Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcγRIIA receptors
Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcγRIIA receptors
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DOI：
10.1074/jbc.274.40.28436
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发表时间：
1999-10-01
影响因子：
4.8
通讯作者：
Grinstein, S
中科院分区：
文献类型：
--
作者：
Downey, GP;Botelho, RJ;Grinstein, S
专业吞噬细胞吞噬和杀死微生物病原体是先天免疫反应的重要组成部分。最近，将单个受体异源导入非髓系细胞已被成功地用于阐明吞噬小体形成的早期步骤。然而，目前尚不清楚由这种转基因细胞形成的空泡是否是真正的吞噬小体，能够与内膜融合，管腔酸化，并控制微生物的生长，本研究的目的是确定COS-1和中国仓鼠卵巢细胞是否表达支持吞噬小体成熟所需的细胞机制。免疫定位研究表明，早期的内小体以及晚期的内小体和/或溶酶体依次与转染体中的吞噬小体融合，pH敏感染料标记的颗粒的显微荧光比率成像显示吞噬小体的成熟伴随着管腔酸化，空泡型HC-ATPase抑制剂阻止了pH的下降，达到了与专业吞噬细胞的水平相当的水平。最理想的吞噬体酸化需要提高胞浆[Ca~(2+)]，这表明它是含有质子泵的内膜融合的结果。此外，转基因细胞有效地内化了活细菌。调理作用对于细菌内化是必不可少的，这意味着它是通过Fc-Gamma RIIA介导的吞噬作用发生的，而不是通过入侵。吞噬溶酶体的摄取与细菌生长抑制有关，至少部分是由于吞噬体内的低pH所致。这些研究表明，转导Fc-γRIIA受体的细胞在受体介导的颗粒内化后发生的生化事件与中性粒细胞和巨噬细胞的吞噬成熟过程非常相似，因此，转导Fc-γ-RIIA的细胞可以作为研究吞噬细胞生物学的其他方面的模型。
Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms, The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human Fc gamma RIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants, Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification, The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar type HC-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca2+], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by Fc gamma RIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with Fc gamma RIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages, Fc gamma RIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.