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.

Rapid signaling and structural adaptations to the actin cytoskeleton enable leukocytes to stabilize α4 integrin–mediated adhesion and resist detachment from inflamed endothelium.

Chemokine/chemoattractant G protein-coupled receptors trigger an inside–out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside–in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α–induced upregulation of α4β1 integrin affinity and consequent adhesive events. Affinity upregulation of α4β1 integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1–coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1–coated surfaces. Biochemical analyses revealed that α4β1 expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α4β1 and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α4β1 after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α4β1-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.

No abstract

α4β1 integrin plays an important role in monocyte adhesion and emigration. Activation by chemokines and chemoattractants is required to increase α4β1 integrin affinity and mediate adhesion to VCAM‐1 expressed by cytokine‐activated endothelium. We investigated the role of talin, a cytoskeletal protein that interacts with integrin cytoplasmic tails, in α4β1 integrin‐dependent functions using formyl peptide receptor‐transfected U937 monocytic cells. fMLP‐induced affinity up‐regulation, measured by binding of soluble LDV‐FITC peptide, was unaffected by siRNA knockdown of talin‐1 or sequestration of talin through expression of a dominant inhibitory talin‐binding fragment of phosphatidylinositol phosphate kinase type I‐90. Similarly, talin‐1 knockdown did not inhibit fMLP or SDF‐1‐stimulated adhesion to VCAM‐1. Talin was however, essential for PMA‐induced adhesion to VCAM‐1, but not PMA‐induced α4β1 integrin affinity up‐regulation. In addition, talin knockdown reduced fMLP‐driven, α4β1 integrin‐dependent chemotactic migration across VCAM‐1 coated filters.These results suggest that talin association with the cytoplasmic tail of α4β1 integrin is not involved in chemokine‐induced affinity changes or adhesion. However, talin plays an important role in α4β1 integrin‐mediated chemotactic migration and PMA‐induced adhesion to VCAM‐1, steps that follow affinity modulation. Funded by CIHR.