Dual Functions of Bruton’s Tyrosine Kinase and Tec Kinase during Fcγ Receptor-Induced Signaling and Phagocytosis

Abstract

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)γ, and regulate cytoskeletal rearrangement, yet their role in FcγR-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 FcγR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcγR-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 PLCγ2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCγ2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCγ 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 FcγR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.

Bruton's Tyrosine Kinase Is Required for Apoptotic Cell Uptake via Regulating the Phosphorylation and Localization of Calreticulin

“…Btk has been shown to be activated throughout the process of phagocytosis and can accumulate at the base of the phagocytic cup (24). Our results outlined above demonstrated a clear role for Btk in mediating apoptotic cell uptake in addition to regulating TLR7-induced cytokine responses.…”

“…In addition to its role in regulating TLR-induced cytokine production, Btk is required for appropriate Fcg-mediated phagocytosis in macrophages (13,24). However, the precise involvement of Btk in this process is unclear.…”

Selective Inhibition of BTK Prevents Murine Lupus and Antibody-Mediated Glomerulonephritis

“…Thus, treatment with a BTK inhibitor is unlikely to significantly decrease autoantibody-secreting long-lived plasma cells, although efficacy may be derived from inhibition of IC FcR-driven activation. FcgRdependent phagocytosis of ICs has been reported to require BTK, and it will therefore be important to understand in future studies how the levels of circulating ICs are impacted by treatment with a BTK inhibitor (48,49). In contrast with the aforementioned Ig isotypes, IgG3 levels were significantly reduced compared with baseline levels after treatment of NZBxW_F1 mice with PF-06250112.…”

Ibrutinib interferes with the cell-mediated anti-tumor activities of therapeutic CD20 antibodies: implications for combination therapy

“…These data are in agreement with studies showing that Btk is expressed by myeloid lineage cells and is involved in the activation, cytokine production and chemotaxis of macrophages, granulocytes and mast cells. [44][45][46] The EC 50 for this inhibition was in the range of 0.3 to 3 μM. It is possible that these processes may be inhibited by standard ibrutinib dosing in vivo, although this was not directly demonstrated here.…”