Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes.
Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes.
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DOI：
10.4049/jimmunol.139.2.494
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发表时间：
1987-07
影响因子：
4.4
通讯作者：
F. Levi‐Schaffer;K. Austen;J. Caulfield;A. Hein;P. M. Gravallese;R. Stevens
中科院分区：
文献类型：
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作者：
F. Levi‐Schaffer;K. Austen;J. Caulfield;A. Hein;P. M. Gravallese;R. Stevens
通过蛋白水解处理从肺组织中分离出来的人肥大细胞，通过密度梯度离心富集到23%至68%的纯度，当与小鼠皮肤来源的3T3成纤维细胞在含有10%胎牛血清的RPMI 1640中共培养时，离体维持时间长达13天。人肥大细胞在播种后2 ~ 4小时内粘附在成纤维细胞上，共培养7天后定位在成纤维细胞层之间。肥大细胞和成纤维细胞表面没有形成紧密连接，而是相互靠近在20nm以内。共培养的肥大细胞不分裂；其分泌颗粒呈涡旋状，细胞核呈椭圆形，与体内肥大细胞相似。新分离的肥大细胞和共培养的肥大细胞都通过分泌组胺和产生和释放花生四烯酸代谢物来响应抗人IgE的激活。当新分离的肥大细胞被免疫激活时，它们的总组胺含量的38 +/- 8%被排出，每微克细胞总组胺释放28 +/- 1.9 ng（平均+/-范围，n = 2）前列腺素D2 （PGD2）的免疫反应当量，但没有产生大量的白三烯C4 （LTC4）或白三烯B4 （LTB4）。另一方面，共培养1周的肥大细胞，每微克组胺分别释放86 +/- 10、43 +/- 20和5.2 +/- 5.2 ng（平均+/- SD， n = 4） PGD2、LTC4和LTB4的免疫反应当量。因此，当与成纤维细胞共培养时，人肺源性肥大细胞可以在体外维持，并且当用抗ige处理时，它们通过环氧化酶和5-脂氧化酶途径代谢花生四烯酸。
Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.