老年多发性骨髓瘤患者血清白细胞介素—2的检测及临床意义

目的检测老年多发性骨髓瘤（ＭＭ）患者血清白细胞介素－２（ＩＬ－２）含量，分析其临床意义。方法用固相放射免疫分析（ＲＩＡ）法检测４４例初诊老年ＭＭ患者、２０例健康老年人和４０例健康青中年人血清ＩＬ－２含量，随访患者３年的存活状况。结果ＭＭ患者血清ＩＬ－２为８．１１±２．５４μｇ／Ｌ，显著高于老年健康对照组的２．６８±０．６１μｇ／Ｌ（Ｐ＜０．０１）。血清ＩＬ－２≥５μｇ／Ｌ的患者３年生存率显著提高。血清ＩＬ－２与血清β２微球蛋白（β２－ＭＧ）有关，血清ＩＬ－２≥５μｇ／Ｌ的患者，β２－ＭＧ皆＜６ｍｇ／Ｌ。结论血清ＩＬ－２的检测可作为估计ＭＭ预后的一项指标。     

N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation

Background

Multiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow microenvironment plays a critical role in sustaining the growth and survival of myeloma cells during tumor progression. Identification and functional analysis of the (adhesion) molecules involved in this interaction will provide important insights into the pathogenesis of multiple myeloma.

Design and Methods

Multiple myeloma cell lines and patients’ samples were analyzed for expression of the adhesion molecule N-cadherin by immunoblotting, flow cytometry, immunofluorescence microscopy, immunohistochemistry and expression microarray. In addition, by means of blocking antibodies and inducible RNA interference we studied the functional consequence of N-cadherin expression for the myeloma cells, by analysis of adhesion, migration and growth, and for the bone marrow microenvironment, by analysis of osteogenic differentiation.

Results

The malignant plasma cells in approximately half of the multiple myeloma patients, belonging to specific genetic subgroups, aberrantly expressed the homophilic adhesion molecule N-cad-herin. N-cadherin-mediated cell-substrate or homotypic cell-cell adhesion did not contribute to myeloma cell growth in vitro. However, N-cadherin directly mediated the bone marrow localization/retention of myeloma cells in vivo, and facilitated a close interaction between myeloma cells and N-cadherin-positive osteoblasts. Furthermore, this N-cadherin-mediated interaction contributed to the ability of myeloma cells to inhibit osteoblastogenesis.

Conclusions

Taken together, our data show that myeloma cells frequently display aberrant expression of N-cadherin and that N-cadherin mediates the interaction of myeloma cells with the bone marrow microenvironment, in particular the osteoblasts. This N-cadherin-mediated interaction inhibits osteoblast differentiation and may play an important role in the pathogenesis of myeloma bone disease.     

Hypercalcaemia and increased serum interleukin-6 levels induced by all-trans retinoic acid in patients with multiple myeloma

Summary. All-trans retinoic acid (ATRA) inhibits human myeloma cell growth in vitro, presumably through the down-regulation of interleukin 6 receptors (IL-6R). Based on these and other studies, we initiated a phase II clinical trial using ATRA in patients with advanced refractory multiple myeloma (MM). We report that three out of six treated patients developed severe hypercalcaemia following administration of ATRA, which was accompanied by a significant rise in serum IL-6 levels. Normal calcium levels were restored after the discontinuation of the drug and the administration of standard anti-hypercalcaemic care. We suspect that down-regulation of IL-6R resulted in increased serum IL-6 levels, leading to advanced bone resorption and hypercalcaemia. We conclude that the use of ATRA in patients with advanced MM is not warranted.     

Serum interleukin-6 has no discriminatory role in paraproteinaemia nor a prognostic role in multiple myeloma.

We determined interleukin-6 (IL-6) levels in the serum of 212 well-defined patients with newly diagnosed paraproteinaemia and evaluated its discriminatory value and prognostic role in multiple myeloma (MM). Results were compared with serum neural cell adhesion molecule and beta-2-microglobulin, both established prognostic MM markers. Paraproteinaemia-related diagnoses were: MM (60), other haematological diseases (46), solid tumours (35), autoimmune diseases (17) and monoclonal gammopathy of unknown significance (MGUS) (54). The range of IL-6 levels in all diagnostic groups overlapped widely and did not serve as a discriminatory marker in newly diagnosed paraproteinaemia even when patients with infection or fever (42) were excluded. In MM high IL-6 levels (>/= 50 pg/ml) were not associated with a shorter survival (P = 0.24). We compared our results with 20 published studies on serum IL-6 in paraproteinaemia and/or MM. IL-6 data have to be related to the assay used (bio- or immunoassay) and to the status of MM (newly diagnosed, during therapy, progressive disease). We conclude that serum IL-6 is not specific for paraproteinaemia-related diseases and will not serve as a reliable discriminatory or prognostic marker in paraproteinaemia and MM.     

Interleukin-6 serum levels in patients with multiple myeloma

Summary Studying the prognostic value of serum interleukin-6 (IL-6) levels in multiple myeloma, we observed important daily variations in some patients. Therefore a unique serum IL-6 measurement should be interpreted with caution and requires confirmation by multiple determinations performed over a period of several days.     

Interleukin-1 in multiple myeloma: producer cells and their role in the control of IL-6 production

We studied the role of interleukin (IL)-1β in patients with multiple myeloma. By in situ hybridization and immunochemistry, myeloid and megakaryocytic cells expressed high levels of the IL-1β gene and produced IL-1β. Myeloma cells less potently expressed the IL-1β gene and IL-1β protein. IL-1β gene expression was not constitutive since it was detected in the bone marrow myeloma cells of two patients, unlike circulating tumoural cells. In addition, nine myeloma cell lines failed to express the IL-1β gene and this expression could not be induced by 12 different cytokines. We demonstrated that IL-1 was mainly responsible for IL-6 production in the tumoural environment through a PGE₂ loop. In fact, an IL-1 receptor antagonist (IL-1RA) blocked PGE₂ synthesis and IL-6 production by 80%; this blockage could be reversed by adding synthetic PGE₂. Similar findings were found with indomethacin, an inhibitor of cyclooxygenase that blocks PGE₂ synthesis. Taken together, these data emphasize the possibility of blocking IL-1 by using IL-1RA or other antagonists in order to block IL-6 production, which is a major tumoural survival and proliferation factor.     

IL-6和DKK1在多发性骨髓瘤中表达的意义

目的：研究多发性骨髓瘤（MM）患者中IL-6、DKK1的表达水平,阐明其在MM发病中的临床意义。方法：采用酶链免疫和化学发光法对60例MM患者和50例对照者血清中IL-6、DKK1的表达量检测并进行比较。结果：IL-6、DKK1在对照者中表达低于MM患者,两者比较差异有统计学意义（P〈0.01）;MM患者中IL-6、DKK1的表达水平与临床分期高低有关（P〈0.01）;IL-6和DKK1表达有正相关关系（rs=0.7381,P〈0.01）。结论：IL-6和DKK1的联合检测可作为MM患者病变严重程度的早期诊断指标,为临床治疗提供帮助。     

Serum level of interleukin-16 in multiple myeloma patients and its relationship to disease activity

Interleukin-16 (IL-16) is a chemoattractant of CD4+ lymphocytes, and it has been implicated in the pathogenesis of various inflammatory diseases. There is evidence that it may have a role in multiple myeloma (MM). In the present study, we determined the serum level of IL-16 both before and after treatment of MM and related it to inflammatory markers and survival. Forty-eight newly diagnosed MM patients were included in the study. Disease stage was defined using the Durie-Salmon classification system (10 patients were in stage I, 19 in stage II, and 19 in stage III). After standard treatment, 22 patients reached the plateau phase and were re-evaluated. The following serum parameters were measured: IL-16, IL-6, alpha-1 antitrypsin (alpha1AT), and C-reactive protein (CRP). Survival was determined as the number of months elapsed since original diagnosis. The mean +/- SD of serum IL-16 was 343 +/- 195 pg/ml in the pre-treatment MM group and 101 +/- 30 pg/ml in the control group. All measured parameters were higher in the patient group compared to healthy controls. Furthermore, IL-16, IL-6, alpha1AT, and CRP were significantly increased with increasing stage of disease, from stage I to stage III (P<0.01). All parameters decreased significantly following effective chemotherapy (P<0.002). Patients with a high level of IL-16 (>430 pg/ml) displayed an inferior survival time in comparison to those with lower levels of IL-16. In the pre-treatment group, IL-16 correlated with alpha1AT and IL-6 (r=0.374, P<0.01 and r=0.454, P<0.002, respectively). IL-16 may play a role in multiple myeloma; however, further functional studies are required.     

Recombinant interferon-gamma inhibits the growth of IL-6-dependent human multiple myeloma cell lines in vitro.

Recombinant human IFN-gamma (100-1000 U/ml) inhibited the IL-6-induced growth of 2 human IL-6-dependent multiple myeloma (MM) cell lines U-1958 and U-266-1970 in vitro. In contrast, the U-1996 line, independent of IL-6 for maintenance at a slow growth rate but responding to IL-6 by increased proliferation, and the IL-6-independent U-266-1984 were refractory to the anti-proliferative effect of IFN-gamma. The effect of IFN-gamma in the sensitive MM cell lines was cytostatic in U-266-1970, and cytostatic and cytotoxic in U-1958. Northern blot analysis revealed that the growth inhibition of the IL-6-dependent MM cell line U-1958 was not due to down-regulation of IL-6 receptor mRNA expression and that the differential sensitivity to IFN-gamma was not due to differences in IFN-gamma receptor expression. The growth inhibition was not a consequence of an IFN-gamma-induced terminal differentiation as flow cytometric analyses demonstrated an arrest in all phases of the cell cycle. IFN-alpha inhibited the growth in 3 of the 4 cell lines tested. The results thus suggest that the particular MM phenotype, which includes IL-6 dependency for survival and growth, may also be characterized by IFN-gamma sensitivity. Furthermore, the study demonstrates that MM cell lines are not simultaneously sensitive to IFN-gamma and alpha, indicating that the mechanisms of action of the two types of IFN are distinct.     

Chimaeric anti-interleukin 6 monoclonal antibodies in the treatment of advanced multiple myeloma: a phase I dose-escalating study

Interleukin 6 plays a key role in the pathogenesis of multiple myeloma (MM). Therefore we conducted a phase I dose-escalating study with chimaeric monoclonal anti-IL6 antibodies (cMab) in MM patients resistant to second-line chemotherapy. The cMab (CLB IL6/8; K _d 6.25 × 10⁻¹²  M ) was given in two cycles of 14 daily infusions, starting on day 1 and day 28, repectively, with a daily dose of 5 mg in patients 1–3, 10 mg in patients 4–6, 20 mg in patients 7–9 and 40 mg in patients 10–12 (total dose 140 mg, 280 mg, 560 mg and 1120 mg of anti-IL6, respectively). 11/12 patients had elevated pretreatment IL6 levels.
Except for transient thrombocytopenia in two patients there was no toxicity. There were no changes in haemoglobin levels, granulocyte count, liver enzymes or renal function. No human anti-chimaeric antibodies were induced. This was also reflected in a long half-life time of the cMab (median 17.8 d), resulting in accumulation of the anti-IL6 cMab and high levels of circulating IL6. However, this was in the form of biologically inactive IL6/cMab complexes and did not result in acceleration of the disease. Although C-reactive protein (CRP) levels were decreased to below detection level in 11/12 patients, indicating effective IL6 blocking, none of the patients achieved a response according to the standard criteria. We conclude that this chimaeric anti-IL6 Mab has a low toxicity, low immunogenicity and a long T _1/2. A dose of 40 mg/d for 14 d can safely be used in future phase II studies.     

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.     

Human myeloma cells promote the production of interleukin 6 by primary human osteoblasts

Interleukin-6 (IL-6) is an important growth and survival factor for myeloma cells. However, the identity of the cells producing IL-6 in vivo remains unclear. Myeloma cells are found closely associated with sites of active bone turnover, and cells of the osteogenic lineage, including bone marrow osteoprogenitors, osteoblasts and bone lining cells, may therefore be ideally placed to synthesize IL-6. We have examined the possibility that human osteogenic cells may produce IL-6 in response to stimulation by myeloma cells. Primary human osteoblasts (hOBs) were isolated from normal donors, co-cultured with the human myeloma cell lines, JJN-3, RPMI-8226 and NCI-H929, and the amount of IL-6 released was determined by enzyme-linked immunosorbent assay (ELISA). All myeloma cells stimulated a significant increase in the production of IL-6 when cultured with hOBs (P < 0.05). Prior fixation of hOBs completely abrogated release of IL-6 in the co-cultures. In contrast, fixed myeloma cells retained the ability to induce IL-6 production, suggesting that hOBs were the principal source of IL-6. Physical separation of myeloma cells from hOBs using transwell inserts caused a partial inhibition of IL-6 release (P < 0.05), whereas the addition of media conditioned by myeloma cells to cultures of hOBs stimulated a significant increase in IL-6 production (P < 0.05). hOBs secreted greater amounts of IL-6 than human bone marrow stromal cells (hBMSCs) (2.2- to 3.5-fold, P < 0.05), but incubating hBMSCs with dexamethasone to stimulate osteoblastic differentiation resulted in an increase in their ability to produce IL-6 (1.7- to 4. 8-fold, P < 0.05) and to respond to myeloma cells (P < 0.05). These data clearly indicate that cells of the osteoblast lineage release significant amounts of IL-6 in response to stimulation by myeloma cells and may contribute to the IL-6 that promotes the proliferation and survival of myeloma cells in vivo.     

Fibrinolytic activity in multiple myeloma

The incidence of thromboembolic events is high as a result of disease, disease-related complications, and therapy in multiple myeloma (MM). In patients with hematologic tumors, impaired fibrinolysis may be present and may contribute to the development of thrombotic complications. Therefore, we designed a study to investigate fibrinolytic activity in MM. We compared plasma levels of interleukin (IL)-6, C-reactive protein (CRP), IL-1beta, IL-11, tissue plasminogen activator (tPA) activity, plasminogen activator inhibitor-1 (PAI-1) activity, and global fibrinolytic capacity (GFC) in patients with MM (n = 66) and in control subjects (n = 18). The prevalence of venous thromboembolism was 4.5%, with a median follow-up period of 7 months in our myeloma group. Results are given as mean (median, range). Plasma levels of IL-6 (8.27 +/- 0.74 [9.65, 0.90-13.32] pg/mL versus 2.64 +/- 0.66 [1.80, 0.10-11.86] pg/mL, P < 0.001), CRP (45.57 +/- 9.92 [21.00, 1.34-330.00] mg/L versus 1.96 +/- 0.50 [1.05, 0.19-8.03] mg/L, P < 0.001), PAI-1 (7.40 +/- 0.67 [5.57, 2.40-31.80] IU/mL versus 4.73 +/- 0.65 [3.60, 2.32-11.00] IU/mL, P < 0.01), GFC score (1.90 +/- 0.02 [2, 1-3] versus 2.50 +/- 0.14 [3, 1-3], P < 0.001) were increased compared with controls. In patients with MM, the level of IL-6 was positively correlated with CRP (r = 0.66, P < 0.001), IL-1beta (r = 0.29, P < 0.05), and PAI-1 (r = 0.35, P < 0.01) and negatively correlated with GFC (r = -0.37, P < 0.01). CRP level was positively correlated with plasma PAI-1 level (r = 0.40, P < 0.01) and negatively correlated with GFC (r = -0.44, P < 0.001). A significant negative correlation between PAI-1 level and GFC (r = -0.75, P < 0.001) was also detected. IL-1beta levels were negatively correlated with tPA level (r = -0.26, P < 0.05). These results suggest that patients with myeloma have a decreased fibrinolytic activity mainly because of increased PAI-1 activity. In MM, increased PAI-1 activity seems to be related with elevated IL-6 level. MM should be considered as a hypercoagulable state as a result of both increased procoagulant activity and decreased fibrinolytic activity. Achieving a plateau by means of conventional chemotherapies does not improve the decreased fibrinolytic activity.     

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.     

Activation of sphingosine kinase mediates suppressive effect of interleukin-6 on human multiple myeloma cell apoptosis

Interleukin 6 (IL-6) influences the growth and survival of multiple myeloma (MM) cells via the activation of multiple signalling cascades. Although sphingosine kinase (SPHK) signalling is known to play important roles in the regulation of cell proliferation and apoptosis, the role of SPHK activation in IL-6 signalling and in the pathology of MM remains unclear. This study found that IL-6 activated SPHK in MM cells, which mediates the suppressive effects of IL-6 on MM cell apoptosis. Both MM cell lines and primary MM cells constitutively expressed SPHK, and treatment of MM cells with IL-6 resulted in activation of SPHK in a concentration-dependent manner. Specific inhibitors of the phosphatidylinositol-3 kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways blocked the IL-6-induced activation of SPHK. It was further demonstrated that IL-6-induced activation of SPHK inhibited dexamethasone-induced apoptosis of MM cells. IL-6 stimulation or retroviral-mediated overexpression of SPHK1 in MM cells resulted in increased intracellular SPHK activity and upregulation of myeloid cell leukaemia-1 (Mcl-1), leading to increased cell proliferation and survival. Conversely, inhibition of SPHK1 by small interfering RNA reduced IL-6-induced upregulation of Mcl-1 and blocked the suppressive effect of IL-6 on MM cell apoptosis. Taken together, these results delineate a key role for SPHK activation in IL-6-induced proliferation and survival of MM cells, and suggest that SPHK may be a potential new therapeutic target in MM.     

Serum interleukin-6 (IL-6) and interleukin-4 (IL-4) in patients with multiple myeloma (MM)

In this study we determined, in patients with multiple myeloma (MM), serum levels of IL-4 and IL-6 at diagnosis and during the course of the disease, seeking a correlation with disease activity and prognosis. We studied 54 MM patients, 41 of whom responded to chemotherapy whilst 11 were resistant. At diagnosis, IL-6 was increased in 66% of patients (median 35.5 pg/ml) whereas IL-4 was low (median 4 pg/ml) in 75% of patients. In responding patients, IL-4 increased in remission (median 25 pg/ml), whereas IL-6 decreased (median 4 pg/ml). In chemotherapy-resistant patients, IL-6 and IL-4 values remained stable during the course of the disease.     

小白菊内酯对多发性骨髓瘤细胞蛋白酶体活性及白细胞介素-6表达的影响

目的探讨小白菊内酯(parthenolide,PTL)对多发性骨髓瘤(multiple myeloma,MM)细胞蛋白酶体活性及白细胞介素-6(interleukin-6,IL-6)表达的影响,以期了解PTL抗MM的分子机制。方法2006年5月至2007年3月华中科技大学同济医学院附属协和医院血液病研究所,体外培养人MM细胞系PRMI8266,与不同浓度的PTL作用不同时间。以荧光底物法检测细胞蛋白酶体活性,逆转录聚合酶链反应(RT-PCR)检测IL-6基因表达,酶联免疫吸附试验(ELISA)法检测MM细胞培养上清中IL-6的质量浓度。结果2~10μmol/L的PTL作用16h对PRMI8266细胞蛋白酶体的糜蛋白酶活性具有明显抑制作用,其效应呈现浓度依赖性;10μmol/L可达到接近50%的活性抑制。RT-PCR检测结果表明,2,5,10μmol/L的PTL作用24h,MM细胞的IL-6基因 mRNA表达强度与对照相比均明显降低;2,5,10μmol/L的PTL作用MM细胞48h后,培养上清中IL-6质量浓度分别为(92.6±4.3)ng/L、(67.1±5.7)ng/L、(43.5±4.9)ng/L,与对照组(148.7±8.2)ng/L相比,差异有显著性意义(P<0.01)。结论PTL能明显抑制PRMI8266细胞的蛋白酶体活性,降低IL-6基因表达,减少MM细胞IL-6的自分泌。提示PTL抑制蛋白酶体活性及IL-6表达可能是其抗MM的机制之一。     

Tumour necrosis factor-α and interleukin 4 promote the differentiation of myeloma cell precursors in multiple myeloma

Summary. The effects of tumour necrosis factor-α (TNF-α) and interleukin 4 (IL-4) on peripheral blood mononuclear cells (PBMC) from 36 patients with multiple myeloma (MM), 12 with monoclonal gammopathy of undetermined significance (MGUS) and 21 normal controls, were investigated. In 16/36 patients with MM, monoclonal plasma cells appeared after 4d in cultures containing TNF-α and IL-4. These changes were not observed in PBMC from patients with MGUS or from normal controls. These findings suggest that myeloma cell precursors do exist in the peripheral blood of MM patients and differentiate into plasma cells in the presence of TNF-α and IL-4. Based on these observations, we think that the variation in the number of myeloma cell precursors in peripheral blood could be used as a prognostic parameter of response to chemotherapy in myeloma patients. In addition, this assay may be useful to distinguish early-stage MM from MGUS.