Abstract
Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.
Highlights
Macrophages, which are scattered throughout every tissue, represent a key component of the immune system through their ability to phagocytose foreign material and dying cells, release cytokines and function as antigen-presenting cells
The presence or absence of WASP family verprolinhomologous (WAVE) transcripts in the murine monocyte/macrophage RAW/LR5 cell line and in primary murine thioglycollateelicited peritoneal macrophages was examined by Reverse transcriptase-polymerase chain reaction (RT-PCR)
To determine whether the detected transcripts were indicative of protein expression, WAVE1 and WAVE2 expression was evaluated by western blotting and compared with Wiskott-Aldrich Syndrome protein (WASP) expression (Fig. 1B,C)
Summary
Macrophages, which are scattered throughout every tissue, represent a key component of the immune system through their ability to phagocytose foreign material and dying cells, release cytokines and function as antigen-presenting cells. CSF-1 treatment of macrophages leads to a rapid reorganization of the actin cytoskeleton leading to profound cell morphological changes such as extension of membrane protrusions These early events are required for and followed by the directed migration of macrophages toward the source of CSF-1 (Allen et al, 1997; Boocock et al, 1989; Webb et al, 1996). While the cell signaling events important for macrophage chemotaxis to CSF-1 remain incompletely elucidated, several molecules have been identified as critical components in various steps of this process. These include the p110 catalytic subunit isoforms  and ␥ of class IA phosphatidylinositide 3kinases (PI 3-kinase) (Vanhaesebroeck et al, 1999) and the
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