Dong Fujing, Song Xiangdi, Xing Jing, Tang Xiaoqian, Sheng Xiuzhen, Chi Heng, Zhan Wenbin
Laboratory of pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, Qingdao, 266003, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071, China.
Fish Shellfish Immunol Rep. 2021 Oct 11;2:100030. doi: 10.1016/j.fsirep.2021.100030. eCollection 2021 Dec.
Dendritic cells (DCs) are the professional antigen presenting cells, which play important roles in regulating the immune response and tolerance. In mammals, CD83 is considered as a maturation marker for DCs, which is a type I membrane glycoprotein. In this research, the gene sequence of the CD83 in was obtained on NCBI, the sequence characteristics were studied and CD83 was detected in the head kidney, spleen, gut, gill, liver and muscle, with lower expression in the liver and muscle and higher expression in the head kidney and spleen. And then the CD83 recombinant protein was expressed and the specific antibodies (Abs) were prepared, transiently expressed CD83 eukaryotic proteins could be identified by western blot and indirect immunofluorescence. Then single cell suspensions of head kidney were obtained and DCs were collected by density gradient centrifugation after 7 days of cultivation. Giemsa staining revealed that the nuclei were kidney and dumbbell shaped, and the cell surfaces had dendritic or pseudopod-like protrusions. In addition, the DCs could phagocytose fluorescent microspheres, and the phagocytosis rate was 79.27±1.01%. After incubation with allogeneic lymphocytes, it was found that the lymphocytes clustered and proliferated significantly with the ratio ranging from 39.0% to 59.8% in a dose-dependent manner, which proved that the DCs could elicit mixed lymphocyte responses. After LPS immunization , the relative expression of CD83 in the head kidney and spleen showed a trend of increasing and then decreasing, peaking at 6 h. Meanwhile, the expression of CD83, MHC II, CD80/86 and CD40 were significantly up-regulated in DCs after stimulated with LPS for 24 h . And then IIF revealed that CD83 cells could be detected in the LPS-stimulated group using CD83 Abs, while no positive signal was detected in the control group, which suggested that CD83 protein may be considered as a specific marker for the maturation of DCs in teleost.
树突状细胞（DCs）是专职抗原呈递细胞，在调节免疫反应和免疫耐受中发挥重要作用。在哺乳动物中，CD83被认为是DCs的成熟标志物，它是一种I型膜糖蛋白。本研究在NCBI上获得了[具体物种]的CD83基因序列，研究了其序列特征，并在头肾、脾脏、肠道、鳃、肝脏和肌肉中检测了CD83，其中肝脏和肌肉中表达较低，头肾和脾脏中表达较高。随后表达了CD83重组蛋白并制备了特异性抗体（Abs），通过蛋白质免疫印迹法和间接免疫荧光法可鉴定瞬时表达的CD83真核蛋白。然后获得头肾单细胞悬液，培养7天后通过密度梯度离心收集DCs。吉姆萨染色显示细胞核呈肾形和哑铃形，细胞表面有树突状或伪足样突起。此外，DCs能够吞噬荧光微球，吞噬率为79.27±1.01%。与同种异体淋巴细胞孵育后，发现淋巴细胞显著聚集和增殖，比例在39.0%至59.8%之间呈剂量依赖性，这证明DCs能够引发混合淋巴细胞反应。经脂多糖（LPS）免疫后，头肾和脾脏中CD83的相对表达呈先升高后降低的趋势，在6小时达到峰值。同时，LPS刺激24小时后，DCs中CD83、主要组织相容性复合体II类分子（MHC II）、CD80/86和CD40的表达显著上调。然后间接免疫荧光法显示，使用CD83抗体在LPS刺激组中可检测到CD83细胞，而对照组未检测到阳性信号，这表明CD83蛋白可能被视为硬骨鱼中DCs成熟的特异性标志物。
