Transient activation of the NADPH oxidase through Fc gamma RI. Oxidase deactivation precedes internalization of cross-linked receptors.
It is well known that Fc gamma R mediate the rapid release of agents of inflammation and, in addition, play an important role in the uptake of stimulatory antibody complexes. Activation of the FcR for human IgG1 (Fc gamma RI) on human monocytic cells triggers a transient activation of the NADPH oxidase. In this study, we tested the possibility that transience of the NADPH oxidase activation might have been the result of rapid internalization of cross-linked Fc gamma RI. Stimulatory receptor moieties were formed by cross-linking Fc gamma RI with receptor-specific mAb that are known to trigger superoxide anion release. The formation of the stimulatory receptor units was determined by quantitating the rate of superoxide anion production through its reduction of cytochrome c. This rate has been found to correlate with the rate of binding of cross-linking antibody and, therefore, the rate of formation of the stimulatory moieties (receptor aggregates). Internalization of cross-linked Fc gamma RI was measured by quantitation of cell-associated FITC-labeled Fc gamma RI-specific mAb resistant to acid elution. We found that cross-linking antibody bound to Fc gamma RI continued to be taken up by the cells well after cessation of oxidase activity. The constant rate of uptake and the differential effect of temperature on these two functions suggested that they are separately regulated. Quantitation of cross-linked receptors that were inactive, i.e., no longer stimulating superoxide anion production, indicated that 50% of internalizable, and therefore cross-linked, Fc gamma RI remained on the surface after oxidase activity had ceased. This evidence of cessation of oxidase activity before the endocytic uptake of mAb/R stimulatory units indicates that the activated state of surface cross-linked Fc gamma RI is of brief duration and that occupation of the receptors by cross-linking-ligand does not sustain the activated state of the receptor. Thus, Fc gamma RI-mediated oxidase activation is temporally limited to the formation of the stimulatory receptor moiety.
Pfefferkorn
L C
LC
Department of Microbiology, Dartmouth Medical School, Hanover, NH 03756.
Fanger
M W
MW
D000643:Ammonium Chloride / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000502:physiology; D000943:Antigens, Differentiation / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D001666:Binding Sites, Antibody; D002460:Cell Line; D003432:Cross-Linking Reagents; D004705:Endocytosis; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D009000:Monocytes / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D011961:Receptors, Fc / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D017452:Receptors, IgG; D013481:Superoxides / Q000378:metabolism; D013696:Temperature; D013861:Thiocyanates
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D003432:Cross-Linking Reagents; D005452:Fluoresceins; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D013861:Thiocyanates; D000643:Ammonium Chloride; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D016650:Fluorescein-5-isothiocyanate