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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 89-99
Transient Thrombocytopenia Produced by Administration of Macrophage
Colony-Stimulating Factor: Investigations of the Mechanism
By
Georgiann R. Baker and
Jack Levin
From the Departments of Laboratory Medicine and Medicine, University
of California School of Medicine and the Veterans Affairs Medical
Center, San Francisco, CA.
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ABSTRACT |
Administration of macrophage colony-stimulating factor (M-CSF) to
mice (2 to 8 mg/kg/d × 5d) produced dose-dependent thrombocytopenia,
which reached its nadir on days 4 to 5, followed by rapid recovery.
Surprisingly, when administration of M-CSF was prolonged, the
thrombocytopenia completely resolved, despite continued treatment.
Splenectomy did not prevent the thrombocytopenia. Readministration of
M-CSF after various intervals continued to produce the thrombocytopenic
effect, even after 35 days. Measurements of Meg-CFC and
megakaryocyte ploidy during the periods of M-CSF treatment and recovery
of normal platelet levels showed no evidence of bone marrow
suppression. Platelet survival was markedly decreased after 5 days of
M-CSF (at the platelet count nadir) and after 9 days of continued M-CSF
treatment, when the platelet count had returned to normal. Platelets
from M-CSF-treated donors demonstrated normal survival when transfused
into normal recipients. We concluded that thrombocytopenia produced by
M-CSF was not due to suppression of thrombopoiesis, but to increased
activity of the monocyte/macrophage system, which caused shortened
platelet survival, and that subsequently, increased platelet production
compensated for ongoing platelet destruction and resulted in normal
platelet levels.
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INTRODUCTION |
MACROPHAGE colony-stimulating factor
(M-CSF; CSF-1) is essential for the survival, growth, and development
of the monocyte/macrophage cell lineage.1-5 Experiments in
normal6 and M-CSF-deficient (op/op) mice7,8
suggest that M-CSF has a major regulatory role in the development and
maintenance of mononuclear phagocytes in liver, spleen, and kidney.
Most circulating M-CSF is cleared by binding to its receptor on the
macrophages of the liver and spleen, with subsequent endocytosis of the
receptor-ligand complex and intracellular degradation.9,10
M-CSF has been shown to acutely increase monocyte levels when
administered to various animals and man. The magnitude of reported
increases has been variable, but dose/response relationships have been
demonstrated.5,6,11-14 Some investigations also have
reported a return toward normal of elevated monocyte levels with
continued M-CSF administration.12,13,15,16 Bone marrow
monocyte production has been shown to be increased12;
therefore, the decrease in circulating monocytes during prolonged M-CSF
administration may be due to increased movement of monocytes into
tissues.17 M-CSF administration has been shown to cause
increases in the weights of livers and spleens, with both livers and
spleens exhibiting increased infiltrates of
macrophages.18,19 No consistent changes in the total white
blood cell (WBC), lymphocyte, or neutrophil counts are
produced by M-CSF, but occasionally increases in these cells are
reported.12,15,16 These effects may be secondary to the
induction of other cytokines produced by the responding
macrophages.20 A puzzling and major adverse effect of M-CSF
administration has been the production of thrombocytopenia in all
species studied,12,15,16,21,22 which sometimes resolves
despite continued M-CSF administration.16,22-24
Thrombocytopenia was dose-related, but was not sufficiently severe to
cause bleeding. Several reports document a temporal relationship
between the nadir of the platelet count and the maximum increase in
monocyte counts.12,13,15,20,24,25 In this investigation, we
have characterized the thrombocytopenic effect of M-CSF and have sought
to determine the mechanism of this unpredicted physiologic result and
its resolution despite continued administration of M-CSF.
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MATERIALS AND METHODS |
General techniques.
Female Swiss Webster (SW) mice, 27 to 30 g, and female C57BL/6N (C57BL)
mice, 22 to 25 g, (Simonsen Laboratories, Gilroy, CA) were used for
these studies. Uninjected mice from the same shipments were used as
normal controls. Mice were housed in an American Association for
Accreditation of Laboratory Animal Care approved facility in filter
cages and fed standard rodent chow and tap water ad libitum. All
experimental protocols were approved by the Committee for Animal
Experimentation of the VAMC. In conducting research using animals, the
investigators adhered to the "Guide for the Care and Use of
Laboratory Animals" prepared by the Committee on Care and Use of
Laboratory Animals of the Institute of Laboratory Animal Resources,
National Research Council (National Academy Press, Washington, DC,
1996). Blood samples were obtained from the retroorbital venous plexus,
with the use of 70 µL heparinized EDTA-coated glass capillary tubes
(Drummond Scientific Co, Broomall, PA), on the days indicated,
immediately before M-CSF injection. Splenectomy was performed under
anesthesia with methoxyflurane vapor (Metofane; Pitman-Moore, Inc,
Mundelein, IL). Mice were allowed to recover from surgery for at least
1 month before experimentation. Mice were killed by cervical
dislocation.
Reagents.
Recombinant human macrophage colony-stimulating factor (M-CSF; CSF-1)
was a generous gift from the Cetus and Chiron Corporations, Emeryville,
CA. Dilutions were made in pyrogen-free 0.9% saline for injection
(Abbott Laboratories, Inc, North Chicago, IL). M-CSF was administered
by intraperitoneal (IP) injection twice daily, 8 hours apart, at the
doses indicated, in volumes of approximately 0.3 mL, beginning on day
1.
Blood cell counts.
Platelet counts, total WBC counts, and hematocrit values were
determined in whole blood diluted 1:2 (vol/vol) in isotonic saline
solution (Hematall, Fisher Scientific Co, Pittsburgh, PA) and analyzed
with an automated flow cytometric whole blood counter (Technicon H-1
System, Technicon Instruments, Tarrytown, NY), as previously
described.26
The small size of rodent white blood cells makes the differentiation
between atypical lymphocytes and monocytes imprecise by Wright's
stain, the most common method reported. To unambiguously quantify the
monocytes, we established a new method based on the receptor for M-CSF,
which among cells of the blood is present only on cells of the
monocyte/macrophage lineage.27 Differential WBC counts in
peripheral blood were determined after incubating buffy coat cells for
1 hour at 4°C with 0.1 mL of supernatant from an M-CSF-producing
cell line (Rat2 pAPtag1 MCSF clone C5) (a generous gift of Drs Larry
Rohrschneider and Gary Myles, Fred Hutchinson Cancer Research Center,
Seattle, WA). The supernatant contained a secreted fusion protein that
consisted of alkaline phosphatase fused in frame to amino acids 33-180
of rmM-CSF,28 prepared according to the method of Flanagan
and Leder.29 Cells were then cytofuged and fixed for 30
seconds in a solution of 4% paraformaldehyde, 1.4 mmol/L
Na2HPO4, 7.3 mmol/L
KH2PO4, and 45% acetone, pH 6.6, at 4°C.
An Alkaline Phosphatase Substrate Kit IV (BCIP/BNT) (Vector
Laboratories, Burlingame, CA) was used to produce a blue precipitated
reaction product, that indicated M-CSF binding to its receptor on
monocytes. Cells were counterstained using 0.5% neutral red,
dehydrated, and mounted. One thousand nucleated blood cells per animal
were enumerated to obtain the differential cell count. Lymphocytes and
neutrophils were identified by standard morphologic criteria, and
monocytes were identified by the presence of a blue precipitate in or
on cells.
Proplatelet quantification.
Platelet morphology was quantified as previously
described.30 Briefly, blood was obtained by cardiac
puncture and anticoagulated with acid-citrate dextrose containing
prostaglandin E1, pH 6.7. Platelet-rich plasma (PRP) was
prepared by centrifugation. Platelet morphology was observed by
phase-contrast microscopy. Differential counts of platelet forms were
performed with platelets drifting slowly between the coverslip and an
ordinary glass slide using 400x magnification.
Tissue weights.
At the end of selected experiments, livers, spleens, and lungs were
removed from normal or M-CSF-treated mice and tissue weights were
recorded.
Cell culture.
Soft agar cultures of spleen and bone marrow cells from normal and
M-CSF-treated mice were prepared for quantification of
granulocyte-macrophage colony-forming cells (GM-CFC) and megakaryocyte
colony-forming cells (Meg-CFC), as previously described,31
except for the following modifications: 20% horse serum was used
instead of fetal calf serum and 0.1 mL (instead of 0.2 mL) of pokeweed
mitogen spleen cell conditioned medium was used as the source of growth
factors in each 1-mL culture. Control values were bone marrow: GM-CFC,
116 colonies/5 × 104 cells and Meg-CFC, 16 colonies/5
× 104 cells; spleen: GM-CFC, 62
colonies/106 cells, and Meg-CFC, 52
colonies/106 cells.
DNA measurements.
The ploidy distribution (DNA content) of megakaryocytes from the bone
marrow of C57BL mice was measured using two-color flow cytometry, as
previously described,32 with the following modifications: a
FACScan with Lysis II software (Becton-Dickinson, Inc, San Jose, CA)
was used for analyses. C57BL mice were used because normal SW mice
demonstrate a ploidy distribution that is too variable for precise
studies of changes in ploidy (J. Levin, unpublished observation, March
1992).
Platelet survival.
Normal SW mice, or SW mice treated with M-CSF, were used as platelet
donors and recipients for these studies. Platelet survival studies were
performed as previously described.33 Briefly, platelets
pooled from donor mice were fluorescently labeled with
5-chloromethylfluorescein diacetate (CMFDA) (Molecular Probes, Eugene,
OR) and injected into the tail veins of recipient mice. After infusion
of labeled platelets, blood samples were obtained from the retroorbital
venous plexus at 2, 4, and 6 hours and then approximately every 12
hours for the next 4 days. Blood samples were analyzed by flow
cytometry, using a FACScan, to determine the proportion of labeled
platelets present at each time point. Survival curves were constructed,
and the circulating half-life (T1/2) of the labeled
platelets was determined graphically. In addition, platelet survival
times were determined by using the multiple hit model (gamma
function)34,35 and the best fit estimate, derived from the
use of both linear and exponential sum of squares
calculations.33
Carbon clearance.
The rate of disappearance of carbon particles from the circulating
blood of normal or M-CSF-treated mice was measured as previously
described.36 Briefly, a solution of India Ink (Difco
Laboratories, Detroit, MI) was injected intravenously into either
normal or M-CSF-treated mice. Blood samples were obtained at various
times from 1 to 11 minutes after injection. Blood was lysed, and
duplicate aliquots of each sample were read spectrophotometrically to
determine the absorbance at 620 nm. The background values, obtained
from lysed blood from mice not injected with carbon particles, were
subtracted. Resultant values were used to determine the disappearance
rate (T1/2) of carbon particles from the blood.
Levels of M-CSF and anti-M-SF antibodies in serum.
Serum samples were obtained 4 and 8 hours after administration of 4
mg/kg/d M-CSF on days 1, 3, and 5 for determination of circulating
M-CSF levels. Samples were stored at  70°C until assayed.
Radioimmunoassays were performed in duplicate in a two-step procedure
as previously described by Stanley37 with
modifications.38,39
For detection of murine anti-human M-CSF antibodies, 4 mg/kg/d M-CSF
was administered for 5 days. Serum samples were obtained on days 5, 10,
12, and 15, and stored at 70°C. Samples were assayed using
conditions described for the M-CSF radioimmunoassay37 with
10% normal rabbit serum replacing anti-CSF-antiserum.
Statistical analysis.
Statistical analyses were performed with a two-tailed Student's
t-test, using StatView (Abacus Concepts, Berkeley, CA).
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RESULTS |
Effect of M-CSF on platelet levels.
The effect of different doses of M-CSF on the platelet counts of normal
mice was examined. M-CSF was administered to SW mice for 5 days, in 2
daily IP injections, at doses of 2, 4, or 8 mg/kg/d. The platelet
counts gradually decreased in a dose-dependent manner during the period
of administration, reached a nadir on days 4 to 5, and immediately
began to increase to above normal levels on discontinuation of
treatment (Fig 1A). Blood sampling alone
had no effect on the platelet count (data not shown).
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| Fig 1.
(A) The effect of administration for 5 days of different
doses of M-CSF on the platelet counts of SW mice. M-CSF was
administered to SW mice at the indicated total daily dosages, given in
two IP injections, 8 hours apart, for 5 days. On days 1 to 5, platelet
counts were obtained immediately before the morning M-CSF injection.
The mean platelet count ± 1 SE is shown. The number of determinations
at each time point was at least 3, and usually ranged from 5 to 28
(mean, 13). The symbols indicate significant differences between the
values (P < .05) ( , 2 mg/kg/d v 4 mg/kg/d;  ,
2 mg/kg/d v 8 mg/kg/d; #, 4 mg/kg/d v 8 mg/kg/d). (B)
The effect of administration for 11 days of different doses of M-CSF on
the platelet counts of SW mice. M-CSF was administered to SW mice at
the indicated dosages, given in two daily IP injections, 8 hours apart,
for 11 days. Platelet counts were obtained immediately before the
morning M-CSF injection. The mean platelet count ± 1 SE is shown. The
number of determinations ranged from 5 to 21 at each time point (mean,
11). The symbols indicate significant differences between the values
(P < .05) (, 2 mg/kg/d v 4 mg/kg/d; , 2
mg/kg/d v 8 mg/kg/d; #, 4 mg/kg/d v 8 mg/kg/d).
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To further investigate the relationship of dosage and duration of M-CSF
administration to thrombocytopenia, 2, 4, or 8 mg/kg/d M-CSF was
administered to normal SW mice for a period of 11 days. Platelet levels
decreased in a dose-dependent manner up to day 5, after which levels
began to increase despite continued administration (Fig 1B). The
increase of platelet levels during the latter period of M-CSF
administration also was dose-related; the largest dose allowed the
least platelet recovery. At these doses, rebound thrombocytosis did not
occur during the treatment period. To rule out the production of a
neutralizing antibody against M-CSF as a potential cause of
refractoriness to M-CSF, serum was obtained on days 5, 10, 12, and 15
from mice that had been treated with M-CSF for 5 days and analyzed for
the presence of antibodies to M-CSF. No antibodies against M-CSF were
detected after 5 days of M-CSF administration. Although anti-M-CSF
antibodies subsequently became detectable 10 days after initiation of
M-CSF administration in most animals, the titers were low.
To determine the effect of long-term administration and repeated
exposure to M-CSF, mice received a dose of 8 mg/kg/d for 11 days (Fig
2). Platelet levels increased after
reaching their nadir on day 5, but rebound thrombocytosis did not occur
during this first course of treatment until after M-CSF administration
was discontinued. Following various lengths of time between repeat
challenges of M-CSF, despite the detection of low levels of anti-M-CSF
antibodies 10 to 15 days after the initial injection, thrombocytopenia
still occurred within 5 days of M-CSF treatment, and rebound
thrombocytosis now occurred during administration of M-CSF.
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| Fig 2.
The effect of multiple courses of M-CSF administration on
the platelet counts of SW mice. M-CSF, 8 mg/kg/d, was administered to
SW mice in two daily IP injections, 8 hours apart. Mice initially
received M-CSF for 11 days, were rested for 4 days, and then received
M-CSF on days 16 to 24 and 36 to 43. The mean platelet count ±
1 SE is shown, n = 3 to 4.
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Effect of M-CSF on circulating blood cell levels.
The effect of M-CSF, 4 mg/kg/d, on the blood cell counts of normal mice
was documented. Blood samples were obtained on days 3 and 5 of M-CSF
treatment, and days 8 and 10, after completion of the 5 days of M-CSF
treatment. Total nucleated WBC counts, platelet counts, and hematocrit
values were obtained. Differential cell counts were performed on
cytocentrifuged buffy coat preparations in which monocytes had been
specifically stained (see Materials and Methods) (Fig
3), and neutrophils and lymphocytes were
identified by standard morphologic characteristics. Total monocytes
were significantly increased over control levels of 0.28 ± 0.4
× 103/µL (mean ± 1 standard error
[SE]) on days 3 (0.85 ± 0.17 ×
103/µL) (P < .05) and 5 (0.99 ± 0.25
× 103/µL) (P < .05) of M-CSF
treatment (Fig 4). The platelet count was
significantly and maximally decreased from normal levels at the time of
maximum absolute monocytosis. The total WBC count was significantly
increased only on day 8, from the control level of 6.0 ± 0.3
× 103/µL to 8.9 ± 0.8 ×
103/µL (P < .05). The hematocrit fell from an
initial value of 52.0% ± 0.7% to a nadir of 44.6% ± 1.3% on
day 5 of M-CSF treatment, and then gradually returned to normal.
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| Fig 3.
(A) Representative field from a cytospin preparation of
buffy coat from a normal mouse, stained as described in Materials and
Methods. Black arrow indicates a monocyte identified by the dark blue
precipitate. White arrow indicates a neutrophil nucleus (final
magnification × 1,000). (B) Representative field from a cytospin
preparation of buffy coat from a mouse treated with M-CSF, 4 mg/kg/d,
for 4 days. Cells were stained as described in Materials and Methods.
Black arrows indicate six monocytes identified by the dark blue
precipitate. Note the relative increase in the number of monocytes
(final magnification × 1,000).
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| Fig 4.
The effect of M-CSF on the differential WBC counts of SW
mice. M-CSF, 4 mg/kg/d, was administered to SW mice for 5 days, on days
1 to 5. Blood samples were obtained and the total WBC and platelet
counts were determined. Percentages of monocytes, neutrophils, and
lymphocytes were determined from buffy coat cytofuge preparations.
Neutrophils and lymphocytes were identified by standard morphologic
characteristics. Monocytes were identified by histochemical staining,
using M-CSF conjugated to alkaline phosphatase to bind to the M-CSF
receptor. Absolute total numbers of nucleated WBC are indicated by the
height of the bars. The total WBC count was significantly increased
only on day 8 (#, P < .05). The percentages of monocytes on
each day are indicated in parentheses at the top of each bar. On day 3
(n = 6) and day 5 (n = 5), the total numbers of monocytes were
significantly different from the control (n = 4) (, P <
.05). On day 8 (n = 6) and day 10 (n = 6), total numbers of
monocytes were not different from the control.
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Effect of splenectomy on M-CSF-induced thrombocytopenia.
The possible role of splenic platelet sequestration in the production
of thrombocytopenia after M-CSF administration was examined.
Splenectomized animals were given M-CSF, 2 mg/kg/d, and the platelet
counts were compared with those of intact animals treated with the same
dosage. Platelet counts fell to equivalent levels in intact and
splenectomized SW mice during M-CSF treatment, and splenectomy did not
affect either the degree of the thrombocytopenia or subsequent rebound
thrombocytosis produced (Fig 5).
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| Fig 5.
The effect of M-CSF administration on the platelet counts
of eusplenic and asplenic SW mice. M-CSF, 2 mg/kg/d, was administered
for 5 days, in two daily IP injections, 8 hours apart, to normal mice,
or mice that had been splenectomized at least 4 weeks previously. Blood
samples were obtained immediately before the morning M-CSF injection on
days 1 to 5. The mean ± 1 SE is shown. The number of determinations
at each time point was at least 3, and usually ranged from 4 to 28
(mean, 12).
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Effect of M-CSF on colony-forming cells.
Soft agar cultures were performed to determine the total number of
GM-CFC and Meg-CFC in spleen and bone marrow. M-CSF, 2 mg/kg/d, was
administered to mice for up to 5 days, and total colony numbers were
determined on days 2, 4, 5, 6, and 8. Both GM and Meg colony numbers
remained normal on day 2 and then were increased twofold to sixfold
over controls in the spleens of M-CSF-treated mice on days 4 to 8. The
maximum increase in the spleen occurred on days 5 and 6. However, there
were no differences between the total numbers of detectable GM-CFC or
Meg-CFC present in the bone marrows of M-CSF-treated and normal mice
(data not shown).
Effect of M-CSF on DNA levels in megakaryocytes.
The effect of M-CSF on the ploidy distribution of megakaryocytes in
C57BL mice was analyzed. M-CSF, 2 mg/kg/d, was administered on days 1
to 5. DNA content (ploidy) of bone marrow megakaryocytes was analyzed
on days 3 to 7 (Fig 6). On days 4 to 7, the
proportion of 32N, 64N, and 128N megakaryocytes was significantly
increased over control levels (Fig 6C through F). However, although the
proportion of 16N megakaryocytes decreased on days 6 and 7 as the
higher ploidy megakaryocytes were increasing, 16N megakaryocytes
remained the modal class. A higher dose of M-CSF, 8 mg/kg/d, which
produced more severe thrombocytopenia, also significantly increased the
proportion of 32N, 64N, and 128N classes on days 6 and 7 (Fig 6G
through H), and in addition, 32N became the modal class on day 7.
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| Fig 6.
The ploidy distribution of bone marrow megakaryocytes
after M-CSF administration. M-CSF was administered at the indicated
doses to C57BL mice, starting on day 1, for up to 5 days. Bone marrow
was harvested on the morning of the indicated days, before M-CSF
administration. DNA levels (ploidy) of megakaryocytes were determined
by flow cytometry. Each panel shows the DNA distribution (ploidy class)
on the abscissa and the mean frequency ± 1 SE of each ploidy class as
a percentage of all megakaryocytes on the ordinate. One million bone
marrow cells were analyzed from each animal. The number of
determinations at each time point, representing individual animals, is
shown on each panel. (A) Illustrates the ploidy distribution of normal
C57BL mice. (B through F) Demonstrate the ploidy distributions on the
indicated days following administration of 2 mg/kg/d M-CSF. (G through
H) Demonstrate the ploidy distributions on days 6 and 7 following the
administration of 8 mg/kg/d M-CSF. The asterisks in panels C through H
indicate that the frequencies of these ploidy classes were
significantly different from control (P < .05).
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Effect of M-CSF on platelet survival.
To more thoroughly investigate the possible cause of thrombocytopenia
and the role of the spleen, platelet survival in M-CSF-treated
eusplenic and splenectomized mice was determined. M-CSF, 4 mg/kg/d, was
administered to groups of eusplenic and asplenic SW mice starting on
day 1. On day 4, pooled platelets harvested from normal mice and
labeled with CMFDA were injected intravenously into the treated mice
and a control group. M-CSF injections continued through day 5, and
platelet survival was measured for 93 hours after injection of labeled
platelets (Fig 7). The circulating
half-life (T1/2) of the labeled platelets in the control
group was 32.9 ± 2.3 hours, and platelet survival, as measured by
the multiple hit model (gamma function) was 2.58 ± 0.18
days. The T1/2 of the labeled platelets was markedly
reduced to 19.0 ± 0.7 hours in the eusplenic M-CSF-treated mice,
and to 16.3 ± 1.7 hours in the asplenic mice (for both,
P < .001 v control). Platelet survival was
significantly shorter in M-CSF-treated recipient animals (1.19 ±
0.07 days in eusplenics and 1.06 ± 0.15 days in asplenics [for
both, P < .001 v control]). There was no difference
between the circulating half-life (T1/2) or platelet
survival in eusplenic and asplenic animals. Platelet survival also was
examined in animals in which M-CSF, 4 mg/kg/d, was administered for 8
days before the injection of labeled platelets and was continued
throughout the experiment. The T1/2 of the labeled
platelets in these animals was 26.2 ± 3.1 hours, and the platelet
survival was 2.07 ± 0.43 days (P < .01 v control)
(data not shown). Similar results were obtained for all these
experimental groups when platelet survival estimates also were
calculated using the best fit method (data not shown).
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| Fig 7.
Effect of M-CSF on platelet survival. Eusplenic SW mice,
or SW mice that had been splenectomized at least 4 weeks previously,
were treated with M-CSF, 4 mg/kg/d for 3 days. On the fourth day, M-CSF
was administered in the morning, and 3 hours later approximately 2.5
× 108 platelets that had been fluorescently labeled with
CMFDA were injected into the tail veins of these mice. A control group
of mice that had not received M-CSF also was injected with the same
number of platelets. The percent of the maximum number of circulating
labeled platelets was serially determined for 93 hours by flow
cytometry, and the means ± 1 SE are shown by the closed symbols. The
mean T1/2 for platelet survival in the control animals
( ) was 32.9 hours (n = 5). The T1/2 in the eusplenic
M-CSF treated animals ( ) (19.0 hours; n = 7) and in the asplenic
M-CSF-treated animals ( ) (16.3 hours; n = 7) were both
significantly shorter than that of the controls (P < .001).
The T1/2 values for platelet survival in the 2 groups of
M-CSF-treated animals were not significantly different from each
other. Serial platelet counts for each group were obtained and are
shown with the corresponding open symbols in the inset graph.
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These results indicated faster clearance of platelets from the
circulation in mice that had received M-CSF, that the spleen was not
the primary mediator of this process, and that with prolonged
administration of M-CSF, platelet survival remained significantly
shortened, at a time when the platelet counts had returned to normal
levels (Fig 1B). However, it was unclear whether the normal donor
platelets were modified by exposure to M-CSF present in the circulation
of the recipient mice, and thus had demonstrated shortened survival.
Therefore, we performed additional platelet survival studies, using
platelets from M-CSF-treated donors or normal donors. Donor mice
received M-CSF, 4 mg/kg/d, for 2 days. On the morning of the third day,
a dose of 2 mg/kg M-CSF was administered, and 2 hours later platelets
were harvested from these and a group of untreated mice. Each pool of
platelets was labeled with CMFDA and injected into normal mice, and
survival of the untreated and M-CSF exposed platelets was compared (Fig
8). The M-CSF-exposed donor platelets
exhibited a normal or slightly prolonged T1/2 in normal
mice in comparison to untreated platelets, indicating that the
M-CSF-treated platelets had not been altered by exposure to M-CSF in a
manner that rendered them more susceptible to clearance.
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| Fig 8.
Survival of normal or M-CSF-exposed CMFDA labeled
platelets in normal SW mice. SW mice received M-CSF, 4 mg/kg/d,
administered in two daily IP injections, 8 hours apart. Two hours after
the fifth dose (on the third morning), platelets were harvested from
these animals and from additional nontreated control animals. The two
pools of platelets were then fluorescently labeled with CMFDA and
approximately 2.5 × 108 platelets were injected into the
tail veins of two groups of normal recipient mice (n = 9 for the
control group and 10 for the M-CSF group). The percent of the maximum
number of circulating labeled platelets was serially determined for 93
hours by flow cytometry. The mean ± 1 SE is shown for each time
point. The mean T1/2 for control platelet survival was 35.3
± 0.9 hours ( ± 1 SE) and for M-CSF-exposed platelet
survival was 45.0 ± 1.4 hours ( ± 1 SE) (P <
.0001). The asterisks indicate a significant difference in the percent
of circulating labeled platelets at each indicated time point
(P < .05).
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Effect of M-CSF on the monocyte/macrophage (reticuloendothelial)
system.
To evaluate the effect of M-CSF on the monocyte/macrophage system,
carbon clearance experiments were performed in mice that had been
treated with M-CSF, 4 mg/kg/d, for various lengths of time. Absorbance
(at 620 nm) due to the presence of carbon particles was monitored
beginning at 1 minute after bolus injection to ensure homogeneous
intravascular distribution of particles. Clearance of intravenously
injected India Ink particles was significantly faster in mice that had
been treated with M-CSF for 3, 5, or 9 days (Fig
9). The T1/2 of the carbon
particles in mice treated for 3 days was 3.7 ± 0.4 minutes; for 5
days, 2.8 ± 0.2 minutes; and for 9 days, 3.9 ± 0.5 minutes,
compared with the control value of 12.0 ± 0.7 minutes (for all,
P < .0001). Furthermore, the measured 1-minute absorbance
values (A620) were 1.091 for the control, 0.717 for the 3-day group,
0.566 for the 5-day group, and 0.637 for the day 9 group, indicating
that there was greater clearance of carbon particles during the first
minute in M-CSF-treated mice than in controls. Carbon clearance
remained faster than normal in mice treated for 9 days with M-CSF,
indicating that the monocyte/macrophage system remained hyperactive
despite recovery of the platelet count.
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| Fig 9.
The effect of 3, 5, or 9 days of M-CSF administration on
carbon particle clearance in SW mice. Mice were injected with 4 mg/kg/d
M-CSF, administered in two daily IP injections, 8 hours apart, starting
on day 1. Mice received the final injection of M-CSF on the morning of
the indicated day, approximately 2 hours before the injection of the
carbon particles on day 3, 5, or 9. The mean absolute absorbance of
lysed blood at 620 nm at 1 minute was 1.091 (n = 12) for the control
animals, 0.717 (n = 6) for mice treated with 3 days, 0.566 (n = 6)
for mice treated with 5 days, and 0.637 (n = 6) for mice treated with
9 days of M-CSF. These values were designated as 100%, and subsequent
results were calculated as a percentage of the 1-minute values. The
means ± 1 SE are shown. The T1/2 values obtained after 3
days (3.7 ± 0.4 minutes), 5 days (2.8 ± 0.2 minutes), and
9 days (3.9 ± 0.5 minutes) of M-CSF treatment were significantly
shorter (faster clearance) than the T1/2 in the control
mice (12.0 ± 0.7 minutes) (P < .0001), but were not
significantly different from each other.
|
|
Effect of M-CSF on the liver, spleen and lung weights.
In selected experiments, livers, spleens, and lungs were removed from
M-CSF-treated animals after sacrifice. After administration of M-CSF,
4 mg/kg/d for 5 days, the livers and spleens of treated mice were
significantly larger than those of the control animals. On day 5, liver
weights were increased to 2.0 g (n = 14), from the control value of 1.3
g (n = 19) (P < .001), but by day 9, they were 1.4 g, almost
normal. Spleen weights were increased to 250 mg on day 5 (n = 14), in
contrast to the control value of 136 mg (n = 19) (P < .001)
and remained increased (208 mg) on day 9 (n = 9). Total lung weights
were not significantly increased on day 5 or day 9 from the control
value of 157 mg (n = 8). After 9 days of administration of the same
dose of M-CSF, liver weights were comparably increased to 2.0 g (n =
4), and spleen weights were increased further to 289 mg (n = 4).
Effect of M-CSF on proplatelet formation.
Proplatelet formation was quantified as a potential indicator of
stimulation of platelet production. M-CSF, 4 mg/kg/d, was administered
for up to 5 days, and platelet differentials were performed on days 3,
5, and 9. At these times, when platelet levels were either falling (day
3), at their nadir (day 5), or increasing (day 9), the proportion of
proplatelets was not increased (Table 1).
However, the absolute numbers of proplatelets circulating on day 5 were
significantly decreased from control levels, and conversely, were
significantly increased on day 9 (Table 1).
| |
DISCUSSION |
Our studies have confirmed previous reports that M-CSF can produce
thrombocytopenia.11,12,15,16,21,22 In addition, we have
attempted to define the mechanisms of the production of
thrombocytopenia by M-CSF and of the recovery of platelet levels during
its continued administration. Treatment with M-CSF for 5 days produced
dose-dependent thrombocytopenia, with rapid recovery of normal platelet
levels and subsequent development of rebound thrombocytosis occurring
after discontinuation of treatment. However, in experiments in which
M-CSF was administered over a longer period, platelet levels
surprisingly began to recover after 5 days, despite continued
administration of M-CSF, strongly suggesting that thrombocytopenia was
not the result of suppression of platelet production. Additional
evidence for this hypothesis was that the ploidy distribution of
megakaryocytes in the bone marrow remained essentially normal during
the period of falling platelet levels, which reached their nadir on
days 4 to 5. In contrast, in a model of bone marrow damage, produced by
administration of 5-fluorouracil (5-FU) to mice, the proportion of
higher ploidy megakaryocytes decreased initially, with 8N becoming the
modal class on day 4.40 After M-CSF, we did not observe a
decrease in the proportion of 16N and 32N megakaryocytes, but rather
observed a slight right shift in ploidy. No changes were observed in
the total GM-CFC and Meg-CFC in the bone marrow following M-CSF, in
contrast to the marked increases observed during recovery from bone
marrow damage produced by 5-FU.41 The rapidity of the
recovery to normal or above normal platelet levels after termination of
M-CSF treatment also argues against bone marrow suppression.
Additionally, we saw evidence of delayed stimulation of platelet
production. The increased frequency of high ploidy megakaryocytes on
days 4 to 7 is consistent with previous observations that ploidy levels
shift upward in response to different degrees of thrombocytopenia
produced by peripheral destruction of platelets, and that maximal
changes in ploidy occur 48 to 72 hours after the stimulus of
thrombocytopenia, preceding the recovery of normal platelet
levels.42 The delayed increase in splenic Meg-CFC (and
GM-CFC) is also consistent with the previously reported
response of normal murine hematopoiesis to the stimulus of peripheral
platelet destruction.31,43 Preliminary data have indicated
that the mean platelet volume is increased after 3 or 5 days of M-CSF
administration, consistent with stimulation of thrombopoiesis. The
occurrence of rebound thrombocytosis during recovery of platelet
levels, as well as the less severe nadir of thrombocytopenia observed
after repeated exposure to M-CSF are consistent with the development of
an expanded pool of megakaryocytes that resulted from this stimulation.
There was a gradual decrease in maximum observed M-CSF levels during
the treatment period; however, M-CSF remained detectable after 5 days
of administration, and no anti-M-CSF antibodies were present at this
time. Clearance rates increased after repeated exposure to M-CSF, in
agreement with previous reports.11,13,14,44 These
observations are consistent with an increased monocyte/macrophage
population and the proposed mechanism of monocyte/macrophage-mediated
clearance of M-CSF.11 However, M-CSF remains efficacious
after continued administration. Increased macrophage activity was still
present after prolonged administration of M-CSF, as evidenced by our
platelet survival and carbon clearance data. The ability to repeatedly
produce thrombocytopenia during multiple courses of M-CSF
administration further indicated that development of neutralizing
antibody against M-CSF was not responsible for the phenomenon of
recovery of platelet levels during short-term M-CSF administration.
The most likely cause for the thrombocytopenia appeared to be either
peripheral destruction or platelet sequestration. We have documented a
maximum increase in the level of circulating monocytes after M-CSF
administration at the time of the platelet nadir, an inverse
relationship that has been previously reported.12,13,25
Evidence for increased removal of platelets from the circulation is the
observation that platelet survival was decreased in M-CSF-treated
mice. The role of the spleen in the sequestration or destruction of
platelets in this model, though, is minimal or absent, because asplenic
animals exhibited the same degree of thrombocytopenia as eusplenic mice
and equivalently shortened platelet survival after M-CSF
administration. However, in humans, M-CSF administration resulted in
shortened platelet survival with marked platelet uptake occurring in
the spleen.25 Because platelet survival remained shortened
during prolonged M-CSF administration, at a time when platelet levels
were increasing, it appeared that increased platelet production,
stimulated by thrombocytopenia, had compensated for the increased
platelet clearance. Exposure of platelets to M-CSF did not appear to
produce a platelet defect, because platelets obtained from
M-CSF-treated donors did not exhibit shorter survival than nonexposed
platelets prepared in the same manner, after transfusion into normal
mice.
M-CSF treatment increased the size of the liver and spleen in treated
animals, a common result of stimulation of the monocyte/macrophage
system, which may have contributed to the observed thrombocytopenia.
Our detection of increased rates of carbon particle clearance after
M-CSF administration is consistent with a mechanism for the development
of thrombocytopenia that includes the production of activated
macrophages. There is precedence for a nonimmune process of platelet
removal by macrophages. In the reactive hemophagocytic syndrome,
activated macrophages (designated histiocytes) have been shown to
phagocytose blood cells, including platelets.45,46
Interestingly, M-CSF levels are markedly increased in at least some
patients with this syndrome.47 Following administration of
M-CSF, macrophages from bone marrow aspirates in patients25
and circulating macrophage-like cells in rabbits15 have
been reported to demonstrate phagocytosis of platelets. We attempted to
determine if Kupffer cells were responsible for the clearance of
platelets, by injecting mice with carrageenan, because these cells can
be selectively blocked with carrageenan.10 However, this
reagent cannot be used to evaluate the role of Kupffer cell activity in
production of thrombocytopenia, because we confirmed that carrageenan
itself causes acute, severe thrombocytopenia.48
Administration of other cytokines, such as granulocyte-macrophage
colony-stimulating factor (GM-CSF),49,50
G-CSF,51,52 interleukin (IL)-1,53,54
IL-2,55,56 and stem cell factor (SCF)57,58 also
has been shown to produce dose-dependent thrombocytopenia, which has
been reported in some studies to resolve during continued
administration,50,51,54,57 indicating that this is a
phenomenon not unique to M-CSF. Although the mechanism(s) of
thrombocytopenia associated with these other cytokines has not been
established, descriptions of normal bone marrow function after cytokine
treatment,49,51,55,59 data suggesting peripheral
destruction of platelets,49-51,55 and reports of large
platelets or increased mean platelet volume (MPV) at the
platelet count nadir51,59 suggest that suppression of
megakaryocytopoiesis is probably not the mechanism of the
thrombocytopenia. In a single dog, GM-CSF-induced thrombocytopenia was
not prevented by splenectomy, similar to our finding in mice, but
thrombocytopenia persisted for 4 to 5 weeks after discontinuation of
treatment,50 in contrast to our results in mice. However,
the thrombocytopenia produced by IL-1 administration to mice was
prevented by splenectomy,53 suggesting a different role for
the spleen in that model.
We have concluded that thrombocytopenia produced by M-CSF was due to
increased activity of the monocyte/macrophage system and that increased
removal of platelets from the circulation, primarily due to
phagocytosis by the liver (and perhaps bone marrow) macrophages,
occurred as a result of some as yet unidentified nonimmune process. The
return to normal platelet levels despite continued administration of
M-CSF suggested that increased platelet production, stimulated by
thrombocytopenia, was able to compensate for the increased rate of
removal of platelets from the circulation. Further experiments are
needed to determine whether M-CSF-induced thrombocytopenia can be
prevented in normal or bone marrow damaged individuals by prior or
concurrent administration of a stimulator of thrombopoiesis, such as
thrombopoietin.60,61 This might eliminate the dose-limiting
toxicity of M-CSF and permit further investigation of its clinical
potential.
| |
FOOTNOTES |
Submitted April 7, 1997;
accepted August 28, 1997.
Supported in part by US Army Medical Research, Development,
Acquisition, and Logistics Command, Fort Detrick, MD, Research Contract
MIPR No. MM4585HL7. Also supported in part by the Department of
Veterans Affairs, Washington, DC.
Opinions, interpretations, conclusions, and recommendations are those
of the authors and are not necessarily endorsed by the US
Army.
Address reprint requests to Jack Levin, MD, Veterans Administration
Medical Center (111 H2), 4150 Clement St, San Francisco, CA 94121.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr Jolanda Schreuers (formerly of Chiron Corp) for
helpful discussions and assistance in making these studies possible,
and Dr E. Richard Stanley, Albert Einstein College of Medicine, whose
laboratory determined the M-CSF levels and anti-M-CSF antibody titers.
| |
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Abstract
Introduction
Abstract