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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 CR3and CR4 in non-adherent neutrophils,we designed a novel assay system utilizing dual optical traps,respectively,hold-ing a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface.We chose anti-CD18 asan example ligand,mimicking the bacterial opsonizing complement fragment iC3b.Presentation of anti-CD 18-coatedbeads elicited both pseudopodial protrusion and subsequent phagocytosis.This is in sharp contrast to previously reportedresponses of adherent neutrophils,which phagocytize opsonized particles without pseudopod formation.We used thissame new assay to probe actomyosin pathways in the neutrophil’s pseudopodial and phagocytic response.Disruptionof actin or inhibition of myosin light-chain kinase dose-dependently reduced pseudopod formation and phagocytosisrates.In summary,i)the new dual trap assay can be used to study the responses of suspended neutrophils to a variety ofligands,and ii)in a first application of this technique,we found that local ligation of CR3/4 in unactivated neutrophilsin suspension induces pseudopod formation and phagocytosis at that site,and that these events occur via an actomyosin-dependent pathway.
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, hold-ing a suspended unactivated cell and presenting a specific ligand-coated bead to the cell surface. We chose anti-CD18 asan example ligand, mimicking the bacterial opsonizing complement fragment iC3b.Presentation of anti-CD 18- coatedbeads elicited both pseudopodial protrusion and subsequent phagocytosis.This is in sharp contrast to previously reportedresponses of adherent neutrophils, which phagocytize opsonized particles without pseudopod formation. We used thissame 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 pseu dopod formation and phagocytosisrates. 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.