Báo cáo Y học: Interferon-alpha inhibits Stat5 DNA-binding in IL-2 stimulated primary T-lymphocytes doc

Interferon-alpha inhibits Stat5 DNA-binding in IL-2 stimulated
primary T-lymphocytes
Sven Erickson
1,
*, Sampsa Matikainen
2,
*, Lena Thyrell
1
, Olle Sangfelt
1
, Ilkka Julkunen
2
,
Stefan Einhorn
1
and Dan Grande
Â
r
1
1
Department of Oncology and Pathology, Cancer Centre Karolinska (CCK), Karolinska Hospital and Institute, Stockholm, Sweden;
2
Department of Virology, Mannerheimintie 166, Helsinki, Finland
It has previously been shown that IFN-a is a potent inhibitor
of IL-2 indu ced proliferation in p rimary T-lymphocytes, by
selectively a brogating t he downstream eects of IL-2 on the
core cell cycle machinery regulating the G1/S transition.
Theoretically this could be mediated through cross-talk
between the signalling cascades activated by these cytokines,
as several signalling components are known to be shared.
IL-2 activates multiple signalling pathways that are impor-

tant for T-cell proliferation and dierentiation. In the pre-
sent study, the eects of IFN-a on IL-2 signal transduction
was investigated. The IFN-a induced inhibition of IL-2
induced proliferation in activated T-lymphocytes, was
associated with a suppressed Jak3 protein expression as well
as an inhibited prolonged Stat5 DNA binding, and a par-
tially reduced expression of the Stat5 inducible gene IL-2Ra.
Our results provide a possible molecular link between the
prominent antiproliferative eects of IFN-a on IL-2 induced
T-cell proliferation and the signal transduction pathways
emerging from the IL-2 receptor.
Keywords: interferon-alpha, T-lymphocytes; Stat5; inter-
leukin-2; proliferation.
Interferons (IFNs) constitute a family of proteins ®rst
isolated because of their antiviral abilities [1]. Today, IFNs
are known to be therapeutically effective in t he treatment of
a number of malignancies [2], viral diseases, as well as in
immunorelated disor ders such a s multiple s clerosis. IFNs
mechanism o f a ction i n t hese diseases is unclear, but its well
known ability to inhibit proliferation has been suggested to
be of major importance. The antiproliferative effect of
IFN-a in malignant cell lines has recently been linked to
potent effects on the basic cell cycle machinery [3±5]. The
investigated cell lines are commonly arrested by IFN in the
G1 phase of the cell cycle, an effect that is coinciding with
the up-regulation of the cyclin-dependent kinase inhibitors
(CKIs), such as p15, p21 and p27 [4,5]. We have recently
shown that IFN-a also inhibits interleukin-2 (IL-2) induced
proliferation in nontransformed human peripheral T-lym-
phocytes, by inhibiting the activation of key cell cycle

molecules [6]. This ®nding has prompted us to investigate
whether this effect may relate to interactions with the
signalling from the IL-2 receptor.
The lymphocyte-derived cytokine IL-2 plays a pivotal
role in the regulation of immune responses, and is a major
regulator of T-lymphocyte proliferation [7,8]. IL-2 signalling
is mediated by a multimeric receptor (IL-2R), consisting of
two obligate signalling subunits, IL-2Rb and c
c
and the
variably expressed IL-2Ra subunit, which regulates the
af®nity for IL-2 [8]. The IL-2 receptor exhibits no intrinsic
kinase activity but rather relies on the association of
intracellular protein kinases for signalling. Ligation of IL-2
to the receptor triggers several signal transduction path-
ways, including the Jak-Stat pathway as well as alternative
pathways leading to the activation of molecules such as
c-myc, and phosphatidylinositol-3-kinase (PtdIns3K) [7,8].
Heterodimerization of the IL-2Rb and c
c
chains results in
the rapid activation of several protein tyrosine kinases
including J ak1, Jak3 and Syk. Syk activation has been
suggested to be of importance for the up-regulation of
c-myc, thereby being crucial f or the proliferative response t o
IL-2 [7,9]. Jak1, however, seems dispensable f or IL-2
induced proliferation [10], whereas in contrast, Jak3 activa-
tion seem to be essential for IL-2 dependent mitogenic
signalling. This conclusion is supported by a number of
manipulative studies. For example, ®broblasts expressing a

reconstituted IL-2R complex will fail to proliferate in the
presence of IL-2 unless Jak3 is coexpressed [11], and
furthermore, an inactive form of Jak3 severely inhibits IL-2
mediated proliferation in BAF3 cells [12]. Receptor associ-
ated Jak3 will phosphorylate speci®c residues in the
cytoplasmatic domains of the IL-2Rb. These phosphotyro-
sine motifs serve a s docking sites for the SH2 domain i n
Stat5, a member of the Stat family of transcription factors.
Upon binding to the receptor complex, Stat5 will become
phosphorylated, dimerize and translocate into the nucleus
inducing transcription of its target genes [8].
Stat5 exists in two different forms, Stat5a a nd Stat5b,
encoded by separate genes. The two genes are highly
Correspondence to D. Grande
Â
r, Department of Oncology and
Pathology, Cancer Centre Karolinska (CCK), Karolinska Hospital
and Institute, S-171 76 Stockholm, Sweden. Fax: + 46 8 339031,
Tel.: + 46 8 51776262, E-mail:
Abbreviations: I FN, interferon; CKI, cyclin-dependent kinase inhibi-
tor; IL, interleukin; IL-2R, interleukin-2 receptor; PtdIns3K, phos-
phatidylinositol-3-kinase; Jak, Janus kinase; Stat, signal transduction
activator of transcription; EMSA, electrophoretic mobility shift assay;
GAS, gamma activation site; MEK, mitogen-activated/extra-cellular
regulated kinase; ERK, extracellular regulated kinase; Cdk, cyclin
dependent kinase; SOCS, suppressor of cytokine signalling.
*Note: the authors contributed equally to this manuscript.
(Received 17 August 2001, revised 18 October 2001, accepted 19
October 2001)
Eur. J. Biochem. 269, 29±37 (2002) Ó FEBS 2002

homologous but differ in the C-terminus region [13]. Stat5 is
expressed in a variety of tissues and its biological effects are
still incompletely understood. Mice with homozygous
inactivation of the Stat5a gene exhibit a failure in postpar-
tum mammary gland differentiation and lactation, demon-
strating that Stat5 i s r equired for mammopoesis and
lactation. The hematopoietic system is not severely affected
in Stat5a±/±,Stat5b±/± or Stat5a±/± Stat5b±/± double
knockout mice [14]. Although Stat5 is apparently not
required for hematopoiesis, it is however, of crucial
importance for the p roliferative response t o IL-2, as
thymocytes fr om Stat5 double knockout m ice fail to
proliferate in response to IL-2 [14,15].
We have recently demonstrated that IFN-a is a potent
inhibitor of IL-2 induced proliferation in PHA stimulated
human peripheral T-lymphocytes [16] where IFN-a abro-
gated the activation of the basic cell cycle machinery as well
as the inhibited the down-regulation of the CKI p27 [16].
The effect on IL-2 signalling was however, selective, as IFN-
a did not inhibit the IL-2 dependent up-regulation o f c-myc
or Cdc25A. In o rder to obtain a better understanding
whether these previously observed e ffects m ay be due to
cross-talk with the more upstream IL-2 signalling, we have
here investigated how IFN-a affects t he activation of IL-2R
associated molecules, and subsequent activation o f IL-2
respon sive genes.
MATERIALS AND METHODS
T-Lymphocyte isolation and cell culture
Buffy coats from healthy blood donors were heparinized,
mononuclear cells isolated by Lymphoprep gradient cen-

trifugation (Nycomed, Oslo, Norway), and T-lymphocytes
isolated using nylon wool columns, as previously described
[17]. In each experiment performed, > 95% of the cells
were viable, as d etermined by t rypan blue exclusion. The
enriched human T-lymphocytes wer e tested for purity
before stimulation as previously described [17] and were
found to be more then 94% pure.
The lymphocytes were seeded at a density of 5 ´ 10
5
cells
per ml in complete MEM (MEM supplemented with 10%
heat inactivated AB
+
serum, 2 m
M
glutamine 50 mgámL
)1
of streptomycin and 50 mgámL
)1
of penicillin) and kept in a
humid incubator w ith 5% CO
2
. The cells were counted and
reseeded every second day (5 ´ 10
5
cells per mL) when fresh
medium was added. The resting T-lymphocytes were
stimulated to proliferation i n a two-stage process by the
sequential addition of 0.8 lgámL
)1

PHA (Sigma Chemical
Co.) for 72 h and, subsequently IL-2 at a concentration of
100 UámL
)1
[17,18]. Restimulation was performed e very
second day by inclusion of IL-2 (100 UámL
)1
)inthefresh
medium added to the cells. I n cultures receiving IFN -a
(5000 UámL
)1
), it was added to the cultures alone or
together with IL-2.
Each experiment was repeated with cells from two to
seven different donors. All ®gures s how the results from
representative experiments.
Cytokines and antibodies
Recombinant IFN-a2b (from Schering-Plough, Kenil-
worth, NJ, USA) was used in the d ifferent experiments.
Recombinant IL-2 was a generous gift form A. O
È
sterborg
(Karolinska Institute, Stockholm, Sweden). The following
antibodies were used for Western blotting and immunopre-
cipitation in this study. The phosphospeci®c polyclonal Akt
antibodies (Ser473 and Thr308) and the polyclonal Akt
antibody were from New England Biolabs, polyclonal J ak3
from Santa Cruz and monoclonal phosphotyrosine anti-
bodies (4G10) from Upst ate Biotechnology (Lake Placid,
NY, USA).

Electrophoretic mobility shift assay (EMSA)
To prepare nuclear extracts, 10 ´ 10
6
cells were harvested
by centrifugation, 600 g,for8minat4°C, and washed
once in 1 .5 mL ice -cold NaCl/P
i
. Subsequently cells were
resusp end ed i n 4 00 lL i ce-cold Buffer A (10 m
M
Hepes/
KOH pH 7.9, 1.5 m
M
MgCl
2
,10m
M
KCl, 0.5 m
M
dith-
iothreitol and Complete
TM
protease inhibitor cocktail
according t o the manufacturers instructions (Boehringer
Mannheim, Mannheim, Germany) b y ¯icking the tube.
Cells were allowed to s well on ice for 10 min, and then
vortexed for 10 s. Samples were centrifuged for 10 s, and the
supernatant discarded. The pellets were resuspended in
100 lL ice-cold Buffer B (20 m
M

Hepes/KOH pH 7.9,
25% glycerol, 420 m
M
NaCl, 1.5 m
M
MgCl
2
,0.2m
M
EDTA, and Complete
TM
protease inhibitor cocktail
according the manufacturers instructions (Boehringer
Mannheim, Mannheim, Germany) and incubated on ice
for high-salt extraction. Cellular debris was removed by
centrifugation for 2 min at 4 °C, the supernatant removed
and saved at ) 70 °C. Protein concentration was determined
spectrophotometrically with the Bradford method, accord-
ing to instructions of the manufacturer (Bio-Rad). Nuclear
protein/DNA binding reactions were performed as previ-
ously described [19]. The following oligonucleotides were
used: IFP 53 GAS (5¢-GATCAATCACCCAGATTCT
CAGAAACACTT- 3¢), IRF-1 GAS (5¢-AGCTTCAG
CCTGATTTCCCCGAAATGACGGA-3¢)andIL-2Ra
GAS-c/GAS-n (5¢-TTTCTTCTAGGAAGTACCAAA
CATTTCTGATAATAGAA-3¢). The probes were
32
P-labelled b y T4 polynucleotide kinase and the b inding
reaction was performed at room temperature for 30 min.
Samples were separated on 6% nondenaturating low-ionic

strength polyacrylamide gels in 0.25 ´ Tris/borate/EDTA.
Subsequently gels were dried and bands visualized by
autoradiography. Antibodies used in supershift experiments
were purchased from Santa Cruz B iotechnology. The
following antibodies were used: anti-Stat1 (sc-245X),
anti-Stat3 (sc-482X), anti-Stat4 ( sc-486X) a nd anti-Stat5
(sc-835). In supershift experiments, antibodies (1 : 20 dilu-
tion) were incubated with the nuclear extracts for 1 h on ice.
Western blot analysis
Western blot a nalysis was performed essentially as previ-
ously described [4]. Brie¯y, whole extracts were prepared by
lysis through sonication in LSLD buffer containing protease
inhibitors. The protein concentration was determined as
described above. Seventy lg of protein was loaded in each
well. Proteins were resolved by SDS/PAGE on 12% gels
and electroblotted to poly(vinylidene di¯uoride) membranes
(Boerhinger Mannheim GMbH, Germany) by semidry
transfer. For protein detection, the ®lters were hybridized
30 S. Erickson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
for 1 h with t he appropriate antibody. Antibody±antigen
interaction was detected b y incubation with horseradish
peroxidase-conjugated anti-(rabbit IgG)Ig or anti-(mouse
IgG)Ig for one hour, and subsequent detection by enhanced
chemiluminescence, ECL (Amhersham).
Immunoprecipitation
For immunoprecipitation whole protein extracts were
prepared as described a bove. Lysates were p re cleared
with protein A±Seph arose beads (Pharmacia Biotech AB,
Uppsala, Sweden) with gentle agitation for at least 2 h at
4 °C. Precleared extracts representing 400 lgofwhole

protein extract were incubated with polyclonal Jak3 anti-
bodies for 2 h at 4 °C. Antibody associated complexes w ere
then bound to prewashed protein A±Sepharose beads,
washed four times i n LSLD buffer c ontaining protease
inhibitors and denatured as previously described [5].
Precipitated proteins were resolved on an 8% SDS/PAGE,
electroblotted and analysed by sequential immunoblotting
with Jak3 and anti-phosphotyrosine Ig.
Northern blot analysis
Total cellular RNA was isolated using Trizol Reagent
according to the instructions of the manufacturer ( Life
Technologies), and the R NA concentration was measured
spectrophotometricly. Twenty micrograms of total RNA
from each sample was subjected to electrophoresis in a 1.2%
agarose formaldehyde gel. Blotting was performed as
described [4]. Northern ®lters (Hybond C-extra, Amersham)
were hybridized with the cDNA probes encoding IL-2Ra
and pim-1, and probes were labelled a s p reviously described
[4]. EtBr staining of rRNA bands was used to ensure equal
RNA loading.
Quanti®cation of apoptosis using Annexin V staining
Redistribution of plasma membrane phosphatidylserine is a
marker of apoptosis and was assessed using Annexin V
FLUOS and Propidium Iodide staining kit according
to the manufacturer's protocol (Boehringer Mannheim,
Mannheim Germany).
Quantitation of band intensity
For s canning of the ®lms a Luminescent image analyzer
LAS-1000 plus (Fuji Film Co., Ltd) was used. Hybridiza-
tion signals w ere quanti®ed by using I

MAGE
G
AUGE
Ver.
3.12 by Fuji Film Co., Ltd.
RESULTS
IFN-a inhibits IL-2 dependent up-regulation of Jak3,
but not its transient phosphorylation
Puri®ed primary T-lymphocytes from healthy donors, were
treated with P HA for three days, a nd subsequently with
IL-2 alone or IL-2 together with IFN for up to 72 h. Using
the stimulation protocol described i n the materials and
methods section, the initial PHA s timulation provides an
activation signal, but alone, is clearly not suf®cient to induce
proliferation in this setting, in agreement with previous data
from Firpo et al. [18] and subsequent data from our own
group [16]. Exogenous IL-2 addition allows for S-phase
entry and activation the G1 Cdk complexes [16,18]. As
described elsewhere, cotreatment with IFN- a abrogated the
proliferative response to IL-2 a nd severely inhibited t he
activation of the cell cycle machinery [16]. The effect of IFN-
a on the proportion of apoptotic cells was also measured by
¯ow cytometry for DNA content and AnnexinV positivity.
There were no signi®cant differences in the proportion of
apoptotic cells in IL-2 treated or IFN-a plus IL-2 treated
cells up to 48 h of culture as measured by sub-G1 content
(data not shown) or Annexin V staining (Fig. 1).
Recent studies have clearly demonstrated that Jak3 is
required t o activate the IL-2R pathway for T-cell prolifer-
ation. Jak3 is rapidly phosphorylated upon IL-2 stim ula-

tion, an event essential for a proper p roliferative signal
[8,20,21]. In order to investigate if IFN-a affects the IL-2
induced expression of Jak3, Western blotting was per-
formed. IL-2 stimulation for 24 h (day 4) increased t he Jak3
expression signi®cantly from the low levels present in
unstimulated cells, whereas prolonged stimulation did not
lead to any further increase in Jak3 protein levels (Fig. 2A).
IFN-a suppressed the IL-2 dependent Jak3 expression in
cells treated for more than 24 h (Fig. 2A).
Because low levels of Jak3 are present in the cells before
IL-2 stimulation we analysed whether IFN a ffected the
immediate Jak3 tyrosine phosphorylation upon IL-2 addi-
tion. PHA treated T-lymphocytes were treated with I L-2
alone or together with IFN-a for 10 and 30 min, w hereafter
Jak3 was immunoprecipitated and tyrosine phosphoryla-
tion detected by Western blotting. In contrast to the
abrogation of the IL-2 dependent up-regulation of Jak3,
IFN-a did not inhibit the rapid tyrosine phosphorylation at
any of the timepoints investigated (Fig. 2B).
IFN-a effects on Stat5 DNA-binding
Although the role of Stat5 in T-lymphocyte proliferation is
still somewhat controversial, recent s tudies indicate that
Stat5 is indeed required for a proper proliferative response
to IL-2 [15]. T o characterize the effect of IFN-a in the onset
of IL-2 signalling and Stat5 b inding, PHA activated cells
were treated with IL-2 alone, IFN-a alone or IL-2 together
with IFN-a. Nuclear extracts w ere prepared from these cells
and analysed by EMSA, by using a labelled IFP-53 GAS
Fig. 1. Eects of IFN-a on apoptosis as measured by annexin V and
propidium iodide staining. PHA activated T-cells were treated with IL-2

alone or IL-2 together with IFN-a for 48 h. The percentage o f ap op-
totic (annexin V positive) cells in th e samples is shown. Annexin V
staining is represented on the x-axis and propidium iodide staining is
representedonthey-axis.
Ó FEBS 2002 Interferon-a inhibits Stat5 induced transcription (Eur. J. Biochem. 269)31
DNA-element that i s known to bind Stat5 with high af®nity
[22]. EMSA a nalysis revealed that untreated and PHA
treated T-cells contained no DNA binding activity to this
element. Stimulation of the cells with exogenous IL-2 was
repeatedly found to induce a biphasic IFP-53 GAS binding
activity, with the appearance of two clear bandshifts already
0.5 h after stimulation (Fig. 3A). The shifts gradually
decreased at 6±24 h, with a reappearance of the strong shift
at 24±48 h (Fig. 3A). Co-treatment with IFN-a did not
signi®cantly alter the initial wave of IFP-53 GAS binding
activity, whereas it severely inhibited t he reappearance of
bandshifts during the second wave of IL-2 induced binding
at 24±48 h (Fig. 3A). Furthermore, IFN-a cotreatment also
caused the appearance of a n ovel bandshift in-between the
two IL-2 induced bands (Fig. 3A). The composition of the
DNA-binding complexes from T-cells stimulated with IL-2
and/or IFN-a for 0.5 and 48 h, as analysed by supershifts
with speci®c antibodies. The IL-2 induced complexes were
intact following incubation with Stat1 antibodies, while
antibodies recognizing both Stat5a and b shifted the entire
complex ( Fig. 3B). Antibodies for Stat5a and S tat5b,
respectively, partly shifted the complex (data not shown).
In the IFN-a cotreated sample, the additional intermediate
band was c ompletely shifted using antibodies recognizing
Stat1. In other studies, Stat1 has sometimes been found to

form a slightly faster moving complex than S tat5. T he
reason for t his discrepancy is probably due to the f ormation
of Stat5 complexes containing different Stat5 isoforms
depending on celltype and type of stimulation [23,24].
Again, cotreatment with I FN-a for 48 h resulted in a
signi®cantly decreased Stat5 bandshift as well as supershift.
Thus, IFN-a clearly inhibited the binding of Stat5a/b to the
IFP53 element at this timepoint. Total levels of Stat5 protein
on the other hand, were not changed by cotreatment with
IFN-a, as demonstrated by Western blotting for this protein
(data not shown).
Recently an IL-2 responsive element in the IL-2Ra
promoter has been described. T his r egion c ontains a
consensus and a nonconcensus GAS motif (GAS-c/GAS-n),
which has been shown to be e ssential f or proper IL-2
induced IL-2Ra transcription [25]. IL-2 stimulated prolifer-
ating T-lymphocytes have also recently been shown to
contain S tat1, 3, 4 and 5 DNA binding activity to this GAS
element upon costimulation with IFN-a [26]. T o further
characterize the effect of IFN-a on the binding activity also
to this element, further EMSAs were performed using this
GAS motif (GAS-c/GAS-n) as a probe. IL-2 alone induced
one major complex already a fter 0.5 h. IFN-a treated cells
also expressed one single complex, albeit with a broader
migration pattern, i mplicating the comigrat ion of t wo
separate bandshifts. Co-treated cells also expressed both of
these complexes 0.5 h after stimulation (Fig. 4A). At later
timepoints ( 6 h and thereafter), c omplex formation was
signi®cantly decreased in IL-2 treated and cotreated cells,
and only the slowly migrating form was detectable. IFN-a

treated cells alone co ntained n o c omplexes at these later
timepoints (Fig. 4A). Furthermore, as observed using IFP-
53 GAS as a probe, IFN-a clearly i nhibited the complex
formationcomparedtoIL-2treatedcellsaloneatthetwo
latest timepoints (24 and 48 h). To ver ify the c omplex
composition, supershifts with a set of different Stat anti-
bodies were performed. In the I L-2 t reated cells Stat5 w as the
only antibody generating a supershift shifting the majority of
the complex (Fig. 4B). In the extracts from IL-2 /IFN-a
cotreated cells, i ncubation with Stat1, Stat3, Stat4 an d Stat5
antibodies all resulted in supershifts (Fig. 4B), however, the
Stat1 antibody generated the most prominent shift. In
extracts fr om IFN-a treated cells alone Stat1 shifted the
absolute majority of the complex, however, the complexes
also contain ed very small amounts of Stat3 and 4 binding
(Fig. 4B). Taken together these data c on®rm the ®nd in gs
using the IFP53 GAS element, that is, IFN-a cotreatment
inhibited the prolonged IL-2 dependent Stat5 DNA binding
activity but not the immediate one. Furthermore, identical
data on the effects of IFN- a on Stat5 a ctivity w ere a lso
obtained using an IRF-1 GAS element (data not shown).
Effects of IFN-a on Stat5-induced transcription
IL-2 induced T-cell proliferation is associated with the
induction of several IL-2 responsive genes such as IL-2Ra,
pim-1 and c-myc . IL-2Ra and pim-1 expression have also
been shown to b e regulated by Stat5 [26,27]. We have
previously shown that the IL-2 dependent c-myc
up-regulation in T-cells is not abrogated by cotreatment
with IFN-a, but rather slightly enhanced [16]. To analyse
whether the inhibitory effect of IFN-a on IL-2 induced

Stat5 DNA binding correlated with an altered expressio n of
Stat5 inducible genes, Northern blotting and hybridization
using IL-2R-a and pim-1 as probes was performed.
Untreated cells did not show IL-2Ra or pim-1 expression,
Jak3
Jak3
W:Jak3
W:P-Tyr
IFN-α
MinutesB
–
10
+– +
30
IFN-α
HoursA
–
0
+ +––
24 48
Fig. 2. Ee cts of IFN-a on IL-2 induced Jak3 protein expression and
phosphorylation. (A)ImmunoblotanalysisofJak3expressionwas
analysed in quiescent (day 0) or PHA activated T-cells treated w ith
IL-2 or IL-2/IF N-a for 24 or 48 h. (B) PHA activated T-cells were
treated with IL-2 alone or IL-2 together with IFN-a for 10 and 30 min
Whole protein extracts were immunoprecipitated with polyclonal Jak3
antibodies followed by sequential immunob lotting with anti-phosp-
hotyrosine and anti-Jak3 Ig.
32 S. Erickson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
however, PHA treatment for 3 days induced expression of

both these genes. IL-2 stimulation further incr eased the
expression of both genes reaching a plateau between 6 and
24 h (Fig. 5). IFN-a did not inhibit the early IL-2 (2 and
6 h) induced expression and at these timepoints, IFN- a
treated cells alone maintained some expression of both
genes. T-cells stimulated with IL-2 for 24 and 48 h expressed
continued high levels of IL-2R-a and pim-1.IFN-a
cotreated cells exhibited partially reduced expression levels
of IL-2R-a compared to IL-2 treated c ells alone (Fig. 5),
with a 30±40% reduction at 24 h a nd around 20%
reduction at 48 h, as measured by scanning densitometry.
Stat 5
Stat 1
Stat 5
Antibody
α–Stat1

α–Stat5
α–Stat5
α–Stat1
α–Stat5
α–Stat1
α–Stat5
α–Stat1
α–Stat5
α–Stat1
IFNα 0.5h
IL-2/IFNα 0.5h
IL-2 0.5h
PHA

0
PHA
0
IL-2 48h
IL-2/IFNα 48h
B
0.50 PHA 6 24 48
Stat 5
Stat 1
Stat 5
Hours
IL-2
IL-2/IFNα
IFNα
IL-2
IL-2/IFNα
IFNα
IL-2
IL-2/IFNα
IFNα
IL-2
IL-2/IFNα
IFNα
A
Fig. 3. Stat5 b inding to the IF P-53 GAS element. (A) Nuclear extracts fro m qu iescent ( day 0) o r P HA activated T -cells treated w ith, IL -2 (0.5±48 h),
IL-2/IFN-a (0.5±48 h) and IFN-a (0.5±48 h) treated T-cells were p repared and incubated with a
32
P-labelled IFP-53 GAS element. DNA binding
activity was analysed by EMSA. (B) Nuclear extracts from q uiescent (day 0), PHA ( day3), IL-2 (0.5 a nd 48 h), IL-2/IFN-a (0 .5 and 48 h) and IFN-
a (0.5 h) treated T-cells were prepared and incubated for one hour with Stat1 and Stat5 antibodies, f ollowed by binding to a

32
P-labelled IFP-53
GAS element. DNA binding a ctivity was analysed by EMSA.
Ó FEBS 2002 Interferon-a inhibits Stat5 induced transcription (Eur. J. Biochem. 269)33
The effects of IFN-a on pim-1 expression on the o ther hand
were minor. IFN-a treated cells alone did not express either
of th e g enes after 24 h of treatment. The effects of
cotreatment with IFN-a on IL-2 induced Stat5 DNA-
binding activity thus partially seem to translate into effects
on Stat5 dependent transcription of the IL-2Ra gene.
Effects of IFN-a on the PtdIns3K pathway
Recently, the PtdIns3K pathway has been linked to the
activation of the basic cell cycle machinery in primary
T-lymphocytes [28]. IL-2 indu ced activation of this pathway
leads to down-regulation of the CKI p27, an d up-regulation
of D-type cyclins as well as activation of E2F driven
transcription. Furthermore, IL-2 induced PtdIns3K activa-
tion involves phosphorylation of Akt (protein kinase B). In
order to ®nd out whether IFN-a inhibits the activation o f
the PtdIns3K pathway, Akt phosphorylation in IL-2
stimulated T-cells was analysed by immunoblotting. Anti-
bodies recognizing serine 473 phosphorylated Akt revealed
that unstimulated T-cells expressed detectable levels of
phosphorylated Akt. IL-2 stimulation only s lightly
increased the exp ression and I FN-a did not inhibit t he
phosphorylation at any timepoint (d ata not shown). Anti-
bodies recognizing threonine 308 phosphorylated Akt gave
an identical result (data not shown).
DISCUSSION
Induction of proliferation in human primary T-lymphocytes

depends on cytokine stimulation both in vivo and in vitro,
and IL-2 is a central molecule in this response. However,
also IL-4, IL-7, and IL-15 all promote T-cell proliferation in
a m anner similar to IL-2 [8]. These cytokines share the ccin
the receptors as well as intracellular s ignalling molecules
such as the Jak and S tat molecules. Type I interferons signal
through a similar set of Jak/Stat molecules but with
completely different cellular effects. IFN-a is a well-estab-
lished antiproliferative c ytokine, both in malignant and
nonmalignant cells. As mentioned a bove, we have recently
shown that IFN-a inhibits IL-2 induced proliferation in
PHA-activated T-lymphocytes, by abrogating the activation
of the cell cycle mac hinery [16]. The present study investi-
gates whether this effect may be mediated by cross-talk
between the signalling molecules in the IL-2 and IFN-a
pathways. We found that IFN-a did not affect the
immediate IL-2 mediated phosphorylation of Jak3, the
immediate Stat5 DNA binding or the early induction of
Stat5 induced genes. However, Jak3 protein expression was
severely inhibited by IFN-a after 2 4 h of exposure as well as
the prolonged Stat5 DNA-binding. Furthermore, IFN-a
partially inhibited the prolonged expression of the Stat5
respons ive gene, IL-2Ra, although the reduction was not as
prominent as the effect of IFN-a on Stat5 DNA-binding.
The reason f or the discrepancy between the effects o f IFN-a
on Stat5 DNA binding and the steady state mRNA levels of
Stat5 responsive genes, is unclear at present. It is most likely
due to regulation of such genes also by other transcription
factors than the ones investigated in the presen t study. This
is also supported by t he fact that low mRNA levels of these

genes appear well before there is any measurable Stat5
DNA binding activity in the cells (Figs 3,4 and 5).
The links between IL-2 signalling and activation of the
cell cycle in T-cells are poorly understood. Activation of the
PtdIns3K pathway in the human IL-2 dependent T-cell line,
Kit225, induces CyclinD3 expression and down-regulation
of p27, resulting in pRb phosphorylation and E2F driven
transcription [28]. In this s ystem the Stat5 and MEK/ERK2
48
Stat 5
Stat 1
Hours 2 4630.50 PHA
IFNα
IL-2/IFNα
IL-2
IFNα
IL-2/IFNα
IL-2
IFNα
IL-2/IFNα
IL-2
IFNα
IL-2/IFNα
IL-2
IFNα
IL-2/IFNα
IL-2
Stat 5
Stat 1
Antibody

-
α–Stat5
IL-2/IFNα 0.5h

α–Stat4
α–Stat3
α–Stat1
-
α–Stat5
α–Stat4
α–Stat3
α–Stat1
-
α–Stat5
α–Stat4
α–Stat3
α–Stat1
IFNα 0.5hIL-2 0.5h0 PHA
A
B
Fig. 4. Stat binding to IL-2Ra GAS-c/GAS-n.
(A) Nuclear extracts from quiescent (day 0) or
DHA activated T-cells treated with, IL-2
(0.5±48 h), IL-2/IFN-a (0.5±48 h) and IFN-a
(0.5±48 h) were prepared and i ncubated with a
P
32
IL-2Ra GAS-c/GAS-n labelled GAS ele-
ment. DNA binding activity was analysed by
EMSA. ( B) Nuclear extrac ts from quiescent

(day 0), IL-2 (0.5 h), IL-2/IFN-a (0.5 h) an d
IFN-a (0.5 h) -treated T-cells were prepared
and incubated for one hour with Stat1, Stat3,
Stat4 and Stat5 antibodies, followed by bind-
ing to a P
32
IL-2Ra GAS-c/GAS-n labelled
GAS element. DNA binding activity was
analysed by EMSA.
34 S. Erickson et al. (Eur. J. Biochem. 269) Ó FEBS 2002
pathways seem dispensable for cell cycle activation. How-
ever, peripheral T-lymphocytes from Stat5 double-knock-
out mice (Stat5a
±/±
,Stat5b
±/±
) a re profoundly d e®cient in
their ability to proliferate in response to IL-2, whereas this is
not the case for the Stat5a and Stat5b single knockouts [15].
Also, t he incapability of the Stat5a/b de®cient peripheral
T-cells to respond to IL-2 stimulation was not to be rescued
by supraphysiological concentrations of IL-2, which would
bypass the requirement for the expression of IL-2Ra,
suggesting a d irect role of Stat5 for IL-2 induced cell cycle
progression of peripheral T-lymphocytes [15].
The ®nding that IFN-a inhibited the IL-2 induced
expression of Jak3 protein levels and prolonged Stat5
DNA-binding and t ranscription in T-cells may b e of
importance in contributing to the antiproliferative pheno-
type and i nability t o activate t he cell cycle machinery in

these cells. Stat5a/b de®cient T-cells exhibited extensive
defects i n IL-2 induced cell cycle activation, with impaired
induction of CyclinD2, CyclinD3, CyclinE, Cyclin A and
Cdk6. However, downregulation of p27 and induction of
Cdk2 was not affected in the Stat5a/b de®cient mice [15].
Addition of IFN- a together with IL-2 to PHA- activated
T-cells seems to result in a broader effect on the cell cycle
machinery with inhibited induction of CyclinD3, CyclinE,
Cdk2 and Cdk6 as well as an abrogated down-regulation of
p27 [ 16]. It is therefore likely that IFN-a inhibits several
pathways downstream of the IL-2 receptor to generate this
prominent effect. Activation of the PtdIns3K pathway
results in Akt (protein kinase B) phosphorylation and, as
discussed above, a partial activation of the cell cycle
machinery. As IFN-a has b een demonstrated to regulate
this pathway in some systems [29], we analysed whether IFN
affected the IL-2 dependent phosphorylation of Akt.
Quiescent T-cells exhibited quite a clear level of Akt-phos-
phorylation and IL-2 only slightly enhanced this phospho-
rylation. We could not detect any clear differences in
IL-2Rα
Hours
IL-2
IL-2/IFN-α
IFN-α
IL-2
IL-2/IFN-α
IFN-α
IL-2
IL-2/IFN-α

IFN-α
IL-2
IL-2/IFN-α
IFN-α
18S rRNA
28S rRNA
0 PHA 2 6 24 48
Pim-1
Fig. 5. IFN-a eects on IL-2 induced IL-2Ra and pim-1 expression. Analysis of IL-2Ra (A) and pim-1 (B) regulation in quiescent (day 0), and PHA
(day 3), IL-2 (2±48 h), IL-2/IFN-a (2±48 h) and IFN-a (2±48 h) -treated cells by Northern blot analysis. 18S and 28S rRNA serves as a control for
equal loadin g.
Ó FEBS 2002 Interferon-a inhibits Stat5 induced transcription (Eur. J. Biochem. 269)35
phosphorylation levels in the IFN cotreated cells. Based on
these experiments it is unlikely that IFN inhibits cell cycle
activation by inhibiting the PtdIns3K pathway, however,
additional experiments need to be performed to characterize
this pathway greater in detail.
One possible explanation for the almost complete inhi-
bition of the IL-2 induced proliferation could b e that IFN-a
inhibited the formation of a functional IL-2 receptor.
However, based on our ®ndings this seems unlikely. First,
IFN-a did not inhibit the immediate IL-2 dependent Stat5
DNA binding or Jak3 phosphorylation. Secondly, we have
previously shown that the induction of the oncogene c-myc
is not inhibited by cotreatment with IFN-a,ratherIFN
seemed to synergize with IL-2 to enhance c-myc expression
[16]. Taken together these d ata strongly indicate that the
IL-2 receptor is also functional in the presence of IFN-a.
In this study, we repeatedly found a biphasic activation of
Stat5 in response to IL-2 (Figs 3A and 4A). One explanation

for the decrease in Stat5 DNA binding between 3 and 24 h
could be the expression of SOCS (suppressors of cytokine
signalling). SOCS-3 h as be en reporte d t o b e r apidly and
transiently induced in T-lymphocytes following IL-2 stim-
ulation, inhibiting Stat5 phosphorylation by interacting with
Jak1 and IL-2Rb [30].Furthermore,IFN-a was also found
to induce SOCS-3 [31]. One possible explanation for the low
levels of Stat5 DNA binding in the IFN-a cotreated cells
could thus be that SOCS-3 levels do not return to basal levels
and by that inhibit further Stat5 phosphorylation. Another
possibility is that the suppression of J ak3 expression
(Fig. 1A) in the IFN treated cells affects the phosphorylation
of the IL-2Rb chain and thereby inhibits the Stat5 activity.
It has been noted that activation of Stats may a lso
contribute to the pathogenesis of human leukaemia.
Constitutive Stat activation has been reported in cell lines
derived from patients with acute myelogenous leukaemia,
acute lymphoblastic leukaemia, chronic myelogenous
leukaemia and T -cell lymphoma [32±35]. S everal of these
malignancies respond favourably to IFN treatment, an
effect that could theoretically be mediated via an effect on
Stat5 activation. A further elucidation of the interaction
between IFN-signalling a nd Stat5 a ctivation in such cells
may thus also have implications for the understanding of
IFNs antitumor activity in these diseases.
ACKNOWLEDGEMENTS
We are grateful for the excellent technical a ssistance o f M s Elisab et
Anderbring, Ann-Charlotte Bjo
È
rklund, Marika Ylisela

È
and Teija
Westerlund. Dr Martin Corcoran is thanked for careful reading of the
manuscript. This work has been supported by the Swedish Cancer
Society, the Cancer Socie ty of Stockholm, King Gustaf Vth Jubilee
Fund, Alex and Eva Wallstro
È
ms Foundation Medical Research Council
of the Academy of Finland, the Technology Development Center of
Finland, the S igrid Juselius Foundation and t he Finnish Cancer
Foundations.
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