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 and differentiation of erythroid progenitor cells and to produce apoptosis 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 (ECFCs) from human blood in sufficient quantity and purity to measure binding of radioiodinated recombinant human IFN-gamma ([125I]rhIFN-gamma). When [125I]rhIFN-gamma was incubated with day 6 ECFC, 77% of the binding 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 straight line passing through the origin, and equilibrium was reached at 0 degree C by 24-48 hours. Saturation of specific binding occurred at a [125I]rhIFN-gamma concentration of 1.0 nM and internalization was demonstrated with further incubation at 37 degrees C. Scatchard analysis showed a single class of binding sites and at a high ECFC cell purity of 80-89%, 1910-2070 binding sites per ECFC were present with a Kd 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 for [125I]IFN-gamma greatly decreased. These experiments delineate specific binding sites for IFN-gamma on human erythroid progenitor cells and indicate that the enhanced sensitivity to rhIFN-gamma inhibition of mature day 3-day 6 burst-forming units-erythroid may be a result of enhanced specific binding. Human IFN-gamma is a multifunctional lymphokine, secreted by activated T lymphocytes and NK cells, which exerts antiviral, 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 colony-forming units, burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) in vitro [3-7]. Most recently, mature day 3 to day 6 BFU-E have been shown to be most sensitive to the inhibitory effect of recombinant human (rh) IFN-gamma, while primitive day 1 to day 2 cells and later day 7 cells were less affected [7]. Incubation of rhIFN-gamma with mature BFU-E inhibits hemoglobin accumulation and produces apoptosis of the maturing erythroid cells [7]. Moreover, since blood IFN-gamma levels are elevated and vary directly with the degree of the anemia, in patients with hematologic malignancies [8] and HIV-seropositivity [9], IFN-gamma appears to have a prominent role in producing the anemia associated with chronic disease [10,11]. Although characterization of human IFN-gamma receptors has been extensively performed for a variety of human cells including fibroblasts, lymphocytes, monocytes, granulocytes, eosinophiles, platelets, and many tumor cells [12-17], IFN-gamma receptors have not been identified on red cells [12] and the presence plus the extent of IFN-gamma receptors on progenitor cells, including human erythroid progenitor cells, remains unknown. A method has been reported from our laboratory by which human erythroid colony-forming cells (ECFC) can be highly purified, starting with peripheral blood BFU-E, in a sufficient amount for analysis of cytokine binding [18-20]. In this paper, we report the results of [125I]rhIFN-gamma binding to day 6 ECFC in vitro and demonstrate the presence of specific binding that is saturable at 1.0 nM. Scatchard analysis reveals that there are 1910-2070 rhIFN-gamma binding sites per ECFC with a Kd of 0.01-0.02 nM and, as with erythropoietin (EP) and insulin-line growth factor I (IGF-I) receptors, specific binding is highest with the earliest BFU-E studied and declines progressively as the erythroid progenitors mature.
Taniguchi
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S
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6305, USA.
Dai
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CH
Krantz
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SB
D003114:Colony-Forming Units Assay; D004705:Endocytosis; D015672:Erythroid Precursor Cells / Q000378:metabolism; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D017471:Receptors, Interferon / Q000097:blood*; D013997:Time Factors; D000096743:Interferon gamma Receptor
D007371:Interferon-gamma; D017471:Receptors, Interferon; D000096743:Interferon gamma Receptor