Langerhans Cells Are Required for UVR-Induced ... - Core

ORIGINAL ARTICLE

Langerhans Cells Are Required for UVR-Induced Immunosuppression Agatha Schwarz1, Madelon Noordegraaf2,3, Akira Maeda4, Kan Torii4, Bjo¨rn E. Clausen2,3 and Thomas Schwarz1 Painting of haptens onto UVR-exposed skin does not result in sensitization but induces regulatory T cells (Treg). This was explained by UVR-mediated depletion of Langerhans cells (LCs). Furthermore, migration of UVRdamaged but still viable LCs into lymph nodes appears to be essential to induce Treg. Accordingly, the steroid mometasone, which kills LCs, inhibited sensitization but did not induce Treg. In Langerin-diphtheria toxin receptor knock-in (DTR) mice, LCs can be depleted by injection of diphtheria toxin (DT). LC-depleted mice could be sensitized though less pronounced than wild-type mice, but sensitization was not suppressed by UVR. Similarly, Treg did not develop. Langerin is not only expressed in LCs but also in some dermal dendritic cells (dDCs). Langerin-positive dDCs repopulate within 10 days after depletion, whereas LCs are still absent. Langerin-DTR mice treated with DT 10 days before UVR and sensitization were still resistant to UVR-induced inhibition of contact hypersensitivity (CHS). Similarly, Treg did not arise. As in this setting only LCs but not Langerin-positive dDCs are absent, LCs appear to be essential for both the suppression of CHS and the induction of Treg by UVR. This supports the concept that LCs are more important for the downregulation than the induction of immune responses in the skin. Journal of Investigative Dermatology (2010) 130, 1419–1427; doi:10.1038/jid.2009.429; published online 21 January 2010

INTRODUCTION UVR, in particular the mid-wave range (UVB, 290–320 nm), is well known for its immunosuppressive properties that mostly affect T cell-mediated immune reactions. UVRmediated immunosuppression is most convincingly demonstrated by the inhibition of contact hypersensitivity (CHS). Toews et al. (1980) observed that UVR inhibits the sensitization to contact allergens if they are applied directly onto the UVR-exposed skin areas. In addition, the very same animals could not be sensitized against the same hapten at a later time point without further UVR exposure. As other immune reactions were not suppressed, this suggested the development of hapten-specific tolerance (Toews et al., 1980). In those days, Langerhans cells (LCs) were considered as the exclusive antigen-presenting cells in the skin (Stingl et al., 1980). As LCs were found to be depleted on UVR exposure 1

Department of Dermatology, University Kiel, Kiel, Germany; 2Department of Immunology, Erasmus University Rotterdam, Rotterdam, The Netherlands; 3 Department of Cell Biology and Histology, Amsterdam University, Amsterdam, The Netherlands and 4Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Mizuho-ku, Nagoya, Japan Correspondence: Thomas Schwarz, Department of Dermatology, University Kiel, Schittenhelmstrasse 7, Kiel 24105, Germany. E-mail: [email protected] Abbreviations: CHS, contact hypersensitivity; CPD, cyclobutane pyrimidine dimer; dDC, dermal dendritic cell; DT, diphtheria toxin; Langerin-DTR mice, Langerin-diphtheria toxin receptor knock-in mice; LC, Langerhans cell; Treg, regulatory T cells; UVR-Treg, UVR-induced Treg Received 24 June 2009; revised 23 November 2009; accepted 30 November 2009; published online 21 January 2010

& 2010 The Society for Investigative Dermatology

(Toews et al., 1980; Aberer et al., 1981), inhibition of sensitization was explained simply by the absence of LCs. Today the situation appears less clear, as a variety of studies have challenged the exclusive role of LC as antigenpresenting cells of the skin (Allan et al., 2003; Bennett et al., 2005; Kaplan et al., 2005; Kissenpfennig et al., 2005). UVR-induced immunologic unresponsiveness can be adoptively transferred by injection of splenocytes and lymph node cells obtained from UVR-tolerized mice into naive mice, which subsequently cannot be sensitized against the respective hapten (Elmets et al., 1983). This indicated that UVR-induced tolerance is mediated through the induction of hapten-specific T suppressor cells, which are nowadays called regulatory T cells (Treg) (Schwarz, 2008). UVR-induced Treg (UVR-Treg), which suppress haptenmediated CHS, are quite well characterized. They express CD4 and CD25 (Schwarz et al., 2004), the negative regulatory molecule CTLA-4 (Schwarz et al., 2000), glucocorticoid-induced tumor necrosis factor receptor (Maeda et al., 2008a), and bind the lectin dectin-2 (Aragane et al., 2003). In addition, they secrete IL-10 on hapten-specific stimulation (Schwarz et al., 2000, 2004) and may use the apoptosis-related Fas/Fas-ligand system (Schwarz et al., 1998). As UVR-Treg suppress in an antigen-specific manner, they harbor therapeutic potential for the treatment of (auto)immune-mediated disorders. With regard to their antigen specificity, UVR-Treg differ from natural Treg (Sakaguchi et al., 2008) but also from immunosuppressive drugs that cause immunosuppression in a general manner. Therefore, we are interested (i) in www.jidonline.org 1419

A Schwarz et al. Langerhans Cells and UVR-Induced Immunosuppression

RESULTS Mometasone inhibits the induction of CHS but does not induce Treg

Topical corticosteroids are well known to effectively kill LC by inducing apoptosis (Grabbe et al., 1995; Hoetzenecker et al., 2004). Thus, we used the steroid derivative mometasone. Topical application of mometasone (0.1% for 4 days) depleted LC, as demonstrated by immunohistochemical staining of skin sheet preparations obtained 24 hours after the last steroid administration (Figure 1a). To differentiate whether mometasone causes depletion of LC by inducing emigration from the epidermis into the lymph nodes or simply by killing LC, draining lymph nodes were collected after mometasone application. Lymph node cell suspensions were stained with antibodies against Langerin and CD11c. In lymph nodes of untreated mice, a small number of Langerinpositive cells was detected (Figure 1b), confirming previous results (Schwarz et al., 2005). In contrast, no Langerinpositive cells were found in the lymph nodes on steroid application. This indicates that steroids, in contrast to lowdose UVR, deplete LC from the epidermis not through inducing emigration into the lymph nodes but by killing LC. Mice were sensitized by painting DNFB onto mometasone-pretreated skin. Ear challenge was performed 5 days later and ear swelling measured 24 hours thereafter. Positive control mice were sensitized through untreated skin and reacted with a pronounced ear swelling response upon challenge (Figure 2a). Negative control mice were only challenged without sensitization and did not react with ear swelling, excluding a toxic reaction. Mice that were sensitized with DNFB through mometasone-treated skin revealed a significantly reduced ear swelling response, indicating that topical steroids prevent efficient sensitization. To address whether Treg developed in the mometasonetreated mice, adoptive transfer experiments were performed. The above-mentioned experiment was repeated. Five days 1420 Journal of Investigative Dermatology (2010), Volume 130

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further characterizing UVR-Treg and (ii) in clarifying the mechanisms by which UVR-Treg are induced. The latter appears to be critically dependent on the migration of LC into the lymph nodes and the presence of UVR-induced DNA damage in the LC (Schwarz et al., 2005). This was confirmed by the observation that IL-12, which reduces UVR-mediated DNA damage (Schwarz et al., 2002; Meeran et al., 2006a, b), prevents the generation of UVR-Treg (Schmitt et al., 1995; Schwarz et al., 1996). These findings gave rise to the hypothesis that the appearance of UVR-damaged but still viable LC in the regional lymph nodes is an essential event during the generation of UVR-Treg. If this holds true, a stimulus that does not damage but definitely kills LC should prevent the induction of UVR-Treg. To clarify this issue we used the steroid mometasone in this study. To further substantiate the crucial role of LC for the generation of UVR-Treg, Langerin-diphtheria toxin receptor knock-in (Langerin-DTR) mice were used, in which LC can be depleted by the injection of diphtheria toxin (DT). We postulated that according to our hypothesis UVR-Treg should not develop in LC-depleted mice.

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Figure 1. Mometasone depletes LCs. (a) Mice were treated on their ears with 0.1% mometasone for 4 days. Twenty-four hours after the last mometasone application, ears were collected and ear sheets prepared. Ear sheets were stained with an antibody directed against major histocompatibility complex class II. Untreated mice served as controls (Co). Bars ¼ 200 mm. (b) Mometasone was applied on the shaved back for 4 days. Twenty-four hours after the last mometasone application, draining lymph nodes were obtained. Cell suspensions were stained with antibodies directed against Langerin and CD11c and subjected to FACS analysis.

after sensitization, spleen and lymph node cells were obtained from mometasone-treated donors and injected intravenously into naive mice. Recipients were sensitized against DNFB 24 hours after cell transfer, and ear challenge was performed 5 days later. Mice that had received cells obtained from mometasone-treated donors responded with a pronounced ear swelling response comparable to that of positive control mice (Figure 2b). Thus, Treg did not develop in mometasone-treated donors. Together, this indicates that mometasone similar to UVR prevents the induction of CHS but in contrast to UVR does not induce Treg. Langerin-DTR mice are resistant to UVR-induced immunosuppression

The data depicted in Figure 2 support our hypothesis that for the development of Treg, damaged but still viable LC are required. However, one cannot exclude the fact that mometasone affects not only LC but also other cells, which could influence the adoptive transfer experiments. To further elucidate the role of LC in the development of UVR-Treg, we used Langerin-DTR mice. These mice harbor a targeted insertion of the human DTR into the langerin locus (Bennett et al., 2005). As the cytotoxicity of DT is strictly dependent on receptor-mediated endocytosis (Naglich et al., 1992), and as murine cells are resistant to DT due to low affinity of the toxin for the rodent DTR, injection of DT into Langerin-DTR mice results in complete depletion of LC (Figure 3a). Thus, we postulated that if LCs are crucially required for the induction


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Figure 2. Mometasone inhibits the induction of CHS but does not induce Treg. (a) Mice were treated on their razor-shaved back skin with mometasone for 4 days. Twenty-four hours after the last application, mice were sensitized on their backs with DNFB (Mometasone). Five days after sensitization, mice were challenged with DNFB on the left ear. The ear swelling response was measured 24 hours later. Positive control mice (Pos Co) were sensitized and challenged, mice of the negative control (Neg Co) were challenged only. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. (b) Immediately after the measurement of ear swelling, spleens and lymph nodes were obtained from the mometasone-treated mice. Cells were injected intravenously into naive recipients (transfer). After 24 hours, recipients were sensitized against DNFB. Five days later, ear challenge was performed and ear swelling measured 24 hours later. Positive control mice (Pos Co) were sensitized and challenged, negative control mice (Neg Co) were challenged only. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. *Po0.001 versus Pos Co.

of UVR-Treg, they should not develop in DT-treated Langerin-DTR mice. Langerin-DTR mice were injected with DT. After 72 hours, mice were exposed to UVR (150 mJ cm2) on 4 consecutive days. After 24 hours, the last exposure animals were sensitized with DNFB through irradiated skin. After 5 days, animals were challenged on the left ear and ear swelling was measured 24 hours later. DT-treated Langerin-DTR mice, which were either sensitized and challenged or challenged only, were included as controls. Additional control groups included Langerin-DTR mice, which did not receive DT but

were otherwise treated identically. These mice behaved similar to wild-type animals (Figure 3c). The positive control mice responded with a significant ear swelling response to sensitization (#1) in comparison to negative control mice, which were challenged only (#2). Mice that were sensitized through skin exposed to low-dose UVR, which effectively depletes LC from the epidermis (Figure 3b), were significantly suppressed in their CHS response (#3, Figure 3c). Mice in which LC were depleted by DT injection could be sensitized (#4), although the sensitization response was weaker than in mice that did not receive DT (#1), thus confirming previous observations (Bennett et al., 2005, 2007). Surprisingly, UVRexposure of LC-depleted mice did not result in suppression of CHS (#6, Figure 3c). These mice revealed an equally pronounced ear swelling response as DT-treated mice, which were sensitized without UVR exposure (#4). Similar data were obtained when UVR exposure was initiated 24 hours instead of 72 hours after DT injection (data not shown). To determine whether UV-Treg developed in these mice, lymph nodes and spleens were obtained from the UVRexposed mice (#3, #6 of Figure 3c). Cells were injected intravenously into wild-type mice. Recipients were sensitized against DNFB 24 hours later. After 5 days, mice were challenged on the left ear and ear swelling was evaluated 24 hours later. Positive control mice comprised wild-type mice that were sensitized and challenged (Figure 4, #1), whereas negative control mice were challenged only (#2). Mice (#3) injected with cells from UVR-exposed donors, which were not treated with DT (UVR), revealed a remarkably suppressed CHS response, indicating that Treg had developed in the donors (Figure 4, #3). In contrast, recipients of cells from LC-depleted donors which were sensitized through UVR-exposed skin (UVR þ DT) responded with a significant ear swelling response to sensitization (Figure 4, #4). This indicates that in the LC-depleted donors Treg did not develop. Together, these data imply that UVRinduced immunosuppression is critically dependent on LC. As the rodent DT receptor exhibits only low affinity to DT, wild-type mice do not respond to DT (Bennett and Clausen, 2007). However, DT may be contaminated with toll-like receptor ligands and thus could affect the CHS response. To exclude this, C57BL/6 mice were treated with DT, UVexposed, and sensitized, as described above. Positive control mice were treated with DT, sensitized, and challenged (Figure 5, #1). Negative control mice were injected with DT but only challenged (#2). The ear swelling responses of positive control mice (#1) did not differ from those observed in wild-type mice (#4), which were sensitized but had not received DT (Figure 5). Similarly, suppression of the induction of CHS by UVR was not affected by DT in wildtype animals (#6). Langerin-positive dermal dendritic cells are not required for UVR-induced immunosuppression

Although Langerin was initially described as a specific and exclusive marker for LC (Valladeau et al., 2000; Takahara et al., 2002), it recently turned out that other cells than LC can express Langerin as well (Bursch et al., 2007; Ginhoux www.jidonline.org 1421


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Figure 4. UVR-Treg do not develop in LC-depleted mice. Immediately after measurement of ear swelling, spleens and regional lymph nodes were obtained from UVR-exposed Langerin-DTR mice demonstrated in Figure 2 (#3, #6). Single cell suspensions were prepared and cells were injected intravenously into naive C57BL/6 mice. Recipients were sensitized against DNFB 24 hours after injection (#3 UVR, #4 UVR þ DT). Five days later, ear challenge with DNFB was performed. Positive control mice (Pos Co) comprised C57BL/6 mice, which were sensitized and challenged (#1); negative control mice (Neg Co) were challenged only (#2). Ear swelling was measured 24 hours later. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. *Po0.005 versus Pos Co; **Po0.05 versus UVR þ DT; NS, not significant versus Pos Co.

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Figure 3. Sensitization is not suppressed by UVR in LC-depleted mice. (a) Langerin-DTR mice were injected with DT. After 72 hours, ears were obtained and epidermal sheets were stained with an antibody directed against major histocompatibility complex (MHC) class II. Untreated mice served as controls. Bars ¼ 100 mm. (b) Langerin-DTR mice were exposed to UVR on their ears. Twenty-four hours after the last irradiation, ears were obtained and epidermal sheets were stained with an antibody directed against MHC class II. Untreated mice served as controls. Bars ¼ 100 mm. (c) Langerin-DTR mice were injected with DT. After 72 hours, mice were UVR-exposed for 4 consecutive days (UVR, #6). Twenty-four hours after the last UVR exposure, mice were sensitized with DNFB painted onto UV-irradiated skin. After 5 days, animals were challenged on the left ear, and ear swelling was measured 24 hours later. Positive control mice comprised DT-treated Langerin-DTR mice, which were sensitized and challenged (DT, Pos Co, #4); negative control mice were challenged only (DT, Neg Co, #5). As additional controls, Langerin-DTR mice were treated identically but not injected with DT (w/o DT, #1–3). CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. *Po0.002 versus Pos Co; NS, not significant versus Pos Co.

Figure 5. DT does not affect CHS and UVR-induced immunosuppression in wild-type mice. C57BL/6 mice were injected with DT. After 72 hours, mice were exposed to UVR on their backs for 4 days. After 24 hours, the last exposure DNFB was painted onto the UVR-exposed skin. Ear challenge was performed 5 days later. Positive control mice (Pos Co) were injected with DT and sensitized and challenged with DNFB. Negative control mice (Neg Co) were injected with DT and challenged only. Ear swelling responses were compared with groups that were treated identically but did not receive DT (w/o DT). CHS was determined as the amount of swelling of the haptenchallenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103. *Po0.00005 versus Pos Co (w/o DT); **Po0.00002 versus Pos Co (DT).

et al., 2007; Poulin et al., 2007; Nagao et al., 2009). This applies mostly for dendritic cells in the dermis. There is evidence that these cells do not represent LCs emigrating from the epidermis but a separate subtype of dermal dendritic cells (dDCs). Thus, these cells are also eliminated in Langerin-DTR mice on injection of DT. Differences in the behavior of LCs and Langerin-positive dDCs have been

described. After depletion with DT, Langerin-positive dDCs repopulate within 10 days, whereas LCs are still absent (Bursch et al., 2007). Similar kinetic analyses were performed with our Langerin-DTR mice (M Noordegraaf et al., submitted). Forty-eight hours after DT injection, no LC and no dDC could be detected. By day 10, around 22% of the dDCs were found, but no LC. Fourteen days after injection, around

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DISCUSSION UVR-Treg represent a special subtype of Treg (Beissert et al., 2006). They differ from natural and induced Treg in many aspects (Kabelitz et al., 2006; Sakaguchi et al., 2008). In contrast to natural CD4 þ CD25 þ Treg, which suppress in a general manner, UVR-Treg suppress only when activated in an antigen-specific manner. In addition, UVR-Treg have been demonstrated not only to inhibit the induction but also the elicitation phase of CHS, provided they can reach the skin (Schwarz et al., 2004, 2007). Thus, UVR-Treg could represent a suitable tool not only to prevent but also to treat (auto)immune-mediated diseases. To achieve this aim, the mechanisms by which they are induced need to be elucidated.


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Figure 6. Dermal Langerin-positive cells are not involved in UVR-induced immunosuppression. Langerin-DTR mice were injected with DT. Ten days later, mice were UVR-exposed for 4 days (10d-DT, UVR, #6). Twenty-four hours after the last UVR exposure, mice were sensitized with DNFB painted onto UV-irradiated skin. After 5 days, animals were challenged on the left ear, and ear swelling was measured 24 hours later. Positive control mice comprised Langerin-DTR mice, which were injected with DT and sensitized against DNFB 14 days after injection (10d-DTR, Pos Co, #4); negative control mice were treated identically but not sensitized and challenged only (10dDTR, Neg Co, #5). As additional controls, Langerin-DTR mice were treated identically but not injected with DT (w/o DT, #1–3). CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. *Po0.0005 versus Pos Co (w/o DT); NS, not significant versus Pos Co (10d-DTR).

10 NS

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25% of the dDCs had reappeared and a few LC could be visualized, as previously reported (Bennett et al., 2005; Nagao et al., 2009). To discern the role of Langerin-positive dDCs and LCs in UVR-induced immunosuppression, Langerin-DTR mice were treated with DT 10 days before UVR exposure (10d-DT). Ten days were selected as according to our kinetic analysis, it is a time point at which dDCs but no LC had reappeared. Mice were exposed to UVR and sensitized as described above. As expected, untreated UVR-exposed Langerin-DTR mice that did not receive DT (Figure 6, #3) were suppressed in their sensitization response when compared with positive control mice (#1). Langerin-DTR mice that were treated with DT 10 days before the treatment still revealed a significant ear swelling response (#4) when compared with negative control mice (#5). However, the response was still ameliorated when compared with Langerin-DTR mice that did not receive DT (#1). This indicates that Langerin-positive dDCs, which meanwhile have repopulated the skin, cannot fully compensate for an efficient sensitization response. Even more important, 10d-DT Langerin-DTR mice that were sensitized through UVR-exposed skin (#6) were not at all suppressed in their sensitization response when compared with positive control mice (#4). This indicates that UVR-induced inhibition of the induction of CHS does not depend on Langerin-positive dDCs but on LCs. To address whether Treg developed, splenocytes and lymph node cells were obtained from the UVR-exposed mice, displayed in Figure 6 (#3, #6). Cells were injected intravenously into naive wild-type mice that were sensitized 24 hours after injection. Challenge was performed 5 days later. As demonstrated in Figure 7, recipients that had received cells from UVR-exposed donors that were not treated with DT were suppressed in their sensitization response (#3), indicating the development of UV-Treg in the donors (#3, Figure 6). In contrast, mice that had received cells from donors injected with DT 10 days before UVR exposure were not suppressed in their sensitization response (Figure 7, #4). This indicates that UVR-Treg had not developed in the donors (#6, Figure 6). As in these donors dDCs are already present but LCs still absent, one can conclude that LCs are required for the development of UVR-Treg.

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Figure 7. Dermal Langerin-positive cells are not involved in the development of UVR-Treg. Immediately after measurement of ear swelling, spleens and regional lymph nodes were obtained from the UVR-exposed Langerin-DTR mice shown in Figure 4 (#3, #6). Single cell suspensions were prepared, and cells were injected intravenously into naive C57BL/6 mice. Recipients were sensitized against DNFB 24 hours after injection (#3, #4). Five days later, ear challenge with DNFB was performed. Positive control mice (Pos Co) comprised C57BL/6 mice, which were sensitized and challenged (#1); negative control mice (Neg Co) were challenged only (#2). Ear swelling was measured 24 hours later. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103 (mean±SD of one representative of three independent experiments). Each group consisted of at least six mice. *Po0.005 versus Pos Co; NS, not significant versus Pos Co.

We have recently proposed that the appearance of damaged but still viable LC in the regional lymph nodes is required for the development of UVR-Treg (Schwarz et al., 2005). This was based on the observation that www.jidonline.org 1423


Langerin-positive cells harboring UVR-specific DNA damage (cyclobutane pyrimidine dimers, CPDs) could be detected in mice that were sensitized through UVR-exposed skin and subsequently developed UVR-Treg. On injection of IL-12, which prevents the induction of UVR-Treg (Schmitt et al., 1995; Schwarz et al., 1996), Langerin-positive cells were still detected in the lymph nodes but did not contain CPDs; consequently these mice did not develop UVR-Treg. The reducing effect of IL-12 on the amounts of CPDs appears to be mediated by nucleotide excision repair, the major repair system for UVR-induced DNA damage, as IL-12 did not reduce CPDs in xeroderma pigmentosum A knock-out mice, which are deficient in nucleotide excision repair (de Vries et al., 1995). Similarly, IL-12 did not prevent the induction of UVR-Treg in xeroderma pigmentosum A knock-out mice. This provided a first link between UVR-induced DNA damage and the induction of UVR-Treg. In this context, it is important to mention that UVR-induced DNA damage has been favoured as the major molecular trigger for UVR-induced immunosuppression for quite a long time (Kripke et al., 1992; Nishigori et al., 1996). It is striking that UVR-induced immunosuppression is caused by rather low UVR doses (Toews et al., 1980) that are suberythemogenic and thus in the physiological range. For a long time it was thought that UVR depletes LC from the epidermis simply by killing LCs. This does not appear to be the case. The vast majority of LCs emigrate from the epidermis on application of such UVR doses and can be found in the regional lymph nodes (Vink et al., 1996; Schwarz et al., 2005). Initially, it was thought that Langerin is a specific marker for LCs (Valladeau et al., 2000; Takahara et al., 2002), which is not the case as a distinct subtype of dDC was also found to express Langerin (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007; Nagao et al., 2009). Hence, the appearance of Langerin-positive cells in the lymph nodes does not automatically imply that these are exclusively LCs, as they could also represent Langerinpositive dDCs. CPDs, the major DNA damage induced by the mid-wave UVR spectrum (UVB, 290–320 nm), can be detected by a specific antibody. The major part of UVB is absorbed in the epidermis and only a minor fraction penetrates into the dermis. Thus, cells found in the lymph nodes containing high levels of CPDs most likely originate from the epidermis. As these cells coexpress Langerin, they highly likely represent LCs. Antigen-specific Treg can also be induced by extracorporeal photopheresis. In this setting, mononuclear cells obtained from hapten-sensitized mice are exposed in vitro to long wave UVR (UVA, 320–400 nm) in combination with the photosensitizer 8-methoxypsoralen (Dani and Knobler, 2009). Infusion of these cells induces hapten-specific Treg in the recipients (Maeda et al., 2005, 2008b). Extracorporeal photopheresis-induced Treg are less well characterized than UVR-Treg, but as of today they appear to be quite similar. It is known that the combination of UVA plus 8-methoxypsoralen induces apoptosis (Enomoto et al., 1997). However, CD11 þ cells that have been shown to be essential for the induction of Treg (Maeda et al., 2005) undergo apoptosis on UVA plus 1424 Journal of Investigative Dermatology (2010), Volume 130

8-methoxypsoralen exposure much later than T cells; they die only after 72 hours. Hence, in the setting of extracorporeal photopheresis, viable but certainly damaged dendritic cells are infused. This gave rise to the hypothesis that damaged but still viable antigen-presenting cells are crucial for the development of Treg and that the same may apply for the induction of UVR-Treg. This suggested that the induction of UVR-Treg is an active process that requires viable antigen-presenting cells which, however, are damaged and thus impaired in their antigenpresenting function. This does not result in priming and the development of effector T cells but in tolerance through induction of Treg. We postulated that, if this hypothesis is correct, a stimulus that kills LCs should not induce Treg. An obvious option to test this postulate would be to increase the UVR dose beyond the physiological range, which then would immediately kill LCs. However, this experiment cannot be performed as it is known that higher doses of UVR induce the release of immunosuppressive cytokines, such as IL-10 from keratinocytes, which then causes systemic immunosuppression (Ullrich and Schmitt, 2000). Under such conditions, CHS is also suppressed and Treg develop as well. Therefore, we used as an alternative stimulus topical steroids, which are known to induce apoptosis of LCs (Hoetzenecker et al., 2004). Accordingly, no LCs were detected in ear sheets after mometasone application and no Langerin-positive cells were found in the regional lymph nodes. Hence, we hypothesized that steroids due to killing of LCs will prevent sensitization but in contrast to UVR not induce Treg. This appears to be the case. Application of DNFB onto mometasone-pretreated skin did not result in sensitization and Treg failed to develop as demonstrated by adoptive transfer experiments. However, one could argue that mometasone does not exclusively kill LCs and may affect also other cells both in the epidermis and dermis. Although supporting our hypothesis this experimental approach did not definitely prove that LCs are required for the development of UVR-Treg. Hence, we used Langerin-DTR mice. We surmised that if LCs are crucial for the development of UVR-Treg, LangerinDTR mice should not develop Treg on UVR exposure. First, we had to check whether Langerin-DTR mice could be sensitized against DNFB. This was the case, though the response was weaker than in wild-type mice as reported previously (Bennett et al., 2005, 2007). Topical steroids also inhibited sensitization in LC-depleted mice (data not shown). Most surprisingly Langerin-DTR mice were not at all suppressed in their sensitization response on UVR exposure. The response was equal to that of LC-depleted positive control mice, which were only sensitized and challenged. Similarly, UVR-Treg did not develop, confirming the hypothesis that LCs are essentially required for the development of UVR-Treg. However, Langerin is not any longer accepted as an exclusive marker for LCs. It has turned out that Langerin is also expressed by dDC (Merad et al., 2008). On injection with DT, the latter cells are depleted as well. Thus, involvement of dDC in the induction of UVR-Treg could not be excluded. On depletion by DT, dDC repopulate within


10 days, whereas LCs appear in the epidermis after several weeks (Bursch et al., 2007; Ginhoux et al., 2007; Poulin et al., 2007; Nagao et al., 2009; M Noordegraaf et al., submitted). Because of these differential kinetics, it is possible to discern functions of Langerin-positive dDCs and LCs. Langerin-DTR mice were injected with DT 10 days before UVR exposure. Under these conditions, mice already harbor Langerin-positive dDCs but not LCs when exposed to UVR. If dDCs suffice to induce UVR-Treg, cells obtained from such mice should suppress in an adoptive transfer experiment. However, this was not the case. Ten day-DT Langerin-DTR mice behaved identically as compared with Langerin-DTR mice that received DT immediately before UVR exposure. This indicates that LCs are relevant for the induction of UVRTreg. In addition, inhibition of the induction of CHS by UVR was not observed in 10d-DT Langerin-DTR mice. This implies that not only the induction of UVR-Treg but also the inhibition of the induction of CHS is critically dependent on LCs. During the submission of this manuscript, a paper was published which, in contrast to our findings, reported that LCs are not involved in UVR-induced immunosuppression (Wang et al., 2009). However, this publication only referred to the suppression of the induction of CHS by UVR. Adoptive transfer experiments were not performed. Hence, the role of LCs in the induction of Treg remains unclear in this paper. For the depletion of LC, Langerin-DTR knock-in mice were used (Kissenpfennig et al., 2005). In most of the experiments, epicutaneous sensitization against ovalbumin and transfer of OT-I cells were used as an immunological model. There is ample evidence that this system cannot be compared with the hapten-induced CHS (Herrick et al., 2003). Only two experiments were performed with a similar CHS model as we used. However, lower UVR doses and lower hapten concentrations were applied, which might account for the differences in the results. In addition, the authors claimed that UVR does not deplete LC, which is quite in contrast to the literature (Ko¨lgen et al., 2003). The presence of LC was monitored in epidermal cell suspensions but not evaluated in ear sheets. Probably, the UVR dose applied was too low to deplete the LC in this setting. Taken together, it appears that the discrepancies to our results may be mostly due to the differences in the methods. For a long time, LCs have been considered as the exclusive antigen-presenting cells of the skin (Romani and Schuler, 1992). This concept, however, has recently changed quite dramatically. There is accumulating evidence that immune responses can be generated in the skin in the absence of LC (Bennett et al., 2005; Kaplan et al., 2005; Kissenpfennig et al., 2005) and that antigen-presenting cells in the dermis can be more relevant than LCs (Allan et al., 2003; Ritter et al., 2004). Hence, the concept arose that LCs are more involved in downregulating immune responses (Kaplan et al., 2008; Udey and Rosenberg, 2008). From a clinical point of view this would make sense, as T cell responses in the skin appear to be more hazardous than protective. The vast majority of inflammatory dermatoses is T cell-driven (Robert and Kupper, 1999). Patients with T cell defects do not suffer from major

skin infections, as the innate immune system is much more relevant for the skin defense. Accordingly, in contrast to the adaptive, the innate immune response is induced by UVR as recently demonstrated by the induction of antimicrobial peptides, an essential component of the innate immune system (Gla¨ser et al., 2009). In contrast, the skin is constantly exposed to contact allergens and represents an organ prone to autoimmunity (Seery et al., 1997; Mehling et al., 2001). Hence, a constant downtuning of the adaptive immune system may be beneficial (Schwarz, 2010). This may be achieved by physiological UVR doses and, as confirmed by the present findings, LCs appear to be an essential part of this regulatory system. MATERIALS AND METHODS Mice and reagents Eight- to ten-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). Langerin-DTR mice were generated by B.E. Clausen (Bennett et al., 2005) and bred in the central animal facilities of the University Clinics Schleswig-Holstein, Campus Kiel. To deplete Langerin-positive cells, mice were injected with DT (Sigma–Aldrich, Taufkirchen, Germany) intraperitoneally and subcutaneously (400 ng each). Mometasone was obtained from Essex Pharma (Munich, Germany), and applied on razor-shaved skin (0.1% for 4 days). Animal care was provided by expert personnel in compliance with the relevant laws and institutional guidelines.

UVR-induced immunosuppression UVR-induced immunosuppression was induced by irradiating the shaved backs of mice with 150 mJ cm2 UVR daily on 4 consecutive days. UV irradiation was performed using a bank of six fluorescent bulbs (TL12, Philips, Eindhoven, The Netherlands), which emit most of their energy within the UVB range (290–320 nm) with an emission peak at 313 nm. After 24 hours of the last UVR exposure, mice were sensitized by painting 50 ml of DNFB (Sigma–Aldrich) solution (0.5% in acetone/olive oil, 4:1) on their backs. Five days after sensitization, mice were challenged with 20 ml of 0.3% DNFB on the left ear. The ear swelling response was quantified with a spring-loaded micrometer 24 hours later. Positive control mice were sensitized and challenged. Negative controls were challenged only. CHS was determined as the amount of swelling of the hapten-challenged ear compared with the thickness of the vehicle-treated ear and expressed in cm 103.

Generation of UVR-Treg Mice were sensitized against DNFB through UVR-exposed skin as described above. After challenge on day 5 and measurement of ear swelling on day 6, spleens and regional lymph nodes were removed and single cell suspensions prepared. 1 108 cells were injected intravenously through tail veins into naive wild-type mice. Recipients were sensitized against DNFB 24 hours after injection and ear challenge was performed 5 days later. After 24 hours, ear swelling was measured.

Staining of ear sheets and FACS analysis Ears were obtained and mechanically split into dorsal and ventral sides. Sheets were floated on phosphate-buffered saline (PBS) containing 20 mM EDTA (pH 7.4) at 37 1C for 3.5 hours. After www.jidonline.org 1425


washing with PBS, the dermis was removed and the epidermal sheets were spread floating on PBS in a Petri dish and subsequently fixed in acetone for 20 minutes at 20 1C. After briefly washing in PBS, epidermal sheets were blocked with PBS supplemented with 1% fetal calf serum for 1 hour at room temperature. Subsequently a phycoeryhtrin-conjugated mAb recognizing IA/IE (murine major histocompatability complex class II) (clone M5/114, Becton Dickinson, Franklin Lakes, NJ) was added (dilution 1:100) and incubated overnight at 4 1C. An appropriate isotype control was included. After washing in PBS for 1 hour at room temperature, ear sheets were mounted using Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany). Immunofluorescence staining was analyzed using a Zeiss Axiovert 40 CFL microscope (Carl Zeiss, Go¨ttingen, Germany). Twenty-four hours after the last mometasone application draining lymph nodes were obtained. Cell suspensions were stained with FITC-conjugated anti-CD11c mAb (BD Bioscience, San Diego, CA). Subsequently, cells were fixed and permeabilized using BD cytofix/ cytoperm plus kit (BD Bioscience) according to the manufacturer’s instruction, followed by intracellular staining with an allophycocyanin-conjugated anti-Langerin antibody (clone 929F3.01, Dendritics, Lyon, France). Cells were analyzed by an FC 500 CXP FACS analyzer (Beckman Coulter, Krefeld, Germany).

Statistics

de Vries A, van Oostrom CT, Hofhuis FM et al. (1995) Increased susceptibility to ultraviolet-B and carcinogens of mice lacking the DNA excision repair gene XPA. Nature 377:169–73 Elmets CA, Bergstresser PR, Tigelaar RE et al. (1983) Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation. J Exp Med 158:781–94 Enomoto DN, Schellekens PT, Yong SL et al. (1997) Extracorporeal photochemotherapy (photopheresis) induces apoptosis in lymphocytes: a possible mechanism of action of PUVA therapy. Photochem Photobiol 65:177–80 Ginhoux F, Collin MP, Bogunovic M et al. (2007) Blood-derived dermal langerin+ dendritic cells survey the skin in the steady state. J Exp Med 204:3133–46 Gla¨ser R, Navid F, Schuller W et al. (2009) UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo. J Allergy Clin Immunol 123:1117–23 Grabbe S, Steinbrink K, Steinert M et al. (1995) Removal of the majority of epidermal Langerhans cells by topical or systemic steroid application enhances the effector phase of murine contact hypersensitivity. J Immunol 155:4207–17 Herrick CA, Xu L, McKenzie AN et al. (2003) IL-13 is necessary, not simply sufficient, for epicutaneously induced Th2 responses to soluble protein antigen. J Immunol 170:2488–95 Hoetzenecker W, Meingassner JG, Ecker R et al. (2004) Corticosteroids but not pimecrolimus affect viability, maturation and immune function of murine epidermal Langerhans cells. J Invest Dermatol 122:673–84 Kabelitz D, Wesch D, Oberg HH (2006) Regulation of regulatory T cells: role of dendritic cells and toll-like receptors. Crit Rev Immunol 26:291–306

Data were analyzed by Student’s t-test. Differences were considered significant at Po0.05.

Kaplan DH, Jenison MC, Saeland S et al. (2005) Epidermal langerhans celldeficient mice develop enhanced contact hypersensitivity. Immunity 23:611–20

CONFLICT OF INTEREST

Kaplan DH, Kissenpfennig A, Clausen BE (2008) Insights into Langerhans cell function from Langerhans cell ablation models. Eur J Immunol 38:2369–76

The authors state no conflict of interest.

ACKNOWLEDGMENTS We are grateful to Martina Wedler, Susanne Dentel, and Nadine Tu¨xen for excellent technical assistance and to Dr Christian Jantschitsch for technical support in staining ear sheets. This study was supported by grants from the German Research Foundation (SCHW 1177/1-3; SFB 415/A16, SCHW 625/4-1) and the Landsteiner Foundation for Blood Transfusion Research (LSBR, 0414F to B.E.C.). B.E. Clausen is a VIDI fellow of the Netherlands Organization for Scientific Research (NWO, 917-76-365).

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