Boyce J A, Friend D, Matsumoto R, Austen K F, Owen W F
Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA.
J Exp Med. 1995 Jul 1;182(1):49-57. doi: 10.1084/jem.182.1.49.
Granulocytes with the hybrid characteristics of eosinophils and basophils have been identified in the bone marrow and peripheral blood of humans with myeloid leukemias. We now describe a technique by which such hybrid granulocytes can be developed in vitro from normal cord blood precursors cultured in the presence of recombinant human interleukin (rhIL) 3 (350 pM) and rhIL-5 (200 pM) in a plastic vessel coated with Matrigel. After 14 d in culture, 90 +/- 3% (mean +/- standard error of the mean) of the nonadherent cells cultured in the Matrigel-coated flasks contained both eosinophil and basophil granules, as indicated by staining with Wright's and Giemsa stains. Of the nonadherent cells, 93 +/- 1% contained cyanide-resistant peroxidase, and 88 +/- 2% were toluidine blue-positive, characteristic of eosinophil and basophil granules, respectively. Transmission electron micrographs showed hybrid cells containing ultrastructurally distinct eosinophil granules with developing crystalline cores and basophil granules with reticular structures. These 14-d cord blood-derived cell cultures showed strong hybridization signals for eosinophil-derived neurotoxin by RNA blot analysis and contained 78 ng histamine per 10(6) cells. When the granulocytes were removed from cytokine-containing medium and suspended without Matrigel in RPMI 1640 medium containing 10% fetal calf serum (FCS), more than 80% of the granulocytes excluded trypan blue for as long as 5 d, and 93% had developed into eosinophils at 6 d. Conditioned medium prepared over 48 h from the 14-d cell cultures (hybrid granulocytes) sustained the 4-d viability in vitro of 78% of peripheral blood eosinophils from atopic donors. In comparison, 13% survived in RPMI 1640 containing 10% FCS alone. This viability-sustaining activity was nearly completely neutralized by an anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibody and was only minimally reduced by anti-IL-3 or IL-5. Thus, cells possessing both eosinophil and basophil granules by both histochemical and ultrastructural analysis can be developed from normal progenitors in vitro in response to eosinophilopoietic cytokines and Matrigel. Their subsequent spontaneous development into mature eosinophils suggests that hybrid granulocytes are part of a normal developmental sequence during eosinophilopoiesis. Furthermore, these hybrid granulocytes are capable of autoregulation through elaboration of GM-CSF, which sustains their viability.
在患有髓系白血病的人类的骨髓和外周血中，已鉴定出具有嗜酸性粒细胞和嗜碱性粒细胞混合特征的粒细胞。我们现在描述一种技术，通过该技术，在涂有基质胶的塑料容器中，在重组人白细胞介素（rhIL）-3（350 pM）和rhIL-5（200 pM）存在的情况下，从正常脐带血前体细胞体外培养出这种混合粒细胞。培养14天后，在涂有基质胶的培养瓶中培养的非贴壁细胞中，90±3%（平均值±平均值的标准误差）同时含有嗜酸性粒细胞和嗜碱性粒细胞颗粒，用瑞氏和吉姆萨染色显示。在非贴壁细胞中，93±1%含有抗氰过氧化物酶，88±2%甲苯胺蓝阳性，分别为嗜酸性粒细胞和嗜碱性粒细胞颗粒的特征。透射电子显微镜照片显示，杂交细胞含有超微结构上不同的嗜酸性粒细胞颗粒，其结晶核心正在发育，以及具有网状结构的嗜碱性粒细胞颗粒。通过RNA印迹分析，这些源自脐带血的14天细胞培养物对嗜酸性粒细胞衍生的神经毒素显示出强烈的杂交信号，并且每10^6个细胞含有78 ng组胺。当粒细胞从含细胞因子的培养基中取出，在不含基质胶的情况下悬浮于含有10%胎牛血清（FCS）的RPMI 1640培养基中时，超过80%的粒细胞在长达5天的时间内排斥台盼蓝，并且在6天时93%已发育为嗜酸性粒细胞。从14天的细胞培养物（混合粒细胞）中制备的48小时条件培养基维持了78%特应性供体外周血嗜酸性粒细胞在体外4天的活力。相比之下，仅在含有10% FCS的RPMI 1640中，13%存活。这种活力维持活性几乎完全被抗粒细胞/巨噬细胞集落刺激因子（GM-CSF）抗体中和，而被抗IL-3或IL-5仅轻微降低。因此，通过组织化学和超微结构分析同时具有嗜酸性粒细胞和嗜碱性粒细胞颗粒的细胞可以在体外从正常祖细胞发育而来，以响应嗜酸性粒细胞生成细胞因子和基质胶。它们随后自发发育为成熟嗜酸性粒细胞表明，混合粒细胞是嗜酸性粒细胞生成过程中正常发育序列的一部分。此外，这些混合粒细胞能够通过分泌GM-CSF进行自我调节，从而维持其活力。
