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Molecular mechanisms of isotype switch recombination.

同型转换重组的分子机制。

基本信息

批准号：
11470083
负责人：
TAKATSU Kiyoshi
金额：
$ 9.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470083/
关键词：
IL-5 CD38 Switch recombination germline transcripts Stat5 NF-κB Btk Blimp-1 CD40 Blimp-1 stat5 IL-4 DNA組み換え

项目摘要

Mouse B cells express CD38 whose ligation by anti-CD38 antibody induces their proliferation and protection from apoptosis. We previously showed that stimulation of mouse splenic B cells with interleukin 5 (IL-5) together with CS/2, an anti-mouse CD38 monoclonal antibody, induces production of IgG1 and IgM.Here we examined the role of IL-5 and CS/2 in the expression of germline γ1 transcripts and the generation of reciprocal products forming DNA circles as by products of μ-γ1 switch recombination. By itself, CS/2 induced significant expression of germline γ1 transcripts in splenic naive B cells, whereas IL-5 neither induced nor enhanced germline γ1 expression. Increased cellular content of reciprocal product, which is characteristic of μ-γ1 recombination, was not observed after culturing B cells with CS/2, but increased reciprocal product along with high levels of IgG1 secretion was found when B cells were cultured with CS/2 plus IL-5. Although IL-4 did not, by itself, induce μ-γ1 recom … More bination in B cells stimulated with CS/2, in conjunction with CS/2 plus IL-5, IL-4 dramatically enhanced sterile γ1 transcription and IgG1 production. These results demonstrate that CD38 ligation induces only germline γ1 transcription and that IL-5 promotes both μ-γ1 switch recombination and IgG1 secretion in an IL-4 independent manner.Although some post-receptor signaling events of IL-5 in activated B cells have been characterized, the involvement of the Janus kinase/signal transducer and activator (Jak/Stat) pathway in IL-5 signaling has not been thoroughly evaluated. In this study, we examined whether IL-5 activates the Jak/Stat pathway in CD38-activated mouse splenic B cells. Both Stat5a and Stat5b were activated by IL-5 stimulation. The role of Stat5a and Stat5b in IL-5-induced μ-γ1 switch recombination and IgG1 production were documented, as IL-5 did not act on CD38-stimnulated splenic B cells of Stat5a^<-/-> and Stat5b^<-/-> mouse. Expression levels of germline γ1 transcripts to CD38 and of activation-induced cytidine deaminase (AID) in Stat5a^<-/-> and Stat5b^<-/-> B cells upon IL-5 stimulation were comparable to these of wild type B cells. Thus, both Stat5a and Stat5b are essential for at IL-5-dependent μ-γ1 switch recombination, and their targets may not be g1 and AID genes. The impaired μ-γ1 switch recombination by Stat5b^<-/-> B cells, but not by Stat5a^<-/-> B cells, were rescued in part by IL-4, as the addition of IL-4 to the culture of CD38- and IL-5-stimulated B cells did induce μ-γ1 switch recombination leading to significant IgG1 production. Our data support the notion that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function on B cell differentiation. Analysis of cell division cycle number of B cells, examined by using 5-, 6-carboxyfluorescein diacetate, succinimidyl ester (CFSE), revealed that μ-γ1 switch recombination and surface IgG1-positive cells were observed after 5 to 6 division cycles upon CS/2 and IL-5. Stat5a^<-/-> and stat5b^<-/-> B cells showed 5 to 6 cell division cycles, but they could express neither μ-γ1 switching nor surface IgG1. Less

小鼠B细胞表达CD 38，其通过抗CD 38抗体的连接诱导其增殖和保护免于凋亡。我们以前研究过用白细胞介素5（IL-5）和抗小鼠CD 38单克隆抗体CS/2刺激小鼠脾B细胞可诱导IgG 1和IgM的产生，在此我们研究了IL-5和CS/2在生殖系γ1转录本表达和形成DNA环的相互产物（μ-γ1开关重组的副产物）的产生中的作用。CS/2本身诱导脾幼稚B细胞中生殖系γ1转录物的显著表达，而IL-5既不诱导也不增强生殖系γ1表达。在用CS/2培养B细胞后，未观察到作为μ-γ1重组特征的互补产物的细胞含量增加，但当用CS/2加IL-5培养B细胞时，发现互补产物增加沿着高水平的IgG 1分泌。虽然IL-4本身并不诱导μ-γ1受体， ...更多信息在用CS/2刺激的B细胞中，与CS/2 + IL-5、IL-4联合使用显著增强了无菌γ1转录和IgG 1产生。这些结果表明，CD 38连接只诱导生殖系γ1转录，IL-5以IL-4非依赖性方式促进μ-γ1开关重组和IgG 1分泌，尽管IL-5在活化的B细胞中的一些受体后信号传导事件已被表征，但Janus激酶/信号转导和激活剂（Jak/Stat）通路在IL-5信号传导中的参与尚未被彻底评估。在这项研究中，我们研究了IL-5是否激活CD 38激活的小鼠脾B细胞中的Jak/Stat通路。Stat 5a和Stat 5 b均被IL-5刺激激活。由于IL-5不作用于Stat 5 a ^<-/->和Stat 5 B ^<-/->小鼠的CD 38刺激的脾B细胞，因此记录了Stat 5 a和Stat 5 B在IL-5诱导的μ-γ1开关重组和IgG 1产生中的作用。IL-5刺激后，Stat 5 a ^<-/->和Stat 5 B ^<-/-> B细胞中CD 38的生殖系γ1转录物和活化诱导的胞苷脱氨酶（AID）的表达水平与野生型B细胞的表达水平相当。因此，Stat 5a和Stat 5 b对于IL-5依赖性μ-γ1开关重组都是必需的，并且它们的靶标可能不是g1和AID基因。IL-4部分地挽救了Stat 5 B ^<-/-> B细胞而非Stat 5 a ^<-/-> B细胞的受损的μ-γ1开关重组，因为向CD 38和IL-5刺激的B细胞的培养物中添加IL-4确实诱导了μ-γ1开关重组，导致显著的IgG 1产生。我们的数据支持Stat 5a和Stat 5 B b不是冗余的，而是在它们对B细胞分化的功能上至少部分不同的观点。用5-，6-羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）分析B细胞分裂周期数，发现CS/2和IL-5作用5 ~ 6个周期后，可观察到μ-γ1开关重组和表面IgG 1阳性细胞。Stat 5a ^<-/->和stat 5 B ^<-/-> B细胞显示5 - 6个细胞分裂周期，但它们既不能表达μ-γ1开关，也不能表达表面IgG 1。少