Pierini L, Holowka D, Baird B
Department of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
J Cell Biol. 1996 Sep;134(6):1427-39. doi: 10.1083/jcb.134.6.1427.
Cells of the mucosal mast cell line, RBL-2H3, are normally stimulated to degranulate after aggregation of high affinity receptors for IgE (Fc epsilon RI) by soluble cross-linking ligands. This cellular degranulation process requires sustained elevation of cytoplasmic Ca2+. In this study, we investigated the response of RBL-2H3 cells to 6-micron beads coated with IgE-specific ligands. These ligand-coated beads cause only small, transient Ca2+ responses, even though the same ligands added in soluble form cause larger, more sustained Ca2+ responses. The ligand-coated 6-micron beads also fail to stimulate significant degranulation of RBL-2H3 cells, whereas much larger ligand-coated Sepharose beads stimulate ample degranulation. Confocal fluorescence microscopy shows that the 6-micron beads (but not the Sepharose beads) are phagocytosed by RBL-2H3 cells and that, beginning with the initial stages of bead engulfment, there is exclusion of many plasma membrane components from the 6-micron bead/cell interface, including p53/56lyn and several other markers for detergent-resistant membrane domains, as well as an integrin and unliganded IgE-Fc epsilon RI. The fluorescent lipid probe DiIC16 is a marker for the membrane domains that is excluded from the cell/bead interface, whereas a structural analogue, fast DiI, which differs from DiIC16 by the presence of unsaturated acyl chains, is not substantially excluded from the interface. None of these components are excluded from the interface of RBL-2H3 cells and the large Sepharose beads. Additional confocal microscopy analysis indicates that microfilaments are involved in the exclusion of plasma membrane components from the cell/bead interface. These results suggest that initiation of phagocytosis diverts normal signaling pathways in a cytoskeleton-driven membrane clearance process that alters the physiological response of the cells.
黏膜肥大细胞系RBL-2H3的细胞通常在通过可溶性交联配体使IgE高亲和力受体（FcεRI）聚集后被刺激而脱颗粒。这种细胞脱颗粒过程需要细胞质Ca2+持续升高。在本研究中，我们研究了RBL-2H3细胞对包被有IgE特异性配体的6微米珠子的反应。这些包被配体的珠子仅引起小的、短暂的Ca2+反应，尽管以可溶形式添加相同的配体可引起更大、更持久的Ca2+反应。包被配体的6微米珠子也未能刺激RBL-2H3细胞发生显著脱颗粒，而大得多的包被配体的琼脂糖珠子则刺激充分脱颗粒。共聚焦荧光显微镜显示，6微米珠子（但不是琼脂糖珠子）被RBL-2H3细胞吞噬，并且从珠子吞噬的初始阶段开始，许多质膜成分被排除在6微米珠子/细胞界面之外，包括p53/56lyn和其他几种抗去污剂膜结构域的标记物，以及一种整合素和未结合配体的IgE-FcεRI。荧光脂质探针DiIC16是被排除在细胞/珠子界面之外的膜结构域的标记物，而一种结构类似物快速DiI，它与DiIC16的不同之处在于存在不饱和酰基链，并未从界面大量排除。这些成分均未从RBL-2H3细胞与大的琼脂糖珠子的界面排除。额外的共聚焦显微镜分析表明，微丝参与了质膜成分从细胞/珠子界面的排除。这些结果表明，吞噬作用的启动在由细胞骨架驱动的膜清除过程中改变了正常的信号通路，从而改变了细胞的生理反应。
