Syk Regulates Multiple Signaling Pathways Leading to CX3CL1 Chemotaxis in Macrophages

Abstract

Several studies have clearly established the importance of the interaction between macrophages and CX3CL1 in the progression of disease. A previous study demonstrated that Syk was required for CX3CL1-mediated actin polymerization and chemotaxis. Here, we delineated the signaling cascade of Syk-mediated cell migration in response to CX3CL1. Inhibition of Syk in bone marrow-derived macrophages or reduction of Syk expression using siRNA in RAW/LR5 cells indicated that Syk was required for the activation of PI3K, Cdc42, and Rac1. Also, reduction in WASP or WAVE2 levels, common downstream effectors of Cdc42 or Rac1, resulted in impaired cell migration to CX3CL1. Syk indirectly regulated WASP tyrosine phosphorylation through Cdc42 activation. Altogether, our data identify that Syk mediated chemotaxis toward CX3CL1 by regulating both Rac1/WAVE2 and Cdc42/WASP pathways, whereas Src family kinases were required for proper WASP tyrosine phosphorylation.Chemokines were first described as chemoattractant cytokines synthesized at sites of inflammation that stimulate the directional migration of leukocytes and mediate inflammation (1, 2). Among chemokines, CX3CL1 (also called fractalkine) is the only chemokine that functions not only as a chemoattractant but also as an adhesion molecule, and it is likely that CX3CL1 is involved in the extravasation of leukocytes into inflamed tissues (2-4). Inappropriate expression or function of CX3CL1 has also been implicated in inflammatory conditions leading to vascular and tissue damage (2), and CX3CL1 has now emerged as important mediator in the pathogenesis of various clinical diseases, including rheumatoid arthritis (5-7), atherosclerosis, and cardiovascular disease (8 -11).The recruitment of monocyte/macrophages in response to CX3CL1 has been shown to significantly correlate with the extent of disease (12). The direct effects of CX3CL1 at the wound site has been tested using disruption of the receptor for CX3CL1, CX3CR1, and included marked reduction of macrophages and macrophage products such as TGF-␤1 and vascular endothelial growth factor (13). Indeed, ApoE Ϫ/Ϫ CX3CR1 Ϫ/Ϫ mice have significantly reduced atherosclerotic lesion size and macrophage recruitment (9). In addition, deletion of CX3CL1 in CCR2 (Ϫ/Ϫ) mice dramatically reduced macrophage accumulation in the artery wall and the subsequent development of atherosclerosis (14). These multiple studies have clearly established the importance of the interaction between macrophages and CX3CL1. However, the mechanistic details underlying macrophage to chemotaxis toward CX3CL1 are not well understood. CX3CL1 exerts its activities upon interaction with a G protein-coupled receptor, CX3CR1, that is highly expressed on different lymphocyte populations, including monocytes, T cells, and natural killer cells (15, 16). Cambien et al.(1) have identified activation of both Src and Syk tyrosine kinases under soluble CX3CL1 stimulation in MonoMac6 cells. Also, Syk was rapidly activated and required for proper formation of membrane protrusions a...

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

“…WT BMM cells showed an increased in WASP phosphorylation after CX3CL1 or EIgG treatment, peaking at 1 or 5 min, respectively (Fig. 1A), consistent with results published previously (14,15). WASP phosphorylation was significantly reduced in Hck Ϫ/Ϫ BMM cells under the same conditions (Fig.…”

“…Hck Deficiency Results in a Defect of WASP-mediated Macrophage Functions-Our previous work established that macrophage functions such as phagocytosis, chemotaxis, and matrix degradation depend on tyrosine phosphorylation of WASP for optimal function (9,14,15). Therefore, we investigated the effect of Hck deficiency or reduced expression of Hck on macrophage functions.…”

“…Hck Mediates WASP Tyrosine Phosphorylation-We previously showed that tyrosine phosphorylation of WASP was required for macrophage chemotaxis to CX3CL1 (15) and Fc ␥ R-mediated phagocytosis (14). Because SFKs are required for tyrosine phosphorylation of WASP (15), we speculated that Hck might be the major kinase mediating WASP tyrosine phosphorylation.…”

“…Chemotaxis was measured using 8-m pore size inserts (Falcon; BD Biosciences) according to the manufacturer's instructions and described in Park and Cox (15). Briefly, the inserts were placed into 24-well plates containing RPMI 1640 for RAW/LR5 cells or ␣-MEM for BMMs in the presence or absence of CX3CL1 (50 ng/ml).…”

“…WASP activity is required for several macrophage functions, in addition to podosome formation such as efficient fibronectin degradation, phagocytosis, and chemotaxis (9,(13)(14)(15). Remarkably, all of these WASP-mediated functions also require phosphorylation of WASP on tyro-sine 291 (human numbering) (9,14,15).…”