FcRn facilitates PMN internalization of IgG-opsonized bacteria. (A) Antigen-binding capacity of a WT IgG1 mAb (GDob1) and its H435A IgG1 derivative to polysaccharide was indistinguishable by ELISA. IgG1 concentrations were quantified by anti-kappa capturing, and anti-Fc detection sandwich ELISA. Equal amounts of IgG were subsequently allowed to bind to pneumococcal polysaccharide 6B-coated plates, and bound IgG was detected with anti-kappa antisera. (B) Alexa488-labeled pneumococci (green) were ingested efficiently by human PMNs when incubated with WT IgG1 but not on incubation with the H435A IgG1 derivative. PMNs were visualized by red membrane staining using MAC-1 mAb (CR3/CD11b). (C) Binding (4°C) and phagocytosis (37°C) of IgG-opsonized FITC-labeled Streptococcus pneumoniae serogroup 6B measured by FACS on incubation with human PMNs in the absence (no IgG) or presence of 5 μg/mL human IgG1 (IgG1) or a mutated H435A IgG1 (H435A IgG1) variant. For evaluation of ingested bacteria (□), fluorescence of PMNs was measured after quenching of extracellular bacteria. Data are represented as phagocytic index (PI).¹⁵  (D) Ingestion of FITC-labeled pneumococci incubated at 37°C with PMNs from WT, β2M-, and FcRn-knockout mice in the presence of IgG1. Fluorescence of bound but not ingested bacteria were quenched as described in “Phagocytosis experiments.” NS indicates not significant; ^*P < .05 when compared with WT. Data are presented as means plus standard deviations from 2 (A) or 4 (D) experiments, respectively. Experiments (B-C) were performed 3 times, yielding similar results.