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Stem Cells, Vol. 14, No. 6, 661-677, November 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Recombinant Human Ligand for MPL, Megakaryocyte Growth and Development Factor (MGDF), Stimulates Thrombopoiesis in Vivo in Normal and Myelosuppressed Baboons

Robert G. Andrewsa,b, Aaron Winklera, David Myersona, Robert A Briddellc, Glenn H. Knitterd, Ian K. McNiecec, Pamela Huntc

a Division of Clinical Research, Fred Hutchinson Cancer Research Center and
b Department of Pediatrics. University of Washington School of Medicine. Seattle, Washington, USA;
c Developmental Hematology Department, Amgen, Thousand Oaks, California, USA; and
d University of Washington Regional Primate Research Center, Seattle, Washington, USA

Key Words. Thrombopoietin • Hematopoiesis • Chemotherapy • Bone Marrow • Platelets • Colony-forming cells • Baboons (papio species)

Dr. Robert G. Andrews, Pediatric Oncology Program, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, WA 98104, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Megakaryocyte growth and development factor (MGDF) is a ligand for c-mpl and a member of the hematopoietic growth factor superfamily. Recombinant murine MGDF specifically stimulates thrombopoiesis in mice. Recombinant human (rHu) MGDF stimulates megakaryocytic differentiation of baboon CD 34+ marrow cells in vitro. Therefore, we determined the in vivo biological effects of rHuMGDF administered to normal baboons in the absence and presence of myelosuppression with 5-fluorouracil (5-FU). rHuMGDF was administered to normal baboons as single s.c. injection at doses of 1, 10, 25 and 50 µg/kg/day for 10 days and, as a control, heat-inactivated MGDF was administered at a dose of 10 µg/kg/day. Platelet counts were markedly increased in all animals administered native rHuMGDF but not in animals given heat-inactivated rHuMGDF. Platelet counts began to increase between three and six days after starting rHuMGDF administration and the maximum average increases were 1.7-, 3.4-, 5.1- and 4.0-fold above baseline in animals administered 1, 10, 25 and 50 µg/kg/day, respectively. Maximum platelet counts were reached between 7 and 10 days after starting rHuMGDF and maintained for four days after the last dose. Thereafter, platelet counts decreased, reaching stable pretreatment values between 11 and 14 days after the last dose of rHuMGDF. No changes in red cell mass, peripheral blood white blood cell counts or differentials were observed during rHuMGDF treatment. For animals administered 10, 25 and 50 µg/kg/day of rHuMGDF, megakaryocytes increased more than threefold in marrow, were markedly enlarged, and had increased numbers of lobes. Overall marrow cellularity remained unchanged, as did red cell and white cell morphology. No marrow fibrosis was detected. Progenitor cells were not increased in marrow but did increase modestly in the peripheral blood, associated with increased numbers of CD34+ cells in circulation.

Following a single dose of 5-FU (120 mg/kg) animals were given either saline or pegylated (PEG) rHuMGDF (25 µg/kg/day) for 14 days. Platelet counts recovered to baseline by 13.8 ± 1.8 days for PEG-rHuMGDF-treated baboons compared with 16.8 ± 0.6 days for saline treated controls. Marrow biopsies revealed more rapid recovery of overall marrow cellularity and megakaryocytes in PEG-rHuMGDF-treated animals compared with controls. Thus, rHuMGDF specifically stimulates thrombopoiesis in normal and myelosuppressed baboons. rHuMGDF may be useful for stimulating thrombopoiesis in humans in clinical settings after myelosuppression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The mpl receptor and its ligand have recently been identified as playing a major role in the regulation of thrombopoiesis [1-8]. Wendling initially described the myeloproliferative leukemia virus as inducing a myeloproliferative syndrome in mice that involved thrombopoiesis, granulopoiesis and erythropoiesis [9]. Megakaryocyte progenitor cells, as well as granulocyte-macrophage and pluripotent progenitor cells infected by the myeloproliferative leukemia virus could display factor-independent proliferation in vitro [10, 11]. Subsequently the v-mpl oncogene was identified as a truncated receptor belonging to a family of hematopoietic cytokine receptors [12, 13]. The normal cellular homolog, the proto-oncogene c-mpl, was demonstrated to belong to the hematopoietic growth factor superfamily [14-16]. It was expressed in normal hematopoietic tissues and expression was amplified in a significant portion of acute myeloid leukemias and myelodysplastic syndromes [17-19]. An important finding was that antisense oligonucleotides that target mpl's message specifically inhibited in vitro growth and differentiation of human CD34+ megakaryocyte progenitor cells, whereas the proliferation of erythroid and myelocytic progenitor cells was unaffected [1]. Targeted disruption of murine c-mpl gene results in the production of thrombocytopenic mice that have abnormal thrombopoiesis while the other hematopoietic lineages are normal [7]. The cloning and expression of the recombinant murine, human, and canine ligands for the mpl receptor, have been reported [2-6]. The mpl ligand specifically stimulates the in vitro proliferation of human and murine megakaryocyte progenitors and can support complete in vitro differentiation of newly formed human megakaryocytes to produce phenotypically, morphologically and functionally normal platelets in vitro [2-5, 20-23]. The administration of recombinant murine mpl ligand to mice produces a thrombocytosis and an increase in number and size of megakaryocytes in spleen and marrow [2, 6] and decreases the period of thrombocytopenia after treatment with carboplatin [24]. Farese et. al. have recently reported that megakaryocyte growth and development factor (MGDF) stimulates thrombocytosis in normal rhesus monkeys [25]. In this paper we report that recombinant human (rHu) MGDF stimulates thrombopoiesis in vivo in normal and myelosuppressed baboons while having little apparent effect on granulopoiesis, erythropoiesis or lymphopoiesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Healthy juvenile baboons (Papio cynocephalus cynocephalus or P. cynocephalus anubis) were housed at the University of Washington Regional Primate Research Center, under the American Association for Accreditation of Laboratory Animal Care-approved conditions. Studies were conducted under Institutional Review Board and Animal Care and Use Committee-approved protocols. All animals were provided with water, biscuits and fruit ad libitum throughout the study. All procedures, including bone marrow biopsies and blood draws, were performed after animals had been anesthetized with a combination of ketamine-HCI (Aveco; Fort Dodge, IA) and Xylazine (Haver; Shawnee, KS). A total of 24 animals was studied as part of these experiments. Peripheral blood samples, 1 to 2 ml, for colony-forming cell (CFC) assays and analysis of expression of the CD34 antigen were collected in preservative-free heparin. One to two-ml blood samples were also collected in EDTA for CBC. Differential cell counts (>=100 cells) on peripheral blood smears stained with Wright-Giemsa were determined for each CBC. Marrow biopsies were obtained from sites on the iliac crest, distal femur, proximal humerus, and proximal tibia using an 11-gauge Jamshidi biopsy needle. The marrow biopsy site was the same for each animal at each time point, and each site was biopsied only once. Marrow biopsies were fixed in either B-5 fixative or buffered formalin. Multiple sections from marrow core biopsies were stained with hematolyxin and eosin, periodic acid schiff, and for reticulin. Slides were examined by the hematopathologist (David Myerson) without prior knowledge of the treatment the animals had received. Sections from normal cellular baboon marrows had a cellularity of 50% to 60% (cell to fat ratio of approximately 1:1).

rHuMGDF

Preparation and in Vivo Administration   rHuMGDF, the ligand for c-mpl, was prepared as previously described [2] and was used as either the unmodified or pegylated protein. Purified material was stored at 4°C until used. In some instances, the rHuMGDF was diluted 1:5 or 1:10 with sterile 0.9% (w/v) saline immediately prior to administration in order to have a volume of material that could be easily measured for s.c. injection, i.e., a volume greater than 0.1 ml.

rHuMGDF was administered daily for 10 days as a single s.c. injection, with the volume of injection being less than 1.5 ml per injection site. The injections were administered in the lateral aspect of the thigh or midline of the back which had been shaved prior to the start of the study. Two animals were administered 50 µg/kg/day of rHuMGDF: three animals were administered 25 µg/kg/day of rHuMGDF: three animals were administered 10 µg/kg/day of rHuMGDF: two animals were administered 1.0 µg/kg/day of rHuMGDF: and as controls, two animals were administered 10 µg/kg/day of heat-inactivated rHuMGDF (100°C x 30 minutes).

Administration of 5-Fluorouracil (5-FU)
5-FU was administered to 11 animals at a dose of 120 mg/kg as a single i.v. infusion over 30 min. Beginning 48 h after the 5-FU dose, animals received either pegylated (PEG)-rhMGDF or saline s.c. once daily for 14 days. Animals were treated with Ceftazidime i.m. twice daily beginning on day 7 until day 21, during the period of neutropenia. Marrow biopsies were obtained from each animal before starting on study and then weekly for 4 weeks.

Monoclonal Antibodies and Immunofluorescent Staining
Monoclonal antibody 12-8 (CD34) and the isotype-matched control antibody H12C12 (antimouse Thy 1.2) were used to label peripheral blood and marrow cells, and were stained with phycoerythrin-conjugated goat antimouse IgM (mu chain specific) antisera (Calbiochem; La Jolla, CA or Biomeda; Foster City, CA), as previously described [26].

CFC Assays
Baboon peripheral blood buffy coat and marrow buffy coat cells were isolated, residual red blood cells were lysed with ammonium chloride buffer, and the cells were cultured at 5.0 or 10.0 x 104 per 35 mm culture dishes (Nunc; Naperville, IL) using a two-layer agar culture technique [26]. Culture plates were placed in humidified polystyrene boxes in which the atmosphere was replaced by gassing with a 5% O2, 5% CO2, 90% nitrogen gas mixture (Shumway Medical Supplies; Seattle, WA), after which the boxes were sealed with gas-impermeable tape (3M; St. Paul, MN) and placed in a 37°C incubator. At day 14 of culture colony forming units-granulocyte/macrophage (CFU-GM) and BFU-E colonies were enumerated using an inverted microscope as previously described.

Two classes of baboon megakaryocyte (MK) CFC, including the more primitive burst-forming unit-megakaryocyte (BFU-MK) and the more differentiated CFU-MK, were assayed from 1.0 x 105 peripheral blood or bone marrow buffy coat cells using a serum-depleted fibrin clot assay system, as previously described [26]. The 35 mm dishes were inverted and their base areas completely scanned at 100x using a microscope with a reflected fluorescent light attachment. Both BFU-MK-and CFU-MK-derived colonies were identified by previously described criteria [27]. All cultures were performed in triplicate unless otherwise indicated. Data are reported as the mean ± standard deviation per 1 x 105 cells in culture.

Liquid Cultures
Purified CD34+ marrow cells were cultured in triplicate at 104 per flat bottom 1/2-area well in 96-well plates in long-term culture medium [28] to which exogenous rHuMGDF was added at 0, 0.1, 1, 10 and 100 ng/ml. After 14 days, the entire contents of each well were harvested the cells counted, and cytospin slides prepared and stained with Wright-Giemsa stain.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
rHuMGDF Stimulates Proliferation and Megakaryocyte Differentiation of Baboon Marrow CD34+ Cells
CD34+ marrow cells, isolated by cell sorting, were cultured for 14 days in serum-containing liquid cultures in the absence or presence of rHuMGDF, as well as with the combination of recombinant human interleukin 3 (rHuIL-3), rHuIL-6 and recombinant human stem cell factor (rHuSCF). Cultures stimulated with rHuMGDF showed increased cell numbers compared with medium controls, and a marked increase in megakaryocytes was observed in cultures stimulated with rHuMGDF (Fig. 1Go) as compared with controls and cultures stimulated with IL-3, IL-6 and SCF. Thus, rHuMGDF stimulated the in vitro differentiation of CD34+ baboon marrow cells into megakaryocytes, similar to the reported effects of rHuMGDF on human CD34+ marrow cells in liquid culture [2, 21].



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  		Figure 1. rHuMGDF stimulates megakaryocytic differentation by baboon CD 34+ marrow cells cultured in liquid cultures. Cytospin preparation stained with Wright-Giemsa were photographed under 40 x magnification. Panel AA: CD 34+ marrow cells purified by flurescence-activated cell sorting (>98% CD34+) prior to culture: Panel B: these same purified CD34+ cells after 10 days of culture in serum-containing culture in the absence of rHuMGDF: Panels C and D: these same purified CD34+ cells after 10 days of cultures in the presence of 100 mg ml rHuMGDF. Immature and maturing megakaryocytes were seen only in cultures stimulated with rHuMGDF. Some of these megakaryocytes showed evidence of cytoplasmic budding suggestive of platelet formation.

 
Administration of rHuMGDF Stimulates Increased Peripheral Blood Platelet Counts in Baboons
rHuMGDF was administered to healthy juvenile baboons as a single daily s.c. dose for a total of 10 days at 50 [n = 2], 25 [n = 3], 10 [n = 3] and 1 [n = 2] µg/kg/day. Control animals [n = 2] received 10 µg/kg/day of heat-denatured rHuMGDF. Compared with pretreatment values, platelet counts increased markedly for all animals given 50, 25 and 10 µg/kg/day of rHuMGDF, while animals administered 1 µg/kg/day of rHuMGDF had only modest increases in platelets and animals administered heat-inactivated rHuMGDF showed no change in platelet counts (Fig. 2Go). Platelet counts began to increase between three and six days after starting rHuMGDF and reached maximum values between 7 and 10 days after starting treatment. Platelet counts remained at the maximally attained value for a period of four to five days following the last dose of rHuMGDF, after which they returned to pretreatment values within 14 days. The highest platelet counts were observed in animals administered 25 µg/kg/day where the maximum platelet counts ranged between 1,761 and 3,192 x109/1 (pretreatment range 310 to 482 x 109/1), 3.7-to 6.5-fold increases over baseline. Animals administered 10 and 50 µg/kg/day had similar increases in platelet counts with maximal counts ranging from 887 to 1,612 x109/1 (baseline range from 195 to 570 109/1) representing 2.0-to 4.6-fold increases. At 1.0 µg/kg, platelet counts increased to only 1.4-and 1.7-fold above baseline. These findings suggest that rHuMGDF stimulates dose-dependent increases in circulating platelet counts and that a dose of rHuMGDF as low as 25 µg/kg/day produced maximal biological effects in baboons. While the platelet counts were somewhat lower in animals administered 50 as compared with 25 µg/kg/day rHuMGDF, these differences were not statistically significant. Platelet volume was not altered in experimental animals as compared with controls (data not shown).



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  		Figure 2. Peripheral blood platelet counts increase in baboons administered rHuMGDF s.c. once daily for 10 days. Panel A: platelet count x 10-9/l of blood; Panel B: the change in platelet counts relative to pretreatment values is shown. All values for a given animal are normalized by dividing by the initial platelet count obtained at day - 7. Data are the mean for animals administered 50 ug/kg/day [{blacktriangleup}, n = 2] 25 µg/kg/day [•, n = 3], 10 µg/kg/day [{Delta}, n = 3], 1 µg/kg/day [{square}, n = 2] and control animals administered heat-denatured rHuMGDF at the equivalent of 10 µg/kg/day[{bigcirc}, n = 2].

 
In contrast with the marked increases in circulating platelets observed in animals administered rHuMGDF, the total number of red blood cells, white blood cells (WBC), neutrophils and monocytes in the peripheral blood did not change (Fig. 3Go), nor did the proportion of WBC represented by lymphocytes, eosinophils and basophils change in any of the animals (data not shown).



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  		Figure 3. Changes in numbers of total white blood cells (Panel A), neutrophils (Panel B), monocytes (Panel C) and red blood cells (Panel D) in baboons administered rHuMGDF. Data are the mean for animals administered 50 (µg/kg/day [{blacktriangleup}, n = 2], 25 µg/kg/day [•, n = 3] µg/kg/day [{Delta}, n = 3], 1 µg/kg/day [{square}, n = 2] and control animals administered heat-denatured rHuMGDF at the equivalent of 10 µg/kg/day [{bigcirc}, n = 2].

 
Administration of rHuMGDF Increases the Size and Numbers of Megakaryocytes in Marrow
Bone marrow biopsies were obtained immediately prior to the first dose of rHuMGDF, day 0; on days 3, 5, 7 and 14; and between days 28 and 31 of the study (Table 1Go). Animals treated with 10, 25 and 50 µg/kg/day of rHuMGDF all showed similar changes in their marrows. First, given inherent variability due to sampling, the overall marrow cellularity did not appear to change as a result of rHuMGDF administration. Second, after three days of rHuMGDF administration, before any significant increases in circulating platelet numbers were detected, megakaryocytes increased at least threefold and developed morphologic changes characterized by increased cytoplasmic volume and increased nuclear lobes or ploidy (Fig. 4Go). These morphologic changes were present in biopsies obtained during and shortly after the period of rHuMGDF administration but were absent from marrow biopsies obtained 18 to 21 days after the last dose. Reticulin stains of the marrow biopsies obtained during and after rHuMGDF administration showed no evidence of fibrosis. No changes in morphology of granulocytic or erythrocytic elements were noted in the marrow biopsies obtained during or after rHuMGDF administration. The two animals administered 1 µg/kg/day of rHuMGDF had evidence of increased numbers of megakaryocytes in marrow biopsies (Table 1Go), but these megakaryocytes did not demonstrate the morphologic changes observed in animals administered higher doses. In contrast, no changes in megakaryocyte number of morphology were observed in the marrows of the two control animals administered heat-inactivated rHuMGDF.


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  		Table 1. Effects of rHuMGDF on marrow cellularity, megakaryocyte morphology and frequency, and myeloid-to-erythroid (M:E) ratio
 


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  		Figure 4. Changes in the number and size of marrow megakaryocytes are shown for a representative baboon administered 25 µg/kg/day of rHuMGDF in biopsy to rHuMGDF treatment (Panel A), after three days of rHuMGDF (Panel B), four days after the las dose of rHuMGDF (Panel C), and 21 days after the last dose of rHuMGDF (Panel D). Megakaryocytes in Panels B and C show characteristic morphologic changes including increased cytoplasmic volume and increased nuclear lobes.

 
Changes in Marrow and Peripheral Blood Progenitor Cells Stimulated by rHuMGDF
We asked if administration of rHuMGDF altered circulation of CD34+ progenitor cells as administration of other hematopoietic growth factors including rHuG-CSF, rHuSCF, and rHuGM-CSF are known to elicit circulation of hematopoietic progenitor cells in nonhuman primates [26, 27, 29]. Peripheral blood and marrow buffy coat cells obtained at different times during rHuMGDF treatment were cultured to ascertain the frequency of progenitor cells. In peripheral blood we noted a transient, modest increase in circulating CD34+ cells, CFU-GM and BFU-E (Fig. 5Go) but not of CFU-MK or BFU-MK (data not shown) during the period of rHuMGDF administration. To determine if the CD34+ cells in the blood represented progenitor cells, CD34+ and CD34– cells were isolated from the blood, on day 7 of rHuMGDF (25 µg/kg), of two animals by fluorescence-activated cell sorting and compared for growth of progenitor cells compared to unfractionated blood cells (Fig. 6Go). The CD34+ cells isolated were enriched for progenitor activity while progenitors were depleted from the CD34– population, thus confirming the presence of progenitor cells within the circulating CD34+ cells induced by rHuMGDF treatment. In contrast, in the marrow, the frequency of detectable CFU-GM and BFU-E during rHuMGDF administration either remained the same or declined (Fig. 7Go). In cultures of marrow cells for CFU-MK and BFU-MK there was a dramatic increase in large single megakaryocytes in the cultures, although the number of CFU- and BFU-MEG did not change.



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  		Figure 5. Changes in peripheral blood of CD34+ cells (Panel A), total colony-forming cells (Panel B), colony forming unites-granulocyte/macrophage (Panel C), and burst-forming units-erythroid (Panel D) stimulated by administration of rHuMGDF for 10 days. Data are the mean ± standard deviation for animals administered 50 µg/kg/day [{blacktriangleup}, n = 2], 25 µg/kg/day [•, n = 3], 10 µg/kg/day [{Delta}, n = 2], and control animals administered heat-denatured rHuMGDF at the equivalent of 10 µg/kg/day [{bigcirc}, n = 2]. PBL: peripheral blood lymphocytes.

 


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  		Figure 6. Progenitor cells stimulated to circulate by treatment with rHuMGDF express CD34. Unseparated, CD34-, and CD34+ cells were isolated from the blood of two animals, administered 25 µg/kg/day of rHuMGDF A and B, on day 10 of growth factor administration. Virtually all myelocytic and erythrocytic progenitor cells were present in the CD34+ population in blood.

 


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  		Figure 7. Changes in bone marrow CD34+cells (Panel A); total colony-forming cells (= colony-forming units-granulocyte/macrophage [CFU-GM] + BFU-E) (Panel B); CFU-MEG (Panel C), and large megakaryocytes in culture (Panel D) stimulated in animals administered rHuMGDF for 10 days. Data are the mean ± SD for animals administered 50 µg/kg/day [{blacktriangleup}, n = 2], 25 µg/kg/day [•, n = 3], 10 µg/kg/day [{Delta}, n = 3], 1 µg/kg/day [{square}, n = 2], and control animals administered heat-denatured rHuMGDF at the equivalent of 10 µg/kg/day [{bigcirc}, n = 2].

 
MGDF Stimulates Rapid Recovery From 5-FU-Induced Thrombocytopenia
Administration of a single dose of 5-FU at 120 mg/kg i.v. causes reproducible neutropenia and thrombocytopenia. We asked if rHuMGDF would abrogate the 5-FU-induced thrombocytopenia. Beginning 48 h after the 5-FU dose, animals received either PEG-rHuMGDF at 25 µg/kg/day (experimental group, n = 5) or saline (control group, n = 6) s.c. as a single daily dose for 14 days (Fig. 8Go). All animals became thrombocytopenic whether they received PEG-rHuMGDF or saline. The nadir platelet count as well as the day at which the nadir occurred were similar for both the PEG-rHuMGDF and saline-treated animals. The rate of platelet count recovery was slightly faster for animals administered PEG-rHuMGDF, reaching pretreatment values by 13.8 ± 1.8 days as compared with 16.8 ± 0.6 days for controls administered saline. The PEG-rHuMGDF-treated animals had a more exaggerated thrombocytosis between days 17 and 24 as compared with controls. There was no difference between experimental and control animals in the rate of recovery for neutrophils, monocytes, or lymphocytes.



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  		Figure 8. Administration of PEG-rHuMGDF, 25 µg/kg/day, for 14 days after a single dose of 5-FU (120 mg/kg) stimulates more rapid recovery of platelets. Panel A shows the changes in platelet counts (mean ± standard deviation) after 5-FU for animals that were administered either PEG-rHuMGDF • or saline {bigcirc}. Panels B and C show the changes in neutrophil and monocyte counts, respectively, for these animals.

 
Bone marrow biopsies were obtained before, during and after treatment in experimental and control animals. Prior to treatment with 5-FU, the marrow cellularity and megakaryocyte numbers were similar for both groups of animals (Fig. 9Go). One week later, marrow biopsies obtained from two animals in each group showed similar findings diminished cellularity and decreased proportion of megakaryocytes. However, marrow biopsies from PEG-rHuMGDF-treated animals suggested a more rapid recovery of marrow cellularity and increased numbers of megakaryocytes by three weeks after the 5-FU compared with saline treated controls. This more rapid recovery suggests that in addition to the effects of rHuMGDF on megakaryocytes and their immediate progenitors, rHuMGDF also may have an effect on cells' more primitive progenitor cells that may be reflected in other hematopoietic lineages. By day 28, 12 days after the last dose of PEG-rHuMGDF, the marrow biopsies were again similar in both experimental and control groups of animals. There were no clear differences between groups with regard to white cell or red cell morphology in the marrow biopsies obtained at different times during the study. In this regard, the leukopenia, neutropenia and anemia that occurred as a result of 5-FU treatment as well as the rate of recovery of these cell lineages was not different between the PEG-rHuMGDF- and saline-treated groups.



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  		Figure 9. Recovery of megakaryocytes, Panel A, and marrow cellularity, Panel B, in marrow of animals administered PEG-rHuMGDF after treatment with 5-FU. The value for individual animals administered PEG-rHuMGDF is presented by a • and the value for individual control animals administered saline is represented by a {bigcirc}. BM: bone marrow.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we have shown that rHuMGDF, a ligand for the mpl receptor, stimulates in vitro megakaryocytopoiesis by baboon CD34+ marrow cells. Administration of rHuMGDF to normal baboons stimulated in vivo thrombopoiesis but had no detectable effects on production of cells in the lymphoid, erythroid, granulocytic, monocytic, eosinophilic, or basophilic lineages. Changes in the number and size of marrow megakaryocytes preceded the rise in peripheral blood platelet counts. The elevations in platelet counts and changes in megakaryocyte morphology persisted for at least four days after the last dose of rHuMGDF, and it took up to 14 days for peripheral blood platelet counts to return to baseline. Although low titers of anti-rHuMGDF antibodies were detected in some animals after treatment (data not shown), none of the animals in the study developed evidence of thrombocytopenia. In blood, there was evidence of a transient increase in circulating CD34+ granulocytic and erythrocytic progenitors but not megakaryocytic progenitors during rHuMGDF treatment. This finding suggests that the mpl ligand may have additional biological effects on progenitors other than those committed to megakaryocytic differentiation, as has been suggested by studies of Alexander et al. and Kaushansky et al. [30, 31]. Importantly, rHuMGDF stimulated more rapid recovery of thrombocyte production after myelosuppressive chemotherapy with 5-FU. Although it did not abrogate the thrombocytopenia in this model.

It had long been hypothesized that humoral factors were involved in regulating platelet production [32-34]. It was possible to detect activity in the serum and urine of thrombocytopenic animals that stimulated the growth of megakaryocytic progenitors in vitro, yet purification of thrombopoietin remained elusive. Factors that can promote the in vitro proliferation of megakaryocytic progenitor cells, and to a lesser extent, indirectly stimulate in vivo thrombocytopoiesis, have been identified, including IL-3 [35], IL-6 [36-38], IL-1 [35], IL-11 [39, 40] and GM-CSF [41]. However, stimulation of the terminal differentiative events in steady-state thrombocytopoiesis does not appear to be a primary role for most of these factors, as experiments with knock-out mice have demonstrated [42-45]. In contrast, mice with disruption of their normal c-mpl receptor genes are severely thrombocytopenic but do not have overt abnormalities in other hematopoietic lineages [7]. The ligand for mpl fulfills many of the characteristics of the hypothesized thrombopoietin [32-34]. Recombinant murine mpl ligand stimulates thrombopoiesis in mice [26-, 24]. The recombinant human mpl ligand stimulates the proliferation and differentiation of human megakaryocytic progenitors in vitro [2], including terminal differentiation of megakaryocytes with production of functional platelets [21, 22]. The present study, as well as that of Farese et al. [25], demonstrates that recombinant human mpl ligand will specifically stimulate thrombopoiesis in vivo in nonhuman primates. Importantly, Farese et al. demonstrated that rHuMGDF stimulates more rapid recovery of platelet counts in sublethally irradiated primates [46]. Similarly, in the present study we have shown that rHuMGDF stimulates more rapid recovery of platelets after significant myelosuppressive chemotherapy. In this manner, the response to rHuMGDF appears similar to that of granulocyte production to rHuG-CSF in that neither factor abrogates the nadir of induced cytopenia but promotes a more rapid recovery of the mature cell type in blood. Studies reported by Harker et al., also in nonhuman primates, have demonstrated that the platelets produced in response to in vivo administration of recombinant human mpl ligand are functionally normal [47, 48]. None of the animals in the present study or in the studies of Farese et al. and Harker et al. have demonstrated any adverse toxicities. Thus, rHuMGDF, the ligand for human mpl, may provide a means for specifically stimulating thrombopoiesis in clinical settings where thrombocytopenia occurs, including after nonmarrow-ablative chemotherapy and radiation therapy. Whether rHuMGDF will have a similar effect on the recovery of platelet production following marrow and peripheral blood stem cell transplantation remains to be determined.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
We gratefully acknowledge the assistance of Ray Colby in caring for all animals, and the assistance of the staff of the Clinical Hematology and the Pathology Shared Resources Laboratories at the Fred Hutchinson Cancer Research Center.

Supported by Amgen, Inc. and in part by grants and contracts NO1-AI-35191, NIHRR00166 and CA-18029.


   Footnotes
 
Provisionally accepted May 21, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Methia N, Louache F, Vainchenker W et al. Oligodeoxynucleotides antisense to the protooncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis. Blood 1993;82:1395-1401.[Abstract/Free Full Text]

  2. Bartley TD, Bogenberger J, Hunt P et al. Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 1994;77:1117-1124.[Medline]

  3. Wendling F, Maraskovsky E, Debili N et al. cMpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 1994;369:571-574.[Medline]

  4. Kaushansky K, Lok S, Holly RD et al. Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 1994;369:568-571.[Medline]

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Received April 12, 1996; accepted for publication July 5, 1996.




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