重组人白细胞介素-18表达载体的构建及其在大肠杆菌中的表达

目的克隆人白细胞介素 18(IL 18)基因 ,构建重组表达质粒 ,在大肠杆菌中表达重组人白细胞介素 18(rhIL 18) ,并对其生物学活性进行初步鉴定。方法利用RT PCR方法从人外周血单个核细胞中扩增出成熟肽编码区cDNA(496bp) ,克隆到表达载体pBV2 2 0质粒中 ,Western印迹证实rhIL 18在大肠杆菌DH5α中获得表达。经分子筛层析法初步纯化后 ,用溴化四唑蓝 (MTT)比色法测定复性的rhIL 18的体外活性。结果rhIL 18表达量占总菌体蛋白的 2 7%左右 ,经纯化得到纯度达 96 %以上的rhIL 18。实验显示rhIL 18协同IL 2 (10U/ml)诱导NK细胞细胞毒活性。结论rhIL 18具有较好的生物学功能 ,应予进一步的研究     

吲哚美辛对脂多糖引起的人类风湿性关节炎滑膜细胞中白介素-6表达的影响

目的　研究吲哚美辛对脂多糖引起的人类风湿性关节炎 (RA)成纤维状滑膜细胞 (FLS)中白介素 6 (IL 6 )表达的影响。方法　用放免分析法检测IL 6蛋白的表达水平 ;RT PCR方法测定IL 6mRNA的表达水平。结果LPS处理对FLS中IL 6表达无显著影响 ;LPS刺激的U937细胞培养液上清液可明显增强FLS中IL 6蛋白分泌及mRNA表达 ;吲哚美辛可显著抑制上述变化 ,且其抑制作用随浓度的增加而增强。结论　吲哚美辛可抑制LPS刺激的U937细胞培养上清液引起的FLS中IL 6的表达     

重组人白细胞介素-11对2,4-二硝基氟苯致小鼠接触性皮炎和炎症细胞因子水平的影响

目的观察重组人白细胞介素11(rhIL11)对接触性皮炎有无抗炎作用。方法采用2,4二硝基氟苯(DNFB)致小鼠耳部皮肤接触性皮炎试验模型,观察scrhIL110.375～1.5mg·kg-1·d-1,连续10d后皮肤炎症反应程度,同时采用放射免疫分析法测定血清中肿瘤坏死因子α(TNFα)和白细胞介素6(IL6)的水平,采用生物化学检测法测定血清一氧化氮(NO)的水平,采用免疫组化法测定皮肤中细胞间粘附分子(ICAM1)的表达。结果rhIL11可显著降低炎性细胞浸润,减轻炎症反应程度,并显著降低血清中TNFα,NO,IL6及皮肤中ICAM1的水平。结论rhIL11可抑制DNFB诱发的炎症反应,并明显降低血清中促发因子TNFα,NO,IL6等的水平及皮肤中ICAM1的表达。     

白介素-1受体相关激酶-2反义寡核苷酸对白介素-1和肿瘤坏死因子激活核因子-κB的不同影响

目的　研究白介素 1受体相关激酶 2 (IRAK 2 )在白介素 1(IL 1)与肿瘤坏死因子 (TNF)激活核因子 κB(NF κB)过程中的作用。方法　IRAK 2反义寡核苷酸转染人胚肾细胞 (2 93细胞 ) ,阻断IRAK 2的表达 ,用IL 1及肿瘤坏死因子刺激细胞后 ,用夹心ELISA方法检测NF κB活性。结果　以IRAK 2反义寡核苷酸预处理可明显减弱IL 1诱导的NF κB活性 ,此效应强度存在时间和浓度依赖性 ,但I RAK 2反义寡核苷酸对肿瘤坏死因子诱导的NF κB活性无影响。结论　IRAK 2反义寡核苷酸对IL 1和TNF激活NF κB有不同影响。IRAK 2在IL 1诱导的NF κB信号转导通路中起关键作用     

以GLP-1受体为靶点的药物筛选模型的建立及功能鉴定

目的以GLP-1受体为靶点,建立GLP-1类似物活性检测的细胞模型,为GLP-1类似物以及GLP-1受体激动剂的药物筛选提供一种简单可靠的评价方法。方法首先将人源GLP-1受体基因插入pEGFP-N3,构建真核表达载体pEGFP-GLP-1R,并转染至HEK293A细胞中,经G418压力筛选后获得稳定表达GLP-1R-GFP的293A细胞株,然后通过GFP荧光信号和Western blot检测GLP-1R-GFP融合蛋白在该细胞中的表达与分布;最后利用GLP-1类似物利拉鲁肽刺激细胞,均相时间分辨荧光法检测细胞内cAMP含量变化。结果成功构建了GLP-1R-GFP-293A稳转细胞株,GLP-1RGFP蛋白主要分布在细胞膜表面,该细胞对GLP-1类似物利拉鲁肽具有高灵敏度和产生cAMP的特异性反应。结论利用所构建的细胞模型,可对小分子和GLP-1类似物进行体外活性分析,为筛选GLP-1受体激动剂奠定了模型基础。     

重组人白细胞介素15表达载体的构建及其在大肠杆菌中的高效表达

目的 :获得重组人白细胞介素 1 5 (rhIL 1 5 )高效表达菌株。方法 :经细菌脂多糖 +γ干扰素活化的人外周血单个核细胞提取细胞总RNA ,用RT PCR方法扩增出编码人IL 1 5cDNA的基因片段 ,采用 pBV2 2 0表达载体 ,经DNA重组技术构建IL 1 5基因工程菌。结果 :核酸序列测定与预期一致 ,所表达的IL 1 5经SDS PAGE证明分子量约 1 5kD ,表达量占菌体总蛋白的 2 8% ,经CTLl2 细胞检测 ,表达产物粗提物 1∶1 0 0复性后效价可达到1 0 6IU /ml。结论 :构建的基因工程菌为IL 1 5高效表达菌株。     

rhIL-10对银屑病动物模型的治疗作用及免疫功能的影响

目的　研究重组人白细胞介素 10 (rhIL 10 )对银屑病动物模型的治疗作用以及免疫功能的影响。方法　用小鼠雌激素期阴道上皮模型、鼠尾鳞片表皮模型观察重组人白细胞介素 10的抗银屑病作用 ,并检测ConA诱导T淋巴细胞增殖、LPS诱导B淋巴细胞增殖、NK细胞活性及细胞因子(IFN γ、IL 2、IL 1、TNF α)水平。结果　rhIL 10 (5 ,2 0 ,80μg·kg-1·d-1,sc)对小鼠阴道上皮细胞有丝分裂有抑制作用 ,促进小鼠尾鳞片颗粒层的形成。对ConA诱导T淋巴细胞增殖反应及IL 2、IFN γ产生有抑制作用 ;对LPS诱导小鼠腹腔巨噬细胞产生IL 1、TNF α具有抑制作用 ;对NK细胞活性有抑制作用 ;对LPS诱导B淋巴细胞增殖有促进作用。结论　rhIL 10可能是通过抑制表皮细胞增殖与促进其分化 ,并参与抗炎与免疫调节过程而发挥治疗银屑病动物的作用     

Graves病甲亢患者细胞因子/可溶性受体测定及其意义

目的：研究Graves病（GD）甲亢患外周血细胞因子及其可溶性受体水平的变化，并探讨其在Graves病免疫病理中的意义。方法：应用夹心酶联免疫吸附法（ELISA）动态检测了40例GD外周血中白介素－2（IL－2）、白介素－15（IL－15）、可溶性白介素2受体（sIL-2R)、白介素－6（IL－6）和可溶性白介素6受体（sIL-6R，包括sgp130、spg80)水平变化，并且随访其中20例治疗后缓解。结果：与正常对照组相比，治疗前初发GD患外周血细胞因子及其可溶性受体水平异常改变，经治疗后细胞因子及其可溶性受体水平与临床转归密切相关，但细胞因子水平恢复滞后于临床缓解。结论：T细胞生长因子IL－2／IL－15的失平衡参与GD的免疫病理过程，可溶性受体sIL-2R和sIL-6R可作为其病情变化的监测指标。     

重组人白细胞介素-1受体拮抗剂对大鼠胶原性关节炎的治疗作用

目的　研究重组人白细胞介素 1受体拮抗剂 (recom binanthumaninterleukin1receptorantagonist, rhIL 1ra)对大鼠胶原性关节炎 (collagen inducedarthritis,CIA)的治疗作用。方法　SD大鼠随机分为 6组:正常对照组、模型组、rhIL 1ra3个剂量组和进口IL 1ra组;采用Ⅱ型胶原(CⅡ)乳剂皮内注射诱导大鼠CIA模型;每周测体重观察体重变化;足爪容积法测量大鼠足爪肿胀度;测定CⅡ的迟发性变态反应(DTH);ELISA法检测血清中抗Ⅱ型胶原抗体水平;同时进行病理组织学的检查。结果　CIA大鼠在致炎d10,出现关节红、肿,体重减轻,血清抗CⅡ抗体水平升高,关节病理可见滑膜增生,炎性细胞浸润,血管翳形成,软骨和骨的破坏等。rhIL 1ra(7 5, 30, 120mg·kg-1 )和阳性对照组anakinra(120mg·kg-1 )皮下注射,连续 7d,能明显抑制CIA大鼠足肿胀;rhIL 1ra(30, 120mg·kg-1 )皮下注射,连续 7d,可以明显减轻CⅡ诱发的迟发性变态反应 (DTH);另外,rhIL 1ra也可明显降低CIA大鼠血清中抗CⅡ抗体的水平;病理学检查表明,rhIL 1ra大剂量能明显减轻CIA大鼠关节滑膜炎症和滑膜增生,减轻血管翳和软骨破坏。结论　rhIL 1ra对CIA具有治疗作用。     

重组人红细胞生成素对维持性血液透析患者抗感染免疫功能的影响

目的　探讨重组人红细胞生成素 (r HuEPO)对维持性血液透析 (MHD)患者抗感染免疫功能的影响。方法　选取 17例MHD患者于静脉注射r HuEPO前和注射后取血清检测白介素 2 (IL 2 )、可溶性白介素 2受体 (sIL 2R)、白介素1(IL 1)、白介素 6 (IL 6 )、肿瘤坏死因子 ((TNF α)的含量和测血液中T淋巴细胞亚群的比例。另取 13例未注射r HuEPO治疗的MHD患者作为对照。结果　r HuEPO可提高MHD患者血清IL 2的含量、CD+ 4T细胞的比例、CD4 /CD8的比值 ;降低血清sIL 2R、IL 1和TNF α的含量。但对CD+ 8细胞比例、血清IL 6的含量没有明显的影响。结论　r HuEPO在改善贫血的同时可通过改变血清中细胞因子的含量、血中T细胞亚群的比例来增强机体的抗感染免疫功能。     

Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor

Aim:

Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists.

Methods:

HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z′-factor.

Results:

RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC₅₀ value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z′-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC₅₀=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells.

Conclusion:

A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.     

Anti-IL-6 receptor antibody increases blood IL-6 level via the blockade of IL-6 clearance, but not via the induction of IL-6 production

We explored the mechanism for the increase of blood IL-6 level after anti-IL-6 receptor (IL-6R) antibody injection. First, we examined whether anti-IL-6R antibody stimulates IL-6 production. Single injection of tocilizumab (anti-IL-6R antibody) in monkeys with collagen-induced arthritis (CIA) caused a marked increase in blood IL-6 and IL-6R levels, but did not increase IL-6 mRNA and IL-6R mRNA expression in liver, spleen, lymph nodes, synovium or whole blood 1, 3 and 7 days later. This suggests that tocilizumab did not induce IL-6 and IL-6R production. Second, we investigated whether anti-IL-6R antibody releases IL-6 from IL-6 complexes in the blood. When plasma from CIA monkeys was incubated with tocilizumab, the IL-6 concentration was not affected. Finally, we studied whether anti-IL-6R antibody affects the clearance of IL-6 from the blood. When MR16-1 (anti-mouse IL-6R antibody) was injected into IL-6-deficient mice continuously infused with human IL-6, blood human IL-6 levels significantly increased. These results suggest that the elevation of blood IL-6 after the administration of anti-IL-6R antibody is the result of inhibition of the clearance of IL-6 due to IL-6R blockade, and that it is not the result of induction of IL-6 production or release of IL-6 from complexes.     

pCEP4/hIL-17真核表达载体的构建及表达

目的构建pCEP4/hIL-17载体及在真核细胞中表达hIL-17/mFc融合蛋白;初步研究IL-17生物学特性。方法采用RT-PCR的方法克隆hIL-17CDS段基因序列;将测序正确的hIL-17序列插入pCEP4质粒构建pCEP4/IL-17真核表达载体,转染中华仓鼠卵巢(CHO)细胞后,筛选阳性表达细胞株;并用RT-PCR、ELISA和Western blot等法鉴定IL-17基因的mRNA和蛋白表达;流式细胞术分析纯化的蛋白对Raji细胞表达的hIL-17受体的结合能力;以体外实验验证其促炎症作用。结果成功构建了pCEP4/hIL-17重组载体,并在CHO细胞中稳定表达;所获得hIL-17重组蛋白能稳定结合Raji细胞上的IL-17受体;体外刺激HeLa细胞,能明显促进IL-6等炎症因子的分泌。结论稳定表达hIL-17重组蛋白的CHO细胞系的建立,为进一步研究hIL-17的生物学功能奠定了良好的基础。     

Anti-interleukin-6 therapy for Crohn's disease

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis and physiopathology of various chronic inflammatory conditions including Crohn's disease (CD). Among these cytokines, interleukin-6 (IL-6) must be especially important because increased serum concentrations of acute phase proteins, reduced level of serum albumin, and remarkable thrombocytosis are all well-explained by the increased level of IL-6. Moreover, IL-6 is capable of stimulating even IL-6 receptor (IL-6R) negative cells such as vascular endothelial cells when complexed to soluble form of IL-6R (sIL-6R), and serum level of IL-6 as well as sIL-6R has been demonstrated to increase during inflammation. To investigate the therapeutic potential of IL-6 signaling blockade for CD, anti-IL-6R monoclonal antibody (mAb) was introduced to various murine models of colitis. Anti-IL-6R mAb successfully prevented wasting disease and the development of macroscopic and histological lesions. It suppressed the accumulation of ICAM-1 positive and Mac-1 positive cells in the lamina propria (LP) and the expression of ICAM-1 and VCAM-1 by vascular endothelial cells. Expansion of colonic and splenic CD4(+) T cells was reduced as well as the colonic expression of tumor necrosis factor alpha (TNF-alpha), IL-1beta, and interferon gamma (IFN-gamma) mRNA without affecting the production of transforming growth factor beta (TGF-beta), IL-10, and IL-4 mRNA. The treatment also suppressed established colitis by inducing LP T cell apoptosis. These results strongly suggest that specific targeting of IL-6/sIL-6R pathway will be a promising new approach for the treatment of CD, and the clinical trial of humanized anti-IL-6R mAb is now under way.     

Proliferation of spleen cells in culture stimulated by 7-thia-8-oxoguanosine: evidence that both B- and T-cells are the targets of its action

7-thia-8-oxoguanosine (immunosine) is a nucleoside analog showing efficient antiviral activity in rodent models as a consequence of enhancement of the immune response. However, little is known about the mechanisms of its action. In this work the effect of immunosine on proliferation of mouse and rat splenocytes in culture was studied. It was found that the compound stimulated proliferation of lymphocytes in a dose-dependent manner without any additional stimuli. The effect is predominantly mediated by interleukin-2 (IL-2) as judged by increased IL-2 production, upregulation of IL-2 receptor alpha (IL-2R alpha) expression and by significant inhibition (60-75%) of cell proliferation by anti-IL-2R alpha monoclonal antibodies (mAbs). Immunosine also stimulated proliferation both of T- and B-cells purified by immunomagnetic sorting. The response of B-cells was much higher than that of T-cells. The stimulatory effect of immunosine on both lymphocyte subpopulations was further increased by the addition of enriched splenic antigen-presenting cells or purified dendritic cells. Proliferation of purified T-cells to immunosine was also significantly potentiated by an anti-alpha beta T-cell receptor mAb (R 73). All these data suggest that T-, B- and accessory cells in splenic cultures are the targets of immunosine action.     

Novel therapy for Crohn's disease targeting IL-6 signalling

The aetiology of Crohn's disease (CD) remains unknown, however, the importance of pro-inflammatory cytokines including IL-6 has been shown. IL-6 can transduce its signal into the cells lacking membrane-bound receptors by forming a complex with soluble IL-6R (sIL-6R). To pursue the therapeutic potential of IL-6 signalling blockade for CD, anti-IL-6R monoclonal antibody (mAb) was introduced to experimental colitis, and successfully prevented and treated intestinal inflammation by suppressing vascular adhesion molecules and by inducing lamina propria T cell apoptosis. Based on these results, the clinical trial of humanised anti-IL-6R mAb, MRA, was carried out. Eighty per cent of the patients given twice-weekly MRA had a significantly better clinical response rate as compared to 31% of the placebo-treated patients. The response rate of the every-4-weeks regimen was 42%. The incidence of adverse events was similar in all groups. These data strongly suggest a therapeutic potential of MRA for CD.     

Histamine influences the expression of the interleukin-6 receptor on human lymphoid, monocytoid and hepatoma cell lines.

Based on our earlier data on the enhancing effect of histamine on the action of interleukin-6 (IL-6), we have studied the molecular mechanisms of these interactions. The effect of histamine was investigated on the binding of 125I-IL-6 by B lymphoma cell line CESS, monocytoid cell line U937 and hepatoma cell line HepG2. Histamine increases the IL-6 binding by CESS cells and inhibits that by U937 and HepG2 cells. Using H1 receptor (cetirizin and loderix) and H2 receptor (cimetidine and ranitidine) specific antagonists, an H1-dependent stimulation of IL-6 binding by CESS cells was found. In contrast, down-regulation of IL-6 binding by histamine was clearly mediated through H2 receptors. On U937 cells, using a monoclonal antibody reacting with the 80 kd chain of the human IL-6 receptor, and H2-receptor mediated inhibition of IL-6 receptor expression was found by FACS analysis.     

Tocilizumab inhibits signal transduction mediated by both mIL-6R and sIL-6R, but not by the receptors of other members of IL-6 cytokine family

To characterize the biological activity of tocilizumab, a humanized anti-human interleukin-6 receptor (IL-6R) monoclonal antibody, we examined its binding activity to both soluble IL-6R (sIL-6R) and membrane bound IL-6R (mIL-6R) and its neutralizing activity to other IL-6 family cytokines. ELISA assay demonstrated that tocilizumab bound to sIL-6R and inhibited IL-6 binding to sIL-6R in a dose-dependent manner. The dissociation constant (Kd value) for IL-6R was determined to be 2.54+/-0.12 nmol/L by Scatchard analysis. In addition, tocilizumab had the ability to dissociate IL-6 and sIL-6R from their preformed complex. The immune complex of tocilizumab and sIL-6R did not transmit signaling. Moreover, tocilizumab suppressed the IL-6/sIL-6R complex-induced proliferation of human gp130-transfected cell, BAF-h130. In addition, tocilizumab had the ability to bind to human IL-6R expressing COS-7 cells and to suppress the growth of the IL-6-dependent myeloma cell line, KPMM2. Finally, to analyze the specificity of this antibody, the effects on signal transduction of IL-6 family cytokines such as interleukin-11 (IL-11), oncostatin M (OSM), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) were examined using murine transfectant cell lines (BaF/IL-6R, BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR) that proliferate depending on IL-6, IL-11, OSM, LIF and human CNTF, respectively. Tocilizumab inhibited the proliferation of BaF/IL-6R induced by IL-6, but did not inhibit the proliferation of BaF/IL-11R, BaF/OSMR, BaF/LIFR and BaF/CNTFR cells induced by their corresponding cytokines. These lines of evidence indicate that tocilizumab is able to bind to both sIL-6R and mIL-6R and to inhibit IL-6 binding to its receptors, leading to the blockade of the IL-6 signaling through both sIL-6R and mIL-6R, but not block the signaling of other IL-6 family cytokines.     

Interleukin-8 as a mediator of sympathetic pain.

1. The hyperalgesic effects of interleukin-8 (IL-8), interleukin-1 beta (IL-1 beta) and carrageenin were measured in a rat paw pressure test. 2. IL-8 evoked a dose-dependent hyperalgesia which was attenuated by a specific antiserum, the beta-adrenoceptor antagonists atenolol and propranolol, the dopamine receptor antagonist SCH 23390 and the adrenergic neurone-blocking agent guanethidine. The hyperalgesia was not attenuated by the cyclooxygenase inhibitor indomethacin or the IL-1 beta analogue Lys-D-Pro-Thr. 3. IL-1 beta-evoked hyperalgesia was attenuated by indomethacin and Lys-D-Pro-Thr but not by atenolol or SCH 23390. 4. Carrageenin-evoked hyperalgesia was attenuated by atenolol, indomethacin and anti-IL-8 serum. The effects of atenolol and anti-IL-8 serum were not additive. The effects of indomethacin and anti-IL-8 serum were additive: this combination abolished carrageenin-evoked hyperalgesia. 5. A new biological activity of IL-8 is described, namely the capacity to evoke hyperalgesia by a prostaglandin-independent mechanism. IL-8 is the first endogenous mediator to be identified as evoking hyperalgesia involving the sympathetic nervous system. Since IL-8 is released by activated macrophages and endothelial cells it may be a humoral link between tissue injury and sympathetic hyperalgesia.