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INTRODUCTION |
UV radiation and, in particular, UVB with a wavelength range
between 290 and 320 nm, represents one of, if not the most important environmental danger to human health. Its hazardous effects include the
induction of skin cancer (1), suppression of the immune system (2), and
chronic skin damage e.g. premature skin aging (3). The effects of UV on the cellular level include the induction of
apoptotic cell death (4), the induction of inflammatory processes via
the release of inflammatory cytokines (5), and the inhibition of
cellular immune responses (2). In particular, the immunosuppressive
properties of UV are of major biological relevance, because suppression
of the immune system by UV is not only responsible for the exacerbation
of infectious diseases following UV exposure,
e.g. herpes simplex (6), but also contributes to
the induction of skin cancer (7). Hence, understanding of the
mechanisms by which UV suppresses the immune system is of primary
importance. UV suppresses the immune system in multiple ways (2). Just
to name a few, it inhibits antigen presentation by down-regulating the
surface expression of accessory molecules (8), it induces the
generation of T suppressor cells, which inhibit antigen-specific immune
responses (9), and it induces the release of immunosuppressive
cytokines e.g. interleukin
(IL)1-10, tumor necrosis
factor
, and transforming growth factor
(reviewed in Refs. 10
and 11).
Recently, we obtained the first evidence that UV does not only have the
ability to influence the release of cytokines but that it can also
interfere with the biological activity of immunomodulatory mediators.
Specifically, we demonstrated that UV is able to hinder the
immunomodulatory cytokine interferon-
(IFN
) from exerting its
biological effects (12). As a consequence of the interaction of IFN
with its receptor, the signal transducer and activator of transcription
protein STAT1 (13, 14) becomes tyrosine-phosphorylated. Phosphorylated
STAT1 dimerizes, translocates to the nucleus where it binds to the
IFN
-activated sequences (GAS) in various promoters, and ultimately
induces specific gene transcription. We observed that UV inhibits
IFN
-induced STAT1 phosphorylation and consequently STAT1 binding
capacities (15). Because phosphorylation of STAT1 is a critical event
in IFN
signaling, UV inhibits IFN
from exerting its biological
effects via this mechanism. Interestingly, UV had no effect on the
phosphorylation of STAT3, which is involved in signaling of IL-6, a
potent proinflammatory cytokine whose release is also induced by UV
(16). Because IL-6 is an inflammatory mediator and IFN
is an
immunomodulatory molecule, we postulated that the differential effects
on cytokine signaling by UV may explain the diverse biological effects
of UV, which on the one hand causes inflammation (via induction of the
release of inflammatory cytokines) but, on the other hand, inhibits
immune reactions (presumably by interruption of the signal transduction
of immunomodulatory cytokines). If this hypothesis holds true, the
inhibitory effect of UV should not only affect IFN
signaling but
also other immunostimulatory cytokines. Hence, we were interested in
finding out whether UV also interferes with IL-2 signaling.
IL-2 is a key regulator of normal immune function and acts on a variety
of lymphoid cells including T lymphocytes, B lymphocytes, and natural
killer cells (17, 18). Blockade of IL-2 or the IL-2 receptor (IL-2R)
results in pronounced impairment of antigen-specific proliferative T
cell responses (17, 18). To exert its biological effects, IL-2 must
interact with the specific IL-2 receptor, which consists of at least
two subunits. Heterodimerization of the constitutively expressed
IL-2R
chain and the common cytokine receptor
chain (
c) forms an intermediate affinity receptor for
IL-2-mediated signaling. Although the IL-2R
chain is not necessarily
required for IL-2 binding or signaling, it is essential for forming
high affinity receptors (19, 20). IL-2R
expression is tightly regulated by the extracellular binding of IL-2 to the intermediate affinity receptor (21), resulting in intracellular phosphorylation of
the activated
chain or
c. Recruitment of the janus
kinases Jak1 to IL-2R
and Jak3 to
c results in
tyrosine phosphorylation, which in turn triggers downstream tyrosine
phosphorylation of STAT5 proteins (22, 23). Upon phosphorylation, the
STAT5 molecules dimerize and translocate to the nucleus where they
serve as transcription factors for their responsive elements. Positive
regulatory elements responsive for STAT5 binding are located in the
IL-2R
chain gene promotor region and serve as enhancers of IL-2R
gene expression (24, 25).
Here, we demonstrate that UV inhibits IL-2-mediated tyrosine
phosphorylation of STAT5 and consequently inhibits STAT5 binding activity. In contrast, phosphorylation of the upstream-located kinases
Jak1 and Jak3 was not affected by UV. As a consequence of impaired
STAT5 phosphorylation, IL-2-mediated up-regulation of the IL-2R
chain was reduced in T cells exposed to UV at both the mRNA and the
protein level. Hence, UV may inhibit immune responses by interfering
with the signaling of IL-2. Together, these data indicate that
interference of UV with the signal transduction of
immunostimulatory cytokines represents an additional pathway by
which UV compromises the immune system.
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EXPERIMENTAL PROCEDURES |
Cells and Reagents--
The murine T cell line CTLL
(ATCC, Manassas, VA) was cultured in RPMI 1640 supplemented with
10% fetal calf serum, 5 × 10
5 M
-mercaptoethanol and IL-2. Supernatants obtained from concanavalin A-stimulated rat splenocytes were used as a source for IL-2 at a final
concentration of 10% (26). IL-2 containing supernatant was added to
the culture medium every 24 h. IL-2 (100 units/ml) supplementation
was stopped 60 h before experiments were performed. For
stimulation, recombinant murine IL-2 was used (R & D Systems Inc.,
Minneapolis, MN). The tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) and the serine
phosphatase inhibitor ocadaic acid were purchased from Sigma.
UV Irradiation of Cells--
UV irradiation was performed as
described previously with slight modifications (5). Briefly, cells
(1 × 106/ml) were washed with phosphate-buffered
saline and exposed to UV radiation through colorless medium without
fetal calf serum. For UV irradiation a bank of six fluorescent bulbs
(TL12; Philips, Eindhoven, The Netherlands) was used that emit most of
their energy within the UVB range (290-320 nm) with an emission peak
at 313 nm. Throughout this study a dose of 400 J/m2 was
used. This dose was used, because it is in the range of the physiologically relevant doses usually used for in vitro
studies when investigating UVB (27). Control cells were subjected to the identical procedure without being exposed to UV.
Electrophoretic Mobility Shift Assay (EMSA)--
15 min after
stimulation, nuclear proteins were extracted as described previously
(12). Binding reactions were carried out in the presence of 2 µg of
poly(dI-dC) (Roche Molecular Biochemicals), 104 cpm
of 32P-labeled double stranded oligonucleotide with a
consensus binding site for STAT5 (sc-2565; Santa Cruz Biotechnology,
Santa Cruz, CA), and 20 µg of nuclear protein extracts for 20 min at
22 °C. Reaction samples were separated electrophoretically on native high ionic gels at 150 V for 1.5 h and detected by
autoradiography. Competition analysis was performed by the addition of
the unlabeled consensus oligonucleotide in a 50-fold molar
excess. Supershifts were carried out utilizing specific antibodies
directed against either STAT5a (sc-108X) or STAT5b (sc-835X; both Santa Cruz).
Immunoprecipitation and Western Blot Analysis--
15 min after
stimulation cells were lysed in lysis buffer (50 mM Hepes,
pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM pyrophosphate, 0.01% NaN3, and CompleteTM protease inhibitor
mixture) by sonication. After centrifugation, supernatants were
collected, and the protein content was measured using a Bio-Rad protein
assay kit (Bio-Rad, Hercules, CA). Immunoprecipitations were carried
out with 1 ml of a 1 mg/ml protein solution supplemented with 5 µg of
the corresponding antibody directed against Jak1 (AF602; R & D
Systems), Jak3 (05-406; Upstate Biotechnology, Lake Placid, NY), or
STAT5 (PA-ST5A; R & D Systems) and with 50 µl of protein A/G-agarose
(Santa Cruz) at 4 °C overnight. Samples were washed 3 times in lysis
buffer with centrifugation being performed at 13,000 rpm for 5 s.
Precipitated proteins were released from protein A/G-agarose beads at
95 °C in sample buffer containing 5%
-mercaptoethanol. The
protein samples were subjected to 6% SDS polyacrylamide gel
electrophoresis, blotted onto nitrocellulose membranes, and incubated
with an antibody directed against phosphotyrosine (sc-7020; Santa
Cruz). Membranes were stripped and reprobed with different antibodies
detecting Jak1 (sc-1677; Santa Cruz), Jak3 (06-667, Upstate
Biotechnology), or STAT5 (sc-1656, Santa Cruz). Signals were visualized
with an ECLTM kit (Amersham Pharmacia Biotech).
Semiquantitative Reverse Transcription Polymerase Chain Reaction
(PCR)--
2 h after stimulation total RNA was extracted from cells
according to the protocol described by Chomczynski and Sacchi
(28). 1 µg of total RNA was reverse transcribed with SuperScript
RNase H reverse transcriptase (Life Technologies, Inc.). The amount of
template needed was titrated by
-actin PCR in a 20-µl reaction utilizing the RedTaq polymerase system from Sigma and evaluated densitometrically. A murine IL-2 receptor
-amplimer set from CLONTECH (Palo Alto, CA) was used as primers for
the amplification of the IL-2 receptor
chain.
Flow Cytometry--
Aliquots of 2 × 105 cells
were incubated with fluorescein isothiocyanate-conjugated rat
anti-mouse antibodies directed against either the IL-2 receptor
-chain (CD25) or the IL-2 receptor
-chain (CD122; both from
Southern Biotechnology Associates Inc., Birmingham, AL) for 45 min on
ice. Purified rat IgG was used as an isotype control. Cells were then
washed twice in phosphate-buffered saline, propidium iodide (100 µM) was added, and cells were analyzed in a flow
cytometer (Epics XL; Coulter, Miami, FL).
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RESULTS |
UV Interferes with Phosphorylation of STAT5--
To address the
question whether UV interferes with the phosphorylation of STAT5,
proteins from lysates of CTLL cells were immunoprecipitated with an
antibody against STAT5, and Western blot analysis was performed using
antibodies against phosphotyrosine and STAT5, respectively (Fig.
1). In untreated CTLL cells (lane 1) STAT5 was barely phosphorylated if at all, whereas stimulation with IL-2 induced a strong phosphorylation of STAT5 (lane
2). However, in cells that, in addition to IL-2, were also exposed to UV, IL-2-induced STAT5 phosphorylation was remarkably reduced (lane 4). Exposure of CTLL cells to UV alone did not affect
the phosphorylation status of STAT5 (lane 3).

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Fig. 1.
UV radiation inhibits IL-2-induced tyrosine
phosphorylation of STAT5. A, CTLL cells were left
untreated (lane 1), stimulated with 100 units/ml IL-2
(lane 2), irradiated with 400 J/m2 UV
(lane 3), or treated with UV plus IL-2 (lane 4).
Samples in lanes 5 and 6 were preincubated with 1 mM Na3VO4 for 2 h before
stimulation with IL-2 (lane 5) or with UV plus IL-2
(lane 6). 15 min after stimulation immunoprecipitations were
performed using an antibody directed against STAT5. Western blot
analyses were performed using an antibody against phosphotyrosine
(P-Tyr), followed by reprobing of the membranes with an
antibody directed against STAT5. Experiments were performed three
times. One representative experiment is shown. B, the
phosphotyrosine blots were analyzed densitometrically. The
bars shown represent the mean density ± S.D. of three
independently performed experiments.
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Reduced STAT5 Binding Activity in UV-exposed CTLL Cells--
To
determine whether inhibition of phosphorylation of STAT5 by UV is
functionally relevant, EMSAs were performed using nuclear protein
extracts from CTLL cells that were exposed to IL-2 alone or to IL-2
plus UV (Fig. 2). Low constitutive
binding activity was observed in untreated cells (lane 1),
and this activity was considerably induced upon stimulation of cells
with IL-2 (lane 2). UV treatment did not alter the low
constitutive binding activity (lane 3) but significantly
reduced IL-2-induced STAT5 binding (lane 4). Competition
analysis using excess amounts of unlabeled specific oligonucleotides is
shown in lane 7. In addition, as demonstrated in Fig.
3, the specificity of the binding
activity was proven by incubating the extracts with antibodies directed against either STAT5a (lane 5) or STAT5b (lane
6). Both antibodies caused a supershift of the activated STAT5
bound to its specific consensus oligonucleotide, indicating that both
STAT5a and STAT5b proteins heterodimerize upon tyrosine phosphorylation
to form active STAT5.

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Fig. 2.
UV radiation inhibits IL-2-induced binding of
STAT5 to its responsive consensus sequence. A, CTLL
cells were left untreated (lane 1), stimulated with 100 units/ml IL-2 (lane 2), irradiated with 400 J/m2
UV (lane 3), or treated with UV plus IL-2 (lane
4). Samples in lanes 5 and 6 were
preincubated with 1 mM Na3VO4 for
2 h before stimulation with IL-2 (lane 5) or with UV
plus IL-2 (lane 6). 15 min after stimulation nuclear
proteins were extracted, and EMSA was performed using the consensus
STAT5 oligonucleotide. Competition analysis was performed with
IL-2-stimulated nuclear protein extracts by addition of 50-fold
molar excess of unlabeled consensus oligonucleotide (lane
7). Experiments were performed three times. One representative
experiment is shown. B, the bands were analyzed
densitometrically. The bars shown represent the mean
density ± S.D. of three independently performed
experiments.
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Fig. 3.
IL-2-induced phosphorylation of STAT5 is
stabilized by the tyrosine phosphatase inhibitor
Na3VO4 but not by the serine phosphatase
inhibitor ocadaic acid. CTLL cells were left untreated (lane
1), stimulated with 100 units/ml IL-2 (lane 2), or
stimulated with IL-2 after preincubation with 1 mM
Na3VO4 (lane 3) or with 1 mM ocadaic acid for 2 h (lane 4).
Supershifts with antibodies specifically directed against STAT5a
(lane 5) or STAT5b (lane 6) were performed with
nuclear protein extracts of cells stimulated with IL-2 in the presence
of ocadaic acid (lane 5) or Na3VO4
(lane 6).
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Suppression of IL-2-induced Activation of STAT5 by UV Is Prevented
by the Phosphatase Inhibitor Orthovanadate--
There is evidence that
the inactivation of STAT molecules appears to be dependent on protein
tyrosine phosphatase(s) (29, 30). Hence, EMSA was performed in the
presence of the tyrosine phosphatase inhibitor orthovanadate (Fig. 3).
Orthovanadate enhanced IL-2-induced binding activity of STAT5
(lane 3), whereas the serine phosphatase inhibitor ocadaic
acid did not augment IL-2-mediated STAT5 binding (lane 4).
The inhibitory effect of UV on STAT5 binding was also reduced by
orthovanadate (Fig. 2, lane 6). The same effect was observed
when checking for STAT5 phosphorylation. Inhibition of IL-2-induced
STAT5 phosphorylation was prevented in the presence of orthovanadate
(Fig. 1, lane 6). Together, these data suggest that the
activation of a phosphatase may be involved in the inhibitory effect of
UV on IL-2-induced STAT5 phosphorylation and binding.
UV Does Not Inhibit IL-2-induced Jak1 and Jak3
Phosphorylation--
Because phosphorylation of STAT5 is a consequence
of the binding of IL-2 to the intermediate receptor consisting of
IL-2R
and
c (19, 20), UV could indirectly inhibit
STAT5 phosphorylation simply by down-regulating IL-2R
and
c. To determine whether IL-2-mediated phosphorylation of
STAT5 is directly inhibited by UV or rather a consequence of
down-regulation of IL-2R
and
c, the effect of UV on
the receptor-associated tyrosine kinases Jak1 and Jak3 was analyzed.
Jak1 and Jak3 are both required for the IL-2 response and are
tyrosine-phosphorylated upon ligand binding (22). Proteins extracted
from CTLL cells were immunoprecipitated with an antibody directed
against either Jak1 or Jak3, and Western blot was performed using an
antibody directed against phosphotyrosine (Fig.
4). Jak1 and Jak3 were found to be
tyrosine-phosphorylated upon IL-2 stimulation. However, neither Jak1
nor Jak3 phosphorylation was inhibited or reduced upon UV
irradiation.

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Fig. 4.
UV radiation does not inhibit IL-2-induced
tyrosine phosphorylation of Jak1 and Jak3. CTLL cells were left
untreated (lane 1), stimulated with 100 units/ml IL-2
(lane 2), irradiated with 400 J/m2 UV
(lane 3), or treated with UV plus IL-2 (lane 4).
Samples in lanes 5 and 6 were preincubated with 1 mM Na3VO4 for 2 h before
stimulation with IL-2 (lane 5) or with UV plus IL-2
(lane 6). 15 min after stimulation immunoprecipitations were
performed using antibodies directed against Jak1 or Jak3. Western blot
analyses were performed using an antibody against phosphotyrosine
(P-Tyr), followed by reprobing of the membranes with the
respective anti-Jak antibodies.
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UV Suppresses IL-2-induced Up-regulation of the IL-2R
Chain--
In contrast to the constitutively expressed IL-2R
and
c molecules, which form the intermediate-affinity
receptor, the inducible IL-2R
chain contains a STAT5-responsive
positive regulatory element within its promoter region (24). Hence,
inhibition of STAT5 phosphorylation by UV should result in a reduced
transcription rate of the IL-2R
chain gene. To prove this
hypothesis, semiquantitative PCR analysis using specific primers for
the IL-2R
chain was performed (Fig.
5). Untreated CTLL cells expressed low
amounts of IL-2R
chain transcripts, which were enhanced upon
stimulation with IL-2 within 2 h. IL-2-induced up-regulation of
the IL-2R
chain transcripts was remarkably reduced following UV
exposure of IL-2-treated CTLL cells. Again, orthovanadate prevented the
inhibitory effect of UV on IL-2R
chain mRNA expression.

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Fig. 5.
UV radiation inhibits IL-2-induced
up-regulation of IL-2R chain mRNA.
CTLL cells were left untreated (lane 1), stimulated with 100 units/ml IL-2 (lane 2), irradiated with 400 J/m2
UV (lane 3), or treated with UV plus IL-2 (lane
4). Samples in lanes 5 and 6 were
preincubated with 1 mM Na3VO4 for
2 h before stimulation with IL-2 (lane 5) or with UV
plus IL-2 (lane 6). 2 h after stimulation, RNA was
extracted, and reverse transcription PCR was performed using primers
amplifying either IL-2R or -actin.
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To determine whether mRNA expression correlates with the cell
surface expression of the IL-2R
chain, flow cytometric analysis using an antibody directed against the IL-2R
chain was performed. In
accordance with the PCR results, IL-2 caused an up-regulation of the
IL-2R
chain, which was reduced to control levels upon exposure to UV
(Fig. 6). UV alone did not affect
IL-2R
expression. Exposure of cells to orthovanadate prevented the
inhibitory effect of UV on IL-2-induced IL-2R
expression. Neither
IL-2 nor UV had an effect on the constitutive expression of the
IL-2R
chain (data not shown).

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Fig. 6.
UV irradiation inhibits IL-2-induced surface
expression of the IL-2R chain on CTLL
cells. CTLL cells were left untreated (Co), stimulated
with 100 units/ml IL-2, irradiated with 400 J/m2 UV, or
treated with UV plus IL-2 (IL-2/UV). Histograms presented in
the bottom lane are from cells that were preincubated with 1 mM Na3VO4 for 2 h before
stimulation with IL-2 (IL-2/Na3VO4)
or with UV plus IL-2
(IL-2/Na3VO4/UV). 8 h after
stimulation cells were incubated with a fluorescein
isothiocyanate-conjugated antibody directed against the IL-2R chain
(CD25). Purified rat IgG was used as an isotype control (dotted
lines). Histograms show fluorescence intensity (x axis)
versus cell number (y axis).
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DISCUSSION |
STAT proteins are cytoplasmatically located transcription factors
that, upon activation by tyrosine phosphorylation, dimerize, translocate to the nucleus, and bind to specific regulatory elements that control gene expression. Several of the STAT members are involved
in the signal transduction of immunomodulatory cytokines (31);
e.g. immune responses to IL-4 and IL-12 are
mediated by STAT6 and STAT4, respectively. IFN
signaling is
critically dependent on STAT1, and STAT5 plays an important role in the
signal transduction of IL-2 (25). Hence, disruption of or interference
with STAT signaling may be an effective way to compromise an immune
response. Recently, we observed that UV radiation, a potent
immunosuppressor, may interfere with STAT signal transduction and
thereby antagonizes the biological effects of IFN
(12, 15). Here, we
show that the same seems to apply to IL-2 signaling.
A crucial step in IL-2 signal transduction is the phosphorylation,
subsequent dimerization, and nuclear translocation of STAT5 (25).
Hence, disturbances in the STAT5 pathway results in severe alterations
of immune responses as the phenotypes of STAT5a
/
,
STAT5b
/
, and
STAT5a
/
/STAT5b
/
double knockout mice
clearly show (24, 32-34). Our results demonstrate that UV interferes
with IL-2-mediated STAT5 signaling by inhibiting tyrosine
phosphorylation of STAT5. Accordingly, STAT5 binding activity as
demonstrated by EMSA is significantly reduced when CTLL cells are
exposed to UV in the presence of IL-2.
Jak1 and Jak3 are required for normal IL-2-mediated STAT5 activation
(35-38). In contrast to STAT5 phosphorylation, IL-2-induced tyrosine
phosphorylation of Jak1 and Jak3 was not affected by UV. This excludes
the possibility that UV affects components in the IL-2 signaling
pathway located upstream of STAT5 phosphorylation. Hence, it is
unlikely that UV exerts its inhibitory effect by down-regulating the
IL-2 receptor, e.g. by inducing shedding of one
of the IL-2 receptor chains. Along this line, STAT3 phosphorylation, which also appears to be involved in IL-2 signaling, was not
significantly affected by UV (data not shown). In this context it is
important to mention that it was previously observed that IL-6-mediated phosphorylation of STAT3 was also not inhibited by UV (15). These
findings and the fact that UV is a well known activator of nuclear
factor-
B and AP-1 both in vitro and in vivo
(39-41) excludes the notion that inhibition of STAT5 activation
by UV is by a general impairment of transcription factors.
UV exhibits the ability to induce the release of a variety of
cytokines, among these transforming growth factor-
(11). Transforming growth factor-
was recently found to exert its
immunosuppressive effects by inhibiting IL-2-induced tyrosine
phosphorylation and the activation of Jak1 and STAT5 in T lymphocytes
(42). However, it is unlikely that the inhibitory effect of UV on IL-2
signaling observed in our system is indirectly mediated by transforming growth factor-
, because in our hands, phosphorylation of Jak1 was
not affected by UV. In addition, UV did not significantly induce the
release of transforming growth factor-
in CTLL cells (data not shown).
IL-2-induced up-regulation of the IL-2R
chain is largely dependent
on STAT5 signaling (24). Hence, we analyzed IL-2R
expression to
determine whether the inhibition of STAT5 signaling by UV is also
biologically relevant. Indeed, RT-PCR revealed that UV inhibits IL-2-mediated up-regulation of IL-2R
chain transcripts. In addition, as determined by fluorescence-activated cell sorter analysis, IL-2R
protein expression was affected in a similar fashion; IL-2-induced up-regulation was completely prevented when cells were exposed to UV.
However, the effects of UV on IL-2R
protein expression were less
pronounced than the inhibitory effects on STAT5 phosphorylation and its
binding activity. This may be because of the fact that the CTLL cells
constitutively express relatively high amounts of the IL-2R
chain.
Because CTLL cells are strictly IL-2 dependent, they are permanently
cultured in the presence of IL-2. To yield IL-2 responsiveness, IL-2
supplementation was stopped 60 h before performance of
experiments. This time interval was too short to allow complete loss of
the IL-2R
chain. This explanation would imply that CD25 expressed on
the surface of CTLL cells has quite a long half-life. In this context
it is important to mention that Nakajima et al. (24) showed
that anti-CD3-induced expression of CD25 on CD4+ splenocytes is only
minimally reduced if the cells are cultured for an additional 48 h
without any further stimulus. Accordingly, we presume that because of
the high constitutive expression of CD25 the effect of IL-2 on IL-2R
chain induction was not as pronounced as one would have expected from
the immunoprecipitations and EMSAs, both of which revealed remarkable
phosphorylation and activation of STAT5 upon stimulation with IL-2.
Nevertheless, although moderate, the induction of IL-2R
expression
by IL-2 was completely prevented by UV, indicating that the inhibition of IL-2 signaling by UV may also be functionally relevant.
The observation that UV inhibits IL-2-induced STAT5 phosphorylation but
leaves Jak1 and Jak3 phosphorylation unaffected points to the STAT5
protein as a direct target for UV. However, we have no evidence as yet
whether phosphorylation of STAT5 is directly inhibited by UV. Data are
accumulating that show that the amount of phosphorylated STAT proteins
does not only depend on the rate of phosphorylation but also on the
rate of dephosphorylation. It has been demonstrated that STAT1
signaling can be abbrogated by dephosphorylation of the conserved
tyrosines through tyrosine phosphatases (29). Similar observations were
recently made for STAT5 phosphorylation. Using CTLL-20 cells as a model
system, Yu et al. (30) provided evidence that tyrosine
dephosphorylation of STAT5 after IL-2-induced phosphorylation occurs in
the absence of nuclear translocation and new protein synthesis,
indicating the constitutive presence of a STAT5-specific tyrosine
phosphatase activity in the cytosolic compartment. In addition, using
an in vitro tyrosine phosphatase assay with purified
proteins Yu et al. (30) demonstrated that SHP-2, but not
SHP-1, directly dephosphorylates STAT5. UV-mediated suppression of
STAT5 phosphorylation was inhibited by adding the phosphatase inhibitor
sodium orthovanadate, suggesting involvement of a tyrosine phosphatase.
However, these findings should be interpreted with caution, because
orthovanadate also had a slight stabilizing effect on IL-2-activated
STAT5 itself (Figs. 1-3). Therefore, in this experimental design it is
difficult to differentiate whether the increase of binding active STAT5 in the UV sample by orthovanadate is because of inhibition of the UV
effect or depends on the inhibition of dephosphorylation of
phosphorylated STAT5. If UV mediates this effect via dephosphorylation this would imply that UV activates a vanadate-inhibitable phosphatase. The effect of UV on phosphatases, however, appears to be heterogenous. Using protein-tyrosine phosphatase-overexpressing cells, Gross et
al. (43) recently observed that four defined protein-tyrosine phosphatases, SHP-1, RPTP
, RPTP
, and DEP-1, are partially
inactivated upon UV irradiation. How SHP-2, the phosphatase most likely
to be involved in STAT5 dephosphorylation (30), is affected by UV
remains to be determined.
STAT5 proteins have also been reported to be phosphorylated at serine
residues (44). However, it is not yet clear whether serine
phosphorylation is important for transactivation activity (25) as
reported for STAT1 and STAT3 (45). The addition of ocadaic acid, an
inhibitor of serine phosphatases, did not stabilize STAT5 binding
activity in our hands. Thus, it appears unlikely that UV targets serine
phosphorylation of STAT5 resulting in its inhibitory effect.
The UVC component (200-280 nm) of solar radiation is completely
absorbed by the ozone layer and is therefore biologically irrelevant.
We therefore used the UVB range (290-320 nm) that causes a variety of
biological effects in vivo including immunosuppression, inflammation, and induction of skin cancer. UVB is primarily absorbed in the epidermis, and therefore keratinocytes are the major cellular target in vivo for UVB. However, there is clear-cut evidence
that UVB also reaches the dermis where lymphocytes and endothelial cells reside. (46, 47). Young et al. (48) recently observed pyrimidine dimers in dermal nuclei induced by 300 nm of UV, providing clear evidence that UVB enters the dermis and can therefore directly affect lymphocytes.
The immunosuppressive properties of UV/solar radiation may have
important biological implications. Chronic UV exposure can lead to
exacerbation of infectious disease (6). UV-induced immunosuppression,
however, may also contribute to carcinogenesis, because it impairs the
immune response of the host against tumor cells (7). Accordingly,
chronically immunosuppressed individuals, e.g.
renal or cardiac transplant patients, exhibit a dramatically increased
risk for cancer, and this risk strongly correlates with the intensity
of solar exposure (49). On the other hand, UV radiation is used
therapeutically to treat numerous inflammatory skin diseases with great
success. Although the exact mechanisms for this beneficial effect still
remain to be determined, it is the immunosuppressive effect of UV
radiation that is regarded to be most important, because the majority
of diseases responsive to UV can also be treated with steroids or other
immunosuppressive drugs like cyclosporin A (50). In this context,
it is important to mention that one mechanism by which steroids
suppress immune reactions is by inhibiting IL-2-induced STAT5
phosphorylation (51). In psoriasis, a common chronic inflammatory
dermatosis responding favorably to UV therapy, IL-2 appears to be
crucially involved, because cyclosporin A, which targets IL-2
transcription (52), or the application of antibodies directed against
the IL-2R
chain (anti-CD25) are effective treatments (53, 54). Interruption of IL-2 signaling by UV as demonstrated in this study may
represent a mechanism that explains why phototherapy improves psoriasis
so effectively. In addition, the present observation that UV can
antagonize IL-2 effects may also explain why UV preferentially and very
effectively suppresses T cell-mediated immune reactions.