B CELL ACTIVATION INDUCED BY OLIGODEOXYNUCLEOTIDES CONTAINING CPG MOTIFS

含有两侧为两个嘌呤的未甲基化CpG二核苷酸的DNA 在5'侧上的两个嘧啶和在3'侧上的两个嘧啶（CpG基序”）引起 有效的B细胞活化。这表现在早期的 激活基因如p53 -1和c-fos，免疫球蛋白的分泌， IL-6，并在体外和体内进入细胞周期。这 活化与通过B细胞抗原受体的信号协同作用 （巴塞尔公约区域）。这种未甲基化的CpG基序出现的频率是 在微生物的DNA中也是如此。细菌DNA激活B细胞，但 而脊椎动物的DNA却没有。因此，由CpG基序激活的淋巴细胞可能 是一种重要的免疫防御机制，因为它区分了 微生物和自身DNA，似乎有效地促进 抗原特异性免疫 该CpG基序与CREB/ATF的结合位点几乎相同 转录因子家族，CRE。 CREB/ATF蛋白可以 转录调节许多基因，包括IL-1，c-fos和IL-6。 CpG ODN可与一种或多种CREB/ATF蛋白结合，并特异性地竞争 CREB/ATF与CRE的结合。这些数据提出了一种可能性， CpG ODN的作用可能是由于它们与一种或多种细胞因子相互作用， 更多CREB/ATF蛋白。 本提案的主要目标是，首先，确定如何 CpG DNA和BCR信号通路之间发生协同作用;以及 第二，确定CpG DNA 诱导IL-1、c-fos和IL-6的转录。第一特定 目的是确定CpG DNA和BCR信号通路是否 通过近端或更远端激活步骤相互作用。 第二 一个特定的目标将阐明CpG ODN诱导IL-6、c-fos和IL-1基因转录的体外研究 转录测定和启动子报告基因的转染 构建到B细胞中。第三个具体目标将首先确定， 表征，如果需要，克隆CREB/ATF或其它B细胞蛋白 结合CpG ODN;第二，确定这种结合如何影响 这些蛋白质的性质使用凝胶位移和超位移测定， Western和Southwestern印迹，免疫沉淀， 磷酸化、相关蛋白或DNA结合活性的变化。 完成这些研究将增进对 调节淋巴细胞活化的机制。 这些研究也有 可能导致意外免疫激活的影响， “反义”ODN在人基因治疗和DNA疫苗中的应用 含有CpG基序。 这些研究将确定 通过一类有前途的新型免疫调节剂进行免疫调节。

DNA containing an unmethylated CpG dinucleotide flanked by two purines on the 5' side and two pyrimidines on the 3' side (CpG motif") causes potent B cell activation. This is manifested by expression of early activation genes such as egr-1 and c-fos, secretion of immunoglobulin and IL-6, and entry into the cell cycle both in vitro and in vivo. This activation synergizes with signals through the B cell antigen receptor (BCR). Such unmethylated CpG motifs occur more than twenty times as often in microbial DNA as in vertebrates. Bacterial DNA activates B cells, but vertebrate DNA does not. Thus, lymphocyte activation by CpG motifs may be an important immune defense mechanism since it distinguishes between microbial and self DNA and appears to effectively promote antigen-specific immunity. This CpG motif is nearly identical to the binding site for the CREB/ATF family of transcription factors, the CRE. CREB/ATF proteins can transcriptionally regulate many genes, including egr-1, c-fos, and IL-6. CpG ODN can bind one or more CREB/ATF proteins, and specifically compete the binding of CREB/ATF to the CRE. These data raise the possibility that the effects of CpG ODN may result from their interactions with one or more CREB/ATF proteins. The broad goals of the present proposal are first, to determine how synergy occurs between the CpG DNA and the BCR signaling pathways; and second, to determine the molecular mechanism through which CpG DNA induces the transcription of egr-1, c-fos, and IL-6. The first specific aim will determine whether the CpG DNA and BCR signaling pathways interact through proximal or more distal activation steps. The second specific aim will elucidate the molecular mechanism(s) through which CpG ODN induces egr-1, c-fos, and IL-6 transcription using in vitro transcription assays, and transfection of promoter reporter gene constructs into B cells. The third specific aim will first, identify, characterize, and, if necessary, clone CREB/ATF or other B cell proteins that bind CpG ODN; and second, determine how this binding affects the properties of these proteins using gel shift and supershift assays, western and southwestern blots, immunoprecipitation, and assessment of changes in phosphorylation, associated proteins, or DNA binding activity. Completion of these studies will improve the understanding of the mechanisms regulating lymphocyte activation. These studies also have implications for possible unintended immune activation resulting from the use of "antisense' ODN and for human gene therapy and DNA vaccines containing CpG motifs. These studies will determine the mechanism of immune regulation by a promising new class of immunomodulators.