FcγR-Mediated Phagocytosis Stimulates Localized Pinocytosis in Human Neutrophils

Abstract

Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcγR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcγR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca2+ concentration were prevented. Because stimulation of FcγR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcγR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca2+ dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcγR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation.

Quantitative Analysis of Membrane Remodeling at the Phagocytic Cup

“…More recent data, however, suggest that actin polymerization at the phagocytic cup and pseudopod extension can be affected separately, supporting the notion that membrane remodeling is an independent and important component of the phagocytic response (Lowry et al, 1998;Cox et al, 1999). Indeed, it is now appreciated that membrane remodeling during phagocytosis is an active and complex process that involves localized pinocytosis (Botelho et al, 2002), segregation of membrane components into lipid rafts (Kwiatkowska et al, 2003), lateral diffusion of signaling molecules (Henry et al, 2004), and the insertion of endomembranes by focal exocytosis (Bajno et al, 2000;Braun et al, 2004).…”

“…Endocytosis at the cup is conceivable because neutrophils, which are also professional phagocytes, were reported to perform active pinocytosis during phagocytosis (Botelho et al, 2002). We devised an assay to detect endocytosis at the cup by labeling the plasma membrane of RAW cells with the amphiphilic fluorophore FM4 -64.…”

Regulation of Rab5 GTPase activity during Pseudomonas aeruginosa-macrophage interaction

“…Overall, many reviewers suggested that the pivotal role of cell surface receptors is to generate inflammatory or apoptotic signals whereas the phagosome formation and maturation can cross common paths (Vieira, Botelho et al 2002;Underhill 2005;Kinchen, Doukoumetzidis et al 2008). The organized signaling stages in FcR mediated phagocytosis follows the zipper model proposed by Griffin and Silverstein (Griffin and Silverstein 1974).…”

“…Actin polymerization at the phagocytic cup and pseudopod extension can be affected separately, indicating that membrane remodeling is an independent component of the phagocytic response (Lowry, Duchemin et al 1998;Cox, Tseng et al 1999). Furthermore, membrane remodeling during phagocytosis is an active and complex process that involves localized pinocytosis (Botelho, Tapper et al 2002), segregation of membrane components into lipid rafts (Kwiatkowska, Frey et al 2003), lateral diffusion of signaling molecules (Henry, Hoppe et al 2004), and the insertion of endomembranes by focal exocytosis (Bajno, Peng et al 2000;Braun, Wendt et al 2004). …”

“…Syntaxin13 located on recycling endosomes and syntaxin 7 on late endosomes have been related to phagosome maturation process in macrophage (Vieira, Botelho et al 2002).…”