Molecular mechanism and possible therapeutic target of BAFF upregulation in SLE

SLE中BAFF上调的分子机制和可能的治疗靶点

• 批准号: 14570426
• 负责人: TAKEUCHI Tsutomu
• 金额: $ 2.56万
• 依托单位: Saitama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570426/
• 关键词: BAFF; autoantibody; TCRζ chain; splicing variants; microarray; BAFF; TCRζ chain; 3'untranslated region

BAFF is a member of the tumor necrosis factor (TNF) ligand family and co-activates B cells in vitro and in vivo-. BAFF-BAFF receptor system has a key role in the pathogenesis of the lupus-like disease in mice. In human, serum concentration of BAFF in SLE patients had been shown to be elevated compared with that in normal healthy controls. However, the mechanism of elevated serum BAFF in SLE patients has not been fully elucidated. To explore the mechanism, we attempt to examine mRNA level of BAFF in SLE patients.BAFF open reading frame cDNA was amplified by RT-PCR from PBL of SLE patients and healthy controls. Aliquots were taken from the reaction mixture every three cycles, starting with cycle 21. After electrophoresis in an agarose gels with ethidium bromide, UV-induced fluorescence of specific bands was quantitated as relative O.D. values. We calculated the difference in the number of cycles (Δn) needed to reach identical-yields of specific PCR products as compared to the internal G3 … More PDH standard. We then compared BAFF mRNA expression levels with clinical manifestations and laboratory findings in SLE patients. The experiments showed that BAFF mRNA was upregulated in SLE PBL, and it was closely associated with the increased level of serum anti-dsDNA antibody, but not serum immunoglobulin nor complement concentration, suggesting that BAFF has an important role in overproduction of autoantibody in SLE.In the earlier studies, we demonstrated the defective expression of TCR z chain in more than half of the patients with SLE. Since the defects appear to be central in dysfunction of SLE T cells, we, next attempt to investigate the molecular mechanism of BAFF expression, particularly focusing on its relationship to aberrant expression of TCR z. Chain. We constructed the mammalian expression vector containing two spliced variants form of TCR z chain, which are exclusively observed in SLE. We transfected these vectors into mouse T cell hybridoma, designated MA5.2, which lacks TCR z chain, and established stable transfectants expressing two spliced variants such as short 3' UTR and exon 7(-). Not only surface expression, but also cytoplasmic expression of TCR z chain was significantly downregulated in the two transfectants. MRNA stability assay clearly demonstrated that the downregulated expression of TCR z chain in the transfectants is resulted from the unstable mRNSA for both short 3'UTR and exon 7(-) variants. Microarray analysis showed that a series of mRNA are upregulated and downregulated in these two transfectants, when compared to MA5.2 transfected with wild form TCR z chain. The experiment showing that the mRNA of BAFF was not changed in the two TCR z variants, raises a possibility that increased BAFF expression observed in SLE T cells is independent abnormality, but not secondary to TCR z defect in SLE. Less

BAFF是肿瘤坏死因子（TNF）配体家族的成员，在体外和体内共激活B细胞。BAFF-BAFF受体系统在小鼠狼疮样疾病的发病机制中起关键作用。SLE患者血清BAFF浓度高于正常人。然而，SLE患者血清BAFF升高的机制尚未完全阐明。本研究采用RT-PCR方法从SLE患者外周血淋巴细胞中扩增出BAFF开放阅读框cDNA，并与正常对照组进行比较。从循环21开始，每三个循环从反应混合物中取出等分试样。用溴化乙锭在琼脂糖凝胶中电泳后，将特定条带的UV诱导荧光定量为相对O.D.。价值观我们计算了与内部G3相比，达到特异性PCR产物的相同产量所需的循环数（Δn）的差异 ...更多信息 PDH标准。然后，我们比较了BAFF mRNA表达水平与SLE患者的临床表现和实验室检查结果。实验结果表明，BAFF mRNA在SLE患者外周血淋巴细胞中表达上调，且与血清抗dsDNA抗体水平升高密切相关，而与血清免疫球蛋白和补体浓度无关，提示BAFF在SLE自身抗体的过度产生中起重要作用。由于这些缺陷似乎是SLE T细胞功能障碍的核心，我们接下来试图研究BAFF表达的分子机制，特别是关注其与TCR z异常表达的关系。链我们构建了哺乳动物表达载体，含有两个剪接形式的TCR z链，这是唯一观察到的SLE。我们将这些载体转染到缺乏TCR z链的小鼠T细胞杂交瘤（命名为MA5.2）中，并建立了表达两种剪接变体如短3' UTR和外显子7（-）的稳定转染子。在两种转染子中，TCR z链不仅表面表达，而且胞质表达均显著下调。MRNA稳定性测定清楚地表明，转染子中TCR z链的表达下调是由于短3 'UTR和外显子7（-）变体的不稳定mRNSA造成的。微阵列分析显示，当与用野生型TCR z链转染的MA5.2相比时，一系列mRNA在这两种转染子中上调和下调。实验表明，BAFF的mRNA在两种TCR z变体中没有改变，提出了在SLE T细胞中观察到的BAFF表达增加是独立异常的可能性，而不是继发于SLE中的TCR z缺陷。少

项目成果

Tsutomu Takeuchi, Tohru Abe.: "Role of adhesion molecules in Vasculitis Syndrome."Internal Medicne. 41. 41-44 (2002)

Tsutomu Takeuchi，Tohru Abe.：“粘附分子在血管炎综合征中的作用。”内科。

Takeuchi T, Tsuzaka K, Abe T.: "Altered expression of the T cell receptor-CD3 complex in systemic lupus erythematosus."Int Rev Immunol. (In press). (2004)

Takeuchi T、Tsuzaka K、Abe T.：“系统性红斑狼疮中 T 细胞受体-CD3 复合物的表达发生改变。”

竹内 勤, 天野宏一, 津坂憲政, 亀田秀人, 安倍達: "膠原病・リウマチ性疾患"日本内科学会雑誌. 93. 96-102 (2004)

Tsutomu Takeuchi、Koichi Amano、Norimasa Tsusaka、Hideto Kameda、Tatsu Abe：“胶原蛋白疾病和风湿病”日本内科学会杂志 93. 96-102 (2004)。

亀田秀人, 竹内 勤: "TNFを標的分子とした慢性炎症性疾患の治療 -抗TNF生物製剤による関節リウマチの寛解導入-"医学のあゆみ. 208. 336-342 (2004)

Hideto Kameda、Tsutomu Takeuchi：“使用 TNF 作为靶分子治疗慢性炎症疾病 - 使用抗 TNF 生物制剂诱导缓解类风湿性关节炎”，《医学史》208。336-342 (2004)。

亀田秀人, 竹内 勤: "Annual Review 呼吸器(工藤翔二、土屋了介、金沢実、大田健 編集)"中外医学社(東京). 290 (2004)

Hideto Kameda、Tsutomu Takeuchi：“年度回顾呼吸系统（由 Shoji Kudo、Ryosuke Tsuchiya、Minoru Kanazawa、Ken Ota 编辑）”Chugai Igakusha（东京）290（2004）。