Cytokine productions from murine and human mast cells

为了建立小鼠和人肥大细胞的大规模培养方法，我们研究了多种细胞因子对小鼠骨髓细胞和人脐带血CD34^+细胞肥大细胞发育的影响。在小鼠中，将白细胞介素-3 （IL-3）添加到骨髓细胞培养物中可以获得纯肥大细胞（骨髓源性肥大细胞：BMMC）。加入干细胞因子（SCF）、IL-4和IL-10与IL-3共同培养可促进BMMC的发育。虽然干细胞因子（SCF）、IL-4和IL-10单独不能支持BMMC的发展，但SCF与IL-4或IL-10联合可显著促进BMMC的增殖。与BMMC相比，单独IL-3不能支持CTMC的增殖，CTMC是从小鼠腹膜细胞中纯化的，以及IL-4或IL-10。SCF单独支持CTMC的少量菌落。IL-3和IL-4能促进scf依赖性CTMC的增殖，而IL-10不能促进CTMC的增殖。在人体内，我们用细胞分选器从脐带血单个核细胞中分离出CD34^+细胞，在SCF和IL-6或IL-11存在的情况下，通过培养获得了胰酶阳性和乳糜酶阳性的肥大细胞。当CD34^+细胞与SCF和IL-6或IL-11一起培养时，在培养2周后首次检测到具有胰蛋白酶阳性颗粒的肥大细胞，在培养100天后，当20-30%的细胞进行免疫组织染色进行乳糜酶染色时，肥大细胞的纯度几乎达到100%。我们培养的人肥大细胞含有大量的组胺和胰蛋白酶，并在细胞表面表达功能性IgE受体。克隆分选法单细胞培养CD34^+细胞表明，胰酶阳性肥大细胞和乳糜酶阳性肥大细胞均来源于同一造血祖细胞。利用克隆分选系统对人肥大细胞祖细胞进行表型分析，发现承诺的是CD34^+CD38^+，未承诺的是CD34^+CD38^-。利用逆转录酶(RT)-PCR，我们发现小鼠BMMC和培养的CTMC，以及培养的人肥大细胞，经IgE和抗IgE单克隆抗体预处理后，表达TNF、GM-CSF、IL-3、IL-4、IL-6和IL-13的mrna。这些结果表明，小鼠和人类肥大细胞不仅可以释放多种化学介质，如组胺和白垩烯，还可以释放多种细胞因子。少

To establish a large scale culture method for murine and human mast cells, we have examined the effects of various cytokines on the development of mast cells from murine bone marrow cells and human cord blood CD34^+ cells. In murine, the addition of interleukin-3 (IL-3) to cultures of bone marrow cells gives rise to pure cultures of mast cells (bone marrow-derived maust cells : BMMC). Addition of stem cell factor (SCF), IL-4 and IL-10 to culture with IL-3 stimulated the development of BMMC.Although stem cell factor (SCF), IL-4 and IL-10 alone failed to support BMMC development, SCF in combinations with IL-4 or IL-10 significantly stimulated the proliferation of BMMC.In contrast with BMMC,IL-3 alone could not support the proliferation of CTMC,which were purified from mouse peritoneal cells, as well as IL-4 or IL-10. SCF alone supported a small number of colonies from CTMC.IL-3 and IL-4 but not IL-10 enhanced SCF-dependent CTMC proliferation IL-3 stimulated CTMC proliferation in the pres … More ence of IL-4 or IL-10.In human, we obtained both tryptase-positive and chymase-positive mast cells by culturing CD34^+ cells, which were isolated from cord blood mononuclear cells using a cell sortor, in the presense of SCF and IL-6 or IL-11. When CD34^+ cells were cultured with SCF and IL-6 or IL-11, mast cells with tryptase-positive granules were first detected after 2 weeks of culture, and reached to almost 100% purity after 100 days of culture when 20-30% of the cells were immunohistologically stained for chymase. Our cultured human mast cells contained a large amount of histamine and tryptase, and expressed functional IgE receptors on their cell surface. Single cell culture of CD34^+ cells with a clone-sorting method indicated that both tryptase-positive and chymase-positive mast cells were originated from a common hemopoietic progenitor. Phenotipical analyzes of human mast cell progenitors using a clone-sorting system indicated that committed ones were CD34^+CD38^+ and uncommitted ones were CD34^+CD38^-.Using reverse transcriptase (RT)-PCR,we demonstrated that murine BMMC and cultured CTMC,and cultured human mast cells, which were pretreated with IgE and anti-IgE monoclonal antibodies, express mRNAs of TNF,GM-CSF,IL-3, IL-4, IL-6 and IL-13. These results suggest that both murine and human mast cells can release not only a variety of chemical mediators such as histamine and leukotorienes but also various cytokines. Less