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Receptor-Induced Signaling and Phagocytosis1
,
* Toronto General Research Institute, University Health Network, and
Department of Immunology and
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario Canada; and
Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden
Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)
, and regulate cytoskeletal rearrangement, yet their role in FcR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton’s tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLC2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLC2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLC product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant RGPIN 194609-3 from the Natural Sciences and Engineering Research Council of Canada (to J.J.-B.) and Grant MOP-14151 from the Canadian Institutes of Health Research (to M.I.C.). C.I.E.S. is supported by the Swedish Cancer Foundation. M.I.C. is a career investigator of the Heart and Stroke Foundation of Ontario.
2 Address correspondence and reprint requests to Dr. Jenny Jongstra-Bilen, Max Bell Research Centre, Room 1R-408, Toronto General Research Hospital, University Health Network, 200 Elizabeth Street, Toronto, Ontario M5G 2C4, Canada. E-mail address: jbilen{at}uhnres.utoronto.ca
3 Abbreviations used in this paper: PIP2, phosphatidylinositol 4,5-bisphosphate; aggIgG, aggregated IgG; Btk, Bruton’s tyrosine kinase; DAG, diacylglycerol; DIC, differential interference contrast; PKC, protein kinase C; XLA, X-linked agammaglobulinemia; siRNA, small interfering RNA; PLC, phospholipase C; PIP3, phosphatidylinositol 3,4,5-trisphosphate.
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