Cytosolic free Ca(2+) changes and calpain activation are required for beta integrin-accelerated phagocytosis by human neutrophils.
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(2+) imaging, the temporal relationship between changes in cytosolic free Ca(2+) concentration and phagocytosis were correlated. Here we show that whereas small, localized Ca(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(2+) which resulted from Ca(2+) influx. This latter rise in cytosolic free Ca(2+) concentration also liberated a fraction of beta2 integrin receptors which were initially immobile on the neutrophil surface, as demonstrable by both fluorescence recovery after laser bleaching and by visualization of localized beta2 integrin labelling. Inhibitors of calpain activation prevented both the Ca(2+)-induced liberation of beta2 integrin and the rapid stage of phagocytosis, despite the persistence of the global Ca(2+) signal. Therefore, we propose that Ca(2+) activation of calpain causes beta2 integrin liberation, and that this signal plays a key role in the acceleration of beta2 integrin-mediated phagocytosis.
Dewitt
Sharon
S
Neutrophil Signalling Group, University Department of Surgery, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom.
Hallett
Maurice B
MB
D039481:CD11b Antigen / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048429:Cell Size; D002614:Chelating Agents / Q000378:metabolism; D003179:Complement C3b / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D009504:Neutrophils / Q000166:cytology / Q000502:physiology*; D010587:Phagocytosis / Q000502:physiology*; D015054:Zymosan / Q000378:metabolism
D039481:CD11b Antigen; D018821:CD18 Antigens; D002614:Chelating Agents; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D003179:Complement C3b; D015054:Zymosan; D002154:Calpain; D002118:Calcium; D016257:Fura-2