Internalization and degradation of receptor-bound interferon-γ by murine macrophages. Demonstration of receptor recycling

Although the interferon-γ (IFN-γ) receptor on murine and human mononuclear phagocytes has been defined and partially characterized, very little data exists which describes the ultimate fate of receptor-bound ligand. The current studies were specifically designed to define the metabolic processes which act on murine recombinant IFN-γ following its interaction with murine macrophages at physiologic temperatures. Ligand internalization was demonstrated by comparing binding of [¹²⁵I]IFN-γ to macrophages at 4°C and 37°C. When binding was carried out at 4° C, 96% of the cell-associated [¹²⁵I]IFN-γ remained accessible at the plasma membrane and could be stripped from the cell by exposure to pronase. In contrast, at 37°C, only 35% of the cell-associated radioactivity was pronase strippable. Macrophages degraded [¹²⁵I]IFN-γ into trichloroacetic acid-soluble material at 37°C at a constant rate of 7000 molecules/cell/hr over a 12-hr time period. The amount of IFN-γ degraded correlated with the amount of IFN-γ bound to the cell surface. The receptor was neither up- nor down-regulated by ligand or by other agents known to regulate macrophage functional activity such as IFN-α, IN-β, lipopolysaccharide, or phorbol myristate acetate. The constant uptake of IFN-γ by macrophages was due to the presence of an intracellular receptor pool (62% of the total receptor number) and to a mechanism of receptor recycling. Evidence for the latter was obtained using lysosomotropic agents which blocked degradation but not binding and internalization of ligand and caused the intracellular accumulation of receptor. By comparing the relationship between receptor occupancy and biologic response induction, two activation mechanisms became apparent. Induction of certain functions, such as H₂O₂ secretion, appeared to require only a single round of receptor occupancy. However, induction of more complex functions such as nonspecific tumoricidal activity appeared to require three to four rounds of receptor occupancy. These results thus support the concept that IFN-γ internalization and receptor recycling are essential in the induction of nonspecific tumoricidal activity by macrophages.