体外LPS刺激及CD40的配基化对sCD40基因修饰DCs TLR4-MD2表达及IL-12分泌的影响

目的： 研究体外LPS刺激及CD40的配基化对可溶性CD40（sCD40）基因修饰树突状细胞TLR4-MD2表达及IL-12分泌的影响,为有效利用树突状细胞诱导特异性移植免疫耐受提供实验依据。方法: 脂质体法将质粒pEGFP-N1/sCD40及空质粒pEGFP-N1转染DC2.4细胞株;应用LPS及抗CD40单抗刺激6 h,流式细胞仪检测DC表面TLR4-MD2的表达,RT-PCR法检测DC 的TLR4 mRNA 表达水平,并用ELISA法检测细胞因子IL-12p70的分泌。结果: LPS刺激下调DC表面TLR4-MD2的表达,同时给予CD40配基化可引起TLR4-MD2的表达显著增高;CD40配基化对DC TLR4mRNA 水平表达无影响，但可部分地增高LPS引起的TLR4mRNA 表达降低;此外,CD40的配基化可显著诱导LPS刺激后IL-12分泌增加。sCD40基因修饰DC可拮抗以上作用。结论: 体外LPS及抗CD40单抗刺激下,sCD40基因修饰树突状细胞可显著下调其表面TLR4-MD2的表达,IL-12p70分泌减少，可能与阻断胞浆内的TLR4-MD2的转运过程有关。     

小鼠髓系树突状细胞4-1BB及4-1BBL表达调节

探讨小鼠髓系树突状细胞(bone marrow-derived dendritic cells,BMDC)共刺激分子4-1BB及其配体4-1BBL表达的变化。将促DC成熟活化因子CD40L-CHO、TNF-α、LPS和IFN-γ,免疫负性调节因子IL-10以及各成熟活化因子与IL-10联合加入BMDC中,观察BMDC上4-1BB及4-1BBL表达的变化。结果显示,加入成熟活化因子的各组BMDC上4-1BB及4-1BBL表达与对照组相比有显著上调(P<0.05)。而IL-10组与对照组相比两者的表达显著下调(P<0.05),且各成熟活化因子与IL-10联合应用与单用IL-10组相比,BMDC上4-1BB和4-1BBL表达上调,有显著性差异(P<0.05)。提示成熟活化因子不仅能上调BMDC上4-1BB和4-1BBL的表达并且能有效拮抗mIL-10对BMDC上4-1BB和4-1BBL表达的下调作用。     

α-MSH对脂多糖诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA 表达的影响

目的：观察α-黑色素细胞刺激素(α-MSH)对脂多糖（LPS）诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达的影响，探讨α-MSH拮抗LPS的作用机制。方法：用半定量逆转录多聚酶链反应（RT-PCR）的方法检测LPS诱导体外培养的小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达水平和给予α-MSH后对CD14和TLR4 mRNA表达的影响。结果：正常静息小鼠腹腔巨噬细胞只表达少量的CD14和TLR4 mRNA,给予LPS刺激后6 h，两者表达明显强于正常对照（P<0.01），并且其表达量随着LPS刺激时间的增加维持在高水平，24 h达到峰值，在48 h CD14 mRNA的表达降到正常水平，而TLR4 mRNA的表达仍然维持在高水平。在LPS刺激的同时给予α-MSH，CD14和TLR4 mRNA的表达则明显低于LPS组（P<0.05），而且α-MSH这种效应与其使用浓度有关，0.1 nmol/L α-MSH不影响LPS诱导的CD14和TLR4 mRNA的表达，而当α-MSH的浓度达到1、10、100 nmol/L则能显著影响CD14和TLR4 mRNA的表达（P<0.05），但各个浓度组之间的作用没有明显差别（P>0.05）。结论：α-MSH抗LPS的效应可能与其下调LPS信号转导通路关键受体CD14和TLR4 mRNA的表达有关，从而干扰LPS跨膜信号转导，阻碍巨噬细胞活化。     

Lewis肺癌细胞TLR4对Foxp3表达的调控

目的:探讨小鼠Lewis肺癌(Lewis lung cancer,LLC)细胞中TLR4对Foxp3表达的调控作用。方法:选择LPS为配体活化TLR4,采用RT-PCR方法检测LPS在不同浓度(0,1,10μg/ml)和不同时间点(12,24,36,48小时)Foxp3 mRNA的表达量的变化,流式细胞术检测有效浓度LPS 10μg/ml和最佳作用时间点24小时Foxp3蛋白和TLR4蛋白表达量的变化,RT-PCR和流式细胞术检测anti-TLR4/MD2抗体阻断TLR4后再用LPS刺激LLC细胞Foxp3表达量的变化。结果:LPS在10μg/ml浓度作用LLC细胞后可显著上调Foxp3 mRNA的表达量,与未刺激组相比差异显著(P<0.05);LPS最佳作用时间为24小时;LPS在10μg/ml浓度作用LLC细胞24小时后可显著上调Foxp3蛋白和TLR4蛋白的表达量,与对照组相比差异显著(P<0.05);阻断TLR4后再用LPS刺激LLC细胞,Foxp3的表达量较未阻断组明显降低(P<0.05)。结论:LLC细胞TLR4蛋白参与对Foxp3的表达调控,TLR4可能是Foxp3的上游信号分子。     

汉滩病毒感染血管内皮细胞引起TLR4表达上调

目的 探讨汉滩病毒感染血管内皮细胞后,Toll样受体(TRY)分子的表达变化,为抗感染免疫和致病机制研究提供重要资料.方法 分别用LPS,CL097,Poly I:C以及汉滩病毒76-118刺激血管内皮细胞,6 h后提取总RNA,进行反转录获得cDNA,再分别用各自的引物进行PCR反应,产物用1%琼脂糖凝胶电泳分析,并用间接免疫荧光测定汉滩病毒感染血管内皮细胞后TLR4的表达.结果 汉滩病毒刺激感染后,血管内皮细胞TLR2、TLR4转录水平增高,TLR3转录水平降低,细胞膜表面的TLR4表达上调.结论汉滩病毒76-118感染血管内皮细胞后,可以引起TLRs分子转录水平发生改变,其中TLR4表达上调,固有免疫参与了汉滩病毒致病过程.     

脂多糖通过TLR4-Src信号介导微囊胞吞血管内皮钙黏蛋白和增加血管通透性

目的:探讨脂多糖(LPS)诱导微囊胞吞血管内皮钙黏蛋白(VE-Cad)的可能机制。方法:培养人血管内皮细胞株CRL-2922,当其生长至融合状态时分为正常对照组(不予再处理)、LPS处理组(采用10μg/ml LPS分别与CRL-2922再培养1h、2h、4h和6h)、LPS+抑制剂组包括Toll样受体4(TLR4)抑制剂CLI-095和Src抑制剂SU6566[在LPS培养细胞时分别加入CLI-095(5μg/ml)和SU6566(2μmol/L)再培养4h]。采用Western Blotting法检测各组Src蛋白表达、Cav1磷酸化和VE-Cad质膜蛋白表达,以及Cav1与VE-Cad共沉淀水平;采用培养小室半透膜培养细胞,并检测相关各组细胞荧光透过率,以反映血管通透性。结果:LPS处理不同时间组Src蛋白表达均较正常对照组升高(P0.05);与正常对照组比较,LPS处理4h组Cav1磷酸化增强(P0.05)、VE-Cad质膜蛋白表达下调、Cav1与VE-Cad共沉淀水平升高(P0.05),单层细胞荧光透光率增加(P0.05);TLR4抑制剂和Src抑制剂可显著降低LPS增高的Src蛋白高表达和Cav1高度磷酸化(P0.05),上调VE-Cad质膜蛋白表达(P0.05),下调Cav1与VE-Cad共沉淀水平(P0.05),改善单层内皮细胞通透性(P0.05)。结论:LPS可能通过TLR4-Src信号途径诱导微囊胞吞VE-Cad和增加血管通透性。     

UⅡ／UT系统对LPS刺激枯否细胞固有免疫炎症信号通路TLR4-IRF3的影响

目的：探讨Urotensin Ⅱ（ UⅡ）/UT系统对脂多糖（ Lipopolysaccharide ,LPS）刺激枯否细胞（ Kupffer cell ,KC）固有免疫炎症信号通路Toll样受体4（Toll-like receptor 4,TLR4）-干扰素调节因子3（Interferon regulatory factor 3,IRF3）的影响。方法：体外分离培养大鼠肝KCs,KCs培养上清液促炎性细胞因子IL-6、IFN-β和IFN-γ分泌水平采用ELISA分析检测,细胞表面TLR4的表达采用流式细胞技术分析,IRF3基因和蛋白表达情况分别采用real-time PCR和Western blot 分析方法检测。结果：LPS刺激后,KCs培养上清液IL-6、IFN-β和IFN-γ分泌水平、细胞表面TLR4表达阳性细胞率及细胞内IRF3 mRNA表达水平均显著升高,UT拮抗剂urantide预处理抑制了LPS刺激诱导KCs对上述分子的上调表达;LPS的应用也造成了KCs胞核内IRF3蛋白表达水平升高,而使胞浆内IRF3蛋白表达水平降低,urantide预处理后,抑制了LPS诱导KCs核内IRF3蛋白上调和胞浆水平下调。结论：UⅡ/UT系统通过对TLR4-IRF3通路的正性调控作用,介导了或至少部分介导了LPS刺激KCs的免疫性炎症分泌效应。     

地塞米松对小鼠脾脏巨噬细胞内TLR4和TLR2表达的影响

为观察小鼠脾巨噬细胞内的TLR4和TLR2在脂多糖 (LPS )刺激后 ,地塞米松 (Dx )对其表达量的影响 ,在向BALB/c小鼠腹腔内注射LPS和Dx后采用贴壁法分离小鼠脾巨噬细胞 ,通过半定量RT PCR方法测定了TLR4和TLR2mRNA的表达量。结果显示 :①LPS刺激后 ,小鼠脾巨噬细胞内TLR4mRNA显著上调 (吸光度比为 0 11) ,TLR2上调不明显 (吸光度比为0 0 0 8) ;②预先注射不同剂量Dx ,分别为 0 1mg/kg、 1mg/kg、 10mg/kg ,再用LPS刺激小鼠。注射了Dx后巨噬细胞TLR4mRNA表达量较单纯注射LPS的小鼠显著降低 (吸光度比分别为 0 0 93、 0 0 5 0和 0 0 14 ) ;但TLR2mRNA表达量随着Dx使用剂量增加而增加 (吸光度比分别为 0 0 5 4、 0 0 80和 0 16 1)。该项研究表明TLR4是参与LPS引起炎症反应的主要Toll样受体 ,而TLR2不起主要作用 ;Dx对LPS刺激后TLR 4mRNA的表达有抑制作用 ,而对TLR2mRNA的表达有增强作用 ;Dx抑制LPS引起的炎症反应可能与抑制TLR4的表达有关     

人参二醇组皂苷对内毒素休克大鼠脑皮质TLR4 mRNA表达的影响

目的：通过观察内毒素休克大鼠脑皮质中NOS活性、NO含量和TLR4 mRNA的表达及人参二醇组皂苷（PDS）对其的影响，探讨内毒素引起脑组织损伤的分子机制。 方法： 大鼠随机分为实验对照（control）组、内毒素休克（LPS）组、地塞米松（LPS+Dex）组和人参二醇组皂苷（LPS+PDS）组。大鼠静脉注射内毒素（4 mg/kg）4 h后测定脑组织中NOS活性、NO含量及TLR4 mRNA的表达。 结果： LPS+Dex组和LPS+PDS组NOS活性、NO2-/NO3-含量显著低于LPS组（P＜0.05），TLR4 mRNA表达亦明显低于LPS组。 结论： PDS能够下调脑组织中TLR4 mRNA的表达，降低NOS活性、NO含量，对中枢神经系统具有保护作用。     

CD40-mediated up-regulation of Toll-like receptor 4-MD2 complex on the surface of murine dendritic cells

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, which are non-self macromolecular components of pathogens that allow the innate-immune system to recognize infection. TLRs are expressed on macrophages and dendritic cells (DC). TLR stimulation or CD40 agonists can induce inflammatory cytokine secretion from macrophages and DC, and promote DC maturation. The regulation of TLR expression by inflammation has begun to be explored. Our studies have focused on the regulation of TLR4 surface expression on DC. TLR4, along with the adaptor molecule MD2, is involved in the recognition of lipopolysaccharide (LPS). CD40 stimulation via cross-linked anti-CD40 monoclonal antibody (mAb) up-regulates TLR4-MD2 surface expression on a DC cell line (DC2.4) and on ex vivo-cultured splenic DC. LPS treatment down-regulated surface TLR4-MD2 on DC2.4 cells, but if combined with anti-CD40 mAb, increased TLR4-MD2 expression was observed. The increased TLR4-MD2 surface expression by any treatment did not correlate with TLR4 mRNA levels. The functional consequence of increased TLR4-MD2 expression following LPS and anti-CD40 treatment was examined. Although CD40 prestimulation did slightly enhance interleukin-12p70 secretion after LPS restimulation, simultaneous anti-CD40 mAb and LPS treatment, which up-regulates TLR4-MD2 complex, does not restore DC responsiveness to subsequent LPS.     

Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.     

Expression and function of 4-1BB and 4-1BB ligand on murine dendritic cells

4-1BB (CDw137) and its ligand (4-1BBL) have been implicated in cellular immune responses. To further characterize the expression and function of 4-1BBL, we newly generated an anti-mouse 4-1BBL mAb (TKS-1), which can inhibit the interaction of 4-1BBL with 4-1BB. Flow cytometric analyses using TKS-1 and an anti-mouse 4-1BB mAb indicated that 4-1BB was inducible on both CD4(+) and CD8(+) splenic T cells by stimulation with immobilized anti-CD3 mAb, but 4-1BBL was not expressed on resting or activated T cells. 4-1BBL expression was inducible on splenic B cells by stimulation with anti-IgM antibody plus anti-CD40 mAb, on peritoneal macrophages by stimulation with lipopolysaccharide (LPS) and on splenic dendritic cells (DC) by stimulation with anti-CD40 mAb or LPS. Interestingly, splenic DC expressed 4-1BB constitutively, which was down-regulated by anti-CD40 stimulation. Co-culture of splenic DC with 4-1BBL-transfected cells or 4-1BBL-expressing tumor cell lines led to cytokine (IL-6 and IL-12) production and co-stimulatory molecule up-regulation by splenic DC, indicating that 4-1BBL can directly activate DC. Moreover, IL-12 production by anti-CD40-stimulated DC was partially inhibited by TKS-1. These results suggest that 4-1BB expressed on DC may be involved in DC activation through DC--tumor interaction and DC--DC interaction.     

TLR9 cooperates with TLR4 to increase IL-12 release by murine dendritic cells

Toll-like receptors (TLR) are expressed on the surface or intracellularly by dendritic cells (DC) and recognize specifically different pathogen-associated molecular patterns (PAMPs). Increasing evidence suggests that TLR expressed by DC can cooperate to synergize their functions. Here, we describe the cooperation of TLR9 and TLR4 triggering of murine bone marrow derived DC by CpG oligonucleotides and LPS, respectively. The simultaneous DC stimulation of LPS and CpG showed additive effects on the production of IL-12 but not on other cytokines, such as TNF, IL-6 or IL-10. CpG pretreatment before LPS induced five times more IL-12p40 and IL-12p70 production by DC, whereas LPS pretreatment before CpG showed no effect. The optimal time interval between CpG and LPS treatment was 4h and the synergistic effects were dependent on myeloid differentiation factor 88 (MyD88) but independent from the DNA backbone and did not mediate by nucleosome remodeling. The stimulatory effect could be further enhanced by addition of IFN-gamma but not anti-CD40 antibodies. These data show, that TLR4 and TLR9 can cooperate to increase selectively IL-12 production by DC.     

转化生长因子β1抑制树突状细胞的成熟及下调TLR4的表达

目的：研究转化生长因子β1（TGF-β1）对小鼠来源树突状细胞（DC）功能的影响。 方法: 在培养体系中同时应用GM-CSF和TGF-β1培养的TGF β-DC，用脂多糖（LPS）观察其对外源刺激的反应，流式细胞仪（FCM）检测细胞表型，应用BrdU ELISA法通过96 h混合淋巴细胞反应（MLR）检测其同种异基因刺激能力，ELISA法测IL-12 p70的分泌水平，分别用半定量RT-PCR法和FCM检测Toll-like受体4（TLR4）表达。 结果: TGF β-DC与常规培养的未成熟DC（imDC）相比，CD80、CD86、I-Ab、CD40表达更低。LPS对TGF β-DC的促成熟作用反应不明显，其表面共刺激分子升高的幅度不大，异基因的刺激能力提高不显著，且IL-12 p70的分泌下降。RT-PCR与FCM都显示TGF β-DC较imDC弱表达TLR4。 结论: TGF β1能抑制DC共刺激分子的表达，TGF β-DC能抵抗LPS的促成熟作用，并可能与其TLR4表达下降有关。     

Involvement of ERK, p38 and NF-kappaB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll-like receptors (TLR) are sentinel receptors capable of recognizing pathogen-associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG-containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram-positive and Gram-negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up-regulation of co-stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up-regulated following LPS stimulation. The up-regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor-alpha induced by LPS plus CpG ODN. While inhibition of extracellular signal-related kinase and NF-kappaB activation suppressed the up-regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up-regulation of TLR2 and TLR4 mRNA expression but enhanced the up-regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up-regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.     

DC‐SIGN reacts with TLR‐4 and regulates inflammatory cytokine expression via NF‐κB activation in renal tubular epithelial cells during acute renal injury

In the pathological process of acute kidney injury (AKI), innate immune receptors are essential in inflammatory response modulation; however, the precise molecular mechanisms are still unclear. Our study sought to demonstrate the inflammatory response mechanisms in renal tubular epithelial cells via Toll‐like receptor‐4 (TLR‐4) and dendritic cell‐specific intercellular adhesion molecule 3‐grabbing non‐integrin 1 (DC‐SIGN) signalling. We found that DC‐SIGN exhibited strong expression in renal tubular epithelial cells of human acute renal injury tissues. DC‐SIGN protein expression was increased significantly when renal tubular epithelial cells were exposed to lipopolysaccharide (LPS) for a short period. Furthermore, DC‐SIGN was involved in the activation of p65 by TLR‐4, which excluded p38 and c‐Jun N‐terminal kinases (JNK). Interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α expression was decreased after DC‐SIGN knock‐down, and LPS induced endogenous interactions and plasma membrane co‐expression between TLR‐4 and DC‐SIGN. These results show that DC‐SIGN and TLR‐4 interactions regulate inflammatory responses in renal tubular epithelial cells and participate in AKI pathogenesis.