1. Art and Diseño

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Interleukin-l2 Enhances Peripheral Hematopoiesis In Vivo
By John D. Jackson, Yun Yan, Michael J. Brunda, Linda S. Kelsey, and James E. Talmadge
lnterleukin 12 (IL-12) is a cytokine
that supports the proliferation and activation of cytotoxic T lymphocytes and natural
killer (NK) cells. Recent evidence has suggested that IL-12
also has hematopoietic activities in vitro. We report studies
that show that IL-12 has significant in vivo hematopoietic
stimulating activity that includes enhancementof peripheral
(splenic) hematopoiesis and mobilization of hematopoietic
progenitor cells to the peripheral circulation.A single injection of recombinant murine IL-12 significantly reduced the
number of bone marrow (BM) colony-forming unit granulocyte-macrophage (CFU-GM) in a time-dependent manner,
while concomitantly stimulating high proliferativepotential.
In contrast, splenic CFU-GM and HPP were increased in a
time- and dose-dependent manner. Chronic administration
of IL-12 resulted in significant splenic hyperplasia with increasedprogenitorcells,increasedcirculatingprogenitor
cells, and BM hypoplasia with decreased progenitor cells.
These data show that IL-12 has significant in vivo hematopoietic effectsthat includethe ability to mobilize progenitor
cells to the peripheral circulation, which
may prove to be of
significantbenefit for peripheral blood
stem cell transplantation. Thus, IL-12 has potential to be an important agent for
clinicaltransplantationbecause of its hematopoieticmobilization and its previously shown immune augmenting and
therapeuticactivities.Thiscombinationofhematopoietic
and immune functions is unique and not achievable with
currently used hematopoietic growth factors.
0 1995 by The American Societyof Hematology.
I
toneal injections of 1 pglanimal IL-12 for 3 or 7 days. Organ cellularities and hematopoietic progenitor cells were evaluated as described
below.
Tissue preparation. Femurs were removed from the mice and
the marrow plug aseptically flushed into RPM1 1640 containing 3%
fetal bovine serum (FBS), 1 0 0 U penicillin, and 100 pg streptomycin
using a syringe fitted with a 2 l-gauge needle. A single cell suspension was prepared by repeated gentle aspirations of the marrow plug
using the same syringe. The cells were counted and plated in colony
assays.
Spleens were removed aseptically and extraneous tissue was dissected away, placed in cold Hanks’ balanced salt solution (HBSS)
and minced into a tissue sieve. A disposable syringe plunger was
used to press the cells through a stainless steel grid to form a cell
suspension. A single cell suspension of nucleated cells was obtained
after filtration through a Nytex filter. The cells were counted and
plated in colony assays.
Peripheral blood was obtained via the retro-orbital plexus using
heparinized Pasteur pipettes from anesthetized mice. To facilitate
blood collection, the mice were injected with 100 units heparin 30
minutes before bleeding. White blood cell (WBC), redblood cell
(RBC), and platelet counts were determined using a Serono Baker
Model 9000 blood analyzer. A peripheral blood smear was prepared
and stained with Wright’s-Giemsa stain for differential counts. For
the determination of hematopoietic progenitor mobilization to the
peripheral circulation, mice were exsanguinated via the brachial artery. Peripheral blood from two mice were combined and mononuclear cells isolated by centrifugation through Lymphocyte M density
gradient (Cedarlane, Homby, Ontario, Canada). Cells were washed
and plated in colony assays.
NTERLEUKIN-12 (IL-12) is a recently identified cytokine that isproduced by macrophages and B cells.’ Originally, IL-12 was described as a natural killer cell stimulating
factor and cytotoxic lymphocyte maturation factor.2z3Recent
evidence suggests that IL-12 is important in controlling immune responses? IL- 12 enhances cell-mediated responses
while suppressing antibody responses by preferentially stimulating Thl cell populations and inhibiting the induction of
Th2 cell population^.^" In mouse metastatic tumor models,
IL-12 was effective in reducing the growth and metastases
of several tumors.’ Inaddition to its immunologic and antitumor activities, IL-12 has been shown to have in vitro effects
on hematopoietic stem and progenitor cells.’ Although IL12 alone cannot stimulate hematopoietic progenitor colony
formation in vitro, synergy in colony formation was noted
when combined with other cytokines.’”’ In short-term liquid
cultures of hematopoietic cells, IL-12 interacted with other
cytokines in a synergistic manner to enhance the production
of hematopoietic stem and progenitor cells.” Therefore, not
only is IL- 12 an immunologically active cytokine, but IL12 is also a hematopoietically active cytokine in vitro. In
this study, we examined the in vivo hematopoietic effects
of IL-12 in normal and splenectomized hematopoietically
competent mice and show that IL-12 is a potent agent for
the in vivo mobilization of hematopoietic progenitor cells to
the periphery.
MATERIALS AND METHODS
Animals. Normaland splenectomized female Balblc-ANN mice
were purchased from Charles River (Wilmington, MA). The mice
were housed in laminar flow units and given irradiated foodand
acidified water (pH 2.5) ad libitum. Mice were acclimated for at
least 2 weeks before use in any experiments.
IL-12. Recombinant mouse IL-12 was supplied by HoffmannLaRoche (Nutley, NJ). In the time course study, mice received a
single intraperitoneal injection with 1 pg IL-12.At various times
( I , 2, 3, or 4 days) after IL-12 treatment, mice were killed and
hematopoietic parameters from bone marrow, spleen, and peripheral
blood were determined. In the dose response experiment, mice received a single intraperitoneal injection with 0.001, 0.01, 0.1, I , or
3 &animal of IL- 12. Control mice received saline injections. Three
days after treatment, femurs and spleens were removed and organ
cellularities and progenitor content were assessed. To determine the
effects of multiple exposures to IL-12, mice received daily intraperi-
Blood, Vol85, No 9 (May l ) , 1995 pp 2371-2376
From the Department of Pathology and Microbiology, University
of Nebraska Medical Center and Department of Oncology, Hoffmann-LaRoche Inc.
Submitted June 13, 1994; accepted December 8, 1994.
Supported in part by Nebraska Department of Health Grant No.
93-49.
Address reprint requests to John D. Jackson, PhD, Department
of Pathology and Microbiology, University of Nebraska Medical
Center, 600 S 42nd St, Omaha, NE 68198-3135.
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 I8 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8509-06$3.00/0
2371
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JACKSON ET AL
2312
- 200
Colony-forming unit g r a n u l ~ ~ c ~ t e - m a c r o p(CFU-GM)
l~a~~e
c~ssny.
Bone marrow (BM), spleen, or peripheral blood cells were plated
at a concentration of I X IO'. S X I o ' or I X IO" cells/dish, respectively in 35-mm dishes containing Iscove's modified Dulbecco's
medium (IMDM). 0.3% agar. 15% FBS. I O 0 U penicillin. I O 0 pg
streptomycin, 5 X
M 2-mercaptoethanol and I O nglmL recombinant mouse 1L-3 (Biosource International, Camarillo. CA) as a
source of colony-stimulating activity. Dishes were incubated for 7
days at 37°C in a humidified atmosphere containing 5% COz in air.
The colonies were enumerated using an inverted microscope. Groups
of SO or more cells were counted as colonies.
High proliferative potential( H P P ) ossay. BM, spleen. or peripheral blood cells were plated at a concentration of I X IO', S X IO'.
or I X IO5 cellddish respectively, in 35-mm dishes containing
IMDM. 0.3% agar. 15% FBS. S X IO" M2-mercaptoethanol, IO
nglmL IL-3 (Biosource International, Camarillo. CA), and 10% Lcell-conditioned medium as sources of colony-stimulating activity.
Dishes were incubated for 1 1 days at 37°C in a humidified atmosphere containing S 8 CO2 in air. The colonies were enumerated
using an inverted microscope. Cell aggregates greater than 1 mm in
diameter were counted as HPP colonies.
Stntistics. Each study was repeated two or more times and each
group in each study was examined in triplicate. All samples were
plated in triplicate for the in vitro colony assays. Statistical significance between groups was determined by one-way analysis of variance.and all pairwise mulfiple comparisons were determined by
using the Student-Newman-Keds method. A P value of .OS or less
was considered significant.
l0
8
l00
6
-50
zk
35000
L
Q)
.-gg
0
0
30000
25000
20000
15000
10000
5000
0
2
P
2.-
Q)
O
0%
Q)
Q)
P
v)
2
0
40000
='(0C -
4
RESULTS
In studies to determine the time course of IL-12hematopoietic effects, mice received a single bolus injection of I
yglanimal of recombinant murine IL- and
12 were evaluated
for hematopoietic parameters I , 2, 3, and 4 days postinjection. No effect was noted on WBC, RBC, or platelets. However, the percentof monocytes increased on days 2 and 3 post
IL-12 treatment (data not shown). Neither BM norspleen
cellularities were altered by a single injection of IL-12(Fig
IA). No effect on BM CFU-GM was noted except at the
later time point (day 4),which showed a significant decrease
(Fig IB). In contrast, BM HPP increased on day 3 after IL12 administration suggesting differential effects of IL-12on
early versus late hematopoietic progenitor cells. Furthermore, the number of splenic CFU-GM and HPP increased
in a time-dependent manner. The number of progenitor cells
peaked on day 3 postinjection and began decreasing on day
4 (Fig IC).
Using the time-dependent results from the previous experiment, a dose-response study was performed. Normal mice
received a single bolus intraperitoneal injection with doses
of IL-12 ranging from 0.001 to 3 pg/animal. Three days
after IL-12treatment, the peripheral blood, BM, and spleen
were examined for hematologic and hematopoietic parameters. No significant effects were noted on peripheral WBC,
RBC, or platelets numbers. The percent of monocytes significantly increased to 6% at doses of 1 and 3 pg IL-12as
compared to I % in control mice. No significant change in
BM or spleen cellularity was noted (Fig 2A). BM CFU-GM
was not affected by a single injection of IL-12at any dose;
however, BM HPP was significantly increased at the higher
-
-0
,
D
l
i
7
0CFU-GM
m HPP
4000
3000
Q)
-g
2000
0
0 1000
control l
2
3
4
Days Post IL-l2 Treatment
Fig 1. Timecourse study of 11-12 in vivo. Mice received a single
intraperitoneal injection of 1 pglanimal 11-12 At various times postinjection, the number of CFU-GM, HPP, and cellularities from bone
marrow and spleen was determined. (A) BMand spleen cellularities,
(B) BM CFU-GM andHPP, IC) spleen CFU-GM andHPP. Data are
presented as mean t SEM. 'Represents P I.OS.
doses ( I and 3 pglanimal) of IL-12. Again, this indicates a
differential effect of IL-12 on early versus more mature BM
progenitor cell populations. Although there was no effect on
spleen cellularity, the number of splenic CFU-GM and HPP
increased at higher IL-12doses (Fig 2C).
In the chronic administration studies, micereceived IL12 for 3 or 7 consecutive days by intraperitoneal injection
of I ,&animal IL-12.There was a 50% decrease in peripheral WBC (4.4X IO' to 2.4 X I03/mm3)after 7 days of IL12 treatment, but no effect on RBC or platelets was noted.
The percent of peripheralblood lymphocytes was signifi-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2373
IL-12 MOBILIZATION
, 300
14 ,
B
0CFUGM
m HPP
T
*
number of circulating progenitor cells in peripheral blood
was determined. Because the spleen is a hematopoietic organ
in the mouse, splenectomized mice were also used to show
the mobilization properties of IL-12. The number of circulating hematopoietic progenitor cells was assessed in the peripheral blood of normal and splenectomized mice after 7
days of 1L-12 treatment. Circulating progenitor cells were
not detected in the control mice because of the low frequency
of circulating progenitor cells. When the spleen-containing
mice were treated with IL-12, the number of CFU-GM and
HPP increased to S6 and 80 colonies per total blood volume,
respectively (Fig S ) . In splenectomized mice,the baseline
CFU-GM and HPP were 2 and 4 colonies per total blood
volume, respectively. When the splenectomized micere-
A
0Control
3 Days of IL-l2 Treatment
7 Days of IL-l2 Treatment
Granulocyte
C
0CFU-GM
m HPP
h
T
0
20
10
40
30
50
60
70
80
90 100
Cell Differential (%)
Control 0.001 0.01 0.1
l
3 Days of IL-l2 Treatment
7 Days of IL-12 Treatment
IL-12 Dose (pg)
Fig 2. Dose-response study ofIL-12 in vivo. Mice received a single
intraperitoneal injection of the various doses of IL-12. Three days
postinjection, the number of CFU-GM, HPP, and organ cellularities
were determined. (AI bone marrowand spleen cellularities, (B) bone
marrow CFU-GM and HPP, (C) spleen CFU-GM and HPP. Data are
presented as mean ? SEM. *Represents P I .05.
cantly decreased, whereas the percent of granulocytes and
monocytes were significantly increased at the two treatment
times (Fig 3A). The absolute number of monocytes was
significantly increased and the absolute number of lymphocytes was significantly decreased, whereas the absolute number of granulocytes did not change significantly after 3 and
7 days of IL-12 treatment (Fig 3B). Femur cellularities were
decreased and spleen cellularities were increased in a timedependent manner (Fig 4A). The number of CFU-GM and
HPP per femur was decreased after 3 and 7 days treatment
with IL-12 (Fig 4B) with a concomitant increase in spleen
CFU-GM and HPP (Fig 4C) suggesting a mobilization effect
of IL-12.
To directly evaluate the mobilizing effects of IL-12, the
B
0Control
3
Granulocyte
P
Lymphocyte
I
0
l
I
l
I
l
3000 4000 5000 6000 71
Absolute Numberof Cells
1000
2000
K)
Fig 3. Peripheral blood differential and absolute numbers after
chronic 11-12treatment. Micereceived daily intraperitoneal injections
of 1 pglanimal of IL-l2 3for
or 7 days. Peripheral blood was removed
from the retro-orbital plexus form
anesthetized mice and blood
smears prepared. Slide were stained using Wright's
and Giemsa stain
and a 200-cell differential counted. Absolute cell numbers were calculated from the differential and
WBC counts. (A) Peripheral blood cell
differential, and (B) absolute peripheral blood cellnumbers. Data are
presented as mean ? SEM. Represents P I .05.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
JACKSON ET AL
2374
ceived IL-12for 7 days, the number of CFU-GM and HPP
colonies per total blood volume increased to 140 and 163,
respectively (Fig 5). The total blood volume calculation was
basedon 5.85 mL bloodper 100 g body weight for the
mouse.I3 The dramatic increase in circulating progenitor
cells after IL-I2 treatment shows the potentmobilizing activity of IL-12.
0CNOY
Fma HPP
Control
IL-l2
I
Splenectomkd
Control
DISCUSSION
I
l
In these studies, we examined the in vivo effects of IL- I2 Splenoctornlzed
IL-l2
in hematopoietically intact mice. Time- and dose-response
studies as well as chronic administration studies were per-
*I
b--"------l
40
80
120
180
200
240
Colonies/Total Peripheral
Blood Volume
12
.
r
500
Fig 5. Effects of chronic exposureof IL-12 on circulating
peripheral
blood hematopoietic progenitor cells in normal and splenectomized
mice. Mice received daily intraperitoneal injections of 1 pglanimal
of IL-l2 for 7 days. Data are presented as mean f SEM. *Represents
P S .05.
-0
50000
0CFU-GM
HPP
4
l0000
0
0
3
7
Days of IL-l2Treatment
Fig 4. Effects of chronic exposure of 11-12 on bone manow and
spleen hematopoietic progenitor cells. Mice received daily intraperitoneal injections of 1 pglanimal of11-12 for 3 or 7 days. (A) bone
marrow and spleen cellularities,(B) bone marrow CFU-GM and HPP,
(C) spleen CFU-GM and HPP. Data are presented as mean SEM.
*Represents P I .05.
*
formed. We report that the administration of a single bolus
injection of 1 pg/animal of IL-12 reduced the numberof
CFU-GM and increased the number of HPP in the BM and
increased the number of splenic CFU-GM and HPP at 3
days postinjection (Fig 1, B and C, and 2,B and C). These
findings confirm the observations from another study that
showed a decrease in BM C N - G M and anincrease in spleen
CFU-GM after multiple injections of IL-l2.I4.l5
Additionally,
the data show that IL-12 has differential effects on early
(HPP) versus late (CFU-GM)BM progenitor populations.
Initially, the administration of IL-12reduced the number of
late (CN-GM) progenitor cells and increased the number
of early (HPP) progenitor cells in the BM. These data support
the in vitro differential effects of IL-12on the proliferation
of early versus late progenitor cells as reported by Jacobsen
et aL9 Using highly enriched hematopoietic stem cells
(Lin-Sca'), they showed that IL-12had significant HPP colony-stimulating activity, but less activity on more mature
populations (Lin-Sca-). Both the numberand size of the
colonies were increased in the presence of IL-12.9In addition, they showed that IL- was
12 active on single early hematopoietic progenitor cells? Hirayama et all' and Ploemacher
et all2 also showed effects of IL-12on early multipotential
hematopoietic progenitor cells in vitro. These studies illustrate that IL-12in combination with SCF have the ability to
support the proliferation and differentiation of early multilineage hematopoietic stem cells. Therefore, based on our single injection studies, we speculate that the in vivo activity
of IL-12is associated with either an increase in the proliferation ofearly BM hematopoietic progenitor cells or a differential movement of progenitor cells from the BM, where the
more mature BM progenitor cells are the first to mobilize to
the periphery. The early hematopoietic progenitor cells appear to remain in the BM longer after a single exposure
to IL-12.However, chronic exposure to IL-12induced the
mobilization of both mature and immature BM progenitor
populations to the periphery. In these studies, we cannot
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2375
IL-12 MOBILIZATION
directly differentiate between enhanced in situ proliferation
of peripheral progenitor cells or mobilization of progenitor
cells to the periphery. However, Grzegorzewski et a l l 6
showed that L 7 reduced BM progenitor cells and increased
spleen progenitor cells and this redistribution was caused by
the migration of progenitor cells from the BM to the periphery (spleen, blood, and liver). When the total number of
hematopoietic progenitor cells (Cm-GM and HPP) is calculated in control mice and compared with the total number
of progenitors in IL-12-treated mice, there is an overall
reduction of ~ 3 0 %after 7 days of IL-12 exposure. Total
marrow cellularity was used to determine the marrow progenitor cell content." The 30% reduction may represent the
redistribution of progenitor cells from the BM to other organs such as liver, kidney, and lung. Therefore, we speculate
that the decrease in BM and the increase in spleen and peripheral blood progenitor cells after chronic in vivo administration of IL-12 is the result of cytokine-induced mobilization. However, we cannot rule out the possibility of
myelosuppression in the BM by secondary effects from IL12-induced cytokines such as interferon y (INFy). Ploemacher et al showed that the L-12-induced myelosuppression during the first week of treatment in long-term bone
marrow cultures was at least caused in part by INFy."
The hematopoietic effects after IL-12 administration did
not develop immediately after a single injection of IL-12,
but required at least 3 days for significant bone marrow and
maximum splenic effects to occur. These data suggest that
other factors may be involved in the IL-12-induced in vivo
hematopoietic effects. However, in vitro studies suggest that
IL-12, in combination with other cytokines, may act directly
on hematopoietic progenitor cells?
Other immunologically active cytokines have been found
to exhibit hematopoietic effects, primarily in vivo. In vivo
administration of IL-2 was shown to increase the number of
progenitor cells in the bone marrow of normal mice and
to enhance hematopoietic recovery after myelosuppressive
doses of chemotherapy andirradiation.''
However, IL-2
treatment in vitro inhibited the formation of hematopoietic
progenitor colony formation?' IL-2 also mobilized hematopoietic progenitor cells to the peripheral circulation in renal
carcinoma patients." IL-7 has also been found to exhibit
hematopoietic effects in vitrozzand in vivo and to mobilize
hematopoietic progenitor cells after treatment in vivo with
IL-7.16 Note, however, that unlike IL-216, IL-722 and IL12' appear to have direct hematopoietic activity in vitro.
Therefore, it is possible that a common mechanism or induction pathway may be involved with the immunologically
active cytokines for hematopoietic activities.
These findings have significant implications for the use
of IL-12 in a peripheral blood-derived stem cell transplant
setting. IL-12 has both immune augmenting and hematopoietic activities which make it an ideal candidate for inclusion
in a transplant protocol both for mobilization and antitumor
therapie~.'~." This combination of antitumor
immune augmentationz3and hematopoietic mobilizing activity
(Fig 5), suggest that IL-12 will be an important cytokine in
future clinical studies.
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1995 85: 2371-2376
Interleukin-12 enhances peripheral hematopoiesis in vivo
JD Jackson, Y Yan, MJ Brunda, LS Kelsey and JE Talmadge
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