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First published online 27 May 2003
doi: 10.1242/jcs.00499

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Phagosomal oxidative activity during ß2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca²⁺ signal: Ca²⁺ controls time not space

Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK

^* Author for correspondence (e-mail: hallettmb{at}cf.ac.uk)

Accepted 21 March 2003

The temporal and spatial relationship between particle binding to the neutrophil by ß2 integrin (CR3), the Ca²⁺ elevation and subsequent oxidase activation has been unclear. This is because of the difficulty in studying the time course of individual phagocytic events in individual neutrophils. Here, we have used a micromanipulation technique to present C3bi-opsonised zymosan particles to the neutrophil under observation. In this way, the moment of particle contact, pseudopod formation and internalisation has been established and cytosolic free Ca²⁺ and oxidation of dichlorodihydrofluorescein (DCDHF)-labelled particles determined simultaneously. Using this approach, we have found that the Ca²⁺ signal, which is triggered by CR3-mediated phagocytosis, can be resolved into two temporally separated components. The first Ca²⁺ signal occurs during ß2 integrin engagement as the phagocytic cup forms but does not trigger oxidation of the particle. The second global Ca²⁺ signal, which is triggered about the time of phagosomal closure, causes an abrupt activation of the oxidase. This second Ca²⁺ signal was not restricted to the region of the phagosome yet only triggered the oxidase activation locally in the phagosome, with no evidence of activation at other sites in the neutrophil. This points to a dual control of oxidase activation, with Ca²⁺ controlling the timing of oxidase activation but slower and more localised molecular events, perhaps involving oxidase assembly and phosphatidylinositol 3-phosphate generation, determining the site of oxidase activation.

Key words: Neutrophils, Phagocytosis, Cytosolic free Ca²⁺, Oxidase activation, ß2 integrin

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