Compound 48/80活化的肥大细胞对树突状细胞归巢的影响

Impact of compound 48/80 activated mast cell on dendritic cell homing to lymph nodes

目的:检测compound 48/80(C48/80)活化的肥大细胞对树突状细胞(DC)向局部引流淋巴结迁移的影响,初步探讨C48/80增强适应性免疫应答的机制。方法:体外培养C57BL/6小鼠骨髓来源的DC(BM-DC)和MC/9肥大细胞,Transwell趋化小室实验检测C48/80处理的MC/9细胞对BM-DC向趋化因子CCL21迁移的影响。甲苯胺蓝染色法检测小鼠肩背部皮肤注射C48/80后肥大细胞脱颗粒状态;流式细胞术检测肩上和腋窝淋巴结CD11c+DC的数量。小鼠肩背部两侧各注射荧光微球标记的BM-DC,制备局部引流淋巴结冷冻切片,荧光显微镜观察C48/80处理对外源性DC归巢的影响。采用负载卵白蛋白的BM-DC(OVA-DC)免疫C48/80处理及对照小鼠,WST-8试剂检测局部引流淋巴结淋巴细胞的增殖活性。结果:采用小鼠骨髓细胞成功培养并获得大量典型的BM-DC。MC/9肥大细胞活化上清可促进BM-DC向CCL21迁移(P<0.01)。皮内注射C48/80后30 min可诱导局部皮肤明显肥大细胞脱颗粒。C48/80注射部位24 h出现明显炎细胞浸润,局部引流淋巴结细胞总数和DC细胞数量增多。注射荧光微球标记的BM-DC后48 h淋巴结冷冻切片显示C48/80处理侧荧光阳性的DC细胞数量明显多于C48/80未处理侧。OVA-DC免疫小鼠显示C48/80处理组抗原特异性淋巴细胞增殖能力较对照组明显增高(P<0.05)。结论:C48/80诱导的肥大细胞活化能够促进DC向局部引流淋巴结归巢,增强特异性淋巴细胞增殖能力,参与适应性免疫应答。

Objective ：To investigate the effect of compound 48/80（ C48/80） activated mast cell on dendritic cell homing tdraining lymph nodes（DLN） in order to explore the potential mechanism of C48/80 promoting adaptive immune response. MethodsC57BL/6 mice bone marrow derived dendritic cells（BM-DC） and MC/9 mast cell line were propagated in vitro. Chemotaxis of BM-DCsupplemented with supernatant of C48/80 treated MC/9 to CCL21 was measured by transwell chemotaxis assay. C48/80 was injectedinto murine scapular site,local skin mast cell degranulation was detected by toluidine blue staining method. The number of CD11c＋ DCin draining lymph nodes （ DLN） was detected by Flow cytometry. Exogenous green microbeads labeled BM-DC homing to DLN wadetected by fluromicroscope. The mice pretreated with or without C48/80 were vaccinated by ovalbumin pulsed DC（OVA-DC）,DLNlymphocyte proliferation was detected by WST-8 reagent. Results： A large amount of typical BM-DC were haryested from media supplemented with murine recombinant IL-4 and GM-CSF. Product of activated MC/9 enhanced chemotaxis of BM-DC to CCL21（ P〈0. 01）.Intradernal injection of C48/80 induced local mast cell obvious degranulation and local inflammation. Mice pretreated with C48/80demonstrated high number of total cells and DC cells in DLN. The number of fluorescent positive BM-DC in DLN also increased in C4880 pretreated mice. Antigen specific lymphocyte proliferation enhanced in OVA-DC inoculated C48/80 pretreated mice. Conclusion：Mast cell activation induced by C48/80 can enhance DC homing to DLN and increase specific lymphocyte proliferation,which may beone of a mechanism of C48/80 in promoting adaptive immune response.