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，在体外和体内共同激活 B 细胞，BAFF-BAFF 受体系统在小鼠狼疮样疾病的发病机制中发挥关键作用。与正常健康对照相比，SLE 患者血清 BAFF 浓度升高，但 SLE 患者血清 BAFF 升高的机制尚未完全阐明。为了探讨其机制，我们尝试检查 mRNA 水平。 SLE 患者中的 BAFF。通过 RT-PCR 从 SLE 患者和健康对照的 PBL 中扩增 BAFF 开放阅读框 cDNA，从第 21 个循环开始，每三个循环从反应混合物中取出等分试样。在含有乙锭的琼脂糖凝胶中进行电泳后。溴化物，特定条带的紫外线诱导荧光被定量为相对 O.D 值，我们计算了达到特定 PCR 相同产量所需的循环数差异 (Δn)。然后我们将 BAFF mRNA 表达水平与 SLE 患者的临床表现和实验室检查结果进行比较。实验表明，BAFF mRNA 在 SLE PBL 中上调，并且与水平升高密切相关。血清抗 dsDNA 抗体的浓度，但不是血清免疫球蛋白或补体浓度，表明 BAFF 在 SLE 自身抗体过量产生中发挥重要作用。由于缺陷似乎是 SLE T 细胞功能障碍的核心，我们接下来尝试研究 BAFF 表达的分子机制，特别关注其与 TCR z 链异常表达的关系。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，并建立了稳定的 TCR z 链剪接变体形式的哺乳动物表达载体。表达两个剪接的转染子，例如短3'UTR和外显子7(-)，不仅表面表达变体，而且TCR z链的细胞质表达在两个转染子中均显着下调。转染子中TCR z链的变化是由短3'UTR和外显子7(-)变异体的不稳定mRNA引起的。 微阵列分析表明一系列mRNA被上调和下调。在这两种转染子中，与用野生型TCR z链转染的MA5.2相比，该实验表明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表达增加的可能性。独立异常，但不是继发于 SLE 的 TCR z 缺陷。

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。”

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）。

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)。