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Interleukin-9 Is a Major Anti-Apoptotic Factor for Thymic Lymphomas
By Jean-Christophe Renauld, Anne Vink, Jamila Louahed, and Jacques Van Snick
We have recently shown that interleukin-9 (IL-9) strongly
stimulates the proliferation of mouse thymic lymphomas in
vitro. Here we report that this factor is also one ofthe most
potent inhibitors of apoptosis induced by dexamethasone
(DEX) in such cell lines,even if they do not depend on exogenous factors for growth. For the murine thymic lymphoma
BW5147, protection against apoptosis was also obtained
with IL-4 and less strongly with IL-6, whereas IL-2, IL-7, and
IL-10 were inactive. Moreover, IL-4 and IL-9 maintained proliferation of these cells in the presence of DEX. Analysis of
eight other factor-independent thymic lymphoma lines
showed significant protection in seven and six celllines with
IL-9 and IL-4, respectively, whereas only three were protected by IL-7 and only two by IL-2. Comparison of the responses to IL-2 and IL-9 in a factor-dependent cell line that
responds to both cytokines showed that IL-2 is a stronger
inducer of proliferation, while IL-9 is more efficient in protecting the cells against apoptosis. Taken together, our observations suggest that, for thymic lymphomas, proliferation and apoptosis involve distinct regulatory mechanisms
and can be differentially regulated by cytokines.
0 1995 by The American Society of Hematology.
I
ence intrathymic T-cell development, not only by stimulating
cell proliferation but also by controlling apoptosis.
Thymic lymphomas retain many characteristics of normal
thymocytes. They proliferate in response to T-cell growth
factors such as 1L-2. 1L-4, and IL-75 and frequently undergo
apoptosis in the presenceofDEX.I4Cell lines that proliferate
autonomously but retain
sensitivity
to DEX-induced
apoptosis can be derived from such tumors, thus providing
a unique opportunity to investigate cytokine anti-apoptotic
properties without interference by stimulation of cell growth.
By protectingthymic
lymphoma cellsagainstapoptotic
death, cytokines could play an important role in the development of T-cell tumors that do not require any cytokine for
proliferation but are sensitive to inducers of apoptosis. Here
we report that IL-9 and IL-4 are potent inhibitors of glucocorticoid-induced apoptosis and cell growth arrest in factorindependent thymic lymphoma cell lines. By contrast, IL-2
seems to be more efficient in triggering proliferation than in
protecting against apoptosis. suggesting that distinct mechanisms are involved in these processes.
NTERLEUKIN-9(IL-9)
is aT-cell-derived
cytokine
withpleiotropicactivities
on variouscell types.' Although it was initially described as a
T-cell growth factor
for mouse T-helper cell clones,' the role of this cytokine in
T-cell development and function remains elusive. With the
exception of day 15 fetal thymocytes, wherea strong synergy
is observed between IL-2 and IL-9, freshly isolated normal
T cells do not proliferate in response to this cytokine.'.' By
of
contrast,IL-9 hasbeen showntobe
apotentinducer
proliferation for mouse thymic lymphomas in vitro.' Moreover, dysregulated expression of the IL-9 gene in transgenic
miceresults in thesporadicdevelopment
of spontaneous
thymic lymphomas and in a dramatic increase in susceptibility to theinductionof such tumors by mutagens or x-ray
irradiation." In the human, recent observations have shown
that IL-9 responsiveness is highly restricted by the stage of
activation of T-cell lines,' and aputativeinvolvement
in
tumorigenesis has been suggested by the demonstration of
an autocrine loopina
Hodgkin'slymphoma cell line in
vitro.x
Apoptosis plays an essential role in thymic development,
as immature cells are constantly eliminated by this process
as a result of antigen-receptor stimulation""' and exposure to
glucocorticoids." Although their physiologic role in thymic
ontogeny is notcompletely clear, cytokines have
been shown
to interfere with this process in vitro. IL-1 has been shown
to inhibit CD3-induced T-cell death, while IL-4 was found
to protect double-negative and CD4 single-positive thymocytes against the glucocorticoid analoguedexamethasone
(DEX)."." These results suggest that cytokines may influ-
From the Ludwig Instiiute for Cancer Research, Brussels: and
the Experimental Medicine Unit, UniveruitC Catholique de Luuvuin,
Brussels,Belgium.
Submitted May 9, 1994: accepted October 22, 1994.
Supported in pari by the Belgian Stute Prime Minister's Ofice
.Sc,ieucr Policy Programming (Grunt No. 82/87-39). J.C.R. is a Re.wcrrch Associate and J.L. a Scientijic Associute (TilCvie) with the
b'im1.s Natiolzal de la Recherche Scientijique, Belgiurn.
Arldress reprint requests t o Jean-Chrisiophe Renauld, MD, Ludwig Ir~,stitutefr,r Cuncer Reseurch, Ave Hippocrate,
74, B-l200Brus.seis, Belgiurn.
The publication cost,s of this urticle were defrayed in purr by puge
chargepuyment.Thisarticlemustthereforebehereby
marked
"advertisement" in accordance with 18 U.S.C. section 1734 solel? t o
indicate this ,fitcf.
0 l995 by The American Society of' Hernutology.
0006-4971/9~~/~505-0028$3.00/0
1300
MATERIALS AND METHODS
Turners m d cell lines. Thymic lymphoma cell lines BWS147"
andWEHI 22.1'' were obtained from the AmericanTypeCulture
Collection(Rockville,MD). TH 1821 was derived from a y-rayinduced CBAlHt thymic lymphoma.'"The other cell lines were established in our laboratory (Ludwig Institute for Cancer Research,
Brussels. Belgium): NM2TI and NM3T2 from DBA-2 mice treated
with N-methyl-N-nitrosourea (MNU), BR14T2from a C57BL/6
mouse submitted to fractionated x-ray irradiation, and 9T4A2 and
9T7 from thymic lymphomas that spontaneously arosein IL-9transgenic mice hut had lost the IL-9 transgene.' Cell line 9T7.9C I
is an autonomous subclone from the [L-2-dependent 9T7
lymphoma. All cells were cultured in Iscove's medium supplemented
with 10% fetal calf serum, I .5 nImol/L L-glutamine, 0.24 mmol/L
L-asparagine. 0.55 mmol/L L-arginine, and S0 pmollL 2-mercaptoethanol.
Cytokirzes arzd otherreagents.
The followingcytokines
were
provided as indicated: human recombinant IL- la from Dr P. Lomedico (Roche Research Center. Nutley, NJ); human IL-2, mouse interferon-y (IFN-y), and mouse tumor necrosis factor-cu (TNF-a) from
Dr W. Fiers(StateUniversity of Ghent, Ghent, Belgium); human
recombinant IL-7 from R & D Systems (Minneapolis,MN); mouse
IL-10 from Dr P. Vieira and K.W. Moore (DNAX Research Institute
of Molecular andCellular Biology, Palo Alto, CA);and mouse
IL-13 from Dr J . Banchereau (Schering-Plough, Dardilly. France).
Murine IL-4, IL-6, and IL-9 were produced by expression in baculovirus and purified by affinity chromatography at Ludwig Institute
for Cancer Research. DEX was used at 0.25 p m o l n concentration
in all experiments. Staurosporin (Biomol Research Laboratories Inc,
Blood, Vol 85, No 5 (March l), 1995: pp 1300-1305
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1301
ANTI-APOPTOTICACTIVITY OF IL-9
I L-9
A
JI3
-
+
ofDEX. thymic lymphoma cell lines were incubated overnight in
triplicate microtiter wells with or without cytokines before addition
ofDEX to a final 0.25 X IO-’ mollL concentration in a 200-pL
volume. Unless stated otherwise, the cytokine concentrations used
were as follows: IL-la, 20 UlmL: IL-2. 50 UlmL; IL-4. 250 UlmL;
IL-6. 1,000 UlmL: IL-7. 10 ng/mL; IL-9, 500 UlmL: IL-IO. 10 ngl
mL; IL-13, 1 0 0 nglmL; IFNy, 1 0 0 UlmL; and TNFa, 1 0 0 UlmL.
These concentrations were at least 10-fold higher than doses giving
a maximal activity of the factors in their respective bioassays. Cultures werepulsed 2 to 4 days later for 6 hours with 0.5 pCi ‘Hthymidine. Alternatively, cell proliferation was measured by an hexosaminidase assay, as described.’
RESULTS
- +
105Fluorrseenct Inttasit{
Fig 1. Inhibition by IL-9 of DEX-induced apoptosis in BW5147 cells.
(A) Electrophoresis of DNA extracted from BW5147 cells cultured for
3 days in control medium or in thepresence of IL-9 (500 UlmL).DEX
(0.25 pmol/L) was added for the last 30 hours of the culture. (B)
Propidium iodide staining and FACSanalysisof BW5147 cells cultured for 24 hours in control medium, in DEX, or in DEX and 11-9. Y
axis corresponds to the cell numbers and X axis, to fluorescence
intensity. Living cells, which showed no DNAstaining, were differentiated from dead cells, whose DNA was stained.
Plymouth Meeting, PA) and cyclosporin A (CsA) were usedat 5
nglmL and 1 pglmL, respectively.
Assessment of DNA frapnenmtion. BW5147 cells were incubated for 2 days in normal culture medium or in the presence of IL9 (0.5 X IO-’ mol/L) before addition of DEX (0.25 X IO-” mol/L).
Cells were centrifuged 24 hours later; resuspended in lysis buffer
containing 0.15 mol/L NaCI, 10 mmol/L Tris pH 8, 12.5 mmol/L
EDTA, 0.5% sodium dodecyl sulfate (SDS), and 0.5 mglmL proteinase K;and incubated overnight at 56°C. After extraction with phenol
and chloroform, the DNA was precipitated with isopropyl alcohol,
resuspended in TE buffer (10 mmol/L Tris pH 8, 1 mmol/L EDTA)
and incubated at 37°Cwith0.5 mglmL RNAse for I hour before
electrophoresis in a 2% agarose gel.
Viability and proliferation assq.v. Cell viability was determined
using a propidium iodide incorporation assay as described.” Briefly,
cells were incubated for 30 minutes with propidium iodide (125 pgl
mL) at room temperature before fluorescence-activated cell sorting
(FACS) analysis with an ATC3000 flow cytometer (Odam, Wissembourg, France). Under these conditions, dead cells are brightly
stained, while live cells are not. A minimum of 5,OOO cells were
counted per sample.
To test the influence of cytokines on proliferation in the presence
IL-9 inhibits DEX-induced apoptosis in B W5147 thynic
/vn7phorna cell line. IL-9 is a potent stimulator of thymic
tumor cell proliferation, but its ability to inhibit apoptosis has
not yet been investigated. To evaluate possible antiapoptotic
activity mediated by IL-9, we selected BWS147, a thymic
lymphomathat proliferates actively in vitro without cytokines and undergoes apoptotic cell death in the presence of
DEX. Cells incubated with or without a saturating dose of
IL-9 were exposed to DEX for 30 hours. Genomic DNA
was extracted and analyzed by gel electrophoresis to detect
fragmentation into 200-bp fragments as a reflection of
apoptosis. Cell viability was also measured by FACS after
staining with propidium iodide. As indicated in Fig I , exposure to DEX induced a strong DNA fragmentation and approximately 30% cell death after 30 hours. Both processes
were almost completely inhibited by IL-9 (Fig l). Pretreatment of BW5147 with IL-9 was, in fact, not necessary, as
cells were protected as efficiently when the cytokine was
added at the same time as DEX (data not shown).
Activity of other cytokines in protecting BW5147 against
DEX-induced apoptosis. To evaluate the antiapoptotic activity of other cytokines, BW5147 cells were incubated with
a series of different factors before addition of DEX. Cell
mortality was measured by propidium iodide staining after
“
5
“. . ?
?
?
?
2
‘
4
?
:
2
1
2
.
*
2-
i
k
-
Z
k
P
Fig 2. Cytokines protecting BW5147 cells against DEX-induced
cell death. Cells were incubated for 20 hours with saturating concentrations of the indicated cytokines, as described in Materials and
Methods, before addition of DEX (0.25 FmollLI. Cells were stained
24 hours later with propidium iodide, and the proportion of positive
cells was measured by FACS for duplicate cultures. In the absence
of DEX, the mean proportion of stained cells was 2.5% k 1.4%. The
results are representative of three separate experiments.
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RENAULD ET AL
A
1 ,
I”
I
I
0.75-
B
B
0.5-
e,
B
Fig 3. Cytokine-induced proliferation of BW5147 cells in the presence of DEX. Cells ( I O 3 per well) were incubated with saturating
concentrations of the indicated cytokines, as described in Materials
and Methods, and DEX (0.25 pmollL1 for 3 days. Cell proliferation
was evaluated by measuring thymidine incorporation (A) or by an
hexosaminidase colorimetric assay (B). Standard deviations were calculated from triplicate cultures. In the absence of DEX, thymidine
incorporation was 127,370 2 8,081 cpm (A), and absorbance at 405
nm was1.142 (B);cytokines had no stimulatoryeffects on thisproliferation (data not shown). Results are representative of three independent experiments evaluated by thymidine
incorporation and t w o
independent experiments using the hexosaminidase assay.
a 24-hour exposure to DEX. This analysis showed that, IL4 and IL-6, in addition to IL-9, also inhibited DEX-induced
mortality, whereas IL-l, IL-2,IL-7, and IL-IO were inactive.
By contrast, TNF-a and, to a lesser extent, IFN-y increased
DEX-induced mortality (Fig 2). Interestingly, IL-9 also inhibited the increment in mortality induced by these factors
(data not shown).
As inhibition of DEX-induced apoptosis does not necessarily result in cell proliferation, we evaluated the growthstimulatoryactivity of cytokines on BWS147cells in the
presence of DEX. With DEX alone, the
Proliferation was
inhibited by 90% to 95% (data not shown). By contrast, in
the presence of IL-9, a strong proliferation was observed in
thymidine incorporation or hexosaminidase assays. Lower
but significant levels of protection were obtained with IL-4
and IL-6, whereas other cytokines, such as IL-la, 1L-2, IL10, and IL-13, were inactive (Fig 3). Concentrations of IL4 required to maintain proliferation were much higher than
those of IL-9. Half-maximal protection required SO0 pmol/
L for IL-4, while the same result was obtained with 5 pmol/
L IL-9, this sensitivity making BW5147 in the presence of
DEX equivalent to the most sensitive biologic assay for IL-
9 described so far.2 For IL-6, the half-maximal effect was
obtained ata concentration on the orderof 10 pmol/L, which
is similar to that which induces half-maximal responses in
fresh mouse T cells” (Fig 4).
To test whether the protective activities of IL-4 and 1L-9
againstDEXwere
long-lasting, cellcultures were established with or without DEX and successively diluted for 2
weeks. As shown in Fig S, the protective activities of either
factor allowed for permanentgrowth in the presenceof DEX.
Over a 12-day period, the number of divisions was 39 in the
absence of DEX and 29 or 26 with DEX and XL-9 or IL-4,
respectively. With DEX in the absence of cytokines, viable
cellscouldnolongerbe
detectedafter 1 week. Without
DEX, cytokines do not affect cell growth (data not shown).
Eject .f staurosporin and CsA on rescue meclzunisms
triggered by cytokinesinDEX-treuted BW5147 cells. To
begintoanalyzethe
signaltransduction mechanismsinvolved in the anti-apoptotic activity of IL-9, IL-4, and IL6, we evaluated the effect of staurosporin (an inhibitor for
a large variety of kinases) and CsA. Although these reagents
did not affect cell viability in normal medium, they significantly accelerated DEX-induced cell death. In the presence
of S ng/mL of staurosporin, the activities of IL-4 and 1L-9
were completely blocked, while IL-6 had still a weak but
reproducible activity. By contrast, CsA at l pg/mL did not
affect the protection conferred
by IL-4 or IL-9 but significantly reduced the effect of IL-6 (Fig 6). These results suggest that distinct signaling pathways are activated by IL-9
and IL-4 on the one hand and IL-6 on the other.
Activity of T-cellgrowth ,factors againstDEX-induced
upoptosis qf other thymic lymphoma cell lines. The ability
of IL-2, IL-4, IL-7, and IL-9 toprevent apoptosis was examined on eight other factor-independent, DEX-sensitive thymic lymphoma cell lines obtained under distinct conditions
in different mouse strains. These cells were incubated with
‘”
I
-
IL-6
L 4
O+
a
a?
’
,a
Cytokine
Concentration
,aa
aaaa
(PM)
Fig 4. Dose-response curves of anti-DEX activities of IL-4,IL-6.
and IL-9 on BW5147 cell line. Cells ( I O 3 per well) were incubated with
the indicatedcytokines for 3 days in thepresence of DEX (0.25 pmoll
L). Thymidine incorporations were measured in triplicate cultures.
Concentrations used in this experiment ranged from 0.26 x lo3 to 4
x I O 3 pmollL for IL-6, and from 0.6 to I O 4 pmol/L for IL-4 and IL-9.
Results are representative of two independent experiments.
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ANTI-APOPTOTICACTIVITY OF IL-9
1303
DISCUSSION
1010
/I
I
I
I
I
I
0
4
7
9
12
1
Days
Fig 5. Prolonged growth of SW5147 cells in the presence of DEX
and 11-4 or IL-9: (W), control; (0).DEX; (0).
DEX IL-9; (A), DEX
IL-4. Cultureswere started with 1,000 cells permicrowell and diluted
when appropriate. Concentrations were 0.25 pmol/L for DEX, 500 U/
mL for IL-9, and 250 U/mL for IL-4. The curves represent the means
of triplicate cultures and are representative of two independent experiments. In the absenceof DEX, IL-9 and IL-4 do not affect cell
growth, and corresponding curves are similar to the control (data
not shown).
+
+
or without cytokines for 24 or 48 hours before addition of
DEX. Significant inhibition (at least 20%) of DEX-induced
cell death was observed in seven cell lines with IL-9, in six
with IL-4, in three with 1L-7. and in two with IL-2 (Fig 7).
However, reversal of the antiproliferative activity of DEX
by cytokines was not observed in all thymic lymphoma cell
lines where significant inhibition of apoptosis had been detected: in three of the seven cell lines tested, wedidnot
detect significant growth in the presence of DEX with IL-2,
1L-4, IL-7, or IL-9 (Table l ) . The failure of cytokines to
maintain proliferation in the presence of DEX in these cells
contrasted with their strong anti-apoptotic activities observed
with the same cells, indicating that protection against
apoptosis does not automatically correlate with cellproliferation.
To compare the efficiency of IL-9 and IL-2 to induce
proliferation and to protect against apoptosis, we used the
lymphomacell line 9T7, which proliferates autonomously
butwhose growth can still be stimulated by IL-2. In the
absence of DEX, IL-9 stimulates 9T7 proliferation only
poorly, whereas IL-2 induces a 100% increase in thymidine
incorporation. By contrast, in the presence of DEX, IL-9
wasfoundtobemore
efficient than IL-2 to protect cells
against apoptosis (35% U 17% of cytokine-induced protection). Moreover, when IL-9 and IL-2 were combined, additive effects were observed both for proliferation and protection against apoptosis (Fig 8), further suggesting that the
mechanisms involved in the activities of these cytokines are
distinct.
The results presented here show that IL-9 is a potent inhibitor of glucocorticoid-induced apoptosis in thymic
lymphoma cell lines that do not depend on cytokines for
growth. For the BW5147 lymphoma cell line, IL-4 was an
equally potent inhibitor of apoptosis as 1L-9, but with a lower
specific activity. IL-6 also reduced DEX-induced cell death,
but less than the two other factors. Attempts to prevent the
anti-apoptotic activity of IL-9, IL-4, and IL-6 with staurosporin or CsA showed a definite difference: whereas the
anti-apoptotic activity of IL-4 and IL-9 was inhibited by
staurosporin and not by CsA, the converse was true for IL6. The opposite effects of these two reagents suggest that the
anti-apoptotic activity of IL-6 involves different mechanisms
than those triggered by IL-4 and IL-9. Comparison of the
anti-apoptotic properties of various cytokines on different
cell lines indicates that IL-4 and IL-9 give the bestprotection
against DEX. Protection was also observed, butless frequently, with IL-7, while IL-2 was poorly active in this kind
of assay.
The strong inhibition by IL-9 of DEX-induced apoptosis
in thymic lymphoma cell lines contrasts with the absence of
proliferative activity observed with freshly isolated thymocytes and further shows the preferential action of IL-9 on
activated or transformed T cells. Recent observations have
.
..
.
...
5 npllrnl
60
+
m
PIODD:
No Faclor
+
IL-4
+
IL-6
+
IL-9
Fig 6. Effect of staurosporinand CsA on the antiapoptotic activity
of IL-4,11-6. and 11-9. Cells were incubatedfor 24 hours with saturating concentrations ofthe indicated cytokines, as described
in Materials and Methods, with or without DEX (0.25 pmol/L) before evaluation of cellviability by propidium iodide staining of duplicatecultures.
Top panel, normal medium; middle panel, with staurosporin (5 ngl
mL); bottom panel, with CsA (1 pg/mLI. The results are representative of two separate experiments.
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RENAULD ET AL
IO0
loo
1
loo
m
loo
n
50
0
IL2
IL4
IL7
IL9
NM3n
WEHl 22.1
IL2 IL4 IL7 IL9
924A2
IL2 IL4 1L7 IL9
9’17.9f3
IL
IL
I2L47
BR13
IL2 IL4 I L 7 IL9
NM2Tl
IL9
IL7
IL4
IL2
IL9
loo
m1821
IL9
IL7
IL4
IL2
Fig 7. Cytokine-supported resistance t o DEX of various factor-independent and DEX-sensitive thymic lymphoma cell lines. Cell viability
was measured by FACS analysis of 10‘ propidium iodide-stainedcells 24 hours after exposure t o DEX (0.25 pmollL). Cytokine concentrations
were as follows: IL-2, 50 UlmL; IL-4, 250 UlmL; IL-7, 10 nglmL; and IL-9, 500 UlmL. For TH1821, cell viability was measured after 48 hours.
Results are expressed as percent inhibition of DEX-induced death measured in the absence of cytokines and correspond t o the mean of
duplicate samples. Standard deviations were less than 10%. In the absence of cytokines, DEX-induced cell death was 36% for NM3T2, 4146
for WEHl 22.1, 81% for 9T4A2, 60% for 9T7.9Cl. 13.2% for BRl4T2, 45% for TH18.21, 74% for BR13, and 63% for NM2T1. Similar results were
obtained in at least one additional experiment foreach cell line.
indeed shown that resting human T cells are clearly unresponsive to IL-9 but become responsive soon after in vitro
preactivation.’ The fact that thymic progenitors similarly acquire IL-9 responsiveness upon transformation waspreviously reported for freshly isolated tumoral samples.’ Moreover, we found recently that constitutive IL-9 expression in
vivo does not affect the normal thymic development but
results in an abnormally high incidence of thymic lymphomas.‘ The present demonstration that IL-9 is particularly
efficient in the inhibition of apoptosis. in addition toits
ability to stimulate cell growth. provides a potential explanation for these results, as apoptosis is a characteristic of proliferating cells.
Contrary to IL-9, IL-2 exhibits a poor anti-apoptotic activity on the thymic lymphoma cell lines used in this study. A
simple explanation for this observation would be the loss of
IL-2 receptor-a expression by some tumors. Nevertheless.
the levels of protection against DEX provided by IL-2 on
cells that express functional IL-2 receptors are rather week
as compared with those conferred by IL-4 or IL-9. Moreover,
when we usedthe 9T7 lymphoma cellline, where IL-2 receptor-a was expressed andwhose spontaneous proliferation
rate could still be enhanced by IL-2, only minimal protection
against DEX was obtained with this factor. IL-9, on the
contrary. had a negligible stimulatory effect on cell growth
butinhibited apoptosis more clearly. The factthat IL-9
strongly protected several cell lines against apoptosis while
failing tomaintaincell
proliferation further suggests that
distinct regulatory elements are involved in these two processes.
Interestingly, with 9T7 cells, combination of IL-2and
Prnliferalinn
Proleclion against DEX
Table 1. Proliferation of Autonomous Thymic LymphomaCell Lines
in the Presence of DEX and Cytokines
Controll
DEX
No
Cell Line
Factor
NM2Tl
96,504
50,566
NM3T2
213,126
BR13
88,720
TH1821
71,493
9T4A2
10,417
9T7.9c1
BR 1 4T2 36,384
No
Factor
c200
3,943
9,486
185
206
<l00
<l00
IL-2
IL-4
3,572
4,275
11,545
c100
146
<l00
<l00
400
355
13.245
28,429
1,638
289
<l00
<l00
5,014
80,451
<l00
354
<l00
<l00
IL-7
IL-9
2,627
16,943
45,743
2,380
<l00
<l00
<l00
Cells (3,000BR13,9T4A2, and TH1821 cells; 5,0009T7.9C1 cells;
7,000 NMZTl cells: and 30,000 NM3T2 and BR14T2 cells) were incubated in microwells in the presence or absence of DEX and the indicated cytokines for 2 to 3 days before measuring 3H-thymidine incorporation. Results are expressed in cpm and correspond to the mean
of triplicate cultures. Standard deviations were less than 10%.
Fig 8. Comparison of the activities of 11-2 and 11-9 for induction
of proliferationor protection against apoptosis in 9T7 lymphoma cell
line. Left panel shows thymidine incorporation of9 l 7 cells (500 per
well) after a 4-day incubation in control medium or with cytokines
as indicated. Right panel depicts
9T7 cell mortalitymeasured by propidium iodide staining after a 24-hour exposure t o DEX in the presence of the indicatedcytokines. Standard deviations werecalculated
from triplicatecultures for the left panel
and duplicate cultures for the
right panel. Results are representative of two separate experiments.
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ANTI-APOPTOTICACTIVITY OF IL-9
IL-9 showed an additive activity both for proliferation and
protection against apoptosis. In another cell line, proliferation in the presence of DEX required the combination of
IL-2 and IL-9 (data not shown), further demonstrating the
synergistic interaction between IL-2 and E - 9 originally observed for the proliferation of fetal thymocyte^.^ In light of
these results, an attractive explanation for this synergy might
be that IL-2 and IL-9 act preferentially on two distinct and
complementary aspects of cell growth: cell division for IL2 and cell survival for IL-9.
The present study indicates that the putative role of cytokines, and particularly of IL-9, in the development of thymic
lymphomas is not limited to cells that depend on these factors
for proliferation. This possibility enlarges the spectrum of
activities of these molecules with respect to tumors that are
sensitive to inducers of apoptosis in vivo. The importance
of this function has been clearly demonstrated by the finding
that IFN-y, which is constitutively produced in B-cell
chronic lymphocytic leukemia, protects these cells against
apoptosis without stimulating their proliferation.” Further
studies will have to assess the hypothesis of a similar role
for IL-9 in lymphoid malignancies in which an IL-9 autocrine loop has been suggested.8
ACKNOWLEDGMENT
We are grateful to B. de LestrC and A. Tonon for expert technical
assistance and to Dr T. Gajewski for critical reading of the manuscript.
REFERENCES
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Uyttenhove C: Interleukin 9. Adv Immunol 54:75, 1993
2. Uyttenhove C, Simpson R, Van Snick J: Functional and structural characterization of P40, a mouse glycoprotein with T cell
growth factor activity. Proc Natl Acad Sci USA 85:6934, 1988
3. Schmitt E, van Brandwijk R,Van Snick J, Siebold B, Rude
E: TCGFIIUP40 is produced by naive murine CD4+ T cells but is
not a general T cell growth factor. Eur J Immunol 19:2167, 1989
4. Suda T, Murray R, Fischer M, Tokota T, Zlotnik A: Tumor
necrosis factor-a and P40 induce day 15 murine fetal thymocyte
proliferation in combination with IL-2. J Immunol 144:1783, 1990
5. Vink A, Renauld J-C, Warnier G, Van Snick J: Interleukin-9
1305
stimulates in vitro growth of mouse thymic lymphomas. Eur J Immuno1 23:1134, 1993
6. Renauld J-C, van der Lugt N, Vink A, van Roon M, Godfraind
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1995 85: 1300-1305
Interleukin-9 is a major anti-apoptotic factor for thymic lymphomas
JC Renauld, A Vink, J Louahed and J Van Snick
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