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Photoimmunology, DNA repair and photocarcinogenesis
www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 54 (2000) 87–93
Invited Review
Photoimmunology, DNA repair and photocarcinogenesis Mark Berneburg, Jean Krutmann * ¨ Clinical and Experimental Photodermatology, Department of Dermatology, Heinrich-Heine-University, Moorenstraße 5, D-40225 Dusseldorf, Germany Received 2 November 1999; accepted 26 January 2000
Abstract In recent years major progress has been made in identifying the molecular mechanisms by which UV radiation modulates the immune system of the skin. From these studies it appears that the generation of DNA damage and the subsequent activation of DNA repair enzymes play a critical role in the generation of UV-B-induced immunosuppression. These studies have made use of cells from both nucleotide excision repair (NER)-deficient individuals and mice. Results obtained from these studies have important clinical implications for DNA-repairdeficient patients in particular and for effective photoprotection of human skin in general. q2000 Elsevier Science S.A. All rights reserved. Keywords: Photoimmunology; Photocarcinogenesis; DNA-repair deficiency; Chromophore
1. Introduction For more than a century it has been known that sunlight plays an important role in the generation of skin tumours [1]. Sunlight is a complete carcinogen and ultraviolet B (UV-B) radiation is the most effective wavelength range in inducing skin malignancies [2]. Elegant studies in mice, conducted by Kripke et al. [3], showed that skin tumours are highly immunogenic, since such tumours that were transplanted onto normal syngeneic mice induced an immunological response leading to inhibition of tumour growth and ultimately tumour rejection. When these tumours were transplanted into previously UV-B-irradiated animals, no immune response was mounted, leading to tumour growth and ultimately to death of the recipient animals [4]. These discoveries triggered a multitude of investigations about the role of photoimmunology in the process of photocarcinogenesis and within the last two decades our understanding regarding the capacity of ultraviolet radiation to modulate the function of the skin immune system has increased significantly (for review, see Ref. [5]). From these studies it is known that UV light exerts its photoimmunological effects on different levels in the cell. Lipid peroxidation and induction of reactive oxygen species can result from interaction of UV with membrane lipids. Transformation of urocanic acid from its trans to the cis * Corresponding author. Tel.: q49-211-811-7627; fax: q49-211-8118830; e-mail: [email protected]
isomer in the stratum corneum has also been shown to exhibit immunosuppressive properties [6]. Another very important factor is the interplay of different UV wavelengths. Recent evidence demonstrates that UV-A has a protective effect against UV-B-induced immunosuppression in animal models [7,8], and it appears that this UV-A effect is mediated via interferon-g [9]. However, the extent of interplay of UV-A and UV-B in naturally occurring sunlight still remains to be elucidated. The induction of DNA damage appears to play a central role in photoimmunology. Interaction of UV with DNA can give rise to different forms of DNA damage, which is normally removed by nucleotide excision repair (NER). Subunits of the NER machinery are not only involved in removal of DNA damage but also have a role in transcription [10,11], possibly involving immunologically relevant genes. This designated deficiencies in NER as important model systems in the investigation of the role of DNA damage, NER and transcription of immunologically relevant genes in photoimmunology and the link to photocarcinogenesis. In this review we shall focus on the photoimmunological effects of UV-B radiation and the central role of DNA damage and DNA repair in the process of photocarcinogenesis. 2. Induction of T suppressor cells Tumour-associated antigens (TAAs) on UV-irradiated tumours are only poorly characterized. To study photoim-
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munological processes, cell-mediated immune responses such as delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) are therefore widely used as models. These model systems employ haptens to induce allergic reactions. Two models have been studied extensively depending on the dose of applied UV. High doses of UV (5–30 kJ/m2 UV-B) are applied and five days after irradiation mice are exposed to a hapten such as DNCB at a non-irradiated site to induce an immune response. In these studies [12–14] exposure to UV radiation suppressed the ability to mount an appropriate immunological response. In the second model, low doses are applied [15–17]. In this system, hapten application at the irradiated site results in immune suppression, whereas hapten application at distant sites does not. In both of these models hapten-specific transferrable T suppressor (Ts) cells have been found in the spleen of experimental animals.
3. Role of cytokines Apart from the generation of Ts cells in the above models, it has been found that antigen-presenting cells (APCs)of irradiated animals are ineffective in proper presentation of antigen. Therefore it has been proposed that Ts cells may be generated by ineffective presentation of antigen to native T cells [18,19]. Ineffective antigen presentation can occur in different ways. One way would be a defect of APCs per se due to a direct irradiation effect. Another possibility is the induction of paracrine mechanisms in cells distinct from APCs, such as the production of immunosuppressive cytokines. It has been shown that the presentation of TAA critically depends on the sequence and magnitude of expression of several cytokines and that exposure to UV-B light increases several proinflammatory cytokines such as the interleukins IL-1, IL-6 and TNF-a (for review, see Ref. [18]). It has also been reported that TNF-a and IL-10 can suppress contact hypersensitivity locally and systemically [20–22].
4. Antigen-presenting cells: migration and DNA as target for UV-B There is increasing evidence that DNA damage constitutes the molecular basis for these mechanisms. Work by Yarosh et al. has shown that the DNA repair enzyme T4 endonuclease V (T4NV) can be delivered to keratinocytes, epidermal Langerhans cells and dendritic cells located in the local draining lymph node by means of liposomes containing T4NV applied to normal skin of mice [23]. Furthermore, it has been demonstrated that application of T4NV in liposomes is capable of increasing removal of cyclobutane pyrimidine dimers (CPDs) from keratinocytes and skin-derived dendritic cells from local draining lymph nodes [24]. In order to demonstrate a critical role of DNA damage in APCs, it would be necessary to generate DNA damage exclusively in APCs, rather than in total skin, and then observe the
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effect of these cells in the immune system. This has been done in mice by Vink et al., who isolated APCs, generated DNA damage by irradiating them, increased the repair capacity of the cells by offering a repair enzyme in vitro and then reinjected these cells into mice [25]. The capacity of the recipient mice to mount an immunological response after being challenged with the hapten FITC would indicate whether removal of DNA damage also recovers immune responses. Since APCs contain only small nucleotide pools, in their study, Vink et al. exchanged the previously employed repair enzyme T4NV with photolyase, derived from Anacystis nidulans, which specifically removes CPDs (photosomew treatment). This has the advantage that APCs do not have to carry out the repair but the DNA damage is removed entirely by the enzyme. Vink et al. demonstrated that reduction of CPDs by f55% specifically in APCs reconstituted their capacity to induce CHS and T suppressor cells in vivo and interferon-g (IFN-g) production of T cells in vitro. This repair effect appeared to be CPD specific, since treatment with a specific psoralen and UV-A light (IPUVA) did suppress CHS, but this was not restored by photosomew treatment. However, the fact that IPUVA treatment can also suppress an FITC-induced CHS indicates that CPDs are not the only type of DNA damage capable of inducing immunosuppression. The induction of IL-10 in UV-B-irradiated murine keratinocytes, triggered by DNA damage, reported by the same group [26], furthermore indicates that DNA damage influences the development of post-UV-B immune responses specifically in APCs but also may be of photoimmunologic importance in other cell types.
5. DNA-repair deficiencies As demonstrated above, repair of DNA damage is not only of paramount importance in prevention of mutations in the genome, but also plays a critical and direct role in photoimmunological processes. Therefore it is important to investigate photoimmunology and photocarcinogenesis in DNA-repair-deficient backgrounds. The most relevant DNA damage generated after UV-B irradiation is the formation of CPDs. These lesions are primarily repaired by NER, which removes bulky DNA damage in two distinct subpathways [28]. Damage existing in actively transcribed genes is removed by a quick mechanism called transcription-coupled repair (TCR) [27]. Damage prevailing in other parts of the genome is removed in a somewhat slower fashion by socalled global genome repair (GGR). Defects in both subpathways of NER can lead to three distinct diseases [28,29]: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). These diseases provide important model systems for investigating aspects of DNA repair, since they are all deficient in NER but in different subpathways. XP of the complementation group A (XP-A) is deficient in both TCR and GGR, XP complementation
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group C (XP-C) is deficient in GGR only, and CS is deficient in TCR only. 5.1. Mice Knockout mice of the different diseases provide an important in vivo tool to study photocarcinogenesis and mice corresponding to the respective defects have been generated in the last few years. While knocking out some XP genes was deleterious in some mice [30], mice deficient for XP-A, XPC, CS and TTD were viable [31–35]. Model mice for XP-A and XP-C demonstrated an increased risk of developing tumours after exposure to several carcinogens, including UV radiation. Following exposure to UV, XP-A knockout mice showed longer and stronger oedema after UV-B and PUVA treatment as well as more sunburn cell formation, delayed Langerhans cell recovery and increased local and systemic immunosuppression [36]. This is in line with the notion that DNA repair plays a role in UV-B-related immune responses. While XP-A mice are defective in both TCR and GGR, XPC mice are capable of carrying out TCR but not GGR [37]. These mice exhibit an increased skin cancer risk and can only remove DNA damage from actively transcribed genes. Interestingly, in these animals the minimal dose of UV-B needed to generate erythema (MED) and oedema is similar to that in wild-type animals. This is in striking contrast to XP-A mice (deficient in TCR and GGR), which demonstrate increased skin-cancer susceptibility and also a lower MED. These results indicate that GGR and TCR have different implications in photocarcinogenesis and post-UV reactions such as erythema and oedema. Since photoimmunology and photocarcinogenesis appear to be more closely linked than photocarcinogenesis and erythemal responses, it will be interesting to see whether TCR, GGR or both play a role in photoimmunological processes. Human CS and TTD are both not associated with an increased skin-cancer risk [38,39]. However, CS and TTD model mice do show an increased development of skin cancer [34,35], which may be explained by species differences such as a higher rate of spontaneous immortalization in mice. However, this may indicate that experiments conducted with NERdeficient mice, albeit still a very important tool for in vivo study of photoimmunological reactions, need to be evaluated cautiously with regards to photocarcinogenesis. 5.2. Humans It has been postulated earlier that defective NER may play a causative role not only in the induction of DNA damage leading to mutated DNA but also in the immune response to UV radiation, thus being critically involved in the formation of skin tumours. Early work by Norris et al. and Gaspari et al. indicated that patients with XP had normal numbers of T cells and natural killer (NK) cells as well as normal tumour necrosis factor production [40,41]. However, cells from XP patients proved to be low in NK cell activity and deficient in
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the production of the proinflammatory cytokine IFN-g [41]. Another study by Mariani et al. compared cells from XP patients with cells from TTD patients [42] and found, although NK cell lytic activity was low in both groups, the relative proportion of CD3q and CD4q circulating lymphocytes was reduced in XP but not in TTD patients. Furthermore, XP patients presented a low total number of circulating T cells. Earlier work by Norris et al. showed that in CS patients adaptive cell-mediated immunity and natural killer cell function were normal [43]. These findings provided evidence for the hypothesis that deficiencies in the immune response after exposure to UV radiation are critical for the formation of skin tumours in XP patients. Reports that some XP genes involved in NER were also subunits of the basal transcription factor TFIIH [10,11] further supported the link between repair of DNA damage and transcription of photoimmunologically relevant genes. The two diseases XP and TTD are particularly interesting in this context. Mutations in the gene encoding for the XPD gene can give rise to both XP and TTD [44]. Since XP is associated with a high skin-cancer risk and TTD is not, this may be an important background for studying photoimmunological responses. Intercellular adhesion molecule 1 (ICAM-1) is expressed on Langerhans cells, keratinocytes and fibroblasts and is involved in a multitude of immunologically relevant pathways. Studies in cells from NER-deficient individuals are normally carried out in fibroblasts and their constitutive ICAM-1 expression can be induced by IFN-g. If these cells are exposed to UV-B radiation, the IFN-g-induced ICAM-1 expression can be dose-dependently suppressed [45,46]. A study by Ahrens et al. took advantage of this photoimmunological model system. They compared fibroblasts of patients with XP-D or TTD and normal individuals [47]. Repair of DNA damage as measured by unscheduled DNA synthesis did not differ between the XP-D and TTD cells. In this study, TTD cells showed a normal IFN-g-induced ICAM-1 expression following UV-B irradiation, whereas XP-D cells demonstrated an abnormal ICAM-1 response. This suggested that the photoimmunological phenotype (ICAM-1 response) in DNA-repair-deficient individuals is correlated with the risk of developing skin cancer. This study has been extended and corroborated with more TTD patients, more XP complementation groups and the third repair deficiency, CS [48]. When looking specifically at removal of CPDs in DNA-repair deficiencies, it could be shown that the photoimmunological phenotype correlated with the removal of CPDs. This was the case in XP, CS and TTD cells. In the specific case of XP-D combined with CS, it even appears that the coordination between DNA repair and transcription is so fundamentally altered that repair is carried out randomly over the whole genome, possibly at sites of present transcription, leading to rampant incision throughout the whole genome [49]. These results support the above-mentioned hypothesis that unrepaired CPDs, prevailing in the genome, may stall transcribing RNA polymerase II [50], suppressing the transcription of
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the genes required to respond to UV exposure. Taken together, these results support the notion that repair of DNA damage plays a central role in two major pathways (mutagenesis and immunsuppression) involved in photocarcinogenesis (Fig. 1). It has to be mentioned, however, that there exists a subgroup of TTD patients who do exhibit an abnormal photoimmunological phenotype. These patients are mutated at a specific site of the XPD gene. In this subgroup there is room for discussion about whether the skin-cancer risk correlates with the photoimmunological phenotype. On one hand, TTD is normally not associated with increased skin-tumour susceptibility. On the other hand, these TTD patients show a severe clinical phenotype with some of them dying as early as three years of age.
6. New fields of investigation Several groups have investigated in mice and humans whether there is a connection between NER and immunoglobulin-associated hypermutation. However, experiments in NER-deficient backgrounds have not been able to provide any evidence for this link [51–54]. There have been other findings linking immunity and photocarcinogenesis. A report by Svane et al. demonstrated that high MHC class I expression correlates with slow growth in UV-induced skin carcinomas in hairless mice [55]. Work by Hill et al. indicated that the ligand of the apoptosis-inducing Fas/CD95 may have a critical role in the active suppression of systemic immune responses by UV light [56]. Since the apoptogenic effects of UV radiation on the membrane (Fas/CD95 system) and the nucleus (induction of DNA damage) appear to be linked with each other [57], it is tempting to speculate that this cross-connection also exists for UV-mediated immunosuppression.
7. Therapy/outlook Protection against and/or treatment of adverse side effects of sun exposure has become increasingly important not only for the normal population but also for those individuals who do not have endogenous protection (albinism) or repair (xeroderma pigmentosum). Sunscreens are the most widely applied protection against sun damage. A comprehensive review of sunscreens and their role in different aspects of photocarcinogenesis has been presented by Elmets and Anderson [58]. However, it is important to stress that the capacity of a sunscreen to protect against erythemal responses (SPF) does not necessarily reflect its potential to protect against UV-B radiation-induced immunosuppression. In recent years other substances have been investigated for their protective properties against processes related to photocarcinogenesis. Work by Anolik et al. demostrated that isotretinoin therapy reduced the frequency of skin-cancer
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Fig. 1. Model for a central role of DNA repair in photocarcinogenesis. Exposure to UV radiation causes DNA damage, which is removed by DNA repair. Endogenous repair (NER) can be supplemented by exogenous enzymes (T4NV, photolyase), which reduce generation of mutations and restore post-UV immune responses, thus preventing photocarcinogenesis on two different levels.
development [59]. Conversely, however, the NK cell function was decreased in this study. New studies also point in the direction of antioxidants as possible protectors against UV-modulated immune function. Substances such as epigallocatechin-3-gallate, derived from green tea [60], and nicotinamide [61] have been reported to exhibit protective properties against skin cancer. The protective role of these antioxidants indicates a role of reactive oxygen species (ROS). As mentioned above, UV radiation can induce effects directly through induction of DNA damage and indirectly via generation of ROS. The main wavelength range reponsible for ROS induction is the UV-A. Whether the protective properties of antioxidants against skin-tumour formation stems from a role of UV-A radiation in photoimmunosuppression or the capacity of UV-B radiation to induce relevant amounts of ROS, needs to be elucidated further. Nonetheless the use of antioxidants in sunscreens may not only protect against photoaging but also against photocarcinogenesis. All of the mentioned substances are characteristically prophylactic in nature. However, the repair of already generated damage would beneficially add to the line of defence against skin-tumour formation. Work by Vink et al., cited above, has already indicated a possible way to do this. The central role of CPDs in UV-mediated immune suppression [25] and the possibility of their removal by topical application of different repair enzymes lead the way to investigating whether the efficacity of this approach holds true in humans. A multicentre study investigated this for DNA-repair-deficient individuals where the DNA-repair enzyme T4NV proved to be efficient in CPD removal [62]. A very recent study extended these investigations to normal human individuals [63]. Topical application of photolyase to UV-B-irradiated skin decreased the number of CPDs formed by f40%. The removal of CPDs restored two immunological end-points: hypersensitivity reaction to nickel sulfate in sensitive individuals and IFN-g-induced expression of ICAM-1 in normal
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individuals. Furthermore, this treatment also prevented erythemal responses and sunburn cell formation, thus indicating that topical application of a DNA-repair enzyme is effective in dimer removal and exhibits immunoprotective effects in a way that allows already existing damage to be removed. Application of this treatment, e.g., as an ‘after-sun lotion’ in addition to normal application of sunscreens may have a profound impact on the prevalence of sun-induced tumours. 8. Conclusions Exposure to ultraviolet light has a multitude of effects on the skin. The induction of skin tumours by UV light is of major epidemiological relevance, since such tumours are the most prevalent human malignancy with a continuously increasing frequency. The development of tumours is a multistep process involving induction of mutations and escape from immune surveillance. The role of immunosuppressive effects induced by UV light (photoimmunology) has been elucidated significantly in recent years and recent findings have strengthened the link between DNA damage, photoimmunosuppression, DNA repair and photocarcinogenesis. Furthermore, the first therapeutic strategies capitalizing from this connection are emerging, which may open the door not only to protective measures but also to actual repair of pre-existing photodamage, photoimmunosuppression and thus photocarcinogenesis. 9. Abbreviations APC CHS CPD CS DTH GGR ICAM-1 IFN-g NER NK ROS T4NV TAA TCR Ts TTD UV-A UV-B XP (A–G)
antigen-presenting cell contact hypersensitivity cyclobutane pyrimidine dimer Cockayne syndrome delayed-type hypersensitivity global genome repair intercellular adhesion molecule 1 interferon-g nucleotide excision repair natural killer reactive oxygen species T 4 endonuclease V tumour-associated antigen transcription-coupled repair T suppressor trichothiodystrophy ultraviolet A, 315–400 nm ultraviolet B, 290–315 nm xeroderma pigmentosum complementation groups A–G
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[39] M. Stefanini, H. Fawcett, E. Botta, T. Nardo, A.R. Lehmann, Genetic analysis of twenty-two patients with Cockayne syndrome, Hum. Genet. 97 (1996) 418–423. [40] P.G. Norris, G.A. Limb, A.S. Hamblin, J.L. Hawk, Impairment of natural-killer-cell activity in xeroderma pigmentosum [letter], New Engl. J. Med. 319 (1988) 1668–1669. [41] A.A. Gaspari, T.A. Fleisher, K.H. Kraemer, Impaired interferon production and natural killer cell activation in patients with the skin cancer-prone disorder, xeroderma pigmentosum, J. Clin. Invest. 92 (1993) 1135–1142. [42] E. Mariani, A. Facchini, M.C. Honorati, E. Lalli, E. Berardesca, P. Ghetti, S. Marinoni, F. Nuzzo, G.C. Astaldi Ricotti, M. Stefanini, Immune defects in families and patients with xeroderma pigmentosum and trichothiodystrophy, Clin. Exp. Immunol. 88 (1992) 376–382. [43] P.G. Norris, C.F. Arlett, J. Cole, A.R. Lehmann, J.L. Hawk, Abnormal erythemal response and elevated T lymphocyte HRPT mutant frequency in Cockayne’s syndrome, Br. J. Dermatol. 124 (1991) 453– 460. [44] A.R. Lehmann, Dual functions of DNA repair genes: molecular, cellular, and clinical implications, Bioessays 20 (1998) 146–155. [45] J. Krutmann, A. Kock, E. Schauer, F. Parlow, A. Moller, A. Kapp, E. Forster, E. Schopf, T.A. Luger, Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression, J. Invest. Dermatol. 95 (1990) 127–131. [46] J. Krutmann, E. Bohnert, E.G. Jung, Evidence that DNA damage is a mediate in ultraviolet B radiation- induced inhibition of human gene expression: ultraviolet B radiation effects on intercellular adhesion molecule-1 (ICAM-1) expression, J. Invest. Dermatol. 102 (1994) 428–432. [47] C. Ahrens, M. Grewe, M. Berneburg, S. Grether-Beck, X. Quilliet, M. Mezzina, A. Sarasin, A.R. Lehmann, C.F. Arlett, J. Krutmann, Photocarcinogenesis and inhibition of intercellular adhesion molecule 1 expression in cells of DNA-repair-defective individuals, Proc. Natl. Acad. Sci. USA 94 (1997) 6837–6841. [48] M. Berneburg, P.H. Clingen, J.E. Lowe, E.M. Taylor, M.H.L. Green, J. Krutmann, C.F. Arlett, A.R. Lehmann, The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect, Cancer Res., in press. [49] M. Berneburg, J. Lowe, T. Nardo, S. Araujo, M. Fousteri, M.H.L. Green, J. Krutmann, R.D. Wood, M. Stefanini, A.R. Lehmann, UV damage causes uncontrolled breakage in cells from patients with XP combined with Cockayne, EMBO J., in press. [50] B.A. Donahue, S. Yin, J.S. Taylor, D. Reines, P.C. Hanawalt, Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template, Proc. Natl. Acad. Sci. USA 91 (1994) 8502–8506. [51] S.D. Wagner, J.G. Elvin, P. Norris, J.M. McGregor, M.S. Neuberger, Somatic hypermutation of Ig genes in patients with xeroderma pigmentosum (XP-D), Int. Immunol. 8 (1996) 701–705. [52] H.M. Shen, D.L. Cheo, E. Friedberg, U. Storb, The inactivation of the XP-C gene does not affect somatic hypermutation or class switch recombination of immunoglobulin genes, Mol. Immunol. 34 (1997) 527–533. [53] N. Kim, K. Kage, F. Matsuda, M.P. Lefranc, U. Storb, B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes, J. Exp. Med. 186 (1997) 413–419. [54] H. Jacobs, Y. Fukita, G.T. van der Horst, J. de Boer, G. Weeda, J. Essers, N. de Wind, B.P. Engelward, L. Samson, S. Verbeek, J.M. de Murcia, G. de Murcia, H. te Riele, K. Rajewsky, Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice, J. Exp. Med. 187 (1998) 1735–1743. [55] I.M. Svane, A.M. Engel, N.B. Thomsen, H.C. Wulf, O. Werdelin, High MHC class I expression correlates with slow growth in UVinduced skin carcinomas in hairless mice, Apmis 106 (1998) 1101– 1107.
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UVB-induced inflammatory responses and infiltration of leukocytes in human skin, Photochem. Photobiol. 69 (1999) 148–153. [61] H.L. Gensler, Prevention of photoimmunosuppression and photocarcinogenesis by topical nicotinamide, Nutr. Cancer 29 (1997) 157– 162. [62] D. Yarosh, J. Klein, J. Kibitel, L. Alas, A. O’Connor, B. Cummings, D. Grob, D. Gerstein, B.A. Gilchrest, M. Ichihashi, M. Ogoshi, M. Ueda, V. Fernandez, C. Chadwick, C.S. Potten, C.M. Proby, A.R. Young, J.L. Hawk, Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme, Photodermatol. Photoimmunol. Photomed. 12 (1996) 122–130. [63] H. Stege, L. Roza, A.A. Vink, M. Grewe, T. Ruzicka, S. GretherBeck, J. Krutmann, Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin, Proc. Natl. Acad. Sci. USA 97 (2000) 1790–1795.
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Invited Review
Photoimmunology, DNA repair and photocarcinogenesis Mark Berneburg, Jean Krutmann * ¨ Clinical and Experimental Photodermatology, Department of Dermatology, Heinrich-Heine-University, Moorenstraße 5, D-40225 Dusseldorf, Germany Received 2 November 1999; accepted 26 January 2000
Abstract In recent years major progress has been made in identifying the molecular mechanisms by which UV radiation modulates the immune system of the skin. From these studies it appears that the generation of DNA damage and the subsequent activation of DNA repair enzymes play a critical role in the generation of UV-B-induced immunosuppression. These studies have made use of cells from both nucleotide excision repair (NER)-deficient individuals and mice. Results obtained from these studies have important clinical implications for DNA-repairdeficient patients in particular and for effective photoprotection of human skin in general. q2000 Elsevier Science S.A. All rights reserved. Keywords: Photoimmunology; Photocarcinogenesis; DNA-repair deficiency; Chromophore
1. Introduction For more than a century it has been known that sunlight plays an important role in the generation of skin tumours [1]. Sunlight is a complete carcinogen and ultraviolet B (UV-B) radiation is the most effective wavelength range in inducing skin malignancies [2]. Elegant studies in mice, conducted by Kripke et al. [3], showed that skin tumours are highly immunogenic, since such tumours that were transplanted onto normal syngeneic mice induced an immunological response leading to inhibition of tumour growth and ultimately tumour rejection. When these tumours were transplanted into previously UV-B-irradiated animals, no immune response was mounted, leading to tumour growth and ultimately to death of the recipient animals [4]. These discoveries triggered a multitude of investigations about the role of photoimmunology in the process of photocarcinogenesis and within the last two decades our understanding regarding the capacity of ultraviolet radiation to modulate the function of the skin immune system has increased significantly (for review, see Ref. [5]). From these studies it is known that UV light exerts its photoimmunological effects on different levels in the cell. Lipid peroxidation and induction of reactive oxygen species can result from interaction of UV with membrane lipids. Transformation of urocanic acid from its trans to the cis * Corresponding author. Tel.: q49-211-811-7627; fax: q49-211-8118830; e-mail: [email protected]
isomer in the stratum corneum has also been shown to exhibit immunosuppressive properties [6]. Another very important factor is the interplay of different UV wavelengths. Recent evidence demonstrates that UV-A has a protective effect against UV-B-induced immunosuppression in animal models [7,8], and it appears that this UV-A effect is mediated via interferon-g [9]. However, the extent of interplay of UV-A and UV-B in naturally occurring sunlight still remains to be elucidated. The induction of DNA damage appears to play a central role in photoimmunology. Interaction of UV with DNA can give rise to different forms of DNA damage, which is normally removed by nucleotide excision repair (NER). Subunits of the NER machinery are not only involved in removal of DNA damage but also have a role in transcription [10,11], possibly involving immunologically relevant genes. This designated deficiencies in NER as important model systems in the investigation of the role of DNA damage, NER and transcription of immunologically relevant genes in photoimmunology and the link to photocarcinogenesis. In this review we shall focus on the photoimmunological effects of UV-B radiation and the central role of DNA damage and DNA repair in the process of photocarcinogenesis. 2. Induction of T suppressor cells Tumour-associated antigens (TAAs) on UV-irradiated tumours are only poorly characterized. To study photoim-
1011-1344/00/$ - see front matter q2000 Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 0 0 ) 0 0 0 2 4 - 5
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munological processes, cell-mediated immune responses such as delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) are therefore widely used as models. These model systems employ haptens to induce allergic reactions. Two models have been studied extensively depending on the dose of applied UV. High doses of UV (5–30 kJ/m2 UV-B) are applied and five days after irradiation mice are exposed to a hapten such as DNCB at a non-irradiated site to induce an immune response. In these studies [12–14] exposure to UV radiation suppressed the ability to mount an appropriate immunological response. In the second model, low doses are applied [15–17]. In this system, hapten application at the irradiated site results in immune suppression, whereas hapten application at distant sites does not. In both of these models hapten-specific transferrable T suppressor (Ts) cells have been found in the spleen of experimental animals.
3. Role of cytokines Apart from the generation of Ts cells in the above models, it has been found that antigen-presenting cells (APCs)of irradiated animals are ineffective in proper presentation of antigen. Therefore it has been proposed that Ts cells may be generated by ineffective presentation of antigen to native T cells [18,19]. Ineffective antigen presentation can occur in different ways. One way would be a defect of APCs per se due to a direct irradiation effect. Another possibility is the induction of paracrine mechanisms in cells distinct from APCs, such as the production of immunosuppressive cytokines. It has been shown that the presentation of TAA critically depends on the sequence and magnitude of expression of several cytokines and that exposure to UV-B light increases several proinflammatory cytokines such as the interleukins IL-1, IL-6 and TNF-a (for review, see Ref. [18]). It has also been reported that TNF-a and IL-10 can suppress contact hypersensitivity locally and systemically [20–22].
4. Antigen-presenting cells: migration and DNA as target for UV-B There is increasing evidence that DNA damage constitutes the molecular basis for these mechanisms. Work by Yarosh et al. has shown that the DNA repair enzyme T4 endonuclease V (T4NV) can be delivered to keratinocytes, epidermal Langerhans cells and dendritic cells located in the local draining lymph node by means of liposomes containing T4NV applied to normal skin of mice [23]. Furthermore, it has been demonstrated that application of T4NV in liposomes is capable of increasing removal of cyclobutane pyrimidine dimers (CPDs) from keratinocytes and skin-derived dendritic cells from local draining lymph nodes [24]. In order to demonstrate a critical role of DNA damage in APCs, it would be necessary to generate DNA damage exclusively in APCs, rather than in total skin, and then observe the
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effect of these cells in the immune system. This has been done in mice by Vink et al., who isolated APCs, generated DNA damage by irradiating them, increased the repair capacity of the cells by offering a repair enzyme in vitro and then reinjected these cells into mice [25]. The capacity of the recipient mice to mount an immunological response after being challenged with the hapten FITC would indicate whether removal of DNA damage also recovers immune responses. Since APCs contain only small nucleotide pools, in their study, Vink et al. exchanged the previously employed repair enzyme T4NV with photolyase, derived from Anacystis nidulans, which specifically removes CPDs (photosomew treatment). This has the advantage that APCs do not have to carry out the repair but the DNA damage is removed entirely by the enzyme. Vink et al. demonstrated that reduction of CPDs by f55% specifically in APCs reconstituted their capacity to induce CHS and T suppressor cells in vivo and interferon-g (IFN-g) production of T cells in vitro. This repair effect appeared to be CPD specific, since treatment with a specific psoralen and UV-A light (IPUVA) did suppress CHS, but this was not restored by photosomew treatment. However, the fact that IPUVA treatment can also suppress an FITC-induced CHS indicates that CPDs are not the only type of DNA damage capable of inducing immunosuppression. The induction of IL-10 in UV-B-irradiated murine keratinocytes, triggered by DNA damage, reported by the same group [26], furthermore indicates that DNA damage influences the development of post-UV-B immune responses specifically in APCs but also may be of photoimmunologic importance in other cell types.
5. DNA-repair deficiencies As demonstrated above, repair of DNA damage is not only of paramount importance in prevention of mutations in the genome, but also plays a critical and direct role in photoimmunological processes. Therefore it is important to investigate photoimmunology and photocarcinogenesis in DNA-repair-deficient backgrounds. The most relevant DNA damage generated after UV-B irradiation is the formation of CPDs. These lesions are primarily repaired by NER, which removes bulky DNA damage in two distinct subpathways [28]. Damage existing in actively transcribed genes is removed by a quick mechanism called transcription-coupled repair (TCR) [27]. Damage prevailing in other parts of the genome is removed in a somewhat slower fashion by socalled global genome repair (GGR). Defects in both subpathways of NER can lead to three distinct diseases [28,29]: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). These diseases provide important model systems for investigating aspects of DNA repair, since they are all deficient in NER but in different subpathways. XP of the complementation group A (XP-A) is deficient in both TCR and GGR, XP complementation
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group C (XP-C) is deficient in GGR only, and CS is deficient in TCR only. 5.1. Mice Knockout mice of the different diseases provide an important in vivo tool to study photocarcinogenesis and mice corresponding to the respective defects have been generated in the last few years. While knocking out some XP genes was deleterious in some mice [30], mice deficient for XP-A, XPC, CS and TTD were viable [31–35]. Model mice for XP-A and XP-C demonstrated an increased risk of developing tumours after exposure to several carcinogens, including UV radiation. Following exposure to UV, XP-A knockout mice showed longer and stronger oedema after UV-B and PUVA treatment as well as more sunburn cell formation, delayed Langerhans cell recovery and increased local and systemic immunosuppression [36]. This is in line with the notion that DNA repair plays a role in UV-B-related immune responses. While XP-A mice are defective in both TCR and GGR, XPC mice are capable of carrying out TCR but not GGR [37]. These mice exhibit an increased skin cancer risk and can only remove DNA damage from actively transcribed genes. Interestingly, in these animals the minimal dose of UV-B needed to generate erythema (MED) and oedema is similar to that in wild-type animals. This is in striking contrast to XP-A mice (deficient in TCR and GGR), which demonstrate increased skin-cancer susceptibility and also a lower MED. These results indicate that GGR and TCR have different implications in photocarcinogenesis and post-UV reactions such as erythema and oedema. Since photoimmunology and photocarcinogenesis appear to be more closely linked than photocarcinogenesis and erythemal responses, it will be interesting to see whether TCR, GGR or both play a role in photoimmunological processes. Human CS and TTD are both not associated with an increased skin-cancer risk [38,39]. However, CS and TTD model mice do show an increased development of skin cancer [34,35], which may be explained by species differences such as a higher rate of spontaneous immortalization in mice. However, this may indicate that experiments conducted with NERdeficient mice, albeit still a very important tool for in vivo study of photoimmunological reactions, need to be evaluated cautiously with regards to photocarcinogenesis. 5.2. Humans It has been postulated earlier that defective NER may play a causative role not only in the induction of DNA damage leading to mutated DNA but also in the immune response to UV radiation, thus being critically involved in the formation of skin tumours. Early work by Norris et al. and Gaspari et al. indicated that patients with XP had normal numbers of T cells and natural killer (NK) cells as well as normal tumour necrosis factor production [40,41]. However, cells from XP patients proved to be low in NK cell activity and deficient in
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the production of the proinflammatory cytokine IFN-g [41]. Another study by Mariani et al. compared cells from XP patients with cells from TTD patients [42] and found, although NK cell lytic activity was low in both groups, the relative proportion of CD3q and CD4q circulating lymphocytes was reduced in XP but not in TTD patients. Furthermore, XP patients presented a low total number of circulating T cells. Earlier work by Norris et al. showed that in CS patients adaptive cell-mediated immunity and natural killer cell function were normal [43]. These findings provided evidence for the hypothesis that deficiencies in the immune response after exposure to UV radiation are critical for the formation of skin tumours in XP patients. Reports that some XP genes involved in NER were also subunits of the basal transcription factor TFIIH [10,11] further supported the link between repair of DNA damage and transcription of photoimmunologically relevant genes. The two diseases XP and TTD are particularly interesting in this context. Mutations in the gene encoding for the XPD gene can give rise to both XP and TTD [44]. Since XP is associated with a high skin-cancer risk and TTD is not, this may be an important background for studying photoimmunological responses. Intercellular adhesion molecule 1 (ICAM-1) is expressed on Langerhans cells, keratinocytes and fibroblasts and is involved in a multitude of immunologically relevant pathways. Studies in cells from NER-deficient individuals are normally carried out in fibroblasts and their constitutive ICAM-1 expression can be induced by IFN-g. If these cells are exposed to UV-B radiation, the IFN-g-induced ICAM-1 expression can be dose-dependently suppressed [45,46]. A study by Ahrens et al. took advantage of this photoimmunological model system. They compared fibroblasts of patients with XP-D or TTD and normal individuals [47]. Repair of DNA damage as measured by unscheduled DNA synthesis did not differ between the XP-D and TTD cells. In this study, TTD cells showed a normal IFN-g-induced ICAM-1 expression following UV-B irradiation, whereas XP-D cells demonstrated an abnormal ICAM-1 response. This suggested that the photoimmunological phenotype (ICAM-1 response) in DNA-repair-deficient individuals is correlated with the risk of developing skin cancer. This study has been extended and corroborated with more TTD patients, more XP complementation groups and the third repair deficiency, CS [48]. When looking specifically at removal of CPDs in DNA-repair deficiencies, it could be shown that the photoimmunological phenotype correlated with the removal of CPDs. This was the case in XP, CS and TTD cells. In the specific case of XP-D combined with CS, it even appears that the coordination between DNA repair and transcription is so fundamentally altered that repair is carried out randomly over the whole genome, possibly at sites of present transcription, leading to rampant incision throughout the whole genome [49]. These results support the above-mentioned hypothesis that unrepaired CPDs, prevailing in the genome, may stall transcribing RNA polymerase II [50], suppressing the transcription of
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the genes required to respond to UV exposure. Taken together, these results support the notion that repair of DNA damage plays a central role in two major pathways (mutagenesis and immunsuppression) involved in photocarcinogenesis (Fig. 1). It has to be mentioned, however, that there exists a subgroup of TTD patients who do exhibit an abnormal photoimmunological phenotype. These patients are mutated at a specific site of the XPD gene. In this subgroup there is room for discussion about whether the skin-cancer risk correlates with the photoimmunological phenotype. On one hand, TTD is normally not associated with increased skin-tumour susceptibility. On the other hand, these TTD patients show a severe clinical phenotype with some of them dying as early as three years of age.
6. New fields of investigation Several groups have investigated in mice and humans whether there is a connection between NER and immunoglobulin-associated hypermutation. However, experiments in NER-deficient backgrounds have not been able to provide any evidence for this link [51–54]. There have been other findings linking immunity and photocarcinogenesis. A report by Svane et al. demonstrated that high MHC class I expression correlates with slow growth in UV-induced skin carcinomas in hairless mice [55]. Work by Hill et al. indicated that the ligand of the apoptosis-inducing Fas/CD95 may have a critical role in the active suppression of systemic immune responses by UV light [56]. Since the apoptogenic effects of UV radiation on the membrane (Fas/CD95 system) and the nucleus (induction of DNA damage) appear to be linked with each other [57], it is tempting to speculate that this cross-connection also exists for UV-mediated immunosuppression.
7. Therapy/outlook Protection against and/or treatment of adverse side effects of sun exposure has become increasingly important not only for the normal population but also for those individuals who do not have endogenous protection (albinism) or repair (xeroderma pigmentosum). Sunscreens are the most widely applied protection against sun damage. A comprehensive review of sunscreens and their role in different aspects of photocarcinogenesis has been presented by Elmets and Anderson [58]. However, it is important to stress that the capacity of a sunscreen to protect against erythemal responses (SPF) does not necessarily reflect its potential to protect against UV-B radiation-induced immunosuppression. In recent years other substances have been investigated for their protective properties against processes related to photocarcinogenesis. Work by Anolik et al. demostrated that isotretinoin therapy reduced the frequency of skin-cancer
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Fig. 1. Model for a central role of DNA repair in photocarcinogenesis. Exposure to UV radiation causes DNA damage, which is removed by DNA repair. Endogenous repair (NER) can be supplemented by exogenous enzymes (T4NV, photolyase), which reduce generation of mutations and restore post-UV immune responses, thus preventing photocarcinogenesis on two different levels.
development [59]. Conversely, however, the NK cell function was decreased in this study. New studies also point in the direction of antioxidants as possible protectors against UV-modulated immune function. Substances such as epigallocatechin-3-gallate, derived from green tea [60], and nicotinamide [61] have been reported to exhibit protective properties against skin cancer. The protective role of these antioxidants indicates a role of reactive oxygen species (ROS). As mentioned above, UV radiation can induce effects directly through induction of DNA damage and indirectly via generation of ROS. The main wavelength range reponsible for ROS induction is the UV-A. Whether the protective properties of antioxidants against skin-tumour formation stems from a role of UV-A radiation in photoimmunosuppression or the capacity of UV-B radiation to induce relevant amounts of ROS, needs to be elucidated further. Nonetheless the use of antioxidants in sunscreens may not only protect against photoaging but also against photocarcinogenesis. All of the mentioned substances are characteristically prophylactic in nature. However, the repair of already generated damage would beneficially add to the line of defence against skin-tumour formation. Work by Vink et al., cited above, has already indicated a possible way to do this. The central role of CPDs in UV-mediated immune suppression [25] and the possibility of their removal by topical application of different repair enzymes lead the way to investigating whether the efficacity of this approach holds true in humans. A multicentre study investigated this for DNA-repair-deficient individuals where the DNA-repair enzyme T4NV proved to be efficient in CPD removal [62]. A very recent study extended these investigations to normal human individuals [63]. Topical application of photolyase to UV-B-irradiated skin decreased the number of CPDs formed by f40%. The removal of CPDs restored two immunological end-points: hypersensitivity reaction to nickel sulfate in sensitive individuals and IFN-g-induced expression of ICAM-1 in normal
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individuals. Furthermore, this treatment also prevented erythemal responses and sunburn cell formation, thus indicating that topical application of a DNA-repair enzyme is effective in dimer removal and exhibits immunoprotective effects in a way that allows already existing damage to be removed. Application of this treatment, e.g., as an ‘after-sun lotion’ in addition to normal application of sunscreens may have a profound impact on the prevalence of sun-induced tumours. 8. Conclusions Exposure to ultraviolet light has a multitude of effects on the skin. The induction of skin tumours by UV light is of major epidemiological relevance, since such tumours are the most prevalent human malignancy with a continuously increasing frequency. The development of tumours is a multistep process involving induction of mutations and escape from immune surveillance. The role of immunosuppressive effects induced by UV light (photoimmunology) has been elucidated significantly in recent years and recent findings have strengthened the link between DNA damage, photoimmunosuppression, DNA repair and photocarcinogenesis. Furthermore, the first therapeutic strategies capitalizing from this connection are emerging, which may open the door not only to protective measures but also to actual repair of pre-existing photodamage, photoimmunosuppression and thus photocarcinogenesis. 9. Abbreviations APC CHS CPD CS DTH GGR ICAM-1 IFN-g NER NK ROS T4NV TAA TCR Ts TTD UV-A UV-B XP (A–G)
antigen-presenting cell contact hypersensitivity cyclobutane pyrimidine dimer Cockayne syndrome delayed-type hypersensitivity global genome repair intercellular adhesion molecule 1 interferon-g nucleotide excision repair natural killer reactive oxygen species T 4 endonuclease V tumour-associated antigen transcription-coupled repair T suppressor trichothiodystrophy ultraviolet A, 315–400 nm ultraviolet B, 290–315 nm xeroderma pigmentosum complementation groups A–G
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