Megakaryocyte Colony-stimulating Factor Research Articles

Administration of Recombinant Human Thrombopoietin Soon after Irradiation Promotes Survival and Stimulates Hematopoietic Recovery in a Nonhuman Primate Model of Radiation-Induced Severe Acute Radiation Syndrome

Accidental or intentional irradiation causes incidents involving acute radiation syndrome (ARS) victims. Thus, it is urgent to search for effective radioprotectors with no or low toxicity. Recombinant human thrombopoietin (rhTPO) is a megakaryocyte colony-stimulating factor. We have demonstrated previously that early administration of rhTPO soon after irradiation promotes survival of lethal dose irradiated mice. To evaluate the therapeutic effect of rhTPO short-term injection soon after radiation in nonhuman primates exposed to a high dose of gamma ray, rhesus monkeys received subcutaneously of early rhTPO 10 μg/kg injection at 0.5 and 24 h after or WR2721 30 mg/kg intramuscularly administration at 0.5 h before total body irradiated (TBI) with a 7 Gy gamma dose. Survival was monitored and hematopoiesis was evaluated at 40 d following early treatment. rhTPO short-term early injection (0.5 and 24 h) after 7 Gy TBI induced 100.0% survival versus 33.3% in irradiated controls, while 83.3% in WR2721 protected monkeys. rhTPO early treatment significantly promoted hematopoiesis recovery and apparently improved the quality of life, and additionally simplified supportive care in ARS rhesus monkeys. According to the survival and hematopoiesis of irradiated nonhuman primates, radioprotection of rhTPO short-term injection soon after TBI is better than WR2721 administration at 0.5 h before TBI. Thus short-term injection of rhTPO soon after irradiation might be chosen as a first-line therapeutic strategy in the treatment of accidental acute radiation victims.

Protein Profile Changes in Platelet Concentrates According to Storage and Leukoreduction - Analysis Using Proteomics Technology

Knowing how the protein profile of platelet products changes with storage or leukoreduction may give us greater insight into cell physiology and the cause of transfusion reactions other than cytokines and chemokines. We filtered four packs of platelet concentrates (PC) within 24 hr of blood collection and after 120 hrs of storage. Four aliquots of each supernatant in PC were obtained: pre-storage+prefiltration, pre-storage+post-filtration, post-storage+pre-filtration and post-storage+post-filtration. Routine chemistry tests and a two-dimensional electrophoresis (2-DE) were performed. The stained images were analyzed and the significant spots were identified using a peptide mass finger printing (PMF) with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis after trypsin digestion. The protein spots increased with storage and decreased after filtration (P<0.05, prestorage+post-filtration). The spot density of various proteins, including macrophage inflammatory protein-2 alpha, megakaryocyte colony stimulating factor and interleukin-22 changed with storage and leukoreduction. The database of identified protein spots and their changes produced in this study is a useful basic tool for future studies on the mechanism of transfusion reactions. Further studies should validate the significance of each protein spot.

Bone and bone marrow interactions: hematological activity of osteoblastic growth peptide (OGP)-derived carboxy-terminal pentapeptide: II. Action on human hematopoietic stem cells

Osteogenic growth peptide (OGP) is a peptide exerting regulatory effects on the bone and on bone marrow. The carboxy-terminal pentapeptide (OGP10-14) is the biologically active portion of OGP. We evaluated OGP10-14 hematopoietic activity performing colony-forming tests on human stem cells derived by bone marrow, peripheral blood and cord blood. Granulocyte-macrophage colony-forming unit (CFU) were significantly increased in OGP10-14-treated samples, while granulocyte–erythrocyte–monocyte–megakaryocyte CFU and burst-forming unit (BFU) erythroid were increased only in the cord blood test. Moreover, OGP10-14 preserves stem cells self renewal potential in long-term culture (LTC) initiating cells and acts directly on CD34+ enriched cells or increasing activity of stem cell factor (SCF) and granulocyte–megakaryocyte colony-stimulating factor.

Spontaneous proliferative megakaryocytopoiesis and platelet hyperreactivity in essential thrombocythemia: is thrombopoietin the link?

Essential thrombocythemia (ET) is one of the less rare variants of the chronic myeloproliferative disorders (MPD). The present review questions the possible link between spontaneous megakaryocytopoiesis, platelet hyperreactivity, and the occurrence of platelet-mediated vascular manifestations in acquired and hereditary ET. In acquired ET, the role of thrombopoietin (TPO) is crucial to the observed hypermegakaryocytopoiesis, which is characterized by an increased proliferation of megakaryocyte (MK) progenitors, even in conditions of culture without addition of any known megakaryocyte colony-stimulating factors. An increased reactivity of megakaryocyte progenitors to TPO remains to be precisely delineated. A defective clearance of TPO by megakaryocytes and platelets because of a reduced number of TPO receptors is possible. TPO is able to enhance platelet aggregation induced by ADP, thrombin, and collagen. A point mutation in the TPO gene as the cause of increased TPO production in hereditary ET can readily explain both spontaneous megakaryocytopoiesis and platelet-mediated microvascular manifestations simulating the phenotype of acquired ET. Nevertheless, to date, no mutation of the TPO structural gene, as shown in two families with hereditary ET, and no mutations in the TPO receptor have been found in patients with acquired ET. There is good evidence that the microvascular circulation disturbances in ET are caused by intravascular activation and aggregation of hypersensitive platelets, with sludging or occlusion of the endarterial microvasculature. In this process, the generation of platelet-derived products, endothelial cell damage, fibromuscular intimal proliferation, and platelet thrombi are essential and can be inhibited by a platelet-specific regimen of aspirin, thus providing a rationale for using low-dose aspirin as an antithrombotic agent in thrombocythemia. In contrast, the generation of thrombin appears not to be essential for the formation of platelet thrombi, thereby explaining the inefficacy of coumadin derivatives and heparin in the treatment and prevention of microvascular circulation disturbances in hereditary and acquired ET.

Enhancement of megakaryocytopoiesis by Campath-1G-treated natural killer cells.

Campath-1G is a CD52 (rat IgG2b) moAb used in bone marrow transplantation (BMT) to prevent graft-versus-host disease (GVHD) by the elimination of T cells via antibody-dependent cell cytotoxicity (ADCC) in vivo. We have previously reported that Campath-1G induces T cell proliferation, activation, and production of cytokines which in turn causes an enhancement of megakaryocytopoiesis in vitro. In view of the fact that recent studies have indicated that natural killer (NK) cells may also be involved in the regulation of megakaryocytopoiesis, we undertook the study of the in vitro effect of Campath-1G-treated NK cells on the regulation of megakaryocytopoiesis. Early burst-forming BFU-MK and late colony-forming CFU-MK were grown from 2 x 10(5) peripheral blood non-adherent mononuclear cells (NAMC) in plasma clots in the presence of aplastic canine plasma (PICS-J) which was used as megakaryocyte colony-stimulating factor (MK-CSF). The first step in elucidating this series of events was to investigate the direct influence of NK cells on megakaryocytopoiesis. Co-culturing NK cells (>85% CD16+) with autologous NAMC at a ratio of 1:1 resulted in a significant increase in the proliferation of CFU-MK and BFU-MK over NAMC cultured alone. This effect was further enhanced upon exposure of NK to Campath-1G (0.1-3 microg/ml). To investigate the possible influence of soluble factors released from NK cells treated with Campath-1G on MK maturation, conditioned medium (CM) derived from Campath-1G-treated-enriched populations of NK cells was found to enhance MK progenitor growth. Our data demonstrate that resting and Campath-1G-treated NK may be involved in the immunomodulation of megakaryocytopoiesis.

Interferon-gamma enhances megakaryocyte colony-stimulating activity in murine bone marrow cells.

We have demonstrated previously that interferon-gamma (IFN-gamma) accelerates platelet recovery in mice with 5-FU induced-marrow aplasia in vivo. However, the mechanism for the regulation of megakaryocyte development induced by IFN-gamma in bone marrow cells in vivo remains unknown. To further study the effects of IFN-gamma on megakaryocyte development, various steps during IFN-gamma-mediated accelerated differentiation of the megakaryocytes were investigated in serum-free cultures of murine bone marrow cells in vitro. IFN-gamma markedly induced acetylcholine esterase (AChE) activity, a marker of murine megakaryocytic cells, accompanied by increased colony formation of the megakaryocyte lineage. A prominent increase in megakaryocyte number was observed after IFN-gamma treatment. All of these effects were dependent on the presence of IL-3, and, therefore, these results suggest that IFN-gamma acts as a megakaryocyte potentiator (Meg-POT). However, IFN-gamma did not enhance megakaryocyte maturation with respect to increase in cell size. The effects of IFN-gamma on megakaryocyte maturation were similar to those observed after treatment with higher doses of IL-3 alone. Meg-POT is defined as a factor that induces megakaryocyte maturation. Since IFN-gamma enhanced IL-3-dependent megakaryocyte colony formation and proliferation rather than megakaryocyte maturation, the effects on megakaryocyte development, which were induced by IFN-gamma treatment, seem to be different from the effects of a Meg-POT. We, therefore, propose a new function for IFN-gamma as an enhancer of megakaryocyte colony-stimulating factor activity. The effect of IFN-gamma in vitro appears to correlate well with the acceleration of platelet recovery in vivo.

Physiologic role of TPO in thrombopoiesis.

Recombinant human thrombopoietin (rHuTPO) serves as a megakaryocyte colony-stimulating factor and predominantly acts on GPIIb/IIIa+ rat late megakaryocyte progenitor cells, colony forming units-megakaryocyte (CFU-MK). The GPIIb/IIIa+ fraction of CFU-MK differentiates into mature megakaryocytes and further into proplatelets in liquid culture containing rHuTPO. rHuTPO stimulates cultured megakaryocytes generated from rat GPIIb/IIIa+ CFU-MK to enhance proplatelet formation and to increase megakaryocyte size. rHuTPO also induces a big size of megakaryocyte colonies from human cord blood CD34+ cells. rHuTPO does not cause aggregation of platelets from normal mice and mice made thrombocytotic by consecutive administration of rHuTPO, but preincubation with rHuTPO enhances adenosine diphosphate-induced aggregation, suggesting that platelets induced by rHuTPO administration may have a normal function. Administration of rHuTPO to normal mice daily for five days causes a dose-dependent thrombocytosis. On the other hand, rHuTPO induces a significant decrease in hemoglobin concentration and does not affect white blood cell counts. rHuTPO increases the size and number of marrow megakaryocytes and the number of marrow CFU-MK, and also influences the development of other hematopoietic progenitor cells. The effects of rHuTPO on thrombocytopenia associated with myelosuppression were examined in animal models. Following treatment with mitomycin C, mice received daily injections of various doses of rHuTPO. rHuTPO reduced the severity of thrombocytopenia, accelerated the recovery of platelets and improved neutropenia. Similar therapeutic efficacy was observed in cynomolgus monkeys treated with nimustine. These results suggest the clinical usefulness of rHuTPO for the treatment of thrombocytopenia.

The Mpl-Ligand or Thrombopoietin or Megakaryocyte Growth and Differentiative Factor Has Both Direct Proliferative and Differentiative Activities on Human Megakaryocyte Progenitors

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin-6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors

Previously, it was believed that megakaryocytopoiesis was regulated by two types of humoral factors: megakaryocyte colony-stimulating factor (MK-CSF), which acts on progenitors inducing their proliferation, and thrombopoietin (TPO), a megakaryocyte(s) (MK) maturational factor that induces platelet formation. The recently cloned Mpl-ligand (Mpl-L) seems to have both properties in vivo and in vitro and has also been called TPO. However, it cannot be excluded that a part of these activities is due to a synergistic effect with growth factors present in the serum or synthesized by accessory cells. To delineate the precise TPO (Mpl-L) biologic activities, we performed serum-free cultures at limiting cell dilution. Target cells were adult human marrow CD34+CD41+ cells, which represent a highly selected population of late MK progenitor or transitional cells. Cells were purified using a flow cytometer equipped with an automatic cloning design unit. We determined that the recombinant molecule had a biologic activity that reached a plateau at 10 ng/mL. At this concentration, a linear relationship between the average MK number per well and the number of cells seeded (between 1 to 50 cells per well) was observed. At one cell per well, 60% of the wells contained a single MK at day 5 of culture. Half of these wells contained only one large MK, whereas the other half contained several MK (up to 25), demonstrating that TPO has direct proliferative biologic activity. In contrast, at limiting dilution, none of the other cytokines tested (stem cell factor [SCF], interleukin- 6 [IL-6], and erythropoietin [Epo]) were effective, whereas IL-3 showed a mild effect. However, a combination of SCF plus IL-6 plus IL-3 produced similar results as TPO alone. Addition of the other cytokines to TPO did not enhance the cloning efficiency of the CD34+CD41+ cells but increased twofold the average number of MKs per clone. MKs reached a ploidy of 32N and 64N in the presence of TPO. The mean ploidy value was approximately 6 and was not modified by addition of the other cytokines. At the ultrastructural level, a majority of the MKs showed maturational defects related to an imbalance between the synthesis of alpha-granules and demarcation membranes. However, a fraction (about 30%) had a cytoplasmic maturation that exactly mimicked that of marrow MKs. In addition, proplatelet-shedding MKs were observed in the cultures, even at limiting dilution. Such a result was not observed with any other individual cytokines, including the combination of three cytokines.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification and Biologic Characterization of Plasma-Derived Megakaryocyte Growth and Development Factor

The isolation and cloning of the ligand for the cytokine receptor, Mpl, have been recently described. In this report we present details of the purification of this novel cytokine (megakaryocyte growth and development factor [MGDF]) from aplastic canine plasma. Two forms of canine MGDF, with apparent molecular weights of 25 kD and 31 kD and sharing a common N-terminal amino acid sequence, were isolated. The sole contaminant detected in purified 25-kD or 31-kD MGDF was canine Ig. Canine MGDF is characterized as a human megakaryocyte colony-stimulating factor that acts synergistically with human recombinant stem cell factor but not interleukin-3. MGDF also appears to be physiologically regulated in response to platelet demand. In canine and murine models, serum levels of MGDF activity peak during the thrombocytopenic periods after irradiation, 5-fluorouracil, or antiplatelet antisera injections. These data indicate that the megakaryocyte-stimulating activity that accumulates in plasma in response to platelet losses is a novel cytokine that functions through an interaction with the Mpl cytokine receptor.

Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

A Phase I Trial of Recombinant Human Interleukin-6 in Patients With Myelodysplastic Syndromes and Thrombocytopenia

To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts. Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy. Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d. All patients experienced at least grade II fever and all had an increase in acute phase proteins. Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria. Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy. Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively. Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor. Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population. Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway.

C-mpl, the Thrombopoietin Receptor

v-mpl is a mouse oncogene that is part of the genome of the myeloproliferative leukemia virus (MPLV). The cellular homolog gene c-mpl encodes an hematopoietic cytokine receptor mainly restricted to the megakaryocytic lineage of differentiation. Using the Mpl receptor protein, cDNAs corresponding to the receptor ligand were isolated in humans, pigs, dogs and mice. In vivo and in vitro, the recombinant molecule has both the thrombopoietin (TPO) and megakaryocyte-colony stimulating factor (MK-CSF) activities. This molecule (Mpl ligand/TPO/MK-CSF) seems to be the humoral physiological regulator of platelet production.

CMpl ligand is a humoral regulator of megakaryocytopoiesis.

Megakaryocytopoiesis is the cellular developmental process that leads to platelet production. At least two humoral growth factors may be necessary for megakaryocyte proliferation and maturation. One is a megakaryocyte-colony stimulating factor (MK-CSF) which induces the proliferation and differentiation of megakaryocyte progenitors, and the second, thrombopoietin, is a megakaryocyte maturation factor. Neither of these factors has been fully characterized. The proto-oncogene c-mpl, an orphan member of the haematopoietin receptor family, is specifically involved in megakaryocyte regulation. Here we present evidence that the c-mpl-encoded receptor binds a ligand (c-Mpl ligand) which is a humoral factor implicated in platelet homeostasis. Our results suggest that c-Mpl ligand, thrombopoietin and MK-CSF might be the same molecule.

Megakaryocyte colony-stimulating factor (Meg-CSF) is a unique cytokine specific for the megakaryocyte lineage.

The regulation of megakaryocytopoiesis and platelet production has not yet been clearly elucidated. Several cytokines have been shown to be capable of producing megakaryocyte colonies from bone marrow [i.e. Interleukin (IL)-3, granulocyte-macrophage (GM)-colony-stimulating factor (CSF), erythropoietin (Epo)]. In addition, other activities have been reported to stimulate megakaryocyte precursors, yet a megakaryocyte-CSF (Meg-CSF) has not been purified to homogeneity and IL-3, GM-CSF and/or Epo often contaminate purification attempts which could account for the activities. A Meg-CSF has been isolated from the urine of patients with aplastic anaemia and purified by sequential ultrafiltration, cation exchange, G-50 chromatography, preparative PAGE, chromatofocusing and cation exchange HPLC. The activity of this material is 2-4 x 10(4) CFU-Meg/mg as measured in a murine marrow, serum-containing assay. This activity also stimulates CFU-Meg in the absence of adherent accessory cells and in serum-free cultures, indicative of the direct stimulation on CFU-Meg. Immunoassays, colony forming assays, and proliferation assays demonstrate that purified Meg-CSF has no GM-CSF, IL-3, M-CSF, G-CSF or IL-1 alpha, -3, -6, -9 and -11. In confirmation of these results, neutralizing antibody to IL-6 also did not abrogate Meg-CSF activity. Therefore the previously-reported megakaryocyte colony-stimulating activity in purified aplastic anaemia patient urine is due to a unique cytokine: Meg-CSF.

Circulating megakaryocyte progenitors in myeloproliferative disorders are hypersensitive to interleukin-3.

Previous studies have reported that megakaryocyte progenitors in myeloproliferative disorders (MPD) formed spontaneous megakaryocyte colonies without the addition of megakaryocyte colony-stimulating factor (Meg-CSF). To determine whether this spontaneous colony formation is due to autocrine proliferation of MPD megakaryocyte progenitors or to hypersensitivity to Meg-CSF that might exist in the culture system, we investigated colony-forming unit-megakaryocytes (CFU-Meg) in the peripheral blood of 11 MPD patients, using serum-free cultures. Spontaneous megakaryocyte colonies were observed in serum-free cultures of nonadherent mononuclear cells (NAdMNC) obtained from MPD patients with thrombocytosis, whereas the NAdMNC of MPD patients without thrombocytosis, that of patients with reactive thrombocytosis and normal subjects never formed spontaneous colonies. However, the spontaneous colonies from MPD patients with thrombocytosis disappeared in cultures using highly purified CD34-positive cells as target cells. To study the hypersensitivity of megakaryocyte progenitors to Meg-CSF, dose-response experiments were performed with interleukin-3 (IL-3). CFU-Meg from MPD patients with thrombocytosis showed maximal growth at the concentrations of IL-3 lower than those for normal subjects. CFU-Meg of MPD patients without thrombocytosis and that of patients with reactive thrombocytosis showed the same colony growth response to IL-3 as that of normal subjects. This result indicates that the CFU-Meg of MPD patients with thrombocytosis are hypersensitive to IL-3. It also suggests that spontaneous colony formation by NAdMNC is not due to the autocrine growth of megakaryocyte progenitors but is due to the hypersensitivity of megakaryocyte progenitors to Meg-CSF, such as IL-3, released by accessory cells. Furthermore, it is possible that such hypersensitivity of CFU-Meg to IL-3 might be a pathogenic factor in MPD with accompanying thrombocytosis.

Suppression of murine hematopoiesis in vivo after chronic administration of zidovudine: evidence that zidovudine-induced anemia is the result of decreased bone marrow-derived, erythropoietin-responsive progenitor cells.

We studied the long-term effect of continued zidovudine exposure in mice on hematopoiesis, as determined by peripheral blood indices, assays of erythroid (colony-forming unit-erythroid [CFU-E] and burst-forming unit-erythroid [BFU-E]), myeloid (CFU-granulocyte-macrophage [GM]), megakaryocyte (CFU-Meg), and plasma titers of erythropoietin, granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, and tumor necrosis factor-alpha. Dose-escalation of zidovudine (0.1, 1.0, and 2.5 mg/ml) induced a dose-dependent decrease in hematocrit, white blood cells, and platelets. High-dose drug, i.e., greater than 1.0 mg/ml, reduced marrow CFU-E; splenic CFU-E was increased after 1 week, then declined. BFU-E was increased at Weeks 1 and 2, then declined to control levels. Splenic BFU-E rose during the examination period that was dose-dependent. Femoral CFU-GM was cyclic, i.e., low-dose drug, 0.1 mg/ml, was increased gradually, the declined; higher doses of 1.0 and 2.5 mg/ml were lower until Week 5, then were above controls. Splenic CFU-GM was increased initially at Week 2 (1.0 mg/ml), then declined; the higher dose (2.5 mg/ml) increased initially, then declined below controls (Week 6). Femoral CFU-Meg was increased after low-dose drug and inhibited after high dose (2.5 mg/ml). Splenic CFU-Meg was reduced initially, followed by an increase at Week 4. Plasma titer of erythropoietin was elevated, proportional to dose escalation of drug, and inversely proportional to the hematocrit. No difference was observed in plasma levels of granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, or tumor necrosis factor-alpha. This study demonstrates that zidovudine-induced anemia results from: (i) inadequate numbers of bone marrow-derived, erythropoietin-dependent hematopoietic progenitors, i.e., CFU-E; and (ii) a shift in erythropoietin-responsive progenitors from bone marrow to spleen capable of responding to obligatory growth factors.

Purification of human megakaryocyte colony-stimulating factor (Meg-CSF) from urine of aplastic anemia patients

Our laboratory has already documented that extracts of urine from aplastic anemia patients stimulate megakaryocyte and platelet production in vivo in rats and mice. This stimulatory activity was apparently mediated by a unique cytokine. This report describes the purification of the factor likely responsible for this activity because it stimulates megakaryocyte colony formation (CFU-meg) in vitro. The factor, when purified to homogeneity, exhibits a molecular weight of 55,000–57,000 and an isoelectric point of 7.2–7.4. Under physiological conditions, ultrafiltration experiments indicate that human megakaryocyte colony-stimulating factor (hMeg-CSF) exists as a monomer or dimer. In contrast to human urinary erythropoietin (Epo), only a small amount of hMeg-CSF forms complexes of high molecular weight aggregates. In contrast to interleukin (IL)-3, hMeg-CSF induces small colonies in vitro containing three to five megakaryoblasts. Even at concentrations 100-fold greater than the threshold amount required to stimulate CFU-meg colony formation, hMeg-CSF stimulated only CFU-meg colonies, but did not stimulate myeloid or erythroid lineage colonies. We propose that this purified factor is specific for the megakaryocyte lineage, and that it be termed human Meg-CSF.

Interaction of monocytes and T cells in the regulation of normal human megakaryocytopoiesis in vitro: role of IL‐1 and IL‐2

Autologous or allogeneic peripheral blood T cells can stimulate the human megakaryocyte progenitor cell (CFU-Meg)-derived colony formation in a dose-dependent fashion in agar cultures of nonadherent (NA), T cell-depleted (NT) bone marrow (BM) cells. Low concentrations of monocytes and T cells can collaborate in the stimulation of CFU-Meg colony formation or in the production of megakaryocyte colony stimulating factor (Meg-CSF) by T cells in the presence of mitogens or IL-2. Monocytes alone can produce only negligible Meg-CSF under any conditions. When monocyte conditioned medium (CM) was added to T cell-stimulated NA, NT BM cell cultures, CFU-Meg colony growth was appreciably increased compared with that stimulated by T cells alone. Dose-dependent increase in CFU-Meg colony growth was noted when varying concentrations of IL-1 were added to T cell-stimulated NA, NT cell cultures, although IL-1 itself could support no CFU-Meg colony growth in the absence of T cells. These data suggest that a synergistic interaction between T cells and monocytes during the production of Meg-CSF by T cells could be partly mediated by IL-1. IL-2 was found to stimulate Meg-CSF production by T cells in the presence or absence of mitogens. IL-2-stimulated Meg-CSF production by T cells was augmented by the addition of monocytes. Although IL-2 itself had no stimulatory effect on CFU-Meg colony growth, dramatic augmentation in the CFU-Meg colony number was noted when IL-2 was added to T cell-stimulated NA, NT cell cultures. High concentrations of monocytes and prostaglandin E (PGE) inhibited the CFU-Meg colony formation. These results suggest that IL-1 and IL-2 may play a stimulatory role on the normal human in vitro megakaryocytopoiesis, and may be involved in the development of reactive thrombocytosis and bone marrow megakaryocytic hyperplasia in various inflammatory diseases.

Cytokine Regulation of the Human Burst-Forming Unit-Megakaryocyte

The human burst-forming unit-megakaryocyte (BFU-MK) is a primitive megakaryocytic progenitor cell. A marrow cell population enriched for BFU-MK (CD34+ DR-) was obtained by monoclonal antibody labeling and fluorescence-activated cell sorting. CD34+DR- cells were assayed in a serum-depleted, fibrin clot culture system. Recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant interleukin-3 (rIL-3), and megakaryocyte colony-stimulating factor (MK-CSF), partially purified from human plasma, were each individually capable of promoting BFU-MK-derived colony formation. Recombinant erythropoietin, rG-CSF, rIL-4, rIL-6, and thrombocytopiesis stimulating factor, partially purified from human embryonic kidney cell conditioned media, had no stimulatory effect on BFU-MK-derived colony formation when added alone or in various combinations with either GM-CSF, IL-3, or MK-CSF, GM-CSF and IL-3, GM-CSF and MK-CSF, but not IL-3 and MK-CSF had additive actions in promoting BFU-MK-derived colony formation, rIL-1 alpha had no influence alone on BFU-MK cloning efficiency, but had a dose-dependent, synergistic effect with IL-3, but not with GM-CSF or MK-CSF. The synergistic relationship between IL-1 alpha and IL-3 was abrogated by addition of an IL-1 alpha neutralizing antibody but not by a GM-CSF neutralizing antiserum, suggesting that IL-1 alpha acts directly on the BFU-MK and not by stimulating marrow auxiliary cells to secondarily release additional cytokines. Information presented here indicates that the regulatory influence, acting on the different stages of megakaryocyte development, are stage-specific and accomplished by multiple cytokines.