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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1586-1597
A Single Dose of Thrombopoietin Shortly After Myelosuppressive Total
Body Irradiation Prevents Pancytopenia in Mice by Promoting Short-Term
Multilineage Spleen-Repopulating Cells at the Transient Expense of
Bone Marrow-Repopulating Cells
By
Karen J. Neelis,
Trudi P. Visser,
Wati Dimjati,
G. Roger Thomas,
Paul J. Fielder,
Duane Bloedow,
Dan L. Eaton, and
Gerard Wagemaker
From the Institute of Hematology, Erasmus University Rotterdam,
Rotterdam, The Netherlands; and Genentech Inc, South San Francisco, CA.
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ABSTRACT |
Thrombopoietin (TPO) has been used in preclinical myelosuppression
models to evaluate the effect on hematopoietic reconstitution. Here we
report the importance of dose and dose scheduling for multilineage
reconstitution after myelosuppressive total body irradiation (TBI) in
mice. After 6 Gy TBI, a dose of 0.3 µg TPO/mouse (12 µg/kg)
intraperitoneally (IP), 0 to 4 hours after TBI, prevented the severe
thrombopenia observed in control mice, and in addition stimulated red
and white blood cell regeneration. Time course studies showed a gradual
decline in efficacy after an optimum within the first hours after TBI,
accompanied by a replacement of the multilineage effects by lineage
dominant thrombopoietic stimulation. Pharmacokinetic data showed that
IP injection resulted in maximum plasma levels 2 hours after
administration. On the basis of the data, we inferred that a
substantial level of TPO was required at a critical time interval after
TBI to induce multilineage stimulation of residual bone marrow cells. A
more precise estimate of the effect of dose and dose timing was
provided by intravenous administration of TPO, which showed an optimum
immediately after TBI and a sharp decline in efficacy between a dose of
0.1 µg/mouse (4 µg/kg; plasma level 60 ng/mL), which was fully
effective, and a dose of 0.03 µg/mouse (1.2 µg/kg; plasma level 20 ng/mL), which was largely ineffective. This is consistent with a
threshold level of TPO required to overcome initial
c-mpl-mediated clearance and to reach sufficient plasma levels
for a maximum hematopoietic response. In mice exposed to fractionated
TBI (3 × 3 Gy, 24 hours apart), IP administration of 0.3 µg TPO 2 hours after each fraction completely prevented the severe thrombopenia
and anemia that occurred in control mice. Using short-term
transplantation assays, ie, colony-forming unit-spleen (CFU-S) day 13 (CFU-S-13) and the more immature cells with marrow repopulating ability
(MRA), it could be shown that TPO promoted CFU-S-13 and transiently
depleted MRA. The initial depletion of MRA in response to TPO was
replenished during long-term reconstitution followed for a period of 3 months. Apart from demonstrating again that MRA cells and CFU-S-13 are separate functional entities, the data thus showed that TPO promotes short-term multilineage repopulating cells at the expense of more immature ancestral cells, thereby preventing pancytopenia. The short
time interval available after TBI to exert these effects shows that TPO
is able to intervene in mechanisms that result in functional depletion
of its multilineage target cells shortly after TBI and emphasizes the
requirement of dose scheduling of TPO in keeping with these mechanisms
to obtain optimal clinical efficacy.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN (TPO), the ligand for the
cytokine receptor c-mpl, has been cloned and characterized in
19941-3 and shown to be the physiological regulator of
platelet production by generating mice deficient for either TPO or
c-mpl.4,5 Administration of pharmacological doses of TPO to
normal mice and nonhuman primates resulted in dose-dependent increases
in platelets, far exceeding that observed after administration of other
growth factors.1,2,6,7 These observations have led to the
pharmaceutical development of TPO as a therapeutic to counteract
thrombopenic states, such as those resulting from myelosuppression due
to cancer treatment. In myelosuppression models, TPO effectively
alleviated the nadir for platelets and accelerated recovery to normal
values.6,8-11 In several of those models, daily
administration during the pancytopenic phase resulted in an overshoot
in platelet counts to supranormal values.6,8,10,11 Although
in normal experimental animals the response to TPO was dominant along
the megakaryocytic lineage, in myelosuppression models multilineage
effects have been shown such as stimulation of erythroid
recovery,8,11-14 acceleration of immature progenitor cell
reconstitution in bone marrow,8,15 and augmentation of the
responses to granulocyte-macrophage colony-stimulating factor (GM-CSF)
and granulocyte colony-stimulating factor (G-CSF).8,16 We
and others have shown that a single dose of TPO administered 24 hours
after total body irradiation (TBI) was as effective in alleviating the
nadir for thrombocytes as daily dosing,13,16-18 thereby
reducing the need for thrombocyte transfusions in myelosuppressed nonhuman primates and accelerating recovery to normal platelet counts.
The effects on red cell regeneration and immature bone marrow
progenitor cells were retained with single dosing of
TPO.13,16 These results showed that the more conventional
dose schedules that have come into use for growth factors such as
GM-CSF and G-CSF were less appropriate for TPO.
Careful design of growth factor treatment protocols requires
preclinical experiments to gain insight into the mechanisms of action
and in the optimal dose and dose schedule to achieve maximum therapeutic benefit and reductions in the occurrence of side effects, as well as to prevent unnecessary treatment, thereby reducing costs.
Because a delay in the administration of TPO after cytoreductive treatment proved to be detrimental to its efficacy,18,19 we examined in detail the time dependence of TPO efficacy in the initial
12 hours after myelosuppressive TBI, with special emphasis on the
response of immature bone marrow cells and their progeny, to reveal
mechanisms of TPO action on immature repopulating cells important for
efficacy.
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MATERIALS AND METHODS |
Animals.
Female (C57BL × CBA)F1 (BCBA) mice, approximately 12 weeks of age,
were bred at the Experimental Animal Facility of Erasmus University
(Rotterdam, The Netherlands) and maintained under specific pathogen-free conditions. Housing, experiments, and all other conditions were approved by an ethical committee in accordance with
legal regulations in The Netherlands.
Experimental design.
TBI was administered at day 0 using two opposing 137Cs
sources (Gammacell 40; Atomic Energy of Canada, Ottawa, Canada) at a
dose rate between 0.92 and 0.94 Gy/min as described.20
Doses used were 6 Gy for single dose irradiation and a fractionated
dose of 9 Gy in three equal doses with 24-hour intervals. Mice were bled only once; for each data point a random experimental group of
three mice was killed. All parameters were collected for individual mice.
Test drug.
Recombinant full length murine TPO produced by Chinese hamster ovary
cells (Genentech Inc, South San Francisco, CA) was used throughout the
experiments, diluted in phosphate-buffered saline/0.01% Tween 20, and
administered intraperitoneally (IP) or intravenously (IV) in a volume
of 0.5 mL. The dose of TPO used was 0.3 µg/mouse (12 µg/kg, based
on a mean body weight of 25 g at the time of irradiation) unless
otherwise indicated. We have previously shown that this dose was
effective in a similar model for myelosuppression.13
Hematologic examinations.
After ether-anesthesia, the mice were bled by retro-orbital puncture
and killed by cervical dislocation. Blood was collected in EDTA tubes.
Complete blood cell counts were measured using a Sysmex F-800
hematology analyzer (Toa Medical Electronics Co, LTD, Kobe, Japan).
Phenotypic analysis of white blood cells.
For phenotypic analysis blood was collected in EDTA tubes. Samples from
three mice that received the same treatment were pooled to yield
sufficient numbers of cells. Red blood cells were removed by incubating
whole blood in lysing solution (8.26 g ammonium chloride/1.0 g
potassium bicarbonate and 0.037 g EDTA/L) for 10 minutes at 4°C.
After lysing, cells were washed twice with Hanks' buffered Hepes
solution (HHBS) containing 0.5% (vol/vol) bovine serum albumin (BSA;
Sigma, St Louis, MO), 0.05% (wt/vol) sodium azide, and 0.45% (wt/vol)
glucose (Merck, Darmstadt, Germany) (HBN). The cells were
resuspended in 50 µL HBN containing 4% (vol/vol) normal mouse serum
to prevent nonspecific binding of the monoclonal antibodies (MoAbs). To
detect neutrophilic granulocytes and monocytes the MoAb ER-MP20 (rat
IgG2a)21 was added in a volume of 50 µL. ER-MP20 bright cells are monocytes (corresponding with Mac-1-positive cells22) and cells staining intermediate with ER-MP20 are
granulocytes (corresponding with Gr-1-positive cells22).
To detect lymphocytes the anti-CD4 MoAb YTS 191 and the anti-CD8 MoAb
YTS 16923 (a kind gift from Dr H. Waldmann, Department of
Pathology, Cambridge University, UK) were added at a concentration of 2 µg/mL. Cells and MoAbs were incubated for 30 minutes on ice. After
two washes the cells were incubated with a fluorescein-labeled
goat-anti-rat MoAb. After another two washes, the cells were labeled
with propidium iodide and measured by flow cytometry. Ungated list mode
data were collected for 10,000 events and analyzed using Lysis II
software (Becton Dickinson, Mountain View, CA).
TPO levels.
Data for characterization plasma TPO pharmacokinetics were generated at
Genentech Inc as previously described.24 In short, mice
were injected IP with 125I-rmTPO either with a single dose
of 0.9 µg/mouse (36 µg/kg) or with three doses of 0.3 µg/mouse
(12 µg/kg) separated by 24 hours. Citrated blood was collected
immediately after dosing and at intervals thereafter (n = 3 mice per
time point), centrifuged at 2,950g for 10 minutes, plasma
obtained, and TCA-precipitable radioactivity determined.
Pharmacokinetic parameters were estimated after converting trichloroacetic acid-precipitable cpm/mL and fitting the data of
concentration versus time to a two-compartment model with first order
absorption using nonlinear least-squares regression analysis (WIN-NONLIN; Statistical Consultants, Lexington, KY). Area under the
concentration time curves, maximum concentration, terminal half-lives,
and clearance (mL/h/kg) were calculated using coefficients and
exponents obtained from the model fits.
Colony assays.
Serum-free methylcellulose cultures were used in this
study.25-27 Appropriate numbers of bone marrow cells were
suspended in Dulbecco's modified Eagle's medium obtained from GIBCO
(Life Technologies LTD, Paisley, Scotland) supplemented with the amino
acids L-alanine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic
acid, and L-proline (Sigma); vitamin B12, biotin,
Na-pyruvate, glucose, NaHCO3, and antibiotics (penicillin
and streptomycin) at an osmolarity of 300 mOsm/L; supplemented with 1%
BSA (Fraction V, Sigma), 2 × 10 6 mol/L iron-saturated
human transferrin (Intergen Company, New York, NY), 107
mol/L Na2SeO3 (Merck), 104 mol/L
 -mercapto-ethanol (Merck), linoleic acid (Merck), and cholesterol
(Sigma), both at a final concentration of 1.5 × 105
mol/L and 103 g/L nucleosides (cytidine, adenosine,
uridine, guanosine, 2 -deoxycytidine, 2deoxyadenosine, thymidine, and
2deoxyguanosine obtained from Sigma) and 0.8% methylcellulose
(Methocel A4M Premium Grade; Dow Chemical Co, Barendrecht, The
Netherlands). The cultures were plated in 35-mm Falcon 1008 Petri
dishes (Becton Dickinson Labware) in 1-mL aliquots.25
Granulocyte/macrophage colony formation was stimulated by a saturating
concentration of macrophage colony-stimulating factor (M-CSF) purified
from pregnant mouse uteri extract as described before,28,29
supplemented with 100 ng/mL murine stem cell factor (SCF; Immunex
Corporation, Seattle, WA) and 10 ng/mL murine interleukin-3 (IL-3; R&D,
Minneapolis, MN). Granulocyte/macrophage colony-forming unit (GM-CFU)
colonies were counted after 7 days of culture. Burst-forming unit-erythroid (BFU-E) growth was stimulated by 100 ng/mL SCF and 4 U/mL human erythropoietin (EPO; Behringwerke, Marburg, Germany), titrated to an optimal concentration. Colonies were counted after 10 days of culture. The culture medium of the erythroid progenitors also
contained hemine (bovine, type I; Sigma) at a concentration of 2 × 104 mol/L.
Megakaryocyte progenitor cells (CFU-Meg) were cultured in 0.375% agar
cultures. Colony formation was stimulated by 100 ng/mL SCF, 10 ng/mL
IL-3, and 10 ng/mL murine TPO (Genentech Inc). After 10 days, the
cultures were dried, stained for acetylcholinesterase-positive cells,
and counted.30-32 All cultures were grown in duplicate at 37°C in a fully humidified atmosphere with 10% CO2 in
the air. Colony numbers are expressed as a number per femur or per
spleen and represent the mean ± SD of individual mice.
The spleen colony assay.
This assay was performed as described by Till and
McCulloch.33 Briefly, mice were injected with one fifth of
the cell content of a femur in HHBS one day after TBI. Thirteen days
later, mice were killed, and spleens were excised and fixed in
Tellyesniczky's solution (64% ethanol, 5% acetic acid, and 2%
formaldehyde) in H2O. Colony numbers were expressed as a
number per donor femur ± SD. The cells giving rise to the spleen
colonies were designated as day 13 CFU-spleen (CFU-S), shortly
CFU-S-13.
Marrow repopulating ability (MRA).
Bone marrow from mice irradiated with 9 Gy TBI (in 3 equal fractions,
each separated by 24 hours) was collected 24 hours after the last
fraction of TBI, and 10 lethally irradiated recipient mice were
injected with the cellular content of one femur of control mice or mice
treated with 0.3 µg of TPO 2 hours after each fraction of TBI. After
13 days the bone marrow of recipient mice were assayed for the presence
of GM-CFU.34 MRA is expressed as the number of GM-CFU per
recipient femur. Data from two independent experiments with similar
results were pooled. At the time points of 1 and 3 months and for
normal mice, the standard number of 105 bone marrow cells
was injected.
Statistics.
SDs were calculated and are given in the text and the figures on the
assumption of a normal distribution. The significance of a difference
was calculated by one-way analysis of variance followed by a nonpaired
Student's t-test using StatView (Abacus Concepts Inc,
Berkeley, CA). The SD of CFU-S-13 was calculated on the assumption that
crude colony counts are Poisson distributed. Differences in
repopulating abilities were evaluated using Fisher's exact test. All
colony assays were performed in duplicate for individual mice. The
results of the colony assays are expressed as the mean ± SD per femur
or spleen for at least three mice per group.
| |
RESULTS |
Efficacy of TPO after 6 Gy TBI.
Exposure of mice to a TBI dose of 6 Gy resulted in severe bone marrow
suppression and impaired blood cell production for a period of about 3 weeks. The nadir for thrombocytes occurred around day 10 (Fig
1). A single dose of TPO administered 24 hours after TBI was effective in alleviating the thrombocyte nadir.
Platelet counts 10 days after irradiation were 465 ± 142 × 109/L (n = 15) compared with 144 ± 62 × 109/L for control mice (n = 14), (Fig 1 and Table
1). TPO injected 2 hours after exposure was
significantly more effective, thrombocyte levels 10 days after TBI
being 739 ± 165 × 109/L (n = 15,
P < .0001). White blood cell regeneration was not influenced by TPO administered 24 hours after TBI; counts at day 10 were 0.4 ± 0.1 × 109/L, similar to 0.4 ± 0.1 × 109/L for control mice (Table 1). However, if TPO was
administered 2 hours after TBI, white blood cell counts 10 days after
TBI were 1.1 ± 0.4 × 109/L. For red blood cells the
results were: 7.2 ± 0.5 × 1012/L for control mice, 7.5 ± 0.5 × 1012/L for the 24-hour mice, and 9.0 ± 0.6 × 1012/L for mice treated 2 hours after TBI (Table 1).
Clearly, administration earlier than 24 hours after TBI resulted in an
accelerated multilineage peripheral blood reconstitution in contrast to
the lineage dominant effect of TPO when administered 24 hours after
TBI. As is well known from previous observations in mice and nonhuman
primates,9,15 the multilineage effects originated from
accelerated reconstitution of progenitor cells along the neutrophil,
erythroid, and megakaryocytic lineages measured in bone marrow of mice
7 days after irradiation. Femoral GM-CFU, BFU-E, and CFU-Meg reached
consistently higher numbers in the treatment group compared with the
placebo controls. (Fig 2). The results in
the 24-hour treatment group were in between those of the 2-hour and
control groups, although the differences were significant only for
GM-CFU due to the large variance as a result of exponential
reconstitution of progenitor cells at this time interval after TBI. The
higher number of femoral GM-CFU in the 24-hour treatment group compared
with control numbers was not reflected in accelerated leukocyte
regeneration 10 days after TBI (Fig 3),
which is consistent with our previous observation in rhesus monkeys
that the TPO effect on GM progenitors may require administration of G-
or GM-CSF for peripheral blood manifestation.8,16
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| Fig 1.
Regeneration pattern of platelets in mice irradiated with
6 Gy TBI and treated with 0.3 µg/mouse of TPO 24 hours after TBI
( ), 2 hours after TBI ( ), or placebo ( ), data from one
representative experiment, n = 3 per data point. The shaded area
represents the mean platelet counts ±SD of 19 normal mice
(1123 ± 89). *P < .0001 compared with control mice;
#P < .0001 compared with mice treated 24 hours
after TBI.
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| Fig 2.
The effect of 0.3 µg TPO 24 hours or 2 hours after TBI
on the regeneration of bone marrow progenitors after 6 Gy TBI in mice.
GM, GM-CFU; E, BFU-E; and Meg, CFU-Meg per femur at day 7 after TBI
(means ± SE). The open bar () represents control mice
(n = 6), the lightly shaded bar ( ) represents mice treated with
TPO 24 hours after TBI (n = 6), and the dark shaded bar ()
represents mice treated with TPO 2 hours after TBI (n = 6).
*Significant compared with control mice (P < .03),
#significant compared with mice treated with TPO 24 hours
after irradiation (P < .03).
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| Fig 3.
The effect of the time of administration of 0.3 µg TPO
IP on peripheral blood cell counts 10 days after 6 Gy TBI. Values are
means ± SD. Shaded areas represent the mean ± SD of 14 control mice. The closed squares () are counts from mice treated
with 30 µg of mTPO 2 hours before irradiation.
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To establish the optimal time point for TPO administration, mice were
injected IP with a single dose of TPO at various intervals before or
after 6 Gy TBI. Based on Fig 1, the time dependence of the effect of
TPO treatment was evaluated 10 days after TBI. The effect of TPO was
consistently optimal if TPO was administered IP 0 or 2 hours after TBI
(Fig 3). Progressively lower counts were observed when TPO was injected
at later time points; platelet counts at day 10 after TBI did not
significantly differ between mice treated 6 hours or later after TBI,
whereas the difference between later groups and the 0- and 2-hour
groups were highly significant (P < .003).
A similar time dependence was observed for white and red blood cell
counts. For the white blood cells, TPO administration 10 hours or later
after TBI resulted in counts not significantly different from control
mice while being significantly lower than the 0- and 2-hour treatment
groups. The effect on white blood cell regeneration could be attributed
to differences in neutrophilic granulocytes and monocytes. Flow
cytometric analysis of peripheral blood cells 10 days after irradiation
performed in two experiments revealed a major increase in the numbers
of ER-MP20-positive granulocytes and monocytes in the TPO treated
mice, whereas the number of CD4/CD8-positive T cells was similar in TPO
treated mice compared with controls (data not shown), thus showing the
myeloid nature of the white blood cell response. Also for red blood
cells a gradual decline in effectiveness of TPO with later
administration of TPO was observed. We noted that there was no
difference between the effects of TPO administration at 0 hours and at
2 hours after TBI, whereas TPO administration 2 hours before TBI
resulted in consistently lower levels of platelets and white blood
cells at day 10. The hypothesis that pharmacological levels of TPO in
the first hours after TBI are required was initially evaluated by
administration of a very high dose of TPO before TBI to examine whether
an efficacy could be reached similar to that obtained by TPO early
after irradiation. Administration of 30 µg TPO IP 2 hours before TBI
was as effective as 0.3 µg IP 2 hours after TBI (Table 1 and Fig 3).
The dose of 0.3 µg/mouse IP 2 hours before irradiation was
significantly less effective compared with the 30-µg dose in
alleviating the thrombocyte nadir (P = .03) (Table 1 and Fig
3).
The improved efficacy of TPO when administered early and the decline in
the efficacy at time points later than 4 hours after TBI led us to
speculate that relatively high levels of TPO in the first 2 hours after
administration would be of decisive importance for its efficacy. A
pharmacokinetic analysis of plasma levels after IP injection of 0.3 µg TPO revealed that peak levels of 29 ng/mL were reached 2 to 2.5 hours after administration, with a terminal half-life of 35 hours and a
clearance of 27 mL/h/kg. After the maximum value, there was an initial
steep decline followed by a slower wash-out phase (Fig
4). On the basis of these data, in
combination with those of Fig 3, we postulated that the multilineage efficacy of TPO was dependent on a threshold level of TPO within the
first few hours after TBI. The time course and the plasma level
required were accurately assessed using IV administration of TPO (Table
2 and Fig 5). IV
administration also showed that early treatment (0 and 2 hours after
TBI) was considerably more effective in stimulation of platelet level
regeneration than treatment 24 hours after TBI. The multilineage
effects seen with early IP treatment were also observed with early IV
administration, and were similarly lost if TPO was administered 24 hours after TBI (Table 2). Lowering the dose of TPO to 0.1 µg/mouse
did not affect the results, but at 0.03 µg/mouse or lower efficacy
was lost, thus demonstrating a threshold TPO level required to achieve
optimal multilineage efficacy (Fig 5).
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| Fig 4.
Plasma level of TPO after IP administration of 3 doses of
0.3 µg/mouse separated by 24 hours each (dotted lines), and of a
single dose of 0.9 µg/mouse (solid line). For explanation, see
text.
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Table 2.
Major Peripheral Blood Cell Counts 10 Days After 6 Gy
TBI and IV TPO Administration at Different Timepoints After TBI
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| Fig 5.
Dose-response curves for the multilineage
effect of TPO administered IV immediately after 6 Gy TBI. Peripheral
blood cell counts 10 days after 6 Gy TBI. Values are means ± SD, n
= 6 for the doses of 0.01, 0.03, and 0.1 µg and n = 12 for the
dose 0.3 µg/mouse. The vertical line in the upper panel represents
the dose level of 0.15 µg/mouse, which results in 50% saturation of
c-mpl on platelets. The placebo control levels coincide with
the level at which the horizontal axis is set.
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Efficacy of TPO after fractionated irradiation.
To enable assessment of immature hematopoietic cells by short-term
transplantation assays, which is technically not feasible shortly after
6 Gy TBI due to the very low frequency of residual immature cells, we
amplified the TPO response by adjusting the model by fractionation of a
total dose of 9 Gy TBI in three equal doses separated by 24 hours. This
regimen induced a slightly more profound pancytopenia than the single
dose of 6 Gy TBI (Fig 6). Fractionated TBI
would also more closely resemble the protracted cytotoxic insult
administered to chemotherapy patients. Groups of mice received either
no TPO, or were treated with 0.9 µg TPO 2 hours after the last
fraction of TBI, 0.3 µg TPO 2 hours after each fraction of TBI, or
0.9 µg TPO 2 hours before the first fraction of TBI. Pharmacokinetic
analysis of repetitive IP administration (three doses of 0.3 µg/mouse, each separated by 24 hours) did not reveal accumulation of
TPO plasma levels. The dose of 0.9 µg/mouse resulted in higher peak
plasma levels (151 ng/mL) without affecting the terminal half-life or
clearance (Fig 4). TPO administered 2 hours after each fraction of
radiation completely prevented thrombopenia (Fig 6 and Table
3). This schedule did not prevent the
severe reduction in neutrophils, but accelerated their recovery to
normal values. Platelet counts and red and white blood cells were
significantly lower in the group treated with 0.9 µg TPO 2 hours
after the last fraction compared with 0.3 µg TPO 2 hours after each
fraction. Administration of TPO 2 hours before the first fraction in a
dose of 0.9 µg/mouse was largely ineffective. The effect of TPO on
blood cell regeneration was also reflected at the level of progenitors
of different blood cell lineages (Fig 7).
Placebo mice and mice treated with 0.9 µg/mouse 2 hours before TBI
displayed very low levels of progenitors even at day 13 after TBI,
before progenitor cell reconstitution resulted in a large overshoot,
especially manifest in the spleen 3 weeks after TBI. In contrast, the
schedule optimal for blood cell regeneration (2 hours after each
fraction) also led to a rapid normalization of progenitor cells in the
spleen without the characteristic large overshoot in the placebo
controls (P = .01 at day 17) and in the mice treated with the
suboptimal TPO dose schedule. In the fractionated radiation model,
administration of 30 µg of TPO 2 hours before the first radiation
fraction was as effective as TPO administered 2 hours after each dose
of radiation (Table 3 and Fig 6).
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| Fig 6.
The effect of TPO on hematopoietic regeneration after 9 Gy TBI in mice. The mice were irradiated with three fractions ( ) of
3 Gy with 24-hour intervals. In the upper panel platelet regeneration
is depicted, in the middle panel red blood cell regeneration is shown,
and in the lower panel white blood cell regeneration is shown. (),
mice treated with three doses of 0.3 µg/mouse of mTPO IP 2 hours
after each fraction of TBI; ( ), mice treated with 0.9 µg IP 2 hours after the last fraction of TBI (day 0); (), mice treated with
0.9 µg IP 2 hours before the first fraction of TBI; (), control
mice. Data are given as means ± SD, three mice per data point per
group. The closed triangles ( ) are counts from mice treated with 30 µg of mTPO IP 2 hours before irradiation (n = 3).
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Table 3.
Major Peripheral Blood Cell Counts 10 Days After 9 Gy
TBI (3 × 3 Gy, 24 Hours Apart) and IP TPO Administration
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| Fig 7.
Regeneration of progenitor cells in bone marrow (upper
panel) and spleen (lower panel) at 7, 10, 13, and 17 days after the
last fraction of TBI in mice irradiated with three fractions of 3 Gy
with 24 hour intervals. (), GM-CFU per femur or spleen; (),
BFU-E; (), CFU-Megs per femur or spleen. Placebo, control mice;
TPO-2h, mice treated with 0.9 µg TPO 2 hours before the first
fraction of TBI; TPO+ 2h, mice treated with 0.9 µg 2 hours after
the last fraction of TBI; TPO3x+2h, mice treated with three doses of
0.3 µg/mouse 2 hours after each fraction of TBI, three mice per data
point. SDs were calculated for the sum of the individual colonies per
mouse. *Significantly different from time matched controls
(P < .05), **(P < .005).
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Because the progenitor cell data of irradiated TPO-treated mice in this
fractionated TBI regimen indicated a prominent effect of TPO on
immature bone marrow BM cells, CFU-S-13 were enumerated, the MRA of
bone marrow of the TPO-treated mice assessed, and progenitor cells
assayed 24 hours after the last TBI fraction (Table
4). The numbers of CFU-S-13 of mice treated
with TPO 2 hours after each dose of irradiation were approximately
14-fold those of the placebo control mice (Table 4). The progenitor
cell content of the bone marrow 24 hours after TBI was also
significantly increased (Table 4), GM-CFU numbers per femur
approximately 4-fold, BFU-E numbers 65-fold, and CFU-Meg 20-fold those
of placebo control mice, which grossly corresponds to the efficacy of
TPO treatment along those lineages. The MRA of TPO-treated mice,
defined as secondary GM-CFU in the bone marrow of lethally irradiated
recipients, was more than one order of magnitude reduced in the
TPO-treated mice compared with placebo-treated controls (Fig
8). This result showed that the
TPO-stimulated increase of CFU-S-13 and progenitor cells occurred at
the expense of the more immature MRA cells, which were proportionally
depleted. Although day 13, which is the time of the peripheral blood
count nadirs at which white blood cells were undetectable in both
groups of lethally irradiated recipients, is not the most suitable time
interval to study the impact of this shift among the immature bone
marrow cells on peripheral blood cell reconstitution, the difference in
repopulating ability was also clearly reflected in the erythrocyte as
well as the thrombocyte counts of the lethally irradiated recipients
used for the MRA assay (measured in one of the two experiments from
which the upper panel of Fig 8 was derived). The 10 recipients of bone
marrow from TPO-treated mice reached erythrocyte counts all exceeding 4.4 × 1012/L (4.7 ± 0.3), and all but 1 reached
thrombocyte counts of more than 10 × 109/L, as opposed to
the 8 (of 10) surviving recipients of bone marrow from placebo-treated
mice that had erythrocyte counts of 3.4 ± 0.9 × 1012/L
and thrombocyte counts of 5.5 ± 2.8 × 109/L. These
differences are highly significant (P = .002 and
P = .006, respectively; Fisher's exact test), thus
illustrating the importance of spleen-repopulating cells in early
reconstitution of peripheral blood counts in mice. Because a depletion
of immature stem cells, such as measured by the MRA assay, could be
potentially deleterious in the clinical setting, the effect of TPO on
bone marrow progenitor cells was also evaluated after a more prolonged
interval. Peripheral blood count and progenitor cell content of both
spleen and femurs were evaluated 1 and 3 months after the 3 × 3 Gy
irradiation protocol. Peripheral blood counts did not differ between
TPO- or placebo-treated mice at 1 and 3 months, and also the femoral
and spleen content of GM-CFU, BFU-E, and CFU-Meg was not different
between both groups at either time point (data not shown). At these
time intervals, we also measured the MRA of the bone marrow of the TPO-
versus placebo-treated mice (Fig 8). MRA cells were similar in both
groups of mice at 1 month after irradiation although still depleted
compared with normal levels. At 3 months after irradiation, MRA cells
in both groups had returned to subnormal levels, again without a difference between the recipient groups. On this basis, we concluded that the initial depletion of MRA of TPO-treated mice became
replenished during long-term hematopoietic reconstitution from cells
with, by definition, long-term repopulating ability. The protracted nature of MRA reconstitution, which has not been documented before, is
noteworthy.
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Table 4.
Effects of TPO Treatment After 9 Gy TBI (3 × 3 Gy,
24 Hours Apart) on CFU-S-13 and Progenitor Cell Content of Bone Marrow
24 Hours After the Last Dose of TBI in Mice Treated With TPO IP 2 Hours
After Each Dose of TBI Versus Control
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| Fig 8.
Marrow repopulating ability: GM-CFU, ranked in ascending
order, on day 13 in femurs of recipients of bone marrow of mice treated
with 0.3 µg TPO IP 2 hours after each fraction of 3 Gy TBI () in
comparison with those of recipients of control marrow () of mice
that did not receive TPO. Upper panel: a total of 25 mice were injected
with bone marrow (the content of 1 femur, 24 hours after TBI) of
TPO-treated mice of which only 5 mice had more than 10 GM-CFU per
femur, and 17 (surviving of 20 injected) mice with bone marrow of
placebo-treated mice of which 10 had more than 10 GM-CFU per femur.
Results from two experiments. This difference is highly significant
(P = .01, Fisher's exact test). Upper middle panel: 10 mice were injected with the standard 105 bone marrow cells
from each of the groups 1 month after TBI, of which in each recipient
group 9 mice survived and 5 mice had more than 10 GM-CFU per femur.
Lower middle panel: 10 mice were injected with the standard
105 bone marrow cells from each of the groups 3 months
after TBI, of which in the recipient group of the TPO-treated mice 1 mouse died before day 13 and 1 mouse had less than 10 GM-CFU/femur.
Lower panel: 20 mice were injected with the standard 105
bone marrow cells from normal, untreated donors for comparison.
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DISCUSSION |
The present analysis shows that a single administration of TPO is
capable of counteracting radiation-induced pancytopenia mediated
through its effect on immature multilineage repopulating cells and
their direct progeny, thereby promoting peripheral blood reconstitution
of thrombocytes, erythrocytes, and granulocytes. Multilineage efficacy
was critically dependent on time relative to TBI and a relatively high
dose of TPO. In particular, the first few hours after TBI appeared to
be important to elicit the multilineage response to TPO. Mechanisms
involved included prevention of depletion of multilineage cells during
the first 24 hours after TBI, a preferential stimulation of
spleen-repopulating cells with short-term peripheral blood
reconstituting ability at the transient expense of marrow-repopulating cells, and a threshold dose of TPO to overcome initial
c-mpl-mediated clearance.
The mechanism by which TPO makes multilineage cells available for
accelerated hematopoietic reconstitution remains to be further elucidated, as does their apparent functional depletion as a function of time after TBI in the absence of TPO. Multilineage TPO
responsiveness declined sharply as a function of time after TBI,
leaving a lineage dominant thrombopoietic response when administered 24 hours after TBI. This indicates that in the absence of TPO,
multilineage TPO responsive cells are rapidly depleted or become
inaccessible. The loss of the multilineage TPO response may have
diverse and complex causes, which include apoptosis or
radiation-induced cell death, differentiation along other hematopoietic
lineages, inhibition mediated by cytokines produced in response to
radiation injury, or inaccessibility of the immature cells for TPO due
to stromal reactions to radiation. TPO has been shown to prevent
apoptosis of immature hematopoietic cells35 and this might
be a prime candidate mechanism to explain the short time interval
available for optimally effective TPO intervention. In vitro, TPO does
not confer a proliferative response to immature hematopoietic cells, but does so strongly in the presence of suitable other factors, eg, Kit
ligand.36-42 The mechanisms involved might therefore also include activation of one or more cofactors required for the strong proliferative response observed in the present study. We also do not
exclude that the effect of TPO on multilineage cells is augmented by
the release of various cytokines by megakaryocytes43,44 subsequent to stimulation by TPO, although the time frame observed makes such a mechanism not likely.
The lack of a leukocyte response 10 days after 6 Gy TBI in the presence
of significantly more GM-CFU at 7 days in the 24-hour treatment group
is noteworthy. The neutrophil response to TPO has been variable
throughout the reported studies.6,9,11,45,46 We observed in
a myelosuppression model in rhesus monkeys that TPO-stimulated GM-CFU
reconstitution was not reflected in an increase in peripheral blood
granulocytes which, however, could be brought out by coadministration
of G- or GM-CSF.8,16 Thus, without exogenous
CSFs, the myeloid progenitor cell effect of TPO after myelosuppression
may remain unnoticed at the peripheral blood cell level, to which the
short survival of circulating neutrophils might also contribute. These
observations are fully consistent with the mouse data presented here.
It is not inconceivable that some myelosuppression regimens may result
in sufficiently high endogenous CSF levels for a significant neutrophil
response to exogenous TPO alone, as has been observed in some
studies.9,11,45
To establish the TPO dose level needed, a dose titration was performed
using IV administration immediately after irradiation. The doses of 0.3 and 0.1 µg/mouse (12 and 4 µg/kg, respectively) gave identical
results, whereas 0.03 (1.2 µg/kg) and 0.01 µg/mouse were largely
ineffective. This observation is interpreted as evidence for a
threshold level of TPO required for optimal efficacy. It was previously
shown13,24 that IV injection of 125I-rmTPO into
mice results in an initial sharp decline in plasma levels, followed by
steady state clearance approximately 3 hours after IV
injection.24 A lower dose does not influence the terminal half-life.13 After IV bolus injection, the initial rapid
decline in plasma TPO levels is due to binding of TPO to c-mpl
on platelets and on cells in the spleen,24 whereas the
slower terminal decline is likely related to uptake and clearance by
c-mpl on platelets, spleen cells, and megakaryocytes, as well
as nonspecific mechanisms.24,47 The initial binding and
uptake of TPO to c-mpl is concentration dependent and becomes
saturated at higher doses, leading to greater plasma TPO
levels.48 This relationship between TPO pharmacokinetics and c-mpl levels has recently been studied in normal mice,
which showed that an IV dose of approximately 6 µg/kg ( 0.15
µg/mouse) was needed to obtain an occupancy of 50% of c-mpl
sites (indicated in Fig 5). Doses greater than 6 µg/kg began to
saturate this specific clearance mechanism, whereas lower doses failed
to reach 50% receptor occupancy.48 This is consistent with
a threshold level of TPO needed to overcome initial
c-mpl-mediated clearance and to result in sufficient plasma
TPO levels to achieve a maximal hematopoietic response. Doses larger
than 6 µg/kg (0.15 µg/mouse) may not provide any greater efficacy.
These data are consistent with the findings presented in Fig 5, showing
that the hematopoietic recoveries after IV doses of 0.3 and 0.1 µg/mouse (12 and 4 µg/kg, respectively) were not different, whereas
a dose of 0.03 µg/mouse (1.2 µg/kg) was suboptimal in preventing
myelosuppression. Based on the previous IV pharmacokinetic data
comparing TPO plasma levels in c-mpl knockout and normal
mice,24 it was inferred that 40% of the exogenous TPO
binds to c-mpl on platelets and 60% is available as free TPO in the plasma. Assuming a plasma volume of 1 mL in a 25-g mouse, the
suboptimal dose of 0.03 µg/mouse results in a maximum level of 20 ng/mL and the effective dose of 0.1 µg/mouse in 60 ng/mL. Consequently, the minimum effective or threshold plasma level is in
between those levels.
Fractionation of the dose of radiation and appropriate dosing of TPO
was thought to amplify the TPO effect on immature cells to enable
short-term transplantation assays. Such an approach would also be more
representative of clinically used radiation regimens and of the
protracted nature of cytoreductive treatment by means of chemotherapy.
Also after fractionated TBI (3 × 3 Gy, 24 hours apart), the optimal
dose and dose scheduling of TPO derived from the 6 Gy experiments, ie,
0.3 µg/mouse, 2 hours after each TBI fraction, prevented thrombopenia
and promoted erythrocyte and leukocyte reconstitution. We noted that
also the femoral and spleen progenitor cells (Fig 7) normalized rapidly
and lacked the late overshoot in the spleen characteristic of the
placebo controls. Because the range of stimulation by TPO was not
lineage specific, we postulated that the effect was mediated by
stimulation of multilineage cells. After transplantation of bone marrow
into lethally irradiated recipients, the number of CFU-S-13 is a
measure for relatively immature repopulating stem cells,49
associated with the initial, short-term wave of hematopoietic
reconstitution, which lasts for several months.50 By this
assay it was shown that the multilineage effect of TPO administered 2 hours after TBI is mediated through stimulation of these immature cells
and is already manifest 24 hours after the last fraction of TBI. The number of secondary in vitro clonogenic progenitors in the bone marrow
of such recipients is a measure of the MRA of the graft, the primary
cells being closely associated with those that provide sustained
hematopoiesis after bone marrow transplantation.34 MRA,
measured by enumeration of GM-CFU numbers in the bone marrow of mice
injected with cells from TPO treated mice, was one to two orders of
magnitude less than that in control mice. The increase of CFU-S-13 and
the concomitant decrease of cells with MRA most likely indicates
recruitment of multilineage short-term repopulating cells from a more
immature ancestral population. This is the more conceivable from the
magnitude of this effect (14-fold for CFU-S), which suggests three to
four cell doublings. Because these occurred during the 3 days that
elapsed from the first TPO administration to the time of the
measurement, 24 hours after the last fraction of TBI, this would be in
close agreement with the doubling time established
previously27 for such immature cell populations during
hematopoietic reconstitution. The decline in marrow-repopulating cells
with the concomitant increase of spleen-repopulating cells could also
be considered as a shift in homing pattern among these immature cells.
To date, there is no evidence to suggest that such a shift may occur
without cell divisions, whereas the ancestral position of the
marrow-repopulating cells relative to the spleen colony-forming cells
has been well documented.34,51,52
The depletion of MRA cells in the TPO-treated mice, measured 24 hours
after TBI, was transient. Peripheral blood cell regeneration 1 and 3 months after three fractions of 3 Gy was not different in mice treated
with TPO compared with controls and neither were the femoral and spleen
in vitro colony-forming cell numbers. Assessment of the MRA at the same
time intervals also did not show differences between the TPO-treated
mice and the placebo group. We interpret these observations as
replenishment of the MRA cells from a more ancestral cell population
with, by definition, long-term repopulating ability, consistent with a
model in which MRA cells are a transitory population intermediate to
stem cells with long-term repopulating ability and the
spleen-repopulating cells measured by the CFU-S-13 assay. To date, the
effect of TPO treatment on long-term repopulating cells has not been
quantitatively documented. Recently, it was reported that
transplantation of bone marrow from TPO-treated, 3.5-Gy irradiated
donor mice facilitated the 90-day survival of the recipient
mice.53 However, this survival effect had become established already at the short-term hematopoietic reconstitution parameter of 30-day survival (in practice already within 17 days), closely associated50 to the CFU-S-13 assay used in the
present study. Using such an experimental design to establish TPO
effects on long-term repopulating cells would require a genetic marker capable of distinguishing between donor and recipient cells along multiple hematopoietic lineages.
In addition, we showed that TPO is effective if administered at a very
high dose shortly before myelosuppressive TBI. The dose used, 30 µg/mouse (1.2 mg/kg), suprasaturates the c-mpl-mediated clearance mechanism (which makes pharmacokinetic measurements futile)
and is not recommended for clinical use. We did not so far establish
empirically the minimum effective dose required for prophylactic TPO
administration, but such a dose could be derived on the assumption of
maintaining plasma TPO levels of approximately 60 ng/mL in the first
hours after TBI as calculated above. However, the observation has
relevance for the further development of efficacious dose and dose
scheduling regimens, especially in conjunction with chemotherapy, as
well as in the area of radiation protection. The present study did not
address propensity to hemorrhage and prevention of mortality, but
rather was directed at mechanisms important to optimize efficacy. The benefit of TPO treatment shortly after much higher
("supralethal") doses of TBI on survival and prevention of
bleeding will be published separately.54
In vivo studies on TPO efficacy have yielded various results, in that
in normal animals usually only a platelet response was obtained,6,7,55-59 whereas in myelosuppressed animals
multilineage responses was the prevailing
pattern,8,9,11-13,15,16,53,60,61 and after transplantation
of limited numbers of stem cells no response was obtained at
all.62 On the basis of this heterogeneity, it can be
assumed that the response to exogenous TPO is determined by multiple
factors. We already pointed out the importance of cotreatment with
G-CSF or GM-CSF to make the TPO effect on GM-CFU reconstitution
manifest in neutrophil numbers in the peripheral blood. The present
study also identifies time relative to myelosuppression and dose of
exogenous TPO as pivotal factors. In addition, the difference between
normal and myelosuppressed animals indicates that the TPO response of
immature cells might be dependent on the presence or activation of one
or more cofactors. As already pointed out, TPO by itself does not
induce in vitro a proliferative response in immature bone marrow cells,
but does so strongly in synergy with, eg, Kit ligand.36-42
Identification of the cofactor(s) which operate in irradiated or
otherwise myelosuppressed animals to generate a proliferative response
to TPO administration might therefore be highly relevant as well to
improve the clinical TPO response.
Irrespective of the mechanisms involved, the optimal efficacy of TPO if
administered within 2 hours after cytoreductive treatment places
emphasis on the importance of dose and dose scheduling in clinical
protocols of cancer treatment and/or after bone marrow transplantation. Indeed, the initial clinical experience did not demonstrate a major effect similar to those observed in the experiments described here.63-66 It is concluded that dosing similar to
what has become conventional based on the G-CSF and GM-CSF experience is not optimal for TPO and that its efficacy can be substantially improved by achieving relatively high TPO levels shortly after cytoreductive treatment. This was attributable to the dual target cell
nature of the TPO response, of which the important multilineage component is functionally depleted shortly after radiation exposure, and to the identified threshold plasma level of TPO required to overcome its initial c-mpl-mediated clearance.
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FOOTNOTES |
Submitted October 29, 1997;
accepted May 1, 1998.
Supported in part by The Netherlands Cancer Foundation Koningin
Wilhelmina Fonds, the Dutch Organization for Scientific Research NWO,
and Contracts of the Commission of the European Communities.
Address |
Abstract
Introduction
Abstract