HIGH-VOLTAGE IMMUNOELECTRON MICROSCOPY OF COMPLEMENT RECEPTOR-TYPE 3-MEDIATED CAPPING AND INTERNALIZATION OF GROUP-A STREPTOCOCCAL CELL-WALLS BY HUMAN NEUTROPHILS

The mechanism of human neutrophil clearance of peptidoglycan group A-s pecific polysaccharide polymers derived from streptococcal cell walls (PG-APS) was investigated by high voltage immunoelectron microscopy (H VEM) in order to determine how neutrophils process this highly inflamm atory bacterial debris. Neutrophil monolayers were incubated from 5-30 min with serum-opsonized PG-APS. Cells were lightly fixed with 0.5% g lutaraldehyde, and the PG-APS was localized on the neutrophil surface by immunogold using antibodies to N-acetyl-glucosamine and 15 nm collo idal gold coupled to goat anti-rabbit IgG. Neutrophils were viewed uns ectioned by stereo HVEM. Patches of PG-APS were distributed randomly o n the plasmalemma of well-spread neutrophils within 5 min. In polarize d cells, PG-APS was densely localized on the uropod and retraction fib ers. Within 15 min, PG-APS was predominantly concentrated into a large aggregate, measuring approximately 1 mu m in diameter, near the cell margin or nucleus. The aggregate of PG-APS was engulfed in the vicinit y 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 elicit ed an increase in density of complement receptor type 3 (CR3, C3bi rec eptor) on the neutrophil surface as determined by morphometry of immun ogold labeled anti-CR3. When cells were stained for both PG-APS and CR 3, co-localization was observed, and stereomicroscopy revealed cluster s of CR3 in areas associated with phagocytosis. These data suggest tha t neutrophils use an efficient mechanism for removal of bacterial debr is. Unlike whole streptococci which are phagocytosed at multiple sites , these bacterial cell walls are first collected into a large aggregat e, or cap, which is then internalized at one site. (C) 1994 Wiley-Liss , Inc.