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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2515-2524
Tissue Uptake of Circulating Thrombopoietin Is Increased in
Immune-Mediated Compared With Irradiated Thrombocytopenic Mice
By
Mei Chang,
John X. Qian,
Sun min Lee,
John Joubran,
George Fernandez,
Jacqueline Nichols,
Annika Knoppel, and
Jeffrey S. Buzby
From the Children's Hospital of Orange County, Orange, CA.
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ABSTRACT |
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 =  .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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
Idiopathic thrombocytopenic purpura-prone male W/B F1 mice
were produced by cross-breeding NZW female and BXSB male (Jackson Laboratories, Bar Harbor, ME) in the animal facility of CHOC. These
animals were raised under specific pathogen-free conditions.
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).
Whole blood (0.1 mL) or plasma (0.05 mL) was counted to obtain the
total cpm per sample. Whole blood (0.1 mL) was diluted to 1 mL in
phosphate-buffered saline (PBS), vortexed, and centrifuged at
2,950g for 10 minutes, and the remaining blood cell-associated radioactivity was counted after aspiration of the liquid phase. To
determine protein (rm-TPO)-associated radioactivity in the plasma,
plasma samples (0.05 mL) were diluted to 0.5 mL in PBS containing 1.0%
bovine serum albumin, mixed with 0.5 mL of 20% trichloroacetic acid
(TCA), and incubated at 4°C for 15 minutes. After centrifugation at
2,950g for 10 minutes and aspiration of the liquid phase, the
remaining pellets were counted to obtain the TCA-precipitable counts
per 0.05 mL plasma. TCA nonprecipitable counts (degradation products of
125I-rmTPO as free isotope) in the plasma were defined as
the difference between total plasma cpm and TCA-precipitable cpm. The
bound radioactivity per million PLT (cpm/million) was defined as the
ratio between blood cell-associated radioactivity per milliliter of
blood and PLT count × 10 6 per milliliter of
blood, assuming blood cell-associated radioactivity was primarily PLT
associated.23
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.
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RESULTS |
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).
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| Fig 1.
PLT counts in thrombocytopenic CD-1 mice. Mice were
treated with 550 cGy total body irradiation (A) or a single injection
of 50 µL RAMPS (B) or injection of 50 µL RAMPS every other day (at
0, 48, and 96 hours) for a 5-day period (C). Values represent the mean ± SD for at least 3 animals in each group. *P < .05 when
compared with day (hour) 0. K = 103.
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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).
Significantly decreased blood cell-associated radioactivity, compared
with those of controls, was observed in all 4 groups of
thrombocytopenic mice, consistent with their corresponding degrees of
thrombocytopenia (P < .05; Table 1 and
Fig 2A). However, levels of
plasma-associated radioactivity (TCA-precipitable cpm) among these
groups differed significantly (Table 1 and Fig 2B). Irradiated
thrombocytopenic mice had significantly elevated plasma associated
radioactivity compared with control animals, the highest among all
groups (P < .05). In marked contrast, the 5 d RAMPS mice and
W/B F1 males had normal levels of plasma-associated
radioactivity compared with controls. One d RAMPS mice had a
plasma-associated radioactivity significantly higher than that of
control (P < .05) but still much lower than that of
irradiated mice (Table 1 and Fig 2B).
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| Fig 2.
Comparison of blood cell-bound and plasma-associated
radioactivity in control and thrombocytopenic mice. (A) Comparison of
blood cell-bound radioactivity. (B) Comparison of plasma-associated
radioactivity (TCA-precipitable cpm). Blood cell-bound radioactivity is
presented as cpm × 104/mL blood (mean ± SD).
Plasma-associated radioactivity is presented as cpm × 104/mL plasma (mean ± SD). *P < .05 when
compared with CD-1 control.  P < .05 when compared with
F1 female control.
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The tissue distribution pattern of 125I-rmTPO in the
irradiated group was very similar to that observed in
c-mpl/ mice.20 Irradiated mice
had the lowest spleen-associated radioactivity normalized by weight
among the various mice (11% of the normal CD-1 control value,
P < .001; Table 1) and, due to the reduced spleen weight in
this group (Fig 3B), also had whole
spleen-associated radioactivity that was only 3.9% of the control
group (P < .05; Fig 3A). Conversely, the spleen-associated
radioactivities of all 3 groups of mice with immune thrombocytopenia
were much higher than that of the irradiated group when normalized for
tissue weight (Table 1). Both 5 d RAMPS and W/B F1 male
groups had significantly increased spleen size and weight (P < .05), contributing to a whole spleen-associated radioactivity that
was either comparable to (5 d RAMPS) or higher than (W/B F1
male) that of the controls (Fig 3). Whole spleen-associated
radioactivity in the 5 d RAMPS mice was also significantly higher than
that of irradiated mice (P < .05). The 1 d RAMPS mice with
normal spleen weights, again, had a whole spleen-associated
radioactivity intermediate to that of irradiated and 5 d RAMPS mice
(Fig 3). The levels of whole blood-associated radioactivity
varied significantly among the thrombocytopenic groups (Table 1). To
account for possible differences in the amount of radioactivity
associated with blood trapped within the tissue, whole
spleen-associated radioactivity was also expressed as the percentage of
the whole blood-associated radioactivity. This further reduced the
value for the irradiated group to only 2.5% of the control.
Conversely, the value for the 5 d RAMPS group was increased to 22%
higher than that of the control (data not shown). However, the
statistical significance of the normalized differences comparing the
thrombocytopenic groups with controls or with each other (data not
shown) remained the same as the results described in Fig 3.
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| Fig 3.
Comparison of whole spleen-associated radioactivity in
control and thrombocytopenic mice. (A) Comparison of whole
spleen-associated radioactivity in control and thrombocytopenic mice.
(B) Comparison of the spleen weight among control and thrombocytopenic
mice. Whole spleen-associated radioactivity is presented as cpm × 104 (mean ± SD). Spleen weight is presented in grams
(mean ± SD). *P < .05 when compared with CD-1 control.
 P < .05 when compared with irradiated. P < .05 when compared with F1 female control.
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Femur- and sternum-associated radioactivity in the RAMPS and W/B F1
male mice, whether normalized by weight (Table 1) or whole bone
associated (data not shown), was also higher than in irradiated mice.
Femur and sternum weights varied only slightly among different mice
groups (~10% of the total bone weight; data not shown).
Tissue/blood-associated radioactivity of the sternum was decreased and
femur was significantly decreased in the irradiated group, whereas both
were significantly increased in 1 d and 5 d RAMPS (P < .001)
versus the CD-1 control animals (Table 2). The W/B F1 male group, which did not develop thrombocytopenia as severe
as the RAMPS mice, also had increased tissue/blood-associated radioactivity in both the sternum and femur compared with their female
controls (22.5± 4.4 v 13.6 ± 0.3%; 22.4% ± 4.4%
v 13.7% ± 1.55%; P = not significant; Table 2).
The plasma-associated radioactivity appears to be inversely related to
the whole spleen-associated radioactivity (r = .9106, P < .05, n = 6) in the W/B F1 male mice (Figs 2B,
3A, and 4). None of the other soft
organ-associated radioactivity, whether normalized by weight or whole
organ-associated radioactivity, had a significant inverse correlation
with plasma-associated radioactivity (data not shown).
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| Fig 4.
Inverse correlation between plasma-associated
radioactivity (TCA-precipitable cpm) and whole spleen-associated
radioactivity in W/B F1 male mice. Plasma-associated
radioactivity is presented as cpm × 104/mL of plasma.
Whole spleen-associated radioactivity is presented as cpm × 104. A significant correlation between plasma and whole
spleen-associated radioactivity was found (r = .91,
P < .05) using Pearson correlation analysis.
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We also compared the ratio between plasma TCA nonprecipitable
radioactivity (representing low molecular weight degradation products
of 125I-rmTPO) and TCA-precipitable radioactivity
(representing intact 125I-rmTPO) among the 6 different
groups of mice (Fig 5). Whereas 5 d RAMPS
and W/B F1 mice had ratios comparable to those of their corresponding controls (Fig 5), the irradiated group had a
significantly lower ratio of plasma TCA nonprecipitable radioactivity
versus TCA-precipitable radioactivity (P < .05; Fig 5). The
ratio of 1 d RAMPS was, again, intermediate among the 4 groups of
thrombocytopenic mice (Fig 5).
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| Fig 5.
Comparison of degraded versus intact
125I-rmTPO in the plasma of control and thrombocytopenic
mice. Data are presented as the ratio between TCA nonpreciptable
radioactivity and TCA-precipitable radioactivity in plasma (mean ± SD). *P < .05 when compared with CD-1 control.
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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.
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| Fig 6.
Histopathological analysis of megakaryocytes in spleens
of irradiated (A) and 5 d RAMPS mice (B). Megakaryocyte numbers appear
markedly decreased in irradiated mice, whereas the 5 d RAMPS mice
appear to have marked increases in the spleen sections. Increased
numbers of megakaryocytes appear associated with increased size
(hematoxylin and eosin; original magnification × 100).
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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).
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Table 4.
Comparison of Bound Radioactivity Per Million PLT and
Mean PLT Volume in Control and Thrombocytopenic Mice
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| |
DISCUSSION |
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 = .91, P < .05; Fig 4). Bone-associated radioactivity was also higher in immune
thrombocytopenic mice than in irradiated mice (Tables 1 and 2). In
contrast, the amount of 125I-rmTPO in tissues other than
spleen and bone had no inverse correlation with the
125I-rmTPO levels in plasma and was generally proportional
to the corresponding whole blood labeling levels: higher in the
irradiated group and lower in the 5 d RAMPS and W/B F1 male
mice compared with that of the controls (Table 1). These results
suggest that, despite significant thrombocytopenia, specific uptake of
125I-rmTPO still occurs in the spleen and bone of the
immune thrombocytopenic but not of the irradiated mice.
The specific uptake of 125I-rmTPO in the hematopoietic
tissues of immune thrombocytopenic mice appeared to be associated with a higher megakaryocyte mass when the tissue samples were examined using
light microscopy (Table 3). Electron microscopic autoradiography further confirmed the binding and internalization of
125I-rmTPO by megakaryocytes and PLTs in the spleens and
marrows of the immune thrombocytopenic mice (data not shown). Labeled PLTs were also found engulfed by splenic macrophages (data not shown).
However, the spleen of irradiated mice contained no morphologically identifiable PLTs or megakaryocytes. Although it has been previously suggested that other cells, such as endothelial and stromal cells, of
hematopoietic tissue may be involved in the regulation of circulating TPO levels in ITP,31,32 our data did not suggest a
significant association between these cells and the uptake of
125I-rmTPO (data not shown). Taken together, these results
suggest that higher Mpl+ cell mass, consisting of higher
megakaryocyte mass and increased production of PLTs, which are rapidly
cleared by reticulo-endothelial destruction, seems to be responsible
for the higher hematopoietic tissue-based uptake of
125I-rmTPO in 5 d RAMPS and W/B F1 male mice compared with
irradiated mice.
In addition to higher hematopoietic tissue-based uptake, our data also
indicated significantly greater 125I-rmTPO uptake per
million circulating PLT in the 2 RAMPS groups compared with the control
(Table 4). PLTs in W/B F1 male mice also seemed to take up
more 125I-rmTPO than expected (Tables 1 and 4). Because all
of these mice also had higher mean PLT volumes, these results seem to
be consistent with the previous speculation13 that larger
PLTs, also frequently observed during human acute ITP, may take up more TPO and, thus, contribute more to the regulation of circulating TPO
levels than their absolute number would indicate. However, further
comparison of the number and affinity of TPO receptor sites present on
normal PLTs and the larger PLTs from immune thrombocytopenic subjects
is needed to verify this hypothesis.
If the lower circulating levels of 125I-rmTPO in ITP models
are indeed associated with higher total mass of Mpl+ cells
compared with the irradiated mice, one would expect this mechanism to
be independent of the process that triggers an increase in
Mpl+ cell mass. Our data are consistent with such a
mechanism. Despite differences in the cause of immune thrombocytopenia
between the 5 d RAMPS and W/B F1 males, both groups had the
highest total mass of Mpl+ cells and the lowest circulating
125I-rmTPO among all groups of thrombocytopenic mice.
Conversely, although both 1 d and 5 d RAMPS had the same type of immune
thrombocytopenia after receiving the same antibody treatment, 1 d RAMPS
mice only had intermediate levels of Mpl+ cells and
circulating 125I-rmTPO levels to those of the irradiated
and 5 d RAMPS mice. These results, together with the findings that the
tissue distribution pattern of 125I-rmTPO in the irradiated
group was very similar to that observed in
c-mpl/ mice (increased plasma-associated
radioactivity, dramatically reduced blood cell as well as
spleen-associated radioactivity,20 and significantly lower
amounts of 125I-rmTPO degradation products), are all
consistent with the hypothesis that a lack of Mpl+ cells in
mice with thrombocytopenia secondary to megakaryocytic hypoplasia
results in higher circulating TPO levels. Conversely, the
Mpl+ cellular mass, and consequently TPO uptake and
catabolic capacity, in steady-state immune thrombocytopenic mice (5 d
RAMPS and W/B F1 male) remained significantly higher than
that of megakaryocyte hypoplastic mice after a brief TPO surge (1 d
RAMPS) in response to the initial thrombocytopenia that induced
megakaryocytopoiesis and thrombopoiesis. This could account, at least
in part, for the low levels of circulating TPO during the steady state
of immune thrombocytopenia.
The relative significance of megakaryocyte versus PLT receptors for TPO
clearance in immune thrombocytopenic mice remains unclear. Increased
megakaryocyte mass in these mice suggests that megakaryocyte-associated
TPO clearance may also be significantly increased. However,
quantitative evaluation of 125I-rmTPO associated with the
large number of megakaryocytes is needed to determine whether the
increased uptake capacity is indeed quantitatively proportional to the
increased cell mass in either pre-steady-state or steady-state ITP
models. In the current study, the femur- and sternum-associated
radioactivity in 1 d RAMPS mice was comparable to those of the 5 d
RAMPS and W/B F1 male mice (Table 2). However, megakaryocytic mass
within the marrow of those mice was greater than that of the 1 d RAMPS
(data not shown). This raises the question of whether the greater
number of Mpl receptor in steady-state ITP models25 may
result in a shortage of the tracer and, therefore, less saturation of
the available receptor by the labeled ligand, causing the
bone-associated radioactivity in those mice to appear lower than might
be expected. Techniques to scan through larger pieces of tissue, such
as light microscopic autoradiography, may be helpful in future studies
to verify this hypothesis.33 Furthermore, ITP in humans and
mice is associated with accelerated new PLT formation as well as
destruction compared with controls.34 It is possible that
the kinetics of the TPO clearance process in immune thrombocytopenic
humans and mice may be increased35 due to the higher
turnover of PLTs in these hosts that could also account, in part, for
the low circulating TPO levels. Because the current data represent only
a glimpse of this highly dynamic process, PLT-associated TPO clearance
over time is likely to be underestimated. Future studies are needed to
quantitatively evaluate the kinetics of TPO clearance in immune
thrombocytopenic versus control and irradiated models. Finally, our
results are consistent with recent reports that splenic
megakaryocytopoiesis in mice seems to be more responsive to induction
or repression than that of the marrow under certain experimental
conditions.11,36 However, these observations do not
contradict other previous evidence demonstrating that the bone marrow,
not the spleen, is probably the production site for the majority of
megakaryocytes, as well as PLTs, in rodents.37 Because it
has been previously shown that the control spleen contributes less than
2% of the total megakaryocyte mass,37 small changes in
local marrow megakaryocyte production are undoubtedly greatly amplified
throughout the total bone marrow. Therefore, although the megakaryocyte
mass increase within the spleen of steady-state ITP models was higher
than that of the marrow, the total bone marrow still probably produces
a majority of megakaryocytes.
In summary, the data in this study provide evidence that, as in
c-mpl/ mice, decreased Mpl+
cellular mass in mice with thrombocytopenia secondary to megakaryocytic hypoplasia (irradiated mice) is associated with higher circulating TPO
levels, whereas the lack of an inverse correlation of circulating TPO
with PLT counts during steady-state ITP may be due, at least in part,
to its uptake and degradation by higher PLT turnover and the increased
mass of megakaryocytes.
| |
ACKNOWLEDGMENT |
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.
| |
FOOTNOTES |
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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