IntroductionPolymorphonuclear neutrophils (PMNs) play an important role in host defense and inflammation. During the acute inflammatory response, PMNs extravasate from the blood into the tissue, where they migrate toward the sites of lesion by following a gradient of chemoattractants. 1,2 Site-directed migration (chemotaxis) requires 3 different processes 3 : (1) the periodical formation of lamellipodia; (2) the establishment of cell polarity that is characterized by the formation of a posterior pole (uropod) and an anterior pole (leading edge) that forms the lamellipodium with enhanced sensitivity for chemoattractants; and (3) the translation of the external chemotactic gradient into an internal signaling gradient. This process of directional sensing is due to an asymmetric distribution of signaling components within the cell. 3 Accordingly, neutrophil-like differentiated cells, an established model for PMNs, orient their polarity in response to a chemoattractant gradient and perform site-directed migration. 4 Chemoattractants signal via G-protein-coupled receptors that are responsible for directional sensing and the establishment of the polarized sensitivity within the cell. 3 However, ordered leukocyte polarization not only requires the presence of chemoattractants, but also depends on adhesive cell-matrix interactions that are mediated mainly by the leukocyte adhesion molecules of the integrin family. 5 ␤ 2 integrins (CD11/CD18) especially are critically involved in firm adhesion and migration of PMNs. 6 Recently, we and others have shown that the non-receptor tyrosine kinase Syk is constitutively associated with CD18, the ␤ subunit of the ␤ 2 integrins in human PMNs and in HL-60 cells. [7][8][9] Syk is widely expressed in hematopoietic cells and fulfills essential roles in hematopoietic cell functions like Fc receptor 10 and cytokine receptor signaling, 11 phagocytosis, 12 lymphocyte development, 13,14 and platelet activation. 15,16 Syk and ZAP-70, the other member of this protein family, contain 2 Src homology 2 (SH2) domains in a tandem repeat that are separated by the interdomain A. The C-terminal kinase domain is separated from the 2 SH2 domains by the interdomain B. 17 Outside-in signaling of Mac-1 (CD11b/CD18), the most abundant ␤ 2 integrin on PMNs, occurs upon ligand binding to immobilized fibrinogen or the intercellular adhesion molecules-1 and -2. 18 Mac-1-mediated adhesion induces tyrosine phosphorylation of different signaling components, including Syk. 7 A physiologic role for Syk has been demonstrated previously for multiple ␤ 2 integrin-mediated functions of murine PMNs, including the respiratory burst, degranulation, and spreading. 19 Recently, we have shown that endogenous Syk is enriched at the leading edge of both murine PMNs and HL-60 cells, where Syk plays a role in the stabilization of the leading edge of the polarized cell and colocalizes with CD18. 19,20 Syk has several downstream targets, including Pyk2, and Vav. 17,21 Vav is a guanine exchange factor for the Rho family GTPases Ra...

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class IA. Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class IA as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo.

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