流感病毒H1N1和H5N1感染小鼠小胶质细胞与星形胶质细胞诱导趋化因子转录水平上调

目的:探讨胶质细胞感染流感病毒后的天然免疫反应,检测流感病毒H1N1和H5N1体外感染小鼠小胶质细胞和星形胶质细胞,是否会诱导胶质细胞趋化因子转录水平的变化及其规律.方法:从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化小胶质细胞和星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H1N1和H5N1进行体外感染,8小时后用免疫荧光检测流感病毒核蛋白(NP)的表达,以确认感染细胞比例.在感染早期(6小时)和感染中期(24小时)分别提取细胞RNA,检测趋化因子转录水平的变化.结果:分离得到小鼠的小胶质细胞和星形胶质细胞,病毒感染后超过95%的细胞可以被感染,感染后的小胶质细胞与星形胶质细胞的CCI-3、CCL-5、CXCL-2、CXCL-9和CXCL-10的转录水平发生不同程度的上调,其中CXCL-10的上调幅度最为明显,禽流感病毒H5N1感染能诱导更强烈的上调反应.结论:流感病毒H1N1和H5N1感染小鼠小胶质细胞与星形胶质细胞,可诱导趋化因子转录水平上调.     

硫辛酸抑制脂多糖诱导的星形胶质细胞细胞因子及趋化因子的表达

目的探讨硫辛酸(LA)对脂多糖(LPS)激活的星形胶质细胞分泌TNF-α、IL-1β、IL-6和IL-10及相关趋化因子的影响。方法分离并鉴定新生C57BL/6小鼠大脑皮质星形胶质细胞,1μg/mL LPS刺激第2代星形胶质细胞,100μg/mL LA进行干预,Griess法检测NO的分泌,ELISA检测TNF-α、IL-1β、IL-6和IL-10炎性因子的含量,反转录PCR检测炎症趋化因子CC亚族趋化因子配体20(CCL20),单核细胞趋化蛋白1(MCP-1)和巨噬细胞炎性蛋白1α(MIP-1α)mRNA的表达。结果与正常组比较,LPS刺激星形胶质细胞后,NO、TNF-α、IL-1β、IL-6分泌显著升高,IL-10分泌下调(P0.05);LA能抑制LPS诱导的NO、TNF-α、IL-1β、IL-6的分泌,增加IL-10的分泌,与LPS组相比差异有统计学意义(P0.05)。LA能显著下调LPS所致的CCL20、MIP-1α、MCP-1 mRNA的分泌。结论 LA能抑制LPS激活星形胶质细胞所致的炎性反应,其作用与抑制炎性因子及趋化因子的分泌有关。     

外周注射福尔马林诱导前扣带皮层中胶质细胞激活和细胞因子表达增高

为了观察外周注射福尔马林对大鼠前扣带皮层(ACC)中星形胶质细胞标记物胶质纤维酸性蛋白(GFAP)和S100B,小胶质细胞标记物CD14以及与胶质细胞密切相关的炎症前细胞因子白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)表达的影响,本研究结合行为学检测和RT-PCR方法,观察了大鼠单侧足底皮下注射福尔马林后的行为学变化,并检测了在不同时间点ACC中GFAP、S100B、CD14、IL-1β和TNF-α在基因水平的表达变化。结果显示:福尔马林急性疼痛刺激的大鼠出现典型的双时相自发性疼痛反应。ACC中GFAP和S100B mRNAs表达水平在刺激后30min、1h增高,2h恢复正常;CD14 mRNA表达水平在15min开始增高,1h达到高峰,6h恢复正常;IL-1β和TNF-αmRNAs表达水平在刺激后30min、1h、2h增高,6h恢复正常。上述结果表明,福尔马林外周急性伤害性刺激能够诱导ACC内短暂性的星形胶质细胞、小胶质细胞激活及IL-1β、TNF-α表达增高,提示激活的胶质细胞及表达上调的炎症前细胞因子可能参与伤害性信息的调制。     

雷藤舒(LLDT-8)对成纤维样滑膜细胞分泌趋化因子的影响

研究(5R)-5-羟基雷公藤内酯醇(雷藤舒,LLDT-8)对TNF-α联合IL-17诱导类风湿关节炎(rheumatoid arthritis,RA)成纤维样滑膜细胞(fibroblast-like synoviocytes,FLS)分泌趋化因子GRO-α、ENA-78、MCP-1、MIP-1α和RANTES的影响。ELISA法检测FLS上清液中GRO-α、ENA-78、MCP-1、MIP-1α和RANTES的浓度;RT-PCR法检测FLS中GRO-α、ENA-78、MCP-1、MIP-1α和RANTES mRNA的表达。结果显示LLDT-8可显著抑制TNF-α联合IL-17诱导的FLS上清液中GRO-α、ENA-78、MCP-1、MIP-1α和RANTES的表达;也可有效抑制TNF-α联合IL-17诱导的FLS GRO-α、ENA-78、MCP-1、MIP-1α和RANTES mRNA的表达。该研究提示LLDT-8通过抑制FLS产生GRO-α、ENA-78、MCP-1、MIP-1α和RANTES而在RA的治疗中发挥抗炎作用。     

IL-1β影响大鼠星形胶质细胞中SSeCKS表达的研究

目的:研究Src抑制的蛋白激酶C的底物(SSeCKS)在IL-1β激活的星形胶质细胞中的表达.方法:以炎性因子IL-1β刺激大鼠体外培养的星形胶质细胞,观察细胞中SSeCKS的表达及其细胞内的亚定位的改变.结果:IL-1β可上调大鼠原代培养星形胶质细胞中SSeCKS的表达,诱导其丝氨酸磷酸化.在亚细胞水平,IL-1β还能引起SSeCKS向核周、细胞星状突起聚集,而蛋白激酶C(PKC)抑制剂Ro-31-8220能抑制IL-1β对细胞中SSeCKS亚细胞定位及磷酸化水平的影响.结论:在IL-1β的影响下,大鼠星形胶质细胞中SSeCKS的表达增加,磷酸化水平增强,细胞内定位发生改变,这种改变与PKC的功能相关,提示SSeCKS可能作为PKC的下游分子参与到炎性因子引起的星形胶质细胞活化的过程中.     

活化小胶质细胞致星形胶质细胞激活

目的探讨活化小胶质细胞培养液对星形胶质细胞的影响。方法 LPS激活原代培养小胶质细胞,采用活化的小胶质细胞条件培养液刺激星形胶质细胞,观察星形胶质细胞GFAP及IL-1β和TNFα的表达。结果 LPS刺激后,小胶质细胞OX42表达量上升,IL-1β和TNFα的表达量增高;小胶质细胞条件培养液可致星形胶质细胞激活,GFAP表达量上升,IL-1β和TNFα的表达量增加。结论活化小胶质细胞的条件培养液可致星形胶质细胞激活,激活的小胶质细胞和星形胶质细胞表达前炎症介质IL-1β和TNFα。     

甜叶悬钩子苷通过NF-κB和MAPK信号通路抑制小鼠神经炎症细胞模型炎症反应

目的：通过体外实验探究甜叶悬钩子苷(RUB)对脂多糖(LPS)诱导小鼠BV-2小胶质细胞神经炎症反应的影响及改善机制。方法：采用CCK-8法检测RUB对BV-2细胞活力的影响;使用LPS诱导BV-2小胶质细胞建立神经炎症细胞模型,用不同浓度RUB进行干预,将BV-2细胞分为对照组、LPS模型组及LPS+RUB(25、50和100μmol/L)干预组,采用ELISA法检测细胞上清中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的浓度;RTqPCR法检测促炎细胞因子环加氧酶2(COX-2)、诱导型一氧化氮合酶(iNOS)、IL-1β和TNF-α的mRNA表达水平;Western blot法检测NF-κB和MAPK信号通路相关蛋白的表达;免疫荧光染色观察细胞中p65的表达情况。结果：与对照组相比,LPS诱导后细胞上清液中IL-1β和TNF-α的分泌量增加(P<0.01),促炎细胞因子COX-2、iNOS、IL-1β和TNF-α的mRNA水平显著增加(P<0.01),NF-κB和MAPK信号通路蛋白表达显著上调(P<0.05或P<0.01),且p65出现荧光...     

Fasudil通过TLR4通路抑制脂多糖诱导的小鼠BV-2小胶质细胞TNF-α和IL-1β的表达

目的探讨Fasudil对脂多糖(LPS)诱导BV-2小胶质细胞系促炎细胞因子表达中的作用。方法体外培养BV-2小胶质细胞系,实验分为PBS对照组、LPS刺激组、LPS联合Fasudil干预组,ELISA检测细胞TNF-α、IL-1β的释放,Griess法检测NO释放水平,流式细胞术检测Toll样受体4(TLR4)、TLR2蛋白表达。结果 LPS刺激BV-2细胞可导致TNF-α、IL-1β和NO的释放明显增加,还可导致炎性信号通路中的受体TLR4表达明显增加。Fasudil能明显抑制炎性因子的释放和TLR4的表达。结论 Fasudil可抑制LPS诱导的小胶质细胞NO、TNF-α和IL-1β释放,其作用机制可能与Fasudil下调TLR4通路有关。     

Fractalkine对脂多糖激活的小胶质细胞炎症相关因子表达的影响

目的:探讨趋化因子Fractalkine对脂多糖(LPS)诱导的小鼠小胶质细胞(N9)激活时所分泌的TNF-α、IL-1β和一氧化氮(NO)表达的影响.方法:用Fractalkine处理经LPS激活的小鼠小胶质细胞24 h,以ELISA法检测细胞培养上清中TNF-α和IL-1β的浓度,以NO试剂盒检测培养上清中NO的浓度.结果:LPS能够激活小胶质细胞,使IL-1 β、TNF-α和NO的表达量与对照组相比明显升高;Fractalkine能够降低LPS激活的小胶质细胞IL-1β、TNF-α和NO的表达.结论:Fractalkine可能通过抑制炎症相关因子的产生而在中枢神经系统中发挥神经保护作用.     

外周炎症性疼痛刺激诱导丘脑中胶质细胞激活及细胞因子的表达

目的:研究完全弗氏佐剂(CFA)外周刺激后,大鼠丘脑中线核群和板内核群中星形胶质细胞标记物(GFAP)、小胶质细胞标记物(Iba)以及细胞因子(IL-1β,TNF-α)的表达变化.方法:逆转录聚合酶链式反应(RT-PCR),免疫印迹法,免疫组织化学.结果:在炎症刺激后的4 h、3 d和14 d,丘脑中线核群和板内核群中小胶质细胞的标记物CD14、细胞因子IL-1β,TNF-α的mRNA和蛋白均有增加;星形胶质细胞标记物GFAP在炎症后的3 d和14 d,其mRNA和蛋白有显著增加.结论:外周炎症性刺激能够相继激活丘脑中线核群和板内核群的小胶质细胞和星形胶质细胞,并使炎症前细胞因子表达增多.提示该部位的胶质细胞可能通过细胞因子参与对炎症性疼痛的调节.     

Apoptosis and proinflammatory cytokine responses of primary mouse microglia and astrocytes induced by human H1N1 and avian H5N1 influenza viruses

Patients with an influenza virus infection can be complicated by acute encephalopathy and encephalitis. To investigate the immune reactions involved in the neurocomplication, mouse microglia and astrocytes were isolated, infected with human H1N1 and avian H5N1 influenza viruses, and examined for their immune responses. We observed homogeneously distributed viral receptors, sialic acid （SA）-a2,3-Galactose （Gal） and SA-a2,6-Gal, on microglia and astrocytes. Both viruses were replicative and productive in microglia and astrocytes. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection （p.i.）. Expression of IL-1β, IL-6 and TNF-a mRNA examined at 6 h and 24 h p.i. was up-regulated, and their expression levels were considerably higher in H5N1 infection. The amounts of secreted proinflammatory IL-1β, IL-6 and TNF-a at 6 h and 24 h p.i. were also induced, with greater induction by H5N1 infection. This study is the first demonstration that both human H1N1 and avian H5N1 influenza viruses can infect mouse microglia and astrocytes and induce apoptosis, cytopathy, and proinflammatory cytokine production in them in vitro. Our results suggest that the direct cellular damage and the consequences of immunopathological injury in the CNS contribute to the influenza viral pathogenesis. Cellular ＆ Molecular Immunology.     

Innate immune response to H3N2 and H1N1 influenza virus infection in a human lung organ culture model

We studied cytokine responses to influenza virus PR8 (H1N1) and Oklahoma/309/06 (OK/06, H3N2) in a novel human lung tissue model. Exposure of the model to influenza virus rapidly activated the mitogen-activated protein kinase signaling (MAPK) pathways ERK, p38 and JNK. In addition, RNase protection assay demonstrated the induction of several cytokine and chemokine mRNAs by virus. This finding was reflected at the translational level as IL-6, MCP-1, MIP-1α/β, IL-8 and IP-10 proteins were induced as determined by ELISA. Immunohistochemistry for IP-10 and MIP-1α revealed that alveolar epithelial cells and macrophages were the source of these two cytokines. Taken together, both PR8 and OK/06 cause similar induction of cytokines in human lung, although OK/06 is less effective at inducing the chemokines MCP-1 and IL-8. This human organ culture model should thus provide a relevant platform to study the biological responses of human lung to influenza virus infection.     

Induction of inflammatory cytokines and toll-like receptors in human normal respiratory epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, and highly pathogenic H5N1 influenza virus

Respiratory epithelial cells are one of main targets for infections caused by influenza viruses. Recently, the induction of proinflammatory cytokines and toll-like receptors (TLRs) in normal human bronchial/tracheal epithelial cells infected with seasonal H1N1, 2009 pandemic H1N1, seasonal H3N2, or highly pathogenic H5N1 influenza virus were studied to understand the pathogenesis and early immune responses. The cells were productively infected with the viruses. Among the inflammatory cytokines tested, interleukin (IL)-8 was predominantly induced in virus-infected cells. Among the chemokines tested, interferon-γ-inducible protein-10 (IP-10) and growth-related oncogene-α (GRO-α) were predominantly induced in virus-infected cells. TLR-5 was predominantly induced in cells infected with seasonal H1N1, pandemic H1N1, or H5N1 influenza virus, and TLR-3 was predominantly induced in cells infected with seasonal H3N2 influenza virus. Taken together, the results suggest that IL-8, IP-10, and GRO-α are predominantly induced in respiratory epithelial cells infected with influenza A viruses, and that TLR-5 and TLR-3 are involved in the stimulation of virus-infected respiratory epithelial cells.     

An interferon-gamma-related cytokine storm in SARS patients

Fourteen cytokines or chemokines were analyzed on 88 RT-PCR-confirmed severe acute respiratory syndrome (SARS) patients. IFN-gamma, IL-18, TGF-beta, IL-6, IP-10, MCP-1, MIG, and IL-8, but not of TNF-alpha, IL-2, IL-4, IL-10, IL-13, or TNFRI, were highly elevated in the acute phase sera of Taiwan SARS patients. IFN-gamma was significantly higher in the Ab(+) group than in the Ab(-) group. IFN-gamma, IL-18, MCP-1, MIG, and IP-10 were already elevated at early days post fever onset. Furthermore, levels of IL-18, IP-10, MIG, and MCP-1 were significantly higher in the death group than in the survival group. For the survival group, IFN-gamma and MCP-1 were inversely associated with circulating lymphocytes count and monocytes count, but positively associated with circulating neutrophils count. It is concluded that an interferon-gamma-related cytokine storm was induced post SARS coronavirus infection, and this cytokine storm might be involved in the immunopathological damage in SARS patients.     

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechanisms have not yet been fully elucidated. We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS. Cytokine profile of SARS patients showed marked elevation of Th1 cytokine interferon (IFN)-gamma, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least 2 weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumour necrosis factor (TNF)-alpha, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2 and Th2 cytokine IL-4. The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-gamma-inducible protein-10 (IP-10). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 to 8 days after treatment (all P < 0.001). Together, the elevation of Th1 cytokine IFN-gamma, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.     

The pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages

The attenuated equine infectious anemia virus (EIAV) vaccine was the first attenuated lentivirus vaccine to be used in a large-scale application and has been used to successfully control the spread of equine infectious anemia (EIA) in China. To better understand the potential role of cytokines in the pathogenesis of EIAV infection and resulting immune response, we used branched DNA technology to compare the mRNA expression levels of 12 cytokines and chemokines, including IL-1α, IL-1β, IL-4, IL-10, TNF-α, IFN-γ, IP-10, IL-8, MIP-1α, MIP-1β, MCP-1, and MCP-2, in equine monocyte-derived macrophages (eMDMs) infected with the EIAV(DLV121) vaccine strain or the parental EIAV(DLV34) pathogenic strain. Infection with EIAV(DLV34) and EIAV(DLV121) both caused changes in the mRNA levels of various cytokines and chemokines in eMDMs. In the early stage of infection with EIAV(DLV34) (0-24h), the expression of the pro-inflammatory cytokines TNF-α and IL-1β were significantly up-regulated, while with EIAV(DLV121), expression of the anti-inflammatory cytokine IL-4 was markedly up-regulated. The effects on the expression of other cytokines and chemokines were similar between these two strains of virus. During the first 4 days after infection, the expression level of IL-4 in cells infected with the pathogenic strain were significantly higher than that in cells infected with the vaccine strain, but the expression of IL-1α and IL-1β induced by the vaccine strain was significantly higher than that observed with the pathogenic strain. In addition, after 4 days of infection with the pathogenic strain, the expression levels of 5 chemokines, but not IP-10, were markedly increased in eMDMs. In contrast, the vaccine strain did not up-regulate these chemokines to this level. Contrary to our expectation, induced apoptosis in eMDMs infected with the vaccine strain was significantly higher than that infected with the pathogenic strain 4 days and 6 days after infection. Together, these results contribute to a greater understanding of the pathogenesis of EIAV and of the mechanisms by which the immune response is induced after EIAV infection.     

Selective induction of the monocyte-attracting chemokines MCP-1 and IP-10 in vesicular stomatitis virus-infected human monocytes.

It is characteristic of viral infections that monocytes/macrophages and lymphocytes infiltrate infected tissue, and neutrophils are absent. CC and non-ELR CXC chemokines predominantly attract mononuclear leukocytes, whereas the ELR motif-expressing CXC chemokines primarily act on neutrophils. To investigate the general role of chemokines in viral diseases, we determined their release and expression patterns after infection of human monocytes with vesicular stomatitis virus (VSV). Human monocytes were productively infected by VSV. Surprisingly, VSV did not induce the release of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6. In contrast, we found a strong induction of the CC chemokine monocyte chemotactic protein-1 (MCP-1) and the non-ELR CXC chemokine interferon-gamma (IFN-gamma) inducible protein-10 (IP-10) by VSV on the gene and protein level. The expression and release of the neutrophil chemoattractants IL-8 and growth-related oncogene-alpha (GRO-alpha) remained unaffected after VSV infection. Our results indicate that the typical monocyte and lymphocyte-dominated leukocyte infiltration of virus-infected tissue is based on a selective induction of mononuclear leukocyte-attracting chemokines.