1. No category

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1994 84: 1457-1463
Corticosteroid modulation of interleukin-1 hematopoietic effects and
toxicity in a murine system
J Rinehart, EW Delamater, L Keville and J Measel
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Corticosteroid Modulation of Interleukin-l Hematopoietic Effects
and Toxicity in a Murine System
By John Rinehart, Elizabeth W. Delamater, Lisa Keville, and John Measel
Interleukin-l (IL-l)has been shown to ameliorate the hematopoietic toxicities of antitumor chemotherapeutic agents in
both mice and humans. However, IL-l toxicity in humans is
considerable and is similar to the systemic inflammatory
toxicities induced by IL-3,IL-6, and other cytokines with
pleiotropic biologic activities, eg, fever, nausea, malaise,and
hypotension. We hypothesized that corticosteroids may reduce IL-l toxicity without reducing IL-l hematopoietic effects in vivo. C3H/HeJ mice (female, 6 weeks) were treated
for 7 days subcutaneously with cortisone acetate (CA), (0.1.
0.25, or 0.5 mg/d/mouse), intraperitoneally with IL-l (1or 2
pg/d/mouse), or both. Asexpected, IL-l increased white
blood cell counts, splenic granulocyte-macrophage colonyforming units, and spleen cell number, and protected mice
fromlethal dosesof carboplatin (200 mg/kg; Paraplatin,
Bristol Laboratories, Evansville, IN) administered the day
after completion of the 7 days of IL-l administration. CA did
not significantly block the hematopoietic effects of IL-l or
the ability of IL-l to protect mice from the hematopoietic
toxicity of carboplatin. IL-l administered to mice at 8 pg/d/
mouse for 5 days induced decreased activity, roughening of
hair, diarrhea, pancytopenia, multiple metabolic abnormalities, and death in 60% of mice. IL-l at thetherapeutic doses
(0.5 to 2 pg/d) was not toxic. CA in a dose-dependent manner blocked all of the above mentioned toxicities when administered 24 hours and 30 minutes before each IL-l injection. CA also decreased IL-l-induced increase in plasma
tumor necrosis factor levels at the time pointexamined.
0 1994 by The American Societyof Hematology.
H
postorbital venous plexus using a microcapillary tube coated with
5% EDTA in Hanks' balanced salt solution (HBSS; GIBCO-BRL,
Gaithersburg, MD). Sixty microliters of the blood was diluted into
120 pL ofEDTA solution andthewhiteblood
cell (WBC) and
platelet counts were determined using a model S+IV B Coulter
counter (Coulter Electronics, Hialeah, FL). Smears were made of
the blood samples and stained with Wright's stain (Sigma Chemical
CO,St Louis, MO). These slides were examined by light microscopy
and the percentages of lymphocytes. granulocytes, and monocytes
were determined.
Preparation of spleen mononuclear cell suspensions and leukocyte-conditioned media (LCM). Animals were killed by cervical
dislocation, their spleens removed and placed in cold Iscove's modified Dulbecco's Eagle's Medium (IDMEM; GIBCO). Spleens were
disassociated by pressing through a Cellector tissue sieve (PGC
Scientifics, Gaithersburg, MD) into 60-mm tissue culture dishes (Falcon 1007, Becton Dickinson Labware, Lincoln Park, N3) containing
IDMEM supplemented with 20% fetal calf serum (FCS). Cells were
layered on Ficoll-Paque (Pharmacia Diagnostics, Silver Spring, MD)
in 15-mL polypropylene tubes. These were centrifuged at 400g for
25 minutes and interface cells were harvested. Spleen mononuclear
cells were washed three times by centrifugation at 200g. Spleen
mononuclear cells from treated animals were cultured in a methylcellulose hematopoietic progenitor cell assays for CFU-GM (see below). To produce LCM, washed spleen mononuclear cells from normal, spleens from untreated animals were resuspended in IDMEM
+ 20% FCS + 10 yg/mL phytohemagglutinin to a final concentration
EMATOPOIETIC growth factors and cytokines have
shown effectiveness in reducing the toxicity of anticancer chemotherapeutic agents. Both granulocyte colonystimulating factor and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been shown to reduce the
period of granulocytopenia if given after administration of
chemotherapeutic agents that induce damage to hematopoietic precur~ors."~
Other cytokines with activity on early hematopoietic progenitors have been tested in animals or clinical trials and have the potential to reduce suppression by
chemotherapeutic agents of platelet production, and production and function of various lymphocyte subsets, eg, interleukin-l (IL-I), IL-3, IL-6, IL-10, IL-l l , stem cell growth factor, and other^.^"' Many of the cytokines with pleiotropic
biologic activities (eg, IL-l, IL-3, and IL-6) that act on early
hematopoietic precursors share similar systemic toxicities:
fever, chills, malaise, nausea, rashes, hypotension, and fluid
retention. Preliminary data suggest that some of these toxicities may be caused by a secondary release of tumor necrosis
factor (TNF), tumor growth factor (TGFP), and other cytokines which may also negate the positive hematopoietic effects of the administered ~ y t o k i n e . ' ~ " ~
We have shown in vitro that corticosteroids enhance GMCSF, IL- 1, and IL-3 induced formation of granulocyte-macrophage colony forming units (CFU-GM) and inhibit IL-1induced release of TNF by human mononuclear cells.16 We
hypothesized that corticosteroids may block IL- 1 toxicity
and enhance IL-I hematopoietic effects in vivo. We report
the experiments examining this hypothesis in a murine
model.
MATERIALSAND METHODS
Mice. Female C3WHeJ mice were obtained from theAnimal
Production Area, National Cancer Institute-Frederick Cancer Research and Development Center (Frederick, MD). Animals were
routinely maintained at 5 to 8/cage inside filter bonnets in an isolation
room, with food and water provided ad libitum. Mice were quarantined for 1 week before treatment. Experiments were performed
using mice at 5 to 8 weeks old.
Preparation of peripheral blood smears for differential counts.
Animals were anesthetized by Forane (isoflurane) inhalant (Anaquest, Madison, WI), thenblood samples were obtained from the
Blood, Vol 84, No 5 (September l), 1994:pp 1457-1463
From the Divisions of Hematology and Oncology, the Department
of Medicine, Scott & White Clinic & Memorial Hospital: the Scott,
Sherwood and Brindley Foundation; andTexasA&M
University
Health Science Center, College of Medicine, Temple, TX.
Submitted September 7, 1993: accepted April 29, 1994.
Supported by National Institutes of Health Grant No. R01 CA51160-011A and institutional funds from the Scott, Sherwood and
Brindley Foundation and Texas A & M University Health Sciences
Center.
Address reprint requests toJohn Rinehart, MD, Scott & White
Clinic, 2401 S 31st St, Temple, TX 76508.
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 1994 by The American Society of Hematology.
0006-497//94/8405-00$3.00/0
1457
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1458
of 6 X 1Oh/mL. Thecellsuspension was placed in 25-cm2 tissue
culture flasks, then incubated at 37°C for 5 days. After incubation,
thecellsupernatant(ie,LCM)wascentrifugedandtheresultant
supernatant was passed through
a 0.22-pm syringe filter (Gelman
Sciences, Ann Arbor, MI), then stored in 2-mL aliquots at -4°C.
Preparation and cultureof bone marrow (BM). Mice were killed
by cervical dislocation, and tibias placed in chilled HBSS. BM cells
were obtained by flushing cold HBSS through tibiaswith a 27-gauge
needle. Cells were washed once in cold HBSS, then resuspended to
3 X lo6 cells/mL in IDMEMsupplemented with 20%FCS. BM
a
mononuclear cells (3x10') were incubated in methylcellulose at
final concentrationof 1% supplemented with 20%FCSand
l%
antibiotic-antimycoticsolution.Positivecontrolcultures
had 10%
LCM added. The cell cultures were plated in triplicate wells in 6well tissue culture dishes and incubated at37°C in a fully humidified
atmosphere of 5% COz for 7 days. On the seventh day, the number
of CFU-GMweredetermined
by examinationusing an inverted
microscope.
Administration of' biologic response modifiers or rhemotherupy.
IL-I,f? was a gift from Syntex Discovery Research (Palo Alto, CA).
IL-IP was dissolved in sterile saline containing
0.05% bovine serum
albumin (BSA); injections (0.2 mL/mouse) were administered intraperitoneally (IP). Cortisone acetate (CA)(Cortone; Merck, Sharp and
Dohme, West Point, PA) was dissolvedin sterile IDMEM; injections
(0.2
mL/mouse)
were
administered
subcutaneously
(SQ). Carboplatin (Paraplatin, Bristol Laboratories, Evansville, IN) was disto package direcsolved in sterile 0.9% NaCl for injection according
tions. Individual animals in an experimental group were administered
an intravenous (IV) injection of Carboplatin (0. I mL) in the lateral
or dorsal tail vein.
Cell proliferation. Cells were prepared as described in previous
sections, then resuspended to I X IO6 cellslmL in RPMI-1640 containing 20% fetal bovineserum, supplemented with 100 pg/mL penicillin, 0.1 pg/mL streptomycin, 0.25 pg/mL amphotericin-B, and 0.2
mmol/L L-glutamine. The cell suspension was pipetted into quadruplicate wells of a 96-well round-bottomed tissue cultureplate in 100
pL. Proliferation was induced by human recombinant IL-2. A twofold serial dilution of IL-2(10,000 to 156 Cetus U/mL) was prepared
and100 pL wasaddedtotheappropriate
wells. The plates were
incubated at 37"C, 5% COz for 72 hours. After incubation, the wells
were pulsed with 0.5 pCi 'H-thymidine, for 4 hours. The cells were
harvested using a PHD multiple automated sample harvester (Cambridge Technology Inc, Cambridge, MA) and the radioactivity on
theglass filter discsobtainedfromtheharvest
was measured by
as meancountsper
liquidscintillation.Thedatawereexpressed
minute (cprn) from quadruplicate wells of 5,000
the U/mL concentration that induced the highest cpm in all experiments.
Staining of BMor spleen forflow, cytometty. BM cells and spleen
mononuclearcellswereobtainedasdescribed
above. Cellswere
X IO' cells/mL
washed three times in HBSS and resuspended to 2
in HBSS without phenolred containing I % BSA (Fraction V, Sigma)
(staining buffer). Ethidium bromide (5 mg/mL in I O mmoUL TRIS,
1 mmol/L EDTA) was added to the cell suspension. After incubating
20 minutes in an ice bath, the cells were washed
by adding 2 mL
of staining buffer and pelleting at 300g for 6 minutes at 4°C. Stained
cells were resuspended in 400 pL cold staining buffer and held on
ice until quantitation by a flow cytometer.
Enzyme-linked immunosorbent assay (ELISA). ELISA was used
to determine the concentration of putative inhibitors
of hematopoiesis. Human TNFa was assayed using a commercially available
kit
(Predicta;GenzymeCorp,Cambridge,MA).Theabsorbancewas
measured at 450 nm. Concentration of TNFa was determined by
comparing results of test samples with a standard curve generated
by the same assay. The sensitivity of this assay is 10 pg/mL.
Statistical methods. Statistical analysis was performed using the
RINEHART ET AL
12.
t
t
IL-l 1.0pg
IL-l 1.0 pg + CA 0.1 mg
"E 10-
E
"
.8X
8a
4
61
/
l
2
04
0
2
4
6
128
10
Days
t
t
CA 0.1 rng
IL-l 1.0 pg
2i
04
0
l
2
4
6
8
10
12
8
10
12
Days
16,
t
0
Control
IL-l 1.0 pg
2
4
6
Days
Fig1. Six-week-old female C3HIHeJ mice were injected with IL1 IIP at 1.0 pglmouse) or CA IS0 at 0.1 mglmouse) or both IL-l and
CA. Controls were injected with carrier IP and SO for 7 days 16 mice/
group). Mice were bled from the retro-orbital plexus and peripheral
blood counts w w e determined as described in Materials and Methods. A, AGC; B, ALC; C, platelets.
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
INTERACTIVE EFFECTS OF IL-l AND CORTICOSTEROIDS
1459
Table 1. Effect of CA and IL-lp on Hematopoietic Parameters in C3H/Hd Murine Spleens
Dose (mouseld
X
7 d)*
IL-l 2 pg
Control
Spleen weight (mg)
No. mononuclear cells/spleen ( ~ 1 0 ' )
Spleen CFU-GM*
YO Spleen leukocytes in S-phase
Spleen leukocyte proliferation in response to IL-2
153
16.3
807
2.5
1,745
t 10t
t 3.7
2 119
t 0.4
t 41711
CA 0.25 mg
IL-l 2 pg
52 I
35
8.1 t 0.6
61 -t 249
0.7 t 0.15
455 t 46
416 t 155
137 t 12.45
36,696 -t 5,4141
14.9 t 1.65
15,312 t 1,6329
+
CA 0.25 mg
391
128.5
74,422
18.3
14,672
t 365
t 0.25
f 16,6639
t 0.85
t 8415
Mice were killed on day 8 after 7 days of SQ CA, IP IL-Ip, or both at doses indicated.
? SD (n = 15 to 17 mice, three experiments).
Numbers are total CFU-GM/spleen.
5 P < .01 compared with control.
11 Data presented as mean cpm t SD; IL-2 concentration = 5,000 Cetus U/mL.
t Data presented as mean
+
Student's r-test and one-way analysis o f variance. Differences i n
survival betweenvarious treatment groups were analyzedusing Fisher's
test.
x*
RESULTS
In initial experiments, we administered IL-1 either at 1.0
or 2.0 pg/d/mouse IP, CA at 0.1 or 0.25 mg/d/mouse SQ or
both IL-1 and CA for 7 days. Controls received injections
both IP and SQ of appropriate carriers. Peripheral blood
counts were determined the day before, during, and after
treatment as indicated in Fig1. IL-1 alone suppressed the
absolute lymphocyte count (ALC), the absolute granulocyte
count (AGC), and platelet count during treatment, but the
values of these parameters increased above baseline and control levels by day 12. CA alone at 0.1 mg/d/mouse had no
significant effects on these parameters, but CA administered
in combination with IL-1 attenuated the decrease in AGC,
ALC, and platelet count seen during IL-l administration
alone, and attenuated the rebound in these parameters seen
when IL-1 was administered alone. The effect of these treatments on splenic parameters is seen in Table 1. Mice were
killed the day after completing the treatments as described
above and in Table 1. IL- 1 alone increased splenic weight,
cell number, CFU-GM, the fraction of cells in S-phase, and
proliferative response to IL-2. CA decreased all of these
parameters when administered alone. However, when CA
was administered with IL-1, splenic CFU-GM increased in
number compared with IL- 1 alone. The effect of these treat-
ments on BM parameters is presented in Table 2; CA, ILl, and the combination did not significantly change the number of cells or CFU-GM /femur or the fraction of cells in Sphase.
In the next series of experiments, we examined the protective effects of IL-1 alone (2 ,ug/d/mouse), CA alone (0.1,
0.25, or 0.5 mg/d/mouse), and IL-l plus CA when given for
7 days before carboplatin, 200 mgkg; 20/24 control mice
died compared with 0/20 mice after receiving IL-1 alone ( P
< .01) (Fig 2). IL-1 plus CA was as effective as IL-1 alone
in rescuing mice from carboplatin toxicity when all three
doses of CA administered with IL-1 were analyzed together
(1/28 died) and compared with IL-1 alone, or when the three
IL-I plus CA groups were separately compared with IL-l
alone (see Fig 2). Interestingly, CA alone at 0.5 mg/d/mouse
also improved post-carboplatin survival ( P < .01). IL-1 and
IL-1 plus CA reduced, but did not eliminate, the marked
decrease in AGC, ALC, and platelet counts induced by Carboplatin, 200 mgkg (Fig 3).
To determine if CA reduced toxicity of IL- 1, weexamined
the toxicity of increasing doses of IL-1: 2 pgldmouse induced no apparent toxicity by inspection of mice; 4 and 6
pg/d/mouse induced weight loss, roughened fur, diarrhea,
and decreased activity, but no deaths (data not shown). Mice
treated with 8 pg/d/mouse for 5 days died (6/10) or were
moribund by day 6 (Table 3). Administration of CA 24 hours
and then30 to 60 minutes before each K- 1 injection reduced
mortality in a dose-dependent manner; no deaths were ob-
Table 2. Effect of CA and IL-lp on Hematopoietic Parameters in C3HIHe.J Murine Bone Marrow
Dose Imouseld
Control
No. bone marrow cells/femur ( ~ 1 0 ' )
Bone
CFU-GM
marrow
% Bone marrow cells in S-phase
Bone marrow cell proliferation in response to IL-2
6.9
526
581
12.7
9,391
t 0.9t
t 96$
t 0.3
t 1,0615
* Numbers are total CFU-GM/femur.
5 Data presented as mean cpm t SD; IL-2 concentration = 5,000 Cetus U/mL.
d)*
CA 0.25 mg
IL-l 2 pg
IL-l 2 pg +
CA 0.25 mg
7.3 t 0.6
618 t 150
10.9
14.6
I
0.4
13,627 t 1,114
6.4 t 0.6
532 -t 51
13.3
t 0.8
10,378 t 2,148
5.6 t 0.6
t 146
t 0.8
8,302 t 452
Mice were killed on day 8 after 7 days of SQ CA, P; IL-l@,or both at doses indicated.
t Data presented as mean +- SD (n = 15 to 17 mice, three experiments).
X
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
1460
A
-
RINEHART ET AL
ment with CA. CA aloneatthehigher
doses induced expected modest hematologic and biochemical alterations. but
mice receiving CA alone appeared well and no deaths were
observed (Table 3, and Figs 4 and S).
100
80
.-I
g
DISCUSSION
60
The data presented here support the hypothesis we developed; in vivo corticosteroids may blockthetoxic
effects,
2
al
g
40
Control (24)
CAO.lmg (10)
t CA 0.25 mg (10)
-+- CA 0.5 mg (8)
-a-
20
&
t Control
t
0
2
4
6
8
1'0
14
1'2
1
Days
"
I
8
CA0.25mg
t
IL-l 2pg
t
CA 0.25 mg + IL-l 2 pg
4 -
80
2
0-
,
,
,
,
,
-2
0
2
4
6
8
m
,
,
,
,
,
,
10
12
14
16
18
20
Days
c
al
G 40-
-" IL-l 2 ug (20)
a
20 -
8
t
IL-l 2 ug + CA O.lmg (10)
t
IL-l 2 ug + CA 0.25 mg (10)
A
IL-l 2 ug + CA 0.5 mg (8)
6
0
04
0
B
2
8 4
6
1
0
1
2
1
4
Days
E
E
'0
z 4
X
Fig 2. In three separate experiments, 6-week-old female C3H/HeJ
mice were injected with IL-l (IP at 2 pgldlmouse), CA (SO at 0.10,
0.25, or 0.5 mgldlmouse) or both IL-l and CA or carrier only SO and
IP for 7 days (-7 through -1). On day zero, mice were administered
200 mglkg carboplatin IV. Mice were then followed daily for survival
without further manipulation.
The number of micelgroupare as indicated in parenthesis. (In Fig 28, all of the lines except IL-l 2 Ng+CA
0.5 mg are superimposed.)
served in animals receiving CA 0.5 mg/d/mouse plus IL-l.
Preadministration of CA also reduced weight loss induced
by IL-1 and improved activity levels in surviving mice. IL1 at this toxic dose induced multiple hematologic and biochemical abnormalities (Figs 4 and 5); by day 6, total WBC
count,AGC,ALC,and
platelet countwere allmarkedly
reduced. (Compare the"zero", ie, no cortisone controlmice
between Fig 4A (noIL- 1, no CA) and Fig 4B(IL- l , no CA).
CA partially reversed IL- l-induced thrombocytopenia and
neutropenia and, infact, increased AGC above controllevels.
Asexpected,thisschedule
of IL-1also increasedplasma
serum glutamic oxaloacetic transaminase, andblood urea
nitrogen (BUN)and markedlydecreasedplasma
glucose,
iron, and cholesterol. Mice pretreated with CA showed ameliorization of these IL- l-induced hematologic and biochemical abnormalitiesin a dose-dependent manner. IL- 1 administration at 8 pg/d/mouse(Table 3), but not 2 pg/d/mouse,
induced high plasma TNFwhich were decreased by pretreat-
sa
2
r
:
0-2
-
t Control
t
-t
2
0
4
6
8
10
12
CA 0.25 mg
IL-l 2pg
CA 0.25 mg + IL-l 2 pg
14
16
18
4
20
22
20
i
22
Days
12-
C
"c
10~
,
"-2
d
--f
2
4
6
Control
CA 0.25 mg
8 12 10
l4 18 16
Days
Fig 3. Mice (8/group) were treatedas described in the figure legend for days -7 through -1. All mice received carboplatin 200 mglkg
on day 0. Mice were bled on indicated days t o determine peripheral
blood counts. A,AGC; B, ALC; and C, platelets.
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INTERACTIVEEFFECTSOF
1461
IL-l ANDCORTICOSTEROIDS
Table 3. Survival and Plasma TNF Levels After High-Dose IL-1/3 in
C3H/He.J Mica: Effect of Treatment With Cortisone Acetate
No. DeathsFotal
Control
CA 0.25 mg
CA0.5 mg
lL-l*
IL-l CA0.1 mg
IL-l CA 0.25 mg
IL-l CA 0.5 mg
+
+
+
016
013
013
420 6110t
216
2110
0110
Plasma TNF Levels
(pg/rnL)
0
0
0
?
153$
0
0
0
* C3HlHeJ mice received no IL-10 or IL-10 at 8 pglmouseld ondays
2,3,4, and 5 without or with CA, SQ on days 1,2,3,4, and 5. CA was
given 30 to 60 minutes before IL-10.
t Treated animals died on day 5; on day 6 surviving animals were
exsanguinated by cardiac puncture and TNFo levels determined by
ELISA.
Data presented as mean 2 SD of the mean. Two separate experiments were performed.
*
but not the hematopoietic effects of IL-1.Thus, this study
surprisingly showed that CA blocks some immunologic effects of IL-1while sparing the examined hematologic effects
in vivo, and therefore, supports our previously reported in
vitro observations.’6 Corticosteroids induce multiple metabolic and immunologic effects when administered in pharmacologic doses to mice and
Just as complex are
the protean effects induced by administration of IL-1.” Although our present work mayprovide some clues, the mechanisms of these interactions are of undoubted complexity and
will require additional studies to elucidate. IL-I at toxic
doses induced measurable plasma TNF levels, and pretreatmentwith CA blocked development of IL-1 toxicity and
reduced the observed levels of TNF below our assay level
of detection. TNF may induce many of the toxicities associated with IL-1administration in humans and mice; eg, TNF
can induce hypotension, fluid loss into tissue with resultant
hypovolemia and increase in BUN, as well as WBCand
platelet margination, nausea, and other systemic symptom~.’’,’~
TNF may also decrease proliferation of hematopoietic progenitor^.'^ However, in our studies, the lowest dose
of CA used completely eliminated the increased TNF levels
at the single time point examined, but not all IL-1 toxicity.
Several possibilities may explain this paradox; local TNF
release and action maybe important in TNF toxicity and
measurement of plasma levels may underestimate TNF effects. Furthermore, we only obtained TNF levels in plasma
at one time point that may not have been optimal in reflecting
TNF release. Thus, TNF may have been released but undetected at the time point examined. Finally, other cytokines
may play a role and may act alone or synergistically with
TNF at levels below our levels of detection to induce toxicity
observed. For example, IL-6 and interferons may induce
inflammatory-related systemic effects as well as TNF. IL-1
may induce toxicity unrelated to induced release of cytokines; IL-1 may directly enhance margination of oxidantreleasing granulocytes and macrophages into sensitive tissues. The decreased WBC counts observed with toxic doses
of IL-1 supports this possibility. Finally, IL-I may directly
induce alteration in vascular endothelial cells to ultimately
induce tissue hypoperfusion.”
CAdidnot block the hematopoietic effects of IL-I in
these studies and, in fact, appeared to enhance IL- 1 induction
of splenic CFU-GM. We have not explained as yet the mechanism of this latter effect. Moreover, these results must be
regarded with caution because we examined only one time
point and one dose of CA (Table 1). Nevertheless, we did
examine multiple doses of CA in combination with IL-Iin
regard to the effect on carboplatin-induced mortality; doses
of CA that failed to block carboplatin-induced mortality did
not block IL-lreversal of carboplatin-induced mortality (Fig
2 ) . We hypothesize that ( 1 ) IL-1 induces production of inflammatory mediators (eg, TNF, interferons, a , ,& and 7 ,
macrophage inflammatory proteins, prostaglandins, and others) that inhibit hematopoiesis, and (2) that IL-I induces
production of hematopoietic growth factors when given at
nontoxic doses (0.5 to 2 pgldmouse). We further hypothe-
0
+ UMMS
0
0.2
0.1
X
lo5
0.3
0.4
0.5
CA Dose (mg/mouse/day)
No IL-l
18,
l
~ ~ 1X 2io3
~
-e WBC
I
1 0 3
0
04
0
0.1
0.2
0.3
0.4
I
0.5
CA Dose (mglmouselday)
IL-l 8pg/mouse/day
Fig 4. In two separateexperiments, 6-week-old C3HlHe.J mica
were given IL-l (IP at 8 pglmouse), CA (SO at 0.1, 0.25, 0.5 mgl
mouse), or both IL-l and CA for 5 days. Mice surviving were bled on
day 6 by cardiac puncture for determination of peripheral blood
counts illustrated in Fig 4 and plasma parametars illustrated in Fig
5. The number of animals surviving to day 6 and plasma TNF levels
are shown in Table 3.
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
1462
RINEHART ET AL
t Glucose (mgldl
5
0
r
"t
BUN (mgldl)
t
SGOT (UIL)
&
Cholesterol (mgldl)
~
Iron (vgldl)
i
-b
0.1
0.2
0.3
0.4
1
0.5
CA Dose (mg/mouse/day)
No IL-l
Glucose (mg/dl
BUN (mg/dl)
t SGOT(UL)
+ Cholesterol (mg/dl)
++ Iron (pg/dl)
"+
t
B
-6
0.1
0.2
0.3
0.4
0.5
CA Dose (mglmouselday)
IL-l 8CcglmouseIday
Fig 5.
We have also observed that at high doses, CA (0.5 mg/
dlmouse) blocked carboplatin-induced death in our murine
model. We chose carboplatin for use in these atudies because
its only observed toxicity at the doses used are hematologic;
mice died at the nadir of their peripheral blood counts and
bacterial peritonitis was present at death. Furthermore, biochemical profiles obtained 24 hours before death in mice
receiving carboplatin alone showed no decrease in renal or
hepatic function (data not presented). We have n o w shown
that high-dose cortisone acetate does ameliorate the hematopoietic toxicity of carboplatin."' It is likely that the mechanisms by which CA and IL-1 protect mice from the hematopoietictoxicities of carboplatinaredifferentbecause
IL-l
increases the number of hematopoietic precursors in spleen
(CFU-GM), whereas at high doses, CA reduces hematopoieticprecursors. CA may act by increasing the number of
hematopoietic precursors in G , or induction of other protective mechanisms (eg, induction of increased levels of glutathione or metalothiones). IL- 1 may providehematopoietic
protection by increasing the total number of hematopoietic
precursors.
These observations may have clinicalutility. It may be
possible to reduce toxicity of IL- I , IL-3, IL-6, or GM-CSF
by concomitant administration of corticosteroids. We have
shown in vitrothathydrocortisonesuccinate,prednisone,
and dexamethasone enhanceIL- I , IL-3, and GM-CSF induction of human CFU-GM proliferationatconcentrations
readily obtainable clinically with modest dosesof these corticosteroids."."." Furthermore, we have shown that GM-CSF
andIL-l inducehuman mononuclear release of TNF and
this release is blockedby the same concentrations of corticosteroids that enhance CFU-GM induction."' These findings
suggest that a clinical trial to examine thecombination corticosteroids and IL-l or IL-3 may be feasible.
See legend to Fig 4.
ACKNOWLEDGMENT
size that corticosteroids block production of these inflammatory inhibitors, but not hematopoietic growth factors. Data
have been developed to support the first part of our hypothesis,ie, that IL-I administration induces production of inflammatory mediators such as TNF,21.2"2sand hematopoietic
growth factors such as GM-CSF and
IL-6.2h-2X
The second
part of our hypothesis, ie, thatcorticosteroids
enhance
production of hematopoietic growth factors and inhibit production of inflammatory mediators, is not supported by an
observation suggesting that corticosteroidsinvitroinhibit
GM-CSF p r o d ~ c t i o n . ~ ~ have
W e recently examined thepharmacokinetics of CA in C3H/HeJ mice: CA administered SQ
at 0.5 mg induces a cortisol (the active metabolite of CA)
peak level of 50 pg/dL. This concentrationof hydrocortisone
enhances IL-l-induced CFU-GM formation in vitro." We
hypothesize that the concentrationsof corticosteroids needed
to inhibit IL-l-induced hematopoietic growth factor release
may be greater than those required to inhibit release of inflammatory cytokines. In addition, we are currently examining theeffect of corticosteroidson hematopoietic growth
factor and inflammatory mediator mRNA expression in vivo
and in vitro.
We would like to acknowledge the support of William Buhles.
PhD, DVM (Syntex Discovery Research) and Edward Rappaport,
MD (Scott & White), in the development of these data.
REFERENCES
1. Pettengell R, Gurney H, RadfordJA,
Deakin DP,James R,
Wilkinson PM, Kane K.Bentley J, Crowther D: Granulocyte colonystimulating factor to prevent dose-limiting neutropenia in non-HodgBlood 80:1430,
kin'slymphoma: A randomizedcontrolledtrial.
1992
2. Advani R, Chao NJ, Homing SJ. Blume KG, Ahn DK. Lamhorn KR, Fleming NC, Bonnem EM, Greenberg PL: Granulocyteto
macrophage colony-stimulating factor (GM-CSF) as an adjunct
autologous hemopoieticstem cell transplantation for lymphoma. Ann
Intern Med 116: 183, 1992
3. Neidhart JA, Mangalik A. Stidley CA, Tebich SL. Sarmiento
LE, Ptile JE, Oette DH, Oldham FB: Dosing regimen ofgranulocytemacrophage colony-stimulating factorto support dose-intensive chemotherapy. J Clin Oncol 10: 1460, 1992
4. Neidhart JA, Mangalik A. Kohler W, Stidley C, Saiki J, Duncan P, Souza L, Downing M: Granulocyte colony-stimulating factor
stimulates recovery of granulocytes in patients receiving dose-intensive chemotherapy without BM transplantation. J Clin Oncol 7: 1685.
I989
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
INTERACTIVEEFFECTS
OF IL-l AND CORTICOSTEROIDS
5. Rinehart J: Phase 1 trial of IL-3, Carboplatin, and etoposide
in patients with solid tumors. Blood 80:86a, 1992 (abstr, suppl)
6. Samuels B, Bukowski R, Gordon M, Samuel S, Isaac R, Demetri G: Phase 1 study of rhIL-6 with chemotherapy (CT) in advanced
sarcoma. Proc Am Soc Clin Oncol 12:291, 1993
7. Olencki T, Budd GT, Murthy S, Finke J, Herzog P, McLain
D, Tubbs R, Tuason M, Edinger M, Levitt D, Bukowski RM: Immunoregulatory and hematopoietic effects of interleukin-6 (rhIL-6) in
cancer patients. Proc Am SW Clin Oncol 12:292, 1993
8. Smith J W , Longo DL, Alvord WG, Janik JE, Sharfman WH,
Gause BL, Curti BD, Creekmore SP, Holmlund JT, Fenton RC: The
effects of treatment with interleukin-la on platelet recovery after
high-dose Carboplatin. N Engl J Med 328:756, 1993
9. Du XX, Nebem T, Goldman S, Williams DA: Effects of recombinant human interleukin-] 1 on hematopoietic reconstitution in
transplant mice: Acceleration of recovery of peripheral blood neutrophils and platelets. Blood 81:27, 1993
10. Brandt J, Briddell RA, Srour EF, Leemhuis TB, Hoffman R:
Role of c-kitligand in the expansion of human hematopoietic progenitor cells. Blood 79:634, 1992
11. Molineaux G, Migdalska A, Szmitkowski M, Zsebo K, Dexter
TM: The effects of hematopoiesis of recombinant stem cell factor
(ligand for c-kit) administered in vivo to mice either alone or in
combination with granulocyte colony-stimulating factor. Blood
78:961, 1991
12. Hersh EM, Muggia FM, Whitehead RP, Rinehart JJ, Bonnet
JD: Phase I1 studies of recombinant human tumor necrosis factor
alpha (rHuTNFa) in patients with malignant disease. A summary of
the Southwest Oncology Group experience. J Immunother 10:426,
1991
13. Creagan ET, Kovach JS, Moertel CG, Frytak S, Kvols LK:
A phase I clinical trial of recombinant human tumor necrosis factor.
Cancer 62:2467, 1988
14. Eliason JF and Vassalli P: Inhibition of hemopoiesis in murine
marrow cell cultures by recombinant murine tumor necrosis factor
Q: Evidence for long-term effects on stromal cells. Blood Cells
14:339, 1988
15. Hino M, Tojo A, Miyazono K, Urabe A, Takaku F: Effects
of type p transforming growth factor on haematopoietic progenitor
cells. Br J Haematol 70:143, 1988
16. Delamater L, Rinehart J: The effect of corticosteroids on IL-l
induction of hematopoietic progenitor proliferation. Blood 80:413a,
1992 (abstr, suppl)
17. Rinehart JJ, Sagone A, Balcerzak SP, LoBuglio AF: Effect
of corticosteroids on human monocyte function. J Clin Invest
54:1337, 1974
1463
18. Rinehart JJ, Sagone A, Balcerzak SP, Ackerman A, LoBuglio
AF: Effect of corticosteroid therapy on human monocyte function.
N Engl J Med 292:769, 1976
19. Boumpas DT, Paliogianni F, Anastassiou ED, Balow JE: Glucocorticosteroid action on the immune system: Molecular and cellular aspects. Clin Exp Rheumatol 9:413, 1991
20. Bailey JM: New mechanisms for effects of anti-inflammatory
glucocorticoids. Refractors 3:97, 1991
21. Neta N, Oppenheim J: IL-l: Can we exploit Jekyll and subjugate Hyde? Biol Ther Cancer Updates 1O:l. 1992
22. Rajagopalan V, Essex D, Shapiro SS, Konkle BA: TNF-a
modulation of glycoprotein I@ Q expression in human endothelial
and erythroleukemia cells. Blood 80:153, 1992
23. Shalaby MR. Waage A, Aarden L, Espevik T Endotoxin,
tumor necrosis factor Q and interleukin 1 induce interleukin 6 production in vivo. Clin Immunol Immunopathol 53:488, 1989
24. Dinarello CA: Interleukin 1 and its biologically related cytokines. Adv Immunol 44:153, 1989
25. Libert C, Brouckaert P, Shaw A, Fiers W: Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice.
Eur J Immunol 20:691, 1990
26. Bagby GC Jr, Dinarello CA, Wallance P, Wagner C, Hefeneider S, McCall E: Interleukin 1 stimulates granulocyte macrophage
colony-stimulating activity release by vascular endothelial cells. J
Clin Invest 78:1316, 1986
27. Fibbe WE, Van Damme J, Billiau A, Duinkerken N, Lurvink
E, Ralph P, Altrock BW, Kaushansky K, Willemze R, Falkenburg
JHF: Human fibroblasts produce granulocyte-CSF, macrophageCSF, and granulocyte-macrophage-CSFfollowing stimulation by interleukin-l and poly(rI).poly(rC). Blood 72:860,1988
28. Neta R, Vogel SN, Plocinski JM, Tare NS, Benjamin W,
Chizzonite R, Pilcher M: In vivo modulation with anti-interleukin1 (&l) receptor (p80) antibody 35F5 of the response to 1L-l. The
relationship of radioprotection, colony-stimulating factor, and IL-6.
Blood 76:37, 1990
29. Thorens B, Mermod JJ, Vassalli P: Phagocytosis and inflammatory stimuli induced GM-CSF mRNA in macrophages
through posttranscriptional regulation. Cell 48:671, 1987
30. Rinehart J, Delamater E, Keville L: Corticosteroids reduce
hematopoietic toxicity of dose intensive chemotherapy in a murine
model. Blood 82:652a, 1993 (abstr, suppl)
31. DiSanto AR, DeSante KA: Bioavailability and pharmacokinetics of prednisone in humans. J Pharm Sci 64:109, 1975
32. Ferry JJ, Horvath AM,Bekersky I, Heath EC, RyanCF,
Colburn WA: Relative and absolute bioavailability of prednisone
and prednisolone after separate oral and intravenous doses. J Clin
Pharrnacol 28:81. 1988