has been shown to be a pleiotropic cytokine with a wide range of biologic activities such as support of hematopoiesis, induction of acute phase reactions, regulation of immune response, and neural differentiation? One of the most interesting activities of IL-6 is to stimulate the growth of myeloma cells. Kawano et a142first reported that IL-6 is a possible autocrine growth factor for both myeloma cell line U266 and myeloma cells freshly explanted from patients. The evidence supporting this conclusion is threefold. First, recombinant IL-6 was found to induce in vitro growth of myeloma cells. Second, myeloma cells spontaneously produced IL-6 and expressed 1L-6 receptor. Third, in vitro growth of myeloma cells was specifically inhibited by anti-IL-6 antibody. In support of this hypothesis, Schwab et al” found evidence of the growth inhibition of the U266 myeloma cell line by IL-6 antisense oligonucleotides; this inhibition was reversed by addition of recombinant IL-6. Similar findings to support the autocrine growth mechanism mediated by IL-6 have been reported by several groups.29,33, 36, 39, 51, 6o
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On the other hand, Klein et a147have proposed a paracrine instead of an autocrine growth mechanism. They observed a spontaneous proliferation in short-term culture of myeloma cells freshly isolated, but not purified, from patients; this proliferation was also inhibited by neutralizing antibodies to IL-6. They demonstrated, however, that IL-6 mRNA was significantly expressed in bone marrow stromal cells but not in myeloma cells. Moreover, they described that myeloma cell line U266 and RPM18226 did not secrete IL-6 or express mRNA for IL-6; also, anti-IL-6 antibodies did not inhibit their proliferation, nor recombinant IL-6 stimulate it. Several reports have also described IL-6 as a paracrine growth factor 73, 76, 84 This view is specifically supported by the of myeloma cells.7, evidence that macrophages, which are the source of large amounts of IL-6 or recombinant IL-6, can generate the reproducible establishment of myeloma cell lines, which used to be extremely difficult to establish especially from bone marrow.", 84 Because IL-6 production from myeloma cells is much less than that from stromal cells and because myeloma cell lines with IL-6 autocrine growth mechanism are rare, the paracrine hypothesis has been sustained. Close cellular contact between myeloma cells and bone marrow stromal cells trigger marrow stromal cells to produce a large amount of IL-6, which supports the growth and final differentiation of malignant plasma cells.21,s2, Several findings indicate that IL-6 is a likely growth factor in vivo, too. The serum levels of IL-6 reflect the disease severity16, ", 7s* 79* and correlate with bone marrow plasmacytosis and serum levels of lactate dehydrogenase and p2 microglobulin, which are the known prognostic factors of multiple myeloma.75These levels also inversely correlate with h e m ~ g l o b i nIn .~~ addition, patients with high IL-6 levels were found to have more frequent osteolytic bone lesions." Finally, in vivo administration of murine anti-IL-6 monoclonal antibodies to patients with advanced myeloma resulted in a decrease of malignant plasma cells in the S-phase of the DNA cell cycle and an improvement of tumor-associated toxicities such as fever, bone pain, and ~achexia.'~, 48 These findings strongly suggest IL-6 is a major growth factor of myeloma cells both in vitro and in vivo. 583
gpl30-Related Cytokines also Stimulate Myeloma Cell Growth
Recent cytokine studies have revealed that several cytokines have several overlapping multiple biologic functions. This functional redundancy can be explained by the utilization of a common signal transducer. Because IL-6, IL-11, CNTF, LIF, OM, and CT-1 utilize gp130 as a common signal transducer, these cytokines were assumed to be able to augment the proliferation of myeloma cells. In fact, LIF and OM were found to be a growth factor for malignant plasma cells freshly isolated from patients.60Furthermore, Zhang et aP5 showed that IL-11 and CNTF, as
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well as LIF, OM, and IL-6, stimulated the proliferation of myeloma cell lines. The responsiveness of the myeloma cells to these cytokines seems to depend on whether the cell’s express the ligand-binding receptors. Because these cytokines exist in vivo, we need to consider them when devising a therapeutic strategy that utilizes blocking IL-6 signal transduction.
Other Growth Factors for Myeloma Cells
Since Mellstedt et alS5= first reported the efficacy of IFNa in 1979, it has been used as a therapeutic agent for multiple myeloma.l* 23, 63, 66, Most studies, but not all, have proved the overall response to IFNa or its ability, in combination with conventional chemotherapy, to prolong the plateau phase of remission; however, the precise mechanism by which IFNa exerts its therapeutic effect on myeloma has not been elucidated. An autocrine loop of IL-&mediated cell growth was disrupted by IFNa in an IL-6-dependent U266 myeloma cell line through the downregulation of IL-6R and g ~ 1 3 0On . ~ the ~ other hand, two reports state that IFNa augmented the growth of myeloma cell lines and that this augmentation depended on the presence of IL-6 in ~ i t r o .73~More~, over, Blade et all9 found that aggressive plasma cell leukemia developed during IFNa therapy. IFNy has been found to inhibit the growth of myeloma cell lines and freshly explanted myeloma cells.4o, 65 Portier et al,65as well as the present authors,6l reported that the mechanism of this growth inhibition by IFNy operated through the downregulation of IL-6R, although Jemberg-Wiklund et a140could not observe this modulation of IL-6R. Because Quesada et aP6 reported that in vivo administration of recombinant IFNy did not improve the disease activity, further study is required to clarify the exact mechanism of IFNy on the growth of myeloma cells. IL-3 acts synergystically with IL-6 to stimulate proliferation and differentiation of myeloma cells in vitro.18 A large number of malignant plasma cells could be obtained from peripheral blood mononucleax cells cultured in the presence of IL-3 and IL-6, suggesting the presence of myeloma precursors in blood. GM-CSF can accelerate myeloma cell growth through the enhancement of the cells’ response to IL-6, because neutralizing anti-IL-6 antibody abrogates the stimulatory effect of GMCSF and because GM-CSF does not augment IL-6 production from bone marrow cells.83IL-5 has also been reported as a growth factor independent of IL-6.7 Because IL-3, GM-CSF, and IL-5 use a common p signal they may act similarly on myeloma cells. The effect of IL-la and IL-1p on myeloma cell growth remains controversial. According to some investigators, IL-la or IL-1p could accelerate myeloma cell growth through the enhancement of IL-6 production,22,43 but this activity could not be confirmed by other^.^ IL-1 is, however, thought to be an activating factor for osteoclasts responsible 557
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for the osteolytic lesions or hypercalcemia typical of this disease. Therefore, IL-1 must be involved in the development of multiple myeloma. IL-10 is a growth factor for myeloma cells different than IL-6, because the stimulating activity of IL-10 is not affected by either anti-IL6 or anti-IL-6 receptor antib0dies.5~TNF has also been identified as a myeloma cell growth factor through an independent pathway of IL6.20,32 We have also observed a growth-stimulating effect of SCF on the myeloma cells that express c-kit (SCFR) on their surface (unpublished observation). The variety of response patterns of myeloma cells to the previously mentioned growth factors suggests the heterogeneity of myeloma cells. Regulation of Apoptosis in Myeloma Cells
Recently, the clonogenic cell in myeloma has been regarded as a memory B cell or a plasmablast that has already gone through the stage of somatic hypermutation and antigen selection.", 67 These cells result from the rescue of germinal center cells from apoptosis, and bone marrow provides a favorable microenvironment for this step. For the expansion of poorly proliferating myeloma cells, prevention of apoptosis is as important as the stimulation by growth factors. As mentioned earlier, a majority of myeloma cells expressed Fas antigen, which can mediate the apoptosis of various human cells including myeloid cells, as well as T and B lymphoblastoid cells. Fas antigen expressed on myeloma cells can mediate the signal for apoptosis of both freshly isolated myeloma cells and cell lines, although this effect may vary from patient to patient.72 Similar observations have been reported by others.%,81 Whether the cells undergo apoptosis in vitro in response to the signal mediated by the Fas antigen might be related to the relative strength of bcl-2, bcl-XL, and bcl-Xs expression. Another question is how the myeloma cells sensitive to anti-Fas monoclonal antibody in vitro can survive in vivo. Because IL-6 was found to prevent the Fas antigen-mediated apoptosisX as well as thakinduced by dexamethasone30in vitro, IL-6 may play an important role in the prevention of apoptosis in vivo in addition to its function as a growth factor. We also observed in myeloma patients a significant increase of a soluble form of the Fas antigen that may interfere with the interaction between Fas antigen and its ligand (unpublished observation). Cytokines Responsible for the Progressive Bone Resorption Characteristic of Myeloma
In normal individuals, there is a balance between the process of bone formation by osteoblasts and bone resorption by osteoclasts. Such bone remodeling is regulated by local factors, referred to as osteotropic cytukines, that are generated in the microenvironment of the remodeling
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unit. In 1974, Mundy et aP7 reported that the osteoclast stimulating factor produced by marrow cells is responsible for osteolytic bone lesion of multiple myeloma. Garrett et a127found bone-resorbing activity in the culture supernatant of a tumor cell line derived from a myeloma patient with osteolytic bone lesion and hypercalcemia, and they determined that this activity originated from a lymphotoxin (TNF-P). In the supernatant of freshly isolated myeloma compartments, a large amount of IL-1P and a lesser amount of IL-la were detected and found responsible for most of the osteoclast stimulating factor activity.25, Furthermore, IL-1 and TNF as well as parathyroid hormone were found to induce the local production of IL-6 in bone by osteoblasts.26,37, 53 IL-6 is also produced by osteoclasts and stimulates early osteoclast precursor formation from cells present in CFU-GM colonies.50Simultaneous treatment with IL-6 and soluble IL-6 receptors induced osteoclast precursor cells to authentic osteoclasts characterized by the presence of tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation in dentine slices.78These findings, taken together with the evidence that myeloma patients with high IL-6 levels had more frequent osteolytic bone lesions,@show that IL-6 may play a major role in generating osteolytic bone lesion and osteoporosis in multiple myeloma (Fig. 1). NEW THERAPEUTIC APPROACHES UTILIZING INTERFERENCE WITH IL-6 SIGNAL TRANSDUCTION
In comparison with conventional chemotherapy and radiation therapy, early myeloablative therapy supported by autologous stem cells appears effective4,6, lo, 11, 13, 14, 31, 38; however, problems such as residual myeloma cells detectable after autografting still remain.24Therefore, a new therapeutic strategy is still needed. On the basis of the evidence that IL-6 is a potent growth factor for myeloma cells, interference with IL-6 signal transduction may constitute a new therapeutic strategy for this disease. For this purpose, several therapeutic approaches are proposed: (1) inhibition of IL-6 production; (2) neutralization of IL-6; (3) blockade of IL-6 binding onto IL-6R; (4) blockade of IL-6/IL-6R compkx binding to gp130; (5) suppression of IL6R and/or gp130 expression; and (6) blockade of the intracytoplasmic signal from gp130. Klein et a148treated a patient with advanced plasma cell leukemia
Figure 1. Pathological significance of interleukin-6 (IL-6) in multiple myeloma. Cellular contact between myeloma cells and bone marrow stromal cells triggers the latter to produce a large amount of IL-6, which aids the proliferation and final differentiation of myeloma cells. IL-6 may also be responsible for osteolytic bone lesions by activating osteoclasts as well as for renal function disorders by stimulating mesangial cell proliferation.
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with neutralizing murine anti-human IL-6 monoclonal antibodies. In vivo administration of antibodies to the patient alleviated fever and improved the serum M-component, serum calcium, and C-reactive protein in association with a decrease in the number of malignant plasma cells in the S-phase of the DNA cell cycle. This therapeutic efficacy was transient, however, because human antibodies against murine anti-IL-6 monoclonal antibodies appeared. Another explanation for the loss of efficacy was that the concentration of anti-IL-6 monoclonal antibodies was insufficient to neutralize IL-6 that had increased after the treatment. In fact, myeloma cells explanted from the patient after recurrence showed the same IL-Mependent growth in vitro as before treatment. In addition, Bataille et all7 presented clinical data for 10 patients with advanced myeloma who were treated with murine anti-IL-6 monoclonal antibody. Marked inhibition of plasmablastic cell proliferation was observed in two patients, and an antiproliferative effect marked by a significant reduction of the labeling index of bone marrow myeloma cells was found in three patients. None of the patients studied, however, achieved remission or improved outcome as judged by standard clinical criteria. The loss of the efficacy of murine anti-IL-6 monoclonal antibodies in those patients is also likely to have been the result of an increase of in vivo production of IL-6 induced by this treatment, which is too high to be neutralized. It may also be due to the emergence of human antibodies against murine antibodies, especially in patients with treatments longer than 1 month. Although remission could not be achieved, the blocking of IL-6 signal transduction is anticipated to become a new therapeutic strategy for multiple myeloma. Murine antibodies are so highly immunogenic in humans as to limit their therapeutic value for human patients. To overcome this difficulty, murine antibodies have been engineered to look like human antibodies. Reshaped human I'M-1 (rhPM-1) is constructed by grafting the complementarity determining regions from the mouse anti-human IL-6R antibody I'M-1 onto human IgG to recreate a well-functioning antigenbinding site in a reshaped huplan antibody.69rhPM-1 looks very much like a human antibody and can therefore be expected to be a poor immunogen in humans and maintain a prolonged effect when administered in vivo. The inhibitory activity of rhPM-1 on myeloma cell growth in vitro is equivalent to that of both mouse and chimeric I'M-1. Moreover, rhPM-l was found to inhibit both the tumor formation of human plasmacytomas and M-protein synthesis in IL-6 transgenic, severely combined immunodeficient mice, which were inoculated with human myeloma cells (our unpublished results). On the basis of these findings, we administered rhPM-1 to patients with advanced multiple myeloma and observed improvement in tumor associated toxicities (unpublished observation). IL-6 receptor antagonists can be another device acting as a therapeutic agent to block the binding of IL-6 to ILdR as well as to anti-IL-6R antibodies. Factors that decrease production of IL-6 or expression of IL-6R and
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gp130 can be expected to be potential therapeutic agents for multiple myeloma. All-trans retinoic acid (ATRA) reportedly inhibits the growth of myeloma cell line U266 as a result of downregulation of IL-6R and subsequent inhibition of IL-&mediated autocrine We also demonstrated the growth inhibitory activity of ATRA on freshly explanted myeloma cells. This effect is due partly to heterologous or homologous downregulation of IL-6R and gp130 and to suppression of IL-6 production from myeloma as well as bone marrow stromal cells. Steroid therapy and interferon therapy are thought to exert their effect partly through a similar mechanism. A phase I1 clinical trial of ATRA for patients with advanced refractory myeloma was initiated59;however, the results appear to be discouraging because the patients developed severe hypercalcemia following administration of ATRA in association with an increase in serum IL-6 levels, which had previously been observed in ATRA treatment for acute promyelocytic l e ~ k e m i a Further .~ investigation will be required to determine whether this treatment is beneficial. Recent studies of the IL-6 signaling pathway have revealed that binding of IL-6 to IL-6R results in the homodimerization of gp130 and activation of the tyrosine kinase JAK2 and the consequent initiation of the Ras-development MAP kinase cascade. This cascade ultimately leads to the activation of the transcription factor, NF-IL6. In addition, JAK2 may directly tyrosine-phosphorylate the acute-phase response factor (APRF)/signal transductions and activators of transcription 3 (STAT 3), which then rapidly migrates to the nucleus and binds to the DNA. These transcription factors are essential for the expression of biologic activity of IL-6 as an acute-phase protein inducer.45,46 Although the signaling pathway for the growth-stimulatory activity on myeloma cells has not been elucidated yet, the antisense oligonucleotides for these molecules may be effective for blocking the growth signals for myeloma cells. Other therapeutic approaches have been initiated in combination with myeloablative therapy. Monoclonal antibodies specific to myeloma cells are clinically useful not only for characterization of myeloma cells but also for in vivo or in vitro purging of myeloma cells. Goldmacher et alZ8reported the development of a potent anti-CD38 immunotoxin capable of killing myeloma cells. A chimeric anti-CD38 antibody consisting of the Fab portion of the murine monoclonal antibody linked to an Fc molecule derived from human IgGl was constructed to mediate antibody-dependent cellular cytotoxicity, which can kill myeloma cells in vivo.R Finally, IL-6-Pseudomonas exotoxin derivatives could also be effective for ex vivo marrow purging for myeloma patients.49 CONCLUSION
Since the introduction of melphalan for the treatment of multiple myeloma, many therapeutic approaches have been attempted to enhance the remission and survival rates that can be achieved by combination
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chemotherapy with melphalan and predonisolone; however, the results do not seem to be satisfactory. Will it be possible to overcome this refractory disease in the near future? Recent advances in molecular biology have made it possible to identify the precursors for myeloma cells and elucidate the mechanisms of their proliferation and differentiation. In cytokine studies in particular, remarkable progress has been made in the understanding of the pathophysiology of multiple myeloma. We believe that an approach based on these pathophysiologic findings should provide us with a breakthrough to prevail over this so far incurable disease. ACKNOWLEDGMENT We thank Ms. A. Nobuhara for her outstanding secretarial assistance.
References 1. Ahre A, Bjorkholm M, Mellstedt H, et al: Human leukocyte interferon and intermittent high-dose melphalan-prednisone administration in the treatment of multiple myeloma: A randomized clinical trial from the Myeloma Group of Central Sweden. Cancer Treat Rep 68:1331, 1984 2. Akira S, Taga T, Kishimoto T Interleukin-6 in biology and medicine. Adv Immunol 54:1, 1993 3. Akiyama H, Nakamura N, Nagasaka S, et al: Hypercalcaemia due to all-trans retinoic acid [letter]. Lancet 339:308, 1992 4. Alexanian R, Dimopoulos M: The treatment of multiple myeloma. N Engl J Med 330:484, 1994 5. Alexanian R, Dimopoulos MA, Delasalle K, Barlogie B: Primary dexamethasone treatment of multiple myeloma. Blood 80887, 1992 6. Alexanian R, Dimopoulos MA, Hester J, et al: Early myeloablative therapy for multiple myeloma. Blood M4278, 1994 7. Anderson KC, Jones RM, Morimoto C, et al: Response patterns of purified myeloma cells to hematopoietic growth factors Blood 73:1915, 1989 8. Anderson KC, Barut BA, Ritz J, et al: Monoclonal antibody-purged autologous bone marrow transplantation therapy for multiple myeloma. Blood 77712, 1991 9. Anderson KC, Andersen J, Soiffer R, et al: Monoclonal antibody-purged bone marrow transplantation therapy for multiple myeloma. Blood 82:2568, 1993 10. Attal M, Harousseau JL, et al: A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. N Engl J Med 225:91, 1996 11. Attal M, Huguet F, Schlaifer D, et al: Intensive combined therapy for previously untreated aggressive myeloma. Blood 79:1130, 1992 12. Bakkus MH, Heirman C, Van-Riet I, et al: Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 80:2326, 1992 13. Barlogie 8, Epstein J, et al: Plasma Cell Myeloma-New Biological Insights and Advances in Therapy. Blood 73:865, 1989 14. Barlogie 8, Jagannath S, et al: Superiority of tendon autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood, 1997 15. Barut BA, Zon LI, Cochran MK, et a 1 Role of interleukin 6 in the growth of myelomaderived cell lines. Leuk Res 16:951, 1992 16. Bataille R, Jourdan M, Zhang XG, Klein B Serum levels of interleukin 6, a potent
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22. 23.
24. 25. 26. 27. 28. 29. 30. 31.
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' 33. 34. 35. 36. 37. 38. 39.
169
myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias. J Clin Invest 849008, 1989 Bataille R, Barlogie 8, Lu ZY, et al: Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood 86:685, 1995 Bergui L, Schena M, Gaidano G, et al: Interleukin 3 and interleukin 6 synergistically promote the proliferation and differentiation of malignant plasma cell precursors in multiple myeloma. J Exp Med 170:613, 1989 Blade J, Lopez-Guillermo A, Tassies D, et al: Development of aggressive plasma cell leukaemia under interferon-alpha therapy [see comments]. Br J Haematol79:523,1991 Borset M, Waage A, Brekke OL, Helseth E: TNF and IL-6 are potent growth factors for OH-2, a novel human myeloma cell line. Eur J Haematol 53:31, 1994 Caligaris-Cappio F, Bergui L, Gregoretti MG, et al: Role of bone marrow stromal cells in the growth of human multiple myeloma. Blood 772688,1991 Carter A, Merchav S, Silvian-Draxler I, Tatarsky I: The role of interleukin-1 and tumour necrosis factor-alpha in human multiple myeloma. Br J Haematol 74424,1990 Cooper MR, Dear K, McIntyre OR, et al: A randomized clinical trial comparing melphalan/prednisone with or without interferon alfa-2b in newly diagnosed patients with multiple myeloma: A Cancer and Leukemia Group B study. J Clin Oncol 11:155, 1993 Corradini P, Voena C, Astolfi M, et a1 High-dose sequential chemoradiotherapy in multiple myeloma: Residual tumor cells are detectable in bone marrow and peripheral blood cell harvests and after autografting. Blood 85:1596, 1995 Cozzolino F, Torcia M, Aldinucci D, et al: Production of interleukin-1 by bone marrow myeloma cells. Blood 74:380, 1989 Feyen JH, Elford P, Di-Padova FE, Trechsel U Interleukin-6 is produced by bone and modulated by parathyroid hormone. J Bone Miner Res 4:633, 1989 Garrett IR, Durie BG, Nedwin GE, et a1 Production of lymphotoxin, a bone-resorbing cytokine, by cultured human myeloma cells. N Engl J Med 317526, 1987 Goldmacher VS, Bourret LA, Levine BA, et al: Anti-CD38-blocked ricin: An immunotoxin for the treatment of multiple myeloma. Blood 84:3017, 1994 Goto H, Shimazaki C, Tatsumi T, et al: Establishment of a novel myeloma cell line KPMM2 carrying t(3;14)(qZl;q32), which proliferates specifically in response to interleukin-6 through an autocrine mechanism. Leukemia 9:711, 1995 Hardin J, MacLeod S, Grigorieva I, et al: Interleukin-6 prevents dexamethasoneinduced myeloma cell death. Blood 84:3063, 1994 Harousseau JL, Attal M, Divine M, et al: Autologous stem cell transplantation after first remission induction treatment in multiple myeloma: A report of the French Registry on autologous transplantation in multiple myeloma. Blood 85:3077, 1995 Hata H, Matsuzaki H, Takatsuki K: Autocrine growth by two cytokmes, interleukin6 and tumor necrosis factor alpha, in the myeloma cell line KHM-IA. Acta Haematol 83133, 1990 Hata H, Xiao H, Petrucci MT, et a1 Interleukin-6 gene expression in multiple myeloma: A characteristic of immature tumor cells. Blood 81:3357, 1993 Hata H, Matsuzaki H, Takeya M, et a1 Expression of Fas/Apo-1 (CD95)and apoptosis in tumor cells from patients with plasma cell disorders. Blood 86:1939, 1995 Hirano T, Yasukawa K, Harada H, et a1 Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature 32473, 1986 Hitzler JK, Martinez-Valdez H, Bergsagel DB, et al: Role of interleukin-6 in the proliferation of human multiple myeloma cell lines OCI-My 1 to 7 established from patients with advanced stage of the disease. Blood 78:1996, 1991 Ishimi Y, Miyaura C, Jin CH, et a1 IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 145:3297, 1990 Jagannath S, Vesole DH, Glenn L, et al: Low-risk intensive therapy for r'nultiple myeloma with combined autologous bone marrow and blood stem cell support. Blood 80:1666, 1992 Jemberg H, Pettersson M, Kishimoto T, Nilsson K Heterogeneity in response to interleukin 6 (IL-6), expression of IL-6 and IL-6 receptor mRNA in a panel of
170
40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
54. 55. 55a. 56. 57. 58. 59. 60. 61. 62.
NISHIMOTO et a1
established human multiple myeloma cell l i e s [published erratum appears in Leukemia 1991 June 5(6): following 5301 Leukemia 5955, 1991 Jemberg-Wiklund H, Pettersson M, Nilsson K: Recombinant interferon-gamma inhibits the growth of IL-Mependent human multiple myeloma cell lines in vitro. Eur J Haematol46:231, 1991 Jourdan M, Zhang XG, Portier M, et al: IFN-alpha induces autocrine production of IL-6 in myeloma cell l i e s . J Immunol 147:4402, 1991 Kawano M, Hirano T, Matsuda T, et al: Autocrine generation and requirement of BSF-2/IL-6 for human multiple myelomas. Nature 33283, 1988 Kawano M, Tanaka H, Ishikawa H, et a1 Interleukin-1 accelerates autocrine growth of myeloma cells through interleukin-6 in human myeloma. Blood 732145, 1989 Kawano M, Yamamoto I, Iwato K, et al: Interleukin-1 beta rather than lymphotoxin as the major bone resorbing activity in human multiple myeloma. Blood 73:1646,1989 Kishimoto T, Taga T, Akira S Cytokine signal transduction. Cell 76:253, 1994 Kishimoto T, Akira S, Narazaki M, Taga T Interleukin-6 family of cytokines and gp130. Blood 86:1243, 1995 Klein B, Zhang XG, Jourdan M, et al: Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 73517, 1989 Klein B, Wijdenes J, Zhang XG, et al: Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia. Blood 78:1198, 1991 Kreitman RJ, Siegall CB, FitzGerald DJ, et a1 Interleukin-6 fused to a mutant form of Pseudornonas exotoxin kills malignant cells from patients with multiple myeloma. Blood 79:1775, 1992 Kurihara N, Bertolini D, Suda T, et al: IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release. J Immunol 1M4226, 1990 Levy Y, Tsapis A, Brouet J C Interleukin-6 antisense oligonucleotides inhibit the growth of human myeloma cell lines. J Clin Invest 88:696, 1991 Lokhorst HM, Lamme T, de-Smet M, et al: Primary tumor cells of myeloma patients induce interleukin-6 secretion in long-term bone marrow cultures. Blood 842269,1994 Lowik CW, van-der-Ruijm G, Bloys H, et al: Parathyroid hormone (PTH) and PTHlike protein (PLP) stimulate interleukin-6 production by osteogenic cells: A possible role of interleukin-6 in osteoclastogenesis. Biochem Biophys Res Commun 162:1546, 1989 Lu ZY, Zhang XG, Rodriguez C, et al: Interleukin-10 is a proliferation factor but not a differentiation factor for human myeloma cells. Blood 85:2521, 1995 Mandelli F, Awisati G, Amadori S, et al: Maintenance treatment with recombinant interferon alfa-2b in patients with multiple myeloma responding to conventional induction chemotherapy. N Engl J Med 322:1430,1990 Mellstedt H, Ahre A, Bjorkholm M, et al: Interferon therapy in myelomatosis. Lancet s 1945, 1979 Miyajima A, Kitamura T, Harada N, et a1 Cytokine receptors and signal transduction. Annu Rev Immunol10:295,1992 Mundy G R Evidence for the secretion of an osteoclast stimulating factor in myeloma. N Engl J Med 291:1041, 1974 Nachbaur DM, Herold M, Maneschg A, Huber H Serum levels of interleukin-6 in multiple myeloma and other hematological disorders: Correlation with disease activity and other prognostic parameters. Ann Hematol6254, 1991 Niesvizky R, Siege1 DS, Busquets X, et a1 Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma. Br J Haematol 89217, 1995 Nishimoto N, Ogata A, Shima Y, et al: Oncostatin M, leukemia inhibitory factor, and interleukin 6 induce the proliferation of human plasmacytoma cells via the common signal transducer, gp130. J Exp Med 179:1343, 1994 Ogata A, Nishimoto N, Shima Y, et al: Inhibitory effect of all-trans retinoic acid on the growth of freshly isolated myeloma cells via interference with interleukin-6 signal transduction. Blood 843040,1994 Okuno Y, Takahashi T, Suzuki A, et al: Establishment and characterization of four
MYELOMA BIOLOGY AND THERAPY
171
myeloma cell lines which are responsive to interleukin-6 for their growth. Leukemia 5:585, 1991 63. Osterborg A, Bjorkholm M, Bjoreman M, et a1 Natural interferon-alpha in combination with melphalan/prednisone versus melphalan/prednisone in the treatment of multiple myeloma stages I1 and 111: A randomized study from the Myeloma Group of Central Sweden. Blood 81:1428, 1993 64 Pelliiemi TI',lrjala K, Mattila K, et al: Immunoreactive interleukin-6 and acute phase proteins as pro&ostic factors in multiple myeloma. Finnish Leukemia Group. Blood 85:765. 1995 65. Portier M, Zhang XG, Caron E, et a1 Gamma-interferon in multiple myeloma: Inhibition of interleukin-6 (IL-6)-dependent myeloma cell growth and downregulation of IL-6-receptor expression in vitro. Blood 81:3076, 1993 66. Quesada JR, Alexanian R, Hawkins M, et a 1 Treatment of multiple myeloma with recombinant alpha-interferon. Blood 67275, 1986 67. Ralph QM, Brisco MJ, Joshua DE, et al: Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 82202, 1993 68. Salmon SE, Durie BG, Young L, et al: Effects of cloned human leukocyte interferons in the human tumor stem cell assay. J Clin Oncol 1:217, 1983 69. Sat0 K, Tsuchiya M, Saldanha J, et al: Reshaping a human antibody to inhibit the interleukin Mependent tumor cell growth. Cancer Res 53851, 1993 70. Schwab G, Siegall CB, Aarden LA, et al: Characterization of an interleukin-&mediated autocrine growth loop in the human multiple myeloma cell line, U266. Blood 77587,1991 71. Schwabe M, Brini AT, Bosco MC, et al: Disruption by interferon-alpha of an autocrine interleukin-6 growth loop in IL-&dependent U266 myeloma cells by homologous and heterologous down-regulation of the IL-6 receptor alpha- and beta-chains. J Clin Invest 94:2317, 1994 72. Shima Y, Nishimoto N, Ogata A, et al: Myeloma cells express Fas antigen/APO-1 ( 0 9 5 ) but o d y some are sensitive to anti-Fas antibody resulting in apoptosis. Blood 85757,1995 73. Shimizu S, Yoshioka R, Hirose Y, et al: Establishment of two interleukin 6 (B cell stimulatory factor 2/interferon beta 2)-dependent human bone marrow-derived myeloma cell lines. J Exp Med 169:339, 1989 74. Sidell N, Taga T, Hirano T, et a1 Retinoic acid-induced growth inhibition of a human myeloma cell line via down-regulation of IL-6 receptors. J Immunol 146:3809, 1991 75. Solary E, Guiguet M, Zeller V, et al: Radioimmunoassay for the measurement of serum IL-6 and its correlation with tumour cell mass parameters in multiple myeloma. Am J Hematol 39363, 1992 76. Sonneveld P, Schoester M, de-Leeuw K In vitro Ig-synthesis and proliferative activity in multiple myeloma are stimulated by different growth factors. Br J Haematol 79:589, 1991 77. Stevenson FK, Bell AJ, Cusack R, et al: Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD3S antibody. Blood 771071, 1991 78. Tamura T, Udagawa N, Takahashi N, et al: Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proc Natl Acad Sci USA 90:11924, 1993 79. Tienhaara A, Pulkki K, Mattila K, et al: Serum immunoreactive interleukin-6 and Creactive protein levels in patients with multiple myeloma at diagnosis. Br J Haematol 86:391, 1994 80. Uchiyama H, Barut BA, Mohrbacher AF, et al: Adhesion of human myeloma-derived cell lines to bone marrow stromal cells stimulates interleukin-6 secretion. Blood 823712, 1993 81. Westendorf JJ, Lammert JM, Jelinek D F Expression and function of Fas'(AP0-I/ CD95) in patient myeloma cells and myeloma cell lines. Blood 85:3566, 1995 82. Zhang XG, Klein 8, Bataille R Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma. Blood 74:11, 1989 83. Zhang XG, Bataille R, Jourdan M, et al: Granulocyte-macrophage colony-stimulating
172
NISHIMOTO et a1
factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells [see comments]. Blood 76:2599, 1990 84. Zhang XG, Gaillard JP, Robillard N, et al: Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma. Blood 83:3654, 1994 85. Zhang XG, Gu JJ, Lu ZY, et a1 Ciliary neurotropic factor, interleukin 11, leukemia inhibitory factor, and oncostatin M are growth factors for human myeloma cell lines using the interleukin 6 signal transducer gp130. J Exp Med 179:1337, 1994 Address reprint requests to Tadamitsu Kishimoto, MD Department of Medicine 111 Osaka University Medical School 2-2 Yamada-oka, Suita Osaka 565 Japan