1. No category

From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
Interleukin-9 Stimulates the Proliferation of Human Myeloid Leukemic Cells
By Roberto M. Lemoli, Alessandra Fortuna, Agostino Tafuri, Miriam Fogli, Marilina Amabile, Alexis Grande,
Maria Rosaria Ricciardi, Maria Teresa Petrucci, Laura Bonsi, GianPaolo Bagnara, Giuseppe Visani,
Giovanni Martinelli, Sergio Ferrari, and Sante Tura
Human interleukin-9 (IL-9) stimulates the proliferation of
primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia,
we evaluated the proliferative response of three leukemic
cell lines and 32 primary samples from acute myeloblastic
leukemia IAML) patients t o recombinant human Irh)-IL-S
alone and combined with rh-IL-3, granulocyte-macrophage
CSF (GM-CSF), and stem cell factor ([SCFI c-kit ligand). The
colony-forming ability of HL60, K562, and KG1 cells and fresh
AML cell populations upon IL-9 stimulation was assessed by
a clonogenic assay in methylcellulose, whereas the cell-cycle
characteristics of leukemic samples were determined by the
acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the
terminal deoxynucleotidyl transferase assay (TDTA) and
standard analysis of DNA cleavage by gel electrophoresis
were used t o evaluate induction or prevention of apoptosis
by IL-9. 11-9, as a single cytokine, at various concentrations
stimulated the colony formation of the three myeloid cell
lines under serum-containing and serum-free conditions,
and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL-9 resulted in an increase
of blast colony formation in all the cases studied (mean k
SEM: 19 2 10 colony-forming unit-leukemic [CFU-Ll/105
cells plated in control cultures v 107 +. 32 in IL-9-supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned
medium (PHA-LCM), and it was the most effective CSF for
promoting leukemic cell growth among those tested in this
study (ie, SCF, IL-3, and GM-CSF). The proliferative activity
of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the
cytokine. Addition of SCF t o IL-9 had an additive or synergistic effect of the t w o cytokines in five of eight AML cases
tested for CFU-L growth (187 -I: 79 colonies Y 107 r 32 CFUL, P = .05). Positive interaction was also observed when IL9 was combined with IL-3 and GM-CSF. Studies of cell-cycle
distribution of AML samples demonstrated that IL-9 alone
significantly augmented the number of leukemic cefls in Sphase in the majority of cases evaluated. IL-9 and SCF in
combination resulted in a remarkable decrease of the Gocell
fraction (38.2% k 2496 Y 58.640 f 22% of control cultures, P
< .05) and induced an increase of G,- and S-phase cells.
Conversely, neither IL-9 alone nor the combination of IL-9
and SCF had any effect on induction or prevention of
apoptosis of leukemic cells. In summary, our results indicate
that IL-9 may play a role in the development of AML by
stimulating leukemic cells t o enter the S-phase rather than
preventing cell death. Moreover, IL-9 acts synergistically
with SCF for recruiting quiescent leukemic cells in cell cycle.
0 1996 by The American Society of Hematology.
I
Within the myeloid differentiation lineage, IL-9 has
shown burst-promoting activity (BPA) on erythroid clonogenic cells derived from very primitive CD34‘CD33 -DR’
progenitor cells.’ as well as more mature lineage-restricted
precursors (CD34‘ cells).’ More recently, IL-9 has proven
to induce the proliferation of multipotent hematopoietic
CD34’CD33 DR-- cells in combination with c-kit ligand
(SCF).“’ Early studies provided evidence that 1L-9 may also
be involved in the regulation of myeloid leukemic cell
growth. It stimulates murine IL-3-dependent cell lines and
synergizes with SCF to promote proliferation of the human
megakaryoblastic cell line, M07e.’.’ In these cells, IL-9
induces a rapid and transient tyrosine phosphorylation of at
least four proteins different from those activated by SCF.
suggesting that this novel cytokine retains a specific and
unique signal transduction pathway.’
To further define the role of IL-9 in the development of
myeloid leukemia, we have evaluated the colony-forming
ability and changes in cell-cycle distribution of fresh acute
myeloblastic leukemia (AML) cell populations and continuously growing cell lines in response to exogenous IL-9 alone
and combined with SCF, 1L-3, and granulocyte-macrophage
colony-stimulating factor (GM-CSF). Moreover, we assessed the effect of IL-9 on leukemic cell programmed cell
death. The results presented here indicate that IL-9 is a major
proliferative factor for AML by stimulating leukemic cells
to enter the S-phase.
NTERLEUKIN-9 (IL-9) has been originally isolated from
the conditioned medium of a human T-cell leukemia
virus- 1 -transformed T-cell line, based on its ability to stimulate proliferation of the M 0 7 e cell line.’ In vitro biologic
characterization has demonstrated that the growth of T-cell
clones is induced by IL-9, mast cell lines by IL-3,2.3and
mouse fetal thymocytes by IL-2 in the presence of IL-9.‘
More recent data demonstrated the close association of IL9 expression and tumor cells in Hodgkin’s disease and largecell anaplastic lymphoma (LCAL), suggesting an IL-9-mediated autocrine growth loop.”’ Moreover, it has been shown
that this cytokine may influence the development of thymic
lymphomas by preventing apoptosis.’
From the Institutes of Hematology and Histology and Embryology.,
University of Bologna, Bolognu; Institute of Biological Chemistry,
University of Modenu, Modena: and Institute of Hematology, Univer.vity of Rome, Rome, Italy.
Submitted August 7, 199.5; nccepted December 13, 1995.
Supported by MURST 40% and MURST 60%. Itdiun Associution
for Cancer Research, and Italiun Nntioriul Research Council Grunt
No. 94.01200. PF39 (to G.B.).
Address reprint requests to Roberto M . k m o l i , MD, Institute of
Hematology “ L . & A. Seragnoli, Via Massurenti 9, 40138 Bologna,
Italy
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordrmce with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I996 by The American Society of Hemutology
”
0006-4971/96/8709-0007$.3.00/0
3852
’
’
MATERIALS AND METHODS
Recombinant hematopoietic CSF.s. Four human recombinant
growth factors were used in this study. IL-9 and IL-3 were supplied
Blood, Vol 87, No 9 (May 1). 1996:pp 3852-3859
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
3853
INTERLEUKIN-9 STIMULATES AML CELLS
A
L
I
was established by morphologic criteria, cytochemical staining, and
surface-marker analysis using a panel of monoclonal antibodies
(MoAbs). Leukemic specimens were subclassified according to the
French-American-British (FAB) classification system.I5 The mononuclear cell fraction was collected and cryopreserved as previously
described.16 After thawing, viable cells were recovered by FicollHypaque gmhent (Lymphoprep; Nycomed Pharma, Oslo, Norway).
Although the majority of AML samples contained more than 90%
blast cells (Table l),to rule out the possibility that rare contaminating
activated T cells may interfere with the proliferative effects of JL9, some experiments were performed after T-cell depletion by immunomagnetic separation. Briefly, mononuclear cells were incubated
for 30 minutes at 4°C with an excess concentration of an anti-CD3
MoAb (UCHTl, a kind gift from Professor P.C.L. Beverley, ICRF,
London, UK). After two washes, target cells treated with the primary
antibody were incubated with immunomagnetic monodisperse microspheres coated with a sheep antibody to mouse IgG (Dynabeads
M-450; Dynal, Oslo, Norway; bead to cell ratio, 20: 1) for 1 hour at
4°C. The beads, together with the rosetting cells, were retained along
the tube wall with a magnet, and the supematant medium containing
the T-cell-depleted cell fraction was then recovered. A second round
of incubation with the same MoAb followed by immunomagnetic
separation (bead to cell ratio, 1O:l) was performed in case the per-
Table 1. Cllnical Charncterlsticsof the AML Case8
Fig 1. Proliferative response of leukemic cell lines to increasing
concentrntlons of IL-9. Cells ware cultured in the presence (A) end
absence (Bl of exogenous serum.
by Genetics Institute (Cambridge, MA) and were used at a concentration of 10 U/mL and 50 ng/mL, respectively. IL-9 was derived from
the conditioned medium of CHO cells. Its biologic activity by the
M07e proliferation assay was 2.4 X lo5 U/mL. Purified human IL9 of greater than 98% purity based on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (Intergen, Purchase, NY) was
also used in selected experiments. SCF and GM-CSF were provided
by Amgen (Thousand Oaks, CA) and were added to the cultures
at a concentration of 100 ndmL and 1,OOO U/mL. The optimal
concentration of IL-9 was chosen after review of our previous experience” and a preliminary dose-response study of colony formation
from leukemic cell lines (Fig 1). Stock solutions of CSFs were stored
at -80°C. and dilution vials were kept at -20°C (CSFs were diluted
in Iscove’s modified Dulbecco’s medium, [IMDM] with 2% fetal
calf serum ([FCS] Sera Laboratory, Sussex, UK).
Leukemic cell lines. Three human myeloid cell lines maintained
in exponential growth conditions were studied. HL60 is a human
acute promyelocytic leukemia cell line growing with a doubling
time of 24 hours.’* K562 cells derive from a chronic myelogenous
leukemia blastic crisis” with a doubling time of 20 hours. The KGl
myeloblastic leukemic cell line’4 grows with a doubling time of 36
hours. These cell lines were cultured in IMDM supplemented with
10% FCS, 1% conventional antibiotics, and 1%L-glutamine at 37°C
in a fully humidified atmosphere of 5% CO, air. Cell viability was
always more than 90% at the time of study. All cell lines were free
of Mycoplasma contamination.
AML samples. Cells were obtained from peripheral blood or
bone marrow of 32 AML patients (Table 1). The diagnosis of AML
Patient No.
AgalSex
FA6 Classification
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
22lM
34/F
401M
67lF
54/M
57/M
28/M
M2
M4
M2
MI
M4
M4
M5b
M1
M2
M5a
M5a
M5a
M4
M5b
M4
M5a
M5a
M4
M2
M1
M5a
M5b
M2
M2
M4
MI
M3
MI
M2
M4
M3
M3
32/F
17/F
29lF
18/F
52/M
82lF
28/F
49/F
50/M
52/F
27/M
36lM
621F
45/M
631F
32lM
40/M
681F
401F
18/F
35lF
60/F
27/F
41/M
24/M
Blasts (%I*
Source of Cells
95
98
50
92
94
90
96
95
98
84
93
94
91
92
90
95
99
78
70
80
50
98
75
91
98
98
99
90
95
90
100
100
PB
PB
PB
PB
BM
PB
PB
BM
PB
PB
BM
BM
PB
BM
PB
BM
PB
BM
BM
BM
BM
PB
BM
PB
BM
PB
PB
BM
BM
BM
BM
BM
~~
~
Abbreviations: PB, peripheral blood; BM, bone marrow.
* Percentage of blast cells was obtained by counting 200 calls from
May-GrUnwaId-Giemsa-stained cytocentrifuge smears prepared
from thawed light-density mononuclear cell fractions.
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
3854
centage of lymphocytes was more than 1%. The number of contaminating T cells was always less than 0.5% at the end of the procedure.
Clonogenic assay. Colony-forming unit-leukemic (CFU-L)
were assayed as previously described.16 Briefly, the culture medium
consisted of IMDM supplemented with 24% FCS, 0.8% bovine serum albumin (Sigma Chemical, St Louis, MO),
m o l n 2-mercaptoethanol (Sigma), and methylcellulose at a final concentration
of 1%. To measure the optimum cloning efficiency, 20% phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM) was
added to leukemic samples. The number of cells plated was adjusted
to produce approximately 100 colonies per plate. Quadruplicate cultures were incubated at 37°C in 5% C 0 2 air, and colonies (>20
cells) were scored after 10 to 14 days. The clonogenic assay for
myeloid cell lines was the same as that used for CFU-L without
addition of PHA-LCM. Colonies (>50 cells) were recorded after 10
to 14 days of culture. The clonogenic efficiency of HL60, K562,
and KGl cell lines was 25%, 12%, and 18%, respectively.
Where indicated, FCS was replaced by a combination of 1%
deionized-crystallized bovine serum albumin (Sigma), 57 pg/mL
iron-saturated human transferrin (Sigma),4.2 pg/mL sodium selenite
(Sigma), and 4.8 mg/mL lecithin (Sigma) in serum-free cultures."'
Blocking experiments were performed by growing the cell lines,
in the presence and absence of FCS, with and without 5 pg/mL
MoAbs directed to IL-9 (a kind gift from Dr E.M. Alderman, Genetics Institute, Cambridge, MA).
Cell-cycle studies. The percentage of AML cells in active Sphase was determined by the bromodeoxyuridine (BRDU) incorporation assay as described elsewhere." In brief, leukemic cells were
seeded at 5 X lo5 cells/mL in a liquid culture of serum-containing
IMDM. Cultures were maintained for 3 days in the presence of
different growth factors as indicated. At the time of analysis, IO pL
stock BRDU solution (1-mmol/L) were added to the cell suspension
for 30 minutes in 5% COz at 37°C. After two washes, slides were
obtained by cytocentrifugation, air-dried, fixed in a mixture of methanol and acetic acid (3: I), and denaturated in 0.07 mol& NaOH for
12 seconds. For nuclear BRDU staining, slides were first incubated
with 5 pL anti-BRDU MoAb (Becton D i c k " , Mountain View,
CA) diluted 1:15 with phosphate-buffered saline (PBS) with 0.5%
Tween 20 (Sigma) for 30 minutes at room temperature. Finally, they
were treated with TRITC-conjugated antimouse Ig (Dako, Glostrup,
Denmark) diluted 1:30. Preparations were then washed overnight
with PBS, and positive cells were scored with a fluorescence microscope.
To evaluate the effects of CSFs on cell-cycle distribution, an
acridine orange (AO) flow cytometric technique was used. Cellular
DNA and RNA content (percentage of cells in Go, G I , S, and GzM,
mean RNA content per cell in each phase of the cell cycle) was
measured as previously described.I8 RNA content of G I cells was
expressed as an RNA index (RNA-I GI) determined as the ratio of
the mean RNA content of GI cells of the samples times 10, divided
by the median RNA content of control lymphocytes." G,, cells were
defined as cells with RNA content equal to or less than that of
control lymphocytes.
Evaluation ofapoptosis. Cellular apoptosis was determined after
72 hours of liquid suspension culture by AO, in situ terminal deoxynucleotidyl transferase (TdT) assay (TDTA), and assessment of DNA
fragmentation by gel electrophoresis. Using AO, apoptotic cells were
evaluated as a sub-G, peak on DNA-frequency histograms, since
they can be recognized by their diminished stainability with DNAspecific fluorochromes such as AO." Moreover, to discriminate between early and late events in cell death, we used the TDTA technique. TDTA assay allows determination of DNA strand breaks
occurring before (during cell cycle) and after loss of DNA and RNA,
using biotinylated dUTP in the presence of exogenous TdT.2' Briefly,
leukemic cells collected from the culture were fixed in 1% buffered
LEMOLI ET AL
formaldehyde (pH 7.4) for 15 minutes on ice. After two washes in
PBS, the cells were resuspended in 70% ethanol at -20°C for 3
days. After cells were rehydrated in PBS, approximately I x 10'
cells were resuspended in 50 pL 0.1-mom sodium cacodylate (pH
7.0), 0.1 mmol/L dithiothreitol, 0.05 mg/mL bovine albumin, 5 U
TdT, and 0.5 nmol/L biotin-16-dUTP. The cells were incubated for
30 minutes at 37"C, and then rinsed in PBS and resuspended in
100 pL staining buffer containing 2.5 pg/mL fluorescein-avidin, 4X
concentrated saline-sodium citrate buffer, 0.1% Triton X- 100, and
5% (wt/vol) nonfat dry milk. Following 30 minutes of incubation at
room temperature in the dark, the cells were washed in PBS containing 0.1% Triton X-I00 and resuspended in 1 mL PBS with 5
pmol& propidium iodide (PI) and 0.1% RNase. A modified FacScan
(Becton Dickinson) was used to measure fluorescence upon excitation at 488 nm. Five thousand cells were measured for each analysis
at a separate wavelength band for greedDNA and red/RNA (AO)
and greenhiotin-dUTP-avidin-FITCand r e m 1 (TDTA). Samples
were analyzed using a Hewlett Packard microcomputer (San Diego,
CA) and Becton Dickinson software including Consort 32, Cellfit,
and Lysis 11. The activity of study cytokines on induction of
apoptosis was also evaluated by standard analysis of DNA cleavage
by gel electrophoresis as previously described.*' In brief, cells were
suspended in 20 pL IO-mmol/L EDTA plus 50-mmol/L Tris hydrochloride (pH 8.0) containing 0.5% (wt/vol) sodium lauryl sarkosinate
and O S m g * m I - ' proteinase K and then incubated at 50°C for 1
hour. Ten microliters 0.5-mg-mL-' RNase A was added to each
sample, and incubation at 50°C continued for an additional hour.
Leukemic samples were then heated to 70°C, and 10 pL IO-mmol/
L EDTA containing 1% (wt/vol) low -gelling-temperature agarose,
0.25% (wt/vol) bromophenol blue, and 40% (wt/vol) sucrose was
mixed with each sample before loading with a siliconized pipette
tip into the dry wells of a 2% (wt/vol) agarose gel containing 0.1
pg * mL-' ethidium bromide. Electrophoresis was performed in 2
mmol/L EDTA plus 800 mmol/L Tris phosphate (pH 7.8) overnight.
The gels were photographed under UV light.
Statistical analysis. Results are expressed as the mean 2 SEM
of at least three separate experiments. Statistical analysis was performed using the paired nonparametric Wilcoxon rank-sum test.
RESULTS
Proliferative response of leukemic cell lines to IL-9. The
stimulatory activity of IL-9 on three well-documented myeloid cell lines is shown in Fig 1 . Proliferation in response
to the cytokine was dose-dependent, and maximal stimulation of HL60 and KG1 cells was observed at concentrations
of 10 U/mL in the presence and absence of serum (Fig 1A
and B, respectively). KS62 cells were optimally stimulated
by S U/mL IL-9 in serum-containing cultures. The same cell
line did not show clonogenic activity in serum-free medium.
Blocking experiments using an anti-IL-9 MoAb counteracted the proliferative activity of IL-9 on the cell lines and
this effect was reversed by a large excess of IL-9 (SO U/mL;
Fig 2).
IL-9 stimulates CFU-L growth. The clonogenic response
of primary leukemic samples to 10 U/mL IL-9 is reported
in Table 2. To minimize nonspecific cell interactions, each
sample was plated to produce approximately 100 colonies
per dish. Six of eight AML patients showed some spontaneous growth. IL-9 induced significantleukemic colony proliferation in eight of eight cases (107 2 32 v 19 ? 10 colonies
in control samples, P < .OS). IL-9 used as a single cytokine
stimulated 36.8% of CFU-L induced by PHA-LCM, and it
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
INTERLEUKIN-9 STIMULATES AML CELLS
3855
A
600
n
.--
n.. I I
-
20
0
0 1
0
5
1
10
IL-9
CONDITIONED MEDIUM
~~
70
50
Ulml
PURIFIE~
~~
Fig 3. Dose-responsecurve of IL-9 on 4 T-cell-depleted AML samples. Two different preparations of the cytokine were assayed. In this
set of experiments, the mean number of CFU-Ls stimulated by PHALCM was 361 ? 31.
Fig 2. Abrogation of IL-9-mediated colony formation by an anti11-9 MoAb. Clonogenic assays were performed in serum-containing
(A) and serum-free (Bl conditions. The inhibitory effect of anti-IL-9
antibody was reversed by an excess concentration of the cytokine.
CTR, control.
showed a higher blast colony-forming ability than SCF, IL3, and GM-CSF (Table 2). Addition of SCF to IL-9 resulted
in an additive or synergistic effect of the two CSFs in five
of eight cases (no. 4, 9, 5, 10, and 12: 187 -t 79 colonies v
107 2 32 for the whole group, P = .05), whereas no increase
in the size of CFU-L was observed (data not shown). The
combination of IL-9 and IL-3 or GM-CSF produced a higher
number of CFU-Ls, as compared with the same cytokines
used alone, in three cases (no. 9, 5, and 12) and two cases
(no. 13 and IO), respectively.
To rule out the possibility that residual T cells may interfere with the proliferative effect of IL-9. four additional
experiments (no. 25. 26, 27, and 32) were performed after
T-cell depletion (Fig 3). In the same set of experiments we
also demonstrated that highly purified IL-9 has the identical
activity of COS cell-derived cytokine. Taken together. these
results suggest that IL-9 activity is directed to target cells
and is not mediated by accessory cells or unwanted proteins
present in COS cell -derived conditioned medium that could
have an effect on cell growth.
IL-9 increases the proportion of A M L cells in S-phase.
By the BRDU incorporation assay, IL-9 augmented the number of S-phase leukemic cells (>20% increase v control
sample) in five of eight cases (12.9% -+ 4 8 v 9% 5 3% for
the whole group, P < .OS: Table 3). Significant proliferation
was also induced in some cases by IL-3 and GM-CSF (13.7%
Table 2. Effect of Recombinant Human IL-9 on AML Blast Colony Formation
No. of Colonies
Type
M1
M2
M4
M4
M5a
M5a
M5a
M5a
Mean 2 SEM
Patient No.
Medium
(IMDMI
4
9
5
13
10
11
12
16
2
82
3
31
11
0
25
0
19 2 10
PHA-LCM
IL-9
SCF
IL-3
GM-CSF
IL-9. SCF
IL-9, IL-3
IL-9, GM-CSF
65
1,405
70
95
145
473
48
30
291 2 167*
44
267
7
124
82
219
45
67
107 2 32*
20
70
45
25
16
0
10
ND
27 2 9
27
170
52
45
90
24
64
ND
67 2 19*
40
180
143
80
64
16
35
ND
81 2 23'
68
720
70
97
197
214
76
56
187 2 79'
4
428
80
120
138
128
180
56
142 2 45
46
308
125
310
158
130
60
20
145 z 39
Results are expressed as the number of CFU-L/105 cells plated and represent the mean of triplicate counts.
Abbreviation: ND, not determined.
IL-9, IL-3, and GM-CSF induced, as single cytokines, a CFU-L growth rate significantly higher ( P < .05) than that of control (medium) samples.
The IL-9 and SCF combination resulted in an increased blast colony formation as compared with the same CSF used alone ( P < .05 v SCF; P
= .059 v IL-9).
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
LEMOLI ET AL
3856
Table 3. Effect of 11-9 on the Proportion of AML Cells in S-Phase Evaluated by BRDU Incorporation Assay
S-Phase i%P
Medium
(IMDMI
IL-9
SCF
IL-3
M1
8
10
18
18
10
11
M2
M2
1
3
2
6
7
14
15
26
2
2.8
17
4
7
3.5
9 f3
32
ND
ND
12
7
18
4
15.1 f 4 t
31.5
2.4
3.7
18
6
20
3.7
12.9 f 4 t
25
2.5
5
14
7
6
4.3
9.2 t 2
37
3
5
11
9
30
3.5
13.7 f 4 t
Type
M4
M4
M5b
M5b
M4
Mean -c SEM
Patient No.
PHA-LCM
IL-9, SCF
IL-9, IL-3
IL~9,GM-CSF
20
30
2
4
19
7
38
5.8
15.7 fr 5
18
34
2.1
4.3
16
5
35
5.5
14.9 5 5
22
40
3.7
7
23
8
39
4.5
18.4 5 5 t
GM-CSF
16
34
1.5
6
25
5
12
3.3
12.8 f 4
* T h e number of cells in S-phase was determined by the BRDU incorporation assay after 3 days of liquid culture. BRDU-positive cells at day
0.3% 2 1%.
t Statistically significant v control samples or single cytokines ( Pi.05).
? 4% and 12.8% 2 4% BRDU-positive cells, respectively).
Addition of SCF or IL-3 to IL-9 resulted in an increase of
the DNA synthesis rate in two cases (no. 14 and 15). The
two-factor combination consisting of GM-CSF and IL-9 was
the most effective for inducing proliferation of AML cells
( P < .05 v both cytokines used alone).
To further address the effects of IL-9 on cell-cycle distribution of leukemic cells, eight additional AML samples (no.
17 to 24) were studied by AO. The mean results are shown
in Table 4 and confirm the capacity of IL-9 to induce leukemic cells to enter S-phase (14.7% +- 11% v 11.3% 2 lo%,
P = .04). Addition of SCF to IL-9 further increased the
percentage of AML cells undergoing DNA synthesis (20.7%
? 12% v 11.3% ? lo%, P = .01). More interestingly, the
cytokine combination also induced a significant recruitment
of quiescent leukemic cells in cell cycle by decreasing the
Go cell fraction (from 58.6% ? 22% to 38.2% ? 24%, P <
.03) and increasing the RNA content of GI cells (ie, RNAI G I : 19.2 ? 2 v 16.8 '-c 2 , P = .003). A representative
example (case no 18) of kinetic changes induced by IL-9
and SCF in combination is shown in Fig 4.
IL-9 does not affect programmed cell death. Evaluation
of apoptosis by A 0 did not show any significant effect of
IL-9, whereas the combination of IL-9 and SCF provided
some protection from programed cell death, but it was not
statistically significant (Table 4). Moreover, to evaluate early
apoptotic events, AML samples were also analyzed by the
TDTA technique (no. 17 to 24) and by gel electrophoresis
(no. 25 to 32, assessed after T-cell depletion), which confirmed the absence of protective effects of IL-9 on leukemic
blasts (Table 4 and Fig 5).
DISCUSSION
Several investigators have demonstrated that a group of
glycoproteins termed CSFs, which are essential for in vitro
proliferation and differentiation of normal hematopoietic
cells, are also involved in the process of malignant transformation." The constitutive expression of the mRNA of several CSFs (ie, GM-CSF, G-CSF, IL-1, and SCF) and their
production by AML cells have provided evidence of the
pivotal role of growth factors in acute le~kemia?~.'~
Moreover, the recruitment of accessory or stromal cells to increase
cytokine supply by IL- 1 or tumor necrosis factor-alpha secretion has also been rep~rted.'~
The newly described cytokine, IL-9, has been shown to
be expressed by activated CD4+ T cells.28It was first isolated
and characterized for its ability to stimulate a megakaryoblastic cell line'; subsequently, IL-9 has demonstrated BPA
on erythroid burst-forming units derived from immature
CD34TD33-DR' progenitor cells' and colony-promoting
ability on earlier precursors (CD34TD33-DR- cells) before
the determination of erythroid commitment." Previous investigations have shown that IL-9 may also be involved in
the regulation of myeloid leukemic cell growth.'." However,
Table 4. CellGycle Effects of 11-9 and SCF on AML Blast Cells
%APO
Parameter
Day 0
A0
2 2 2
TDTA
%Go
%G,
%S
%G2M
RNA-I G,
NE
67.6 2 19
19.3 f 7
10.7 t 12
2.3 f 2
16.9 t 1
10.4 It 1
11.7 2 2
10.9 2 7
58.6 f 22
61.1 f 19
38.2 2 24"
28.6 f 25
24.7 f 22
39.1 2 25
11.3 -t 10
14.7 2 11*
20.7 f 12*
1.4 f 1
1.5 t 1
1.8 f 1
16.8 C 2
16.7 f 2
19.2 Z 2*
Day 3
CTR
IL-9
IL-9, SCF
18.1 f 17
18.1 2 16
12.5 f 8
Results are expressed as the mean f SEM of 8 different experiments (cases no. 17 to 24 in Table 1).
Abbreviations: NE, not evaluated; APO, apoptotic.
* Statistically significant v control samples at day 3.
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
INTERLEUKIN-9 STIMULATES AML CELLS
Fig 4. Representativeexample of AML cells (case no. 181recruited
in cell cycle by 11-9 and SCF. BM. bone marrow.
those preliminary studies were performed on continuously
growing CSF-dependent cell lines. which may not be fully
representative of the stem cell compartment of primary leukemic samples.
The aim of the present study was to investigate the proliferative activity of fresh AML cells upon stimulation with
IL-9 alone and combined with other cytokines. SCF, IL-3,
and GM-CSF were chosen because earlier studies demonstrated a positive interaction with IL-9 for stimulating the
growth of both normal and leukemic myeloid cells."',"
Our results demonstrated that IL-9, as a single cytokine,
promotes the growth of three myeloid cell lines under serumcontaining and serum-free conditions and stimulates the proliferation of a majority of fresh AML samples studied. The
degree of response to IL-9 did not appear to correlate with
the FAB classification or any clinical manifestation. Results
of the experiments performed using T-cell-depleted leukemic populations (Fig 3) and data derived from serum-free
medium clonogenic assays (Figs I and 2) ruled out that 1L9 activity might be mediated by accessory cells or exogenous
proteins contained in FCS. Furthermore, two different IL-9
Fig 5. DNA fragmentation of 3 representative leukemic samples.
Cells were incubated in the absence (lanes2,7, and 9) and presence
(lanes 3,6,and 11) of IL-9 and IL-9 and SCF (lanes4, 5, and 12). DNA
fragmentation before liquid culture is shown in lanes 1, 8, and 10.
3857
preparations were compared to evaluate whether contaminant proteins in COS-conditioned medium may interfere
with IL-9-mediated proliferative activity (Fig 3).
We have also used 16 AML samples for studying the
effects of IL-9, singly and combined with c-kit ligand, on
apoptosis and cell-cycle status. Through kinetic analysis. we
have been able to demonstrate that the greatest fraction of
S-phase cells were found in the presence of IL-9 and SCF.
Moreover, this cytokine combination induced a significant
recruitment of leukemic cells out of GI, phase. This is in
agreement with mapping and kinetic studies of normal blast
cell colonies that suggested that the transition of cells from
GI' depends on the simultaneous presence of two or more
CSFs including an early-acting cytokine."' However, IL-9
alone augmented the clonogenic efficiency and active synthesis of DNA in 12 of I2 and I3 of I6 AML cases tested,
respectively. In contrast to previous reports' indicating IL-9
as a major antiapoptotic factor for thymic lymphomas, we
were not able to demonstrate significant protection against
apoptosis in AML cells after incubation with 1L-9 or IL-9
and SCF. Thus, the putative role of IL-9 in the development
of AML seems to be restricted to cells dependent on this
factor for proliferation.
Taken together, these results suggest that IL-9 effects on
AML-derived cells differ from those exerted on their normal
counterparts. IL-9 individually showed little if any proliferative activity on highly purified hematopoietic progenitor
cells."' In the same report, we also showed that simultaneous
stimulation with IL-9 and SCF or IL-3 or GM-CSF in the
presence of erythropoietin remarkably enhanced the proliferation of early precursors. In this study, we observed a strong
stimulatory effect of IL-9 alone on AML samples that was
higher than that of SCF, IL-3, and GM-CSF. Moreover,
addition of IL-9 to the other CSFs resulted in an additive or
synergistic stimulation in only half the cases. One potential
explanation for the activity of IL-9 alone is the endogenous
production of other cytokines by leukemic cells. Thus far,
we have detected the presence of high concentrations of IL-6,
GM-CSF, and IL-3 in the supernatant of some AML samples
stimulated with IL-9 (manuscript in preparation). If the differences between normal and leukemic cells are confirmed
by additional studies. the use of IL-9 might be clinically
relevant for entering AML blasts into cell cycle before exposure to S-phase drugs. Several reports have documented that
certain human recombinant hematopoietic growth factors,
including IL-3. GM-CSF, and G-CSF. can enhance cytosine
arabinoside cytotoxicity on leukemic cells by recruiting quiescent AML cells in S-phase."."' However, the responsiveness of leukemic progenitors to CSFs seems to reflect
that of normal bone marrow cells.'' Thus, their clinical use
may be limited by potential enhancement of the myelotoxic
effect of antileukemic drugs. Recent clinical trials that did
not show an improved response rate when GM-CSF or GCSF were administered before induction chemotherapy in
AML patients may partly reflect these limitations."." In this
regard, IL-9 may offer some advantages in comparison to
other cytokines because of its negligible activity on normal
marrow cells.
Early investigations have demonstrated a consistent pat-
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
LEMOLI ET AL
tern of expression of IL-9 in human malignant lymphomas,
suggesting a possible role for this cytokine as an autocrine
growth factor for Hodgkin's disease and LCAL.h Furthermore, the constitutive expression of IL-9 in vivo resulted in
a high incidence of thymic lymphomas in transgenic mice.73
Conversely, it has been recently reported that there is a lack
of constitutive expression of IL-9 in bone marrow and peripheral blood of healthy individuak3' In this regard, preliminary data from our laboratory indicate that IL-9 mRNA is
not expressed in four myeloid cell lines studied (ie, K562,
HL60, KG1, and U937; data not shown), suggesting that a
paracrine rather than an autocrine growth stimulation may
be operative in AML.
In summary, this report provides the first evidence that
IL-9 may play a role in the development of AML cells
by triggering cells to enter S-phase. Moreover, IL-9 acts
synergistically with SCF to recruit quiescent leukemic cells
out of GO phase. Further studies are in progress to investigate
whether IL-9 mRNA is present in fresh AML samples and
whether a biologically active form of the protein is secreted.
REFERENCES
I . Yang YC, Ricciardi S, Ciarletta A, Calvetti J, Kelleher K,
Clark SC: Expression cloning of a cDNA encoding a novel human
hematopoietic growth factor: Human homologue of murine T-cell
growth factor P40. Blood 74: 1880, 1989
2. Van Snick J, Goethals A, Renauld JC, Van Roost E, Uyttenhove C, Rubira MR, Moritz RL, Simpson RJ: Cloning and characterization of a cDNA for a new mouse T-cell growth factor (p40).J
Exp Med 169:363, 1989
3. Hultner L, Druez C, Moeller J, Schmitt E, Uyttenhove C, Rude
E, Dormer P, Van Snick J: Mast cell growth-enhancing activity
(MEA) is structurally related and functionally identical to a novel
mouse T-cell growth factor p40/TCGF 111 (interleukin 9). Eur J
Immunol 20:1413, 1990
4. Suda T, Murray R, Fischer M, Yokota R, Zlotnik A: Tumor
necrosis factor and p40 induce day 15 murine fetal thymocyte proliferation in combination with IL-2. J Immunol 144:1783, 1990
5 . Merz H, Houssiau FA, Orscheschek K, Renauld JC, Fliedner
A, Herin M, Noel H, Kadin M, Mueller-Hermelink HK, Van Snick J,
Feller AC: Interleukin-9 expression in human malignant lymphomas:
Unique association with Hodgkin's disease and large cell anaplastic
lymphoma. Blood 78: 13 11, 199 1
6. Gruss HJ, Brach MA, Drexler HG, Bross KJ, Hermann F:
Interleukin 9 is expressed by primary and cultured Hodgkin and
Reed-Stemberg cells. Cancer Res 52: 1026, 1992
7. Renauld JC, Vink A, Louahed J, Van Snick J: Interleukin-9 is
a major anti-apoptotic factor for thymic lymphomas. Blood 85: 1300,
1995
8. Lu L, Leemhuis T, Srour EF, Yang YC: Human interleukin
(1L)-9 specifically stimulates proliferation of CD34+ + + DR+
CD33- erythroid progenitors in normal human bone marrow in the
absence of serum. Exp Hematol 20:418, 1992
9. Donhaue RE, Yang YC, Clark SC: Human P40 T-cell growth
factor (interleukin-9) supports erythroid colony formation. Blood
75:2271, 1990
10. Lemoli RM, Fortuna A, Fogli M, Motta MR, Rizzi S, Benini
C, Tura S: Stem cell factor (c-kit ligand) enhances the interleukin9-dependent proliferation of human CD34+ and CD34+ CD33DR- cells. Exp Hematol 22:919, 1994
1 I . Miyazawa K, Hendrie PC, Kim YK, Mantel C, Yang YC,
Kwon BS. Broxmeyer HE: Recombinant human interleukin-9 induces protein tyrosine phosphorylation and synergizes with steel
factor to stimulate proliferation on the human factor-dependent cell
line M07e. Blood 80:1685, 1992
12. Collins SJ, Gallo RC, Gallagher RE: Continuous growth and
differentiation of human myeloid leukemia cells in suspension CUIture. Nature 270:347, 1977
13. Lozzio CB, Lozzio BB: Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome. Blood 45:32 I ,
I975
14. Koeffler HP, Golde DW: Acute myelogenous leukemia: A
human cell line responsive to colony-stimulating activity. Science
200:1153, 1978
15. Bennet JM, Catovsky D, Daniel MT, Fladrin G, Galton DAG,
Gralnick HR, Sultan C: Position papers: Proposed revised criteria
for the classification of acute myeloid leukemia. Ann lntem Med
103:620, 1985
16. Lemoli RM, Gulati SC, Strife A, Lambek C, Perez A,
Clarkson BD: Proliferative response of human acute myeloid leukemia cells and normal marrow enriched progenitor cells to human
recombinant growth factors IL-3, GM-CSF and G-CSF alone and
in combination. Leukemia 5:386, 1991
17. Lemoli RM, Fogli M, Fortuna A, Amabile M, Zucchini P,
Grande A, Martinelli G. Ferrari S, Tura S: Interleukin 1 1 (IL-I I )
acts as a synergistic factor for the proliferation of human myeloid
leukemic cells. Br J Haematol 91:319, 199.5
18. Tafuri A, Lemoli RM, Chen R, Gulati SC, Clarkson BD,
Andreeff M: Combination of hematopoietic growth factors containing IL-3 induce acute myeloid leukemia cells sensitization to
cycle specific and cycle non-specific drugs. Leukemia 5:749, 1994
19. Andreeff M, Assing G, Cinincione C: Prognostic value of
DNA/RNA flow cytometry in myeloblastic and lymphoblastic leukemia in adults: RNA content and S-phase predict remission duration
and survival in multivariate analysis. Ann NY Acad Sci 406:387.
1984
20. Darzynkiewicz Z, Bruno S, Del Bino G, Gorezyca W, Hotz
MA, Lassota P, Traganos F: Features of apoptotic cells measured
by flow cytometry. Cytometry 13:795, 1992
21. Zinzani PL. Tosi P, Visani G, Martinelli G, Farabegoli P,
Buzzi M, Ottaviani E, Salvucci M, Bendandi M, Zaccaria A, Tura
S: Apoptosis induction with three nucleoside analogs on freshly
isolated B-chronic lymphocytic leukemia cells. Am J Hematol
47:301, 1994
22. Oster W, Mertelsmann R, Herrmann F: Role of colony-stimuk i n g factors in the biology of acute myelogenous leukemia. Int J
Cell Cloning 7: 13, 1989
23. Young DC, Demetri GD, Ernst TJ, Cannistra SA, Griffin JD:
In vitro expression of colony-stimulating factor genes by human
acute myeloblastic leukemia. Exp Hematol 16378, 1988
1-4.Young DC. Wagner K, Griffin JD: Constitutive expression
of the granulocyte-macrophage colony-stimulating factor gene in
acute myeloblastic leukemia. J Clin Invest 79:100, 1987
2.5. Cozzolino F, Rubartelli A, Aldinucci D, Sitia R, Torcia M,
Shaw A, Di Guglielmo R: Interleukin-1 as an autocrine growth factor
for acute myeloid leukemia cells. Proc Natl Acad Sci USA 86:2369,
1989
26. Pietsch T, Kyas U, Steffens U, Yakisan E, Hadam MR, Ludwig WD, Zsebo K, Welte K: Effects of human stem cell factor (ckit ligand) on proliferation of myeloid leukemia cells: Heterogeneity
in response and synergy with other hematopoietic growth factors.
Blood 80:1199, 1992
27. Griffin JD, Rambaldi A, Vellenga E, Young DC, Ostapovicz
D, Cannistra SA: Secretion of interleukin-I by acute myeloblastic
leukemia cells in vitro induced endothelial cells to secrete colony
stimulating factors. Blood 70:1218, 1987
28. Renauld JC, Goethals A, Houssiau F, Merz H, Van Roost E,
Van Snick J: Human P40nL-9 expression in activated CD4+ cells,
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
INTERLEUKIN-9 STIMULATES AML CELLS
genomic organization and comparison with the mouse gene. J Immuno1 144:4235, 1990
29. Ogawa M: Differentiation and proliferation of hematopoietic
stem cells. Blood 81:2844, 1993
30. Tafuri A, Andreeff M: Kinetic rationale for cytosine-induced
recruitment of myeloblastic leukemia followed by cycle-specific chemotherapy in vitro. Leukemia 12:826, 1990
31. Estey E, Thall PF, Kantarjian H, O'Brien S, Koller C, Beran
M, Gutterman J, Deisseroth A, Keating M: Treatment of newly
diagnosed acute myelogenous leukemia with granulocyte-macrophage colony-stimulating factor (GM-CSF) before and during continuous-infusion high-dose ara-C + daunorubicin: Comparison with
patients treated without GM-CSF. Blood 79:2246, 1992
32. Estey E, Thall P, Andreeff M, Beran M, Kantarjian H,
3859
O'Brien S, Escudier S, Robertson LE, Koller C, Komblau S, Pierce
S, Freireich El,Deisseroth A, Keating M: Use of granulocyte colonystimulating factor before, during and after fludarabine plus cytarabine induction therapy of newly diagnosed acute myelogenous leukemia or myelodysplastic syndromes: Comparison with fludarabine
plus cytarabine without granulocyte colony-stimulating factor. J Clin
Oncol 12:671, 1994
33. Renauld JC, van der Lugt N, Vink A, van Roon M, Godfraind
C, Warnier G, Merz H, Feller A, Bems A, Van Snick J: Thymic
lymphomas in IL-9 transgenic mice. Oncogene 9: 1327, 1994
34. Cluitmans FHM, Esendam BHJ, Landegent JE, Willemze R,
Falkenburg FJH: Constitutive in vivo cytokine and hematopoietic
growth factor gene expression in the bone marrow and peripheral
blood of healthy individuals. Blood 85:2038, 1995
From www.bloodjournal.org by guest on January 21, 2015. For personal use only.
1996 87: 3852-3859
Interleukin-9 stimulates the proliferation of human myeloid leukemic
cells
RM Lemoli, A Fortuna, A Tafuri, M Fogli, M Amabile, A Grande, MR Ricciardi, MT Petrucci, L
Bonsi, G Bagnara, G Visani, G Martinelli, S Ferrari and S Tura
Updated information and services can be found at:
http://www.bloodjournal.org/content/87/9/3852.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.