Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection
Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection
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DOI：
10.1016/j.dci.2008.09.012
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发表时间：
2009-01-01
影响因子：
2.9
通讯作者：
Watanabe, Tasuku
中科院分区：
文献类型：
--
作者：
Komatsu, Koichiro;Tsutsui, Shigeyuki;Watanabe, Tasuku
为了确定鱼类肠上皮细胞 (IEC) 是否有助于粘膜免疫，我们建立了一种从虹鳟 Oncorhynchus mykiss 中分离 IEC 的方法，并检查了这些细胞中细胞因子的产生。通过用 1 mM 二硫苏糖醇 (DTT)/乙二胺四乙酸 (EDTA) 孵育肠片，释放肠上皮成分。通过在 35%/40% Percoll 梯度上离心，然后使用抗鳟鱼 IgM 抗血清进行磁性细胞分选，获得富含 IEC 的部分（纯度 >90%；存活率类似于 95%）。在有或没有致病菌杀鲑气单胞菌培养细胞 6 小时后，研究了鳟鱼 IEC 中 14 种细胞因子的基因表达谱。鳟鱼 IEC 可以产生多种细胞因子，其中当用活杀鲑鱼刺激细胞时，IL-1 β 和 TNF α 2 上调。使用抗鳟鱼 TNF 抗体进行的免疫组织化学分析证实，在肛门内用活杀鲑鱼攻击的鳟鱼的 IEC 中存在 TNF 蛋白。这些结果表明鳟鱼 IEC 是肠道免疫系统的重要触发因素。此外，福尔马林灭活的杀鲑鱼、该细菌的条件培养基或活的非致病性大肠杆菌不能上调这些细胞因子的表达。这些结果表明，IEC 产生的炎性细胞因子是由杀鲑鱼的粘附引起的，但不仅仅是由于 IEC 表面分子与细菌之间简单的配体-受体相互作用或对细菌分泌物的反应。 (C) 2008 Elsevier Ltd. 保留所有权利。
To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1 mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate similar to 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6 h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1 beta and TNF alpha 2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions. (C) 2008 Elsevier Ltd. All rights reserved.