The international standard for interleukin-6 Evaluation in an international collaborative study

Three ampouled preparations of interleukin-6 (IL-6) were evaluated by 12 laboratories in seven countries for their suitability to serve as the international standard of IL-6. The preparations were assayed using in vitro bioassays and immunoassays. On the basis of the results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (coded 89/548) was established as the international standard of IL-6.     

雌激素与IL-6、IL-8在卵巢癌细胞中的调节作用

目的 探讨雌激素与IL-6、IL-8在卵巢癌细胞中的交互调节作用及作用机制.方法 选择兼有雌激素受体(estrogen receptor,ER)及IL-6、IL-8受体表达的卵巢癌细胞系CAOV-3和OVCAR-3作为研究模型,分别探讨17B-雌二醇(estradiol,E2)对IL-6、IL-8及其受体表达的作用以及IL-6、IL-8对EB表达及ER转录活性的作用.结果 一方面E2不仅可经NF-κB途径促进卵巢癌细胞IL-6、IL-8分泌,而且还对二者受体的表达具有一定的调节作用.E2诱导的促IL-6、IL-8分泌作用可被其受体阻断剂他莫昔芬(tamoxifen,Txf)完全阻断.另一方面在无雌激素的条件下,IL-6、IL-8能上调卵巢癌细胞Erα表达及下调ERB表达,且还能分别通过丝裂原活化蛋白激酶(MAPK)信号通路和Src活化增强卵巢癌细胞ER的转录活性,该作用可被Txf完全封闭.结论 雌激素与IL-6、IL-8两种细胞因子在卵巢癌细胞中交互调节,由此通过产生的放大信号通路促进卵巢癌的生长和发展.     

CyPA对类风湿关节炎中性粒细胞的趋化和产生IL-8的作用及其意义

目的:观察细胞环亲素A(CyPA)对人中性粒细胞HL-60株和类风湿关节炎(RA)患者外周血中性粒细胞的趋化和产生IL-8的作用.方法:选择12例RA患者,分离外周血中性粒细胞,采用Boyden小室趋化实验比较CyPA和/或IL-8对HL-60和RA患者粒细胞的趋化作用,采用ELISA检测CyPA促进培养的粒细胞产生IL-8作用,并与健康人比较.结果:CyPA组、 IL-8组及CyPA+IL-8组较阴性对照组趋化性显著增强( P <0.05);与IL-8组相比,CyPA抗体+IL-8组趋化指数降低( P <0.05);与CyPA组或IL-8组相比,CyPA+IL-8组趋化指数有所增加.RA患者中性粒细胞培养上清中IL-8水平较健康人显著增加( P <0.05);经CyPA刺激后,RA患者中性粒细胞培养上清中IL-8水平较CyPA刺激前增加,差异有统计学意义( P <0.05);阻断CyPA后,RA患者中性粒细胞培养上清中IL-8水平较CyPA刺激前无明显差异.结论:CyPA影响IL-8对中性粒细胞的趋化作用并对其分泌有影响.     

Inhibition of human neutrophil activity by an RNA aptamer bound to interleukin-8

Interleukin-8 (IL-8) is a proinflammatory CXC chemokine that has been associated with the promotion of neutrophil chemotaxis, degranulation, and the pathogenesis of several neutrophil-infiltrating chronic inflammatory diseases. In the current study, we generated and characterized a 2′-fluoro-pyrimidine modified RNA aptamer (8A-35) against human IL-8. The 8A-35 aptamer binds to IL-8 with high specificity and affinity, yielding an estimated K_D of 1.72 pM. NMR data revealed that the residues of Lys8, Leu10, Val63, Val66, Lys69 and Ala74 of IL-8 interact with aptamer. Moreover, the 8A-35 aptamer has a potent IL-8-neutralizing activity that can modulate multiple biological activities of IL-8 in human neutrophils, including migration, intracellular signaling, and intracellular Ca²⁺ mobilization. Our results suggest that the 8A-35 aptamer has great potential to be a lead structure in the development of effective therapeutic agents against inflammatory diseases.     

一种新型重组人源性白细胞介素-8活性检测方法的建立

目的通过蛋白质片段互补检测（proteinfragmentcomplementationassay,PCA）技术检测重组人源性IL-8的生物学活性。方法利用PCA方法构建了Split—TEVGPCR激活检测系统,为了验证其有效性,在不同的宿主系统中表达和纯化了各种亚型的人源性IL-8重组蛋白用于进行活性检测。蛋白样品包括：①IL-8亚型I（残基21-99）,通过在哺乳动物细胞HEK293中分泌表达前体IL-8基因获得,其N端信号肽（残基1—20）被切除;②IL-8亚型Ⅱ（残基23—99）和亚型Ⅲ（残基28—99）,通过在大肠杆菌BL21（DE3）中表达获得。结果Split—TEVGPCR激活检测系统证明纯化后的重组人源性IL．8样品对天然受体IL8RB都具有明显的激活活性,其EC50值分别为：12．32±0．89ng／mL（亚型I）、15．14±1．84ng／mL（亚型Ⅱ）和2．854-0．50ng／mL（亚型Ⅲ）。结论成功建立了-种新型的重组人IL-8活性检测方法,为进一步的理论研究和IL-8中和抗体的高通量筛选奠定了基础。     

Attenuated interleukin-8/leukocyte immunoresponse in preterm infants compared with term infants hospitalized with respiratory syncytial virus bronchiolitis: a pilot study

Decreased transplacental transfer of antibodies and altered immunoresponsiveness may place preterm (PT) infants at higher risk for serious consequences from respiratory syncytial virus (RSV) bronchiolitis. We hypothesize that among infants hospitalized with RSV bronchiolitis, immune response in PT infants may be different when compared with that of term infants. Nasal-wash samples were collected from 11 PT (<37 weeks of gestation) and 13 term infants (≥37 weeks of gestation) hospitalized with RSV bronchiolitis. Severity of illness (clinical score [CS]), admission peripheral oxygen saturation, and days subjects required supplemental oxygen were compared. Nasal-wash leukocyte count as well as cytokines for interleukin (IL)-8, IL-4, and interferon-γ (IFN-γ) were assayed. No significant differences in CS, admission SaO(2), and O(2) days were seen between PT and term infants. Nasal-wash leukocyte counts and IL-8 levels were higher in term infants compared with PT and correlated with severity (higher CS) in term (p < 0.05) but not in PT (p > 0.05) infants. IL-4 and IFN-γ levels did not differ between the 2 groups (p > 0.05). PT infants hospitalized with RSV bronchiolitis have lower nasal-wash leukocyte counts and a less robust IL-8 response than term infants, and only in term infants did IL-8 levels correlate with clinical disease severity.     

IL-8对OX-LDL诱导的RAW264.7株作用的研究

目的探讨白细胞介素8（IL-8）对OX-LDL诱导的小鼠单核巨噬细胞264.7细胞株（RAW264.7）的作用。方法以IL-8作用于OX-LDL诱导RAW264.7细胞株,48 h后收集细胞和上清培养液,分别用流式细胞仪检测细胞表位CD14、CD34、CD86及其早、晚期凋亡率;用RT-PCR法检测细胞内IL-8 mRNA的表达水平;用Western bot检测IL-8 mRNA表达蛋白的量;用ELISA方法检测上清液IL-1β、IL-10的量。结果与对照组相比,实验组的CD14、CD34表位表达及晚期凋亡明显增多（P〈0.05）,而对CD86、早期凋亡影响不明显（P〉0.05）,IL-8 mRNA及其表达的蛋白量明显升高,上清培养液中IL-1β增多（P〈0.05）,IL-10减少（P〈0.05）。结论 IL-8致动脉粥样硬化（AS）作用可能是部分通过上调单核巨噬细胞表面CD14、CD34表位表达,促进IL-8 mRNA及其表达蛋白的量,促进IL-1β、抑制IL-10的分泌,并促进其晚期凋亡,参与AS的炎症反应。     

微量酶联夹心杂交法定量检测hIL-8 mRNA PCR扩增产物

目的:建立一种灵敏度高、特异性强、定量、精确、操作简便的微孔板酶联夹心杂交技术,定量检测人IL-8 mR-NA。方法:针对人IL-8 mRNA逆转录聚合酶链反应产物一条链的不同区域序列设计一对特异探针,其中一条为捕获探针,5′端用活性氨基修饰,与微量DNA结合板表面的NOS基团共价结合,“竖直”地包被在微孔板内;另一条检测探针的3′端标记生物素,和辣根过氧化物酶结合。提取人外周血单个核细胞总RNA,进行RT-PCR扩增IL-8 mRNA,双链DNA产物经热变性后加入已包被捕获探针的微孔板内进行杂交,加入检测探针与已杂交的产物结合,经亲和素-辣根过氧化物酶系统检测杂交信号。结果:该法灵敏度为检出16个循环的PCR产物、5×103个PBMCs中的IL-8 mRNA、检测PCR终产物的最高稀释倍数为1∶256阳性;特异性试验非目的扩增片段酶联杂交检测未检出阳性杂交信号;精密度试验CV为5.2%。结论:该方法具有操作简便、灵敏度高、特异性强等优点,适合IL-8 mRNA PCR扩增产物的定量检测。     

Phagocytosis and endocytosis of silver nanoparticles induce interleukin-8 production in human macrophages

Phagocytosis or endocytosis by macrophages is critical to the uptake of fine particles, including nanoparticles, in order to initiate toxic effects in cells. Here, our data enhance the understanding of the process of internalization of silver nanoparticles by macrophages. When macrophages were pre-treated with inhibitors to phagocytosis, caveolin-mediated endocytosis, or clathrin-mediated endocytosis, prior to exposure to silver nanoparticles, Interleukin-8 (IL-8) production was inhibited. Although cell death was not reduced, the inflammatory response by macrophages was compromised by phagocytosis and endocytosis inhibitors.     

CXCL8 of Scophthalmus maximus: expression, biological activity and immunoregulatory effect

CXCL8, or interleukin-8, is a CXC chemokine that promotes neutrophil migration in response to inflammatory stimuli. In this study, we identified and analyzed a CXCL8 orthologue, SmCXCL8, from turbot (Scophthalmus maximus). The deduced amino acid sequence of SmCXCL8 is 99-residue in length and shares 52-83% overall identities with the lineage 1 CXCL8 of a number of teleost. SmCXCL8 possesses a CXC chemokine domain that contains the conserved CXC motif preceded by the tripeptide sequence EMH. Purified recombinant SmCXCL8 (rSmCXCL8) induced chemotaxis in peripheral blood neutrophils and, to lesser extents, head kidney (HK) lymphocytes and macrophages in a dose-dependent manner. Mutation of the EMH motif by alanine substitution reduced the chemoattractive effect of rSmCXCL8. Expression of SmCXCL8 as determined by quantitative real time RT-PCR (qRT-PCR) was detected mainly in immune organs under normal physiological conditions and was upregulated by experiment challenges with bacterial pathogen and poly(I:C). In addition, SmCXCL8 expression was also induced to significant extents following vaccination of turbot with a subunit vaccine. When rSmCXCL8 was added to the cell cultures of peripheral blood leukocytes and HK lymphocytes and macrophages, it stimulated the proliferation of these cells and enhanced cellular resistance against intracellular bacterial survival. qRT-PCR analysis showed that rSmCXCL8 induced the expression of TNF-α and suppressor of cytokine signaling 3 in HK lymphocytes in different time frames. On the other hand, SmCXCL8 expression was also upregulated by TNF-α. Taken together, these results indicate that SmCXCL8 is a functional CXC chemokine with immunomodulatory effect and plays a role in inflammatory response induced by bacterial infection.     

原发性肝癌患者免疫原快速激活红细胞调控IL-8和球蛋白变化仿真实验研究

目的 研究原发性肝癌患者血浆中红细胞固有免疫反应与来自白细胞的IL-8和球蛋白之间规律变化的关系.方法 灭活癌细胞(S180株5×106/ml)或生理盐水(对照组)0.2 ml加到枸橼酸抗凝的0.2 ml全血细胞悬液(自然实验组)或白细胞悬液(白细胞分离实验组)和新鲜血浆0.3 ml中,混匀后放37℃温育1 h,测定反应液中IL-8和球蛋白含量.结果 原发性肝癌患者自然实验组IL-8含量(376.35 ±243.96)Pg/ml和球蛋白(7.86 ±2.01)g/L,明显高于正常人自然实验组的IL-8含量(11.36±6.93)Pg/ml和球蛋白含量(7.21 ±1.75)g/L,(P＜0.01),正常人自然实验组IL-8含量(11.36 ±6.93)pg/ml和球蛋白含量(7.21 ±1.75)g/L明显高于正常人自然对照组IL-8含量(4.98 ±4.35)pg/ml和球蛋白含量(5.38±1.61)g/L,(P＜0.05),正常人自然实验组IL-8的激活指数2.49 ±2.33明显高于原发性肝癌患者自然实验组IL-8的激活指数0.22 ±0.24,(P＜0.05).结论 红细胞能快速调控血浆中的IL-8和球蛋白含量,但原发性肝癌患者红细胞调控IL-8和球蛋白的能力明显低于正常人,在原发性肝癌患者系统血液固有免疫反应中,红细胞固有免疫反应活性与白细胞固有免疫反应活性之间的关系处于失衡状态.     

Virulence characteristics of translocating Escherichia coli and the interleukin-8 response to infection

Four efficiently translocating Escherichia coli (TEC) strains isolated from the blood of humans (HMLN-1), pigs (PC-1) and rats (KIC-1 and KIC-2) were tested for their ability to adhere and translocate across human gut epithelial Caco-2 and HT-29 cells, to elicit a proinflammatory response and for the presence of 47 pathogenic E. coli virulence genes. HMLN-1 and PC-1 were more efficient in adhesion and translocation than rat strains, had identical biochemical phenotype (BPT) and serotype (O77:H18) and phylogenetic group (D). KIC-2 adhered more than KIC-1, belonged to different BPT and serotype but the same phylogenetic group as KIC-1. TEC strains elicited significantly higher IL-8 response in both cell lines (P < 0.05) and monocytic THP-1 (P < 0.0001) cells than non-TEC strains. KIC-2 induced the highest IL-8 response which may be associated with its immunostimulatory flagellin. Apart from adhesin genes fimH and bmaE that were carried by all strains, HMLN-1 and PC-1 carried capsule synthesis gene kpsMT III and KIC-2 carried the EAST1 toxin gene. The lack of known virulence genes and the ability of TEC to efficiently adhere and translocate whilst causing proinflammatory response suggests that these strains may carry as yet unidentified genes that enable their translocating ability.     

细胞因子IL6、IL8及IL-10与变应性鼻炎的关系研究

目的探讨白细胞介素-6、8、10与变应性鼻炎（allergicrhinitis,AR）发病的相关性,为指导临床治疗提供实验依据。方法应用酶联免疫吸附测定（enzymelinkedimmu—nosorbentassay,ELISA）方法检测35例正常健康对照组以及60例变应性鼻炎患者脱敏治疗前后血清标本中IL-6、IL-8、IL-10的表达水平,并应用SPSSl3．0软件分析治疗前后各指标的表达差异,以及三者之间的相关性。结果IL-6、IL-8在AR患者血清中高表达,与健康对照组相比差异具有显著性（t=15．213,P〈0．01;t=12．231,P〈0．01）,脱敏治疗后表达均下降,治疗前后差异具有统计学意义（t=21．995,P〈0．01;t=19．766,P〈0．01）;IL-10在患者血清中表达低于对照组（t=7．446,P〈0．01）,脱敏治疗后其表达上升,与治疗前相比差异显著（t=10．228,P〈0．01）;IL-6、IL-8在AR患者中的表达呈正相关（r=0．523。P〈0．01）,而二者与IL-10的表达呈负相关（r=-0．482,P〈0．01）。结论IL-6、IL-8及IL-10与变应性鼻炎发病过程密切相关,相应的细胞因子或拮抗剂将可能成为候选的治疗药物。     

A highly sensitive enzyme-linked immunosorbent assay for the measurement of interleukin-8 in biological fluids.

Interleukin-8 (IL-8) is a chemotactic and activating cytokine for neutrophils, which plays an important role in acute inflammatory responses. We aimed to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for IL-8 and established 18 clones of anti-IL-8 monoclonal antibodies (mAbs). These mAbs were evaluated in terms of their antigen-binding affinities, and five clones were selected and used for the comparative study of various combinations of antibodies in sandwich ELISA. Affinity purified rabbit polyclonal antibody was also used in this study. One antibody pair, which showed relatively high sensitivity and which was not severely interfered with blood components, was selected and the assay conditions were optimized by choosing the appropriate buffer for sample dilution and by directly labeling the second antibody with enzyme. The finalized ELISA, using polyclonal antibody as first (coated) antibody and horseradish peroxidase-labeled mAb (clone EL139) Fab' fragment as second antibody, could detect as low as 2.5 pg/ml (0.125 pg/well) of IL-8 by in total 2 h incubation, without being affected by body fluid components. The ELISA was specific to IL-8, showing no cross-reactivity with other cytokines or various IL-8 family proteins which share some amino acid sequence homology with IL-8. As an example of its application to clinical specimens, plasma samples from patients with septic shock were measured. The results showed that sepsis patients contain significantly higher levels of plasma IL-8 compared to normal controls. When analyzed by gel-filtration chromatography, IL-8 in sepsis plasma was eluted in a molecular weight (M(r) region corresponding to the monomer form. The ELISA established here is expected to be effectively used for further investigations on the relationship between IL-8 and various diseases.     

Apoptosis of rat gastric mucosa and of primary cultures of gastric epithelial cells by indomethacin: role of inducible nitric oxide synthase and interleukin-8

In order to gain insights into indomethacin-induced gastric injury, rats were fed with indomethacin (20 mg/kg), or alternatively, the primary cultures of rat gastric epithelial cells were cultured with different doses of indomethacin (1-1000 microM). Light microscopy, electron microscopy, fluorescence microscopy, TdT-mediated dUTP-biotin nick end labelling staining, ssDNA staining and DNA fragmentation assay were employed to evaluate the levels of gastric injury and apoptosis. Cells expressing inducible nitric oxide synthase (iNOS) and interleukin (IL)-8 were localized at the rat gastric mucosa by immunohistochemistry. Administration of indomethacin to rats caused apoptosis and injury of the gastric mucosal epithelial cells. Indomethacin also induced apoptosis of primary cultures of gastric epithelial cells in a dose-dependent manner. Cells expressing iNOS and IL-8 were detected at and around the sites of gastric injury in the indomethacin-fed rats, but not in the control rats. The induction of apoptosis by indomethacin in the primary cultures of gastric epithelial cells suggests that the direct apoptotic capacity of indomethacin. iNOS and IL-8 may be involved in this process.     

谷氨酰胺、地塞米松对内毒素血症大鼠血清IL-8,IL-10的影响

目的通过观察谷氨酰胺、地塞米松对内毒素血症大鼠血清IL-8,IL-10的影响,探讨地塞米松、谷氨酰胺对内毒素血症大鼠的保护途径.方法选健康18日龄Wistar大鼠120只,随机取8只腹腔注射生理盐水作对照组(0h),余大鼠按腹腔注射药物不同分成内毒素(L)、谷氨酰胺预防(Gln)地塞米松治疗(D1)组,每组40只,各组于加LPS后2、4、6、24及72h分别断头取血,用放免法测定IL-8,IL-10.结果(1)L组IL-8在2、24h出现两次升高,前者升高程度更明显,差异显著,P<0.01;D1和C1n组各时间点IL-8均低于L组,尤其以2h抑制作用更强,差异显著,P<0.01;Gln组第一次高峰在6h.(2)L组IL-10在2、72h出现两次升高,后者升高程度更明显,差异显著,P<0.01;D1和Gln组第二次升高均提前至24h,增幅最高的为D1组,而其它各时间点IL-10均以Gln组升高明显,差异显著,P<0.01.结论细胞因子IL-8/IL-10在内毒素血症中起主要的损害与抗损害作用,地塞米松治疗对机体能抑制损伤/促进修复;谷氨酰胺不仅有与地塞米松相似的作用,而且可使损伤延迟,二者都能使修复作用提前.     

细胞因子IL6、IL8及IL-10与变应性鼻炎的关系研究

目的 探讨白细胞介素-6、8、10与变应性鼻炎( allergic rhinitis,AR)发病的相关性,为指导临床治疗提供实验依据.方法 应用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)方法检测35例正常健康对照组以及60例变应性鼻炎患者脱敏治疗前后血清标本中IL-6、IL-8、IL-10的表达水平,并应用SPSS 13.0软件分析治疗前后各指标的表达差异,以及三者之间的相关性.结果 IL-6、IL-8在AR患者血清中高表达,与健康对照组相比差异具有显著性(t=15.213,P＜0.01;t=12.231,P＜0.01),脱敏治疗后表达均下降,治疗前后差异具有统计学意义(t=21.995,P＜0.01;t =19.766,P＜0.01);IL-10在患者血清中表达低于对照组(t=7.446,P＜0.01),脱敏治疗后其表达上升,与治疗前相比差异显著(t=10.228,P＜0.01);IL-6、IL-8在AR患者中的表达呈正相关(r=0.523,P＜0.01),而二者与IL-10的表达呈负相关(r=-0.482,P＜0.01).结论 IL-6、IL-8及IL-10与变应性鼻炎发病过程密切相关,相应的细胞因子或拮抗剂将可能成为候选的治疗药物.     

去白细胞处理对全血保存中IL-2和IL-8水平的影响

目的:研究全血在保存过程中是否存在IL-2和IL-8的积累,保存前白细胞滤除对其保存水平的影响。方法:选10名健康捐血者,各采集 200ml全血分成2袋,其中一袋经去白细胞处理。在4℃保存不同时期测定血液中 IL-2、IL-8含量的动态变化。结果:随着保存,全血中IL-2和IL-8的浓度不断增高。经去白细胞处理后,IL-8水平在保存中无明显变化,IL-2的含量仍然增高,但在保存期末低于正常全血中的相应含量。结论:IL-2和IL-8在全血保存中存在产生和累积,而去白细胞过滤可以抑制IL-2和IL-8在全血保存中存在产生和累积,而去白细胞过滤可以抑制IL-8的升高,对预防发热性非溶血性输血反应有一定作用。