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Recombinant Human Interleukin-1 1 Stimulates Megakaryocytopoiesis
and Increases Peripheral Platelets in Normal and Splenectomized Mice
By Tamlyn Yee Neben, Jean Loebelenz, Lori Hayes, Kyle McCarthy,
John Stoudemire, Robert Schaub, and Samuel J. Goldman
The effects of recombinant human interleukin-1 1 (rhlL-11)
on in vivo mouse megakaryocytopoeisis were examined.
Normal C57B1/6 mice and splenectomized C57B1/6 mice
were treated for 7 days with 150 ag/kg rhlL-11 administered subcutaneously. In normal mice, peripheral platelet
counts were elevated compared with vehicle-treated controls after 3 days of rhlL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhlL11 showed elevated peripheral platelet counts that were
similar in magnitude to normal rhlL-1 1-treated mice.
However, on day 10the platelet counts in rhlL-11-treated,
splenectomized mice were no longer elevated. Analysis of
bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the
percentage of 32N megakaryocytes in both normal and
splenectomized animals treated with rhlL-11. In normal
mice, the number of spleen megakaryocyte colony-forming
cells (MEG-CFC) were increased twofold to threefold relative t o controls after 3 and 7 days of rhlL-11 treatment,
whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone
marrow of rhlL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3
and 7 of the study. These data show that in vivo treatment
of normal or splenectomized mice with rhlL-11 increased
megakaryocyte progenitors, stimulated endoreplication of
bone marrow megakaryocytes, and increased peripheral
platelet counts. In addition, results in splenectomized mice
showed that splenic hematopoiesis was not essential for
the observed increases in peripheral platelets in response
to rhlL-11 administration.
0 1993 by The American Society of Hematology.
I
activity of rhIL- 1 1 in splenectomized animals was very similar
to the activity in normal mice, demonstrating that splenic
megakaryocytopoiesis was not necessary for the observed increase in peripheral platelets that occurred in response to
rhIL-11 treatment.
NTERLEUKIN- 1 1 (IL- 1 1) was originally identified as a
factor produced by IL- I-stimulated PU-34 bone marrow
stromal cells that could stimulate proliferation of the IL-6dependent T1165 plasmacytoma.' This biological activity was
used to screen a cDNA library generated from IL-l-stimulated PU-34 mRNA expressed in COS cells. A primate IL1 I probe was then used to screen a cDNA library generated
from PMA and IL-I-stimulated MRC 5, a human fetal lung
fibroblast cell line. The isolated human IL-I 1 cDNA contained a unique open reading frame of 579 nucleotides encoding a predicted polypeptide of 199 amino acids that included a putative protein secretory sequence of 20 amino
acids.
Preliminary characterization of biological activity has
shown that recombinant human (rh)IL-lI is a multifunctional hematopoietic ~ytokine.**~
In addition to stimulating
proliferation of the T1165 plasmacytoma, rhIL-11 could
stimulate immunoglobulin-producing B cells in vitro and in
vivo.',4It has also been shown to synergize with IL-3 and IL4 in vitro to enhance the proliferation of early progenitors
by shortening the Go period.5,6Secondary cultures of IL-3generated blast colonies in the presence of rhIL- 1 1 produced
pure macrophage colonies5
A number of reports have described multiple effects of
rhIL-1 I on megakaryocytopoieisis in vitro.7-" In serum-depleted murine bone marrow cultures, addition of rhIL-I1
increased megakaryocyte size, megakaryocyte ploidy, and
acetylcholinesterase activity.8It also has been shown to synergize with IL-3 to stimulate murine and human megakaryocyte colony formation.7s9210
Similar megakaryocytic stimulatory activities have been described for other cytokines,
including IL-6 and leukemia inhibitory factor (LIF).8,'2-'6
To date, little is known about the in vivo activities of rhIL1 I. We have examined the effects of rhIL- 1 1 on hematologic
cell counts and megakaryocytopoiesis in normal and splenectomized mice. The results show that rhIL- 1 I increased
peripheral platelet counts in normal mice. In addition, there
was a stimulation of both early and later stages of megakaryocytopoiesis in bone marrow and spleen. The in vivo
'
Blood, Vol81, No 4 (February 15). 1993: pp 901-908
MATERIALS AND METHODS
Mice. Adult female C57B1/6 mice, 8 to 12 weeks old were purchased from Jackson Laboratories, Bar Harbor, ME. The mice were
housed five to a cage and fed standard rodent chow diet with water
supplied ad libitum. The average weight of the animals was approximately 20 g. During the study, the animals were monitored daily
for clinical signs of ill health, behavioral changes, or reaction to treatment. Splenectomies were performed as previously described except
that methoxyflurane was used for anesthesia."
Recombinant cytokine. rhIL-11 purified to homogeneity from
Escherichia coli was used in these studies. The protein was diluted
with saline to appropriate concentration for injection supplemented
with 0.5% homologous mouse serum. The control article was saline
for injection (Abbott Laboratories, North Chicago, IL) supplemented
with 0.5% homologous mouse serum. Mice were treated for 7 consecutive days with rhIL-11 at a dose of 75 pg/kg twice a day (150
pg/kg/d). The cytokine was administered by subcutaneous injection
(intrascapular).
Hematology. Hematologic evaluations consisting of complete
blood counts and white blood cell (WBC)differentialswere performed
on animals from each group on days 1.5, 3, 7, 10, and 15. Animals
were anesthetized with isoflurane and blood samples were collected
by intracardiac puncture into EDTA-rinsed 1 mL tuberculin syringes.
The samples were immediately transferred into a tube containing
From the Division of Preclinical Biology, Genetics Institute, Cambridge, MA.
Submitted June 29, 1992; accepted October 9, 1992.
Address reprint requests to Samuel J. Goldman, PhD, Preclinical
Biology, 87 Cambridge Park Dr, Cambridge, MA 02140.
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.
0 1993 by The American Society of Hematology.
0006-49 71/93/8104-0022$3.00/0
90 1
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902
NEBEN ET AL
lyophilized EDTA. Automated hematologic analyses were performed
on a Baker 9000 hematology analyzer (Serono Baker Diagnostics,
Allentown, PA) using mouse-specific discriminator settings. The
analyses included: hematocrit, WBC counts, hemoglobin, red blood
cell (RBC) counts, and platelet counts. Blood smears were prepared
and stained with Wright-Geisma for WBC differentials based on a
100-cellcount.
Histology. Samples for histology, with the exception of the sternum, were fixed in 10%neutral buffered formalin. The sternum was
simultaneously fixed and decalcified for 48 hours in Calex-I1(Fisher,
Pittsburgh, PA). The tissues were then processed in a Tissue Tek
vacuum infiltration processor (Miles Inc, Dallas, TX) for paraffin
embedding. The samples were sectioned at 6 pm and stained with
hematoxylin and eosin.
Preparation of bone marrow cells ,forflow cytometry and colonyforming cell (CFC) assays. The femurs were cleaned of any surrounding tissue and the marrow was flushed from the femurs with
mol/L theophylline,
CATCH buffer (0.38% sodium citrate, 2 X
IX
mol/L adenosine in Hank’s Balanced Salt Solution, pH 7.2)
supplemented with 1 pg/mL prostaglandin E, (supplemented
CATCH) and 3% bovine serum albumin (BSA) (Sigma Chemical
CO,St Louis, MO) using a 25-gauge needle on a 10-mL syringe. A
single cell suspension was made by passing the marrow through an
IS-gauge needle three times. The cells were centrifuged at 1,200 rpm
for 10 minutes and resuspended in I O mL of supplemented CATCH
buffer, counted on the Baker hematology analyzer, and directly aliquoted for flow cytometric analysis. Marrow cells for colony assay
were centrifuged at 1,200 rpm for 5 minutes. The pellets were resuspended in 10 mL of RBC lysis buffer (0.14 mol/L NH,CI, 17
mmol/L Tris-HCI, pH 7.2) and incubated for 10 minutes at room
temperature. The cells were then washed once in CATCH buffer and
were resuspended in Iscove’s modified Dulbecco’s Modified Eagle’s
(DME) supplemented with 10% heat-inactivated (HI) fetal bovine
serum (FBS).
Preparation of spleen cells for flow cytometry and CFC assays. The spleen pieces were teased between two forceps into 5 mL
CATCH buffer (no supplements). Large tissue pieces were removed
from the suspension by allowing them to settle, and the cells were
passed through an IS-gauge needle three times to form a single cell
suspension. The spleen cells were centrifuged at 1,200 rpm for 5
minutes. The pellets were resuspended in Iscove’s modified DME
supplemented with 10% HI FBS and counted on the Baker hematology analyzer.
Quantitation of megakaryocyte progenitors. Megakaryocytecolony-forming cells (Meg-CFC) were quantitated in semisolid medium.
Meg-CFC were established by the addition of 1 X IO5 marrow cells
or 1 X IO6 spleen cells to a mixture of 0.325% agar (Difco, Detroit,
MI), Iscove’s modified DME supplemented with 10% FBS, 10%
WEHI-3b conditioned medium (as a source of IL-3), and 30 U/mL
of rhIL-1 I . Control wells contained the same volume of medium in
place of the WEHI-3b and rhIL-11. Gels were allowed to form in 1mL aliquots in 35-mm wells for 10 minutes before incubation for 6
days at 37°C in 5% COz, 95% humidified air. The gels were then air
dried and stained for the presence of acetylch~linesterase.~~~~~
A
megakaryocyte colony was defined as a positively stained group of
three or more cells.
Determination of megakaryocyte fyequency and ploidy. Megakaryocyte ploidy was determined using a modification of the method
of Corash et
Bone marrow cells ( I to 4 X 106/mL) in I mL of
supplemented CATCH plus 3% BSA were incubated with 200 pg/
mL of rat Ig (to block F, receptor) and I to 2 pg/mL of the megakaryocyte-specific monoclonal antibody, 4A5 (12), on ice for 60 to
90 minutes. After incubation, samples were underlayed with 1 mL
of supplemented CATCH buffer plus 5% BSA and spun at 1,200
rpm (350g) for 6 minutes. The supernatants were removed and discarded. To each sample, 500 pL of supplemented CATCH buffer
plus 3% BSA containing 0.5 pg/mL of streptavidin fluorescein was
added, and samples were incubated on ice for 30 to 40 minutes. After
incubation, samples were underlayed with 750 pL of supplemented
CATCH plus 5% BSA, centrifuged as above, and supernatants were
discarded. To each sample, 500 pL of supplemented CATCH plus
3% BSA and 0.5% Tween 20 were added, samples were vortexed to
resuspend the pellets and placed at 4°C for 30 to 40 minutes. After
incubation, 500 pL of 1% paraformaldehyde in phosphate-buffered
saline (PBS) containing 0.5% Tween 20 was added to each sample
and samples were incubated at 4°C for an additional 10 minutes.
Samples were spun at 1,200 rpm for 6 minutes and supernatants
were discarded. Pellets were resuspended in 1 mL 0.5% citrate in
PBS plus 1% BSA. For ploidy determinations, 50 pg/mL propidium
iodide and 2 to 5 pg/mL of RNase were added to appropriate samples
and incubated for 90 minutes at room temperature. All samples were
filtered through 100 micron nytex before FACS analysis. A minimum
of 1,000 megakaryocytes were analyzed for ploidy determinations
and frequency determinations were based on 500 megakaryocytes.
RESULTS
The animals in both the rhIL- 1 1-treated group and the
vehicle-treated control group exhibited normal activity and
seemed healthy throughout the study. There were no notable
Table 1. Peripheral Hematology and Spleen Weights in Control and rhlL-1 1-Treated Mice
rhlL-l 1 Treated
Vehicle Control
Normal
Spleen weight (mg)
Platelets (10-3pL)
WBC (X 1 0 - 3 p ~ )
Splenectomized
Platelets (10-3pL)
WBC (X 10-3 r ~ )
Day 3
Day 7
Day 10
Day 3
Day 7
Day 10
7 0 (3)
8 7 4 ( 1 3)
2.6 (0.5)
6 6 (10)
871 (38)
3 . 3 (0.5)
6 6 (7)
7 9 8 (41)
3.7 (1.1)
9 8 (10)
1,034 (105)
3 . 5 (0.9)
9 8 (20)
1,165 (101)
3.4 (1.5)
7 4 (18)
1,068 (78)
4.3 (1.8)
9 4 3 (74)
9.6 (2.4)
9 2 7 (1 14)
7 . 7 (1.2)
9 1 6 (49)
9.8 (1.1)
1,148 (1 18)
4 . 1 (1.0)
1,312 (68)
4.9 (0.5)
9 4 6 (130)
9.4 (3.9)
Spleen weights in IL-1 1-treated mice were significantly greater than in control mice (P < .005) on days 3 and 7 . Blood was collected by cardiac
puncture as described in Materials and Methods. WBC and platelet analyses were performed on an automated Baker 9 0 0 0 hematology analyzer
using mouse-specific discriminator settings. Mean WBC counts of IL-1 1-treated, splenectomized mice were significantly different than those of
control mice on day 3 ( P i .01) and day 7 (P < .01). Data are presented as mean ( S D ) , n = 5 for normal mice and n = 4 for splenectomized mice.
From www.bloodjournal.org by guest on July 28, 2017. For personal use only.
IN VIVO ACTIVITIES OF RHIL-~1
changes in body weights in either group. Gross pathology was
unremarkable in both groups. Compared with vehicle-treated
controls, there was an increase in spleen weight in rhIL-11treated animals of 29% and 33%after 3 days and 7 days of
dosing, respectively (Table 1). There was also an increase in
splenic cellularity that was not statistically significant (data
not shown).
Hematology. Administration of rhIL- I 1 to normal mice
resulted in a significant increase in peripheral platelet counts
(Table 1 and Fig IA). After 3 days of dosing, the platelet
counts of rhIL-1 1-treated animals were 17%above vehicletreated controls. The platelet counts in rhIL-l I-treated animals were 31% above vehicle-treated controls on day 7. On
day 10 peripheral platelets in rhIL-l 1-treated animals remained 33%above vehicle control, and on day 15 there was
no significantdifference in the number of peripheral platelets
between treated and control animals. Comparing mice treated
with rhIL-11 with vehicle-treated controls, there were no
consistent changes during the course of the study in RBC,
reticulocyte, or WBC counts (data not shown). There were
no changes during the course of the study in the WBC differential in either group (data not shown).
Administration of rhIL- 1 1 to splenectomized mice also
resulted in significant increases in peripheral platelet counts
compared with vehicle-treated controls (Table 1 and Fig 1B).
The magnitude of the platelet responses in rhIL-l I-treated,
splenectomized animals was very similar to the responses
observed in normal rhIL-l 1-treated mice during the first 7
days of the study (Fig 1). However, on day 10 the platelet
counts in rhIL-l I-treated, splenectomized mice were no
longer elevated. This contrasts with the result observed in
normal mice treated with rhIL-I1 in which the day 10 platelet
counts were still elevated.
The WBC count in splenectomized mice was about twofold
higher than in normal mice. There was no consistent change
in the W C count in vehicle-treated, splenectomized controls
during the course of the study (Table I). However, the WBC
counts of rhIL- 1 1-treated, splenectomized mice were significantly decreased compared with controls on days 3 and 7,
and returned to control levels on day IO (Table 1). There
were no consistent changes in the WBC differential of either
vehicle-treated control or rhIL-I1 -treated mice during the
course of the study (data not shown). No other changes in
hematology were observed.
Megakaryocyte frequency. Quantitation of megakaryocytes in the bone marrow by flow cytometry showed no
changes in the frequency of megakaryocytes between rhIL1 I -treated animals and vehicle-treated controls in either
normal or splenectomized mice (Table 2). This observation
was confirmed by histologic examination of bone marrow
megakaryocytes (data not shown). However, histologic evaluation of megakaryocyte frequency in the spleen showed an
increase in the number of splenic megakaryocytes in rhILIl-treated normal mice (Fig 2). This trend was apparent
after 3 days of dosing with rhIL- 1 1 and persisted throughout
the course of the study.
Megakaryocyte ploidy. The ploidy distribution of bone
marrow megakaryocytes was determined by two-color flow
903
A
“0°1
1300
-
I
v
1
1
70
600 0
1
2
3
4
5
6
7
8
9 1 0 1 1 1 2 1 3 1 4 1 5
STUDY DAY
B
1400T
T
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-1
F?
x
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800-
700-
Fig 1. Platelet counts of control versus rhlL-1 1-treated mice.
Blood was collected by cardiac puncture as described in Materials
and Methods. Platelet analyses were performed on an automated
Baker 9000 hematology analyzer using mouse-specificdiscriminator
settings. (A) Platelet counts in normal mice. (B) Platelet counts in
splenectomized mice. Mean platelet counts of IL-11-treated normal
mice were significantly greater than those of control mice on days
3. 7 . and 10 (P < .005). Mean platelet counts of IL-1 1-treated
splenectomized mice were significantly greater than those of control
mice on day 3 (P < .04) and day 7 (P < .OOl). Data are presented
as mean (+SD) n = 5 for normal mice and n = 4 for splenectomized
mice: (0)
vehicle treated control; (0)rhlL-11, 1 5 0 pg/kg/d.
cytometry. Normal mice and vehicle-treated control mice
had a modal megakaryocyte ploidy of 16N (40% to 50% of
megakaryocytes) with 15% to 20%of megakaryocytes in the
8N ploidy class and 10% to 15% of megakaryocytes at 32N
(Fig 3). After 3 days of dosing with rhIL-11 there was a clear
increase in 32N megakaryocyte ploidy in mice treated with
rhIL-11 compared with vehicle-treated controls (28% compared with 13%).There was a corresponding decrease in the
percentage of 8N megakaryocytes (9% in rhIL-1 1-treated
mice compared with 17% in vehicle-treated controls) (Fig
4A). Stimulation of endoreplication of megakaryocytes to
higher ploidy classes in rhIL-l 1-treated mice was also observed on day 7. The rhIL-l 1-treated mice had an average
of 26%of megakaryocytes in the 32N ploidy class compared
with 16% in vehicle-treated control. The percentage of 8N
and 16N megakaryocytes on day 7 in rhIL-1 1-treated mice
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904
NEBEN ET AL
Table 2. Megakaryocyte Frequency in Bone Marrow of Normal Mice
Megakaryocytes(%)
Day 3
Day 7
Day 10
0.16 (0.02)
0.19 (0.03)
0.13 (0.03)
0.1 1 (0.02)
0.12 (0.05)
0.12 (0.06)
0.092 (0.01 9)
0.095 (0.029)
0.106 (0.011)
0.100 (0.007)
0.088 (0.019)
0.067 (0.019)
Day 1
Normal
Saline control
IL-11 treated
Splenectomized
Saline control
rhlL-11 treated
0.15 (0.03)
0.18(0.01)
.
ND
ND
Bone marrow cells were processedand stained with the megakaryocyte-specificmonoclonalantibody, 4A5, as describedin Materialsand Methods.
Megakaryocyteswere analyzed by flow cytometry and frequency determinations were based on collection of a minimum of 400 megakaryocytes
per animal. Data are presented as mean (2SD). n = 5 for normal mice and n = 4 for SDlenectomized mice.
Abbreviations: ND, S/B not determined.
was 15% and 35%. respectively (Fig 4B). On day IO there was
no difference in the ploidy distribution between treated and
control animals (data not shown).
A similar pattern of stimulation of bone marrow megakaryocyte endoreplication was observed in splenectomized
mice. Vehicle-treated, splenectomized mice had a modal
ploidy of 16N (55% to 60% of megakaryocytes) with 15% to
20% of megakaryocytes in the 8 N ploidy class and about 10%
of megakaryocytes at 32N. Figure 4 also shows that megakaryocyte endoreplication was stimulated by rhlL- I 1 treatment on both day 3 (Fig 4C) and day 7 (Fig4D). The relative
increase in 32N megakaryocytes was similar to the increase
observed in normal mice (compare Fig 4A and B with C and
D). At day I O there was no difference in the ploidy distribution
between treated and control splenectomized mice (data not
shown).
Megakaryocyfe progenitors. Treatment of normal or
splenectomized mice with rhlL-l I resulted in stimulation of
megakaryocyte progenitors. There was no difference in MEG-
CFC in the bone marrow between vehicle-treated control
and rhlL-l I-treated normal mice on day 3 (Fig SA). However, on day 7 there was a threefold increase, relative to vehicle-treated controls, in MEG-CFC in the bone marrow of
rhlL-l I-treated mice. In splenectomized mice there was a
twofold increase, relative to vehicle controls, in bone marrow
MEG-CFC in rhlL-l I-treated animals on both days 3 and
7 (Fig 5B). On day I O there were no differences in bone marrow MEG-CFC between vehicle-treated control and rhlL1 I -treated animals in either normal or splenectomized mice.
In the spleens of rhlL-1 I-treated normal mice there was
a threefold increase, relative to vehicle-treated controls, in
MEG-CFC on day 3 (Fig 5C). After 7 days of rhlL-11 dosing,
MEG-CFC in the spleens of rhlL-l I-treated mice were still
elevated compared with vehicle-treated controls. On day I O
there was no difference in spleen MEG-CFC between vehicletreated control and rhlL- 1 I -treated mice. Although there
20
18
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G
16
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14
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A
DAY 3
DAY 7
DAY 10
DAY 15
STUDY D A Y
Fig 2. Histologically identifiable megakaryocytes in the spleen
of control and rhlL-11-treated mice. (A) Paraffin embedded sections
of the spleen from days 1.5, 3,7, 10, and 1 5 were stained with
hematoxylin and eosin. The average number of megakaryocytes
per spleen section was determined by counting the number of histologically identifiable megakaryocytes in a total of four randomly
chosen, nonoverlapping spleen sections per mouse (n = 5 mice/
group): (0)vehicle-treated controls; (m) rhlL-11, 150 pg/kg/d.
FLUORESCENCE INTENSITY
Fig 3. Bone marrow megakaryocyte ploidy in control mice.
Megakaryocyte ploidy was quantitated by two-colorRow cyhmetry
as described in Materials and Methods. Megakaryocytesform discrete ploidy classes that were quantitated by setting markers in the
valleys between peaks. The location of 2N and 4 N ploidy classes
was confirmed by examining unfractionated bone marrow. The histogram shown is representative of the ploidy distribution observed
in vehicle-treated mice.
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IN VIVO ACTIVITIES OF ~ ~ l L - 1 1
905
DAY 3
M
E
G
A
DAY 7
f
50-1
45
40
R
Y
Y
T
E
35
y"
c
30
30
25
y
25
T
35
20
2o
15
r4 lo
(?h)l5
10
5
6
0
2N
n
"
4N
8N
16N
32N
64N
2N
4N
PLOIDY
32N
64N
32N*
64N
DAY 7
c
LT
55.-
M
16N
PLOIDY
DAY 3
60.-
8N
50.-
E
G 45.A
K 40.-
;35.30-
c
Y 25-
T
E 20-151-
5
0
2N
4N
8N
16N
32N
64N
PLOIDY
2N
4N
8N*
16N
PLOIDY
Fig 4. Megakaryocyte ploidy in control and rhlL-I 1-treated mice. Megakaryocyte ploidy was quantitated by two-color flow cytometry
as described in Materials and Methods. A minimum of 1,000 megakaryocytes were collected for each animal and the percentage of cells
in each ploidy class was determined as described in Fig 3. (A and B) Normal mice, days 3 and 7, respectively. (C and D) Splenectomized
mice, days 3 and 7, respectively. Compared with vehicle-treated controls there were statistically significant increases (P < .01) in the
percentage of 32N megakaryocytes in rhlL-1I-treated normal mice and rhlL-I 1-treated splenectomized mice on days 3 and 7. Data are
presented as the mean ( S D ) n = 5 for normal mice and n = 4 for splenectomized mice: (0)
vehicle-treated controls; (0)
rhlL-11, 150 r g /
kg/d.
were clear differencesin spleen and bone marrow MEG-CFCs
between vehicle-treated control and rhIL- 1 1-treated normal
mice on day 7 (Fig 5A and C ) ,the absolute number of megakaryocyte colonies in vehicle-treated controls was lower
(compared with days 3 and 10) in both groups of animals.
DISCUSSION
In this report we have examined the effects of in vivo administration of rhIL- 1 1 to normal and splenectomized mice.
Administration of rhIL-11 to normal mice resulted in a
marked stimulation of megakaryocytopoiesis and a corre-
sponding increase in peripheral platelet counts. The magnitude of the increase in peripheral platelets in these studies
was modest (30%to 40%);however, platelet increases of this
magnitude could be maintained throughout the dosing period
when rhIL-11 was administered for 14 days (data not shown).
In vivo administration of I L 6 in mice resulted in platelet
increases of similar magnitude.2',22
There was no change in the frequency of bone marrow
megakaryocytes in normal mice following rhIL- 1 1 treatment.
However, there were increased numbers of bone marrow
MEG-CFC. Endoreplication of bone marrow megakaryocytes
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NEBEN ET AL
906
30
A
21
.
c
26
24
I
DAY 3
B
DAY 7
=I
DAY 1 0
38
C
32
#
22
0
20
L
0
N
I
E
S
I
W
E
L
L
DAY 3
DAY 7
DAY 1 0
T
;I
C
10
DAY 3
DAY 7
DAY 10
Fig 5. Quantitation of MEG-CFC in control and rhlL-11-treated mice. Bone marrow and or spleen cells were harvested from each mouse
and assayed in triplicate for MEG-CFC as described in Materials and Methods. (A) Bone marrow MEG-CFC in normal mice. The number of
bone marrow MEG-CFC in the IL-11-treated mice was significantly greater than in control mice on day 7 (P < .005). (B) Bone marrow
MEG-CFC in splenectomized mice. The number of MEG-CFC in the IL-11-treated mice was significantly greater than in control mice on
days 3 and 7 (P < .01). (C) Splenic MEG-CFCin the normal mouse. The number of splenic MEG-CFC in IL-11-treated mice was significantly
greater than in control mice on days 3 and 7 (P < .001). On day 3 the number of MEG-CFC/spleen was 420 f 63 and 1,355 f 265 for
vehicle control and rhlL-11-treated mice, respectively. On day 7 the number of MEG-CFC/spleen was 36 f 18 and 318 f 166 for vehicle
control and rhlL-11-treated mice, respectively. On day 10 the number of MEG-CFC/spleen was 675 f 196 and 873 f 270 for vehicle
control and rhlL-11-treated mice, respectively. Data are presented as mean (fSD), n = 5 for normal mice and n = 4 for splenectomized
mice; (0)vehicle-treated controls; (9)rhlL-11, 150 pg/kg/d.
was also stimulated by rhlL- I 1 treatment. These data suggest
that early as well as later stages of megakaryocytopoiesiswere
affected by rhlL-I 1 treatment. In addition, there were a
number ofprofound changes in the spleens ofrhlLl 1-treated
animals. Spleen weights increased by 35% and there was a
trend toward increased splenic cellularity. Assay of megakaryocyte progenitors in the spleen showed an increase in
MEG-CFC and histologic examination of the spleens also
showed a trend of increased numbers of morphologically
identifiable megakaryocytes throughout the dosing period.
There was also a modest (twofold to threefold) increase in
macrophage progenitors in the spleens of rhlL- I I-treated
animals (J. Loebelenz and S. Goldman, unpublished observation). These data suggested that rhlL-I1 could stimulate
splenic hematopoiesis and megakaryocytopoiesis in normal
mice.
In normal mice, evidence suggests that the spleen does not
play a role in platelet production.*' To address the role of
the spleen in the in vivo response to rhlL-I 1 we examined
the effect of rhlL-l I in splenectomized mice. Analyses of
peripheral platelets, MEG-CFC, and megakaryocyte endoreplication produced results that were similar to the results
observed in normal mice. In addition, there was a 15% to
25% increase in the cross-sectional area of mature bone marrow megakaryocytes in rhIL- l l-treated, splenectomized mice
compared with megakaryocytes from vehicle-treated, splenectomized controls (T. Neben, unpublished observation).
These data indicated that splenic megakaryocytopoiesiswas
not essential for the observed increase in peripheral platelets
in response to in vivo rhlL- I I treatment. The ability of rhlL1 I treatment to stimulate both early and later stages of megakaryocytopoiesis was also unaffected by splenectomy.
Some minor differences were observed between splenectomized mice and normal mice in the kinetics of the above
responses to rhlL-l I . Increased numbers of bone marrow
MEG-CFC appeared earlier in splenectomized mice than in
normal animals (day 3 v day 7, Fig 5 ) , whereas the decline
of peripheral platelets to control levels was faster in splenectomized mice than in normal animals (day IO v day 15, Fig
I). The accelerated rate of decline in peripheral platelet counts
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IN VIVO ACTIVITIES OF RHIL-11
in splenectomized mice is consistent with the suggestion that
splenic megakaryocytopieisis may play a role in sustaining
the platelet response after dosing with rhIL- 1 1 has stopped.
However, subsequent studies have shown that treatment of
splenectomized mice with higher doses of rhIL-11 (ie, 250
pg/kg/d) resulted in sustained platelet increases on day 10
similar to those observed in normal mice (data not shown).
In vitro characterization of rhIL-11 has shown that this
cytokine can exhibit multiple biological activities on hematopoietic cells from the myeloid, lymphoid, and erythroid
lineages.1,2,5,6,’1,’4,’5
In some of these cases rhIL- 1 1 mediated
effects may be indirect. For example, the ability of rhIL- 1 1
to stimulate B cell immunoglobulin production was dependent on the presence of CD4 T c e k 4 One possible explanation for rhIL-11 effects on B cells was suggested by the
observation that r h I L l 1 can stimulate CD4/CD45RA T cells
to produce IL-6 mRNA.25The observation that rhIL- 1 1 could
induce IL-6 mRNA raised the possibility that some of rhIL11 effects on megakaryocytes may result, at least in part,
from induction of IL-6. However, the ability of rhIL-11 to
enhance in vitro megakaryocyte endoreplication in mouses
and in human was not blocked by the addition of a neutralizing IL-6 antibody, nor did rhIL-11 induce IL-6 production in murine short-term liquid marrow cultures.’’ In a
preliminary experiment using the B9 bioassay for IL-6, we
were unable to detect any serum IL-6 bioactivity on either
day 3 or day 7 in mice that received subcutaneous injection
of rhIL-ll(250 pg/kg/d) for 7 days (unpublished observation).
Most in vitro activities of rhIL-11 are seen as synergistic
responses of rhIL-11 with other cytokines. One possible explanation for the apparent single-lineage effects of rhIL- 1 1
in vivo is that the complement of cytokines present in the
bone marrow of normal mice may channel the synergistic
activity of rhIL- 1 1 toward the megakaryocyte lineage. However, it should be noted that stimulation of other lineages has
been seen following in vivo rhIL-11 treatment in mice. Increased neutrophil and platelet counts have been reported in
normal mice of a different strain (B6DF1) following rhIL- 1 1
treatment.’ In addition, D u et
have shown that in vivo
rhIL- 1 1 treatment can accelerate both neutrophil and platelet
recovery in lethally irradiated, bone marrow transplanted
mice. These data are consistent with the suggestion that the
synergistic interactions of rhIL-11 with other cytokines in
vivo may differ in various animal models leading to different
experimental outcomes.
In vivo treatment of normal mice affected predominantly
cells of the megakaryocyte lineage. No consistent changes
were observed in the RBC, reticulocyte, or WBC counts, or
WBC differentials. In contrast to these results in normal mice,
a transient decrease in the WBC count was observed in splenectomized mice during dosing with rhIL-11. The nature of
this effect is unclear, although the observation that the WBC
differential did not change in rhIL- 1 1-treated, splenectomized
mice suggests that this effect is global rather than targeted to
a specific compartment of leukocytes. Further studies will be
necessary to address the mechanism of this transient decrease
in WBC count.
907
The data presented in this report show that treatment of
normal or splenectomized mice with rhIL- 1 1 stimulated
megakaryocyte progenitors, endoreplication of bone marrow
megakaryocytes, and increased peripheral platelet counts. The
responses of normal and splenectomized animals were similar. These studies suggest that rhIL-11 may be useful in the
management of thrombocytopenia associated with disease
states or marrow ablative therapies.
ACKNOWLEDGMENT
We thank Dr Samuel Burstein for the generous gift of the 4A5
monoclonal antibody, Dr Shirley Ebbe for the use of the Zeiss Mop
3 image analysis system, and Andy Long for performing the B9
bioassay. rhIL-11 was provided by Ed LaVallie and Kathy Grant.
We also thank Dr Katherine Turner for a critical reading of the
manuscript.
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1993 81: 901-908
Recombinant human interleukin-11 stimulates megakaryocytopoiesis
and increases peripheral platelets in normal and splenectomized mice
TY Neben, J Loebelenz, L Hayes, K McCarthy, J Stoudemire, R Schaub and SJ Goldman
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