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Quantal release of free radicals during exocytosis of phagosomes

Nature Cell Biology volume 4, pages 279–285 (2002)Cite this article

Abstract

Secretion of lysosomes and related organelles is important for immune system function. High-resolution membrane capacitance techniques were used to track changes in membrane area in single phagocytes during opsonized polystyrene bead uptake and release. Secretagogue stimulation of cells preloaded with beads resulted in immediate vesicle discharge, visualized as step increases in capacitance. The size of the increases were consistent with phagosome size. This hypothesis was confirmed by direct observation of dye release from bead-containing phagosomes after secretagogue stimulation. Capacitance recordings of exocytosis were correlated with quantal free radical release, as determined by amperometry. Thus, phagosomes undergo regulated secretion in macrophages, one function of which may be to deliver sequestered free radicals to the extracellular space.

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Figure 1: Ultrastructural and electrophysiological analysis of hydrophobic polystyrene bead uptake in J774 macrophages.
Figure 2: Comparison of the time course and amplitude of GTP-γS stimulated secretory response in resting and polystyrene bead-loaded cells.
Figure 3: Comparative ultrastructure of cells loaded with latex beads and cells which have been stimulated to release beads after loading.
Figure 4: Quinacrine staining of J774.1 cells.
Figure 5: Macrophages exocytose phagosomes containing beads after stimulation with human HAIGG.
Figure 6: Simultaneous recording of membrane capacitance changes and superoxide-generated amperometric current in a J774 macrophage.
Figure 7: Correlation between superoxide release and capacitance increase in GTP-γS stimulated J774 macrophages preloaded with beads.

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Acknowledgements

This work was supported by NIH/NIGMS R01 GM36823 to DJN, and the University of Chicago DDRCC (DK42086).

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Authors and Affiliations

  1. Dept. of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, 60637, IL, USA

    Anke Di, Boris Krupa, Vytas P. Bindokas, Mary E. Brown, H. Clive Palfrey & Deborah J. Nelson

  2. Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, 60637, IL, USA

    Yimei Chen

  3. Department of Physiology and Biophysics, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Blvd., MCLM 985, Birmingham, 35294, AL, USA

    Anjaparavanda P. Naren & Kevin L. Kirk

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Supplementary information

Movie 1

J774.1 macrophage cell loaded with polystyrene beads and quinacrine. All quinacrine-loaded vesicles were typically discharged within 20 sec of HAIGG addition. Numbers adjacent to the vesicles mark denote sequence of quinacrine-stained vesicle release. (AVI 1628 kb)

Movie 2

The surface plane of a J774.1 macrophage cell pre-loaded with polystyrene beads shown following HAIGG application. Beads are observed to rise to the surface over a time course of 128 sec. Arrows mark appearance of beads with time at the cell surface.view). (AVI 1935 kb)

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Di, A., Krupa, B., Bindokas, V. et al. Quantal release of free radicals during exocytosis of phagosomes. Nat Cell Biol 4, 279–285 (2002). https://doi.org/10.1038/ncb771

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