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Phagocytosis (and endocytosis) is an unusual cellular process that results in the formation of a novel subcellular organelle, the phagosome. This phagosome contains not only the internalised target of phagocytosis but also the external medium, creating a new border between extracellular and intracellular environments. The boundary at the plasma membrane is, of course, tightly controlled and exploited in ionic cell signalling events. Although there has been much work on the control of phagocytosis by ions, notably, Ca2+ ions influxing across the plasma membrane, increasing our understanding of the mechanism enormously, very little work has been done exploring the phagosome/cytosol boundary. In this paper, we explored the changes in the intra-phagosomal Ca2+ ion content that occur during phagocytosis and phagosome formation in human neutrophils. Measuring Ca2+ ion concentration in the phagosome is potentially prone to artefacts as the intra-phagosomal environment experiences changes in pH and oxidation. However, by excluding such artefacts, we conclude that there are open Ca2+ channels on the phagosome that allow Ca2+ ions to "drain" into the surrounding cytosol. This conclusion was confirmed by monitoring the translocation of the intracellularly expressed YFP-tagged C2 domain of PKC-γ. This approach marked regions of membrane at which Ca2+ influx occurred, the earliest being the phagocytic cup, and then the whole cell. This paper therefore presents data that have novel implications for understanding phagocytic Ca2+ signalling events, such as peri-phagosomal Ca2+ hotspots, and other phenomena. [ABSTRACT FROM AUTHOR]

Phagocytosis of microbes coated with opsonins such as the complement component C3bi is the key activity of neutrophils. However, the mechanism by which opsonins enhance the rate of phagocytosis by these cells is unknown and has been difficult to study, partly because of the problem of observing and quantifying the events associated with phagocytosis. In this study, C3bi-opsonized particles were presented to neutrophils with a micromanipulator, so that the events of binding, pseudopod cup formation, engulfment, and completion of phagocytosis were clearly defined and distinguished from those involved with chemotaxis. Using this approach in combination with simultaneous phase contrast and [Ca.sup.2+] imaging, the temporal relationship between changes in cytosolic free [Ca.sup.2+] concentration and phagocytosis were correlated. Here we show that whereas small, localized [Ca.sup.2+] changes occur at the site of particle attachment and cup formation as a result of store release, rapid engulfment of the particle required a global change in cytosolic free [Ca.sup.2+] which resulted from [Ca.sup.2+] influx. This latter rise in cytosolic free [Ca.sup.2+] concentration also liberated a fraction of [beta]2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized [beta]2 integrin labelling. Inhibitors of calpain activation prevented both the [Ca.sup.2+]-induced liberation of [beta]2 integrin and the rapid stage of phagocytosis, despite the persistence of the global [Ca.sup.2+] signal. Therefore, we propose that [Ca.sup.2+] activation of calpain causes [beta]2 integrin liberation, and that this signal plays a key role in the acceleration of [beta]2 integrin-mediated phagocytosis.

The 18-20 kD glycoprotein CD59 is attached to the cell membrane via glycosyl phosphatidylinositol (GPI) to induce the activation of T cells and neutrophils by binding with an antibody. Studies revealed that the signaling capacity of CD59 is acquired upon cross-linking with protein kinases. Association with protein kinases also induced a large calcium transient to enable CD59 to function efficiently as a signal-transducing molecule.