Hackam D J, Rotstein O D, Schreiber A, Zhang W j, Grinstein S
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, M5G 1X8, Canada.
J Exp Med. 1997 Sep 15;186(6):955-66. doi: 10.1084/jem.186.6.955.
Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcgamma receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor-mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcgamma receptor-mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcgammaRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcgamma receptor-mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fcgamma-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcgamma receptor-mediated phagocytosis by leukocytes.
巨噬细胞和中性粒细胞对细菌的吞噬作用是宿主抵御感染的重要组成部分。尽管酪氨酸激酶的激活被认为是早期步骤，但调理素化颗粒与Fcγ受体相互作用触发调理素化颗粒吞噬的机制仍未完全明了。其他系统的近期研究表明，酪氨酸激酶可依次信号传导ras超家族小GTP酶的激活。因此，我们研究了Rho在Fc受体介导的吞噬作用中的可能作用。为此，我们向J774巨噬细胞显微注射了来自肉毒梭菌的C3外毒素，该毒素可进行ADP核糖基化并使Rho失活。C3外毒素诱导丝状伪足回缩、粘着斑复合体消失以及J774细胞F-肌动蛋白含量整体下降。此外，这些细胞呈现出铺展增加和液泡结构形成。重要的是，Rho失活导致吞噬作用完全消除。在转染FcγRIIA受体后具有吞噬能力的COS细胞中证实了C3外毒素对Fcγ受体介导的吞噬作用的抑制。发现Rho对于吞噬杯周围磷酸酪氨酸和F-肌动蛋白的积累以及Fcγ受体介导的Ca2+信号传导至关重要。Fcγ诱导信号传导的关键步骤，即对调理素产生反应的受体聚集，是最早显示受C3外毒素抑制的事件。毒素的作用具有特异性，因为转铁蛋白受体的聚集和内化不受C3显微注射的影响。这些数据确定了小GTP酶在白细胞Fcγ受体介导的吞噬作用中的作用。
