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Syk activation initiates downstream signaling events during human polymorphonuclear leukocyte phagocytosis
E M Raeder1, P J Mansfield, V Hinkovska-Galcheva
1Department of Pediatrics, Division of Hematology/Oncology, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
We investigated the requirement for Syk activation to initiate downstream signaling events during polymorphonuclear leukocyte (PMN) phagocytosis of Ab-coated erythrocytes (EIgG). When PMN were challenged with EIgG, Syk phosphorylation increased in a time-dependent manner, paralleling the response of PMN phagocytosis. Pretreatment of PMN with piceatannol, a Syk-selective inhibitor, blocked EIgG phagocytosis and Syk phosphorylation. We found that piceatannol inhibited protein kinase Cdelta (PKCdelta) and Raf-1 translocation from cytosol to plasma membrane by >90%. Extracellular signal-regulated protein kinase-1 and -2 (ERK1 and ERK2) phosphorylation was similarly blocked. We also investigated phosphatidylinositide 3-kinase (PI 3-kinase) activity and Syk phosphorylation using piceatannol, wortmannin, and LY294002, inhibitors of PI 3-kinase. The phosphorylation of Syk preceded the activation of PI 3-kinase. Both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk. In contrast to piceatannol, wortmannin did not inhibit PKCdelta and Raf-1 translocation. To elucidate signaling downstream of Syk activation, we assessed whether the cell-permeable diacylglycerol analogue didecanoylglycerol could normalize PMN phagocytosis, PKCdelta and Raf-1 translocation, and ERK1 and ERK2 phosphorylation inhibited by piceatannol. The addition of didecanoylglycerol to the Syk-inhibited phagocytosing PMN normalized all three without a concomitant effect on PI 3-kinase activity and Syk phosphorylation. We conclude that Syk activation following Fcgamma receptor engagement initiates downstream signaling events leading to mitogen-activated protein kinase activation independent of PI 3-kinase activation.
Insights
Spleen tyrosine kinase (Syk) activation is crucial for polymorphonuclear leukocyte (PMN) phagocytosis of antibody-coated targets. Syk initiates downstream signaling, including mitogen-activated protein kinase activation, independently of PI 3-kinase.
Area of Science:
- Immunology
- Cell Signaling
- Molecular Biology
Background:
- Polymorphonuclear leukocytes (PMNs) are critical immune cells involved in phagocytosis.
- Antibody-coated erythrocytes (EIgG) serve as a model for studying phagocytosis initiation.
- Spleen tyrosine kinase (Syk) is a key signaling molecule in immune cell activation.
Purpose of the Study:
- To investigate the role of Syk activation in initiating downstream signaling during PMN phagocytosis of EIgG.
- To determine the relationship between Syk activation, PI 3-kinase activity, and downstream signaling events.
- To elucidate the signaling pathway downstream of Syk activation.
Main Methods:
- PMN phagocytosis assays using EIgG.
- Western blotting to assess protein phosphorylation (Syk, ERK1/2).
- Pharmacological inhibition of Syk (piceatannol) and PI 3-kinase (wortmannin, LY294002).
- Assessment of protein translocation (PKCdelta, Raf-1) and PI 3-kinase activity.
- Rescue experiments using diacylglycerol analogue (didecanoylglycerol).
Main Results:
- Syk phosphorylation increased time-dependently with EIgG challenge, paralleling phagocytosis.
- Piceatannol inhibited EIgG phagocytosis, Syk phosphorylation, PKCdelta and Raf-1 translocation, and ERK1/2 phosphorylation.
- Syk phosphorylation preceded PI 3-kinase activation; both wortmannin and piceatannol inhibited PI 3-kinase, but only piceatannol inhibited Syk.
- Wortmannin did not inhibit PKCdelta and Raf-1 translocation, unlike piceatannol.
- Didecanoylglycerol rescued PMN phagocytosis, PKCdelta/Raf-1 translocation, and ERK1/2 phosphorylation in Syk-inhibited cells, independent of PI 3-kinase activity and Syk phosphorylation.
Conclusions:
- Syk activation is a prerequisite for initiating downstream signaling in PMN phagocytosis of EIgG.
- Downstream signaling, including mitogen-activated protein kinase activation, is initiated by Syk activation independently of PI 3-kinase activation.
- Syk acts upstream of PKCdelta, Raf-1, and ERK1/2 activation in this phagocytic pathway.