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Suppression of Juvenile Chronic Myelogenous Leukemia Colony Growth
by
Interleukin-l Receptor Antagonist
By Raffaella Schir6, Daniela Longoni, VincenzoRossi, Oscar Maglia, Alberta Doni, Marcello Arsura, Giovanna Carrara,
Giuseppe Masera, Edouard Vannier, Charles A. Dinarello, Alessandro Rarnbaldi, and Andrea Biondi
Bone marrow (BM) and peripheral blood (PS) cells from patients with juvenile chronic myelogenous leukemia (JCML)
exhibit spontaneous in vitro proliferation. Several cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-18 (IL-la), and tumor necrosis factor alpha (TNFa) have been implicated in
supporting the growthof leukemic monocyte-macrophage
colonies either by autocrine or paracrine pathways. In
seven untreated JCML patients, w e investigated the role
of I L - l in the spontaneous growth of these cells by specifically blocking I L - l receptors. The I L - l receptor antagonist
(IL-lRa) wasadded to theclonogenic assays, and in each
case significant (mean = 63%. range = 35% t o 82%)inhibition of spontaneous proliferation was observed. Uncultured circulating cells from PB or BM of fourout of five pa-
tients expressed IL-18-specific mRNA and secreted the
protein into theculture supernatants. Moreover, by means
of reverse transcriptase-polymerase chain reaction (RTPCR). we demonstrated that most of the spontaneously
growing leukemic colony-forming unit cells (CFU-C) obtained fromBM cells of t w o patients were positive for the
presence of the IL-18-specific mRNA. Despite the presence of a measurable amount of GM-CSF in JCML cell culture supernatants, GM-CSF-specific mRNA in CFU-C cells
of four cases was notdetected by RT-PCR. These data further support a central role for IL-18in thepathogenesis of
JCML and suggest that the
use of I L - l Ra could represent a
novel therapeutic strategy against this disorder.
0 1994 by The American Society of Hematology.
J
of the JCML cell proliferation remains unclear; moreover,
no definitive evidence has been provided on thecell source
of these growth factor molecules. A naturally occurring IL1 inhibitor has been purified, cloned, and renamed IL-Ireceptor antagonist(IL- 1Ra).6 This molecule has been shown
to inhibit the in vitro growth of leukemic cells isolated from
both AML and CML during the accelerated phase of the
disea~e.”~
Based on these findings, we investigated gene and
protein expression of IL- 1p and GM-CSF in cells and clonogenic progenitors in these patients and the effect of IL-l
blockade on spontaneous growth using IL- I Ra.
UVENILE CHRONIC myelogenous leukemia (JCML)
is a rare hemopoietic malignancy of early childhood
distinct from the adult type of chronic myelogenous leukemia (CML).’ Characteristic of JCML are the absence of the
Philadelphia chromosome (Ph”), elevated levels of fetal hemoglobin, hepatosplenomegaly, thrombocytopenia, and
prominent monocytosis.2 In JCML patients, bone marrow
(BM) progenitors show a unique ability to proliferate “spontaneously’’ in the absence of exogenous stimuli. The colonies derived from these patients give riseto a predominance
of monocyte/macrophages over the granulocytic aggreg a t e ~ .Interleukin-l
~
(IL-l),tumor necrosis factoralpha
(TNFa), and granulocyte-macrophage colony-stimulating
factor (GM-CSF) have been reported to be involved in autocrine or paracrine pathways of growth stimulationin
acute myelogenous leukemias (AMLs) and CMLs including
JCML.4 Bagby et a15 reported that monocytes from JCML
produce high levels ofIL- 1, which in turn stimulate the release of other CSFs by normal accessory hemopoietic cells.
However, which of these cytokines is the primary regulator
From the Clinica Pediatrica Universitridi Milano-H.S. Gerardo,
Monza; the Istituto di Ricerche Farmacologiche “M. Negri,” Milano and Bergamo; the Divisione di Ematologia, Ospedali Riuniti
di Bergamo, Italy; and the Department ofMedicine, Tufis University and New England Medical Center, Boston, MA.
Submitted March 11, 1993; accepted September 1, 1993.
Supported in part by the Associazione Italiana per la Ricerca sul
Cancro, Fondazione Tettamanti, byConsiglio Nazionale delle Ricerche PF ACRO (92.02140.PF.39 to A.B. and 92.02392.PF39 to
,4.R.). and by National Institutes of Health Grant No. AI15614
(C.A.D.).
Address reprint requeststo Raffaella Schir6, MD, Clinica Pediatrica Universitridi Milano, H.S. Gerardo, via Donizetti 106, 20052
Monza, italy.
The publication costs ofthis article were defrayed in part by page
charge payment. This article rnusi therefore be hereby marked
“advertisement” in accordance with18 U.S.C. section 1734 solelyto
indicate thisfact.
01994 by The American Society of Hematology.
0006-4971/94/8302-0025$3.00/0
460
MATERIALS AND METHODS
Patients. Between January 1986 and December 1992,the eight
patients in this study were referred to our clinic. Table 1 summarizes the main clinical and laboratory data that fit into the classic
description of JCML.* Thesepatients had varying degreesof hepatosplenomegaly, skinmanifestations,and hemorrhagic signs. Most
had leukocytosis, monocytosis,
anemia, and a high level of fetal hemoglobin. BM aspirates showed normal cellularity with myeloid
hyperplasia when BM smears were stained with May-Griinwald
Giemsa. Cytogenetic analysis of BM cells revealed an apparently
normal karyotype. For control studies, BM was obtained from five
pediatric patients with classic Ph” CML and 10 normal children
undergoing marrow harvesting for allogeneic
BM transplantation.
Approval was obtained from the Institutional Review Board for
these studies. Informed
consent was obtained according to the Declaration of Helsinki.
Cell preparations. Peripheral blood (PB) or BM cells were isolated on Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradients and used for cell cultures and molecular studies.In selected
experiments, PB or BM mononuclear adherent cells were removed
by overnight incubation in plastic flasks(Falcon, Becton Dickinson,
Plymouth, UK) in Iscove’smodified Dulbecco’smedium ([IMDM]
Gibco, Paisley, Scotland, UK) with 10% fetal calfsemm at 37°C to
achieve a satisfactorydepletion of adherent cells. Nonadherent cells
were removed and contained less than 5% of monocytes as assessed
by detecting the presence ofthe CD 14 antigen usingthe M02 antibody (kindly provided by Dr R.F. Todd, University of Michigan,
Ann Arbor, MI)by flow cytometry. The light-density cell fractions
and depleted cellpopulations were then plated
in clonogenic assays.
Clonogenic assays. BM and PB cells at a density of 105/mLand
fftood,Vol83, No 2 (January 15). 1994:pp 460-465
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SUPPRESSION OF JCML CELL GROWTH BY IL-IRA
46 1
Table 1. Clinical Profileof JCML Patients
Case No.
1
Age at diagnosis (mol
Sex
Blood counts
Hemoglobin (g/dL)
WBC (x 1 0 9 / ~ )
Blasts (%)
Monocytes (%)
Platelets (X 1 @/L)
%M
Cellularity'
Colony growth
Karyotype
LAP scoret
Hemoglobin Flevel (%)
Hepato/splenomegaly
Treatment status at time
of cell culture studies
Survival (mo)
19
2
9
3
M
M
M
10.8
26
5
8.9
42
3
28
30
8.0
13
11
5
189
9
52
>l
4:1
>l
CFU-M
46,XY
CFU-M
46,XY
CFU-GM/CFU-M
46.XY
0
32
+/+
140
21
102
18
+/+
Relapse
Relapse
16
+l+
2
Diagnosis
3
5
4
3
6
7
8
29
M
36
25
26
42
M
F
M
M
8.5
48
0
15
99
7.5
25
8
4
43
8.3
43.6
0
13
70
19:l
>l
>l
CFU-M
46,XY
CFU-M
46,XY
t(12;13)
17
40
3
10
95
1
+/+
+/+
Diagnosis
Diagnosis
Alive (38)
BMT
CFU-M
46,XX,-12,
+der( 12)
31
11.9
34
9.0
29
2
2
16
133
3
78
5:l
6:1
CFU-M
46.XY
46,XY
ND
ND
20
+/+
10
58
+/-
Diagnosis
Diagnosis
Diagnosis
Alive (4)
Alive (9)
BMT
+/+
18
Abbreviations:WBC, white blood cells; LAP, leukocyte alkaline phosphatase;8MT. bone marow transplant; ND, not determined.
Myeloid/erythroid ratio.
t Normal values, 1 1 to 94.
5 X 105/mL,respectively, were plated
in IMDM supplementedwith
20% fetal calfserum, +lo% 5637 conditioned medium, and 0.33%
final concentration of Agar Bacto (Difco
Laboratories,Detroit, MI)
as previously described?The conditioned medium from the 5637
tumor cell line was usedas a source of CSFs at a final concentration
of 10%. All samples were cultured in the presence and absence of
CSFs. The culture mixture was plated in a volume of 1 mL in 35mm Petri dishes
in triplicate. After a I4day incubation at 37°Cin a
fully humidifiedatmosphere of 5% CO2in air, colony-forming unit
cells (Cm-C) were scored under an inverted microscope. Aggregates ofmore than 50 cells wereevaluated as colonies.
To test the ability ofthe IL1Ra to inhibit the clonogenic growth
of JCML cells, recombinant IL- 1Ra (kindly provided by Dr Daniel
Tracey, Upjohn, Kalamazoo, MI) was added to the plates at concentrations ranging from 100 ng/mLto 1 &mL in the absence of
5637 conditioned medium. The specificity of the inhibitory effett
of IL-lRa and the lack of toxic contaminants in the preparation
used was confirmed by clonogenic assay performed with normal
BM stimulated with 5637conditioned medium. To characterize the
aggregates,cytocentrifuged preparations of individual colonies
plugged from the plate were stained with May-Griinwald Giemsa
for morphologic evaluation. Nonspecific a-naphthyl acetate and
chloroacetate esterase staining and immunocytochemical analysis
were used to identify the nature of colony-forming cells (Sigma
Italia, Milano, Italy). The expression of surface antigens was assessedby
the alkaline phosphatase anti-alkaline phosphatase
(APAAP) technique, as previouslydescribed." A neutralizing
monoclonal anti-human GM-CSF (clone 3092)'' was provided by
Dr J. Griffin (Dana Farber Cancer Institute, Boston, MA).
Molecular studies. Northern blotanalysis was performedas
previously described." The cDNA forthe IL- l Ra was obtained by
polymerase chain reaction (PCR) using mRNA from AML193
cells as substrate.13 The IL-lB and GM-CSF cDNA probes were
kindly providedby Dr Steven Clark (GeneticsInstitute, Cambridge.
MA). cDNA probes were labeled with 32P-deoxycytidine triphosphate by the method of Feinberg and Vogel~tein.'~ selected
in
experiments, total mRNA was directly isolatedfrom JCML colonies
after 14 days of culture, using the acid guanidium thiocyanateBriefly, 200
phenol-chloroform method as previously desc~ibed.'~
p L guanidium buffer and 4 pg MS2 phage RNA (Boehringer, Mannheim, Germany) as a carrier were added to individual colonies
following removalfrom agar. RNA was
then extracted once by mixing with 200 pL
phenol40 pL chloroform-isoamylic alcohol. After
Table 2. PB andBM CFU-C Growth in JCML Patients
CFU-C
Without CSA
Case No.
Time
Source
With CSA
1
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Relapse
Diagnosis
Diagnosis
Diagnosis
PBMC
PBMC
BM
PBMC
BM
BM
BM
BM
PBMC
20+-1
Jf1
1462 1
1020
21 f 1
122 1
252 1
181 f 1 1
50f 17
752 1
192k 14
664247
Diagnosis
BM
91 f 3
621
BM
8722
0
2
3
4
5
6
7
Ph'
3525
16+3
11425
12*2
4023
2022
+ CML
(5cases)
nBM
(1 0 cases)
Data are presented as the mean of triplicate determinations.
Abbreviations: BM, bone marrow mononuclear cells; Ph'+ CML, Philadelphia-positive CML; nBM, bone marrow from hematologically normal
patients; CSA, colony-stimulating activity.
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SCHIRO ET AL
462
-
Fig 1. BM smear of JCML
patient showing typical monocytoid blast cells with indented
nuclei. May-Griinwald Giemsa
stain ( X 200. A). Photomicrograph ( X 400) of atypical JCML
monocyte-macrophage colony
as it appears in a methylcellulose culture system in the absence of any deliberate source
of exogenous growth factors
(B). Cytocentrifuged smears of
JCML colony-derived cells (C).
CD14 expression as assessed
by APAAP technique (D).
1
,.
..
.~
centrifugation at 17,000g for 20 minutes at 4"C, supernatant was
precipitated with 2 v01 100%ethanol and finally resuspendedin 20
pL of buffer for cDNA synthesis.In vitro reverse transcription of
RNA to cDNA was performed in a 20-pL volume for 15 minutes
at 42°C containing 2.5 U cloned Moloney murine leukemia virus
reverse transcriptaseand oligo deoxyribosylthymineas primers, using a commercial kit (GeneAmp RNAPCR kit, Perkin ElmerCetus, Norwalk, CT) according to manufacturer's conditions. A
volume of 5 pL was then diluted in 100 pL final volume of buffer
mixture containing 50 mmol/L KCl, I O mmol/L Tris hydrochloride (pH8.3 at 25"C), 2 mmol/L MgCI2, 200pmol/L deoxyadenosine triphosphate, deoxyguanosinetriphosphate, deoxycytidinetriphosphate, and deoxyribosylthyminetriphosphate, 30 pmolofeach
primer, and 2.5 U Thermus Aquaticus DNA polymerase.The reaction was performed ina Thermal Cycler (Perkin Elmer-Cetus, Norwalk, CT) for 45 cycles; after the first 30 cycles, one fifth of the
reaction mixture was removed and substituted with fresh reagents
for the successive 15 cycles. The PCR temperatures used for each
cycle were as follows:95°C for5 minutes as the denaturation step;
then 95T, 60'C, and 72°C for 1 minute each; and finally 10minutes
at 72°C. The primers used for IL-lP, included in a commercial kit
(Perkin Elmer-Cetus), were5'AAACAGATGAAGTGCTCCTTCCAGG-3' and 5"TGGAGAAACACCACTTGTTGCTCCA-3'.
The primers used for GM-CSF were5'-CTGCACCCGCCCGCTCG-3' correspondingto the 442 through 426 nucleotide residues and 5'-CACTCCTGGACTGGCTC-3' corresponding to residues 58 to 74 ofthe published GM-CSF sequence.16
Ten microliters
of PCR mixture was run on a 1.8% Nusieve agarose gel(FMC BioProducts, Rockland, ME) stained with ethidium bromide and visualized under a UV lamp. In selectedexperiments, IO pL PCR products fractionatedby electrophoresisthrough a 1.8%agarose gel was
transferred to nylon membranes (Genescreen Plus, New England
Nuclear, Boston, MA).Prehybridization,hybridization, and washings were performed according to manufacturer's instructions as
previously reported."
Measuremenl of cytokine produdion. IL-Ip and GM-CSF levels weremeasured by specific, non-cross-reacting radioimmunoas-
says (RIAs).""* Each RIA has been
validated usingthe cytokines in
their respective biologic assay.
RESULTS
Spontaneous JCML cell proliferation in vitro. As shown
in Table 2, when either PB or BM cells from JCML patients
were cultured in the CFU-C assay, a significant number of
colonies were observed in all cases in the absence of any
deliberate source ofcolony-stimulating activity. By comparison, no significant growth of CFU-C was observed under
the same conditions when normal or Phf CML BM cells
were plated. Moreover, neither the total number nor the
cell
type of the colonies changed significantly whenJCML cells
were cultured in the presence of colony-stimulating factors.
After careful depletion of adherent cells in selected experiments, a complete abrogation of the spontaneous colony
growthwasobserved(cases
no. 3, 4, 6, and 7; data not
Table 3. Effect of 11-1 Raon CFU-C Growth in JCML
Patients
CFU-C
Case No.
Time
Source
Medium
1
2
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Diagnosis
Relapse
Diagnosis
Diagnosis
PBMC
PBMC
BM
PBMC
BM
BM
BM
PBMC
35c5
16k3
114+5
12k2
4023
20c2
752 1
664 k 47
+IL-l Ra'
10k2
3+ 1
55k 1
3+ 1
12? 1
135 1
40? 2
156 14
Data are presented
as the meanof triplicate determinations.
Abbreviation: BM, bone marrow mononuclear cells.
11-1 Ra at 100 ng/mL.
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SUPPRESSION OF JCML CELL GROWTH BY IL-1Rn
Pt. N o
IL-
463
1
2
3
4
5
1
2
3
4
5
P
A
Pt. N o
IL-l ra
-
B
Fig 2. Expressionof 11-18 andIL-l Ra genes in JCML cells. Total
cellular RNA was purified from uncultured PB cells ofthe indicated
JCML patients, blotted, and hybridized to IL-18-and IL-1Ra-specific probes.
shown). The monocyte feature of JCML cells, which were
growing in the absence of colony-stimulating activity, CFUM, as indicated in Table I , was confirmed by the positive
nonspecific esterase staining of a single JCML CFU-C aggregate (as shown in Fig 1B) and by the expression of C D I4
antigen on a colony as assessed by immunochemistry
(Fig 1D).
IL-1Ra inhibits spontaneous JCML cell proliferation in
vitro. To block IL- I , we considered IL- 1Ra based on our
previous findings on the reduction in the spontaneousproliferation of AML cells.’ As shown in Table 3, when IL-l Ra
was added to each clonogenic assay, we observed a significant inhibition of spontaneous JCML cell proliferation in
each case (ranging from 35% to 82%). Increasing the concentration of IL-I Ra by I pg/mL did not result in a further
abrogation of the spontaneous growth.
Cytokinegene exprcxsion in JCML cells. In light of the
central role of IL-Ip in supporting JCML cells, we investigated whether JCML cells expressed the IL-I@ gene.As
shown in Fig 2A, Northern analysis revealed IL-l@-specific
mRNA in most cases (four of five). It is noteworthy that ILI @ gene expression was readily observed in RNAs purified
from uncultured PB. Due to the
comparable kinetics of ILI @ and IL-l Ra gene expression in normal human blood
monocytes,’’ we investigated the presence of IL-IRa-specific mRNA. As indicated in the representative Northern
analysis shown in Fig 2B, IL- I Ra mRNA was detected in
the samecases that were positive for IL- I @ gene expression.
To investigate whether clonogenic cells were indeed the cellular source of IL-IB mRNA, PCR analysis of IL-I@ gene
expression was performed in selected cases. Figure 3 shows
a representative experiment of PCR analysis for IL- I @ gene
expression conducted on six single BM colonies of patient
no. 4. Superimposable results were obtained when the same
experiment was performed on 12BM colonies of patient
no. 2 that were found to be consistently positive for IL-1s
message. By comparison, a similar pattern of IL-IS gene expression was observed in CFU-C obtained from BM cells of
normal individuals but in the presence of colony-stimulating factors (data not shown).
Because GM-CSF hasalso been reported to be involved in
the regulation of JCML colony growth?&*’we investigated
GM-CSF gene expression in PB or BM JCML cells. Specific
GM-CSF mRNAwas not detected by Northern blot experiments when the same mRNA
preparations, foundto be positive for IL-I@or IL-l Ra gene expression, were hybridized
with a GM-CSF cDNAprobe (data not shown). To increase
our capacity to detect GM-CSF-specific transcripts, PCR
analysis was performed on total cellular RNA purified from
PB and BM mononuclear cells of patients no. I, 2,4, and7
(Fig 4). Since the GM-CSF transcriptwas found in BM and
PB cells of patient no. 4 only, we speculated that this could
be due tocontamination of a minor proportion of cells. Interestingly, when PCR analysis for GM-CSF expression was
performed on six colonies from patients no. I and 4 and 12
from patient no. 2, no GM-CSF-specific mRNA was detected (data not shown). Despite the absence of GM-CSF
mRNA in single JCML colony preparations, GM-CSFwas
detected in JCML cell culture supernatantderived from 24and 48-hour liquid culturespecimensfrom
case no. 1
(>2.50 pg/mL). Moreover, anti-human GM-CSF induced a
significant suppression (40%) of JCML colony growth in
case no. 8 (data not shown).
DISCUSSION
The in vitro “autonomous” proliferation of BM or PB
cells with monocyte-macrophage features has emerged as
one of the peculiar biological features of JCML.3 Similar to
the studies of Estrov et a13 and Freedman et.al,’ we confirmed the proliferation of monocyte-macrophage clonogenic cells of untreated JCML patients in the absence of any
deliberate source of growth factors and that the depletion
of adherent cells abrogated spontaneous colony formation,
furthersupporting the central role of monocyte-macrophage cells in the mechanism of the disease.
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SCHIRO ET AL
464
m
a,
-I
+
U
0
Y
Z m N
m [L I
Q
z o
a
c
e
388 bp +
W
~
rx
Fig 3. Detection of human
IL-lpmRNA by PCR analysisin
six CFU-C BM colonies of patient no. 4. Ascontrols, PCR
analysis was performedon total
RNA extracted from BMmononuclear cells of the same patient (no. 4) and on total RNA
from normal PBmononuclear
cells stimulated with bacterial
LPS). The
endotoxin (PBMC
amplified products (10% of the
reaction mixture) were analyzed on a l .8% agarosegel,
visualized by ethidium bromide
staining, blotted onto synthetic
membrane, and hybridized with
an IL-1ScDNA.
+
Several cytokines including GM-CSF. TNFcu, and IL-l
have been shown to support the abnormal growth of malignant cells of monocyte-macrophage lineage by either autocrine or paracrine pathways. In the present study. we found
that IL-l Ra inhibited spontaneous JCML cell proliferation
and thatin most cases the inhibition was profound. Similar
observations were recently reported by Rambaldi et al' and
Estrov et al.* who described the suppression of acute myeloblastic leukemia and CML colony growth by IL-lRa. Because of the role of IL-I in the multiple autocrine circuits
operating within a single malignant myeloid clone, IL- I Ra
likely exerts its suppressive action on colony growth by
blocking theinteraction between the IL-l endogenously
produced and theleukemic cells.22Bagby et als first reported
that the abnormalproliferative activity of JCML cells was
dependent in part on the abnormalproduction of IL-l,
Fig 4. Detection of human
GM-CSF mRNA by PCR analysis. Total RNA (1 *g) was extracted either from PB or BM
mononuclearcells.As
a positive control,
RNA
from PB
mononuclear cells of a healthy
donor was stimulated with
phorbol 12-myristate 13-acetate(1
ng/mL) and phytohemagglutinin (10 rg/mL) for 24
hours.The amplified products
(10% of the reaction mixture)
were analyzed on a l .8% agarose
gel,
blotted onto nylon
membrane, and hybridizedwith
a oligonucleotide complementary to the GM-CSF sequence
according to established procedure.
which in turn stimulated the production ofcolony-stimulating activity by accessory cells.5In this context,it isnoteworthy that thefindings that either freshly obtained circulating
cells or CFU-C from either PB or BM cells ofJCML patients
expressed IL- I@-specificmRNA and secreted the protein in
the short-term culture (data not shown). It has also been
shown that IL- I cu could act directly to support JCML colony
growth, but not to the same magnitude as TNFa or GMCSF.4
Alternatively, IL-l could exert its effect by inducing the
secretion of GM-CSF and/or othergrowth factors, as shown
for a variety of target ~ e l l s . * ~Several
- * ~ studies have implicated GM-CSF as the primaryendogenous regulator of
JCML cell proliferation.20 We were not able to detect GMCSF transcripts in mRNA preparations from single JCML
colonies, but, as previously reported." GM-CSF was de-
U
W
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SUPPRESSION OF JCML CELL GROWTH BY I L - ~ R A
tected in the culture supernatants from either PB or BM
JCML cells. Since anti-human GM-CSF consistently inhibited cell proliferation, it is likely that theautocrine secretion
of TNFa and/or IL-Ip by the proliferating population of
malignant monocyte-macrophages stimulates the production of GM-CSF by accessory c e k 4
Under the same experimental conditions where IL-Ip
mRNA was detected in PB or BM JCML cells, we observed
significant IL- 1 Ra gene expression. This finding is not surprising, since comparable kinetics in IL- 1p and IL- 1Ra induction are observed in normal PB mononuclear cells and
GM-CSF is a potent stimulusfor the production of IL- l Ra
by normal human monocytes.”
In conclusion, our dataimplicate a central role for IL-Ip
in JCML and suggest that blocking IL-I with molecules
such as IL- I Ra could provide a novel therapeutic strategy
against this disorder.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1994 83: 460-465
Suppression of juvenile chronic myelogenous leukemia colony growth
by interleukin-1 receptor antagonist [see comments]
R Schiro, D Longoni, V Rossi, O Maglia, A Doni, M Arsura, G Carrara, G Masera, E Vannier and
CA Dinarello
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