Stem Cells, Vol. 15, No. 1, 50-55,
January 1997
© 1997 AlphaMed Press
Leukemia Inhibitory Factor Induces In Vivo Expansion of Bone Marrow Progenitor Cells that Accelerate Hematopoietic Reconstitution but Do Not Enhance Radioprotection in Lethally Irradiated Mice
Johannes F.M. Pruijta,
Ivan J.D. Lindleyb,
Diana P.M. Heemskerka,
Roel Willemzea,
Willem E. Fibbea
a Laboratory of Experimental Hematology, Department of Hematology, Leiden University Hospital, The Netherlands;
b Sandoz Forschungsinstitut, Vienna, Austria
Key Words. LIF • Hematopoietic progenitor cells • Bone marrow transplantation • Hematopoietic reconstitution
Dr. Johannes F.M. Pruijt, Department of Hematology, Leiden University Hospital, Building 1: C2-R, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
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Abstract
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Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with distinct hematopoietic activities. In vivo treatment of mice with recombinant murine LIF induces thrombocytosis and increases the number of hematopoietic progenitor cells (HPCs) in spleen and bone marrow (BM). In this study, we applied LIF to expand HPCs in vivo prior to syngeneic BM transplantation. BALB/c donor mice were treated with recombinant human LIF at a dose of 2.5 µg/day s.c. for seven days. This resulted in a 1.6-fold increment in platelet counts from 941 to 1,470 x 109/l (mean, n = 20). Mean spleen weight increased from 120 mg to 160 mg (n = 5). The total numbers of HPCs in the spleen as well as in the BM, as assessed in a CFU-GM (colony forming unit-granulocyte-macrophage) assay, were significantly higher in LIF-treated donors than in saline-treated controls (30.1 ± 14.5 versus 7.4 ± 5.3 x 103 per spleen; mean ± SD, n = 22, p < 0.001 and 74.4 ± 17.1 versus 55.3 ± 16.1 x 103 per femur, p < 0.001). Recipient mice were lethally (8.5 Gy) irradiated and transplanted with 3 x 105 BM cells derived from LIF- or saline-treated donors. Hematopoietic reconstitution was monitored by tail bleeding at three-day intervals. Platelet and WBC nadir counts in control animals were reached at day 9 (31 ± 25 x 109/l for platelets and 0.40 ± 0.10 x 109/l for WBC; mean ± SD, n = 29 per treatment group); in animals transplanted with LIF-treated BM cells, these counts were 44 ± 25 x 109/l for platelets, p < 0.05 and 0.60 ± 0.38 x 109/l for WBC, p < 0.01. In addition, platelet reconstitution was faster in recipients of LIF-treated BM cells (226 ± 118 versus 126 ± 62 x 109/l at day 12 and 633 ± 174 versus 434 ± 180 x 109/l at day 15, p < 0.001). Similarly, the reconstitution of WBC was also significantly enhanced. The radioprotection rate of lethally irradiated recipients with increasing cell doses of BM cells derived from LIF-treated donors was higher at all cell doses tested then of control animals, but did not reach statistical significance. These results show that in vivo treatment with LIF expands the number of committed progenitor cells and BM repopulating cells that accelerate short-term hematopoietic reconstitution without increasing radioprotection. Our data do not support a major role for LIF as a single factor inducing expansion of hematopoietic stem cells in vivo.
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Introduction
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Leukemia inhibitory factor (LIF) is a pleiotropic cytokine with a wide spectrum of biologic activities. It was initially described and purified on the basis of its ability to suppress proliferation and induce differentiation of the murine myeloid leukemic cell line M1 [1, 2]. LIF receptors have been demonstrated on monocytes, macrophages, megakaryocytes, a small subset of lymphocytes and various nonhematopoietic cells [3, 4].
Several reports indicate that LIF may be involved in the regulation and/or proliferation of primitive hematopoietic stem cells (HSCs). In vitro, LIF has differentiation-inducing effects on some murine and human leukemic cell lines, and proliferative effects on others [1, 5-7]. Furthermore, LIF prevents differentiation commitment with maintenance of totipotentiality of murine embryonic stem cells [8]. As a single factor, LIF shows no direct colony-stimulating activity in semisolid as well as long-term cultures of normal bone marrow (BM) [9, 10]. However, LIF enhances the growth of colony-forming unit-megakaryocyte (CFU-Meg) in the presence of interleukin 3 (IL-3) in mice [11]. In man, it stimulates IL-3-dependent growth of primitive HSCs [12, 13]. LIF-deficient mice have dramatically decreased numbers of stem cells in the BM and spleen, which can be restored by administration of LIF. In addition, these animals exhibit reduced numbers of committed progenitor cells in the spleen but not in the BM [14]. Normal mice treated with LIF show thrombocytosis and an increase in HPCs in the BM and spleen [15]. In nonhuman primates, administration of LIF results in an increase in blood platelet counts [16]. Furthermore, it decreases the duration of thrombocytopenia and increases the neutrophil nadir in rhesus monkeys with radiation-induced marrow aplasia [17].
Summarizing, despite its lack of direct proliferative effects as a single factor, LIF has significant potentiating effects on the proliferation of primitive hematopoietic progenitor cells (HPC) in vitro [12, 13]. Moreover, LIF accelerated early hematopoietic regeneration after sublethal irradiation. These results indicate that LIF may be involved in the regulation of proliferation of primitive HPCs. In the present study, we therefore administered LIF to donor mice in an attempt to expand the number of HPCs. Using a syngeneic mouse transplant model, we studied the effect of these in vivo modified BM cells on hematopoietic reconstitution.
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Materials and Methods
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Mice
BALB/c mice with ages ranging between 8-12 weeks, were purchased from Broekman BV (Someren, The Netherlands). Donor animals were fed commercial rodent chow and acidified water ad libitum. Recipient animals were maintained in a pathogen-free environment and fed water containing ciprofloxacin 1 mg/ml (Bayer Nederland BV; Mijdrecht, The Netherlands), polymyxin-B 70 µg/ml and saccharose 2 g/100 ml. In transplantation experiments, recipient mice were placed in a polymethylmetaacetate box and given TBI (8.5 Gy, Philips SL 75-5/6 mV linear accelerator; Philips Medical Systems; Best, The Netherlands), divided in two parts in posterior-anterior and anterior-posterior position at a dose rate of 4 Gy/min. In reconstitution experiments, a fixed number of 3 x 105 unseparated BM cells, derived from LIF- or saline-pretreated donor mice, were injected in the tail vein of lethally irradiated recipients. In survival experiments, an increasing number of 3 x 103, 1 x 104 and 3 x 104 unseparated BM cells were injected.
Source of LIF
Recombinant E. coli-derived human LIF was provided by Sandoz Pharma AG (Basel, Switzerland). The in vitro biologic activity of the highly purified recombinant human LIF analyzed in a proliferation assay of the DA1 murine leukemic cell line was 11.8 x 10–6 LU/mg protein. The endotoxin content was below 60 pg/ml as determined by the Limulus amoebocyte lysate assay. For in vivo experiments, LIF was diluted to the desired concentration in endotoxin-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin. Two hundred microliters of the LIF solution or PBS was administered s.c. to the donor animals once daily over a period of seven days.
Osmotic Pumps
Mini-osmotic pumps (Alza Co.; Palo Alto, CA) with a delivery rate of 1 or 0.5 µl/h during one or two weeks, respectively, were filled with the desired LIF solution or saline alone. They were implanted s.c. on the dorsal side of the mice under general anesthesia. The skin was closed with N-butyl-2-cyanoacrylate monomer (HistoacrylR; B. BraunMelsungen AG; Melsungen, Germany). Anesthesia was performed with 2-xylidino dihydro thiazine-hydrochloride (RompunR; Bayer; Leverkusen, Germany) 5 mg/kg, and ketamine (Aesculaap B.V.; Boxtel, The Netherlands) 750 mg/kg.
Peripheral Blood Cell Counts
Peripheral blood from donor animals was obtained by intracardiac puncture, and cell counts were performed on a Sysmex® F800 (TOA Medical Electronics Co. Ltd.; Kobe, Japan). Peripheral blood samples of each recipient were obtained sequentially every third day by means of a tail-vein incision, and the total counts of RBCs, WBCs and platelets were determined.
Preparation of Cell Suspensions
Donor mice were killed by CO2 asphyxiation, and BM cells were obtained by flushing the femur under sterile conditions with RPMI 1640 containing 500 µg/ml penicillin, 250 µg/ml streptomycin and 5% fetal bovine serum (FBS) (GIBCO; Grand Island, NY). Single cell suspensions of spleen cells were prepared by mashing the spleen and washing once in RPMI 1640 with 5% FBS. Blood-derived mononuclear cell suspensions were obtained by Ficoll separation as described earlier [18].
Progenitor Cell Assays
CFUs-GM were cultured as described previously [18]. Briefly, BM cells were cultured in microtiter plates containing 105 cells per milliliter in semisolid medium in the presence of murine GM-CSF (1.25 ng/ml). Peripheral blood mononuclear cells and spleen cells were cultured in 3.5 cm dishes containing 5 x 105 and 5 x 106 cells per milliliter semisolid medium, respectively. After six days of culture in a fully humidified atmosphere of 37°C containing 5% CO2, the number of colonies (defined as aggregates of >20 cells) were scored using an inverted microscope.
Statistical Analysis
Differences were evaluated using the Student's t-test. In the ANOVA analysis, groups were compared with respect to their course over time. The analysis was performed on the log values of the data. Values of less than 0.05 are considered statistically significant.
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Results
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Effect of LIF on Donor Animals
Peripheral Blood Cell Counts
In order to establish the optimal dose, we first applied continuous administration of LIF with different dosing schedules (ranging from 1.25 to 6.25 µg/day for 5 to 14 days). The level of thrombocytosis was used as a readout. A similar increment in platelet counts was seen in all dosage regimens used. Thrombocytosis started at day 5 and was maximal at day 7 after start of the infusion, with an increase of the platelet counts of approximately 1.5-fold (data not shown). Subcutaneous administration of LIF at a dose of 2.5 µg/day for seven days resulted in a similar 1.5-fold increase in platelet counts. In subsequent experiments, mice were treated with either LIF (2.5 µg per day) or saline, administered s.c. for seven days. The mice were then killed and blood was obtained by cardiac puncture. The number of platelets in LIF-treated animals was increased compared to the saline-treated controls (1470 ± 376 x 109/l versus 941 ± 118 x 109/l, n = 22 in five experiments for each group, p < 0.001). The number of WBCs or erythrocytes was not significantly different (Table 1
). No reduction in body weight or behavioral changes were seen during LIF treatment.
Effect of LIF Treatment on Numbers of Progenitor Cells in the Hematopoietic Organs
To study the hematopoietic effects of LIF in more detail, we determined the numbers of nucleated cells and progenitor cells in the BM, spleen and peripheral blood of animals treated with LIF s.c. for seven days. Saline-treated animals served as controls. The numbers of progenitor cells as determined in the CFU-GM assay increased from 55,263 ± 16,132 to 74,412 ± 17,136 per femur (mean ± SD, n = 22 per treatment group, p < 0.001), whereas the total number of nucleated cells was not different. All LIF-treated animals developed splenomegaly reflected by an increase in median weight from 120 to 160 mg (n = 5) and also by a significant increment in the total number of nucleated cells from 150 ± 43 x 106 to 214 ± 40 x 106 (mean ± SD, n = 22 per treatment group, p < 0.001). In addition, a fourfold increase in the number of progenitor cells per spleen (30,127 ± 14,463 versus 7,358 ± 5,293, mean ± SD, n = 22, p < 0.001) was observed (Table 2). No significant difference was found in the number of circulating progenitor cells.
Hematopoietic Reconstitution following Transplantation of In Vivo Modified BM Cells
Lethally irradiated recipient animals were transplanted with 3 x 105 BM cells from donors that had been pretreated with LIF at a dose of 2.5 µg/day s.c. for seven consecutive days. The reconstitution of platelets in these recipients was significantly accelerated in comparison to control animals transplanted with an equal number of BM cells derived from saline-treated control donors (226 ± 118 versus 126 ± 62 x 109/l at day 12 and 633 ± 174 versus 434 ± 180 x 109/l at day 15; mean of 29 mice per treatment group in three experiments, p < 0.001, Fig. 1). Platelet and WBC nadir counts in control animals were reached at day 9 (31 ± 25 x 109/l for platelets and 0.40 ± 0.10 x 109/l for WBC; mean ± SD, n = 29). In contrast, platelet and WBC counts at day 9 in animals transplanted with LIF-treated BM cells were 44 ± 25 x 109/l for platelets, p < 0.05, Figure 1, and 0.60 ± 0.38 x 109/l for WBC, p < 0.01, Figure 2; mean ± SD, n = 29. Furthermore, the reconstitution rate of WBCs was significantly enhanced (p < 0.01, Fig. 2). No effect on the reconstitution of erythrocytes was observed (data not shown).
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Figure 1. Transplantation of LIF-modified BM cells accelerates platelet reconstitution. Recipient mice were lethally irradiated (8.5 Gy) and transplanted with 3 x 105 BM cells derived from donor animals treated with LIF (2.5 µg/d/s.c.) or saline for seven days. Data are presented as mean of 30 mice in three experiments. *p < .05; **p < .001. Distribution width of the curves: p < .001.
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Figure 2. Transplantation of LIF-modified BM cells accelerates WBC reconstitution. Recipient mice were lethally irradiated (8.5 Gy) and transplanted with 3 x 105 BM cells derived from donor animals treated with LIF (2.5 µg/d/s.c.) or saline for seven days. Data are presented as mean of 30 mice in three experiments. *p < .05. Distribution width of the curves: p < .001.
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Radioprotective Capacity of LIF-Treated BM Cells
To study the radioprotective capacity of LIF-treated BM cells, recipient mice were lethally-irradiated (8.5 Gy), and transplanted with 3 x 103, 104 or 3 x 104 BM cells derived from LIF-treated donors or saline-treated controls. Cell doses were chosen to result in survival rates of approximately 20%, 50% and 90% for recipients receiving unmodified BM cells [19]. At all cell doses tested, the radioprotection rate of lethally-irradiated recipients of BM cells obtained from LIF-pretreated donor animals was higher than that of control animals transplanted with equal numbers of unmodified BM cells (three experiments, n = 30 per cell dose per treatment condition, Fig. 3), although the differences did not reach statistical significance.
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Figure 3. Radioprotection rate after transplantation of increasing cell numbers of 3 x 103, 104 and 3 x 104 BM cells derived from donors treated with LIF (2.5 µg/d/s.c.) or saline for seven days. Survival data are expressed as absolute percentages of three experiments with 10 mice transplanted per cell dose in each experiment.
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Discussion
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In the present study, we have expanded BM cells by LIF treatment of donor mice. Using a transplant model, we studied the repopulating ability of these cells. Administration of LIF for seven days to healthy donor mice resulted in a 50% increase in the number of circulating platelets. This increment is in agreement with earlier reports [16]. The hemoglobin level and total WBC counts were unaltered. In comparison with saline-treated controls, LIF-treated mice had significantly increased numbers of HPCs in the BM and spleen without a concomitant decrease in marrow cellularity. Metcalf et al. showed an increase in HPCs, in particular megakaryocytic progenitor cells, mainly in the spleen, but also in the BM [16]. However, due to a decrease in marrow cellularity, the total progenitor cell numbers in the BM were essentially unaltered. The discrepancy in HPC numbers in the BM may be related to differences in the dosage regimens. Their initial regimen was 2 µg of LIF i.p. three times daily for 14 days, which appeared to be close to the toxic limit. Lower doses of LIF were still able to induce changes in megakaryocytes and platelets without toxic effects, but the effect on HPC numbers was not reported. At the reported high-dose level, the mice showed weight loss, thymus atrophy, hypermobility and irritability, and death occurred in the smallest mice. These findings were consistent with initial studies from the same group in which mice, transplanted with FDC-P1 cells engineered to produce high LIF levels, developed a fatal syndrome with multiorgan pathology [20, 21]. We have applied a relatively low dose of LIF (2.5 µg/day/s.c.) that induced no toxic side effects, but still resulted in increased platelet counts and HPC numbers. Thus, the dose, route of administration and duration of treatment appear to be important factors determining the hematopoietic and toxic effects of LIF.
Transplantation of BM cells derived from LIF-treated donors into lethally irradiated recipients resulted in significantly accelerated platelet reconstitution and higher platelet nadir counts compared to recipients of unmodified BM cells. In addition, transplantation of in vivo modified BM cells also accelerated the reconstitution of WBCs. The radioprotective capacity of in vivo LIF-modified BM cells was slightly enhanced, albeit not statistically significant. Thus, LIF treatment of mice resulted in expansion of committed HPCs and BM repopulating cells. This increase appeared to be modest since transplantation of in vivo LIF-expanded BM cells resulted in accelerated hematopoietic reconstitution without a significant increase in radioprotection rate.
In vitro, LIF has been reported to promote proliferation of primitive HSCs only when used in combination with other cytokines known to stimulate primitive hematopoietic progenitors (i.e., IL-3, IL-6 and GM-CSF) [12, 13]. In vivo, normal mice treated with LIF showed an increase in the frequency of committed HPCs in the spleen and BM [15]. Furthermore, dramatically decreased numbers of stem cells in the BM and spleen have been found in LIF-deficient mice which could be restored by administration of exogenous LIF [14]. In the same study of Escary et al., primitive HSCs of LIF-deficient mice, although reduced in number, appeared to be functional since their long-term repopulating ability was similar to wild-type cells [14]. Thus, when used in combination with other cytokines, LIF induces proliferation of primitive HSCs in vitro and appears to be required for the survival of HSCs in vivo. However, no proliferative effects on primitive HSCs have been reported for LIF as a single agent.
Since we observed a significant effect on short-term hematopoietic reconstitution without a concomitant increase of radioprotection, our data do not support a major role in vivo for LIF as a single factor inducing expansion of HSCs. Combinations of LIF with other early-acting cytokines (i.e., IL-1, IL-3, IL-6, G-CSF and stem cell factor) that are capable of in vivo and/or ex vivo expansion of HSCs may further optimize the hematopoietic activities of LIF in the setting of stem cell transplantation.
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Acknowledgments
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The authors thank Arie Boon of the Department of Radiotherapy for his technical assistance in irradiating the animals and Peter de Jong of the Facilities for Laboratory Animals for his excellent animal care. This work was in part supported by the Sandoz Forschungsinstitut and the J.A. Cohen Institute for Radiopathology and Radiation Protection (IRS) (grant no. 3.4.16).
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Received June 10, 1996;
accepted for publication September 16, 1996.
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Abstract
Introduction
