| Co-Investigator(Kenkyū-buntansha) |
HORIKAWA Keisuke Institute of Medical Science, The University of Tokyo Research Associate, 医科学研究所, 教務職員 (60313095)
KARIYONE Ai Institute of Medical Science, The University of Tokyo Professor, 医科学研究所, 教務職員 (50114450)
TAKAKI Satoshi Institute of Medical Science, The University of Tokyo Professor, 医科学研究所, 助教授 (10242116)
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| Research Abstract |
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
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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
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