High voltage immunoelectron microscopy of complement receptor type 3-mediated capping and internalization of group A...

The mechanism of human neutrophil clearance of peptidoglycan group A-specific polysaccharide polymers derived from streptococcal cell walls (PG-APS) was investigated by high voltage immunoelectron microscopy (HVEM) in order to determine how neutrophils process this highly inflammatory bacterial debris. Neutrophil monolayers were incubated from 5-30 min with serum-opsonized PG-APS. Cells were lightly fixed with 0.5% glutaraldehyde, and the PG-APS was localized on the neutrophil surface by immunogold using antibodies to N-acetyl-glucosamine and 15 nm colloidal gold coupled to goat anti-rabbit IgG. Neutrophils were viewed unsectioned by stereo HVEM. Patches of PG-APS were distributed randomly on the plasmalemma of well-spread neutrophils within 5 min. In polarized cells, PG-APS was densely localized on the uropod and retraction fibers. Within 15 min, PG-APS was predominantly concentrated into a large aggregate, measuring approximately 1 micron in diameter, near the cell margin or nucleus. The aggregate of PG-APS was engulfed in the vicinity of the indentation of the nucleus (hof). Intact microfilaments were required for aggregation and internalization of PG-APS. Binding of PG-APS was dependent upon complement fixation. Furthermore, PG-APS elicited an increase in density of complement receptor type 3 (CR3, C3bi receptor) on the neutrophil surface as determined by morphometry of immunogold labeled anti-CR3. When cells were stained for both PG-APS and CR3, co-localization was observed, and stereomicroscopy revealed clusters of CR3 in areas associated with phagocytosis. These data suggest that neutrophils use an efficient mechanism for removal of bacterial debris. Unlike whole streptococci which are phagocytosed at multiple sites, these bacterial cell walls are first collected into a large aggregate, or cap, which is then internalized at one site.