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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2515-2524
By
From the Children's Hospital of Orange County, Orange, CA.
We have previously demonstrated a significant inverse correlation
between circulating thrombopoietin (TPO) levels and peripheral platelet
(PLT) counts in patients with thrombocytopenia secondary to
megakaryocytic hypoplasia but not in patients with immune
thrombocytopenic purpura (ITP; Chang et al, Blood 88:3354,
1996). To test the hypothesis that the differences in the circulating
TPO levels in these two types of thrombocytopenia are caused by
differences in the total capacity of Mpl receptor-mediated TPO
clearance, thrombocytopenia was induced in female CD-1 mice either by
sublethal irradiation (irradiated) or rabbit antimouse PLT serum
(RAMPS) for 1 day (1 d RAMPS) and 5 days (5 d RAMPS). A
well-characterized murine model of autoimmune thrombocytopenic purpura,
male (NZW × BXSB) F1 mice (W/B F1), was also
included in this study. All thrombocytopenic mice and their controls
received trace amounts of 125I-recombinant murine TPO
(125I-rmTPO) intravenously and were killed 3 hours postinjection. Blood cell-associated radioactivity was
significantly decreased in all 4 groups of thrombocytopenic mice.
Significantly increased plasma and decreased whole spleen-associated
radioactivity was observed in the irradiated group compared with
controls (P < .05). While a lesser but still significant
increase in plasma and decrease in whole spleen-associated
radioactivity was observed in the 1 d RAMPS mice (P < .05),
there were no significant differences between the 5 d RAMPS nor the W/B
F1 male mice compared with controls, although whole
spleen-associated radioactivity was higher in the W/B F1
male. A significant inverse correlation of plasma and whole spleen-associated radioactivity was demonstrated in W/B F1
male mice (r =
THE PRIMARY REGULATOR of platelet (PLT)
production, thrombopoietin (TPO), identified as the ligand for the
c-mpl proto-oncogene product, has been isolated and cloned from several
species.1-6 Recombinant TPO enhances megakaryocyte colony
formation; increases the size, number, and ploidy of developing
megakaryocytes; and results in increased PLT production in vitro and in
vivo.3-5,7,8 Injection of recombinant TPO into mice,
neonatal rats, and nonhuman primates causes a 400% increase in the
circulating PLT count and increases spleen and bone marrow
megakaryocytes and their precursors.4,7,8 Additionally, in
both c-mpl and TPO-deficient mice, there is a significant decrease in
both circulating PLTs and spleen and marrow megakaryocytes.9-11
If TPO is the primary physiological regulator of PLT
production, circulating TPO levels would be expected to vary inversely with PLT demand. We have previously measured circulating TPO levels in
patients with comparable degrees of thrombocytopenia secondary to two
different mechanisms: (1) decreased PLT production due to
megakaryocytic hypoplasia (myeloablative therapy, submyeloablative therapy, and Fanconi's Anemia) and (2) decreased PLT life span with
normal or increased megakaryocyte mass (immune thrombocytopenic purpura
[ITP]).12 Circulating levels of TPO in the
plasma of all of the thrombocytopenic patients with megakaryocytic
hypoplasia were markedly elevated. A significant inverse correlation
between endogenous TPO levels and peripheral PLT counts was
observed in this group of patients. However, the circulating levels of
TPO present in the plasma of ITP patients with severe
thrombocytopenia remained undetectable. Similar differences in
circulating TPO levels between megakaryocyte hypoplastic and ITP
patients have been independently reported by other
laboratories.13-15
One of several different mechanisms that may regulate circulating TPO
levels16-18 was originally proposed by de Gabriele and Penington,19 and then by Kuter and Rosenberg16
after TPO was isolated, and suggested that TPO production is
constitutive and that plasma levels are controlled by the circulating
PLT levels. Based on the recent finding that PLTs express Mpl receptors
for TPO, Fielder et al20 further demonstrated that plasma
TPO levels are controlled by the circulating PLT levels through Mpl
receptor-mediated uptake and metabolism using mice lacking the Mpl
receptor. Consequently, a lower circulating PLT mass would have less
capacity for TPO uptake and metabolism, resulting in higher circulating
TPO levels. However, recent findings that Mpl receptor is expressed not
only on PLTs, but also on the megakaryocyte and its
progenitors21-23 suggest that circulating TPO levels may
not always depend exclusively on the absolute numbers of circulating
PLTs but may actually be regulated by the total cell mass of the
megakaryocyte lineage that expresses Mpl receptor. Therefore,
circulating TPO levels would be significantly increased in
thrombocytopenic patients with megakaryocytic hypoplasia due to the low
quantity of total Mpl receptor-expressing cellular mass. In contrast,
despite similar degrees of acute thrombocytopenia, the total number of
Mpl-positive cells, and consequently TPO uptake capacity, would be
higher in ITP patients due to increased numbers of megakaryocyte
progenitor cells and megakaryocytes and the continuous production of
new PLTs, which may still be able to take up TPO before
antibody-facilitated reticuloendothelial clearance, resulting in low or
undetectable levels of TPO.
To test this hypothesis, we developed several murine models that mimic
various forms of human thrombocytopenia. CD-1 female mice were treated
with either sublethal irradiation, which induced thrombocytopenia
secondary to megakaryocytic hypoplasia, or rabbit antimouse PLT serum
(RAMPS) for 1 day and 5 days, which shortened PLT half-life. One-day
RAMPS treatment represents a model of pre-steady-state ITP conditions.
Five-day RAMPS treatment more closely mimics steady-state conditions of
ITP in which megakaryocytopoiesis and thrombopoiesis are already
significantly accelerated in response to immune
thrombocytopenia.24,25 In addition, another
well-characterized murine model of autoimmune thrombocytopenic purpura,
male W/B F1 mice, which spontaneously develop
thrombocytopenia with age, showing reduced PLT life-spans, increased
PLT-associated autoantibodies, and PLT-binding serum antibodies,26,27 was also included in the study together
with their nonthrombocytopenic female littermates as controls.
Radiolabeled TPO uptake capacity and organ distribution between normal
controls and these thrombocytopenic models were compared. The results
are consistent with the hypothesis that TPO uptake and, consequently, catabolic capacity during steady states of immune thrombocytopenia remained significantly higher than that of megakaryocyte hypoplastic hosts, which could account, at least in part, for the low levels of
circulating TPO demonstrated in humans during steady-state immune-mediated thrombocytopenia.
Animals.
Eight-week-old female CD-1 mice (Charles River Laboratories, Hollister,
CA) were maintained at constant room temperature with free access to
food and water for at least 1 week before use. Approval for this study
was granted by the Vivarium Committee at Children's Hospital of Orange
County (CHOC; Orange, CA). Thrombocytopenia was induced in 1 group of
CD-1 mice by 550 cGy total body irradiation from a 6 mV x-ray linear
accelerator at 60 cGy/min (Clinac 6/100, Palo Alto, CA).
Thrombocytopenia was induced in a second group of mice by
intraperitoneal injection for 1 day or every other day for a 5-day
period with 50 µL RAMPS that had been adsorbed against erythrocytes
and leukocytes (Inter-Cell Technologies Inc, Hopewell,
NJ). Immediately before irradiation or antiserum injection, and at
intervals thereafter, 10 µL of mice blood was collected by nicking
the tail veins of the animals with a sterile 27-gauge needle. Blood
samples were electronically counted (Serano-Baker Diagnostics,
Allentown, PA) using a mouse cell discriminator to determine PLT counts
and mean PLT volume.
Tissue distribution of 125I-recombinant murine
TPO (125I-rmTPO).
Full-length, biologically active rmTPO was iodinated using the Indirect
Iodogen method described previously.20,23 The specific activity of the 125I-rmTPO was 80 µCi/µg protein.
Iodinated rmTPO retained approximately full biological activity as
determined by its ability to bind to the c-Mpl receptor on mouse PLTs
(data not shown).23 To test the stability of the labeled
protein, purified 125I-rmTPO was incubated with murine
PLT-poor plasma for more than 40 hours at 37°C. Examination using
10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and autoradiography showed virtually no degradation (data not shown).20 Administration of sodium iodine to
prevent accumulation of 125I in the thyroid of the animals
and administration of 125I-rmTPO intravenously into control
and thrombocytopenic mice were as described
previously.20,23 CD-1 thrombocytopenic mice received 125I-rmTPO either 10 days postirradiation (irradiated
group), 1 day (20 to 24 hours) after single injection of RAMPS (1 d
RAMPS group), or 5 days after initial injection of RAMPS, which was
repeated every other day during that period of time. W/B F1
male and female mice received 125I-rmTPO at 4 to 5 months
of age. All mice received 0.1 mL of 125I-rmTPO (4 µCi)
and were killed 3 hours postinjection. The following tissues were
collected immediately after the animals were killed: blood (0.38%
citrate), femur, heart, kidney, liver, lung, spleen, and sternum.
Tissues were sectioned, rinsed, and weighed as described previously.20,23 Tissue-associated radioactivity was
determined by counting for 1 minute in a gamma counter (LKB/Wallac
1282; Wallac, Inc, Turku, Finland).
Histopathology.
Zinc formalin-fixed, paraffin-embedded tissues were sectioned (5-µm
thickness) and stained with hematoxylin and eosin. Histologic sections
were examined by light microscopy with a Zeiss microscope (standard
16). In spleen and marrow sections, the number of megakaryocytes was
evaluated using the 40× objective and 10× eyepiece (~0.1
mm2 per field) with random selection and counting in a
minimum of 40 fields. Megakaryocyte counts were performed by observers
blinded to the different subgroups of animals. Counts are expressed as number of megakaryocytes per 10 high power fields (hpf).
Electron microscopic autoradiography.
Spleen and sternum collected at autopsy from both control and
thrombocytopenic mice that received a single intravenous bolus injection of 125I-rmTPO were processed for electron
microscopy autoradiography.23,28 Briefly, the tissues were
cut into approximately 2-mm cubes, fixed in Karnovsky's fixative (2%
paraformaldehyde, 2.5% glutaraldehyde in cacodylate buffer), postfixed
in osmium tetroxide, dehydrated, and embedded in Eponate 12 media (Ted
Pella Inc, Redding, CA). Thin sections were coated with Ilford L4 EM
autoradiography emulsion (Ilford, Warrington, PA) and exposed for 3 to
8 weeks. Developed sections were stained with lead citrate and uranyl
acetate before observation in a CM12 Philips electron microscope
(Philips Corp, Eindhoven, The Netherlands).
Statistical analysis.
All results are expressed as the mean values plus or minus standard
deviation (SD) of three or more samples. The probability of significant
differences when examining multiple treatments was determined using
one-way analysis of variance (ANOVA) or analogous nonparametric tests
(Kruskal-Wallis test), followed by Tukey's or Dunn's multiple
comparison test to define unique subsets within the study. The
correlation between two sets of variables was determined by linear
correlation test (Pearson's test). P values less than .05 were
considered significant. Statistical analyses were performed using the
Instat (Graphpad, San Diego, CA) or Sigmastat (Jandel Scientific, San
Rafael, CA) statistical software programs.
PLT values in normal and thrombocytopenic mice.
Control CD-1 mice had a mean PLT count of 1,157.5 ± 91.6 × 103/µL (n = 8). The posttreatment PLT counts from
irradiated and 2 RAMPS mice are shown in
Fig 1. Both radiation and RAMPS treatments resulted in severe thrombocytopenia. W/B F1 males at an age
of about 4 months showed a marked reduction in PLT count (476.7 ± 260.94 × 103/µL; range, 836 to 232 × 103/µL) compared with those at an age of 1 month (1,105 ± 134 × 103/µL),29 which was also
significantly lower than that of their nonthrombocytopenic female
littermates (1,313.2 ± 287.2 × 103/µL, n = 11, P < .0001) and CD-1 controls (1,157.5 ± 91.64 × 103/µL, n = 8, P < .05).
Tissue distribution of 125I-rmTPO in normal and
thrombocytopenic mice.
The in vivo tissue content of 125I-rmTPO in various organs
of both control and thrombocytopenic mice is presented in
Table 1. Tissue distribution of
125I-rmTPO in both control groups was very similar to that
reported previously.23 The whole blood and the spleen
contained the most radioactivity, and greater than half of the
radioactivity in the whole blood was due to 125I-rmTPO
binding to washed blood cell fractions. Although radioactivity was
found in all of the highly perfused tissues, the spleen was the only
organ which contained a greater amount per gram than did blood (Table
1).
Megakaryocytes in the spleens and marrows of control and
thrombocytopenic mice.
We next examined the number and morphology of megakaryocytes within the
spleens and marrows of the various thrombocytopenic mice. Compared with
the control spleen samples, the spleens of the irradiated groups were
hypocellular. Thorough examination of the entire spleen sections from
the irradiated mice demonstrated very few, if any, visible
megakaryocytes (Fig 6 and
Table 3). In contrast, examination of
spleen sections taken from both 5 d RAMPS and W/B F1 males
showed a substantial increase in overall cellularity and megakaryocyte
numbers compared with controls (Fig 6 and Table 3). This, together with
their larger megakaryocyte size and increased spleen weight (Fig 3),
indicated that megakaryocyte mass in the entire spleens of these mice
was the highest among the thrombocytopenic mice. Examination of the
spleens from 1 d RAMPS mice showed a slightly increased number of
megakaryocytes per 10 hpf compared with that of controls (Table 3). The
number of megakaryocytes per field within the marrow of the control
mice was markedly higher than that of the spleen. Megakaryocytes within the marrow of 5 d RAMPS and W/B F1 males were also approximately 90%
and 75% greater in number, respectively, and larger in size than
controls (data not shown), consistent with earlier
studies,25,26 whereas megakaryocyte numbers were only
approximately 40% higher in 1 d RAMPS mice than in controls (data not
shown). Although megakaryocytes could be observed in some portions of
the marrow in the irradiated mice, the overall number was reduced
compared with controls (data not shown). In general, changes in the
marrow megakaryocyte mass of thrombocytopenic mice seemed to be less pronounced than in the spleens.
Electron microscopic autoradiograph.
The localization of 125I-rmTPO retained in the spleen and
sternum of control and thrombocytopenic mice was analyzed using
ultrastructural autoradiography. Autoradiographic silver grains
indicating the presence of 125I-rmTPO were found associated
mainly with PLTs in the spleen and megakaryocytes in the marrow of W/B
F1 female control mice, consistent with a previous
report.23 Close examination of the autoradiographs indicated that 125I-rmTPO had been internalized by the PLTs
(data not shown). In W/B F1 male mice,
125I-rmTPO was also associated with the PLTs and the
megakaryocytes in the spleen and marrow. The labeled PLTs could be
found within splenic macrophages (data not shown). The spleens of
irradiated mice contained no morphologically identifiable PLTs or
megakaryocytes and, as expected, only background levels of
autoradiographic silver grains were found in these spleens (data not
shown). 125I-rmTPO could not be specifically detected in
neutrophils, lymphocytes, erythrocytes, stromal cells, or endothelial
cells (data not shown).23
Relative PLT 125I-rmTPO binding capacity of control and
thrombocytopenic mice.
To test the hypothesis that accelerated thrombopoiesis in ITP mice
could lead to the formation of abnormally large PLTs, as in human
ITP,30 and that these larger PLTs may bind and take up more
TPO than those of controls,13 the mean PLT volumes of the
control, irradiated, and various immune thrombocytopenic mice were
measured. The mean PLT volumes in all 3 groups of immune thrombocytopenic mice, but not in the irradiated mice, were indeed significantly higher than that of the control (P < .05;
Table 4). We further compared the bound
radioactivity per million PLT of each of these groups. Bound
radioactivity per million PLT was significantly higher in the 2 RAMPS
groups compared with the control (P < .05; Table 4). Similar
to spleen and bone, PLTs in W/B F1 male mice also seemed to
take up an amount of 125I-rmTPO comparable to that of
controls (Table 4), even though the labeling levels of their whole
blood and their nonhematopoietic tissues were only half of the female
controls (Table 1).
We hypothesized that differences between the circulating TPO levels in
thrombocytopenia secondary to megakaryocyte hypoplasia versus shortened
platelet life span are caused by differences in the total mass of
residual Mpl+ cells and, consequently, total capacity of
Mpl receptor-mediated TPO uptake and clearance during thrombocytopenia.
To test this hypothesis, we compared the 125I-rmTPO uptake
capacity and organ distribution in control, irradiated, and various
forms of immune thrombocytopenic mice. As in thrombocytopenic humans,
the level of plasma-associated 125I-rmTPO in mice with
thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated
group) was significantly higher than that of the immune
thrombocytopenic mice (Table 1 and Fig 2B), despite the absence of
significant differences in the PLT counts or the blood
cell/PLT-associated 125I-rmTPO between the irradiated and
immunothrombocytopenic mice (Table 1 and Fig 2A). Among all of the
tissues studied, only the spleen- and plasma-associated radioactivity
levels were inversely correlated (r = The authors gratefully acknowledge Dr Mitchell S. Cairo (Georgetown
University) for his support, laboratory facilities, and helpful
discussions; Drs Paul Fielder, Eric Stenfanich, Gilbert-Andre Keller,
and Ramon Widmer (Genentech) for the generous gift of 125I-rmTPO, assistance with electron microscopic
autoradiography, and helpful discussions; Dr Yu Suen, Eva Knoppel,
Azita Nourani, and Sara Fernandez for their technical expertise in
animal experiments; Dr Mark Lones for evaluation and photography of the
histological sections of murine hematopoietic tissues; Judy Petella for
preparation of histopathological specimens; Lorie Higgins for scoring
of megakaryocyte numbers; Dr Bruce Liming for assisting with the animal
irradiation; Carmella van de Ven, Sandra Kulczyk, and Dr Francisco
Bracho for helpful discussions; and Sally Anderson and Linda Rahl for
editorial assistance in the preparation of this manuscript.
Submitted August 28, 1998; accepted December 1, 1998.
Supported by grants from the Pediatric Cancer Research
Foundation, the Walden W. and Jean Young Shaw Foundation, and
the Children's Hospital of Orange County Research and Education Foundation.
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.
Presented in part at the Annual Meeting of the American Society of
Hematology, December 1996, Orlando, FL, and December 1997, San Diego,
CA. Address reprint requests to Mei Chang, PhD, Hematology/Oncology
Research, Children's Hospital of Orange County, 455 S Main St, Orange,
CA 92868.
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TOP
ABSTRACT
INTRODUCTION
.91, n = 6, P < .05). There was
also a decrease in bone (femur)/blood-associated radioactivity in the
irradiated group compared with controls (P < .05), but a
significant increase in 1 d and 5 d RAMPS mice (P < .01).
Furthermore, the 125I-rmTPO uptake capacity within the
spleen and marrow of immune thrombocytopenic mice appeared to be
associated with a higher megakaryocytic mass when tissue samples were
examined by light microscopy. Internalization of 125I-rmTPO
by megakaryocytes and PLTs in the spleens and marrows of ITP mice was
also demonstrated directly using electron microscopic autoradiography.
Labeled PLTs were also found within splenic macrophages. Additionally,
the mean PLT volumes of RAMPS mice were significantly higher than those
of the control and irradiated mice (P < .05), as was the
bound 125I-rmTPO (cpm) per million PLT (P < .05).
Finally, significantly decreased 125I-rmTPO degradation
products were only found in the plasma of the irradiated mice compared
with control animals (P < .05). These data suggest that the
lack of Mpl+ cells in the mice with thrombocytopenia
secondary to megakaryocytic hypoplasia (irradiated) results in
decreased uptake and degradation of TPO and higher circulating TPO
levels. Furthermore, these data also suggest that, after a brief TPO
surge in response to immune thrombocytopenia (1 d RAMPS), the lack of
an inverse correlation of circulating TPO with PLT counts during
steady-state immune thrombocytopenic mice (5 d RAMPS + W/B
F1 male) is due, at least in part, to its uptake and
degradation by the high PLT turnover and increased mass of megakaryocytes.
6 per milliliter of
blood, assuming blood cell-associated radioactivity was primarily PLT
associated.
P < .05 when compared with
F1 female control.
P < .05 when compared with irradiated. 
