Stockdale A M, Dul J L, Wiest D L, Digel M, Argon Y
Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710.
J Immunol. 1987 Nov 15;139(10):3527-35.
The expression of membrane and secreted IgM was analyzed during mitogen-induced differentiation of the murine B cell lymphoma CH12. To characterize the Ig genes used by CH12, the nucleotide sequences of the variable gene segments (V mu and V kappa) were determined. The expressed V mu gene segment belongs to the VHII NPb-related family. The D (FL16.1a) and J (JH2) segments are the same as those used by the NP-specific hybridoma B1-8. The V kappa used by CH12 is almost identical to those used by the oxazolone-specific hybridomas NQ5.89.4 and NQ7.7.1. Treatment with lipopolysaccharide (LPS) induces up to 80% of CH12 cells to secrete IgM within 48 hr of culture. The steady state levels of secreted mu (mu s) and kappa mRNA increase four to fivefold over this period in cells stimulated with LPS compared with unstimulated cells. The kinetics are similar for both mRNA and parallel the increase in IgM secretion. EL-4 supernatants induce comparable changes in m mu s and kappa transcript levels. The simultaneous increase in m mu s and kappa transcripts suggests that coordinate control of RNA levels is used to increase the synthesis of secretory IgM during differentiation. The level of mRNA encoding the membrane form of mu (mu m) remains constant in stimulated cells and increases slightly in unstimulated cells. While the net rates of synthesis of membrane-bound mu-chains remain similar during LPS stimulation, the level of surface IgM on secreting cells is reduced three to fivefold. These observations suggest that the level of surface IgM expression during differentiation of CH12 is controlled largely by post-translational mechanisms. Our results demonstrate that the CH12 cell line regulates the expression of membrane and secreted IgM differently during its differentiation. The changes in IgM expression in CH12 parallel those occurring in normal B cells after mitogen or antigen challenge. Thus, the in vitro differentiation of CH12 is a good model for the analysis of late stages of B cell development.
在丝裂原诱导的小鼠B细胞淋巴瘤CH12分化过程中，对膜结合型和分泌型IgM的表达进行了分析。为了鉴定CH12所使用的Ig基因，测定了可变基因片段（Vμ和Vκ）的核苷酸序列。表达的Vμ基因片段属于VHII NPb相关家族。D（FL16.1a）和J（JH2）片段与NP特异性杂交瘤B1-8所使用的片段相同。CH12所使用的Vκ与恶唑酮特异性杂交瘤NQ5.89.4和NQ7.7.1所使用的几乎相同。用脂多糖（LPS）处理可使高达80%的CH12细胞在培养48小时内分泌IgM。与未刺激的细胞相比，在用LPS刺激的细胞中，分泌型μ（μs）和κ mRNA的稳态水平在此期间增加了四到五倍。mRNA的动力学相似，且与IgM分泌的增加平行。EL-4上清液可诱导μm s和κ转录水平发生类似变化。μm s和κ转录本的同时增加表明，在分化过程中，RNA水平的协调控制被用于增加分泌型IgM的合成。编码膜结合型μ（μm）的mRNA水平在受刺激的细胞中保持恒定，而在未刺激的细胞中略有增加。虽然在LPS刺激期间，膜结合型μ链的净合成速率保持相似，但分泌细胞表面IgM的水平降低了三到五倍。这些观察结果表明，CH12分化过程中表面IgM的表达水平在很大程度上受翻译后机制的控制。我们的结果表明，CH12细胞系在其分化过程中对膜结合型和分泌型IgM的表达调控方式不同。CH12中IgM表达的变化与正常B细胞在丝裂原或抗原刺激后发生的变化相似。因此，CH12的体外分化是分析B细胞发育后期的良好模型。
