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，我们研究了各种细胞因子对小鼠骨髓细胞和人脐带血CD 34 ^+细胞肥大细胞发育的影响。在小鼠中，向骨髓细胞培养物中加入白细胞介素-3（IL-3）产生肥大细胞（骨髓源性肥大细胞：BMMC）的纯培养物。在IL-3的作用下加入干细胞因子（SCF）、IL-4和IL-10可促进BMMC的增殖，SCF、IL-4和IL-10单独作用不能促进BMMC的增殖，但SCF与IL-4或IL-10联合作用可显著促进BMMC的增殖，而IL-3单独作用不能促进CTMC的增殖，其从小鼠腹膜细胞中纯化，以及IL-4或IL-10。SCF单独支持少量CTMC集落形成，IL-3和IL-4促进SCF依赖性CTMC增殖，IL-10不促进SCF依赖性CTMC增殖。 关于我们 在人类中，我们通过在SCF和IL-6或IL-11存在下培养CD 34 ^+细胞获得类胰蛋白酶阳性和糜蛋白酶阳性的肥大细胞，所述CD 34 ^+细胞使用细胞分选仪从脐带血单个核细胞中分离。当CD 34 ^+细胞与SCF和IL-6或IL-11共同培养时，在培养2周后首次检测到具有类胰蛋白酶阳性颗粒的肥大细胞，在培养100天后，当20-30%的细胞被糜蛋白酶免疫组织学染色时，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类胰蛋白酶，并在其细胞表面表达功能性IgE受体。用克隆分选法对CD 34 ^+细胞进行单细胞培养，结果表明类胰蛋白酶阳性和糜蛋白酶阳性肥大细胞都来自同一个造血祖细胞。用克隆分选系统对人肥大细胞祖细胞进行表型分析，结果显示定型祖细胞为CD 34 ^+ CD 38 ^+，未定型祖细胞为CD 34 ^+ CD 38 ^-。用逆转录酶（RT）-PCR方法，我们证实了小鼠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。这些结果表明，小鼠和人肥大细胞不仅可以释放各种化学介质，如组胺和leukotorienes，但也有各种细胞因子。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