1. No category

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1995 85: 2393-2401
Recombinant human interleukin-1 receptor antagonist protects early
myeloid progenitors in a murine model of cyclophosphamide-induced
myelotoxicity
UH Chudgar, CH Rundus and VM Peterson
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Recombinant Human Interleukin-l Receptor Antagonist Protects
Early Myeloid Progenitors in a Murine Model of
Cyclophosphamide-Induced Myelotoxicity
By Urrnish H. Chudgar, Christine H. Rundus, and Verlyn M. Peterson
Expression of hematoregulatory cytokines such as interleukin-l (IL-l) in response t o cytotoxic chemotherapy hastens
hematopoietic recovery, but may also potentiate myelotoxicity if myeloid
progenitors enter cell cycle before drug clearrecombinant human
ance. In the present study, the ability of
IL-l receptor antagonist (IL-lra) t o protect hematopoietic
progenitors was studiedin a murine modelof cyclophosphamide (CPA)-induced myelotoxicity. CF-1 female mice received 200 mg/kg CPA and either10 mg/kg IL-lraor an equal
(HSA) intraperitonevolume of 0.05% human serum albumin
ally (i.p.), followed 12 hours later by IL-lra or HSA. CPA and
IL-lra increased absolute neutrophil counts (ANCs) at days
2 ( P = .001) and 14 ( P = .0025) after CPA. In IL-lra-treated
mice, colony-forming units granulocyte-macrophage (CFUGM)/tibia were increased twofold and threefoldat days 2 ( P
= .0047) and 7 ( P = .023),respectively, whereas high
proliferative potential colony-forming cells (HPP-CFC)/tibia
were decreased twofold tothreefold at 8 hours (P = .039)
and 24 hours ( P = .0033), but were approximately threefold
higher than HSA-treated mice at day 7 after CPA. Coadministration of CPA and 11-1 enhanced myelotoxicity compared
with mice injected with CPA and IL-lra or HSA. In vivo, ILI r a protected HPP-CFC, but not CFU-GM, from hydroxyurea
suicide after a single dose of CPA, suggesting that IL-lra
inhibited cycling of HPP-CFC. In vitro, IL-lra did not alter
proliferation of CFU-GM, but inhibited IL-l-enhanced proliieration of HPP-CFC. These data suggest that IL-lra acts as
an indirectnegative regulator of hematopoiesis and protects
HPP-CFC from CPA, possibly by inhibiting IL-l-enhanced
proliferation of early myeloid progenitors.
0 1995 by The American Societyof Hematology.
E
fact that prolonged abrogation of endogenous IL-1 activity
with monoclonal antibodies that are specific for IL-l receptors retards BM recovery and reduces survival in lethally
irradiated mice.26
IL-l receptor antagonist (IL-lra) is a 17-kDpolypeptide
that is produced by several cell types, including adherent
monocyte^.'^*^^ IL-lra is a naturally occurring inhibitor of IL1 activity, and the molecule has been cloned and expressed in
Escherichia coli as a recombinant p r ~ t e i n . ~IL-lra
~ " ~ has the
unique capability of competing with IL-1 for occupancy of
IL- 1 receptors without being internalized or inducing second
signals.28Because IL-I expression may induce cycling of
BM progenitors after administration of myeloablative chemotherapy, it was hypothesized that administration of IL-lra
would attenuate CPA-induced myelotoxicity, and a murine
model of CPA-induced myelotoxicity was created to test this
hypothesis.
CF- 1 female mice received a single, sublethal, intraperito-
FFICACY OF CHEMOTHERAPY in the clinical setting often depends upon dose intensity, but bone marrow (BM) toxicity frequently limits the opportunity for dose
escalation.' Administration of recombinant colony-stimulating factors (CSFs), such as granulocyte CSF (G-CSF) or
granulocyte-macrophage CSF (GM-CSF), alleviates chemotherapy-induced myelotoxicity by augmenting proliferation
of late myeloid progenitors and decreasing the depth and
duration of ne~tropenia.~.~
However, studies in mice indicate
that repeated cycles of chemotherapy prolong the quiescent
state of early hematopoietic progenitors and that the addition
of myelorestorative cytokines may deplete BM of progenitors with long-term repopulating ability?35Such concerns
about the long-term consequences of the use of myelorestorative cytokines after chemotherapy have fostered interest in
alternative strategies. One approach has been to coadminister
agents that prevent or ameliorate chemotherapy-induced myelotoxicity. For example, proliferation of myeloid progenitors after BM ablation coincides with expression of hematoregulatory cytokines,6and several studies have suggested that
coadministration of negative regulators of hematopoiesis can
reduce the number of BM progenitors that enter cell cycle,
thus minimizing myeIot~xicity.~"~
Cyclophosphamide (CPA) and its metabolites persist for
several hours after administration, and, if proliferation of
BM progenitors is induced prematurely by the expression of
positive hematoregulatory cytokines, myelotoxicity may be
enhan~ed.'".'~
Of the cytokines implicated in cycling of hematopoietic cells, interleukin-l (IL-l) is the most pleiotropic.I8IL-1 modulates hematopoiesis by several different
mechanism^.'^^^^ It can shorten Go for quiescent early hematopoietic progenitors and possibly, stem cells, andwhen
combined with G-CSF, M-CSF, or IL-3, IL-1 can synergistically increase proliferation of high proliferative potential
colony-forming cells (HPP-cFCS).~~.~~
IL-1 can also augment CSF production and upregulate expression of CSF receptors on myeloid progenitor^.^*-^^ The importance of the
expression of IL-1 after BM ablation is suggested by the
Blood, Vol 85, No 9 (May I ) , 1995: pp 2393-2401
From the Division of Plastic and Reconstructive Surgery, Department of Surgery and Division of Pediatric Hematology/Oncology,
Department of Pediatrics, and The Children's Hospital, University
of Colorado School of Medicine, Denver, CO.
Submitted March 7, 1994; accepted December 13, 1994.
Supported by a grant from the Cancer League of Colorado.
Presented in part at the Keystone Symposium, January 31-February 6, 1993, Keystone, CO; theannual meeting ofthe Society of
Pediatric Research, April 30-May 3,1993, Washington,DC; and the
annual meeting of theAmerican Society of Hematology, December 26, 1994, Nashville, TN.
Address reprint requests to Verlyn M. Peterson, MD, University
of Colorado Health Sciences Center, Campus Box B-171, 4200 E
Ninth Ave. Denver, CO 80262.
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 1995 by The American Society of Hematology.
0006-4971/95/8509-0007$3.00/0
2393
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2394
CHUDGAR, RUNDUS, ANDPETERSON
neal (IP) injection of CPA, followed immediately by IL-lra
or the diluent, human serum albumin (HSA). Twelve hours
later, mice received a second IP injection of IL-lra or HSA.
Kinetics of the recovery of white blood cells (WBCs), CFUGM, and HPP-CFC in BM and spleen suggested that the
administration of IL-lra protected myeloid progenitors from
CPA, possibly by preventing entrance of early hematopoietic
progenitors into cell cycle. In contrast with IL-Ira, coadministration of IL-la and CPA enhanced myelotoxicity, presumably by driving hematopoietic progenitors into cell cycle
before CPA or its myelotoxic metabolites were cleared from
the system.
MATERIALS ANDMETHODS
Animals
Specific pathogen-free female CF-l mice (Charles River Laboratories, Stone Ridge, NY), 8 to 16 weeks old, were housed in the
AAALAC-accredited animal care facility of the University of Colorado Health Sciences Center and provided acidified water and chow
ad libitum. Animal care conformed to animal welfare guidelines set
forth by the United States Public Health Service. All research was
approved in advance by the Institutional Animal Care and Use Committee.
Cytokines and Reagents
Recombinant cytokines were generous gifts from the following
individuals: Recombinant murine GM-CSF (rMuGM-CSF) and recombinant murine mast cell growth factor (rMuMGF), Drs Steven
Gillis and Michael Widmer, Immunex, Inc, Seattle, WA; recombinant human M-CSF (rhM-CSF), Dr Peter Ralph, Cetus Laboratories,
Emeryville, CA; rhIL-6, Lori Myers, Sandoz Research Institute, East
Hanover, NJ; recombinant human interleukin-la (rhIL-la), Dr Peter
Lomedico, Hoffman-LaRoche, Inc. Nutley, NJ; and rhIL-lra, Dr
James Vannice, Synergen Inc, Boulder, CO. CPA for injection (Elkins-Sinn, Inc, Cherry Hill, NJ) and hydroxyurea (HU; Sigma Chemical CO, St Louis, MO), both reconstituted in sterile water, and 5%
HSA (Baxter Healthcare Corp, Glendale, CA), diluted to 0.05%
in 0.9% NaCI, were purchased commercially. All solutions for IP
injections and in vitro tissue culture were prepared from sterile,
pyrogen-free reagents, and the bacterial lipopolysaccharide content
of all working solutions was 50.05 IU/mL.
Cell and Plasma Harvest
Blood, BM, and spleen were harvested using previously described
techniques.?’ After methoxyflurane anesthesia, the inferior vena cava
was exposed via a midline abdominal incision and mice were exsanguinated, using sterile technique; plasma was separated by centrifugation at X3,OOOg for 10 minutes and frozen at -70°C until assayed
for CSF activity. Bilateral tibial BM cavities were flushed with 1
mL McCoy’s 5a tissue-culture media (GIBCO Laboratories, Grand
Island, NY) supplemented with sodium bicarbonate, sodium pyruvate, Eagle’s minimal essential medium vitamins, essential and
nonessential amino acid mixtures, L-glutamine, L-serine, L-asparagine, and diethylaminoethyl dextran (500 kD). Spleens were gently
passed through a wire mesh and monodispersed in supplemented
McCoy’s 5a tissue culture media by passing the cell suspensions
twice through a 20-gauge needle. Bone marrow andspleen cells
were washed once, resuspended in supplemented McCoy’s Sa, and
held on ice until cultured.
Cell Counts and Morphologic Evaluation
WBC and tibial BM and spleen cell counts were performed with
a ZBI Coulter counter (Coulter Electronics, Inc, Hialeah, FL). When
automated counts were less than l,OOO/pL, manual cell counts were
performed to reduce counting errors. Differential counts of 100 cells
were made on Wright-stained smears of peripheral blood. Differential counts of 500 nucleated BM and splenic cells were madeon
Wright-stained cytospin preparations.
Plusmu CSF Bioussuy
Plasma CSF activity was measured using a previously described
single-layer, soft agar culture te~hnique.~’
Triplicate serial dilutions
of test plasma were added to 35-mm petri dishes and covered with
I mLof 0.3% agar in supplemented McCoy’s 5a with 15% fetal
calf serum (FCS) containing 1 X IO5 BM cells from normal CF-I
female mice. After incubation for 7 days at 37°C in 7.5% CO2 and
100% humidity, myeloid progenitor colonies containing 2 50 cells
were enumerated with a dissecting microscope. CSF activity, expressed as units per milliliter, was calculated as the product of the
reciprocal of the sample dilution and the mean number of colonies
per dish.
Myeloid Progenitor Assays
CFU-GM. Bone marrow and spleen CFU-GM and BM HPPCFC were cultured in soft agar, using slight modifications of previously described te~hniques.~’.~’
For CFU-GM, a single-layer soft
agar technique was used. Viable nucleated BM cells, 1 X I o ” per
Murine Model of Cyclophosphamide-Induced
dish, or spleen cells, 1 X IOh per dish, were plated in triplicate 35-mm
Myelotoxicily
plastic petri dishes containing 1 mLof 0.3% agar in supplemented
McCoy’s 5a and 15% FCS. Recombinant human M-CSF, 22.5 ng/
In the initial phase of the study, mice received a single i.p. injection of 200 mgkg CPA, using a previously described te~hnique.~” plate, served as the stimulus. Cells were incubated at 37°C in 7.5%
CO2 and humidified air for 7 days. At the end of 7 days, colonies
At days I , 2, 3, 7, 10, 14, and 21 after CPA administration, groups
containing 250 cells were enumerated with a dissecting microscope.
of three mice were anesthetized and exsanguinated, and peripheral
HPP-CFC. HPP-CFC are a population of early myeloid progeniWBC and differential counts, nucleated BM and splenic cellularity,
tors that have the ability to survive a single dose of 5-Ruorouracil
plasma CSF activity, and BM and splenic myeloid progenitors were
in vivo and, in the presence of multiple hematopoietic growth factors,
quantified in individual animals. In the second phase of the study,
form large (20.5-mm diameter), highly cellular colonies in soft agar
mice received concomitant TP injections of 200 mgkg CPA and
that generate secondary, more mature colonies upon replating in the
either 10 mgkg IL-Ira or an equivalent volume (200 pL) of 0.05%
presence of CSFs.” HPP-CFC were cultured using slight modificaHSAat time 0 in an attempt to provide myeloprotection. Twelve
tions of a previously described double-layered soft agar system.”
hours later, IP injections of either IL-Ira or HSA were repeated.
The 1 -mL feeder layer initially contained I O ng each of rhIL-6, rhMGroups of three mice were then studied as above at days 2, 7, 10,
CSF, and rMuGM-CSF in 0.5% agar-Iscove’s modified Dulbecco’s
14, and 21. Experiments were repeated twice, and, unless otherwise
medium (IMDM). In subsequent in vivo HU suicide experiments,
indicated, results are based on data from 6 to 9 individually studied
rMuMGF (c-kir ligand) was also incorporated into the feeder layer.
mice per datum point.
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IL-IRA-INDUCED
MYELOPROTECTION
2395
Table 1. Kinetics of Blood Leukocyte, BM, and Spleen Cellularity After CPA
Days After CPA
Cell
Counts
x
108
ANC
WBC
BM
SPL
0
0.52
2.64
9.41
78
~~
t 0.1
0.07
t 0.38
t 0.87
-c 7.48
~
1
2
3
t 0.02*
0.02
1.15 t 0.2t
1.64 t 0.22*
43.6 t 9.3*
t O*
0.46 t 0.05*
0.5 t 0.07'
36.64 t 3.63'
0 f o*
0.03 ? 0.01*
0.07 -t 0.01*
14.8 IT 2.56*
7
0.16
1.28
3.03
66.1
10
0.37
1.8
5.48
102.3
t 0.05*
t 0.25t
t 0.36*
t 19.3
t0.34
0.11
t 0.34
t 0.74t
-c 8.0*
14
21
2 0.03t
2.15 2 0.41
5.24 t 0.59t
119.7 t 7.2*
1.05 t 0.07t
6.05 t 0.92t
80.4 t 7.0
ND
~
~
Abbreviation: ND, not done.
Mice received 200 mgtkg CPA as a single IP injection at day 0. Data expressed as mean t SEM nucleated cell counts per milliliter for ANC
and WBC or per organ for BM and spleen (SPL). Six mice per experimental group and 12 normal pretreatment control mice (day 0)were
individually studied per datum point.
P < ,0005 compared with the respective day 0 value.
t P < ,005compared with the respective day 0 value.
P < .05 compared with the respective day 0 value.
*
The feeder was overlaid with l mL 0.3% agar-IMDM supplemented
with 15% FCS, and contained 5 X IO4 or l X lo5 viable nucleated
BM cells. Cells were then incubated at 37°Cin 7.5% CO,and
humidified air for 10 days. At the end of 10 days, colonies 2 0.5
mm in diameter were scored as HPP-CFC.3'
In Vivo HU Suicide
The ability of IL-lra to protect myeloid progenitors in vivo after
CPA injection was studied using modifications of the in vivo HU
suicide technique described by Vassort et al." Briefly, mice received
concomitant IP injections of 200 mgkg CPA and either l0 mgkg
IL-lra or an equivalent volume (200 pL) of 0.05% HSA at time 0.
Twelve hours later, all mice were injected intravenously by tail vein
with 900 mg/kgHU and either IP IL-lra or HSA. BM cellularity and
BM CFU-GM and HPP-CFC were studied at day 2 after HU injection. Three different experiments with 3 to 4 mice per datum point
were performed, and the data were pooled.
turned to pretreatment levels within 3 weeks. In the spleen,
trilineage hematopoiesis, which was predominantly erythrocytic, became evident by day 7 after CPA.
Latehematopoietic progenitor (CFU-GM) and plasma
CSF response. Injection of CPA was followed by a precipitous decline in both the frequency and absolute number of
bipotential myeloid progenitors (CFU-GM) in BMand
spleen, reaching a nadir 24 hours after CPA (Fig 1). Increases
in the absolute number of CFU-GM were first noted at day
2, but did not reach pretreatment levels until day 21 (data
not shown). Splenic CFU-GM were undetectable at day 3,
but by day 7, the number of CFU-GM surpassed mean pretreatment values by threefold to fourfold before returning to
normal ranges at day 21 (data not shown). Plasma CSF activity was inversely related to the absolute number of CFU-
-
Statistical Analysis
Data were calculated as mean 2 SEM. Statistically significant
differences over time in the same treatment group, or among different
treatment groups at a single time point were determined by one-way
analysis of variance and a post hoc t-test. Statistically significant
differences among in vitro cultures of CFU-GM and HPP-CFC that
were incubated under varying culture conditions were identified by
Student's t-test. Statistical significance was assumed for P values S
12000
In Vivo Response to Cyclophosphamide
Kinetics of the hematologicresponse. Nadirs in nucleated cell counts of peripheral blood, BM, and spleen were
consistently observed at day 3 after a single 200-mgkg IP
injection of CPA. Mean cell counts were reduced approximately 10-fold for WBC and BM and fivefold for spleen.
(Table l). At day 3 after CPA, neutrophils were undetectable
in peripheral blood smears, and differential cell counts of
BM cytospin preparations showed the following distribution
of precursors: neutrophilic, 53.3%; erythroid, 33.3%;
lymphoid, 8.6%; and monocytic, 4.8%. Cytospin preparations of spleen at day 3 after CPA consisted of ~ 9 9 %
lymphocytes. Peripheral blood, BM, and splenic cellularity re-
p--!\
i
-600
\
- I
c
CFU-GM
-CSF
\
8000-fr
- 400
\
\
.05.
RESULTS
7
4000
\
' I
:.i
01
I
I
0123
1
7
10
14
1
1
1
DAYS AFTER CPA
Fig 1. Increases in plasmalevels of CSF ectivity in response to
decreases in proliferation of late BM progenitors (CFU-GM) after a
single IP injection of CPA. After 200 mg/kg CPA inmion, CFU-GM
per tibia were serially quantitated in BM(01; corresponding plasma
levals of CSF activity [V)ware alto determined. Data represent mean
f SEMvaluesfor air individually studied mice
in two separate experiments. * P < .OOO5,compared with the respective mean pretreatment
Iday 0 ) value.
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2396
AND
RUNDUS,
PETERSON
CHUDGAR,
Table 2. Effects of IL-lra or HSA on theKinetics of Recovery of Peripheral Blood, BM, and Splenic Cellular-w After CPA
Davs After CPA
Cells Counted x lo6
WBCImL
ANCImL
Cells/tibia
Cells/spleen
Treatment Group
2
IL-lra
HSA
IL-lra
HSA
IL-lra
HSA
IL-lra
HSA
1.4
1.17 t 0.15
0.87 2 0.18
0.1 2 0.021
0.03 2 0.01
1.04 2 0.09
0.91 -c 0.14
30.08 2 6.52*
15.6
56.4
2 2.16
7
2.11
0.95
0.14
0.1
5.67
4.37
58.80
10
2 0.26
14
1.48 t 0.29
2.05 lr 0.34
0.32 t 0.09
0.31 t 0.07
6.63 t 0.5
6.93 t 0.48
88.28 2 12.6
80.4
t 15.12
t 0.17
t 0.09
t 0.04
t 0.74
t 0.69
2 11.56
104.24
2 10.56
1.23
0.53
0.34
6.58
7.35
90.96
t 0.21
-C 0.26
t 0.05t
t 0.03
2 0.35
t 0.43
5 10.92
t 7.0
Mice received IP injection of CPA and either IL-lra orHSA as described in Materials and Methods, and serial nucleated cell counts, expressed
as the mean 2 SEM per milliliter for WBC counts and ANCs, or per organ for BM and spleen (SPL), were determined. Data represent values
that were pooled from nine individually studied mice in three separate experiments. Mean values for 12 normal, untreated mice depicted as
data for day 0.
P = ,001 compared with respective, same day HSA controls.
t P = .0025 compared with respective, same day HSA controls.
P = ,032 compared with respective, same day HSA controls.
*
GM in BM and spleen (Fig l ) . At 24 hours after CPA administration, plasma CSF activity had increased sixfold over
pretreatment values andremained elevated until day IO,
when hematopoietic repopulation of the BM and spleen was
well under way.
Myeloprotective Eflects Of In Vivo IL-Ira
Hematologic efects. The kinetics of the hematologic response to CPA in IL- 1 ra-treated and HSA-treated mice were
qualitatively similar to that observed in mice treated with
CPA alone. However, statistically significant quantitative
differences were notedbetween IL-Ira-treated andHSAtreated mice (Table 2). Mean WBC counts were consistently
higher in IL-Ira-treated mice, compared with HSA-treated
mice, but the differences were not statistically significant.
Absolute neutrophil counts (ANCs) were also consistently
higher in IL-Ira-treated mice, and these differences achieved
statistical significance at days 2 and 14. BM cellularity was
comparable in both groups at all time points, and, although
splenic cellularity was significantly higher at day 2 in ILIra-treated mice ( P = .032), IL-Ira administration did not
enhance splenic cellularity at later time points compared
with HSA-treated mice.
.Eflects on CFU-GM. Recovery ofBM CFU-GM after
CPA administration in IL-Ira-treated and HSA-treated mice
paralleled the qualitative pattern observed when CPA was
administered alone. However, the absolute number of CFUGM were twofoldand threefold higher in IL-Ira-treated
mice than in HSA-treated mice at days 2 and 7, respectively,
after CPA (Fig 2). This proliferative advantage continued at
days I O and 14, but the differences among IL- Ira and HSA
treatment groups were not statistically significant. Interestingly, the proliferative advantage for BM CFU-GM that was
associated with IL-Ira administration was not observed in
the spleens of IL-Ira-treated mice (data not shown).
E.ect.7 on early myeloid progenitors (HPP-CFC). Bone
marrow HPP-CFC were quantitated at 8, 24, and 48 hours
and 7 days after CPA injection in mice that were also treated
with either IL-Ira or HSA at 0 and 12 hours after CPA
injection. In contrast with the increased number of CFU-GM
per tibia that were observed in IL-Ira-treated mice at days
2 and 7 after CPA, HPP-CFC per tibia were decreased twofold to threefold during the first 24 hours after CPA injection
in IL-lra-treated mice (Fig 3). Thereafter, the number of
HPP-CFC per tibia remained relatively constant in IL-Iratreated mice, whereas HPP-CFC per tibia exhibited a steady
decrease in HSA-treated mice. Consequently, by day 7 after
CPA, BM HPP-CFC were approximately threefold higher in
IL-ra-treated mice than in HSA-treated mice.
0 NORMAL
(n=4)
0
2
7
10
14
DAYS AFTER CPA
Fig 2. Comparison of theeffects of IL-lra andHSA on thekinetics
of the late BM progenitor
(CFU-GM) response t o a single IP injection
of CPA. Mice received IP injections of either200 p L of 0.05% HSA or
10 mg/kg of IL-lra with, and12 hours after200 mglkg CPA. Normal
mice (n = 12) received no injections. Data, expressed as mean 2
SEM, represent values that are pooled from individually studied
mice
(number of mice per datum point shownin parentheses above error
bars) in three separate experiments. * P = .047, and **P = .023, compared with HSA-treated mice.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
IL-1RA-INDUCEDMYELOPROTECTION
2397
60
0 NORMAL
IIL-lra
HSA
50
40
30
20
10
NORhAL
8 'Hr
24 'Hr
48
h
n
7 LAYS
I
I
I
I
CFL
CFL
CFL
CFL
NaCl
NaCl
IL-l
IL-l
IL-lra
+
TIMES POST CPA
I
BF
+
+
+
+
+
Fig 3. Comparison of the effects of IL-lra and
HSA on thekinetics
of the early BM progenitor(HPP-CFC)response t o a single IP injection
IL-l ra
of CPA. Mice received IP injections of either 200 pL 0.05% HSA or 10
mglkg IL-lra withand l 2 hours after 200 mglkg CPA. Normal mice
(n = 12) received no injections. Data, expressed as mean 2 SEM,
represent values that are pooled from nine individually studied mice
Fig 4. Comparison of the effects of IL-lra and IL-la on in vitro
in three separate experiments. * P < .039, **P = .0033, and ***P =
proliferation of early BM progenitors. Day 4 post-5-FU BMcells, 2.5
.0005, compared with HSA-treated mice.
x 10'lculture dish, were incorporatedinto softagar and layered over
a cytokine-impregnated feeder layer (CFL) of soft agar containing 10
ng each of rhlL-6, rhM-CSF, and rMuGM-CSF, and 20 ng ofrMuMGF;
additionally, 10 p g of rhlL-la andlor 10 p g of rhlL-lra were added
HU suicide studies. Effects of IL- 1ra or HSA on survival
t o the CFL as indicated. Blank feeders (BFsl contained an equivalent
ofBM HPP-CFC after a single intravenous (W)injection
volume of 0.9% NaCl in place of cytokines. Data are calculated as the
of HU 12 hours after CPA are shown in Table 3. When the
mean ? SEM and expressed as the number of HPP-CFC or LPP-CFC
effects of HSA and IL-Ira on survival of HPP-CFC after
per 2.5 x 10' BM cells. The number of observations for a given set
HU (groups I1 and IV, respectively) were compared, HPPin parentheses. Addition of rhlL-lu
of culture conditions is indicated
andlor rhlL-lra t o BFs yielded no colony growth(data not shown).
CFC/tibia were 27% higher ( P = .0173) in IL-I ra-treated
* P < .0005, compared with all other HPP-CFC cultures; and * * P <
mice 2 days after HU suicide. The HPP-CFC sparing effect
.002, compared with allother LPP-CFC cultures.
In Vitro Culture Conditions
of IL-Ira was further shown by comparing the fractional
inhibition of HPP-CFC seen in HSA-treated mice (group I
v group 11, 84%) with that observed in IL-Ira-treated mice
(group Ill v group IV, 44%). CFU-GM/tibia were comparable in IL-Ira and HSA treatment groups after HU suicide,
suggesting that IL-lra did not protect late myeloid progenitors (data not shown).
Table 3. In Vivo Effech of IL-lra orHSA on Survival and Fractional
lnhibkion of HPP-CFC After IV HU in CPA-Treated Mice
Treatment Groups
1. CPA
980
+ HSA
11. CPA
+ HSA + HU
111. CPA
+ IL-lra
IV. CPA
+ IL-lra c HU
HPP-CFCTTibia
84%2 161
(n = 6)
158 2 19
(n = 11)
387 2 61
(n = 6)
216 2 18"
(n = 11)
Inhibition %
Group I v II
44%
Group 111 v IV
HU was administered IV 12 hours after i p CPA. Data for groups II
and IV (CPA, followed by HU suicide) represent day 2 post-HU values
expressed as mean 2 SEM. Datafor groupsI and 111 represent proliferation of HPP-CFC after CPA alone (CPA without subsequent HU suicide).
P = .0173 compared with group II.
Mselopoietic Effects
of In Vitro IL-Ira
BM cells from normal CF-I female micewere cultured
in 0.3% agar-McCoy's 5a supplemented with 15% FCS in
the presence of 0, 22, or 45 ng rhM-CSF/culture dish and
increasing concentrations of 0 to 100 pg IL-lra/culture dish.
IL-Ira was incapable of supporting CFU-GM proliferation
when used alone; when added to cultures containing various
concentrations of rhM-CSF, IL-Ira did not significantly alter
the frequency of CFU-GM, regardless of the concentration
of IL-Ira used (data not shown). Similarly, addition of 10'
to IO4 ng IL-l ra/culture dish neither enhanced nor suppressed
the proliferative capacity of HPP-CFC or LPP-CFC cultured
from BMof CF-I female mice thathadbeeninjected
96
hours earlier with 5-fluorouracil (5-FU), 150 mgkg (Fig 4).
However, whenused in excess to saturate IL-I receptors
(ie, I O pg/culture dish), IL- 1 ra abrogated IL- 1 a-associated
increases in HPP-CFC proliferation when 10 ng to 1,000 ng
rhlL-Idculture dish was added as a fourth cytokine in the
feeder layers of these cultures.
Comparative Mvelopoietic Effects of In Vivo IL-l
and IL- I ra
Mice received CPA and either I O mgkg IL-l ra, 20 pg/
kg IL-l, or an equivalent volume (200 pL) of 0.05% HSA,
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
CHUDGAR, RUNDUS, AND PETERSON
2398
along with and 12 hours after CPA. Mice were then killed
48 hours later, and BM cellularity, CFU-GM, and HPP-CFC
were quantitated (Fig 5). Bone marrow cellularity and the
absolute number of HPP-CFC and CFU-GM per tibia were
significantly greater at 48 hours after CPA in IL-Ira-treated
mice ( P < .005) than in IL-la-treated mice. CPA-injected
mice that received HSA exhibited values that were intermediate to those found in mice treated with IL-Ira or IL-la.
A. CELLULARITY
l
*
DISCUSSION
In this study, a single, sublethal, IP injection of CPA
was sufficient to induce myelotoxicity manifested by hypoplasia of myeloid and lymphoid organs, depletion of hematopoietic progenitors, and neutropenia. These data concur
with in vivo murine studies from other laboratories in which
a single, sublethal injection of CPA was shown to be myelotoxic.31133.35 The CPA-induced BM hypoplasia and neutropenia appeared to be physiologically significant, because
an approximately sixfold compensatory increase in plasma
levels of CSF activity occurred within 24 hours of injection
of CPA. Plasma CSF activity subsequently returned to
steady-state levels as the BM recovered.
Coadministration of IL-Ira ameliorated CPA-induced myelotoxicity, as evidenced by statistically significant increases
in peripheral blood ANC andBM CFU-GM. IL-1 ra has been
mentioned as a possible myeloprotective agent, but it has
not, to our knowledge, been previously studied as a myeloprotectant. Our data appear to contradict several in vivo
murine studies in which IL-I was found to bean effective
myeloprotective cytokine.3".33-36
However, a careful review
of these studies showed that IL-l can exert a myeloprotective, myelorestorative, or myelotoxic effect, depending upon
the time at which IL-l is administered relative to CPA. When
administered 20 to 24 hours before CPA injection or irradiation exposure in mice, IL-l protects BM progenitors, and
when injected 48 hours after BM injury, IL-l hastens BM
recoveryand enhances short-term survival in
However, when given as a single injection at any time during
the first 24 hours after CPA, IL-I enhances CPA-induced
myelotoxicity.3'),33
Neta et ally reported that a single IP injection of IL-I into normal mice increased the number of actively cycling BM cells. It has been suggested that the myelotoxic effect of IL-I is caused by an increase in the number
of cycling hematopoietic progenitors.33 In contrast, when
administered 20 hours before a cytotoxic agent, IL-I places
hematopoietic progenitors in a resistant state.36
In determining treatment schedules for the in vivo studies,
IL-Irainjections were timed to provide optimal antagonism
of IL-I while CPA and its myelosuppressive metabolites
remained at toxic levels. Previous studies of the pharmacokinetics of CPA metabolism in mice and rats have indicated
that CPA is hydroxylated in the liver to 4-hydroxycyclophosphamide, which, in turn, is converted to several metabolites,
including the potent myelotoxin, phosphoramide mustard."
Although the half-life of 4-hydroxycyclophosphamide was
less than 1 hour in these studies, serum levels of phosphoramide mustard continued to increase for at least 4 hours after
CPA injection.''.''
B. CFU-GM
sOOl
C.HPP-CFC
Fig 5. Comparison of the effects of IL-lra, 11-1, and HSA on BM
cellularity and BM progenitors (CFU-GM and HPP-CFCI4S hours after
a single IP injection of CPA. Mice received IP injections of 200 p L
0.05% HSA and either 10 mg/kg IL-lra or 20 pg/kg IL-lu with and
12 hours after 200 mg/kg CPA. BM cellularity is shown in A, and
absolute numbers of late (CFU-GM) and early (HPP-CFC)progenitors
per tibia are depicted in B and C, respectively. Data represent mean
f SEM values for six mice studied individuallyin two separate experiments. *P < ,005. compared with respective IL-l treatment group;
**P< .05, compared with respective HSAtreatment group; and***P
< .05, compared with respective IL-lra treatment group.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
2399
IL-1RA-INDUCED MYELOPROTECTION
IL- Ira is cleared very rapidly from the circulation in mice
and has a plasma half-life that is less than 4 hours after a
single, subcutaneous injection (D. Bloedow, personal communication, March 1993). However, IL-lra is cleared less
rapidly from the peritoneal cavity, when injected IP, as in
our study. Additional experiments in our laboratory showed
that IP injections of 10 mgkg IL-lra, at 4-hour intervals
over the first 12 hours, were no more myeloprotective than
the standard schedule used in this study (ie, two injections,
12 hours apart). The pharmacokinetics of rhIL-la after bolus
injection in various animals, is similar to that of IL-1ra.l' In
addition to dose scheduling, the choice of rhIL-la and rhILIra doses may also be important variables when attempting
to achieve optimal myeloprotection. The dose of rhIL-la
selected for use in the present study, 20 pg/kg/d, was comparable with the doses used by others for myelorestoration
and myeloprote~tion.~~.~~.~~
Though not previously used as a
myeloprotective agent, the dose of IL-lra selected for use
in the present study, 20 mgkg/d, has been used to successfully block IL-l-mediated physiologic events in animal models of endotoxemia and sepsis.18
The mechanisms involved in IL- Ira-mediated myeloprotection in our in vivo model are uncertain. Because IL-lra
was administered twice in the first 12 hours after CPA treatment and is cleared within a few hours of injection, it is
unlikely that the enhanced proliferation of CFU-GM that
was observed in IL-lra-treated mice at day 7 was caused
by a direct, long-term stimulatory effect of IL-lra. In vitro
experiments with IL-lra showed no direct effects on the
proliferation of early or late BM progenitors. However, the
ability of IL-lra to indirectly inhibit IL-la-induced proliferation of HPP-CFC was confirmed. The decreased number of
BM HPP-CFC in IL-lra-treated mice during the first 24
hours after CPA administration was temporally related to
administration of IL-lra and recapitulated the in vitro data.
The initial decrease in HPP-CFC after coadministration of
CPA and IL-lra was followed by a twofold increase in CFUGM over the next 24 hours. The prompt appearance of CFUGM after a decrease in HPP-CFC suggests a transient, ILIra-mediated inhibition of HPP-CFC. If the combination of
CPA and rhIL-lra had resulted in a permanent loss of HPPCFC, it is unlikely that the CFU-GM pool could have been
replenished within 24 hours from a population of progenitors
that preceded HPP-CFC in the myelopoietic differentiation
pathway. HU suicide confirmed that IL-lra protected HPPCFC by preventing their entry into cell cycle. HU kills a
high proportion of cells in S-phase without any appreciable
effect on quiescent cells."HUwas
administered intravenously 12 hours after CPA, and we chose to studyBM
HPP-CFC and CFU-GM at 48 hours post-HU based on the
observations made by Lord et al.9 The increase in absolute
number of BM HPP-CFC at day 2 after HU suicide in ILIra-treated mice, as well as the marked difference in fractional inhibition of HPP-CFC in HSA and IL-lra treatment
groups provides strong in vivo evidence that IL-lra protects
HPP-CFC by slowing their entry into cell cycle after CPA
administration.
The ability of IL-lra to inhibit proliferation of HPP-CFC
suggests that IL-lra is a negative regulator of hematopoiesis.
Fibbe et a137have shown that a continuous infusion of rhILIra in normal mice blocks IL-l-induced mobilization of
hematopoietic progenitors from the BM. Other investigators
have shown that negative regulators of hematopoiesis can
successfully function as myeloprotective agent^."^.^^-^ For
example, macrophage inflammatory protein- 1 a (MIP- la),
when given to mice 1 day before and 2 days after a single
injection of 5-fluorouracil, inhibits in vivo proliferation of
late myeloid progenitors7 MIP-la also protects myeloid progenitors when used in conjunction with cytosine arabinoside
or HU.839 The myeloprotective potential of MIP-la is restricted to late myeloid progenitors7 In contrast, transforming growth factor D l (TGF-Dl) selectively inhibits in
vitro proliferation of HPP-CFC, and its potential asan in
vivo myeloprotective agent is well r e c ~ g n i z e d . The
' ~ ~ ex~~~~~
act mechanism of action for TGF-P1 is unknown, but TGFP 1 can inhibit expression of IL- 1 receptors on hematopoietic
progenitor cells!* Such observations provide further indirect
evidence that IL-1 plays an important role in initiating proliferation of BM progenitors after chemotherapy and that abrogation of IL-1 activity results in myeloprotection.
IL-lra is well tolerated clinically and has a short half-life,
making it an ideal myeloprotective agent for use in the early
postchemotherapy setting. Although negative regulators of
hematopoiesis, such as IL-lra, may protect committed myeloid progenitors, complete restoration of BM function after
high-dose chemotherapy requires that lymphomyelopoietic
stem cells with long-term repopulating ability are also prot e ~ t e d . " .The
~ ~ in vitro progenitor assays used in the present
study (ie, CFU-GM and HPP-CFC) provide no direct insight
into the capability of IL-lra to protect long-term repopulating cells. Competitive repopulation assays will be necessary
to determine whether IL- Ira protects long-term repopulating
cells in this murine model of CPA-induced myelotoxicity."
However, based uponthese initial observations, further studies to determine the utility of IL-lra as a myeloprotective
agent appear warranted.
ACKNOWLEDGMENT
We thank Drs William A. Robinson and Linda C. Stork for their
critical reviewof this manuscript and Jennifer Wiler for her
excellent
technical assistance.
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