Ultrastructural, immunocytochemical localization of presumptive erythropoietin binding sites on developing erythrocytic cells of normal human bone marrow.

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Abstract
In spite of the severe restrictions imposed by the lack of high purity erythropoietin (Ep) and Ep-specific antiserum, an attempt was made to demonstrate presumptive Ep binding sites on the surface of developmental forms of erythrocytic cells of normal human bone marrow using ultrastructural immunocytochemical methodology. Cells were exposed to exogenous Ep and subsequently incubated in an Ep antiserum. The surface bound Ep-Ep antiserum complex was then visualized with an Au-labeled IgG reagent. Two Ep preparations of differing potency (55.7 and 9000 U/mg protein) and rabbit anti-Ep (produced by injecting rabbits with human urinary Ep) were employed; the Ep antisera were demonstrated in earlier studies to be specific for Ep. Both morphological and quantitative evaluations of surface labeling densities of the cells of the erythrocytic cell series demonstrated that these cells labeled differentially and that the degree of labeling was related to their stage of maturation. In this regard, basophilic erythroblasts and early forms of polychromatophilic erythroblasts were more heavily labeled than were late forms of polychromatophilic erythroblasts; reticulocytes and erythrocytes did not label appreciably. One group of proerythroblasts exhibited binding equal to or greater than that which occurred with basophilic and early polychromatophilic erythroblast groups, while a 2nd group of proerythroblasts exhibited more limited surface labeling. Omission of Ep from the reaction sequence gave similar binding profiles, although the mean labeling density was appreciably less than was obtained with exogenous Ep. This differential in labeling values for Ep- and non-Ep-treated cells is apparently due to surface binding of endogenous Ep. Binding specificity of the Ep antiserum was evidenced by various control reactions. Substitution of phosphate buffer-bovine serum albumin for the Ep antiserum, high dilutions of the Ep antiserum, preincubation of the Ep antiserum with an Ep-Sepharose complex, or exposure of the Au-labeled IgG alone resulted in very little surface binding on the erythrocytic cells of the bone marrow.