Soluble interleukin-6 receptor as a prognostic factor in multiple myeloma

Interleukin-6 (IL-6) is a major growth factor for the clonal malignant plasma cells in multiple myeloma (MM). The effect of IL-6 may be enhanced by soluble IL-6 receptor (sIL-6R). As there is a clinical need for improved stratification of MM patients at diagnosis, we have studied the role of sIL-6R as a prognostic marker in 207 newly diagnosed MM patients. Serum sIL-6R concentration was above the upper reference limit in 47% of the patients at diagnosis. The concentrations of sIL-6R and two other prognostic factors, IL-6 and β-2 microglobulin (β2M), were all significantly higher in the patients who died within 3 years compared with those who survived. However, serum sIL-6R did not show linear correlation with IL-6 or β2M levels. In univariate logistic regression analysis sIL-6R was a significant predictor of 3-year mortality. Kaplan-Meier analysis showed that raised levels of sIL-6R were associated with shorter survival. When the patients were stratified into four groups according to their serum IL-6 and sIL-6R levels, the patients with normal serum levels of both parameters had clear survival benefit. As β2M was the most powerful prognostic factor in the multivariate analysis, the patients were also stratified according to their serum β2M and sIL-6R levels. The patients with raised levels of both β2M and sIL-6R had shorter survival than the patients in the other three groups. Thus, measurement of these parameters at diagnosis would help to stratify MM patients.     

Paracrine interactions of basic fibroblast growth factor and interleukin-6 in multiple myeloma

Myeloma cells express basic fibroblast growth factor (bFGF), an angiogenic cytokine triggering marrow neovascularization in multiple myeloma (MM). In solid tumors and some lymphohematopoietic malignancies, angiogenic cytokines have also been shown to stimulate tumor growth via paracrine pathways. Since interleukin-6 (IL-6) is a potent growth and survival factor for myeloma cells, we have studied the effects of bFGF on IL-6 secretion by bone marrow stromal cells (BMSCs) and its potential reverse regulation in myeloma cells. Both myeloma-derived cell lines and myeloma cells isolated from the marrow of MM patients were shown to express and secrete bFGF. Cell-sorting studies identified myeloma cells as the predominant source of bFGF in MM marrow. BMSCs from MM patients and control subjects expressed high-affinity FGF receptors R1 through R4. Stimulation of BMSCs with bFGF induced a time- and dose-dependent increase in IL-6 secretion (median, 2-fold; P <.001), which was completely abrogated by anti-bFGF antibodies. Conversely, stimulation with IL-6 enhanced bFGF expression and secretion by myeloma cell lines (2-fold; P =.02) as well as MM patient cells (up to 3.6-fold; median, 1.5-fold; P =.002). This effect was inhibited by anti-IL-6 antibody. When myeloma cells were cocultured with BMSCs in a noncontact transwell system, both IL-6 and bFGF concentrations in coculture supernatants increased 2- to 3-fold over the sum of basal concentrations in the monoculture controls. The IL-6 increase was again partially, but significantly, inhibited by anti-bFGF. The data demonstrate a paracrine interaction between myeloma and marrow stromal cells triggered by mutual stimulation of bFGF and IL-6.     

Interleukin-6 signalling in juvenile idiopathic arthritis is limited by proteolytically cleaved soluble interleukin-6 receptor

OBJECTIVES: Interleukin-6 (IL-6) exerts multiple effects on chondrocytes and fibroblasts within the joint and is associated with disease activity in juvenile idiopathic arthritis (JIA). Although these cells express the ubiquitous signalling receptor for all IL-6-related cytokines, gp130, they do not express a cognate IL-6 receptor. Consequently, IL-6 responses within these cells occur via IL-6 trans-signalling relying on the presence of a soluble receptor (sIL-6R). Levels of sIL-6R in vivo are governed by either proteolytic cleavage (PC) of cognate receptor or by differential sIL-6R mRNA splicing (DS). The aim of this study was to evaluate the contribution of both isoforms to clinical parameters associated with IL-6 signalling in JIA. METHODS: IL-6, sIL-6R and DS-sIL-6R were measured by ELISA in serum and synovial fluid (SF) samples from 86 JIA patients. These data were related to indicators of inflammation-erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and compared between patients stratified by subtype, age and disease duration. RESULTS: SF IL-6 significantly correlated with general indicators of activity (ESR and CRP) and SF PC-sIL-6R to a lesser degree with CRP. When the IL-6:sIL-6R ratio was calculated as an indicator of the potential for IL-6 signalling within the joint, 33% of SF samples showed a ratio >1 indicating saturation of sIL-6R by IL-6. Mean DS-sIL-6R levels were 0.71 ng/ml, whereas PC-sIL-6R levels constituted the majority of sIL-6R at 20.89 ng/ml. CONCLUSIONS: IL-6 trans-signalling within the joints of JIA patients is predominantly governed by the presence of PC-sIL-6R, and the data provided suggest that synovial levels of IL-6 and sIL-6R would be sufficient to drive IL-6 responses in chondrocytes and synovial fibroblasts.     

Evidence of a role for a non-matrix-type metalloproteinase activity in the shedding of syndecan-1 from human myeloma cells

Syndecan-1 is a cell surface proteoglycan that is expressed on human myeloma cells and is thought to act as a co-receptor for certain extracellular matrix proteins and growth factors. The ectodomain of syndecan-1 is thought to be shed from the surface of myeloma cells, although the exact mechanism of release remains unclear. In this study, we used a panel of inhibitors to identify the class of proteinase responsible for shedding the soluble syndecan-1 ectodomain from human myeloma cells. Using enzyme-linked immunosorbent assay, flow cytometry and immunocytochemistry, we demonstrated that myeloma cell lines expressed syndecan-1 on their surface and that this was shed constitutively, but to a varying extent. In addition, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated a marked loss of cell surface syndecan-1 from each of the cell lines and this was associated with a corresponding increase in soluble syndecan-1. Inhibitors of serine and cysteine proteinases, and matrix-type metalloproteinases, did not inhibit constitutive or PMA-stimulated syndecan-1 shedding from JJN3 and RPMI 8226 cells. However, BB-94, a hydroxamate-based, broad-spectrum, metalloproteinase inhibitor, substantially suppressed constitutive and PMA-stimulated syndecan-1 loss from myeloma cells. These data indicate that a non-matrix-type metalloproteinase is responsible for syndecan-1 shedding from the surface of myeloma cells.     

Expression of IL-6 and IL-6 receptors by circulating clonotypic B cells in multiple myeloma: potential for autocrine and paracrine networks

OBJECTIVE: To investigate the participation of clonotypic MM B cells in the IL-6 network in patients with multiple myeloma. METHODS: CD19(+) B cells from 45 patients with multiple myeloma and from 18 healthy donors were sorted and their expression of IL-6, IL-6 receptor (CD126) characterized by flow cytometry, in situ RT-PCR, and ELISA measurement of IL-6 and soluble IL-6R. Expression of CD31 was detected by flow cytometry. RESULTS: Interleukin-6 (IL-6) is a pleiotropic cytokine often overexpressed in multiple myeloma (MM). IL-6 induces growth and inhibits apoptosis of MM plasma cells, and upregulates the activity of osteoclasts. MM plasma cells, the most mature component of the MM clone, secrete IL-6 and induce IL-6 production from other cell types. However, the MM clone also includes circulating clonotypic B lymphocytes. Using ELISA and in situ RT-PCR we demonstrate here that, unlike the healthy control B cells, MM B cells express IL-6 mRNA and secrete IL-6 protein. In vitro, MM B cells were the major producers of IL-6 in peripheral blood mononuclear cells. On average, 50% of MM B cells express the IL-6 receptor (IL-6R, CD126), suggestive of autocrine stimulation. They also express CD31, potentially facilitating their paracrine interactions with osteoclast precursors. CONCLUSION: Secretion of IL-6 by circulating clonotypic B cells in MM may contribute to the autocrine and paracrine cytokine networks that maintain the malignant clone and are responsible for disruption of normal bone metabolism in this incurable disease.     

Soluble interleukin-6 receptor (sIL-6R), a new prognostic factor in multiple myeloma

sIL-6R is a 55 kD soluble molecule mediating the interleukin-6 (IL-6) signal through the IL-6 receptor-associated transmembrane signal transducer, gp130. It has recently been suggested that sIL-6R serum levels may reflect disease severity in multiple myeloma (MM). We determined sIL-6R serum levels in 25 normal controls (NC) and in 80 MM patients at diagnosis and during the course of the disease. Measurements were done by ELISA. In NC, sIL-6R levels ranged from 14 to 40 ng/ml (median 28 ng/ml) whereas in MM patients the range was 10–200 ng/ml (median 38 ng/ml) ( P  < 0.01). 61 patients entered remission and 19 were resistant. Median sIL-6R value at diagnosis was 36 ng/ml (10–120) in responding patients, and 82 ng/ml (20–200) in non-responding patients ( P  < 0.001). During a follow-up from 12 to 89 months, sIL-6R values remained more or less stable in most patients. High sIL-6R levels correlated with poor survival.     

Cytokine stimulated vascular cell adhesion molecule-1 (VCAM-1) ectodomain release is regulated by TIMP-3

OBJECTIVES: Vascular cell adhesion molecule-1 (VCAM-1) is a cell surface adhesion molecule involved in the recruitment of leukocytes to endothelial cells on arterial walls during the pathogenesis of atherosclerosis. The soluble ectodomain of VCAM-1 (sVCAM-1) is proteolytically released from the cell surface into the circulation, a process which is up-regulated in patients with cardiovascular or inflammatory disease. Here we investigate mechanisms involved in sVCAM-1 generation in response to cytokine stimulation. METHODS: VCAM-1 ectodomain release into the conditioned media of MCEC-1 murine endothelial cells and cells grown from primary aortic explants from timp3-/- mice and wild-type littermates was measured by sandwich ELISA and Western blot after stimulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), or the phorbol ester PMA. Protease expression was inhibited (knocked down) with siRNA and validated using real-time PCR. RESULTS: Proinflammatory cytokines IL-1beta and TNFalpha up-regulated VCAM-1 ectodomain release from the MCEC-1 cells, and this was dependant on p38 and mitogen-activated protein kinases (MAP kinases) and inhibited by the matrix metalloproteinase (MMP) inhibitor BB94 and tissue inhibitor of metalloproteinase (TIMP)-3, but not TIMP-1 or TIMP-2. Timp-3-/- cells exhibited greater VCAM-1 ectodomain release following cytokine stimulation than TIMP-3-expressing cells. Additionally, cytokine stimulation of MCEC-1 cells was shown to cause down-regulation of TIMP-3 expression. Knockdown of the metalloproteinase ADAM17, but not ADAM10 or ADAM12, gene expression reduced cytokine-stimulated VCAM-1 shedding. CONCLUSIONS: TIMP-3 regulates the release of sVCAM-1 from cytokine-stimulated endothelial cells, which is mediated by ADAM17.     

Oestrogens prevent the increase of human serum soluble interleukin-6 receptor induced by ovariectomy in vivo and decrease its release in human osteoblastic cells in vitro

OBJECTIVE: Interleukin-6 (IL-6) seems to be a key mediator of the increased bone loss that follows loss of ovarian function. Based on this and on evidence that oestrogen deficiency may also increase cell sensitivity to IL-6, we studied the effects of ovariectomy and of oestrogen replacement therapy on the serum levels of IL-6 and of soluble IL-6 receptor (sIL-6R) in vivo. DESIGN AND PATIENTS: Thirty-seven fertile women undergoing surgery for benign uterine diseases were divided into 3 groups and monitored for 12 months: hysterectomized women (n = 9), ovariectomized untreated women (n = 12) and ovariectomized women starting treatment with transdermal estradiol (E2, 50 microg/d) 1 month after surgery (n = 16). RESULTS: Hysterectomy alone caused no significant changes of sIL6R whereas serum levels of sIL-6R rose progressively after ovariectomy (mean +/- SEM: 31 +/- 9% and 38 +/- 7% over baseline, at 6 and 12 months, respectively; P < 0.01). Oestrogen replacement therapy prevented the increase of sIL6R over a 1-year period. A similar pattern was also found for serum IL-6 but the changes did not reach statistical significance. In ovariectomized (OVX) women there were significant correlations between serum sIL-6R levels and FSH (r = 0.59; P < 0. 01), oestradiol (r = - 0.43; P < 0.01), testosterone (r = - 0.41; P < 0.05), osteocalcin (r = 0.42; P < 0.05) and bone alkaline phosphatase (r = 0.44; P < 0.05). To examine whether oestrogen directly regulates sIL-6R secretion by bone cells, we studied in vitro the basal and phorbol ester (PMA) stimulated release of sIL-6R in a human osteoblastic cell-line (MG-63) and in a tumour-derived osteoclastic cell line (GCT-51). Osteoblastic (but not osteoclastic) cells spontaneously produced considerable amounts of sIL-6R and the protein kinase-C activator PMA (10-8 M) increased the release of sIL-6R by osteoblasts more than 3-fold. More strikingly, 17beta E2 (but not 17alpha) significantly inhibited both the spontaneous- and PMA-induced release of sIL-6R by osteoblastic cells (P < 0.05). CONCLUSIONS: These results indicate that oestrogen loss causes alterations of the IL-6 system, and that sIL-6R is under the direct inhibitory control of oestrogens both in vivo and in vitro.     

The soluble form of interleukin-6 receptor modulates cell proliferation by acute myeloblastic leukemia blast cells

 As interleukin-6 (IL-6) has been shown to have diverse effects on blast cell growth in acute myeloblastic leukemia (AML), and as a soluble (s) form of IL-6 receptor (IL-6R) agonizes IL-6 effects in many cell types, we investigated whether sIL-6R was able to modulate clonogenic blast cell growth in AML. The proliferation responses of eight autonomously growing AML cell lines and eight primary AML blast cell samples were compared with their IL-6 and sIL-6R expression. Only three of the 16 AML samples were influenced by IL-6, two of them being stimulated and one inhibited by it. The sIL-6R-induced responses were more frequent, however, and, in contrast to those by IL-6, always stimulatory: clonogenic cell growth in six of the 16 AML samples was stimulated by sIL-6R treatment. All the cell lines and four of the seven primary blast cell samples analyzed expressed IL-6, and the expression was associated with unresponsiveness to exogenous IL-6. sIL-6R was also frequently expressed by AML cells: only one of the samples was negative for it. However, there was no correlation between sIL-6R expression and the responsiveness of cells to exogenous sIL-6R. The work presented here shows that sIL-6R is able to stimulate blast cell growth in AML. As AML blast cells are provided by exogenous IL-6 and sIL-6R in a bone marrow environment, and as many of them also express IL-6 and sIL-6R themselves in vitro, it is possible that signaling through the IL-6/sIL-6R system plays a role in maintaining their growth also in vivo. Received: February 20, 1998 / Accepted: November 26, 1998     

An intermediate-risk multiple myeloma subgroup is defined by sIL-6r: levels synergistically increase with incidence of SNP rs2228145 and 1q21 amplification

IL-6 signaling can be enhanced through transsignaling by the soluble IL-6 receptor (sIL-6r), allowing for the pleiotropic cytokine to affect cells it would not ordinarily have an effect on. Serum levels of sIL-6r can be used as an independent prognostic indicator and further stratify the GEP 70-gene low-risk group to identify an intermediate-risk group in multiple myeloma (MM). By analyzing more than 600 MM patients with ELISA, genotyping, and gene expression profiling tools, we show how the combination of 2 independent molecular genetic events is related to synergistic increases in sIL-6r levels. We also show that the rs2228145 minor allele is related to increased expression levels of an IL-6r splice variant that purportedly codes exclusively for a sIL-6r isoform. Together, the SNP rs2228145 minor allele C and amplification of chromosome 1q21 are significantly correlated to an increase in sIL-6r levels, which are associated with lower overall survival in 70-gene low-risk disease, and aid in identification of the intermediate-risk MM group.     

Malignancy: Identification of Predictors of Disease Status and Progression in Patients with Myeloma (MM)

Multiple studies have attempted to recognize the best markers of disease activity and outcome in myeloma (MM). Our objective was to identify the best variables that can reflect MM disease status. Design and methods: The data obtained from all the following tests were included in the analysis: serum levels of the 2 growth factors known to be crucial for MM growth (i.e. IL-6, and sIL-6R), routine peripheral blood data (Hb%, serum calcium, albumin, CRP, B2m, LDH) and bone marrow plasma cell (BMPC)%, as well as the age and sex of patients. The study was conducted on 21 cases of MM under chemotherapy (aged 48-74 years; M/F = 13/8) and 12 matched normal individuals. The patients were categorized into 2 groups according to their clinical status: Group#1 (n = 16; cases in plateau/stable phase), and Group#2 (n = 5; advanced/refractory cases). Results: Student t-test confirms that serum IL-6 and sIL-6R are the most statistically different variables upon comparing cases in plateau phase (Group#1) with those of advanced disease (Group#2). Stepwise discriminant analysis of data has resulted in a function that is composed of the 2 most salient variables (i.e. serum IL-6, sIL-6R). The proposed function was highly significant (p = 0.0000) with Wilk's Lambda = 0.02538. The diagnostic capability of the proposed function was very high (percent of grouped cases that were classified correctly= 100%). Conclusion: measurement of serum IL-6 and sIL-6R gives the best prediction of disease activity in patients with MM.     

Soluble interleukin-6 (IL-6) receptor with IL-6 stimulates megakaryopoiesis from human CD34(+) cells through glycoprotein (gp)130 signaling

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.     

Interleukin-6 receptor shedding is enhanced by interleukin-1beta and tumor necrosis factor alpha and is partially mediated by tumor necrosis factor alpha-converting enzyme in osteoblast-like cells

OBJECTIVE: Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process. METHODS: Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding. RESULTS: IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells. This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA. Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels. CONCLUSION: IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation.     

Soluble gp130 is up-regulated in the implantation window and shows altered secretion in patients with primary unexplained infertility

Members of the IL-6 family of cytokines, which includes leukemia inhibitory factor (LIF) and IL-11, play important roles in implantation. The activity of these cytokines is modified by soluble receptors such as the IL-6 receptor (sIL-6R). gp130 is a signal transduction molecule common to the receptor complexes of this family, and its soluble form (sgp130) antagonizes their actions. The purpose of this study was to determine whether secretion of IL-6, LIF, sIL-6R, and sgp130 was different in the endometrium of women with primary unexplained infertility compared with normal fertile women. Endometrial biopsies were taken between d LH+6 and +13 and cultured in serum-free medium for 4 h. Secretion of IL-6, LIF, sIL-6R, and sgp130 was measured in the supernatant by ELISA. We also measured the secretion of IL-6, sIL-6R, and sgp130 by endometrial biopsies taken throughout the menstrual cycle in normal fertile women. Secretion of sgp130 increased 20-fold between d 20 and 26 of the cycle, coinciding with the implantation window (proliferative phase, median, 27.0 pg/ml.mg; range, 23-36; d 20-26, median, 501.5 pg/ml x mg; range, 26.1-1344; P = 0.03). RT-PCR showed that none of the known splice variants of gp130 were present in endometrium, indicating that sgp130 is produced by proteolytic cleavage of the membrane-bound form. IL-6 secretion varied considerably between patients and was greatest during the secretory phase and at menstruation. No significant change was seen in sIL-6R during the cycle. Between LH+6 and +13, secretion of sgp130 was significantly reduced in the infertile group (median, 93.1 pg/ml.mg; range, 28.5-256; compared with the fertile group, median, 223 pg/ml x mg; range, 63-534; U-statistic = 37; P = 0.017). Secretion of IL-6, LIF, and sIL-6R did not differ between the two groups. Immunolocalization of gp130, IL-6R, and the LIF receptor showed that the glandular epithelium and also endothelial cells are targets for IL-6 and LIF. These findings show that during a normal menstrual cycle, sgp130 secretion is greatly increased between d LH+6 and +13, due to proteolytic cleavage of membrane-bound gp130. Infertile patients show reduced secretion of sgp130 compared with fertile controls during this period, which coincides with the implantation window.     

ADAM-9 (MDC-9/meltrin-gamma), a member of the a disintegrin and metalloproteinase family, regulates myeloma-cell-induced interleukin-6 production in osteoblasts by direct interaction with the alpha(v)beta5 integrin

ADAM-9, a member of the a disintegrin and metalloproteinase family, contains both metalloproteinase and disintegrin domains. Myeloma cell lines express ADAM-9; however, its function and role in the pathophysiology of multiple myeloma is unknown. The aim of this study was to establish whether primary myeloma cells express ADAM-9, whether ADAM-9 regulates IL-6 production in human osteoblasts (hOBs), whether ADAM-9 interacts with specific integrin heterodimers, and the identity of downstream signaling pathways. Primary myeloma cells demonstrated increased expression of ADAM-9 (P < .01). ADAM-9 promoted a 5-fold increase in IL-6, but not IL-1beta mRNA, and a dose- and time-dependent increase in IL-6 production by hOBs (P < .01). IL-6 induction was inhibited by an antibody to the alpha(v)beta5 integrin (P < .01) but not by antibodies to other integrin heterodimers. ADAM-9 was shown to bind directly to the alpha(v)beta5 integrin on hOBs. Antibodies to ADAM-9 and alpha(v)beta5 integrin inhibited myeloma cell-induced IL-6 production by hOBs (P < .01). Furthermore, inhibitors of p38 MAPK and cPLA2, but not NF-kappaB and JAK2, signaling pathways inhibited ADAM-9-induced IL-6 production by hOBs (P < .01). These data demonstrate that ADAM-9, expressed by myeloma cells, stimulates IL-6 production in hOBs by binding the alpha(v)beta5 integrin. This may have important consequences for the growth and survival of myeloma cells in bone.     

Influence of humanized anti-IL-6R antibody, tocilizumab on the activity of soluble gp130, natural inhibitor of IL-6 signaling

Tocilizumab, humanized anti-IL-6R antibody is a novel anti-rheumatic drug. In the present study, we examined the influence of tocilizumab on the inhibitory activity of soluble gp130 (sgp130) against IL-6 signaling. BAF-h130 cells, human gp130 transfected mouse pro-B cell line, were cultured with the mixture with IL-6, sIL-6R and sgp130 for 72 h. BAF-h130 cells proliferated by the addition of both IL-6 and sIL-6R, but not IL-6 or sIL-6R alone. The proliferation induced by IL-6/sIL-6R complex was inhibited by sgp130 concentration-dependently. Tocilizumab inhibited IL-6/sIL-6R-induced cell proliferation. On the other hand, it did not affect the suppressive property of sgp130. To examine if tocilizumab can dissociate IL-6/sIL-6R/sgp130 complex, we established an ELISA system to detect IL-6/sIL-6R/sgp130 complex using anti-IL-6R coated ELISA plate. The results clearly indicated that ELISA system established was detectable IL-6/sIL-6R/sgp130 complex in a concentration dependent manner. Tocilizumab was added to IL-6/sIL-6R/sgp130 complex-fixed plate and then remaining IL-6/sIL-6R/sgp130 complexes on the plate were measured. The amount of IL-6/sIL-6R/sgp130 complexes on the plate was not changed by the addition of tocilizumab. In conclusion, tocilizumab exerts its inhibitory effect through the inhibition of IL-6R directly without affecting natural inhibitor sgp130.     

beta-lapachone,a novel plant product,overcomes drug resistance in human multiple myeloma cells

OBJECTIVE: To evaluate the anti-tumor potential of beta-lapachone in multiple myeloma (MM) cell lines (U266, RPMI8226, and MM.1S); MM cell lines resistant to dexamethasone (MM.1R), melphalan (RPMI8226/LR5), doxorubicin (RPMI8226/DOX40), and mitoxantrone (RPMI8226/ MR20); and MM cells from patients (MM1-MM4). MATERIALS AND METHODS: Cytotoxicity of beta-lapachone was assessed by MTT and [3H]-thymidine uptake assays. Apoptosis was analyzed using propidium iodide staining, DNA fragmentation, TUNEL assay, caspase-9 colorimetric assay, and immunoblotting for caspase-3, poly (ADP-ribose) polymerase (PARP), and caspase-8 cleavage products. Paracrine growth of MM cells was assessed by [3H]-thymidine uptake in cultures of bone marrow stromal cells (BMSCs) and MM cells. Interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion in the culture supernatants was measured by specific enzyme-linked immunosorbent assays (ELISAs). RESULTS: beta-lapachone showed significant cytotoxicity in MM cells (IC(50): 4-8 microM). In contrast, normal peripheral blood mononuclear cells (PBMCs) and BMSCs from MM patients were relatively resistant (IC(50): 8-16 microM). IL-6 did not protect against beta-lapachone-induced apoptosis in MM.1S cells, and dexamethasone showed additive cytotoxicity. beta-lapachone also decreased binding of MM.1S cells to BMSCs; abrogated IL-6 and VEGF secretion triggered by adhesion of BMSCs to MM.1S cells; reduced proliferation of MM.1S cells adherent to BMSCs; and decreased intracellular adhesion molecule-1 (ICAM-1) expression on MM.1S cells. Furthermore, beta-lapachone induced typical PARP cleavage, increased caspase-9 proteolytic activity, and activation of caspase-3, without activation of caspase-8 in U266 cells. CONCLUSION: These studies provide a framework for clinical evaluation of beta-lapachone to improve the outcome for patients with MM.     

Clinical significance of elevated soluble interleukin-6 receptor levels in the sera of patients with plasma cell dyscrasias

Summary .We investigated the clinical significance of the serum soluble interleukin-6 receptor (sIL-6R) in 42 patients with plasma cell dyscrasias (27 with multiple myeloma (MM), 13 with monoclonal gammopathy of undetermined significance (MGUS), and two with plasma cell leukaemia (PCL)). Serum levels of sIL-6R in normal individuals were 77 ± 21 ng/ml (mean ± SD, n = 18); those in patients with MGUS and with MM were elevated (102 ± 33 ng/ml, mean ± SD, P < 0–05 and 126 ± 60ng/ml, mean ± SD, P < 0–01, respectively). Significant correlations were not found between the serum levels of sIL-6R and known prognostic factors (C-reactive protein, haemoglobin levels, calcium, creatinine, β₂-microglobulin, amounts of M-protein, or percentages of plasma cells in bone marrow). Elevated serum sIL-6R did not affect the survival of the patients with MM. Serial measurements of sIL-6R together with the clinical course of patients with plasma cell neoplasias revealed a good correlation between the sIL-6R level and disease activity. We conclude that sIL-6R can be used as a clinical factor correlated with the disease activity, at least in some patients with plasma cell neoplasias.     

Incidence of apoptosis and cell proliferation in multiple myeloma: correlation with bcl-2 protein expression and serum levels of interleukin-6 (IL-6) and soluble IL-6 receptor

We evaluated the in vivo incidence of apoptosis and cell proliferation in multiple myeloma (MM) and investigated the correlation of both cellular events with histological tumour stage and grade, bcl-2 protein expression, serum IL-6 and sIL-6R. MATERIAL AND METHODS: The TUNEL method was used to assess apoptosis and immunohistochemistry to assess the expression of proliferating cell nuclear antigen (PCNA) and bcl-2 protein in 30 bone marrow biopsy specimens. The apoptotic index (AI) and proliferative index (PI) were defined as the percentage of TUNEL and PCNA positive plasma cells, respectively. RESULTS: The mean AI was 0.162% and the mean PI 27.44%. A positive correlation between AI and PI was found (r = 0.44, P = 0.017). PI was also correlated with tumour grade (P = 0.015). The mean bcl-2 protein expression was 70% and did not correlate with AI or PI, but was higher in specimens taken at first diagnosis than in specimens taken after response to treatment (P = 0.035). The mean serum IL-6 and sIL-6R values were 9.43 pg mL-1 and 47.27 ng mL-1, respectively. These parameters did not correlate with AI or PI. CONCLUSIONS: The results indicate that MM might be among the malignancies with very low incidence of apoptosis. Proliferative activity increased in parallel with tumour histological grade. A positive correlation between apoptosis and proliferation was observed, but the incidence of these two cellular events seems not to be related to the bcl-2 protein expression and the serum levels of IL-6 and sIL-6R.     

Interleukin-6 is expressed by plasma cells from patients with multiple myeloma and monoclonal gammopathy of undetermined significance

Interleukin-6 (IL-6) is an important growth factor for human myeloma cells in vitro and in vivo . However, the identity of the cells producing IL-6 in vivo in patients with multiple myeloma (MM) remains the subject of debate. We have developed a sensitive dual-colour fluorescence in situ hybridization (FISH) technique to investigate the expression of IL-6 mRNA by individual bone marrow plasma cells from patients with multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS) and healthy subjects. IL-6 mRNA could be identified in all immunoglobulin light chain (IgLC) expressing cells from all patients with MM and MGUS. The IL-6 protein could also be detected by direct immunofluorescence in all plasma cells (cytoplasmic light chain positive) from all patients with MM and MGUS. Furthermore, it was also possible to demonstrate cytoplasmic IL-6 staining of plasma cells from patients with MM by flow cytometric analysis. In contrast, neither the IL-6 mRNA or protein could be detected in normal plasma cells from healthy bone marrow donors. These data demonstrate that plasma cells from patients with MM and MGUS express the IL-6 mRNA and synthesize the IL-6 protein and support the hypothesis that autocrine synthesis of IL-6 is of importance in patients with MM.