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.