The IL-6/sIL-6R fusion protein hyper-IL-6 promotes neurite outgrowth and neuron survival in cultured enteric neurons.

The undisturbed development of the enteric nervous system depends on the supply of various neurotrophic factors during ontogenesis. Besides glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) take part in its development. CNTF and LIF belong to the interleukin-6 (IL-6) family of cytokines. The combination of IL-6 and the soluble IL-6 receptor accelerates peripheral nerve regeneration. In this study, we examined the effect of the fusion protein Hyper-IL-6, which consists of IL-6 and the soluble receptor sIL-6R, on neurite outgrowth and neuronal survival in vitro. Myenteric plexus of newborn rats was dissected and dissociated. Cells were grown in either serum-free chemically defined medium alone or medium supplemented with sIL-6R, IL-6, sIL-6+IL-6, Hyper-IL-6, CNTF, LIF, or GDNF. Average neurite outgrowth per neuron was highest in GDNF-treated and Hyper-IL-6-treated cultures. The number of neurite-bearing neurons was reduced in GDNF cultures compared with Hyper-IL-6-treated cells, so that the total neurite outgrowth was maximal after Hyper-IL-6 stimulation. Hyper-IL-6 furthermore stimulated neuronal survival and morphologic differentiation of the enteric glia.     

Signal through gp130 activated by soluble interleukin (IL)-6 receptor (R) and IL-6 or IL-6R/IL-6 fusion protein enhances ex vivo expansion of human peripheral blood-derived hematopoietic progenitors

This study was designed to investigate the effects of a combination of soluble interleukin (sIL)-6 receptor (R) and IL-6 on the ex vivo expansion of human peripheral blood (PB)-derived hematopoietic progenitor cells in a short-term serum-free liquid suspension culture system, using PB-derived CD34(+)IL-6R(+/-) cells as a target. In combination with stem cell factor (SCF), IL-3, and sIL-6R/IL-6, the expansion efficiency (EE) for granulocyte/macrophage colony-forming unit (CFU-GM) reached a peak level on day 10 of incubation. On the other hand, the EE for erythroid burst (BFU-E) and mixed colony-forming unit (CFU-Mix) reached a peak level on day 7 of incubation. Among the cytokine combinations tested, SCF + IL-3 + sIL-6R/IL-6 + flt3 ligand (FL) most effectively expanded CFU-GM and CFU-Mix. The maximum EEs for CFU-GM and CFU-Mix were 208-fold and 42-fold, respectively. While the EE for BFU-E was 70-90-fold in the presence of SCF + IL-3 + sIL-6R/IL-6, FL significantly augmented the EE for CFU-GM and CFU-Mix. In contrast, thrombopoietin (TPO) significantly augmented the EE for CFU-Mix. Interestingly, in combination with IL-3 and SCF, newly generated IL-6R/IL-6 fusion protein (FP) expanded PB-derived BFU-E and CFU-Mix twice more effectively than a combination of sIL-6R and IL-6. These results demonstrated that human PB-derived committed progenitors were effectively expanded in vitro using sIL-6R/IL-6 or FP, in combination with IL-3, SCF and/or FL or TPO, and that FP may transduce a stronger intracellular signal than a combination of sIL-6R and IL-6.     

Potent antitumor activity of a tumor-specific soluble TCR/IL-2 fusion protein

We previously have generated a single-chain T cell receptor-cytokine fusion protein (264scTCR/IL-2) comprising interleukin-2 genetically linked to a soluble HLA-A2.1-restricted TCR recognizing a peptide of human p53 protein. In this report, we show that 264scTCR/IL-2 inhibits the growth of primary tumors derived from the A375 (p53+/HLA-A2.1+) human melanoma and exhibits significantly better antitumor activity than recombinant human IL-2 alone. Moreover, treatment with 264scTCR/IL-2 results in tumor growth retardation in mice bearing large A375 tumors and other p53+/HLA-A2.1+ human tumors but does not affect tumor outgrowth of HLA-A2.1-negative tumors. This suggests that antigen targeting plays a substantial role in the efficacy of 264scTCR/IL-2 against p53+/HLA-A2+ tumors. Further, the antitumor activity of 264scTCR/IL-2 was found to be likely mediated by NK cell activation and tumor infiltration. A biologically active chimeric version of the molecule (c264scTCR/IL-2) also exhibits favorable pharmacokinetic properties required of a clinical candidate for this novel class of potent antitumor activities and targeted anticancer immunotherapeutics.     

Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence that serum levels of soluble IL-6R are genetically influenced

We have investigated the association of the recently identified IL6R polymorphisms with the serum levels of soluble IL-6 receptor (sIL-6R). sIL-6R is generated by shedding of the membrane-bound receptor (IL-6Ralpha) or alternative mRNA splicing. In total, 115 healthy volunteers were genotyped, with 70 of them analyzed for sIL-6R levels. Using the PCR/RFLP methods, two important polymorphic sites were selected for genotyping: the 48892A/C (D358A) in exon 9 and the -183G/A in the promoter region. In exon 9, C allele carriers had higher sIL-6R level (P<0.0001) showing that this sequence variation, which corresponds to the proteolytic cleavage site of IL-6Ralpha, strongly influences the serum sIL-6R levels. In the promoter region, G allele carriers had lower sIL-6R levels (P<0.0082) compared with the A allele carriers. This could be attributed to the linkage disequilibrium (D'=0.54, chi2=51.3, P<0.0001) between the -183G/A and the 48892A/C gene polymorphisms.     

Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.     

IL-6/IL-6受体与类风湿关节炎关联性研究新进展

IL-6是一种多效性前炎症细胞因子,有多种生物活性,包括介导炎症反应,免疫反应等。IL-6在许多炎症性疾病中高表达,如RA,SLE,Crohn’s病等。IL-6的产生受多种因素的调节如NF-κB,Lin28,let-7 microRNA,IL6阳性反馈环,JunB等。IL-6受体(IL-6R)包括特异性IL-6Rα及IL-6Rβ即IL-6家族成员共有的信号转导蛋白gp130。IL-6可通过两种途径向细胞内传导信号:传统途径及反式信号途径,LMO4可参与反式信号途径的调节。IL-6/IL-6R在T、B细胞的发生中起重要作用。IL-6可抑制Th1、Treg的分化,促进Th2的分化;而且IL-6是CD4+T细胞分化为Th17细胞所必需;在B细胞中IL-6可诱导RAG基因的表达,促进抗体的产生。近年来越来越多的证据表明IL-6/IL-6R与RA存在密切关联性。RA患者血清及滑液中IL-6及sIL-6R的浓度升高,并且IL-6水平与疾病活性及临床表现密切相关,这可以解释RA患者的许多症状。因此可将IL-6信号转导途径相关的分子作为RA的治疗靶点,目前也研制出几种药物,其中之一为抗sIL-6R的人源化抗体Tocilizumab。临床试验中,该药可减轻RA患者的症状,降低疾病活性,可是也带来一定的副作用,这就限制了该药在临床中的应用。正在研制的另一种抗IL-6R的抗体可能为干预RA带来新的希望。     

人可溶性IL-6R及其突变体基因在COS7细胞中的表达及活性分析

目的 研究人可溶性ＩＬ－６Ｒ（ｈｓＩＬ－６Ｒ）的空间构效关系，为小分子拮抗设计提供理论依据。方法：首先利用ＰＣＲ技术扩增天然人ｓＩＬ－６Ｒ基因片段，然后将第２８０位Ｈｉｓ残基突变成Ｉｌｅ。天然基因和突变体基因分别转染ＣＯＳ７细胞，经ＥＬＩＳＡ和Ｗｅｓｔｅｒｂｌｏｔ检测证实转染细胞上清中的表达产物，并利用ＢｉｎｄｉｎｇａｓｓａｙＥＬＩＳＡ和生物学功能分析法检测表达产物与ＩＬ－６的结合能力以及它们所介     

IL-6 and soluble IL-6 receptors (sIL-6R and sgp130) in human pleural effusions: massive IL-6 production independently of underlying diseases

IL-6, soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) levels were measured in sera and pleural effusions from 42 patients with metastatic carcinoma, non-Hodgkin's lymphoma, tuberculosis, cardiac failure and miscellaneous diseases. Pleural IL-6 levels measured by ELISA were very high in all patient groups (mean 34.8 ± 15.3 ng/ml) without significant difference according to diseases. IL-6 was shown to be biologically active in a proliferative assay. Serum IL-6 levels were low (0.049 ± 0.014 ng/ml) and did not correlate with pleural fluid levels. Pleural IL-6 levels correlated with the number of polymorphonuclear cells in pleural fluid (P< 0.03). Pleural sIL-6R levels (76 ± 8 ng/ml) were always lower than serum levels (196 ± 12 ng/ml; P< 0.0001) but correlated with them (P< 0.01). Pleural sIL-6R and albumin levels correlated (P< 0.01), suggesting a transudation of sIL-6R from the serum. Pleural sgp130 levels (10.9 ± 1.0 ng/ml) were lower than serum levels (24.6 ± 2.8 ng/ml; P< 0.002). After gel filtration of pleural fluid, the bulk of IL-6 (>90%) was recovered in a 15 000–30 000 fraction, corresponding to the expected mol. wt of free IL-6. These results suggest a production and a sequestration of IL-6 in the pleural cavity in all studied conditions.     

The effect of interleukin-6 and soluble interleukin-6 receptor protein on the bone resorptive activity of human osteoclasts generated in vitro

The role of interleukin-6 (IL-6) in the regulation of bone resorption is and has not been studied using human tissue in vitro. This study exploits a recently described in vitro model, whereby osteoclasts, defined as cells that resorb bone, can be generated from human bone marrow, and investigated the effect of IL-6 and its soluble receptor on bone resorption, in the presence of 1,25-dihydroxyvitamin D₃[1,25(OH)₂ vitamin D₃]. Human bone marrow was cultured to form a confluent stroma, sedimented onto devitalized bone slices, and recharged with non-adherent bone marrow cells. 1,25(OH)₂ vitamin D₃ increased bone resorption, whereas IL-6 failed to induce a similar stimulatory effect. Both IL-6 at 100 ng/ml and soluble IL-6 receptor protein in the absence of exogenous IL-6 inhibited the stimulatory effect of 1,25(OH)₂ vitamin D₃. Bone resorption was never observed when non-adherent haemopoietic cells were cultured in the absence of stroma but in the presence of IL-6, which indicates that IL-6 cannot replace the stromal factor(s) required for the formation of cells capable of resorbing bone. These results suggest that IL-6 at high concentrations is not a critical cytokine in stimulating osteoclastic bone resorption.     

Serum soluble interleukin-6 receptor in MRL/lpr mice is elevated with age and mediates the interleukin-6 signal

The characteristics of soluble interleukin-6 receptor (sIL-6R) in murine sera were examined. To investigate a relationship between serum sIL-6R level and autoimmune diseases, quantitative analysis of serum sIL-6R in MRL/lpr mice was performed by an enzyme-linked immunosorbent assay. The serum sIL-6R level in MRL/lpr mice of both sexes was below the detection limit (< 1.0 ng/ml) at 8 weeks of age, but it increased in accordance with age and reached 42 ± 9.3 ng/ml in female and 31 ± 13 ng/ml in male mice at 30 weeks of age. In MRL/+ mice, although an age-associated increase in serum sIL-6R level was observed, it was much less extensive than that in MRL/lpr mice. Elevated serum sIL-6R level at the age of 30 weeks was observed in female and male (NZB × NZW)F1 mice (32 ± 10 ng/ml and 17 ± 5.0 ng/ml, respectively), and male BXSB/Mpj Yaa mice (42 ± 18 ng/ml), suggesting that elevated serum sIL-6R in aged mice is one of the characteristics of autoimmune-prone mice. Quantitative analysis of serum IL-6 in MRL/lpr revealed that the serum sIL-6R level correlated well with the serum IL-6 level. We also showed that sIL-6R in the sera from MRL/lpr mice could mediate the IL-6 functions through the IL-6 signal-transducing receptor component gpl30, suggesting that elevated production of sIL-6R may partly contribute to development of autoimmune disease in MRL/lpr mice.     

Endocytosis of interleukin-6—soluble interleukin-6 receptor complex and its intralysosomal degradation

Binding and internalization of interleukin-6—soluble interleukin-6 receptor complex by MDCK and MDCK-gp130 (transfected with gp 130 signal transductor) cells are studied. Binding of labeled complex depends on the concentration of interleukin-6; an effective internalization of the complex is shown. Binding and endocytosis of the complex are demonstrated in human hepatoma cells expressing interleukin-6 receptor and gp 130. These processes depend on the concentration of interleukin-6. The inhibitors of lysosomal functions ammonium chloride, monensin, and leupeptin suppress intralysosomal degradation of the complex, which confirms the important role of intralysosomal cleavage of the complex. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 527–529, November, 1997     

A competitive enzyme-linked immunoassay (ELISA) for the measurement of soluble human interleukin-2 receptors (IL-2R, Tac protein)

A solid-phase, competition enzyme-linked immunosorbent assay (ELISA) was established for the quantitative measurement of soluble (human) interleukin-2 receptors (IL-2R). The ladder of reagents from the solid phase up consisted of: (1) recombinant DNA-derived, purified IL-2R, (2) sample-containing soluble IL-2R and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody, 7G7/B6, directed against the IL-2R, (3) alkaline phosphatase-conjugated rabbit anti-FITC, and (4) substrate. This ELISA was compared with a 'sandwich' ELISA for soluble IL-2R. The competitive ELISA was less sensitive than the 'sandwich' assay, being capable of measuring 5000 versus 31 U/ml, respectively. While both anti-Tac and 7G7/B6 in the IL-2R-containing sample inhibited the 'sandwich' assay, only 7G7/B6 inhibited the competition assay. Anti-mouse immunoglobulin enhanced the 'sandwich' assay and inhibited the competitive assay; both effects could be overcome by the addition of normal mouse immunoglobulin in the sample buffer. Studies of a patient's serum receiving anti-Tac as therapy for the adult T cell leukemia demonstrated that rises in the level of IL-2R occurring with anti-Tac therapy, as measured with the competition assay, were masked in the 'sandwich' assay. This competition ELISA will be useful for measuring soluble IL-2R levels in patients receiving anti-Tac as therapy for various immunologic disorders.     

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12-o-tetradecanoylphorbol 13-acetate for 24–48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin(P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation. Received: 6 July 2002 / Accepted: 18 December 2002 Correspondence to Y. Kawano     

DHT对卵巢癌细胞IL-6、IL-8及其受体表达的调节作用

目的 初步探讨雄激素对卵巢癌细胞IL-6、IL-8及其受体表达的调节作用及作用机制.方法 选择兼有雄激素受体(AR)、IL-6和IL-8及其受体表达的卵巢癌细胞系SKOV-3和OVCAR-3作为研究模型,观察5a-二氢睾酮(DHT)对IL-6、IL-8及其受体表达以及NF-κB加转录的调节作用.结果 DHT可促进卵巢癌细胞IL-6、IL-8分泌及相应mRNA表达,并增强IL-6基因启动子的转录活性,DHT的上述作用可被AR阻断剂氟他胺完全阻断.DHT显著提高卵巢癌细胞NF-κB加亚单位p50、p65(RelA)mRNA的表达水平,其中对后者的作用与对IL-6、IL-8 mRNA的作用相平行.此外,DHT尚能调节IL-6、IL-8受体的表达.结论 雄激素可能通过NF-κB信号传导途径促进卵巢癌细胞IL-6、IL-8的分泌,同时对二者受体的表达也有一定的凋节作用.     

TGF-βRⅡ/Fc融合蛋白的纯化及其生物学活性鉴定

目的 :用重组Bac TRⅡ杆状病毒表达系统大量表达融合蛋白TGF βRⅡ /Fc,并对其进行纯化及鉴定 ;验证该融合蛋白是否能够阻断细胞因子TGF β1的生物学作用。 方法 :采用病毒空斑形成试验测定病毒滴度 ,并对其进行扩增。采用Pro teinG柱和快速蛋白质液相层析法 (FPLC)纯化目的蛋白。采用SDS PAGE分析纯化后的目的蛋白 ,并将蛋白电泳条带进行灰度扫描 ,计算目的蛋白的含量。采用Westernblot和夹心ELISA法 ,鉴定目的蛋白是否表达。采用MTT比色法 ,验证融合蛋白TGF βRⅡ /Fc能否阻断TGF β1对小鼠肺成纤维细胞 (L92 9)生长的作用。采用Westernblot技术 ,验证融合蛋白能否阻断TGF β1对L92 9细胞纤维黏连蛋白 (FN)生成的促进作用。结果 :本实验室构建并保存的重组Bac TRⅡ杆状病毒原液内病毒的滴度为 2× 10 12 pfu/L。蛋白电泳后灰度扫描结果表明 ,目的蛋白约占总蛋白的 10 %。夹心ELISA法和Westernblot分析证明目的蛋白已表达。融合蛋白TGF βRⅡ /Fc能够阻断TGF β1对L92 9细胞生长的抑制作用 ,以及对该细胞FN生成的促进作用。结论 :本室构建的重组Bac TRⅡ杆状病毒表达系统 ,能够表达融合蛋白TGF βRⅡ /Fc ,表达水平为 9mg/L。纯化得到的目的蛋白具有一定的生物学活性 ,可阻断TGF β1对L92 9细胞生长的