A protein-tyrosine kinase, Syk plays a central role in Fc-gamma receptor-mediated phagocytosis in the adaptive immune system. Here we show that Syk also makes an essential role in compliment-mediated phagocytosis in innate immunity. For this purpose, HL60 cells induced to macrophage-like with vitamin D3 and TPA and C3bi-opsonized zymosan were used as the pathogen-phagocyte system. Time-lapse microscopic observation revealed that C3bi-opsonized zymosan particles were promptly attached to and sucked into the cells but non-opsonized zymosan was not, and therefore it was indicated that our phagocytosis system is mediated by the complement receptor 3 (CR3). Syk was tyrosine-phosphorylated by the treatment with C3bi-opsonized zymosan and marked accumulation of Syk was detected around the newly forming phagosomes. To further investigate the effect of Syk on complement-mediated phagocytosis, Syk-siRNA or dominant-negative Syk (DN-Syk) was introduced into HL60 cells. Quantitative analysis by flow cytometry showed that phagocytosis was severely impaired in DN-Syk or Syk-siRNA expressing HL60 cells. To determine whether the impaired capture of zymosan is due to reduced binding to CR3 or reduced engulfment after binding, the cells were treated with a quenching reagent which discriminates fluorescent zymosan particles between inside and outside the cells. In the parental HL60 cells most of the attached zymosan particles were inside the cells, while few particles were ingested in the case of DN-Syk or Syk-siRNA expressing HL60 cells. These data indicated that Syk is required for engulfment of C3bi-opsonized zymosan but not for attachment of zymosan to CR3. To clarify the molecular mechanism how Syk affects the phagosome formation and its engulfment, the behavior of F-actin was searched microscopically. At the early stage of complement-mediated phagocytosis, marked accumulation of F-actin occurred around each phagosome and was followed by rapid depolymerization in the parental HL60 cells. In contrast, phagosomes surrounded by the accumulated actin were significantly decreased in DN-Syk or Syk-siRNA expresssing cells. These results indicate that Syk promotes actin assembly around phagosomes prior to engulfment at the early stage of the phagocytosis. As for the analysis of the downstream pathway of Syk in our phagocytosis system, RhoA-signaling was investigated. Vav-RhoA pathway was activated in HL60 cells but transfer of DN-Syk or Syk-siRNA into HL60 cells reduced the activation of Vav-RhoA signaling. Therefore Syk may act as an activator of the RhoA-pathway in phagocytosis.
Our results demonstrate that Syk is required for complement-mediated phagocytosis by regulating both actin accumulation around phagosomes and RhoA-signaling harmonically and finally leads to phagosome formation and its engulfment.