Adherence to macrophages in erythroblastic islands enhances erythroblast proliferation and increases erythrocyte production by a different mechanism than erythropoietin.

Erythroblasts adhere to central macrophages forming erythroblastic islands in hematopoietic tissues, but the function of these islands is not understood. Murine erythroblastic islands were reconstituted in vitro with macrophages and developmentally synchronous proerythroblasts. Erythroblasts cocultured with macrophages proliferated 3-fold greater than erythroblasts cultured alone. Direct contact with the macrophages was necessary for this enhanced erythroblast proliferation, which resulted from decreased transit time in the G(0)/G(1) phase of cell cycle. Increased erythroblast proliferation in erythroblastic islands occurred over a wide range of erythropoietin concentrations and was the result of a mechanism different from the antiapoptotic effect of erythropoietin. Erythroblasts adherent to macrophages had slightly delayed enucleation, but otherwise differentiation was similar to erythroblasts cultured alone or those that became nonadherent in cocultures. These results suggest a mechanism for the development of anemias associated with abnormal macrophage function and for reduced responsiveness of those anemias to erythropoietin therapy.

In vitro maturation of nascent reticulocytes to erythrocytes.

Most studies of mammalian reticulocyte maturation have used blood reticulocytes. Nascent reticulocytes, as found in bone marrow, have not been available in developmentally synchronized populations. Nascent murine reticulocytes formed in vitro by enucleation of Friend virus-infected erythroblasts were purified and recultured for 110 hours. At 0 hours, all recultured cells were lobulated and contained dense, centralized reticulin. By 110 hours, about 20% to 25% of the cells became biconcave erythrocytes. Most ribosomes and cellular RNAs were degraded within 20 hours, and during that period, heme synthesis declined from a rate equal to that of late erythroblasts to less than 10% of that rate. Many mitochondria appeared normal until they showed outer membrane swelling, degradation, and apparent fusion with intracellular vacuoles at 40 hours of culture. During the period of mitochondrial loss, Bcl-X(L), an antiapoptotic protein that accumulates during erythroblast differentiation and maintains mitochondrial membrane integrity, demonstrated progressive decreases and changes consistent with deamidation. Nevertheless, the reticulocytes did not undergo apoptosis, because their apoptotic machinery was degraded. This experimental system that provides a developmentally synchronized population of nascent murine reticulocytes that mature into biconcave erythrocytes in vitro should be useful in further investigations of the cellular events involved in reticulocyte maturation.

Bcl-x(L) prevents apoptosis of late-stage erythroblasts but does not mediate the antiapoptotic effect of erythropoietin.

The long form of B-cell lymphoma-x (Bcl-x(L)), an outer mitochondrial membrane protein, has been proposed to mediate the antiapoptotic action of erythropoietin on erythroid progenitor cells and to be necessary for heme synthesis in erythroblasts. Mice with conditional knockout of Bcl-x(L) (conditional bcl-x(-/-) mice) develop severe anemia that has been attributed to hemolysis and is accompanied by splenomegaly. We characterized further the anemia of conditional bcl-x(-/-) mice and investigated the role of Bcl-x(L) in the action of erythropoietin and in heme synthesis. We analyzed peripheral blood cells and cultured splenic erythroblasts of conditional bcl-x(-/-) mice and littermates that were rendered anemic by bleeding. Although they had massive splenic erythroblastosis, conditional bcl-x(-/-) mice had decreased circulating reticulocytes compared to littermates even prior to bleeding the littermates. Compared to erythroblasts of bled littermates, bcl-x(-/-) erythroblasts cultured with erythropoietin underwent apoptosis during the later, hemoglobin-synthesizing stages of differentiation. The bcl-x(-/-) erythroblasts synthesized heme, but at reduced rates compared to bled littermate erythroblasts. When cultured without erythropoietin, bcl-x(-/-) erythroblasts underwent apoptosis at early stages of differentiation, prior to hemoglobin synthesis. Bcl-x(L) is not required for heme synthesis and does not mediate the antiapoptotic effects of erythropoietin, but it prevents ineffective erythropoiesis due to apoptosis in late-stage, hemoglobin-synthesizing erythroblasts.