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KINETIC ANALYSIS OF MEGAKARYOPOIESIS INDUCED BY RECOMBINANT HUMAN INTERLEUKIN 11 IN MYELOSUPPRESSED MICE
doi:10.1006/cyto.2000.0832, available online at http://www.idealibrary.com on
KINETIC ANALYSIS OF MEGAKARYOPOIESIS INDUCED BY RECOMBINANT HUMAN INTERLEUKIN 11 IN MYELOSUPPRESSED MICE Minori Saitoh, Katsunari Taguchi, Kazuhiro Momose, Kazutaka Suga, Yumiko Ogawa, 1Shuhei Yasuda, Keiji Miyata Recombinant human interleukin 11 (rhIL-11) has previously been shown to ameliorate thrombocytopenia in several animal models. To elucidate the mechanisms involved in rhIL-11induced hematopoiesis, a kinetic analysis of megakaryopoiesis was performed in mitomycin C (MMC)-induced myelosuppressive mice. Mice intravenously injected with MMC (2 mg/kg) for two consecutive days from day 1 developed severe thrombocytopenia with a nadir of platelet counts at 24104/l on day 12 and neutropenia. Treatment with rhIL-11 (500 g/kg/day) from day 1 to 21 significantly ameliorated the degree and duration of thrombocytopenia and enhanced the platelet recovery, and also enhanced the recovery from neutropenia. In MMC-treated mice, the decreases in bone marrow megakaryocyte progenitors and megakaryocyte counts preceded the decrease in platelet counts by MMC treatment. RhIL-11 induced an increase in the number of megakaryocyte progenitors from day 4 to 14, followed by an increase in the megakaryocytes by day 20. There was a ploidy shift in megakaryocytes towards lower ploidy cells by day 9 in myelosuppressed mice. RhIL-11 caused a shift towards a higher ploidy with 32 and 64N on day 4, and 32N on day 14. These results suggest that rhIL-11 ameliorates the thrombocytopenia via the stimulation of both the maturation and commitment followed by the proliferation of megakaryocytic cells. 2001 Academic Press
Interleukin (IL-) 11 is known as a multifunctional cytokine that was initially identified in medium from the primate stromal cell line PU-34, and was subsequently cloned by bioassay using the IL-6-dependent murine plasmacytoma cell line T1165.1 IL-11 has been reported to have multiple effects on megakaryocyte development. For example, IL-11 stimulates the formation of colony-forming units-megakaryocyte (CFU-Meg), and increases the size and ploidy of immature megakaryocytes in vitro.1–7 Recombinant human IL-11 (rhIL-11) acts on megakaryopoiesis synergistically with the cytokines IL-3 or stem cell factor From the 1Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Correspondence to: Minori Saitoh, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan. E-mail: [email protected] Received 31 July 2000; received in revised form 22 November 2000; accepted for publication 30 December 2000 2001 Academic Press 1043–4666/01/050287+08 $35.00/0 KEY WORDS: chemotherapy/interleukin 11/megakaryopoietic kinetics/mice/thrombocytopenia CYTOKINE, Vol. 13, No. 5 (7 March), 2001: pp 287–294
(SCF).5,8 More recently, it has been reported that rhIL-11 acts on the early stages of megakaryocyte development.5,6 Subcutaneous administration of IL-11 increases platelet counts as well as bone marrow CFUMeg number and megakaryocyte ploidy in normal mice,9 and increases platelet counts associated with the increase in megakaryocyte size in non-human primates.10 The effect of rhIL-11 has also been studied in several myelosuppressed animals.11–14 Thus, in sublethal irradiation and carboplatin-induced myelosuppressed mice, administration of rhIL-11 increases the number of CFU-Meg, which is reflected in the peripheral circulation by a reduction of platelet nadir and a significantly reduced period of thrombocytopenia.11 In addition, rhIL-11 accelerated reconstitution of myeloid progenitors from bone marrow when rhIL-11 increased peripheral platelet and neutrophil counts in BCNU- or cyclophosphamide-induced myelosuppressed mice.12,14 RhIL-11 also accelerated the recovery of periopheral neutrophil and platelet counts in bone marrow and spleen cell transplant mice.15 In clinical trials, rhIL-11 has shortened the duration of thrombocytopenia and decreased the need for platelet transfusion in patients receiving 287
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RESULTS Effect on circulating blood cells MMC injection caused rapid decreases in platelet counts, reaching a nadir of 24104/l 12 days after treatment, followed by gradual recovery, but not to normal levels by day 26 (Fig. 1A). Treatment with rhIL-11 significantly improved the nadir (34104/l, on day 9) and accelerated platelet recovery compared with vehicle-treated animals, reaching normal levels by day 23. Treatment with MMC also resulted in a decrease in the absolute neutrophil count (ANC) (Fig. 1B). The decrease in ANC was sustained from day 5 to 18, and thereafter ANC recovered gradually to reach a normal level by day 26. RhIL-11 slightly but significantly attenuated the severity of neutropenia and accelerated the recovery of ANC. The number of other blood cells, e.g., erythrocytes, lymphocytes and monocytes, were not markedly affected by MMC treatment, and rhIL-11 did not significantly affect the counts of these cells (data not shown).
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chemotherapy,16–18 proving that rhIL-11 treatment is effective in ameliorating thrombocytopenia induced by chemotherapy. In these studies, however, the effects of rhIL-11 on both proliferation and maturation of megakaryocyte lineage cells, i.e., megakaryocytes and their progenitor number and megakaryocyte ploidy, have not been evaluated in detail. Moreover, kinetic analysis of the stimulatory effect of rhIL-11 on these parameters of megakaryopoiesis has not been made, but should be useful for clarifying the role of IL-11 in megakaryopoiesis. In the present study, therefore, the effect of rhIL-11 on bone marrow CFU-Meg, megakaryocyte number and megakaryocyte ploidy was examined chronologically in myelosuppressed animals. Mice kept on a myelosuppressive regimen of mytomycin C (MMC) were used as the myelosuppressed model.
CYTOKINE, Vol. 13, No. 5 (7 March, 2001: 287–294)
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Figure 1. The effects of rhIL-11 on peripheral (A) platelet and (B) absolute neutrophil counts (ANC) in MMC-treated mice. RhIL-11 was given subcutaneously to mice at a dose of 500 g/kg/ day from 1 day after second dosing of MMC (2 mg/kg iv; day 1) to day 21. Each symbol represents the meanSEM from ten animals except where indicated in parenthesis. * and ** show significant differences between the vehicle- and rhIL-11-treated groups (*P<0.05, **P<0.01, Student’s t-test). Normal, — —; vehicle, — —; rhIl-11, — —.
Effect on bone marrow megakaryopoiesis
Number of megakaryocytes MMC decreased the megakaryocyte number on day 4, followed by a nadir, 4.7% of normal, on day 9 (Fig. 3). The megakaryocyte number recovered gradually, but was still at a low level on day 20. Although no significant difference in megakaryocyte number between vehicle- and rhIL-11-treated animals was found on day 14, rhIL-11 significantly increased the number of megakaryocytes on day 20.
Number of CFU-Meg Treatment with MMC led to a dramatic decrease in CFU-Meg number in bone marrow cells on day 4, which preceded the expression of severe thrombocytopenia at least by one day (Fig. 2). Thereafter, the number of CFU-Meg gradually recovered, but did not reach normal levels by day 20. In the rhIL-11 group, there was a significant increase or a tendency to an increase in the number of CFU-Meg in comparison with vehicle-treated animals.
Megakaryocyte ploidy Normal mice had a modal megakaryocyte ploidy of 16N (50.5% to 51.3% of megakaryocytes) with 22.2% to 24.1% of megakaryocytes in the 8N ploidy class and 11.7% to 14.9% at 32N (Fig. 4). The effect of MMC treatment by itself on the megakaryocyte ploidy distribution was complex. Namely, there was a clear decrease in 16N and a simultaneous increase in 2N and 4N megakaryocytes on day 4. On day 9, the modal ploidy class shifted from 16N to 2N and the
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Bone marrow cells harvested from bone marrow on days 4, 9, 14 and 20 were cultured for 7 days, and the number of CFU-Meg then formed was counted. The data represent the meanSEM from eight animals. ** shows a statistical significance between normal and vehicle-treated groups (P<0.01, Student’s t-test). # and ## show a statistical significance between vehicle- and rhIL-11-treated groups (#P<0.05, ##P<0.01, Student’s t-test). Normal, ; vehicle, ; rhIL-11, .
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The number of bone marrow megakaryocytes was determined on days 4, 9, 14 and 20. The data represent the meanSEM from eight to nine animals. ** shows a statistical significance between normal and vehicle-treated groups (P<0.01, Student’s t-test). # shows a statistical significance between vehicle- and rhIL-11-treated groups (P<0.05, Student’s t-test). Normal, ; vehicle, ; rhIL-11, .
distribution pattern was flattened. Thereafter, the modal ploidy class shift to higher ploidy (32N or more) megakaryocytes was shown on days 14 and 20. Treatment with rhIL-11 significantly increased the population of 32N and 64N megakaryocytes on day 4, and that of 32N megakaryocytes on day 14, in comparison with vehicle-treated mice. The distribution pattern in rhIL-11-treated mice was similar to that in vehicle-treated mice on days 9 and 20.
DISCUSSION The effects of rhIL-11 on chemotherapy- or irradiation-induced thrombocytopenia have been investigated. Results demonstrated amelioration of the nadir and accerelation of recovery of thrombocytopenia in pre-clinical studies10–14 as well as a decrease in the need for platelet transfusion in clinical studies.16–18 In the present study, rhIL-11 attenuated the nadir of platelet counts and accelerated the recovery of platelet counts in MMC-induced myelosuppressed mice.
Therefore, the present study confirmed that rhIL-11 ameliorated the depth and duration of thrombocytopenia in various chemotherapy-induced myelosuppressed models. To elucidate the mechanism of the stimulatory effect of rhIL-11 on megakaryopoiesis in a myelosuppressed condition, we quantitated chronological changes in the number of CFU-Megs, megakaryocytes, and megakaryocyte ploidy from bone marrow cells in MMC-treated mice over 20 days after administration of rhIL-11. Treatment with rhIL-11 stimulated megakaryocyte ploidy and CFU-Megs but not megakaryocyte numbers on day 4, suggesting that rhIL-11 exerted its stimulatory effect on megakaryocyte maturation and on the recruitment of progenitors to megakaryocyte lineage at around the same time, which is significantly earlier than that on proliferation of megakaryocytic cells. On day 9, rhIL-11 increased CFU-Meg numbers, but not megakaryocyte number or ploidy. Increases in CFU-Meg number and megakaryocyte ploidy were observed in rhIL-11-treated mice on day 14, but these increases were not detected
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Megakaryocyte ploidy changes in MMC-treated mice that received vehicle and rhIL-11.
Megakaryocyte ploidy was determined by flow cytometer in a double-staining experiment, as described in Materials and Methods. For each measurement, the number of Pm1 + cells was counted in the range between 400 and 1500, and the percentage of cells in each ploidy class vs the total megakaryocyte count was determined. The data represent the meanSEM from seven animals except for the vehicle-treated group on day 9. In the vehicle-treated group on day 9, the frequency of Pm1 + cells in the total of bone marrow cells from one animal was too low to evaluate the megakaryocyte ploidy. Thus, this sample was excluded from the evaluation. * and ** show the statistical significance, for each ploidy class, of the difference between normal and vehicle-treated groups (*P<0.05, **P<0.01, Student’s t-test). # and ## show the statistical significance, for each ploidy class, of the difference between vehicle- and rhIL-11-treated groups (#P<0.05, ##P<0.01, Student’s t-test).
Megakaryopoietic kinetics of rhIL-11 in myelosuppressed mice / 291
on day 20. An increase in megakaryocyte number in rhIL-11-treated animals was detected only on day 20. The increase in megakaryocyte number thus appears to reflect an earlier increase in CFU-Meg number. Taken together, these findings indicate that rhIL-11 exerted a stimulatory effect on the endomitosis of megakaryocytes early in the dosing period as well as the commitment to the megakaryocyte lineage, and thereafter stimulated both proliferation and maturation. Support for this interpretation is provided by studies in normal mice,9 in which rhIL-11 increased megakaryocyte progenitors and stimulated endomitosis of bone marrow megakaryocytes but not bone marrow megakaryocyte frequences. In normal dogs, also, treatment with rhIL-11 increased ploidy number of megakaryocytes in 6/6 adequately evaluated animals, but increased numbers of megakaryocytes in only 3/8,19 demonstrating that rhIL-11 preferentially stimulates endomitosis of megakaryopoiesis rather than proliferation. Further, the increases in marrow and peripheral blood colony-forming units-granulocyte and macrophage (CFU-GM) were obtained in the study using normal dogs.19 Previous studies have demonstrated the effect of rhIL-11 on the recruitment of progenitors to megakaryocyte lineage. Weich et al.6 note that rhIL-11 has the potential to promote murine megakaryocyte development via effects on bone marrow primitive progenitor cells and early and late progenitor cells. RhIL-11 has also been shown to act on human progenitor hematopoietic cells, where it induces an increase in the number of CFU cells derived from CD34 + and CD34 + CD33 DR cells.20,21 Further, an increase in the commitment of stem cells into a multipotential progenitor subpopulation was observed when rhIL-11 was added to human or murine longterm bone marrow cultures.22 These in vitro observations are in agreement with several in vivo studies. RhIL-11 increases the number of CFU-Megs, and the number of multipotential colony-forming unit granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM) progenitor cells during the platelet recovery period in carboplatin- and irradiationtreated mice,11 and BCNU-treated mice,14 respectively. Furthermore, rhIL-11 increases the number of CFUGEMM a little earlier than a thrombopoietic effect appears in cyclophosphamide-treated mice.12 When administered to humans, rhIL-11 stimulates all stages of megakaryopoiesis and, in addition, stimulates precursor cells of different marrow lineages without affecting the number of assayable progenitor cells.23 Our findings that rhIL-11 has an effect on the commitment of stem cells to the megakaryocyte lineage in the early period reflects these findings, and can be considered to represent one of the pluripotent effects of this cytokine on haematopoietic stem cells.
Thrombopoietin (TPO) is a potent stimulant of megakaryopoiesis.24,25 Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of rhTPO derivatized with polyethylene glycol, has been investigated in MMC-induced thrombocytopenic mice, and has been found to inhibit thrombocytopenia dosedependently.26 PEG-rHuMGDF has also been reported to stimulate the recovery of the number of CFU-Megs and of megakaryocytes from 5 and 8 days after the MMC treatment, respectively. In the present study, rhIL-11 four days after MMC treatment stimulated the megakaryocyte endomitosis and the recovery of the number of CFU-Megs that preceded the proliferation of megakaryocytes on day 20. Therefore, it is suggested that two cytokines, rhIL-11 and PEG-rHuMGDF, stimulate the megakaryocyte progenitors in the early period of dosing, with differences in the onset times of the effects on megakaryocyte proliferation. Care is needed in comparing the effects of the two cytokines, because experimental conditions were different, i.e. the dose of MMC. The possibility exists, however, that IL-11 and TPO have distinct effects on megakaryopoiesis. RhIL-11, in combination with IL-3 or SCF, acts synergistically in stimulating human and murine megakaryocyte progenitor-derived colony formation.20,27 RhIL-11 plus IL-3 increased megakaryocyte number and acetylcholinesterase production in murine bone marrow liquid culture compared with IL-3 alone.2 Acting as a single agent, rhIL-11 enhanced the size and ploidy of human and murine bone marrow megakaryocytes.2,3,28 In an in vivo experiment, rhIL-11 increased megakaryocyte progenitors and stimulated endomitosis of bone marrow megakaryocytes, but did not affect bone marrow megakaryocyte frequencies,9 these results being consistent with our results in MMC-treated mice. Hence, rhIL-11 likely has a stimulating effect on megakaryocyte endomitosis and commitment rather than on proliferation. On the other hand, TPO, as a single agent, supports the formation of megakaryocyte colonies and the endomitosis of megakaryocytes.24,25,28,29 A single injection of pegylated murine MGDF acted on megakaryocytic proliferation as well as on megakaryocytic endomitosis in normal mice.30 Consequently, differences may exist in the effect of the two cytokines on megakaryocyte proliferation, and this may influence the onset time of megakaryopoiesis. In addition to the effect on thrombocytopenia, administration of rhIL-11 improved the neutropenia induced by MMC treatment. RhIL-11 stimulates the proliferation of granulocyte progenitor cells in vitro.31,32 Moreover, in in vivo studies rhIL-11 increases ANC in bone marrow-transplanted mice and cyclophosphamide-induced myelosuppressed mice,12,15 while it does not affect ANC in carboplatin- or
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irradiation-induced myelosuppressed mice.11 It is suggested that rhIL-11 has a proliferating effect on ANC by stimulating granulocytopoiesis, but the effect appears to depend on experimental conditions or models. In conclusion, rhIL-11 significantly ameliorated the severity of thrombocytopenia in mice with MMCinduced myelosuppression in the present study. The effect was confirmed to reflect the stimulation by rhIL-11 of both the maturation and commitment to the megakaryocyte lineage, followed by the increase in the megakaryocytes in vivo.
MATERIALS AND METHODS Animals Female Balb/c mice at 7 to 8 weeks of age (17–22 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were housed in autoclaved cages in specific pathogen-free rooms and given sterilized commercial rodent chow and water ad libitum. The animal room was conditioned to 21–25C with a relative humidity of 50–60% and was lit on a 13/11 h light/dark light cycle (7:30 to 20:30). All experiments were approved by the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd.
RhIL-11 RhIL-11 purified to homogeneity from Escherichia coli was obtained from Genetics Institute (Cambridge, MA, USA). The specific bioactivity of rhIL-11 was estimated to be 1.3106 units/mg of protein using a bioassay employing the murine plasmacytoma cell line, T10. RhIL-11 was dissolved in a solution containing 0.004 M sodium dihydrogenphosphate dihydrate, 0.006 M disodium hydrogenphosphate,12H2O, 0.3 M glycine and 0.01% (w/v) polyoxyethylene (20) sorbitan monooleate. This solution, except for the rhIL-11, was used as vehicle.
Thrombocytopenic model A modification of the method described by others26 was used to construct a model of MMC (Kyowa Hakko Kogo Co., Tokyo, Japan)-induced myelosuppression. Mice were given a dose of 2 mg/kg of MMC intravenously on days 1 and 0 to induce severe thrombocytopenia without any animal deaths (see Fig. 1). Subcutaneous treatment of mice with rhIL-11 at a dose of 500 g/kg/day was initiated on day 1 for 21 days.
Peripheral haematology Mice were anesthetized with ether, and peripheral blood was drawn from the inferior vena cava on days 1 (for the normal group), 5, 9, 12, 14, 16, 18, 20, 23 and 26. Blood cell count analysis was performed using a Celltac MEK-5158 (Nihon Kohden, Tokyo, Japan). White blood cell differentials were determined on blood smear preparations stained with May-Giemsa.
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Progenitor cell assay CFU-Meg colony assay was performed as previously described9 with a minor modification. Briefly, bone marrow cells were harvested from mice by flushing the femoral contents on days 4, 9, 14 and 20 with Iscove’s modified Dulbecco’s medium (IMDM; GIBCO BRL, Life Technologies, Inc., Rockville, MD, USA) containing 2% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Inc.). Cells (4105 cells) were suspended in 2.7 ml IMDM supplemented with 3 U/ml recombinant murine IL-3 (rmIL-3; Genzyme Diagnostics, Cambridge, MA, USA), 60 ng/ml rhIL-11 and 10% FBS and preincubated at 37C. Thereafter, the cell suspension was mixed with 300 l of 3.3% agar autoclaved and preincubated at 42C, followed by the addition of 750 l of mixture in two wells per chamber slide (Nalge Nunc International, Naperville, IL, USA). After incubation of the slides at 4C for 20 min to solidify the culture, the chamber slides were incubated for 7 days at 37C in a humidified atmosphere of 5% CO2 in air. The cells were then fixed with 5% glutaraldehyde for 10 min and subjected to histochemical staining for acetylcholinesterase (AchE) using 75 mM phosphate buffer containing 500 g/ml acetylthiocholine iodide (Sigma-Aldrich, St.Louis, MO, USA), 5 mM sodium citrate, 3 mM copper sulfate and 0.5 mM potassium ferricyanide. Megakaryocyte colonies were counted using an inverted microscope. CFU-Meg was defined as comprising three or more AchE + cells. All measurements were carried out in duplicate in each experiment, and the total number of CFU-Megs per femur was calculated as follows: the number of CFU-Megs per well was multiplied by the total number of cells obtained from one femur, which was divided by the number of cells plated per well.
Measurement of the number of megakaryocytes in the femur Bone marrow cells were fixed at 1105 cells per well in 50 l of 7.5% glutaraldehyde in 96-well culture plates for 10 min at room temperature and recovered by centrifugation. The cells were stained for AchE as described above, and the number of AchE + cells was counted as that of megakaryocytes. All measurements were carried out in duplicate in each experiment, and the total number of megakaryocytes per femur was calculated as follows: the number of AchE + megakaryocytes per well was multiplied by total number of cells obtained from one femur which was divided by the number of cells plated per well.
Measurement of megakaryocyte DNA content The experiment was performed according to a previously described method33 with a minor modifications. Briefly, bone marrow cells were harvested from mice by flushing the femoral contents with CATCH solution [Hank’s balanced salt solution (HBSS, GIBCO BRL) containing 0.38% sodium citrate, 1 mM adenosine, 2 mM theophylline and 3.5% bovine serum albumin (BSA, Sigma-Aldrich)]. The cells were incubated with 25 g/ml of murine megakaryocytespecific antibody Pm1,34 which was kindly provided by Dr Nagasawa (University of Tsukuba, Ibaraki, Japan), or 25 g/ ml of control rat IgG antibody (Pharmingen, San Diego, CA,
Megakaryopoietic kinetics of rhIL-11 in myelosuppressed mice / 293
USA) at 4C for 20 min. The cells were washed once and further labeled with 28 g/ml FITC-conjugated goat-anti-rat IgG (Immunotech, Marseille, France) at 4C for 20 min. The cells were washed, incubated in 1.8 ml CATCH containing 0.5% Tween-20 for 40 min at 4C, 200 l 5% paraformaldehyde solution was added, and the mixture was then incubated at 4C for an additional 5 min. For nuclear DNA labeling, the cells were washed and suspended in 0.1% sodium citrate buffer supplemented with 50 g/ml propidium iodide (SigmaAldrich) and 1% BSA. The suspension was stored overnight in the dark at 4C. The following day the cells were washed and incubated with 5 g/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN, USA) at room temperature for 30 min. The DNA content of megakaryocytes was determined by two-colour flow cytometric analysis using a flow cytometer (XL-MCL, Coulter, Miami, FL, USA). The DNA content of the cells labeled with the Pm1 antibody was analyzed by setting markers at the nadirs between the propidium iodide peaks. At least 400 Pm1 + cells were analyzed in each measurement.
Statistical analysis The results are expressed as the meanSEM of one determination from each of seven to ten mice. Experiments were repeated several times to yield the animal number of seven to ten. Significant differences were tested by Student’s t-test. P<0.05 was considered significant.
Acknowledgement We are grateful to Dr T. Nagasawa of University of Tsukuba for helpful suggestions during this study and for providing Pm1 antibody.
REFERENCES 1. Paul SR, Bennett F, Calvetti JA, Kelleher K, Wood CR, O’Hara RM, Jr., Leary AC, Sibley B, Clark SC, Williams DA, Yang Y-C (1990) Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine. Proc Natl Acad Sci USA 87:7512–7516. 2. Yonemura Y, Kawakita M, Masuda T, Fujimoto K, Kato K, Takatsuki K (1992) Synergistic effects of interleukin-3 and interleukin 11 on murine megakaryopoiesis in serum-free culture. Exp Hematol 20:1011–1016. 3. Teramura M, Kobayashi S, Hoshino S, Oshimi K, Mizoguchi H (1992) Interleukin-11 enhances human megakaryocytopoiesis in vitro. Blood 79:327–331. 4. Tsukada J, Misago M, Ogawa R, Oda S, Morimoto I, Eto S, Kikuchi M (1993) Synergism between serum factor(s) and erythropoietin in inducing murine megakaryocyte colony formation: the synergistic factor in serum is distinct from interleukin-11 and stem cell factor (c-kit ligand). Blood 81:866–867. 5. Weich NS, Wang A, Fitzgerald M, Neben TY, Donaldson D, Giannotti J, Yetz-Aldape J, Leven RM, Turner KJ (1997) Recombinant human interleukin-11 directly promotes megakaryocytopoiesis in vitro. Blood 90:3893–3902. 6. Weich NS, Fitzgerald M, Wang A, Calvetti J, Yetz-Aldape Jg, Neben S, Turner KJ (2000) Recombinant human interleukin-11 synergizes with steel factor and interleukin-3 to promote directly the early stages of murine megakaryocyte development in vitro. Blood 95:503–509.
7. Burstein SA, Mei RL, Henthorn J, Friese P, Turner K (1992) Leukemia inhibitory factor and interleukin-11 promote maturation of murine and human megakaryocytes in vitro. J Cellular Physiol 153:305–312. 8. Ariyama Y, Misawa S, Sonoda Y (1995) Synergistic effects of stem cell factor and interleukin 6 or interleukin 11 on the expansion of murine hematopoietic progenitors in liquid suspension culture. Stem Cells 13:404–413. 9. Neben TY, Loebelenz J, Hayes L, McCarthy K, Stoudemire J, Schaub R, Goldman SJ (1993) Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice. Blood 81:901–908. 10. Schlerman FJ, Bree AG, Kaviani MD, Nagle SL, Donnelly LH, Mason LE, Schaub RG, Grupp SA, Goldman SJ (1996) Thrombopoietic activity of recombinant human interleukin 11 (rHuIL-11) in normal and myelosuppressed nonhuman primates. Stem Cells 14:517–532. 11. Leonard JP, Quinto CM, Kozitza MK, Neben TY, Goldman SJ (1994) Recombinant human interleukin-11 stimulates multilineage hematopoietic recovery in mice after a myelosuppressive regimen of sublethal irradiation and carboplatin. Blood 83: 1499–1506. 12. Hangoc G, Yin T, Cooper S, Schendel P, Yang YC, Broxmeyer HE (1993) In vivo effects of recombinant interleukin-11 on myelopoiesis in mice. Blood 81:965–972. 13. Paul SR, Hayes LL, Palmer R, Morris GE, Neben TY, Loebelenz J, Pedneault G, Brooks J, Blue I, Moore MA, et al. (1994) Interleukin-11 expression in donor bone marrow cells improves hematological reconstitution in lethally irradiated recipient mice. Exp Hematol 22:295–301. 14. Maze R, Moritz T, Williams DA (1994) Increased survival and multilineage hematopoietic protection from delayed and severe myelosuppressive effects of a nitrosourea with recombinant interleukin-11. Cancer Res 54:4947–4951. 15. Du XX, Keller D, Maze R, Williams DA (1993) Comparative effects of in vivo treatment using interleukin-11 and stem cell factor on reconstitution in mice after bone marrow transplantation. Blood 82:1016–1022. 16. Gordon MS, McCaskill-Stevens WJ, Battiato LA, Loewy J, Loesch D, Breeden E, Hoffman R, Beach KJ, Kuca B, Kaye J, Sledge GW, Jr. (1996) A phase I trial of recombinant human interleukin-11 (neumega rhIL-11 growth factor) in women with breast cancer receiving chemotherapy. Blood 87:3615–3624. 17. Isaacs C, Robert NJ, Bailey FA, Schuster MW, Overmoyer B, Graham M, Cai B, Beach KJ, Loewy JW, Kaye JA (1997) Randomized placebo-controlled study of recombinant human interleukin-11 to prevent chemotherapy-induced thrombocytopenia in patients with breast cancer receiving dose-intensive cyclophosphamide and doxorubicin. J Clin Oncol 15:3368–3377. 18. Tepler I, Elias L, Smith JW, 2nd, Hussein M, Rosen G, Chang AY-C, Moore JO, Gordon MS, Kuca B, Beach KJ, Loewy JW, Garnick MB, Kaye JA (1996) A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. Blood 87: 3607–3614. 19. Nash R, Seidel K, Storb R, Slichter S, Schuening F, Appelbaum F, Becker A, Bolles L, Deeg H, Graham T, Hackman R, Burstein S (1995) Effects if rhIL-11 on normal dogs and after sublethal radiation. Exp Hematol 23:389–396. 20. Lemoli R, Fogli M, Fortuna A, Motta M, Rizzi S, Benini C, Tura S (1993) Interleukin-11 stimulates the proliferation of human hematopoietic CD34+ and CD34+CD33-DR- cells and synergizes with stem cell factor, interleukin-3, and granulocyte-macrophage colony-stimulating factor. Exp Hematol 21:1668–1672. 21. Leary A, Zeng H, Clark S, Ogawa M (1992) Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors. Proc Natl Acad Sci USA 89:4013–4017. 22. Du X, Scott D, Yang Z, Cooper R, Xiao X, Williams D (1995) Interleukin-11 stimulates multilineage progenitors, but not stem cells in murine and human long-term marrow cultures. Blood 86:128–134. 23. Orazi A, Cooper R, Tong J, Gordon M, Battiato L, Sledge G, Kaye J, Kahsai M, Hoffman R (1996) Effects of recombinant
294 / Saitoh et al. human interleukin-11 (Neumega rhIL-11 growth factor) on megakaryocytopoiesis in human bone marrow. Exp Hematol 24: 1289–1297. 24. Kaushansky K, Lok S, Holly RD, Broudy VC, Lin N, Bailey MC, Forstrom JW, Buddle MM, Oort PJ, Hagen FS, et al. (1994) Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin. Nature 369: 568–571. 25. Kaushansky K (1995) Thrombopoietin: the primary regulator of megakaryocyte and platelet production. Blood 86:419–431. 26. Akahori H, Shibuya K, Ozai M, Ida M, Kabaya K, Kato T, Miyazaki H (1995) Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice. Stem Cells 14:678–689. 27. Bruno E, Briddell RA, Cooper RJ, Hoffman R (1991) Effects of recombinant interleukin 11 on human megakaryocyte progenitor cells. Experimental Hematology 19:378–381. 28. Broudy VC, Lin NL, Kaushansky K (1995) Thrombopoietin (c-mpl ligand) acts synergistically with erythropoietin, stem cell factor, and interleukin-11 to enhance murine megakaryocyte colony growth and increases megakaryocyte ploidy in vitro. Blood 85: 1719–1726. 29. Nichol JL, Hokom MM, Hornkohl A, Sheridan WP, Ohashi H, Kato T, Li YS, Bartley TD, Choi E, Bogenberger J, et al.
CYTOKINE, Vol. 13, No. 5 (7 March, 2001: 287–294) (1995) Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia. J Clin Invest 95:2973–2978. 30. Arnold J, DAW N, Stenberg P, Jayawardene D, Srivastava D, Jackson C (1997) A single injection of pegylated murine megakaryocyte growth and development factor (MGDF) into mice is sufficient to produce a profound stimulation of megakaryocyte frequency, size, and ploidization. Blood 89:823–833. 31. Musashi M, Clark S, Sudo T, Urdal D, Ogawa M (1991) Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice. Blood 78:1448–1451. 32. Musashi M, Yang YC, Paul SR, Clark SC, Sudo T, Ogawa M (1991) Direct and synergistic effects of interleukin 11 on murine hemopoiesis in culture. Proc Natl Acad Sci USA 88:765–769. 33. Corash L, Levin J, Mok Y, Baker G, McDowell J (1989) Measurement of megakaryocyte frequency and ploidy distribution in unfractionated murine bone marrow. Exp Hematol 17:278–286. 34. Nagata Y, Nagahisa H, Aida Y, Okutomi K, Nagasawa T, Todokoro K (1995) Thrombopoietin induces megakaryocyte differentiation in hematopoietic progenitor FDC-P2 cells. Biol Chem 270:19673–19675.
KINETIC ANALYSIS OF MEGAKARYOPOIESIS INDUCED BY RECOMBINANT HUMAN INTERLEUKIN 11 IN MYELOSUPPRESSED MICE Minori Saitoh, Katsunari Taguchi, Kazuhiro Momose, Kazutaka Suga, Yumiko Ogawa, 1Shuhei Yasuda, Keiji Miyata Recombinant human interleukin 11 (rhIL-11) has previously been shown to ameliorate thrombocytopenia in several animal models. To elucidate the mechanisms involved in rhIL-11induced hematopoiesis, a kinetic analysis of megakaryopoiesis was performed in mitomycin C (MMC)-induced myelosuppressive mice. Mice intravenously injected with MMC (2 mg/kg) for two consecutive days from day 1 developed severe thrombocytopenia with a nadir of platelet counts at 24104/l on day 12 and neutropenia. Treatment with rhIL-11 (500 g/kg/day) from day 1 to 21 significantly ameliorated the degree and duration of thrombocytopenia and enhanced the platelet recovery, and also enhanced the recovery from neutropenia. In MMC-treated mice, the decreases in bone marrow megakaryocyte progenitors and megakaryocyte counts preceded the decrease in platelet counts by MMC treatment. RhIL-11 induced an increase in the number of megakaryocyte progenitors from day 4 to 14, followed by an increase in the megakaryocytes by day 20. There was a ploidy shift in megakaryocytes towards lower ploidy cells by day 9 in myelosuppressed mice. RhIL-11 caused a shift towards a higher ploidy with 32 and 64N on day 4, and 32N on day 14. These results suggest that rhIL-11 ameliorates the thrombocytopenia via the stimulation of both the maturation and commitment followed by the proliferation of megakaryocytic cells. 2001 Academic Press
Interleukin (IL-) 11 is known as a multifunctional cytokine that was initially identified in medium from the primate stromal cell line PU-34, and was subsequently cloned by bioassay using the IL-6-dependent murine plasmacytoma cell line T1165.1 IL-11 has been reported to have multiple effects on megakaryocyte development. For example, IL-11 stimulates the formation of colony-forming units-megakaryocyte (CFU-Meg), and increases the size and ploidy of immature megakaryocytes in vitro.1–7 Recombinant human IL-11 (rhIL-11) acts on megakaryopoiesis synergistically with the cytokines IL-3 or stem cell factor From the 1Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Tsukuba, Ibaraki, Japan Correspondence to: Minori Saitoh, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., 21, Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan. E-mail: [email protected] Received 31 July 2000; received in revised form 22 November 2000; accepted for publication 30 December 2000 2001 Academic Press 1043–4666/01/050287+08 $35.00/0 KEY WORDS: chemotherapy/interleukin 11/megakaryopoietic kinetics/mice/thrombocytopenia CYTOKINE, Vol. 13, No. 5 (7 March), 2001: pp 287–294
(SCF).5,8 More recently, it has been reported that rhIL-11 acts on the early stages of megakaryocyte development.5,6 Subcutaneous administration of IL-11 increases platelet counts as well as bone marrow CFUMeg number and megakaryocyte ploidy in normal mice,9 and increases platelet counts associated with the increase in megakaryocyte size in non-human primates.10 The effect of rhIL-11 has also been studied in several myelosuppressed animals.11–14 Thus, in sublethal irradiation and carboplatin-induced myelosuppressed mice, administration of rhIL-11 increases the number of CFU-Meg, which is reflected in the peripheral circulation by a reduction of platelet nadir and a significantly reduced period of thrombocytopenia.11 In addition, rhIL-11 accelerated reconstitution of myeloid progenitors from bone marrow when rhIL-11 increased peripheral platelet and neutrophil counts in BCNU- or cyclophosphamide-induced myelosuppressed mice.12,14 RhIL-11 also accelerated the recovery of periopheral neutrophil and platelet counts in bone marrow and spleen cell transplant mice.15 In clinical trials, rhIL-11 has shortened the duration of thrombocytopenia and decreased the need for platelet transfusion in patients receiving 287
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RESULTS Effect on circulating blood cells MMC injection caused rapid decreases in platelet counts, reaching a nadir of 24104/l 12 days after treatment, followed by gradual recovery, but not to normal levels by day 26 (Fig. 1A). Treatment with rhIL-11 significantly improved the nadir (34104/l, on day 9) and accelerated platelet recovery compared with vehicle-treated animals, reaching normal levels by day 23. Treatment with MMC also resulted in a decrease in the absolute neutrophil count (ANC) (Fig. 1B). The decrease in ANC was sustained from day 5 to 18, and thereafter ANC recovered gradually to reach a normal level by day 26. RhIL-11 slightly but significantly attenuated the severity of neutropenia and accelerated the recovery of ANC. The number of other blood cells, e.g., erythrocytes, lymphocytes and monocytes, were not markedly affected by MMC treatment, and rhIL-11 did not significantly affect the counts of these cells (data not shown).
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chemotherapy,16–18 proving that rhIL-11 treatment is effective in ameliorating thrombocytopenia induced by chemotherapy. In these studies, however, the effects of rhIL-11 on both proliferation and maturation of megakaryocyte lineage cells, i.e., megakaryocytes and their progenitor number and megakaryocyte ploidy, have not been evaluated in detail. Moreover, kinetic analysis of the stimulatory effect of rhIL-11 on these parameters of megakaryopoiesis has not been made, but should be useful for clarifying the role of IL-11 in megakaryopoiesis. In the present study, therefore, the effect of rhIL-11 on bone marrow CFU-Meg, megakaryocyte number and megakaryocyte ploidy was examined chronologically in myelosuppressed animals. Mice kept on a myelosuppressive regimen of mytomycin C (MMC) were used as the myelosuppressed model.
CYTOKINE, Vol. 13, No. 5 (7 March, 2001: 287–294)
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Figure 1. The effects of rhIL-11 on peripheral (A) platelet and (B) absolute neutrophil counts (ANC) in MMC-treated mice. RhIL-11 was given subcutaneously to mice at a dose of 500 g/kg/ day from 1 day after second dosing of MMC (2 mg/kg iv; day 1) to day 21. Each symbol represents the meanSEM from ten animals except where indicated in parenthesis. * and ** show significant differences between the vehicle- and rhIL-11-treated groups (*P<0.05, **P<0.01, Student’s t-test). Normal, — —; vehicle, — —; rhIl-11, — —.
Effect on bone marrow megakaryopoiesis
Number of megakaryocytes MMC decreased the megakaryocyte number on day 4, followed by a nadir, 4.7% of normal, on day 9 (Fig. 3). The megakaryocyte number recovered gradually, but was still at a low level on day 20. Although no significant difference in megakaryocyte number between vehicle- and rhIL-11-treated animals was found on day 14, rhIL-11 significantly increased the number of megakaryocytes on day 20.
Number of CFU-Meg Treatment with MMC led to a dramatic decrease in CFU-Meg number in bone marrow cells on day 4, which preceded the expression of severe thrombocytopenia at least by one day (Fig. 2). Thereafter, the number of CFU-Meg gradually recovered, but did not reach normal levels by day 20. In the rhIL-11 group, there was a significant increase or a tendency to an increase in the number of CFU-Meg in comparison with vehicle-treated animals.
Megakaryocyte ploidy Normal mice had a modal megakaryocyte ploidy of 16N (50.5% to 51.3% of megakaryocytes) with 22.2% to 24.1% of megakaryocytes in the 8N ploidy class and 11.7% to 14.9% at 32N (Fig. 4). The effect of MMC treatment by itself on the megakaryocyte ploidy distribution was complex. Namely, there was a clear decrease in 16N and a simultaneous increase in 2N and 4N megakaryocytes on day 4. On day 9, the modal ploidy class shifted from 16N to 2N and the
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Figure 2.
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Bone marrow cells harvested from bone marrow on days 4, 9, 14 and 20 were cultured for 7 days, and the number of CFU-Meg then formed was counted. The data represent the meanSEM from eight animals. ** shows a statistical significance between normal and vehicle-treated groups (P<0.01, Student’s t-test). # and ## show a statistical significance between vehicle- and rhIL-11-treated groups (#P<0.05, ##P<0.01, Student’s t-test). Normal, ; vehicle, ; rhIL-11, .
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The number of bone marrow megakaryocytes was determined on days 4, 9, 14 and 20. The data represent the meanSEM from eight to nine animals. ** shows a statistical significance between normal and vehicle-treated groups (P<0.01, Student’s t-test). # shows a statistical significance between vehicle- and rhIL-11-treated groups (P<0.05, Student’s t-test). Normal, ; vehicle, ; rhIL-11, .
distribution pattern was flattened. Thereafter, the modal ploidy class shift to higher ploidy (32N or more) megakaryocytes was shown on days 14 and 20. Treatment with rhIL-11 significantly increased the population of 32N and 64N megakaryocytes on day 4, and that of 32N megakaryocytes on day 14, in comparison with vehicle-treated mice. The distribution pattern in rhIL-11-treated mice was similar to that in vehicle-treated mice on days 9 and 20.
DISCUSSION The effects of rhIL-11 on chemotherapy- or irradiation-induced thrombocytopenia have been investigated. Results demonstrated amelioration of the nadir and accerelation of recovery of thrombocytopenia in pre-clinical studies10–14 as well as a decrease in the need for platelet transfusion in clinical studies.16–18 In the present study, rhIL-11 attenuated the nadir of platelet counts and accelerated the recovery of platelet counts in MMC-induced myelosuppressed mice.
Therefore, the present study confirmed that rhIL-11 ameliorated the depth and duration of thrombocytopenia in various chemotherapy-induced myelosuppressed models. To elucidate the mechanism of the stimulatory effect of rhIL-11 on megakaryopoiesis in a myelosuppressed condition, we quantitated chronological changes in the number of CFU-Megs, megakaryocytes, and megakaryocyte ploidy from bone marrow cells in MMC-treated mice over 20 days after administration of rhIL-11. Treatment with rhIL-11 stimulated megakaryocyte ploidy and CFU-Megs but not megakaryocyte numbers on day 4, suggesting that rhIL-11 exerted its stimulatory effect on megakaryocyte maturation and on the recruitment of progenitors to megakaryocyte lineage at around the same time, which is significantly earlier than that on proliferation of megakaryocytic cells. On day 9, rhIL-11 increased CFU-Meg numbers, but not megakaryocyte number or ploidy. Increases in CFU-Meg number and megakaryocyte ploidy were observed in rhIL-11-treated mice on day 14, but these increases were not detected
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Megakaryocyte ploidy changes in MMC-treated mice that received vehicle and rhIL-11.
Megakaryocyte ploidy was determined by flow cytometer in a double-staining experiment, as described in Materials and Methods. For each measurement, the number of Pm1 + cells was counted in the range between 400 and 1500, and the percentage of cells in each ploidy class vs the total megakaryocyte count was determined. The data represent the meanSEM from seven animals except for the vehicle-treated group on day 9. In the vehicle-treated group on day 9, the frequency of Pm1 + cells in the total of bone marrow cells from one animal was too low to evaluate the megakaryocyte ploidy. Thus, this sample was excluded from the evaluation. * and ** show the statistical significance, for each ploidy class, of the difference between normal and vehicle-treated groups (*P<0.05, **P<0.01, Student’s t-test). # and ## show the statistical significance, for each ploidy class, of the difference between vehicle- and rhIL-11-treated groups (#P<0.05, ##P<0.01, Student’s t-test).
Megakaryopoietic kinetics of rhIL-11 in myelosuppressed mice / 291
on day 20. An increase in megakaryocyte number in rhIL-11-treated animals was detected only on day 20. The increase in megakaryocyte number thus appears to reflect an earlier increase in CFU-Meg number. Taken together, these findings indicate that rhIL-11 exerted a stimulatory effect on the endomitosis of megakaryocytes early in the dosing period as well as the commitment to the megakaryocyte lineage, and thereafter stimulated both proliferation and maturation. Support for this interpretation is provided by studies in normal mice,9 in which rhIL-11 increased megakaryocyte progenitors and stimulated endomitosis of bone marrow megakaryocytes but not bone marrow megakaryocyte frequences. In normal dogs, also, treatment with rhIL-11 increased ploidy number of megakaryocytes in 6/6 adequately evaluated animals, but increased numbers of megakaryocytes in only 3/8,19 demonstrating that rhIL-11 preferentially stimulates endomitosis of megakaryopoiesis rather than proliferation. Further, the increases in marrow and peripheral blood colony-forming units-granulocyte and macrophage (CFU-GM) were obtained in the study using normal dogs.19 Previous studies have demonstrated the effect of rhIL-11 on the recruitment of progenitors to megakaryocyte lineage. Weich et al.6 note that rhIL-11 has the potential to promote murine megakaryocyte development via effects on bone marrow primitive progenitor cells and early and late progenitor cells. RhIL-11 has also been shown to act on human progenitor hematopoietic cells, where it induces an increase in the number of CFU cells derived from CD34 + and CD34 + CD33 DR cells.20,21 Further, an increase in the commitment of stem cells into a multipotential progenitor subpopulation was observed when rhIL-11 was added to human or murine longterm bone marrow cultures.22 These in vitro observations are in agreement with several in vivo studies. RhIL-11 increases the number of CFU-Megs, and the number of multipotential colony-forming unit granulocyte, erythroid, macrophage and megakaryocyte (CFU-GEMM) progenitor cells during the platelet recovery period in carboplatin- and irradiationtreated mice,11 and BCNU-treated mice,14 respectively. Furthermore, rhIL-11 increases the number of CFUGEMM a little earlier than a thrombopoietic effect appears in cyclophosphamide-treated mice.12 When administered to humans, rhIL-11 stimulates all stages of megakaryopoiesis and, in addition, stimulates precursor cells of different marrow lineages without affecting the number of assayable progenitor cells.23 Our findings that rhIL-11 has an effect on the commitment of stem cells to the megakaryocyte lineage in the early period reflects these findings, and can be considered to represent one of the pluripotent effects of this cytokine on haematopoietic stem cells.
Thrombopoietin (TPO) is a potent stimulant of megakaryopoiesis.24,25 Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF), a truncated molecule of rhTPO derivatized with polyethylene glycol, has been investigated in MMC-induced thrombocytopenic mice, and has been found to inhibit thrombocytopenia dosedependently.26 PEG-rHuMGDF has also been reported to stimulate the recovery of the number of CFU-Megs and of megakaryocytes from 5 and 8 days after the MMC treatment, respectively. In the present study, rhIL-11 four days after MMC treatment stimulated the megakaryocyte endomitosis and the recovery of the number of CFU-Megs that preceded the proliferation of megakaryocytes on day 20. Therefore, it is suggested that two cytokines, rhIL-11 and PEG-rHuMGDF, stimulate the megakaryocyte progenitors in the early period of dosing, with differences in the onset times of the effects on megakaryocyte proliferation. Care is needed in comparing the effects of the two cytokines, because experimental conditions were different, i.e. the dose of MMC. The possibility exists, however, that IL-11 and TPO have distinct effects on megakaryopoiesis. RhIL-11, in combination with IL-3 or SCF, acts synergistically in stimulating human and murine megakaryocyte progenitor-derived colony formation.20,27 RhIL-11 plus IL-3 increased megakaryocyte number and acetylcholinesterase production in murine bone marrow liquid culture compared with IL-3 alone.2 Acting as a single agent, rhIL-11 enhanced the size and ploidy of human and murine bone marrow megakaryocytes.2,3,28 In an in vivo experiment, rhIL-11 increased megakaryocyte progenitors and stimulated endomitosis of bone marrow megakaryocytes, but did not affect bone marrow megakaryocyte frequencies,9 these results being consistent with our results in MMC-treated mice. Hence, rhIL-11 likely has a stimulating effect on megakaryocyte endomitosis and commitment rather than on proliferation. On the other hand, TPO, as a single agent, supports the formation of megakaryocyte colonies and the endomitosis of megakaryocytes.24,25,28,29 A single injection of pegylated murine MGDF acted on megakaryocytic proliferation as well as on megakaryocytic endomitosis in normal mice.30 Consequently, differences may exist in the effect of the two cytokines on megakaryocyte proliferation, and this may influence the onset time of megakaryopoiesis. In addition to the effect on thrombocytopenia, administration of rhIL-11 improved the neutropenia induced by MMC treatment. RhIL-11 stimulates the proliferation of granulocyte progenitor cells in vitro.31,32 Moreover, in in vivo studies rhIL-11 increases ANC in bone marrow-transplanted mice and cyclophosphamide-induced myelosuppressed mice,12,15 while it does not affect ANC in carboplatin- or
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irradiation-induced myelosuppressed mice.11 It is suggested that rhIL-11 has a proliferating effect on ANC by stimulating granulocytopoiesis, but the effect appears to depend on experimental conditions or models. In conclusion, rhIL-11 significantly ameliorated the severity of thrombocytopenia in mice with MMCinduced myelosuppression in the present study. The effect was confirmed to reflect the stimulation by rhIL-11 of both the maturation and commitment to the megakaryocyte lineage, followed by the increase in the megakaryocytes in vivo.
MATERIALS AND METHODS Animals Female Balb/c mice at 7 to 8 weeks of age (17–22 g) were purchased from Japan SLC, Inc. (Shizuoka, Japan). They were housed in autoclaved cages in specific pathogen-free rooms and given sterilized commercial rodent chow and water ad libitum. The animal room was conditioned to 21–25C with a relative humidity of 50–60% and was lit on a 13/11 h light/dark light cycle (7:30 to 20:30). All experiments were approved by the Animal Ethical Committee of Yamanouchi Pharmaceutical Co., Ltd.
RhIL-11 RhIL-11 purified to homogeneity from Escherichia coli was obtained from Genetics Institute (Cambridge, MA, USA). The specific bioactivity of rhIL-11 was estimated to be 1.3106 units/mg of protein using a bioassay employing the murine plasmacytoma cell line, T10. RhIL-11 was dissolved in a solution containing 0.004 M sodium dihydrogenphosphate dihydrate, 0.006 M disodium hydrogenphosphate,12H2O, 0.3 M glycine and 0.01% (w/v) polyoxyethylene (20) sorbitan monooleate. This solution, except for the rhIL-11, was used as vehicle.
Thrombocytopenic model A modification of the method described by others26 was used to construct a model of MMC (Kyowa Hakko Kogo Co., Tokyo, Japan)-induced myelosuppression. Mice were given a dose of 2 mg/kg of MMC intravenously on days 1 and 0 to induce severe thrombocytopenia without any animal deaths (see Fig. 1). Subcutaneous treatment of mice with rhIL-11 at a dose of 500 g/kg/day was initiated on day 1 for 21 days.
Peripheral haematology Mice were anesthetized with ether, and peripheral blood was drawn from the inferior vena cava on days 1 (for the normal group), 5, 9, 12, 14, 16, 18, 20, 23 and 26. Blood cell count analysis was performed using a Celltac MEK-5158 (Nihon Kohden, Tokyo, Japan). White blood cell differentials were determined on blood smear preparations stained with May-Giemsa.
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Progenitor cell assay CFU-Meg colony assay was performed as previously described9 with a minor modification. Briefly, bone marrow cells were harvested from mice by flushing the femoral contents on days 4, 9, 14 and 20 with Iscove’s modified Dulbecco’s medium (IMDM; GIBCO BRL, Life Technologies, Inc., Rockville, MD, USA) containing 2% fetal bovine serum (FBS; GIBCO BRL, Life Technologies, Inc.). Cells (4105 cells) were suspended in 2.7 ml IMDM supplemented with 3 U/ml recombinant murine IL-3 (rmIL-3; Genzyme Diagnostics, Cambridge, MA, USA), 60 ng/ml rhIL-11 and 10% FBS and preincubated at 37C. Thereafter, the cell suspension was mixed with 300 l of 3.3% agar autoclaved and preincubated at 42C, followed by the addition of 750 l of mixture in two wells per chamber slide (Nalge Nunc International, Naperville, IL, USA). After incubation of the slides at 4C for 20 min to solidify the culture, the chamber slides were incubated for 7 days at 37C in a humidified atmosphere of 5% CO2 in air. The cells were then fixed with 5% glutaraldehyde for 10 min and subjected to histochemical staining for acetylcholinesterase (AchE) using 75 mM phosphate buffer containing 500 g/ml acetylthiocholine iodide (Sigma-Aldrich, St.Louis, MO, USA), 5 mM sodium citrate, 3 mM copper sulfate and 0.5 mM potassium ferricyanide. Megakaryocyte colonies were counted using an inverted microscope. CFU-Meg was defined as comprising three or more AchE + cells. All measurements were carried out in duplicate in each experiment, and the total number of CFU-Megs per femur was calculated as follows: the number of CFU-Megs per well was multiplied by the total number of cells obtained from one femur, which was divided by the number of cells plated per well.
Measurement of the number of megakaryocytes in the femur Bone marrow cells were fixed at 1105 cells per well in 50 l of 7.5% glutaraldehyde in 96-well culture plates for 10 min at room temperature and recovered by centrifugation. The cells were stained for AchE as described above, and the number of AchE + cells was counted as that of megakaryocytes. All measurements were carried out in duplicate in each experiment, and the total number of megakaryocytes per femur was calculated as follows: the number of AchE + megakaryocytes per well was multiplied by total number of cells obtained from one femur which was divided by the number of cells plated per well.
Measurement of megakaryocyte DNA content The experiment was performed according to a previously described method33 with a minor modifications. Briefly, bone marrow cells were harvested from mice by flushing the femoral contents with CATCH solution [Hank’s balanced salt solution (HBSS, GIBCO BRL) containing 0.38% sodium citrate, 1 mM adenosine, 2 mM theophylline and 3.5% bovine serum albumin (BSA, Sigma-Aldrich)]. The cells were incubated with 25 g/ml of murine megakaryocytespecific antibody Pm1,34 which was kindly provided by Dr Nagasawa (University of Tsukuba, Ibaraki, Japan), or 25 g/ ml of control rat IgG antibody (Pharmingen, San Diego, CA,
Megakaryopoietic kinetics of rhIL-11 in myelosuppressed mice / 293
USA) at 4C for 20 min. The cells were washed once and further labeled with 28 g/ml FITC-conjugated goat-anti-rat IgG (Immunotech, Marseille, France) at 4C for 20 min. The cells were washed, incubated in 1.8 ml CATCH containing 0.5% Tween-20 for 40 min at 4C, 200 l 5% paraformaldehyde solution was added, and the mixture was then incubated at 4C for an additional 5 min. For nuclear DNA labeling, the cells were washed and suspended in 0.1% sodium citrate buffer supplemented with 50 g/ml propidium iodide (SigmaAldrich) and 1% BSA. The suspension was stored overnight in the dark at 4C. The following day the cells were washed and incubated with 5 g/ml DNase-free RNase (Boehringer Mannheim, Indianapolis, IN, USA) at room temperature for 30 min. The DNA content of megakaryocytes was determined by two-colour flow cytometric analysis using a flow cytometer (XL-MCL, Coulter, Miami, FL, USA). The DNA content of the cells labeled with the Pm1 antibody was analyzed by setting markers at the nadirs between the propidium iodide peaks. At least 400 Pm1 + cells were analyzed in each measurement.
Statistical analysis The results are expressed as the meanSEM of one determination from each of seven to ten mice. Experiments were repeated several times to yield the animal number of seven to ten. Significant differences were tested by Student’s t-test. P<0.05 was considered significant.
Acknowledgement We are grateful to Dr T. Nagasawa of University of Tsukuba for helpful suggestions during this study and for providing Pm1 antibody.
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