SPECIFIC BINDING OF INTERFERON-GAMMA TO HIGH-AFFINITY RECEPTORS ON HUMAN ERYTHROID COLONY-FORMING CELLS

Interferon-gamma (IFN-gamma) has been shown to inhibit proliferation a nd differentiation of erythroid progenitor cells and to produce apopto sis of erythroid cells, but IFN-gamma receptors are not present on red cells and have never been demonstrated on erythroid progenitor cells. We obtained highly purified day 6 erythroid colony-forming cells (ECF Cs) from human blood in sufficient quantity and purity to measure bind ing of radioiodinated recombinant human IFN-gamma ([I-125]rhIFN-gamma) . When [I-125]rhIFN-gamma was incubated with day 6 ECFC, 77% of the bi nding was inhibited by excess unlabeled rhIFN-gamma, but no inhibition occurred with a variety of growth factors and glycoproteins. Specific binding was directly proportional to the cell concentration with a st raight line passing through the origin, and equilibrium was reached at 0 degrees C by 24-48 hours. Saturation of specific binding occurred a t a [I-125]rhIFN-gamma concentration of 1.0 nM and internalization was demonstrated with further incubation at 37 degrees C. Scatchard analy sis showed a single class of binding sites and at a high ECFC cell pur ity of 80-89%, 1910-2070 binding sites per ECFC were present with a K- d of 0.01-0.02 nM. As day 5 ECFC developed into more mature day 7-day 12 cells, with incubation at 37 degrees C in vitro, specific binding f or [I-125]IFN-gamma greatly decreased. These experiments delineate spe cific binding sites for IFN-gamma on human erythroid progenitor cells and indicate that the enhanced sensitivity to rhIFN-gamma inhibition o f mature day 3-day 6 burst-forming units-erythroid may be a result of enhanced specific binding. Human IFN-gamma is a multifunctional lympho kine, secreted by activated T lymphocytes and NK cells, which exerts a ntiviral, antiproliferative, and immunomodulatory activities on a wide variety of cells [1,2]. With regard to hematopoietic cells, IFN-gamma has been reported to inhibit the growth of granulocyte-macrophage col ony-forming units, burst-forming units-erythroid (BFU-E) and colony-fo rming units-erythroid (CFU-E) in vitro [3-7]. Most recently, mature da y 3 to day 6 BFU-E have been shown to be most sensitive to the inhibit ory effect of recombinant human (rh) IFN-gamma, while primitive day 1 to day 2 cells and later day 7 cells were less affected [7]. Incubatio n of rhIFN-gamma with mature BFU-E inhibits hemoglobin accumulation an d produces apoptosis of the maturing erythroid cells [7]. Moreover, si nce blood IFN-gamma levels are elevated and vary directly with the deg ree of the anemia, in patients with hematologic malignancies [8] and H IV-seropositivity [9], IFN-gamma appears to have a prominent role in p roducing the anemia associated with chronic disease [10,11]. Although characterization of human IFN-gamma receptors has been extensively per formed for a variety of human cells including fibroblasts, lymphocytes , monocytes, granulocytes, eosinophiles, platelets, and many tumor cel ls [12-17], IFN-gamma receptors have not been identified on red cells [12] and the presence plus the extent of IFN-gamma receptors on progen itor cells, including human erythroid progenitor cells, remains unknow n. A method has been reported from our laboratory by which human eryth roid colony-forming cells (ECFC) can be highly purified, starting with peripheral blood BFU-E, in a sufficient amount for analysis of cytoki ne binding [18-20]. In this paper, we report the results of [I-125]rhI FN-gamma binding to day 6 ECFC in vitro and demonstrate the presence o f specific binding that is saturable at 1.0 nM. Scatchard analysis rev eals that there are 1910-2070 rhIFN-gamma binding sites per ECFC with a K-d of 0.01-0.02 nM and, as with erythropoietin (EP) and insulin-lin e growth factor I (IGF-I) receptors, specific binding is highest with the earliest BFU-E studied and declines progressively as the erythroid progenitors mature.