Size of IgG-opsonized particles determines macrophage response during internalization

Abstract

It is generally assumed that particles >1 μm elicit a phagocytic response. To determine whether this is the case, we examined the uptake and transport of IgG-opsonized polystyrene beads of defined size, ranging from 0.2 to 3 μm, by mouse bone marrow-derived macrophages. The kinetics of opsonized bead internalization were comparable for each of the different beads examined. We used rhodamine phalloidin to examine particle-induced assembly of F-actin phagocytic cups by fluorescence microscopy. Phagocytic cup formation was size dependent in a nonlinear fashion. Less than 30% of 0.2- to 0.75-μm particles and greater than 80% of 2- and 3-μm particles were associated with F-actin. Cells treated with 0.25 μm cytochalasin D showed decreased phagocytic cup formation and a linear decrease in bead uptake as a function of particle surface area. In contrast, potassium depletion, which preferentially inhibits clathrin-mediated endocytosis, was more effective at inhibiting the uptake of smaller beads. Thus, with increasing particle size, IgG-opsonized particle uptake became less clathrin dependent and more actin dependent. The kinetics of ligand delivery to lysosomes was measured using an immunoprecipitation assay based on the intermixing of internalized anti-dinitrophenol (DNP) IgG with DNP-derivitized β-glucuronidase (DNP-β-glu) incorporated into lysosomes. Soluble mannosylated anti-DNP IgG was delivered to lysosomes after an 8-min lag period. The kinetics of anti-DNP IgG-opsonized beads showed a size-dependent response, where beads sized 0.2, 0.5, and 0.75 μm showed a lag period prior to delivery to lysosomes. In contrast, beads 1.0 μm or larger showed no lag in delivery to lysosomes. Since beads that had no lag in delivery to lysosomes also showed high levels of phagocytic cup formation, this suggests that phagocytic cups may be important in the rapid delivery of internalized particles to lysosomes.