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Functional expression of granzyme B in human plasmacytoid dendritic cells:a role in allergic inflammation

机译:颗粒酶B在人浆细胞样树突状细胞中的功能性表达:在过敏性炎症中的作用

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Background Plasmacytoid dendritic cells (pDCs) are involved in a variety of immune functions. However, the expression of cytotoxic granule proteins like granzymes and perform in human pDCs is still poorly understood.Objective The aim of this study was to systematically analyse the expression and regulation of cytotoxic granule proteins in human pDCs.Methods The expression of cytotoxic proteins was analysed by RT-PCR, flow cytometry, and fluorescence microscopy. The functional expression of these proteins was confirmed in a flow-cytometry-based cytotoxicity assay using K562 cells as targets. In order to analyse the regulation of pDC-derived cytotoxic proteins in infectious and allergic diseases, human pDCs were analysed after stimulation with toll-like receptor (TLR)7/9 ligands and in the human asthma model of segmental allergen challenge.Results Granzyme B (GrB), but not the granzymes A, H, K, M or perforin, was specifically expressed by human pDCs and this GrB expression was up-regulated by IL-3 stimulation. In addition, IL-3-stimulated pDCs were found to kill K562 cells in a GrB- and caspase-dependent manner. TLR7/9 ligands significantly suppressed GrB expression in pDCs. In contrast, there was an up-regulation of GrB in endobronchial pDCs 24 h after allergen challenge, and this was accompanied by enhanced GrB concentrations in bronchoalveolar lavage fluid. Conclusion We report the selective expression of GrB in human pDCs and show for the first time pDC-mediated GrB- and caspase-dependent cytotoxicity against target cells. In addition, the regulation of GrB expression was investigated in vitro and in vivo providing an evidence for a specific role of pDC-derived GrB in allergic inflammation.
机译:背景浆细胞样树突状细胞(pDC)参与多种免疫功能。然而,细胞毒性颗粒蛋白(如颗粒酶)在人类pDCs中的表达尚不清楚。目的本研究的目的是系统分析人类pDCs中细胞毒性颗粒蛋白的表达和调控。方法分析细胞毒性蛋白的表达通过RT-PCR,流式细胞仪和荧光显微镜检查。这些蛋白的功能表达在以K562细胞为靶标的基于流式细胞仪的细胞毒性试验中得到证实。为了分析pDC衍生的细胞毒性蛋白在传染性和变应性疾病中的调节作用,在使用Toll样受体(TLR)7/9配体刺激后和在人类哮喘的分段性过敏原激发模型中对人pDC进行了分析。结果Granzyme B (grB),但不是颗粒酶A,H,K,M或穿孔素,是由人pDC特异性表达的,而该GrB的表达受IL-3刺激上调。另外,发现IL-3刺激的pDC以依赖于GrB和胱天蛋白酶的方式杀死K562细胞。 TLR7 / 9配体显着抑制了pDC中GrB的表达。相比之下,变应原攻击后24小时,支气管内pDC中的GrB上调,同时支气管肺泡灌洗液中的GrB浓度升高。结论我们报道了在人pDC中GrB的选择性表达,并首次显示了pDC介导的GrB和caspase依赖性对靶细胞的细胞毒性。另外,在体外和体内研究了GrB表达的调节,为pDC衍生的GrB在变应性炎症中的特定作用提供了证据。

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