Phospholipase D1 Regulates Phagocyte Adhesion

Abstract

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation,transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Since phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Using the fluorescent cellular label, calcein, we found that adhesion of primary human neutrophils and monocyte‐derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP‐1) and murine (RAW) myeloid‐macrophage cell lines to fibronectin, fibrinogen, collagen or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy demonstrated that PLD1 colocalized with actin filaments in adherent phagocytes, in proximity to the focal adhesion proteins, paxillin and talin. Reductions in PLD activity by chemical inhibitors or specific siRNA‐induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion. This was accompanied by decreased polymerization of actin at the adhesion interface and reductions in total cellular F‐actin, quantitated using TRITC‐ phalloidin. These data support the hypothesis that PLD1 regulates the initial stages of phagocyte adhesion via promotion of actin polymerization and suggest that stimulation of PLD activity may promote adhesion‐dependent effector responses. Studies supported by NIH GM62302 and AI0559126, and VA Merit Review grants to DJK.

PLD1 rather than PLD2 regulates phorbol-ester-, adhesion-dependent and Fcγ-receptor-stimulated ROS production in neutrophils

“…The results we present are surprising in the context of an accumulation of data over previous years which has implicated PLD in many neutrophil (or neutrophil-like) responses, such as polarisation of the actin cytoskeleton (Zouwail et al, 2005;Nishikimi et al, 2009;Su et al, 2009), migration (Zouwail et al, 2005;Lehman et al, 2006;Powner et al, 2007;Su et al, 2009;Knapek et al, 2010), adhesion and spreading (Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Su et al, 2009) and production of ROS (Cadwallader et al, 2004;Levy et al, 2005). However, much of this published work was carried out using siRNAs (Iyer et al, 2006;Lehman et al, 2006;Chae et al, 2008;Knapek et al, 2010) or primary alcohols as inhibitors of PLDmediated PA production (Cadwallader et al, 2004;Zouwail et al, 2005;Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Knapek et al, 2010).…”

“…However, much of this published work was carried out using siRNAs (Iyer et al, 2006;Lehman et al, 2006;Chae et al, 2008;Knapek et al, 2010) or primary alcohols as inhibitors of PLDmediated PA production (Cadwallader et al, 2004;Zouwail et al, 2005;Iyer et al, 2006;Powner et al, 2007;Chae et al, 2008;Knapek et al, 2010).…”

“…As a major part of the body's antibacterial and fungal defence machinery, neutrophils are required to carry out a range of roles; guided by inflammatory mediators, they migrate from the bloodstream to sites of infection where foreign cells are engulfed and degraded by the release of reactive oxygen species (ROS) and proteolytic granules. A number of key neutrophil functions, such as migration, phagocytosis, actin cytoskeleton remodelling, degranulation and respiratory burst activation have been associated with PLD activity and PA (Agwu et al, 1991;Bauldry et al, 1991;Zhou et al, 1993;Serrander et al, 1996;Cadwallader et al, 2004;Levy et al, 2005;Powner et al, 2005;Zouwail et al, 2005;Corrotte et al, 2006;Iyer et al, 2006;Lehman et al, 2006;Carrigan et al, 2007;Powner et al, 2007;Chae et al, 2008;Nishikimi et al, 2009;Su et al, 2009). …”

Phospholipase D Is Essential for Keratocyte-like Migration of NBT-II Cells

“…These results suggest that localization of PLD to the anterior regions in migrating cells plays important roles in cell migration, pre- sumably through PLD-dependent regulation of the actin cytoskeleton or cell adhesion. This notion is corroborated by the results of earlier studies (Iyer et al, 2006;Santy and Casanova, 2001;Shen et al, 2002).…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…Integrins are known to stimulate PLD and the integrin-induced increase in F-actin requires PLD activity. As such, butanol treatment or knockdown of PLD is associated with defects in the ability to form adhesions (Aguirre Ghiso et al, 1997;Iyer et al, 2006). The speed of migration is largely controlled by the speed at which cells form and detach from these adhesions (Lauffenburger and Horwitz, 1996) and PLD regulates migration speed in wound-healing assays (Mazie et al, 2006).…”