Molecular mechanism and possible therapeutic target of BAFF upregulation in SLE

BAFF是肿瘤坏死因子（TNF）配体家族的一员，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起关键作用。在人体内，与正常健康对照相比，SLE患者血清BAFF浓度已被证明升高。然而，SLE患者血清BAFF升高的机制尚未完全阐明。为了探讨其机制，我们试图检测SLE患者BAFF的mRNA水平。采用RT-PCR方法从SLE患者和健康对照的PBL中扩增出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提取等量。在琼脂糖凝胶中与溴化乙啶电泳后，将特定波段的紫外诱导荧光定量为相对od值。我们计算了与内部G3…更多PDH标准相比，达到相同产量所需的循环次数（Δn）的差异。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结果。实验表明，BAFF mRNA在SLE PBL中表达上调，且与血清抗dsdna抗体水平升高密切相关，但与血清免疫球蛋白和补体浓度升高无关，提示BAFF在SLE自身抗体过量产生中起重要作用。在早期的研究中，我们发现TCR z链在超过一半的SLE患者中存在缺陷表达。由于这些缺陷似乎是SLE T细胞功能障碍的核心，我们下一步将尝试研究BAFF表达的分子机制，特别是关注其与TCR z链异常表达的关系。我们构建了包含两种TCR z链剪接变体形式的哺乳动物表达载体，这两种变体仅在SLE中观察到。我们将这些载体转染到缺乏TCR z链的小鼠T细胞杂杂瘤MA5.2中，并建立了表达短3' UTR和外显子7（-）两种剪接变体的稳定转染物。在两种转染物中，不仅表面表达，胞质中TCR z链的表达也显著下调。MRNA稳定性分析清楚地表明，转染物中TCR z链的表达下调是由于短3'UTR和外显子7（-）变体的mRNSA不稳定所致。微阵列分析显示，与野生型TCR z链转染的MA5.2相比，这两种转染物中一系列mRNA表达上调和下调。实验显示，BAFF mRNA在两种TCR z变异中均未发生改变，提示SLE T细胞中BAFF表达升高可能是独立异常，而非继发于SLE TCR z缺陷。少

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