Department of Genetic Resources Technology, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka, 812-8581, Japan.
Cytotechnology. 2001 Jul;36(1-3):179-86. doi: 10.1023/A:1014001322272.
We have demonstrated that an immature prebasophilic cell line,KU812 cells can be induced to differentiate into basophil-like cells when cultured with hydrocortisone (HC) with enhanced cell surface expression of FcepsilonRI, a high affinity IgE receptor. In this study, we report that sodium nitroprusside (SNP), an intracellular NO donor, also induces cell surface expression of FcepsilonRI on KU812 cells. Cell surface FcepsilonRI expression was detected in about 20% of KU812 cells treated with SNP for 14 days as well as the cells treated with HC for 7 days, while non-treated KU812 cells did not express FcepsilonRI on their cell surface. However, Wright-Giemsa staining and flowcytometry analysis of CD13 and CD15 antigens on HC and SNP treated KU812 cells demonstrated that SNP induced eosinophilic differentiation in KU812 cells differently from HC which induced basophilic differentiation. To further confirm this result, we performed RT-PCR against mRNAs specific for eosinophils, such as eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase(EPO). SNP treated KU812 cells but not HC treated cells expressed EDN and EPO mRNA depending upon the induction of differentiation,clearly demonstrating that SNP induces eosinophilic differentiation in KU812 cells. To clarify that different signaling cascades were activated in HC and SNP treated KU812 cells, we analyzed activities of AP-1, NF-AT and NF-kappaB transcription factors by EMSA, which are known to be involved in signal transduction pathways downstream from the FcepsilonRI molecule of basophils. All these three transcription factors were activated in HC treated KU812 cells,but not in non-treated and SNP treated KU812 cells. These results indicate that KU812 cells are multi-potent precursor cells which can be induced to differentiate into basophils and eosinophils upon exogenous signals, and that NO is an important factor to decide the eosinophilic differentiation in KU812 cells with enhanced surface expression of FcepsilonRI, and further suggest that different signaling cascades can be activated between basophilic and eosinophilic differentiation in KU812 cells.
我们已经证明，在氢化可的松（HC）的作用下，幼稚的前嗜碱性细胞系 KU812 细胞可以分化为类嗜碱性细胞，并增强高亲和力 IgE 受体 FcepsilonRI 的细胞表面表达。在这项研究中，我们报告了一种细胞内 NO 供体硝普钠（SNP）也可以诱导 KU812 细胞表面 FcepsilonRI 的表达。在 SNP 处理 14 天的细胞和 HC 处理 7 天的细胞中，约有 20%的 KU812 细胞检测到细胞表面 FcepsilonRI 的表达，而未经处理的 KU812 细胞则没有表达 FcepsilonRI。然而，对 SNP 处理和 HC 处理的 KU812 细胞的 CD13 和 CD15 抗原进行 Wright-Giemsa 染色和流式细胞术分析表明，SNP 诱导 KU812 细胞的嗜酸性分化与诱导嗜碱性分化的 HC 不同。为了进一步证实这一结果，我们对特异性 mRNAs 进行了 RT-PCR 分析，这些 mRNAs 如嗜酸性粒细胞衍生的神经毒素（EDN）和嗜酸性粒细胞过氧化物酶（EPO）。SNP 处理的 KU812 细胞但不是 HC 处理的细胞表达 EDN 和 EPO mRNA，这取决于分化的诱导，这清楚地表明 SNP 诱导了 KU812 细胞的嗜酸性分化。为了阐明在 HC 和 SNP 处理的 KU812 细胞中激活了不同的信号转导途径，我们通过 EMSA 分析了 AP-1、NF-AT 和 NF-kappaB 转录因子的活性，这些转录因子已知参与了嗜碱性粒细胞 FcepsilonRI 分子下游的信号转导途径。所有这三种转录因子在 HC 处理的 KU812 细胞中均被激活，但在未经处理和 SNP 处理的 KU812 细胞中未被激活。这些结果表明，KU812 细胞是多能前体细胞，在外源信号的作用下可以诱导分化为嗜碱性细胞和嗜酸性细胞，而 NO 是决定增强 FcepsilonRI 细胞表面表达的 KU812 细胞中嗜酸性分化的重要因素，并进一步表明，在 KU812 细胞中，嗜碱性细胞和嗜酸性细胞的分化可以激活不同的信号转导途径。
