Involvement of Dectin-2 in Ultraviolet
Radiation-Induced Tolerance
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J Immunol 2003; 171:3801-3807; ;
doi: 10.4049/jimmunol.171.7.3801
http://www.jimmunol.org/content/171/7/3801
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References
Yoshinori Aragane, Akira Maeda, Agatha Schwarz, Tadashi
Tezuka, Kiyoshi Ariizumi and Thomas Schwarz
The Journal of Immunology
Involvement of Dectin-2 in Ultraviolet Radiation-Induced
Tolerance1
Yoshinori Aragane,2* Akira Maeda,*‡ Agatha Schwarz,‡ Tadashi Tezuka,* Kiyoshi Ariizumi,†
and Thomas Schwarz‡
U
ltraviolet radiation is one of the, if not the most, significant environmental factors affecting humans. Besides
its well-known beneficial and indispensable effects for
human life, UV, in particular the middle wave length range (290 –
320 nm, UVB), can be a hazard to human health by inducing skin
cancer, premature skin aging, inflammation, and cell death (1– 4).
In addition, UV exhibits immunosuppressive properties which may
be relevant for photocarcinogenesis and the exacerbation of infectious diseases following UV exposure (5, 6). The immunosuppressive properties of UV are best illustrated by the inhibition of cellular immune reactions, such as contact hypersensitivity (CHS)3
(7, 8). Epicutaneous sensitization in mice is impaired when the
hapten is applied onto skin which has been exposed to UV immediately before (7). In addition to the failure to generate hapten
sensitization, tolerance develops because mice treated in this way
cannot be resensitized against the same hapten at a later time point
(7). UV-induced tolerance appears to be hapten-specific because
the sensitization against other nonrelated haptens is not affected
(7). Hapten-specific unresponsiveness can be adoptively trans-
*Department of Dermatology, Kinki University School of Medicine, Osaka, Japan;
†
Department of Dermatology, Southwestern Medical Center, University of Texas,
Dallas, TX 75390; and ‡Department of Dermatology, University of Münster, Münster,
Germany
Received for publication March 14, 2003. Accepted for publication July 22, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Grants-in-Aid for Scientific Research
(Category B, No. 14370263) and for Exploratory Research (No. 14657205) from the
Japanese Ministry of Education, Culture, Sports, Science, and Technology (to Y.A.),
and from the German Research Foundation (SFB 293, B9; to T.S.).
2
Address correspondence and reprint requests to Dr. Yoshinori Aragane, Department
of Dermatology, Kinki University, 377-2 Ohnohogashi, Osakasayama City, 589-8511
Osaka, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: CHS, contact hypersensitivity; sDec2, soluble
dectin-2; DHFR, dihydrofolate reductase; DNFB, 2,4-dinitrofluorobenzene; DNBS,
2,4-dinitrobenzene-sulfonic sodium salt.
Copyright © 2003 by The American Association of Immunologists, Inc.
ferred; injection of lymph node cells and splenocytes from UVtolerized mice into syngeneic naive mice inhibits sensitization
against the relevant hapten in the recipients (9). Consequently, it
has been suggested that UV-induced tolerance is mediated via the
induction of hapten-specific T suppressor cells, now renamed regulatory T cells (reviewed in Ref. 10). Although the cellular transfer
of suppression and the hapten specificity were convincingly demonstrated almost two decades ago, the cells transferring haptenspecific unresponsiveness are still poorly characterized and their
mode of action is unclear in major parts.
Using a subtractive cDNA cloning strategy, we previously isolated genes encoding for C-type lectin receptors (termed dectin-1
and dectin-2); dectin-1 is expressed by dendritic cells and macrophages (11). Preliminary in vitro data showed that a soluble receptor of dectin-1 binds to the cell surface of T cells, thereby
delivering T cell costimulatory signals (11). Recently, dectin-1
was reported to bind to ␤-glucan, a polysaccharide synthesized in
yeasts in an opsonin-independent fashion, indicating that dectin-1
is a pattern recognition receptor (12, 13). This ligand does not
block binding of dectin-1 to T cells (12). Thus, dectin-1 has been
well-characterized.
Dectin-2 is also a C-type lectin receptor with a type II configuration, consisting of, from the N terminus, a short cytoplasmic
domain, a transmembrane domain, and an extracellular domain
that contains a neck domain and a single carbohydrate recognition
domain in the C terminus (14). Unlike dectin-1, dectin-2 expression appears more restricted to dendritic cells including epidermal
dendritic cells (Langerhans cells); using a transgenic mouse bearing a dectin-2 promoter-driven luciferase gene, Bonkobara et al.
(15) found the highest luciferase activity in the skin, lower levels
in spleen, lymph node, and thymus and only background levels in
nonlymphoid organs. Despite having a perfect C-type lectin motif,
responsible for recognition of carbohydrates, the ligand for dectin-2 has not yet been identified and even its carbohydrate recognition has not been proven. Accordingly, the function of dectin-2
still remains to be determined.
0022-1767/03/$02.00
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Hapten sensitization through UV-exposed skin induces hapten-specific tolerance which can be adoptively transferred by injecting
T cells into naive recipients. The exact phenotype of the regulatory T cells responsible for inhibiting the immune response and their
mode of action remain largely unclear. Dectin-2 is a C-type lectin receptor expressed on APCs. It was postulated that dectin-2
interacts with its putative ligands on T cells and that the interaction may deliver costimulatory signals in naive T cells. Using a
soluble fusion protein of dectin-2 (sDec2) which should inhibit this interaction, we studied the effect on contact hypersensitivity
(CHS) and its modulation by UV radiation. Injection of sDec2 affected neither the induction nor the elicitation phase of CHS. In
contrast, UV-induced inhibition of the CHS induction was prevented upon injection of sDec2. In addition, hapten-specific tolerance
did not develop. Even more importantly, injection of sDec2 into tolerized mice rendered the recipients susceptible to the specific
hapten, indicating that sDec2 can break established tolerance. FACS analysis of spleen and lymph node cells revealed a significantly increased portion of sDec2-binding T cells in UV-tolerized mice. Furthermore, transfer of UV-mediated suppression was
lost upon depletion of the sDec2-positive T cells. Taken together, these data indicate that dectin-2 and its yet unidentified ligand
may play a crucial role in the mediation of UV-induced immunosuppression. Moreover, sDec2-reactive T cells appear to represent
the regulatory T cells responsible for mediating UV-induced tolerance. The Journal of Immunology, 2003, 171: 3801–3807.
3802
To study the function of dectin-2, we have prepared a soluble
dectin-2 receptor in the format of histidine-fusion proteins, consisting of the extracellular domains (aa 43–209) of dectin-2. This
soluble dectin-2 (sDec2) should bind to the putative ligand and
thus presumably inhibit dectin-2-triggered stimulation. We used
this soluble receptor protein to examine whether dectin-2 is involved in the mediation of CHS. Surprisingly, injection of sDec2
affected neither the induction nor the elicitation of CHS, but
blocked UV-induced suppression of CHS. Even more importantly,
injection of sDec2 was able to break established tolerance. T cells
transferring suppression were found to bind sDec2. Depletion of
the sDec2-binding fraction of T cells was associated with a loss of
transfer of suppression. Taken together, these data indicate that
dectin-2 and its yet unidentified ligand may play a crucial role in
the mediation of UV-induced immunosuppression. In addition,
sDec2 may be a useful tool to further characterize the regulatory T
cells mediating UV-induced tolerance.
Materials and Methods
Recombinant soluble protein of dectin-2 (sDec2) was generated from an
expression vector encoding for 6xHis-tagged extracellular domain of dectin-2 (aa 43–209) as described previously (14). Recombinant soluble protein of dihydrofolate reductatse (DHFR) was derived from 6xHis-tagged
DHFR and used as a control protein. Both proteins were expressed in
Escherichia coli M15 and purified by using a Ni-NTA resin (Life Technologies, Grand Island, NY) followed by refolding.
Contact hypersensitivity
C3H/HeN mice (8- to 10-wk-old) were obtained from Japan Clea
(Hamamatsu, Japan). Mice were sensitized on day 0 by painting 25 ␮l of
0.5% 2,4-dinitrofluorobenzene (DNFB; solved in acetone-olive oil, 4:1)
onto the shaved back. After 5 days, mice were challenged by painting 20
␮l of 0.3% DNFB onto the left ear. The right ear was painted with the
acetone-olive oil solution without DNFB. Ear swelling was quantified 24 h
later using a spring-loaded micrometer. CHS was determined as the amount
of swelling of the hapten-challenged ear compared with the thickness of the
vehicle-treated ear in sensitized animals and expressed in millimeters ⫻
10⫺2 (mean ⫾ SD). After 2 wk, mice were resensitized through nonirradiated abdominal skin and challenged on the right ear after 5 days. Each
group consisted of at least seven mice. Data were analyzed by Student’s t
test, and differences were considered as significant at p ⬍ 0.05.
UV irradiation
Mice were exposed to 1000 J/m2 UV on the shaved back daily for 4 consecutive days. FS-20 fluorescent lamps (Westinghouse Electric, Pittsburgh,
PA) which emit most of their energy within the UVB range (290 –320 nm)
with an emission peak at 311 nm were used. DNFB was applied onto the
surface of the irradiated skin area 24 h after the last UV exposure.
Adoptive transfer
Donor mice were exposed to UV and sensitized with DNFB through UVexposed skin as described above. Five days after sensitization, spleens and
regional lymph nodes were removed, and single cell suspensions were
prepared. T cells were prepared by passing the cell suspension through a
nylon wool column twice, which yields ⬃95% pure T cells as measured by
FACS analysis using Abs directed against T cell markers (Thy 1.2, CD3).
The cell number was adjusted to 1 ⫻ 108 cells/ml, and 200 ␮l were injected
i.v. into naive recipient mice. Recipients were sensitized 24 h later by
epicutaneous application of DNFB on the shaved abdomen. After 5 days,
mice were challenged on the left ear and ear swelling was evaluated 24 h
later. For control purposes, identical numbers of cells obtained from untreated or DNFB-sensitized mice were injected.
Depletion and enrichment of T cell subpopulations
Lymphocytes obtained from regional lymph nodes and spleens were incubated in nylon wool columns. T cells were first reacted with biotinylatedsDec2, -DHFR, or -sDec2 which was boiled. Biotinylation was conducted
by use of the ECL Protein Biotinylation Module (Amersham Biosciences,
Buckinghamshire, U.K.). One hour after the incubation of the cells with the
biotinylated proteins, cells were incubated with streptavidin-coupled mag-
netic beads for 30 min. Magnetoseparation was performed by placing the
tubes into a magnetic field (Dynal, Oslo, Norway) for 4 min. The supernatants containing the cells not binding to sDec2 were removed. Cells were
washed and used for i.v. injection. Cells bound to the magnetobeads were
detached by incubating cells overnight in cell culture medium at 37°C in a
humidified atmosphere containing 5% CO2. Magnetobeads which had
spontaneously detached from the cells after overnight incubation were removed with a magnet. The remaining (dectin-2 binding) cells were harvested, washed, and adjusted to 2.5 ⫻ 107 cells/ml for i.v. injection (200
␮l/mouse). The efficacy of separation was determined by flow cytometry
(FACSCalibur; BD Biosciences, Mountain View, CA).
Flow cytometry
T cells prepared from superficial regional lymph nodes and spleens were
first incubated in PBS supplemented with 1% BSA on ice for 30 min. After
extensive washing in PBS/BSA, T cells were incubated with biotinylated
sDec2 on ice for 1 h. To perform competition of the binding, boiled sDec2,
DHFR, and sDec2 were added to the reaction as a nonspecific and a specific competitor, respectively. After extensive washing in PBS/BSA, the
samples were reacted with streptavidin-coupled with FITC on ice for 15
min. After washing, the samples were analyzed by flow cytometry (FACSCalibur). In some experiments, three-color staining was conducted with
an allophycocyanin-conjugated Ab directed against CD4 (clone RM4-5;
BD Biosciences, San Diego, CA), with a PE-conjugated Ab directed
against CD25 (clone 7D4; BD Biosciences), and with biotinylated sDec2,
which was finally reacted with FITC-coupled streptavidin (BD
Biosciences).
Cytokine induction in vitro
T lymphocytes were prepared from mice which were tolerized by applying
DNFB onto UV-irradiated skin and separated into sDec2⫹ and sDec2⫺
fractions by magnetobead separation. T cells (5 ⫻ 106/ml) were incubated
with dendritic cells (1 ⫻ 106/ml) which were isolated from bone marrow
of syngeneic naive mice as described previously (16). Coincubations were
performed in the absence or presence of the water soluble DNFB analog
2,4-dinitrobenzene-sulfonic sodium salt (DNBS, 0.1 mM). Supernatants
were collected 48 h after stimulation and tested for the amounts of IL-4,
IL-10, IFN-␥, and TGF-␤ using ELISAs (Bender Medsystems, Vienna,
Austria).
Results and Discussion
Injection of sDec-2 does not affect CHS but inhibits UV-induced
suppression of CHS
To elucidate whether interaction of dectin-2 with its putative ligand is of relevance in the generation of CHS, sDec2 was used.
The soluble form of dectin-2 should bind to the putative though yet
unknown ligand, inhibit the interaction of dectin-2 and therefore
act as an antagonist of dectin-2. To test whether dectin-2 is involved in the mediation of CHS, mice received an i.v. injection of
50 ␮g of sDec2 or of DHFR. Three hours later, the animals were
sensitized against DNFB. The ear swelling response caused by the
ear challenge performed 5 days later was not affected by the injection of sDec-2 (data not shown). Likewise, ear challenge was
not altered when sDec2 was injected i.v. into sensitized mice 3 h
before challenge (data not shown). These data suggest that dectin-2
does not play an important role either in the induction or in the
elicitation of CHS.
UV radiation is well known to inhibit the induction of CHS and
to induce hapten-specific tolerance, although the detailed underlying mechanisms still remain to be determined. Therefore, we were
interested in whether interaction of dectin-2 is involved in UVinduced immunosuppression. For that purpose, mice were sensitized against DNFB by epicutaneous application of the hapten on
UV-exposed or unirradiated back skin. Ear challenge was performed 5 days later. In accordance with previous observations, ear
swelling response was significantly suppressed in mice which were
sensitized through UV-exposed skin (Fig. 1). To test whether dectin-2 is involved in UV-induced immunosuppression, mice were
injected i.v. with various amounts of sDec2 3 h before application
of DNFB onto UV-exposed skin. Mice which received 50 ␮g of
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Reagents
DECTIN-2 AND UV-INDUCED TOLERANCE
The Journal of Immunology
sDec2 mounted an ear swelling response upon challenge comparable to that of positive control mice, despite the fact that sensitization was performed in UV-exposed skin (Fig. 1). sDec2 prevented UV-induced immunosuppression in a dose-dependent
manner. Injection of 50 ␮g of the control protein DHFR did not
affect inhibition of the induction of CHS by UV. Together, these
data demonstrate that i.v. injection of sDec2 prevents UV-induced
suppression of CHS, suggesting that interaction of dectin-2 with its
putative ligand may play an important role in UV-induced
immunosuppression.
Injection of sDec2 prevents UV-induced tolerance
Application of haptens onto UV-exposed skin does not only result
in the failure to induce a CHS response, but also induces long-term
immunosuppression because the respective mice cannot be sensitized at a later time against the specific hapten. This tolerance is
hapten-specific because the animals can be sensitized at later time
points against other unrelated haptens (7). Because both impairment of CHS and induction of hapten-specific tolerance are caused
by the identical procedure, i.e., sensitization through UV-exposed
skin, it was postulated for quite a long time that the same mechanisms are involved and that induction of tolerance is the direct
consequence of the inhibition of CHS induction. However, there is
accumulating evidence that the molecular basis of UV-induced
tolerance is different from the mechanism responsible for UV-impaired induction of CHS (17). Whereas a key event in the suppression of CHS by UV appears to be the depletion of Langerhans
cells from the epidermis (7, 18), hapten-specific tolerance seems to
be due to the generation of T suppressor cells (reviewed in Refs.
10 and 19).
Therefore, we next checked whether injection of sDec2 also
prevents UV-mediated tolerance. For this purpose, the mice which
were sensitized through UV-exposed skin after injection of sDec2
were resensitized with DNFB on the abdomen 2 wk after the first
sensitization. Five days after resensitization, the second challenge
was performed on the right ear. There was no difference in the ear
swelling response between sensitized mice and UV-exposed mice
which had initially received sDec2 i.v. (Fig. 2). In contrast, mice
FIGURE 2. sDec2 prevents UV-induced tolerance. The UV-exposed
mice which were untreated or had received 50 ␮g of sDec2 or DHFR
before sensitization (shown in Fig. 1) were resensitized with DNFB applied
on non-UV-exposed abdominal skin 2 wk after first challenge. Challenge
on the right ear was performed 5 days after resensitization. ⴱ, p ⬍ 0.01.
which were sensitized through UV-exposed skin but had not received sDec2 were unresponsive to resensitization, indicating that
tolerance had developed. Taken together, these data demonstrate
that injection of sDec2 prevents the UV-induced inhibition of sensitization and the development of UV-induced tolerance.
sDec2 breaks UV-induced tolerance
We next were interested in whether injection of sDec2 is not only
able to prevent the development of UV-induced tolerance but can
also break tolerance once developed. To address this issue, mice
were tolerized against DNFB by painting the hapten onto UVexposed back skin. As demonstrated above, this procedure induces
tolerance because these animals cannot be resensitized against
DNFB 2 wk later. In one group of mice, sDec2 was injected i.v.
before resensitization, while the other group received DHFR. Mice
FIGURE 3. sDec2 breaks UV-induced tolerance. Mice were tolerized
by application of DNFB onto UV-exposed skin. Two weeks after tolerization, mice were resensitized with DNFB applied on non-UV-exposed abdominal skin. Mice were left untreated or received an i.v. injection of 50
␮g of sDec2 or DFHR 3 h before resensitization. Five days later challenge
was performed on the right ear. Mice which were sensitized without UV
exposure served as positive controls, mice which received only challenge
without sensitization served as negative controls. ⴱ, p ⬍ 0.05.
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FIGURE 1. sDec2 prevents UV-induced immunosuppression. C3H/
HeN mice were sensitized on day 0 by painting 25 ␮l of 0.5% DNFB onto
the shaved back which was exposed to UV or sham-treated. Five days later,
mice were challenged by painting 20 ␮l of 0.3% DNFB onto the left ear.
Ear swelling was quantified 24 h later. To evaluate the involvement of
dectin-2 in this process, various amounts of sDec2 or DHFR were injected
3 h before sensitization. CHS was determined as the amount of swelling of
the hapten-challenged ear compared with the thickness of the vehicletreated ear in sensitized animals and expressed in millimeters ⫻ 10⫺2
(mean ⫾ SD). ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
3803
3804
DECTIN-2 AND UV-INDUCED TOLERANCE
oped at the time of injection of sDec2, one can conclude that injection of sDec2 is able to break established tolerance.
sDec2 prevents the induction of T cells with
regulatory/suppressor activity
which were injected with DHFR did not show an ear swelling
response after resensitization, indicating development of tolerance
against DNFB (Fig. 3). In contrast, mice which received an i.v.
injection of 50 ␮g of sDec2 revealed a specific ear swelling response after resensitization. Because tolerance had already devel-
FIGURE 5. Increase of sDec2⫹ T cells in UV-tolerized mice. T cells were prepared from the lymph nodes and spleens of untreated, DNFB-sensitized,
and UV-tolerized mice. Cells were incubated with biotinylated sDec2. For competition of the binding, excess amounts of unlabeled sDec2, boiled sDec2,
or DHFR were added. Samples were further incubated with streptavidin-coupled FITC and FACS analysis was performed.
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FIGURE 4. Injection of sDec2 inhibits transfer of suppression. Donor mice
were sensitized through UV-exposed skin. Three hours before sensitization
through UV-exposed skin, 50 ␮g of sDec2 or DFHR was injected. Five days
later spleens and regional lymph nodes were removed and T cells were prepared. Cells (2 ⫻ 107) were injected i.v. into naive recipient mice which were
sensitized against DNFB 24 h later. After 5 days, mice were challenged on the
left ear and ear swelling was evaluated 24 h later. ⴱ, p ⬍ 0.01.
UV-induced tolerance is mediated via T cells with suppressor activity because adoptive transfer of T cells obtained from lymph
nodes and spleens of mice which had been UV-tolerized against
DNFB renders the recipient mice unresponsive against DNFB (9).
The mechanism and the phenotype of these T cells with regulatory/
suppressor function still remain to be determined. To address
whether injection of sDec2 prevents the development of these regulatory T cells, donor mice were tolerized against DNFB by painting the hapten onto UV-exposed skin. Five days later, lymph nodes
and spleens were removed and single cell suspensions were prepared. T cells (2 ⫻ 107) were injected i.v. into naive syngeneic
mice. Recipients were sensitized against DNFB 24 h later. Mice
which received cells from UV-tolerized donors could not be sensitized against DNFB (Fig. 4). Mice which received cells from
untreated donors were not impaired in their CHS response (data
not shown). Transfer of cells from mice which received an injection of sDec2 before tolerization against DNFB did not suppress
the CHS response against DNFB in the recipients. In contrast,
transfer of suppression was observed upon injection of cells from
DHFR-treated and UV-tolerized mice (Fig. 4). These data indicate
that injection of sDec2 before tolerization either inhibits the development of UV-induced regulatory T cells or alternatively inhibits their suppressive activity.
The Journal of Immunology
3805
sDec2 binds to regulatory T cells mediating UV-induced
tolerance
Phenotypic characterization of sDec2-binding T cells
To further characterize the phenotype of sDec2-bound T cells,
FACS analysis was performed. T cells obtained from lymph nodes
FIGURE 6. sDec2-binding T cells transfer suppression. A, T cells were
prepared from naive, DNFB-sensitized, or UV-tolerized donors. T cells
were left untreated or depleted of sDec2-binding cells by magnetobead
separation. Subsequently, 200 ␮l of each T cell fraction (1 ⫻ 108 cells/ml)
were injected i.v. into naive recipients. Recipients were sensitized with
DNFB 24 h after injection. Five days later, ear challenge was performed
and ear swelling was evaluated 24 h later. B, sDec2-bound T cells from
UV-tolerized mice cells were obtained by magnetobead separation. Magnetobeads which had spontaneously detached from the cells after overnight
incubation were removed with a magnet and the remaining cells were harvested, 5 ⫻ 106 cells (sDec2-bound T cells) were injected i.v. into naive
recipients, which were then DNFB-sensitized and challenged. In parallel,
“bulk T cells” obtained from UV-tolerized animals were injected. Positive
control mice were sensitized and challenged, negative control mice were
challenged only. ⴱ, p ⬍ 0.05.
and spleens of either naive, DNFB-sensitized, or UV-tolerized
mice were triple-stained with the Abs directed against CD4 and
CD25, respectively, and with biotinylated sDec2. FACS analysis
(Fig. 7) revealed an increase in the number of CD4⫹CD25⫹ T
cells in UV-tolerized animals as compared with naive and sensitized mice. sDec2-binding cells were detected by incubating the
cells with FITC-coupled streptavidin. sDec2-bound CD25⫹ T cells
were almost exclusively observed in UV-tolerized animals, the
same was observed for CD4. Together, these findings indicate that
sDec2-bound T cells also express CD4 and CD25 and thus may
belong to the group of CD4⫹CD25⫹ regulatory T cells. We are
currently investigating whether these cells also express CTLA-4
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The data so far suggested that interaction of dectin-2 with its putative ligand is crucial in the development and mediation of UVinduced tolerance. Because dectin-2 is expressed on dendritic
cells, we postulated that the putative ligand may be expressed on
T cells. To test this hypothesis, T cells were incubated with biotinylated sDec2 followed by incubation with streptavidin-coupled
FITC. After washing, FACS analysis was performed. Although in
naive and DNFB-sensitized animals ⬃0.3 and 2.48% T cells, respectively, bound sDec2, the number of sDec2-positive T cells was
increased (5.95%) in UV-tolerized animals (Fig. 5). The specificity
of the binding was confirmed by competition with excess amounts
of unlabeled sDec2, while the control protein DHFR did not compete (Fig. 5).
Both T cells (10) and NKT cells (20) have been recognized to
mediate UV-induced immunosuppression, the latter being involved in the suppression of delayed-type hypersensitivity and tumor immunity (20). T cells with immunosuppressive properties
have been renamed regulatory T cells (21). Different subsets of
regulatory T cells have been described in mouse and man (reviewed in Ref. 22). An important subtype of regulatory T cells
expresses CD4 and CD25 and comprises 8 –10% of the peripheral
CD4⫹ T cell subset in the mouse (23). In addition, these cells
constitutively express the negative regulatory molecule CTLA-4
(24, 25). How these cells exert their suppressive activity is not yet
clear. It is still under debate whether some regulatory T cells mediate suppression via the release of the immunosuppressive cytokines IL-10 and/or TGF-␤, while others require cell-to-cell contact. There are recent indications that CD4⫹CD25⫹ T cells may
also be involved in the mediation of UV-induced tolerance (26).
Due to the increased binding of sDec2 on T cells from UVtolerized mice, we surmised that sDec2 binds to the regulatory T
cells which are responsible for transferring suppression. Therefore,
T cells obtained from UV-tolerized mice were incubated with biotinylated sDec2. Subsequently, cells were incubated with streptavidin-coupled beads. Finally, UV-tolerized T cells were depleted
of the sDec2-bound fraction. The sDec2-depleted fraction of cells
was injected into naive recipients, which were sensitized against
DNFB 24 h later. Although transfer of bulk T cells from UVtolerized donors inhibited sensitization in the recipients, animals
which received the depleted fraction could be successfully sensitized against DNFB (Fig. 6A). Depletion of sDec2-bound cells
from T cells obtained from DNFB-sensitized donors did not affect
induction of CHS in the recipients.
In turn, sDec2-bound T cells from UV-tolerized mice were obtained by magnetobead separation. Magnetobeads which had spontaneously detached from the cells after overnight incubation were
removed with a magnet and the remaining cells were harvested,
5 ⫻ 106 cells were injected i.v. into naive recipients, which were
then DNFB-sensitized and challenged (Fig. 6B). When compared
with the transfer of equal numbers of bulk T cells obtained from
tolerized mice, inhibition of sensitization was more pronounced
upon injection of sDec2-binding cells. This indicates that sDec2bound T cells are predominantly responsible for transfer of suppression. However, we cannot exclude that only a subfraction of
sDec2-binding T cells transfers suppression, hence it is difficult to
determine the minimum number of cells required for effective
suppression.
3806
DECTIN-2 AND UV-INDUCED TOLERANCE
which has been suggested to be involved in mediating UV-induced
tolerance (25).
To elucidate the cytokine secretion of sDec2-bound T cells, T cells
were obtained from mice which were tolerized against DNFB by UV
and separated into sDec2⫹ and sDec2⫺ fractions. These subtypes
were put into culture with bone marrow-derived DC. Cells were cultured for 48 h in the absence or presence of the water soluble DNFB
analog DNBS. Supernatants were harvested and tested for the
amounts of IL-4, IL-10, IFN-␥, and TGF-␤ (Table I). Stimulation of
sDec2⫹ cells with hapten-coupled DC resulted in the induction of the
release of IL-4, IL-10 and TGF-␤, while IFN-␥ was not affected.
Cytokine induction was hapten-dependent because incubation of DC
in the absence of DNBS did not induce the release of cytokines. In
contrast, no induction was observed when sDec2⫺ cells were stimulated with DC either in the absence or presence of DNBS.
sDec2 inhibits transfer of suppression upon injection into
recipients
Finally, we tested whether injection of sDec2 into recipients of
UV-tolerized T cells prevents transfer of suppression. Therefore, T
cells isolated from UV-tolerized animals were transferred into naive recipients, which had been injected with sDec2 3 h before
transfer. Mice which received T cells from tolerized mice could
not be sensitized, while recipients of the same fraction of cells
which in addition were treated with sDec2 revealed a normal CHS
responses against DNFB (Fig. 8). This indicates that injection of
sDec2 into recipients enables sensitization in the presence of regulatory T cells. When T cells from DNFB-sensitized animals were
transferred, injection of sDec2 did not affect the onset of CHS in
the recipients (data not shown).
Table I. sDec2⫹ T cells release IL-4, IL-10, and TGF-␤ upon hapten-specific stimulation
Incubationa
Cytokine Releaseb (pg/ml)
T cellsc
DC
DNBS
IL-4
IL-10
IFN-␥
TGF-␤
sDec2⫹
sDec2⫹
sDec2⫺
sDec2⫺
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫺
120.1 ⫾ 1.0
37.3 ⫾ 1.5
32.8 ⫾ 1.9
31.0 ⫾ 2.2
891.2 ⫾ 32.2
373.2 ⫾ 9.2
378.2 ⫾ 4.6
220.2 ⫾ 18.5
161.9 ⫾ 8.6
175.6 ⫾ 15.1
251.5 ⫾ 13.0
201.1 ⫾ 16.2
124.5 ⫾ 18.8
55.3 ⫾ 11.4
37.3 ⫾ 24.1
39.2 ⫾ 3.4
a
T cells (5 ⫻ 106/ml) were incubated with bone marrow-derived DC (1 ⫻ 106/ml) in the absence or presence of DNBS
(0.1 mM).
b
Supernatants were harvested 48 h after stimulation and tested for cytokine levels.
c
T cells were obtained from mice that were tolerized against DNFB by applying DNFB onto UV-exposed skin. sDec2⫹ or
sDec2⫺ T cells isolated by magnetobead separation were coincubated with DC.
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FIGURE 7. sDec2-binding T cells express CD4 and CD25. T cells were prepared from the lymph nodes and spleens of untreated, DNFB-sensitized, and
UV-tolerized mice. Cells were incubated with an allophycocyanin-conjugated Ab directed against CD4, with a PE-conjugated Ab directed against CD25
and with biotinylated sDec2, which was finally reacted with FITC-coupled streptavidin. After washing, the samples were analyzed by flow cytometry.
The Journal of Immunology
3807
References
FIGURE 8. Injection of sDec2 into recipients inhibits transfer of suppression. Three hours before i.v. transfer of UV-tolerized T cells (2 ⫻ 107
cells/mouse) into naive recipients, 50 ␮g of sDec2 or DHFR was injected.
Recipients were subsequently DNFB-sensitized and challenged 5 days
later. Ear swelling was evaluated 24 h later. ⴱ, p ⬍ 0.01.
In summary, the present data indicate that dectin-2 and its yet
unidentified ligand may play a crucial role in the mediation of
UV-induced immunosuppression. Interaction between these two
molecules appears to be involved both in the inhibition of the
induction of CHS by UV and in the mediation of UV-induced
tolerance. Because sDec2 binds to the fraction of T cells which are
crucial for transferring suppression, the putative ligand may serve
as a future marker for UV-induced regulatory T cells. Hence, identification of the ligand for dectin-2 on the T cells is urgently required and ongoing in our laboratory.
However, the binding of sDec2 must not be regarded as a specific feature of the cells transferring UV-induced suppression because it cannot be excluded that only a minority of cells transferred
are responsible for mediating hapten-specific unresponsiveness in
the recipients. In contrast, interaction of dectin-2 with its ligand
appears to be functionally relevant for mediating UV-induced immunosuppression because injection of sDec2 in vivo prevented the
suppression of CHS by UV and even broke UV-mediated tolerance. It has been previously reported that established tolerance can
be broken by the injection of IL-12 (27, 28). We do not know
whether these two phenomena are related or independent, but we
are currently investigating whether the binding sites for sDec2 on
T cells are regulated by IL-12. In addition, injection of neutralizing
Abs against CTLA-4 is also able to break UV-induced tolerance
(29). In this context, it was proposed that triggering of CTLA-4 by
B7 molecules expressed on the APCs induces release of IL-10
which subsequently inhibits resensitization, thereby rendering the
respective animal tolerant to the hapten. Accordingly, T cells obtained from UV-tolerized mice were found to release high amounts
of IL-10 upon in vitro stimulation with hapten-coupled APCs (29).
It remains to be determined whether the release of IL-10 by T cells
is suppressed in the presence of sDec2.
UV-induced regulatory T cells are still poorly characterized. A
major obstacle in this context may be the poor proliferative capacity which makes cloning extremely difficult probably even impossible. Therefore, the detection of any further surface marker
expressed on UV-induced regulatory T cells will help to better
characterize these cells and to better understand their mode of
action. The detection of the binding sites for dectin-2 as demonstrated in this study may add to this.
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Conclusion
1. Fisher, G. J., Z. Q. Wang, S. C. Datta, J. Varani, S. Kang, and J. J. Voorhees.
1997. Pathophysiology of premature skin aging induced by ultraviolet light.
N. Engl. J. Med. 373:1419.
2. Gilchrest, B. A. 1990. Actinic injury. Annu. Rev. Med. 41:199.
3. Kraemer, K. H. 1997. Sunlight and skin cancer: another link revealed. Proc. Natl.
Acad. Sci. USA 94:11.
4. Kulms, D., T. Schwarz. 2000. Molecular mechanisms of UV-induced apoptosis.
Photodermatol. Photoimmunol. Photomed. 16:195.
5. Kripke, M. L. 1986. Immunology and photocarcinogenesis. J. Am. Acad. Dermatol. 14:149.
6. Chapman, R. S., K. D. Cooper, E. C. DeFabo, J. E. Frederick, K. N. Gelatt,
S. P. Hammond, P. Hersey, H. S. Koren, R. D. Ley, F. Noonan, et al. 1994. Solar
ultraviolet radiation and the risk of infectious disease. Photochem. Photobiol. 61:
223.
7. Toews, G. B., P. R. Bergstresser, and J. W. Streilein. 1980. Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows skin painting with DNFB. J. Immunol. 124:445.
8. Cooper, K. D., L. Oberhelman, T. A. Hamilton, O. Baadsgaard, M. Terhune,
G. LeVee, T. Anderson, and H. Koren. 1992. UV exposure reduces immunization
rates and promotes tolerance to epicutaneous antigens in humans: relationship to
dose, CD1a-DR⫹ epidermal macrophage induction, and Langerhans cell depletion. Proc. Natl. Acad. Sci. USA 89:8497.
9. Elmets, C. A., P. R. Bergstresser, R. E. Tigelaar, P. J. Wood, and J. W. Streilein.
1983. Analysis of the mechanism of unresponsiveness produced by haptens
painted on skin exposed to low dose ultraviolet radiation. J. Exp. Med. 158:781.
10. Schwarz, T. 1999. UV radiation-induced tolerance. Allergy 54:1252.
11. Ariizumi, A., G. L. Shen, S. Shikano, S. Xu, R. Ritter 3rd, T. Kumamoto,
D. Edelbaum, A. Morita, P. R. Bergstresser, and A. Takashima. 2000. Identification of a novel, dendritic cell-associated molecule, dectin-1, by subtractive
cDNA cloning. J. Biol. Chem. 275:20157.
12. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams,
L. Martinez-Pomares, S. Y. Wong, and S. Gordon. 2002. Dectin-1 is a major
␤-glucan receptor on macrophages. J. Exp. Med. 196:407.
13. Taylor, P. R., G. D. Brown, D. M. Reid, J. A. Willment, L. Martinez-Pomares,
S. Gordon, and S. Y. Wong. 2002. The ␤-glucan receptor, dectin-1, is predominantly expressed on the surface of cells of the monocyte/macrophage and neutrophil lineages. J. Immunol. 169:3876.
14. Ariizumi, K., G. L. Shen, S. Shikano, R. Ritter 3rd, P. Zukas, D. Edelbaum,
A. Morita, and A. Takashima. 2000. Cloning of a second dendritic cell-associated
C-type lectin (dectin-2) and its alternatively spliced isoforms. J. Biol. Chem.
275:11957.
15. Bonkobara, M., P. K. Zukas, S. Shikano, S. Nakamura, P. D. Cruz Jr., and
K. Ariizumi. 2001. Epidermal Langerhans cell-targeted gene expression by a
dectin-2 promoter. J. Immunol. 167:6893.
16. Schwarz A., S. Grabbe, K. Grosse-Heitmeyer, B. Roters, H. Riemann,
T. A. Luger, G. Trinchieri, and T. Schwarz. 1998. Ultraviolet light-induced immune tolerance is mediated via the Fas/Fas-ligand system. J. Immunol. 160:4262.
17. Shimizu, T., and J. W. Streilein. 1994. Evidence that ultraviolet B radiation
induces tolerance and impairs induction of contact hypersensitivity by different
mechanisms. Immunology 82:148.
18. Aberer, W., G. Schuler, G. Stingl, H. Honigsmann, and K. Wolff. 1981. Ultraviolet light depletes surface markers of Langerhans cells. J. Invest. Dermatol.
76:202.
19. Grabbe, S., and T. Schwarz. 1998. Immunoregulatory mechanisms involved in
elicitation of allergic contact hypersensitivity. Immunol. Today 19:37.
20. Moodycliffe, A. M., D. Nghiem, G. Clydesdale, and S. E. Ullrich. 2000. Immune
suppression and skin cancer development: regulation by NKT cells. Nat. Immunol. 1:521.
21. Chatenoud L., B. Salomon, and J. A. Bluestone. 2001. Suppressor T cells–they’re
back and critical for regulation of autoimmunity! Immunol. Rev. 182:149.
22. Roncarolo, M. G., and M. K. Levings. 2000. The role of different subsets of T
regulatory cells in controlling autoimmunity. Curr. Opin. Immunol. 12:676.
23. Shevach, E. M., R. S. McHugh, C. A. Piccirillo, and A. M. Thornton. 2001.
Control of T-cell activation by CD4⫹CD25⫹ suppressor T cells. Immunol. Rev.
182:58.
24. Takahashi, T., T. Tagami, S. Yamazaki, T. Uede, J. Shimizu, N. Sakaguchi,
T. W. Mak, and S. Sakaguchi. 2000. Immunologic self-tolerance maintained by
CD25⫹CD4⫹ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J. Exp. Med. 192:303.
25. Read, S., V. Malmstrom, and F. Powrie. 2000. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25⫹CD4⫹ regulatory
cells that control intestinal inflammation. J. Exp. Med. 192:295.
26. Simon, J. C., H. Hara, R. W. Denfeld, and S. Martin. 2001. UVB-irradiated
dendritic cells induce nonproliferating, regulatory type T cells. Skin Pharmacol.
Appl. Skin Physiol. 15:330.
27. Schwarz, A., S. Grabbe, Y. Aragane, K. Sandkuhl, H. Riemann, T. A. Luger,
M. Kubin, G. Trinchieri, and T. Schwarz. 1996. Interleukin-12 prevents ultraviolet B-induced local immunosuppression and overcomes UVB-induced tolerance.
J. Invest. Dermatol. 106:1187.
28. Schmitt, D. A., L. Owen-Schaub, and S. E. Ullrich. 1995. Effect of IL-12 on
immune suppression and suppressor cell induction by ultraviolet radiation. J. Immunol. 154:5114.
29. Schwarz, A., S. Beissert, K. Grosse-Heitmeyer, M. Gunzer, J. A. Bluestone,
S. Grabbe, and T. Schwarz. 2000. Evidence for functional relevance of CTLA-4
in ultraviolet-radiation-induced tolerance. J. Immunol. 165: 1824.