Photometric assays for FcR I-dependent binding, phagocytosis, and antibody-dependent cellular cytotoxicity mediated by monomeric IgG γ2a in murine peritoneal macrophages

Abstract

Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcR I binds monomeric IgG γ2a, but not monomeric IgG γ2b or IgG γ1 with high affinity at 4°C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4°C of monomeric IgG γ2a to FcR I of mouse peritoneal macrophages using newly developed photometric microassays for quantification of binding, phagocytosis, and antibody dependent cellular cytotoxicity (ADCC) of post-opsonized sheep red blood cell (SRBC) targets. Dose-dependent binding specificity of monomeric IgG γ2a, but not IgG γ2b or IgG γ1 to FcR I of oil-elicited mouse peritoneal macrophages at 4°C for 2 h was confirmed to display typical saturation kinetics both by the photometric assay and by a cellular enzyme-linked immunosorbent assay (CELISA). Binding of monomeric IgG γ2a to macrophage FcR I promoted highly efficient phagocytosis of opsonized SRBC in that most cells that were bound were also rapidly internalized by the phagocytic process during a 1 h incubation at 37°C. Upregulation of FcR I-dependent binding and phagocytosis occurred during 24–48 h in vitro culture of macrophages as shown both by the photometric assays and CELISA. Trypsin treatment of macrophages abrogated FcR I-dependent binding and phagocytosis by monomeric IgG γ2a, but had little effect on FcR II-dependent functions. Cytophilic binding of monomeric IgG γ2a to FcR I failed to trigger ADCC activation. Thus functional characterization of macrophage FcR I-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG γ2a to a trypsin degradable receptor which mediates highly efficient phagocytosis but fails to initiate the signal for ADCC activation. It appears that passively bound immune monomeric IgG γ2a could provide an efficient mechanism by macrophages in vivo for FcR I-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.