Abstract
Phagocytosis of opsonized pathogens by circulating non-adherent neutrophils is an essential step in host defense, which when overwhelmed contributes to sepsis. To investigate the role played by ligation of complement receptors CR3 and CR4 in non-adherent neutrophils, we designed a novel assay system utilizing dual optical traps, respectively, holding a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD18 as an example ligand, mimicking the bacterial opsonizing complement fragment iC3b. Presentation of anti-CD18-coated beads elicited both pseudopodial protrusion and subsequent phagocytosis. This is in sharp contrast to previously reported responses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used this same new assay to probe actomyosin pathways in the neutrophil's pseudopodial and phagocytic response. Disruption of actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosis rates. In summary, i) the new dual trap assay can be used to study the responses of suspended neutrophils to a variety of ligands, and ii) in a first application of this technique, we found that local ligation of CR3/4 in unactivated neutrophils in suspension induces pseudopod formation and phagocytosis at that site, and that these events occur via an actomyosin-dependent pathway.
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Acknowledgements
This work was supported by grants from the Japan Society for the Promotion of Science No. 17590776 to HK. We thank M Sato of Nambu-irika Co. for help in maintaining optimal operating conditions of the DIC microscope, lasers, and optical traps. This work was supported in part by Mombusho Grant No. 12670544, and a JAFAH fellowship from the Japan Foundation for Aging and Health (JPB).
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Dynamics of pseudopod formation and phagocytosis of an anti-CD18 bead at the presentation site. An anti-CD18-coated bead was attached to a round non-adherent neutrophil suspended in the control medium by an optical trap. (MOV 927 kb)
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Suzuki, T., Yanai, M., Kubo, H. et al. Interaction of non-adherent suspended neutrophils to complement opsonized pathogens: a new assay using optical traps. Cell Res 16, 887–894 (2006). https://doi.org/10.1038/sj.cr.7310103
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DOI: https://doi.org/10.1038/sj.cr.7310103
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