Pfeiffer J R, Seagrave J C, Davis B H, Deanin G G, Oliver J M
J Cell Biol. 1985 Dec;101(6):2145-55. doi: 10.1083/jcb.101.6.2145.
Binding of antigen to IgE-receptor complexes on the surface of RBL-2H3 rat basophilic leukemia cells is the first event leading to the release of cellular serotonin, histamine, and other mediators of allergic, asthmatic, and inflammatory responses. We have used dinitrophenol-conjugated bovine serum albumin (DNP-BSA) as well as the fluorescent antigen, DNP-B-phycoerythrin, and the electron-dense antigen, DNP-BSA-gold, to investigate dynamic membrane and cytoskeletal events associated with the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. These multivalent antigens bind rapidly to cell surface IgE-receptor complexes. Their distribution is initially uniform, but within 2 min DNP-BSA-gold is found in coated pits and is subsequently internalized. Antigen internalization occurs in the presence and absence of extracellular Ca2+. The F-actin content of the detergent-extracted cell matrices analyzed by SDS PAGE decreases during the first 10-30 s of antigen binding and then increases by 1 min to almost double the control levels. A rapid and sustained increase is also observed when total F-actin is quantified by flow cytometry after binding of rhodamine-phalloidin. The antigen-stimulated increase in F-actin coincides with (and may cause) the transformation of the cell surface from a finely microvillous to a highly folded or plicated topography. Other early membrane responses include increased cell spreading and a 2-3-fold increase in the uptake of fluorescein-dextran by fluid pinocytosis. The surface and F-actin changes show the same dependence on DNP-protein concentration as stimulated [3H]serotonin release; and both the membrane responses and the release of mediators are terminated by the addition of the non-cross-linking monovalent ligand, DNP-lysine. These data indicate that the same antigen-stimulated transduction pathway controls both the membrane/cytoskeletal and secretory events. However, the membrane and actin responses to IgE-receptor cross-linking are independent of extracellular Ca2+ and are mimicked by phorbol myristate acetate, whereas ligand-dependent mediator release depends on extracellular Ca2+ and is mimicked by the Ca2+ ionophore A23187.
抗原与RBL - 2H3大鼠嗜碱性白血病细胞表面的IgE受体复合物结合，是导致细胞内5 - 羟色胺、组胺及其他过敏、哮喘和炎症反应介质释放的首要事件。我们使用二硝基苯酚偶联的牛血清白蛋白（DNP - BSA）以及荧光抗原DNP - B - 藻红蛋白和电子致密抗原DNP - BSA - 金，来研究与抗DNP - IgE致敏的RBL - 2H3细胞释放[3H]5 - 羟色胺相关的动态膜和细胞骨架事件。这些多价抗原能迅速与细胞表面的IgE受体复合物结合。它们的分布最初是均匀的，但在2分钟内，DNP - BSA - 金出现在被膜小窝中，随后被内化。无论细胞外是否存在Ca2+，抗原都会发生内化。通过SDS - PAGE分析去污剂提取的细胞基质中的F - 肌动蛋白含量，在抗原结合的最初10 - 30秒内会减少，然后在1分钟时增加，几乎是对照水平的两倍。用罗丹明 - 鬼笔环肽结合后通过流式细胞术对总F - 肌动蛋白进行定量时，也观察到快速且持续的增加。抗原刺激引起的F - 肌动蛋白增加与细胞表面从精细的微绒毛状转变为高度折叠或褶皱状的形貌同时发生（且可能导致这种转变）。其他早期膜反应包括细胞铺展增加以及通过液相胞饮作用摄取荧光素 - 葡聚糖增加2 - 3倍。表面和F - 肌动蛋白的变化与刺激的[3H]5 - 羟色胺释放一样，对DNP - 蛋白浓度具有相同的依赖性；并且膜反应和介质释放都可通过添加非交联单价配体DNP - 赖氨酸而终止。这些数据表明，相同的抗原刺激转导途径控制着膜/细胞骨架和分泌事件。然而，对IgE受体交联的膜和肌动蛋白反应不依赖于细胞外Ca2+，并且可被佛波酯肉豆蔻酸乙酸酯模拟，而配体依赖性介质释放则依赖于细胞外Ca2+，并且可被Ca2+离子载体A23187模拟。
