A role for the macrophage in normal hemopoiesis: III. In vitro and in vivo erythropoietin gene expression in macrophages detected by in situ hybridization.

Macrophages derived from unstimulated and unseparated mouse bone marrow cells cultivated on hydrophobic foils can release hemopoietic regulator molecules into the surrounding medium. To prove that one of these regulators exists in macrophages in vitro, in situ hybridization using a 1.2-kb erythropoietin (Epo) gene probe was employed. The probe was biotinylated and the signal developed using a streptavidin-gold reagent. Observation was performed using reflection-contrast microscopy. The results indicate that from a 98% pure population of macrophages, 34% F4/80 (mouse, macrophage-specific antigenic determinant)-positive macrophages exhibited Epo gene expression. The technique was also applied to normal, steady-state mouse bone marrow in which approximately 10% of the cells are F4/80-positive and of which about 3% demonstrated simultaneous Epo gene expression. As positive control, kidneys from anemic mice were hybridized with the biotin-labeled Epo DNA. A second positive control utilized biotin-labeled actin DNA hybridized to cultured macrophages and normal bone marrow cells. The accumulating information, demonstrating that the unstimulated kidney does not express the Epo gene, indicates that Epo is produced in other areas of the body under normal, steady-state conditions. The present results show that 1) macrophages can express the Epo gene, 2) this function is carried out by a subpopulation of macrophages, and 3) bone marrow macrophages in vivo may be responsible for the Epo production-target cell mechanism evoked by short-range and/or cell-to-cell interactions under normal, steady-state conditions.