Analysis of the non-self recognition mechanism of ascidian hemocytes

1)海鞘血细胞基因表达及信号转导途径分析：海鞘血细胞聚集是海鞘损伤后的早期炎症反应。我们已经报道了单克隆抗体A74强烈抑制罗氏嗜血杆菌的血细胞聚集。A74抗原蛋白是一种新型膜糖蛋白，它具有两个免疫受体酪氨酸激活基序（ITAMs）和几个已被提出在信号转导中发挥作用的基序。为了鉴定在H.roretzi血细胞中ITAMs下游发挥作用的蛋白质，我们进行了差异显示分析，以评估血细胞聚集前后mrna的差异表达。分离出4个扩增子（16A、18A、20A和20G-1），它们在诱导血细胞聚集后表达水平升高。结果表明，18A-1克隆体编码谷胱甘肽s -转移酶（GSTO）蛋白，而20G-1克隆体编码一种与其他蛋白不太相似的新蛋白。我们还发现抗tcr抗体可特异性诱导GSTO基因在小鼠t细胞杂交瘤中的表达。2)红细胞补体3受体和罗氏嗜血杆菌血浆中声速因子分析：罗氏嗜血杆菌血浆中含有两种声速因子，半乳糖特异性凝集素（Gal-lectin）和补体3。利用它们的抗体和荧光激活细胞分选器，我们发现gal凝集素仅与SRBC结合，而C3仅与酵母细胞结合。此外，我们发现H.roretzi血细胞具有整合素α亚基蛋白，其抗体抑制H.roretzi血细胞的c3依赖性吞噬。这些结果表明，H.roretzi血细胞具有整合素型受体作为C3受体的功能。少

1) Analysis of gene expression and signal transduction pathway of ascidian hemocytes : Hemocyte aggregation is an early inflammatory response upon injury in the ascidian Halocynthia roretzi. We have already reported that the monoclonal antibody A74 strongly inhibits hemocyte aggregation in H.roretzi. The A74 antigen protein is a novel membrane glycoprotein, and it has two immunoreceptor tyrosine-based activation motifs (ITAMs) and several motifs that have been proposed to play a role in signal transduction. To identify the proteins that function downstream of ITAMs in H.roretzi hemocytes, we carried out a differential display analysis to evaluate mRNAs differentially expressed before and after hemocyte aggregation. Four amplicons (16A, 18A, 20A and 20G-1), the expression levels of which increased after induction of hemocyte aggregation, were isolated. Their expressions were found to be induced by treatment with A74 antibody, but not by control mouse IgG, and were strongly inhibited by … More treatment with BAPTA-AM, Wortmannin and cyclosporin A.From the results of cDNA cloning, it was revealed that 18A-1 clone encoded a glutathione S-transferase omega (GSTO) protein, and 20G-1 clone encoded a novel protein that had little similarity to other proteins. We also found that GSTO gene expression was specifically induced in mouse T-cell hybridoma by treatment with anti-TCR antibody.2) Analyses of opsonic factors in H.roretzi plasma and complement 3 receptor of H.roreti hemocytes : H.roretzi plasma contains two opsonic factors, galactose-specific lectin (Gal-lectin) and complement 3. Using their antibodies and a fluorescence-activated-cell sorter, we found that Gal-lectin bound only to SRBC, while C3 bound only to yeast cells. Furthermore, we found that H.roretzi hemocytes had an integrin α subunit protein and that its antibody inhibited C3-dependent phagocytosis of H.roretzi hemocytes. These results suggest that H.roretzi hemocytes have an integrin-type receptor that functions as a C3 receptor. Less