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）配体系列的成员，并在体外和体内共同激活B细胞。 BAFF-BAFF受体系统在小鼠类似狼疮样疾病的发病机理中具有关键作用。在人类中，与正常健康对照组相比，SLE患者的BAFF的血清浓度已被证明升高。但是，SLE患者血清BAFF升高的机制尚未完全阐明。为了探索该机制，我们尝试检查SLE患者的BAFF的mRNA水平。BAFF开放阅读框架cDNA通过SLE患者PBL和健康对照组的RT-PCR扩增。从每三个周期开始，从循环21开始，从反应混合物中取等分试样。在带有溴化乙锭的琼脂糖凝胶中电泳后，将特异性带的紫外线诱导的荧光定量为相对O.D。值。与内部G3相比，我们计算了达到特定PCR产物相同收益的周期数（ΔN）的差异。然后，我们将BAFF mRNA表达水平与SLE患者的临床表现和实验室发现进行了比较。实验表明，BAFF mRNA在SLE PBL中进行了更新，并且与血清抗DSDNA抗体的水平升高，但没有血清免疫球蛋白或完成浓度相关，这表明BAFF在早期的研究中在SLE的过度生产中具有重要的作用。由于缺陷似乎是SLE T细胞功能障碍的中心，因此我们下一步尝试研究BAFF表达的分子机制，尤其是关注其与TCR Z异常表达的关系。链。我们构建了包含TCR Z链的两种拼接变体形式的哺乳动物表达载体，这些形式仅在SLE中观察到。我们将这些载体转换为缺乏TCR Z链的MA5.2的小鼠T细胞杂交瘤，并建立了表达两个拼接变体的稳定转化体，例如短3'UTR和EXON 7（ - ）。在两个转化体中，不仅表面表达，而且TCR Z链的细胞质表达显着下调。 mRNA稳定性测定清楚地表明，转化体中TCR Z链的下调表达是由短3'UTR和外显子7（ - ）变体的不稳定mRNSA引起的。微阵列分析表明，与MA5.2用野生形式的TCR Z链翻译相比，这两个转化器中的一系列mRNA被上调并下调。该实验表明，在两个TCR Z变体中，BAFF的mRNA并未改变，这提出了一种可能在SLE T细胞中观察到的BAFF表达增加的可能性是独立的，但不是SLE中TCR Z缺陷的继发性。较少的

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)。