The development of blood cells including expansion of megakaryocyte progenitor cells requires the interplay of marrow stromal cells and polypeptide cytokines. Recently, characterization of c-Mpl, the receptor encoded by the proto-oncogene c-mpl, revealed structural homology with the haematopoietic cytokine receptor family, and its involvement in megakaryocyte development. We report here that the ligand for c-Mpl is relatively lineage specific, works both alone and synergistically with early acting cytokines to support megakaryocyte colony formation, and acts at a late stage of development to increase megakaryocyte size, polyploidization and expression of differentiation markers. In vivo, c-Mpl ligand stimulates platelet production by greatly expanding marrow and splenic megakaryocytes and their progenitors, and by shifting the distribution of megakaryocyte ploidy to higher values. Thus, as c-Mpl ligand has the expected characteristics of the major regulator of megakaryocyte development, we propose that it be termed thrombopoietin.
The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that earlyacting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to lateacting MK potentiators, including IL-6 and IL-li. Recently, the Mpl ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.The generation of megakaryocytes (MKs) is a complex process dependent on the interaction of hematopoietic progenitor cells, cytokines, and stromal elements (1-3). Committed MK progenitor cells must undergo a series of mitotic divisions, shift to endomitotic replication, express specific membrane glycoproteins, and undergo cytoplasmic maturation in preparation for platelet shedding. Models of erythropoiesis and granulopoiesis have been established that stress the importance of both early-acting and late-acting cytokines or hormones for completion of the erythroid and granulocytic developmental programs (4-6). A number of investigators have shown that interleukin (IL) 3 and c-kit ligand (KL) can support the production of MK colonies from their progenitors [MK colony-forming units (CFU-MK)] in semisolid medium and of individual MKs in suspension culture (7-10). Moreover, although IL-6, IL-11, and leukemia inhibitory factor are not reported to support MK formation alone, these cytokines augment the MK response to IL-3 or KL (11)(12)(13)(14). Recently, we (15) and other groups (16, 17) have cloned and expressed the ligand for the c-Mpl receptor. Based on its capacity to induce increases in the size, ploidy, and maturation of MKs, its inverse relationship with platelet levels, and its ability to increase platelet production manyfold (18, 19), we proposed that the Mpl ligand is identical to thrombopoietin (Tpo) (20, 21).The publication costs of this article were defrayed in part by page charge payment. This article must therefore...
Thrombopoietin (Tpo), the ligand for the c-mpl receptor, is a major regulator of platelet production in vivo. Treatment of mice with purified recombinant Tpo increases platelet count fourfold and expands colony-forming unit-megakaryocyte (CFU-Meg) numbers. Other cytokines including interleukin-3 (IL-3), IL-6, IL-11, erythropoietin (Epo), and stem cell factor (SCF) can stimulate megakaryopoiesis. Therefore, we examined the effects of recombinant murine Tpo in combination with these cytokines on megakaryopoiesis in vitro. Murine marrow cells were cultured in agar in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% horse serum and beta-mercaptoethanol in the presence of recombinant growth factors, and CFU-Meg colonies were counted on day 5. Megakaryocyte ploidy was analyzed using murine marrow cells cultured for 5 days in IMDM supplemented with 1% nutridoma-SP and recombinant growth factors. Megakaryocytes were identified by labeling with the 4A5 antibody and ploidy was analyzed by flow cytometry. Tpo supported the growth of CFU-Meg in a dose-dependent manner. Although the addition of SCF (50 ng/mL), Epo (2 U/mL), or IL-11 (50 ng/mL) alone exerted only a modest effect on CFU-Meg growth, the combination of SCF plus Tpo, Epo plus Tpo, or IL-11 plus Tpo resulted in a synergistic enhancement of the number of CFU-Meg colonies. IL-3 alone supported CFU- Meg colony growth, and the effects of IL-3 plus Tpo or IL-6 plus Tpo on colony growth appeared to be approximately additive. Fifty percent of megakaryocytes generated in cultures containing IL-3 or Epo displayed < or = 16 N ploidy. In contrast, cultures containing Tpo uniquely generated large numbers (30% to 35% of the total) of megakaryocytes with > or = 64N ploidy. These results show that Tpo stimulates both proliferation of committed megakaryocytic progenitor cells and maturation of megakaryocytes, and that two multipotent cytokines, SCF and IL-11, as well as a late-acting erythroid cytokine, Epo, can synergize with Tpo to stimulate proliferation of CFU-Meg.
Thrombopoietin (TPO), the ligand for the receptor protooncogene c-mpl, has been cloned and shown to be the critical regulator of platelet production. Several features of c-Mpl expression, including its presence on erythroid cell lines, and the panmyeloid transformation characteristic of myeloproliferative leukemia (MPL) viral disease led us to investigate whether this receptor-ligand system may play a role in erythropoiesis. We report that although TPO alone did not support the growth of either early or late erythroid progenitors, it acted in synergy with erythropoietin to expand these populations. Moreover, while the effects on erythropoiesis in normal animals were modest, TPO greatly expanded the number of erythroid progenitors and blood reticulocytes and was associated with accelerated red cell recovery in myelosuppressed mice. Together, these data strongly suggest that erythroid progenitors respond to TPO and that this newly cloned cytokine, critical for platelet production, can augment erythropoiesis in states of marrow failure. (J. Clin. Invest. 1995. 96:1683-1687
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