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Molecular mechanisms of ultraviolet radiation-induced immunosuppression
European Journal of Cell Biology 90 (2011) 560–564
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
Review
Molecular mechanisms of ultraviolet radiation-induced immunosuppression Thomas Schwarz ∗ , Agatha Schwarz Department of Dermatology, University Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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Article history: Received 18 August 2010 Received in revised form 3 September 2010 Accepted 20 September 2010 Keywords: Contact hypersensitivity DNA damage DNA repair Immunosuppression Immunotolerance Interleukins Langerhans cells Photoimmunology Regulatory T cells Ultraviolet radiation
a b s t r a c t Solar ultraviolet radiation (UVR) is well known for its immunosuppressive properties. UVR can suppress immune reactions both in a local and a systemic fashion. One of the major molecular mediators of photoimmunosuppression is UVR-induced DNA damage. In contrast to immunosuppressive drugs, UVR does not act in a general but antigen-specific fashion. This is due to the induction of regulatory T cells. Epidermal Langerhans cells harboring UVR-induced DNA damage appear to be essentially involved in the induction of these cells. Cytokines including interleukin (IL)-12, -18 and -23 exert the capacity to reduce UVRinduced DNA damage via induction of DNA repair. Accordingly, these cytokines prevent UVR-mediated immunosuppression. In contrast to IL-18, IL-12 and IL-23 can also inhibit the suppressive activity of regulatory T cells by a mechanism which still needs to be determined. Clarification of the molecular mechanisms underlying UVR-induced immunosuppression will help to develop new immunosuppressive therapeutic strategies by utilizing UVR-induced regulatory T cells for the treatment of immune-mediated diseases. In addition, these insights will contribute to a better understanding of photocarcinogenesis since suppression of the immune system by UVR essentially contributes to the induction of skin cancer. © 2010 Elsevier GmbH. All rights reserved.
Besides its undisputed beneficial and indispensable effects on human life, solar/ultraviolet radiation (UVR) can be a hazard to human health by inducing skin cancer, premature skin aging, inflammation and cell death (Fisher et al., 1997; Gilchrest, 1990; Kraemer, 1997; Kulms and Schwarz, 2000). This refers mostly to the middle wave length range (290–320 nm, UVB). Over the last three decades it became clear that UVB radiation exerts the capacity to suppress the immune system. UVR inhibits immune reactions locally, but can also affect the immune system in a systemic fashion, provided higher UVR doses are given (de Gruijl, 2008). In contrast to immunosuppressive drugs which suppress the immune system in a general fashion, UVR acts in an antigen-specific fashion. This is mostly due to the generation of T cells which specifically suppress immune reactions. These cells were initially called T suppressor cells but recently were renamed regulatory T cells (Schwarz, 2008). Because of their specific activity they harbor therapeutic potential for the treatment of (auto)immune-mediated diseases. In addition, immunosuppression by UVR plays a crucial role in the induction of UVR-mediated skin cancer, one of the most rapidly growing cancers worldwide (Hanneman et al., 2006). Hence elucidation of the
Abbreviations: CHS, contact hypersensitivity; IL, interleukin; LC, Langerhans cell; NER, nucleotide excision repair; Treg, regulatory T cell; UVR, ultraviolet radiation; UVR-Treg, UVR-induced Treg; wt, wild type. ∗ Corresponding author. Tel.: +49 431 597 1500; fax: +49 431 597 1503. E-mail address: [email protected] (T. Schwarz). 0171-9335/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2010.09.011
molecular mechanisms involved will tremendously contribute to the understanding of photocarcinogenesis and elucidate strategies to more effectively prevent and treat these tumors. In addition, a better phenotypical and functional characterization of regulatory T cells (Treg) and a detailed insight into the mechanisms of their induction by UVR will help to utilize this T cell subtype in a therapeutic setting. The best model to study UVR-induced immunosuppression is the suppression of the induction of contact hypersensitivity (CHS) by UVR. Topical application of potent contact allergens results in antigen-specific sensitization which can be visualized by a specific swelling response upon application of the same allergen on the ear a few days later. Sensitization is suppressed and prevented, respectively, when the contact allergen is painted onto skin which has been preexposed to low doses of UVR (Toews et al., 1980). The very same animals cannot be sensitized against the same allergen at later time points, indicating the induction of immunotolerance. This long-term suppression is antigen-specific since all other immune responses are not affected. This immunotolerance is mediated via antigen-specific Treg which can be demonstrated by adoptive transfer experiments (Elmets et al., 1983). UV-induced Treg (UVR-Treg) have been quite well characterized. They express CD4 and CD25, the negative regulatory molecule CTLA-4, GITR, neuropilin and secrete upon antigen-specific stimulation interleukin (IL)-10 (Schwarz, 2008). Thus they appear to represent a certain subtype of CD4+ CD25+ Treg.
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IL-12, UVR-induced DNA damage and photoimmunosuppression UVR-induced DNA damage has been recognized as the major molecular trigger for UVR-induced immunosuppression (Kripke et al., 1992). UVR induces specific DNA lesions, preferentially cyclobutane pyrimidine dimers and 6–4 photoproducts. Accelerated removal of DNA lesions by exogenous DNA repair enzymes resulted in reduction of UVR-induced immunosuppression, indicating that DNA damage is a crucial molecular mediator in this process (Kripke et al., 1992). IL-12 was the first cytokine being demonstrated to exert the capacity to prevent UVR-induced immunosuppression (Müller et al., 1995; Schmitt et al., 1995; Schwarz et al., 1996). Inhibition of the induction of CHS was prevented upon intraperitoneal injection of IL-12. Likewise Treg were not induced in UVR-exposed mice when they were treated with IL-12. In addition, IL-12 exerts the capacity to inhibit the suppressive activity of Treg since injection of IL-12 into already UVR-tolerized mice enables sensitization of these animals. Furthermore, UVR-Treg lose their suppressive activity upon coincubation with IL-12. How IL-12 inhibits Treg is still unclear. IL-12 was found to exert the capacity to reduce UVR-induced DNA damage (Schwarz et al., 2002) since IL-12 reduced both in vitro and in vivo UVR-induced apoptotic cell death which is mostly driven by the amounts of UVR-induced DNA lesions (Kulms et al., 1999). IL-12 appears to reduce UVR-induced DNA damage via induction or activation of the nucleotide excision repair (NER), the major endogenous DNA repair system, since the effect of IL-12 on UVRinduced cell death was not observed in Xpa knock-out mice which are deficient in NER (Schwarz et al., 2002). Accordingly, IL-12 was not able to prevent UVR-induced immunosuppression, confirming that UVR-induced DNA damage is an essential molecular mediator of immunosuppression caused by UVR (Schwarz et al., 2005). Although the mechanism by which IL-12 affects the NER is still unclear, this observation was quite surprising since until then it was thought that the NER as an essential repair mechanism is just constitutively expressed and not subjected to any regulation. However, Eller et al. (1997) first demonstrated that administration of DNA oligonucleotides induces DNA repair via a p53-dependent mechanism. Accordingly, a recent in vivo study indicated that topical pretreatment with DNA oligonucleotides enhanced the rate of DNA photoproduct removal, decreased UVR-induced mutations, and reduced photocarcinogenesis in UV-irradiated mice (Goukassian et al., 2004). Meanwhile, a variety of additional factors which might influence the NER have been described, including cytokines (IL-18, IL-23 see below), neuropeptides (Böhm et al., 2005), infrared radiation (Jantschitsch et al., 2009) and even vitamin D (Trémezaygues et al., 2009). However, with the exception of IL-12 (Meeran et al., 2006a), these effects have not yet been confirmed by additional independent groups. A hallmark feature of UVR-induced immunosuppression is the depletion of Langerhans cells (LCs) from the epidermis upon UVR exposure (Toews et al., 1980). For a long time it was assumed that UVR eliminates LCs by inducing cell death. This, however, does not appear to be the case, since first the UVR doses required to induce immunosuppression are rather low and second LCs can be found in the regional lymph nodes, indicating that UVR induces emigration of LCs from the epidermis (Schwarz et al., 2005). Injection of IL-12 prevents the emigration of LCs, implying that again UVRinduced DNA damage is the molecular trigger for the emigration. This appears to be the case since IL-12 does not prevent the emigration in Xpa knock-out mice. LCs found in the lymph nodes upon UVR exposure carry significant amounts of DNA damage. Upon injection of IL-12 the amounts of DNA damage in the LCs located in the lymph nodes is significantly reduced. This leads to the hypothesis that UVR-damaged but still viable LCs in the lymph nodes are required
Fig. 1. Effects of IL-12, IL-23 and IL-18 on UVR-induced immunosuppression. IL-12 and IL-23 as well as IL-18 prevent UVR-induced immunosuppression via modulation of DNA repair. Accordingly, suppression of the induction of contact hypersensitivity by UVR, which is mediated via DNA damage, is not observed upon injection of either IL-12, IL-23 or IL-18. In contrast, established UVR-mediated immunotolerance can only be broken by IL-12 and IL-23 but not by IL-18, indicating that IL-12 and IL-23 exert more pronounced immunostimulatory features than IL-18. Though being primarily a proinflammatory cytokine, IL-18 prevents UVR-induced immunosuppression via its capacity to induce DNA repair. Adapted from Schwarz (2008).
for the induction of Treg (Schwarz et al., 2005). One could imagine that the damaged LCs are still able to present the antigen but in an impaired manner which ultimately does not induce T effector cells but Treg. IL-18, another cytokine reducing UVR-mediated DNA damage It turned out that the capacity to reduce DNA damage is not a unique feature of IL-12 but can be achieved also by other mediators including IL-18 (Schwarz et al., 2006). IL-18 is a proinflammatory cytokine which exhibits the unique capacity to induce T helper 1 or T helper 2 polarization, depending on the immunologic context (Reddy, 2004). Although not related to IL-12 structurally, IL-18 shares some biological effects with IL-12, e.g. the induction of interferon-␥ (Okamura et al., 1998; Dinarello and Fantuzzi, 2003). IL-18 was found to reduce UVR-induced apoptosis in a similar fashion like IL-12. This effect was not observed in DNA repair deficient mice, implying that IL-18 reduces UVR-induced DNA damage via the NER. Accordingly IL-18 also prevented UVR-induced immunosuppression in wild type but not in DNA repair-deficient mice (Schwarz et al., 2006). However, in contrast to IL-12, IL-18 was not able to break UVR-induced immunotolerance which is mediated in a DNA damage-independent fashion via UVR-Treg (Schwarz et al., 2006). This indicated that IL-18 though being primarily a proinflammatory cytokine through the capacity to affect DNA repair can restore an immune response which is suppressed by UVR. These findings also identified IL-12 as still unique in its capacity to restore immune responses because of its effect on UVR-Treg (Fig. 1). IL-23 prevents UVR-induced immunosuppression but also inhibits regulatory T cells IL-23 is a heterodimeric cytokine consisting of a p40 and p19 chain. Although it is closely related to IL-12 by sharing the same p40 subunit, IL-23 exerts also different biological activities than IL-12 (Kastelein et al., 2007). The effects of IL-23 have been mainly linked to a T helper 17 cell response (Mills, 2008). Because of the structural similarity but the different biological effects between IL-12 and IL-23 it was obvious to study the effects of IL-23 on UVR-induced DNA damage and immunosuppression. Incubation of cultured keratinocytes with IL-23 before UVR exposure significantly reduced
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the apoptosis rate (Majewski et al., 2010). This was associated with a reduction of DNA damage as demonstrated by Southwestern dot blot analysis. Injection of IL-23 into UVR-exposed mice diminished the number of apoptotic keratinocytes and the amounts of DNA damage. This was not observed in DNA repair-deficient Xpa knockout mice, implying that IL-23 reduces DNA damage via induction of DNA repair. Based on the observations with IL-12 and IL-18 it was predicted that any cytokine which reduces UVR-induced DNA damage should also prevent photoimmunosuppression. This was the case since injection of IL-23 rendered UVR-exposed mice fully susceptible to sensitization. Accordingly, Treg did not develop upon injection of IL-23 in UV-irradiated mice. IL-23 failed to prevent UVR-mediated suppression in Xpa knock-out mice, confirming the involvement of NER. However, in contrast to IL-18, IL-23 also inhibited the activity of UVR-Treg (Majewski et al., 2010). UVR-tolerized mice could be fully sensitized upon injection of IL-23. In addition, UVR-Treg lost their suppressive activity upon incubation with IL-23. Together, this indicates that IL-23 as IL-12 and IL-18 can reduce UVR-induced DNA damage and thereby prevents immunosuppression. However, IL-23 shares with IL-12 the still unique capacity to restore suppressed immune responses because of its effect on Treg (Fig. 1). By which mechanisms IL-12 and IL-23 exert this effect still remains to be determined as well as the question whether they achieve this effect through the same or different pathways. Enhanced photocarcinogenesis in the absence of IL-12 The observation that IL-12 reduces UVR-induced DNA damage via induction of the NER led to the hypothesis that in turn the absence of IL-12 should lead to enhanced amounts of DNA damage and thus support carcinogenesis. To address this issue, we utilized IL-12 knock-out mice which lacked the p40 chain (Magram et al., 1996). IL-12p40 knock-out and wild type (wt) mice were exposed 3×/week to UVR. Skin biopsies obtained after 6 weeks revealed significantly increased numbers of sunburn cells in the knock-out mice (Maeda et al., 2006). Staining of epidermal sheets with an antibody directed against the tumor suppressor gene p53 revealed a higher number of p53 patches in the skin of knock-out mice. After around 200 days first skin tumors developed. Kaplan–Meier analysis indicated a significantly increased probability of tumor development in the knock-out mice. In addition, the number of tumors developing in the individual mice was significantly higher in the knock-out than in wt mice. Furthermore, the tumors in the knock-out mice revealed a more aggressive growth behavior (Maeda et al., 2006). Hence it was concluded that a lack of IL-12 might enhance photocarcinogenesis. However, nowadays this conclusion has to be modified. Since IL-23 also shares the p40 chain these knock-out mice were not deficient in IL-12 as initially anticipated but also in IL-23. Studies utilizing p35 and p19 knock-out mice are currently ongoing. This will clarify whether IL-12, IL-23 or both cytokines exert a protective role during photocarcinogenesis. A recent study confirmed the protective effect of IL-12 by showing p35-deficient mice to be more susceptible to photocarcinogenesis (Meeran et al., 2006b). Defining a new role of Langerhans cells For a long time LCs were suggested to be the most relevant, even the only real antigen-presenting cell of the skin (Stingl et al., 1980). Initial evidence for this assumption was provided by the observation that CHS could not be induced in skin areas that were devoid of LCs (tail, cornea) (Toews et al., 1980). The early photoimmunology studies supported this concept since in those days it was assumed that UVR inhibits sensitization via killing LCs.
Fig. 2. Crosstalk between DNA damage, DNA repair and cytokines. UVR-induced DNA damage is a major molecular trigger for a variety of biologic UVR effects, including the release of cytokines. By their ability to reduce DNA damage, presumably via induction of DNA repair various cytokines may inhibit or reduce some biologic effects of UVR. Taken from Schwarz and Schwarz (2009).
However, other antigen-presenting cells must be able to replace LCs, since transgenic mice in which LCs were completely depleted via the diphtheria toxin receptor technique demonstrated a diminished but not abrogated sensitization response (Bennett et al., 2005). In another similar model, the sensitization response was even completely normal (Kissenpfennig et al., 2005). While the experimental models created for these two studies allowed shortterm inducible ablation of LCs in vivo, a different knockout mouse model was designed characterized by constitutive and durable absence of epidermal LCs (Kaplan et al., 2005). Unexpectedly, these LC-deficient mice actually had an enhanced sensitization response, suggesting that the LCs even may exert regulatory functions. This ‘LC paradigm’ proposes that LCs may act both ways, i.e. tolerogenic when they present antigens under steady-state non-inflammatory conditions, but sensitizing upon stimulation by inflammatory mediators. Which activity is the seminal and original main feature of LCs remains to be determined in the future. Nevertheless, there is accumulating evidence that dermal dendritic cells are equally if not even more important in presenting antigens (Merad et al., 2008). The down-regulating role of LCs is supported by our recent observations. The data obtained with IL-12 mentioned above gave rise to the hypothesis that appearance of UVR-damaged but still viable LCs in the regional lymph nodes is essential for the induction of UVR-Treg (Schwarz et al., 2005). Thus we postulated that a stimulus which does not damage but definitely kills LCs should not induce UVR-Treg. Topical application of mometasone which kills LCs by inducing apoptosis inhibited the induction of CHS but unlike UVR did not induce Treg (Schwarz et al., 2010). To further substantiate the crucial role of LC for the generation of UVR-Treg, Langerin diphtheria toxin receptor knock-in (Langerin-DTR) mice were utilized, in which LC can be depleted by the injection of diphtheria toxin. We postulated that according to our hypothesis UVR-Treg should not develop in LC-depleted mice. This was indeed the case. Adoptive transfer experiments with T cells from UVR-exposed and LC-depleted mice did not cause suppression in the recipients. However, to our great surprise, the induction of CHS in LC-depleted and UVR-exposed mice was not at all suppressed as expected but completely unaffected and comparable to positive control wild type mice which were sensitized and challenged (Schwarz et al., 2010). Kinetic analyses excluded that Langerin-expressing dermal dendrocytes were responsible for the suppression but proved that this was strictly dependent on the presence of LCs. This implies that LCs are not only involved in the induction of UVR-Treg but also in the inhibition of the induction of CHS (Schwarz et al., 2010).
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At the same time 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. It appears that the discrepancies in our results may be mostly due to differences in the methods (UVR dose, antigen used, etc.).
Conclusion The molecular mechanisms involved in UVR-induced immunosuppression are much better understood than many years ago. In particular major achievements have been made in the phenotypical and functional characterization of UVR-Treg. Since these cells suppress immune reactions in an antigen-specific fashion they harbor therapeutic potential. A major step towards this goal has been recently taken by the identification of strategies which alter the migratory behavior of UVR-Treg in such a way that they do not only inhibit the induction but also the effector phase of immune reactions (Schwarz et al., 2007). Another important finding is the observation that cytokines can affect DNA damage (Fig. 2). UVR-induced DNA damage has been recognized as the major trigger for most of the biological effects of UVR. Consequently, for quite a long time this signal transduction pathway was supposed to be unidirectional, since any biological effect should be the consequence of DNA damage. The observation that several cytokines including IL-12, IL-18 and IL23 can, in turn, control DNA repair and consequently UVR-induced DNA damage suggests that this signaling pathway may not be as unidirectional as always thought but demonstrates the existence of a biofeedback mechanism (Schwarz and Schwarz, 2009). This crosstalk may represent a new defense mechanism of the host against UVR-induced immunosuppression and maybe also against carcinogenesis. Finally, the view about the role of LCs in UVR-induced immunosuppression has changed. However, the functional role of LCs in immunology in general is going to be redefined. There is accumulating evidence that LCs may rather act in a down-regulatory than stimulatory fashion. The primary cutaneous antigen presenting cell responsible for the induction of immune responses in the skin appears to be the dermal dendritic cell. Whether LCs act more in a regulatory or stimulatory function may depend on the environmental conditions. According to the most recent studies, LCs seem to be more important for the down-regulation than induction of immune responses in the skin. Our findings that they are essentially involved in mediating UVR-induced immunosuppression add to this concept.
Acknowledgments The studies described in this manuscript were supported by grants from the German Research Foundation (SFB 415, A16; SCHW1177/1-3, SCHW625/4-1).
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European Journal of Cell Biology journal homepage: www.elsevier.de/ejcb
Review
Molecular mechanisms of ultraviolet radiation-induced immunosuppression Thomas Schwarz ∗ , Agatha Schwarz Department of Dermatology, University Kiel, Schittenhelmstrasse 7, 24105 Kiel, Germany
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Article history: Received 18 August 2010 Received in revised form 3 September 2010 Accepted 20 September 2010 Keywords: Contact hypersensitivity DNA damage DNA repair Immunosuppression Immunotolerance Interleukins Langerhans cells Photoimmunology Regulatory T cells Ultraviolet radiation
a b s t r a c t Solar ultraviolet radiation (UVR) is well known for its immunosuppressive properties. UVR can suppress immune reactions both in a local and a systemic fashion. One of the major molecular mediators of photoimmunosuppression is UVR-induced DNA damage. In contrast to immunosuppressive drugs, UVR does not act in a general but antigen-specific fashion. This is due to the induction of regulatory T cells. Epidermal Langerhans cells harboring UVR-induced DNA damage appear to be essentially involved in the induction of these cells. Cytokines including interleukin (IL)-12, -18 and -23 exert the capacity to reduce UVRinduced DNA damage via induction of DNA repair. Accordingly, these cytokines prevent UVR-mediated immunosuppression. In contrast to IL-18, IL-12 and IL-23 can also inhibit the suppressive activity of regulatory T cells by a mechanism which still needs to be determined. Clarification of the molecular mechanisms underlying UVR-induced immunosuppression will help to develop new immunosuppressive therapeutic strategies by utilizing UVR-induced regulatory T cells for the treatment of immune-mediated diseases. In addition, these insights will contribute to a better understanding of photocarcinogenesis since suppression of the immune system by UVR essentially contributes to the induction of skin cancer. © 2010 Elsevier GmbH. All rights reserved.
Besides its undisputed beneficial and indispensable effects on human life, solar/ultraviolet radiation (UVR) can be a hazard to human health by inducing skin cancer, premature skin aging, inflammation and cell death (Fisher et al., 1997; Gilchrest, 1990; Kraemer, 1997; Kulms and Schwarz, 2000). This refers mostly to the middle wave length range (290–320 nm, UVB). Over the last three decades it became clear that UVB radiation exerts the capacity to suppress the immune system. UVR inhibits immune reactions locally, but can also affect the immune system in a systemic fashion, provided higher UVR doses are given (de Gruijl, 2008). In contrast to immunosuppressive drugs which suppress the immune system in a general fashion, UVR acts in an antigen-specific fashion. This is mostly due to the generation of T cells which specifically suppress immune reactions. These cells were initially called T suppressor cells but recently were renamed regulatory T cells (Schwarz, 2008). Because of their specific activity they harbor therapeutic potential for the treatment of (auto)immune-mediated diseases. In addition, immunosuppression by UVR plays a crucial role in the induction of UVR-mediated skin cancer, one of the most rapidly growing cancers worldwide (Hanneman et al., 2006). Hence elucidation of the
Abbreviations: CHS, contact hypersensitivity; IL, interleukin; LC, Langerhans cell; NER, nucleotide excision repair; Treg, regulatory T cell; UVR, ultraviolet radiation; UVR-Treg, UVR-induced Treg; wt, wild type. ∗ Corresponding author. Tel.: +49 431 597 1500; fax: +49 431 597 1503. E-mail address: [email protected] (T. Schwarz). 0171-9335/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2010.09.011
molecular mechanisms involved will tremendously contribute to the understanding of photocarcinogenesis and elucidate strategies to more effectively prevent and treat these tumors. In addition, a better phenotypical and functional characterization of regulatory T cells (Treg) and a detailed insight into the mechanisms of their induction by UVR will help to utilize this T cell subtype in a therapeutic setting. The best model to study UVR-induced immunosuppression is the suppression of the induction of contact hypersensitivity (CHS) by UVR. Topical application of potent contact allergens results in antigen-specific sensitization which can be visualized by a specific swelling response upon application of the same allergen on the ear a few days later. Sensitization is suppressed and prevented, respectively, when the contact allergen is painted onto skin which has been preexposed to low doses of UVR (Toews et al., 1980). The very same animals cannot be sensitized against the same allergen at later time points, indicating the induction of immunotolerance. This long-term suppression is antigen-specific since all other immune responses are not affected. This immunotolerance is mediated via antigen-specific Treg which can be demonstrated by adoptive transfer experiments (Elmets et al., 1983). UV-induced Treg (UVR-Treg) have been quite well characterized. They express CD4 and CD25, the negative regulatory molecule CTLA-4, GITR, neuropilin and secrete upon antigen-specific stimulation interleukin (IL)-10 (Schwarz, 2008). Thus they appear to represent a certain subtype of CD4+ CD25+ Treg.
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IL-12, UVR-induced DNA damage and photoimmunosuppression UVR-induced DNA damage has been recognized as the major molecular trigger for UVR-induced immunosuppression (Kripke et al., 1992). UVR induces specific DNA lesions, preferentially cyclobutane pyrimidine dimers and 6–4 photoproducts. Accelerated removal of DNA lesions by exogenous DNA repair enzymes resulted in reduction of UVR-induced immunosuppression, indicating that DNA damage is a crucial molecular mediator in this process (Kripke et al., 1992). IL-12 was the first cytokine being demonstrated to exert the capacity to prevent UVR-induced immunosuppression (Müller et al., 1995; Schmitt et al., 1995; Schwarz et al., 1996). Inhibition of the induction of CHS was prevented upon intraperitoneal injection of IL-12. Likewise Treg were not induced in UVR-exposed mice when they were treated with IL-12. In addition, IL-12 exerts the capacity to inhibit the suppressive activity of Treg since injection of IL-12 into already UVR-tolerized mice enables sensitization of these animals. Furthermore, UVR-Treg lose their suppressive activity upon coincubation with IL-12. How IL-12 inhibits Treg is still unclear. IL-12 was found to exert the capacity to reduce UVR-induced DNA damage (Schwarz et al., 2002) since IL-12 reduced both in vitro and in vivo UVR-induced apoptotic cell death which is mostly driven by the amounts of UVR-induced DNA lesions (Kulms et al., 1999). IL-12 appears to reduce UVR-induced DNA damage via induction or activation of the nucleotide excision repair (NER), the major endogenous DNA repair system, since the effect of IL-12 on UVRinduced cell death was not observed in Xpa knock-out mice which are deficient in NER (Schwarz et al., 2002). Accordingly, IL-12 was not able to prevent UVR-induced immunosuppression, confirming that UVR-induced DNA damage is an essential molecular mediator of immunosuppression caused by UVR (Schwarz et al., 2005). Although the mechanism by which IL-12 affects the NER is still unclear, this observation was quite surprising since until then it was thought that the NER as an essential repair mechanism is just constitutively expressed and not subjected to any regulation. However, Eller et al. (1997) first demonstrated that administration of DNA oligonucleotides induces DNA repair via a p53-dependent mechanism. Accordingly, a recent in vivo study indicated that topical pretreatment with DNA oligonucleotides enhanced the rate of DNA photoproduct removal, decreased UVR-induced mutations, and reduced photocarcinogenesis in UV-irradiated mice (Goukassian et al., 2004). Meanwhile, a variety of additional factors which might influence the NER have been described, including cytokines (IL-18, IL-23 see below), neuropeptides (Böhm et al., 2005), infrared radiation (Jantschitsch et al., 2009) and even vitamin D (Trémezaygues et al., 2009). However, with the exception of IL-12 (Meeran et al., 2006a), these effects have not yet been confirmed by additional independent groups. A hallmark feature of UVR-induced immunosuppression is the depletion of Langerhans cells (LCs) from the epidermis upon UVR exposure (Toews et al., 1980). For a long time it was assumed that UVR eliminates LCs by inducing cell death. This, however, does not appear to be the case, since first the UVR doses required to induce immunosuppression are rather low and second LCs can be found in the regional lymph nodes, indicating that UVR induces emigration of LCs from the epidermis (Schwarz et al., 2005). Injection of IL-12 prevents the emigration of LCs, implying that again UVRinduced DNA damage is the molecular trigger for the emigration. This appears to be the case since IL-12 does not prevent the emigration in Xpa knock-out mice. LCs found in the lymph nodes upon UVR exposure carry significant amounts of DNA damage. Upon injection of IL-12 the amounts of DNA damage in the LCs located in the lymph nodes is significantly reduced. This leads to the hypothesis that UVR-damaged but still viable LCs in the lymph nodes are required
Fig. 1. Effects of IL-12, IL-23 and IL-18 on UVR-induced immunosuppression. IL-12 and IL-23 as well as IL-18 prevent UVR-induced immunosuppression via modulation of DNA repair. Accordingly, suppression of the induction of contact hypersensitivity by UVR, which is mediated via DNA damage, is not observed upon injection of either IL-12, IL-23 or IL-18. In contrast, established UVR-mediated immunotolerance can only be broken by IL-12 and IL-23 but not by IL-18, indicating that IL-12 and IL-23 exert more pronounced immunostimulatory features than IL-18. Though being primarily a proinflammatory cytokine, IL-18 prevents UVR-induced immunosuppression via its capacity to induce DNA repair. Adapted from Schwarz (2008).
for the induction of Treg (Schwarz et al., 2005). One could imagine that the damaged LCs are still able to present the antigen but in an impaired manner which ultimately does not induce T effector cells but Treg. IL-18, another cytokine reducing UVR-mediated DNA damage It turned out that the capacity to reduce DNA damage is not a unique feature of IL-12 but can be achieved also by other mediators including IL-18 (Schwarz et al., 2006). IL-18 is a proinflammatory cytokine which exhibits the unique capacity to induce T helper 1 or T helper 2 polarization, depending on the immunologic context (Reddy, 2004). Although not related to IL-12 structurally, IL-18 shares some biological effects with IL-12, e.g. the induction of interferon-␥ (Okamura et al., 1998; Dinarello and Fantuzzi, 2003). IL-18 was found to reduce UVR-induced apoptosis in a similar fashion like IL-12. This effect was not observed in DNA repair deficient mice, implying that IL-18 reduces UVR-induced DNA damage via the NER. Accordingly IL-18 also prevented UVR-induced immunosuppression in wild type but not in DNA repair-deficient mice (Schwarz et al., 2006). However, in contrast to IL-12, IL-18 was not able to break UVR-induced immunotolerance which is mediated in a DNA damage-independent fashion via UVR-Treg (Schwarz et al., 2006). This indicated that IL-18 though being primarily a proinflammatory cytokine through the capacity to affect DNA repair can restore an immune response which is suppressed by UVR. These findings also identified IL-12 as still unique in its capacity to restore immune responses because of its effect on UVR-Treg (Fig. 1). IL-23 prevents UVR-induced immunosuppression but also inhibits regulatory T cells IL-23 is a heterodimeric cytokine consisting of a p40 and p19 chain. Although it is closely related to IL-12 by sharing the same p40 subunit, IL-23 exerts also different biological activities than IL-12 (Kastelein et al., 2007). The effects of IL-23 have been mainly linked to a T helper 17 cell response (Mills, 2008). Because of the structural similarity but the different biological effects between IL-12 and IL-23 it was obvious to study the effects of IL-23 on UVR-induced DNA damage and immunosuppression. Incubation of cultured keratinocytes with IL-23 before UVR exposure significantly reduced
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the apoptosis rate (Majewski et al., 2010). This was associated with a reduction of DNA damage as demonstrated by Southwestern dot blot analysis. Injection of IL-23 into UVR-exposed mice diminished the number of apoptotic keratinocytes and the amounts of DNA damage. This was not observed in DNA repair-deficient Xpa knockout mice, implying that IL-23 reduces DNA damage via induction of DNA repair. Based on the observations with IL-12 and IL-18 it was predicted that any cytokine which reduces UVR-induced DNA damage should also prevent photoimmunosuppression. This was the case since injection of IL-23 rendered UVR-exposed mice fully susceptible to sensitization. Accordingly, Treg did not develop upon injection of IL-23 in UV-irradiated mice. IL-23 failed to prevent UVR-mediated suppression in Xpa knock-out mice, confirming the involvement of NER. However, in contrast to IL-18, IL-23 also inhibited the activity of UVR-Treg (Majewski et al., 2010). UVR-tolerized mice could be fully sensitized upon injection of IL-23. In addition, UVR-Treg lost their suppressive activity upon incubation with IL-23. Together, this indicates that IL-23 as IL-12 and IL-18 can reduce UVR-induced DNA damage and thereby prevents immunosuppression. However, IL-23 shares with IL-12 the still unique capacity to restore suppressed immune responses because of its effect on Treg (Fig. 1). By which mechanisms IL-12 and IL-23 exert this effect still remains to be determined as well as the question whether they achieve this effect through the same or different pathways. Enhanced photocarcinogenesis in the absence of IL-12 The observation that IL-12 reduces UVR-induced DNA damage via induction of the NER led to the hypothesis that in turn the absence of IL-12 should lead to enhanced amounts of DNA damage and thus support carcinogenesis. To address this issue, we utilized IL-12 knock-out mice which lacked the p40 chain (Magram et al., 1996). IL-12p40 knock-out and wild type (wt) mice were exposed 3×/week to UVR. Skin biopsies obtained after 6 weeks revealed significantly increased numbers of sunburn cells in the knock-out mice (Maeda et al., 2006). Staining of epidermal sheets with an antibody directed against the tumor suppressor gene p53 revealed a higher number of p53 patches in the skin of knock-out mice. After around 200 days first skin tumors developed. Kaplan–Meier analysis indicated a significantly increased probability of tumor development in the knock-out mice. In addition, the number of tumors developing in the individual mice was significantly higher in the knock-out than in wt mice. Furthermore, the tumors in the knock-out mice revealed a more aggressive growth behavior (Maeda et al., 2006). Hence it was concluded that a lack of IL-12 might enhance photocarcinogenesis. However, nowadays this conclusion has to be modified. Since IL-23 also shares the p40 chain these knock-out mice were not deficient in IL-12 as initially anticipated but also in IL-23. Studies utilizing p35 and p19 knock-out mice are currently ongoing. This will clarify whether IL-12, IL-23 or both cytokines exert a protective role during photocarcinogenesis. A recent study confirmed the protective effect of IL-12 by showing p35-deficient mice to be more susceptible to photocarcinogenesis (Meeran et al., 2006b). Defining a new role of Langerhans cells For a long time LCs were suggested to be the most relevant, even the only real antigen-presenting cell of the skin (Stingl et al., 1980). Initial evidence for this assumption was provided by the observation that CHS could not be induced in skin areas that were devoid of LCs (tail, cornea) (Toews et al., 1980). The early photoimmunology studies supported this concept since in those days it was assumed that UVR inhibits sensitization via killing LCs.
Fig. 2. Crosstalk between DNA damage, DNA repair and cytokines. UVR-induced DNA damage is a major molecular trigger for a variety of biologic UVR effects, including the release of cytokines. By their ability to reduce DNA damage, presumably via induction of DNA repair various cytokines may inhibit or reduce some biologic effects of UVR. Taken from Schwarz and Schwarz (2009).
However, other antigen-presenting cells must be able to replace LCs, since transgenic mice in which LCs were completely depleted via the diphtheria toxin receptor technique demonstrated a diminished but not abrogated sensitization response (Bennett et al., 2005). In another similar model, the sensitization response was even completely normal (Kissenpfennig et al., 2005). While the experimental models created for these two studies allowed shortterm inducible ablation of LCs in vivo, a different knockout mouse model was designed characterized by constitutive and durable absence of epidermal LCs (Kaplan et al., 2005). Unexpectedly, these LC-deficient mice actually had an enhanced sensitization response, suggesting that the LCs even may exert regulatory functions. This ‘LC paradigm’ proposes that LCs may act both ways, i.e. tolerogenic when they present antigens under steady-state non-inflammatory conditions, but sensitizing upon stimulation by inflammatory mediators. Which activity is the seminal and original main feature of LCs remains to be determined in the future. Nevertheless, there is accumulating evidence that dermal dendritic cells are equally if not even more important in presenting antigens (Merad et al., 2008). The down-regulating role of LCs is supported by our recent observations. The data obtained with IL-12 mentioned above gave rise to the hypothesis that appearance of UVR-damaged but still viable LCs in the regional lymph nodes is essential for the induction of UVR-Treg (Schwarz et al., 2005). Thus we postulated that a stimulus which does not damage but definitely kills LCs should not induce UVR-Treg. Topical application of mometasone which kills LCs by inducing apoptosis inhibited the induction of CHS but unlike UVR did not induce Treg (Schwarz et al., 2010). To further substantiate the crucial role of LC for the generation of UVR-Treg, Langerin diphtheria toxin receptor knock-in (Langerin-DTR) mice were utilized, in which LC can be depleted by the injection of diphtheria toxin. We postulated that according to our hypothesis UVR-Treg should not develop in LC-depleted mice. This was indeed the case. Adoptive transfer experiments with T cells from UVR-exposed and LC-depleted mice did not cause suppression in the recipients. However, to our great surprise, the induction of CHS in LC-depleted and UVR-exposed mice was not at all suppressed as expected but completely unaffected and comparable to positive control wild type mice which were sensitized and challenged (Schwarz et al., 2010). Kinetic analyses excluded that Langerin-expressing dermal dendrocytes were responsible for the suppression but proved that this was strictly dependent on the presence of LCs. This implies that LCs are not only involved in the induction of UVR-Treg but also in the inhibition of the induction of CHS (Schwarz et al., 2010).
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At the same time 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. It appears that the discrepancies in our results may be mostly due to differences in the methods (UVR dose, antigen used, etc.).
Conclusion The molecular mechanisms involved in UVR-induced immunosuppression are much better understood than many years ago. In particular major achievements have been made in the phenotypical and functional characterization of UVR-Treg. Since these cells suppress immune reactions in an antigen-specific fashion they harbor therapeutic potential. A major step towards this goal has been recently taken by the identification of strategies which alter the migratory behavior of UVR-Treg in such a way that they do not only inhibit the induction but also the effector phase of immune reactions (Schwarz et al., 2007). Another important finding is the observation that cytokines can affect DNA damage (Fig. 2). UVR-induced DNA damage has been recognized as the major trigger for most of the biological effects of UVR. Consequently, for quite a long time this signal transduction pathway was supposed to be unidirectional, since any biological effect should be the consequence of DNA damage. The observation that several cytokines including IL-12, IL-18 and IL23 can, in turn, control DNA repair and consequently UVR-induced DNA damage suggests that this signaling pathway may not be as unidirectional as always thought but demonstrates the existence of a biofeedback mechanism (Schwarz and Schwarz, 2009). This crosstalk may represent a new defense mechanism of the host against UVR-induced immunosuppression and maybe also against carcinogenesis. Finally, the view about the role of LCs in UVR-induced immunosuppression has changed. However, the functional role of LCs in immunology in general is going to be redefined. There is accumulating evidence that LCs may rather act in a down-regulatory than stimulatory fashion. The primary cutaneous antigen presenting cell responsible for the induction of immune responses in the skin appears to be the dermal dendritic cell. Whether LCs act more in a regulatory or stimulatory function may depend on the environmental conditions. According to the most recent studies, LCs seem to be more important for the down-regulation than induction of immune responses in the skin. Our findings that they are essentially involved in mediating UVR-induced immunosuppression add to this concept.
Acknowledgments The studies described in this manuscript were supported by grants from the German Research Foundation (SFB 415, A16; SCHW1177/1-3, SCHW625/4-1).
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