title: Photometric assays for FcRI-dependent binding, phagocytosis, and antibody-dependent cellular cytotoxicity mediated by monomeric IgG gamma 2a in murine peritoneal macrophages.
abstract: Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcRI binds monomeric IgG gamma 2a, but not monomeric IgG gamma 2b or IgG gamma 1 with high affinity at 4 degrees C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4 degrees C of monomeric IgG gamma 2a to FcRI 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 gamma 2a, but not IgG gamma 2b or IgG gamma 1 to FcRI of oil-elicited mouse peritoneal macrophages at 4 degrees 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 gamma 2a to macrophage FcRI 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 degrees C. Upregulation of FcRI-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 FcRI-dependent binding and phagocytosis by monomeric IgG gamma 2a, but had little effect on FcRII-dependent functions. Cytophilic binding of monomeric IgG gamma 2a to FcRI failed to trigger ADCC activation. Thus functional characterization of macrophage FcRI-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG gamma 2a 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 gamma 2a could provide an efficient mechanism by macrophages in vivo for FcRI-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.
authors.affiliation: Samuel Roberts Noble Foundation, Inc., Biomedical Division, Ardmore, OK 73402.
authors.forename: R W
authors.initials: RW
authors.lastname: Leu
authors.forename: C J
authors.initials: CJ
authors.lastname: Robinson
authors.forename: J A
authors.initials: JA
authors.lastname: Wiggins
authors.forename: B J
authors.initials: BJ
authors.lastname: Shannon
authors.forename: J A
authors.initials: JA
authors.lastname: Rummage
authors.forename: M J
authors.initials: MJ
authors.lastname: Horn
mesh_terms: D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D000943:Antigens, Differentiation / Q000032:analysis* / Q000378:metabolism; D003592:Cytophotometry / Q000379:methods; D007074:Immunoglobulin G / Q000378:metabolism / Q000502:physiology*; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D010529:Peritoneal Cavity / Q000166:cytology; D010587:Phagocytosis / Q000187:drug effects; D011961:Receptors, Fc / Q000032:analysis* / Q000378:metabolism; D017452:Receptors, IgG; D014357:Trypsin
chemical_list: D000943:Antigens, Differentiation; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D014357:Trypsin