Morrison R N, Zou J, Secombes C J, Scapigliati G, Adams M B, Nowak B F
Aquafin CRC, School of Aquaculture, Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Locked Bag 1370, Launceston, Tasmania 7250, Australia.
Fish Shellfish Immunol. 2007 Nov;23(5):1015-31. doi: 10.1016/j.fsi.2007.04.003. Epub 2007 May 3.
Tumour necrosis factor-alpha (TNF-alpha) is a key mediator of inflammation during amoebiasis of humans and mice. Atlantic salmon (Salmo salar L.) are also susceptible to infection by amoebae (Neoparamoeba spp.), inflicting a condition known as amoebic gill disease (AGD). Here, the role of TNF-alpha in AGD-pathogenesis was examined. Two Atlantic salmon TNF-alpha transcripts designated TNF-alpha1 and TNF-alpha2 together with their respective genes were cloned and sequenced. TNF-alpha1 is 1379 bp and consists of a 738 bp open reading frame (ORF) translating into a predicted protein of 246 amino acids. TNF-alpha2 is 1412 bp containing an ORF and translated protein the same lengths as TNF-alpha1. An anti-rainbow trout TNF-alpha polyclonal antibody that bound recombinant Atlantic salmon TNF-alpha1 and TNF-alpha2 was used to detect constitutive and inducible expression of TNF-alpha in various tissues. The anti-TNF-alpha antibody bound to a TNF-like protein approximately 60 kDa that was constitutively expressed in a number of tissues in healthy Atlantic salmon. However, this protein was not detected in lysates from mitogen-stimulated head kidney leucocytes, despite up-regulation of TNF-alpha mRNAs under the same conditions. During the early onset of AGD in Atlantic salmon, there were no demonstrable differences in the gill tissue expression of TNF-alpha1, TNF-alpha2 nor the interleukin-1 beta (IL-1beta), inducible nitric oxide synthase (iNOS) and interferon gamma (IFN-gamma) mRNAs compared to tissue from healthy fish. In Atlantic salmon with advanced AGD, IL-1beta but not TNF-alpha1 or TNF-alpha2 mRNAs was up-regulated and was lesion-restricted. Given that Neoparamoeba spp. modulated both TNF-alpha2 and IL-1beta in head kidney leucocytes in vitro, it appears that rather than being recalcitrant to Neoparamoeba spp.-mediated TNF-alpha expression, either the parasite can influence the cytokine response during infection, there is ineffective signalling for TNF-alpha expression, or there are too few cells at the site of infection with the capacity to produce TNF-alpha. These data support our previous observation that IL-1beta mRNA expression is up-regulated in AGD-affected tissue and that TNF-alpha is not intrinsic in AGD-pathogenesis.
肿瘤坏死因子-α（TNF-α）是人类和小鼠阿米巴病炎症过程中的关键介质。大西洋鲑（Salmo salar L.）也易受变形虫（Neoparamoeba spp.）感染，引发一种称为阿米巴鳃病（AGD）的病症。在此，研究了TNF-α在AGD发病机制中的作用。克隆并测序了两个命名为TNF-α1和TNF-α2的大西洋鲑TNF-α转录本及其各自的基因。TNF-α1为1379 bp，由一个738 bp的开放阅读框（ORF）组成，可翻译成一个预测的246个氨基酸的蛋白质。TNF-α2为1412 bp，包含一个ORF，其翻译的蛋白质长度与TNF-α1相同。一种能与重组大西洋鲑TNF-α1和TNF-α2结合的抗虹鳟TNF-α多克隆抗体，用于检测TNF-α在各种组织中的组成型和诱导型表达。抗TNF-α抗体与一种约60 kDa的TNF样蛋白结合，该蛋白在健康大西洋鲑的许多组织中组成型表达。然而，尽管在相同条件下TNF-α mRNA上调，但在丝裂原刺激的头肾白细胞裂解物中未检测到该蛋白。在大西洋鲑AGD发病初期，与健康鱼的组织相比，鳃组织中TNF-α1、TNF-α2以及白细胞介素-1β（IL-1β）、诱导型一氧化氮合酶（iNOS）和干扰素γ（IFN-γ）mRNA的表达没有明显差异。在患有晚期AGD的大西洋鲑中，IL-1β mRNA上调，但TNF-α1或TNF-α2 mRNA未上调，且上调局限于病变部位。鉴于Neoparamoeba spp.在体外可调节头肾白细胞中的TNF-α2和IL-1β，似乎寄生虫并非难以介导TNF-α表达，而是在感染期间可影响细胞因子反应，或者TNF-α表达的信号传导无效，又或者感染部位产生TNF-α的细胞过少。这些数据支持了我们之前的观察结果，即IL-1β mRNA表达在AGD感染组织中上调，且TNF-α并非AGD发病机制所固有的。
