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Recent advances in urocanic acid photochemistry, photobiology and photoimmunology Neil K. Gibbs,*a Joanne Tyea and Mary Norvalb Received 12th November 2007, Accepted 22nd February 2008 First published as an Advance Article on the web 13th March 2008 DOI: 10.1039/b717398a Urocanic acid (UCA), produced in the upper layers of mammalian skin, is a major absorber of ultraviolet radiation (UVR). Originally thought to be a ‘natural sunscreen’, studies conducted a quarter of a century ago proposed that UCA may be a chromophore for the immunosuppression that follows exposure to UVR. With its intriguing photochemistry, its role in immunosuppression and skin cancer development, and skin barrier function, UCA continues to be the subject of intense research effort. This review summarises the photochemical, photobiological and photoimmunological findings regarding UCA, published since 1998.

Introduction Urocanic acid (UCA) is formed in the upper layers of the epidermis where filaggrin, a histidine-rich filamentous protein produced after caspase-14 cleavage of profilaggrin, is broken down by proteinases into component amino acids.1 It is synthesised as the trans-isomer (trans-UCA) from histidine in a deamination reaction, catalysed by histidase (histidine ammonia-lyase). Trans-UCA accumulates in the skin to high concentration (at least 6 nmol cm−2 of surface skin in most human subjects) as the enzyme urocanase, that catalyses trans-UCA catabolism, is not present in this site. In the inherited metabolic condition, histidinaemia, mutations in the histidase gene result in an accumulation of histidine and lower or non-existent levels of UCA.2 UCA is the principal protondonating species in the stratum corneum and is therefore crucial a Dermatological Sciences, University of Manchester Medical School, Stopford Building, Oxford Road, Manchester, UK M13 9PT. E-mail: [email protected]; Tel: (44) 161-275-5505; Fax: (44) 161-275-5289 b Biomedical Sciences, University of Edinburgh Medical School, Edinburgh, Scotland

to efficient epidermal barrier function.3,4 It is released as terminally differentiated corneocytes slough off and is also eluted from the stratum corneum by sweat. Trans (E)-UCA is a major chromophore in the epidermis. On absorption of ultraviolet radiation (UVR), it converts to the cis (Z)-isomer (see Fig. 1). This reaction is dose-dependent until a photostationary state is reached when the concentration of cisUCA is approximately 60–70%. The action spectrum for trans to cis isomerisation shows a peak at 300–315 nm in mouse skin5,6 and at 280–310 nm in human skin.7 Photoisomerisation still takes place as a result of UVA-II (320–340 nm) or UVA-I (340– 400 nm) radiation.6–8 It has been noted that certain epidermal microflora can degrade L-histidine, trans-UCA and cis-UCA, and one bacterial species that catabolises cis-UCA has been identified as Micrococcus luteus.9 Trans-UCA absorbs erythemogenic UVB (290–315 nm) radiation and was initially proposed to play a role in natural photoprotection, leading to its incorporation in commercial sunscreens and cosmetics from the 1960s until its use was discontinued in the 1990s. More recent evidence demonstrated that trans-UCA is an ineffective sunscreen.10 In 1983, De Fabo and Noonan

Neil Gibbs, PhD, is a Lecturer in Dermatological Sciences, The University of Manchester, UK, with major research interests in human skin photobiology. Joanne Tye, MSc, is a British Skin Foundation funded PhD student in Dermatological Sciences, The University of Manchester, UK, studying immunological aspects of human skin photobiology. Mary Norval, DSc, is Professor Emeritus at the University of Edinburgh in Scotland, with major research interests in the effects of ultraviolet radiation on human health, especially immunological aspects.

Neil K. Gibbs

Mary Norval

This journal is © The Royal Society of Chemistry and Owner Societies 2008

Photochem. Photobiol. Sci., 2008, 7, 655–667 | 655

Fig. 1 The photoisomerisation of naturally occurring trans (E)-urocanic acid to cis (Z)-urocanic acid.

suggested that UCA could act as an initiator in the complex pathway leading to UVR-induced immunosuppression.11 Since that date many experiments in vivo and in vitro have confirmed this role, although, as described in detail below, several intriguing uncertainties remain, especially regarding its mode of action in modulating immune responses. The present review follows from ones published in 199512 and 1999,13 and focuses on recent advances, concentrating on information available since 1998. First there is a section on UCA photochemistry relevant to its photobiological properties, followed by another in which results from the analysis of UCA isomers are brought together. The following section evaluates several proposed mechanisms whereby cis-UCA could downregulate cellmediated immunity. The role of cis-UCA in the immunological control of infectious diseases and photocarcinogenesis are then considered, before a final section summarises strategies to protect against the immunosuppressive effects of cis-UCA.

Photochemistry of UCA On absorbing UVR, trans-UCA isomerises to cis-UCA with an efficiency that is dependent on solvent polarity.14 It has been known for over twenty years that, in an aqueous solution, the quantum yield (U) for this reaction is wavelength dependent with peak photoisomerisation efficiency (U = 0.49) at 310 nm but almost an order of magnitude lower efficiency (U = 0.05) at the trans-UCA absorption maximum of 268 nm.15 The mechanism for this wavelength dependency has been elucidated by Hanson and Simon16 who used pulsed-laser acoustic spectroscopy with excitation at 264 nm and 310 nm. With 264 nm excitation, trans-UCA forms a singlet state and very efficiently undergoes intersystem crossing to a long lived triplet state. In contrast, after 310 nm excitation, singlet trans-UCA intersystem crossing is marginal and the singlet state predominantly undergoes photoisomerisation to cis-UCA. Further studies using two-pulse laser excitation demonstrated that trans-UCA exists as different electronic states that all contribute to the overall absorption spectrum of transUCA but have unique photoreactivity.16 These different states were explored by Ryan and Levy17 who used dispersed emission studies of a supersonic jet of gaseous-phase trans-UCA and identified three distinct regions in its steady-state fluorescence excitation spectrum corresponding to (I) formation of the lowest trans-UCA singlet state, S1 , (II) formation of the S1 states of both trans-UCA and cis-UCA isomers indicating that photoisomerisation occurs efficiently and (III) formation of the S2 trans-UCA excited state which does not result in isomerisation. A consequence of these competing photoprocesses, the wavelength dependent U for UCA photoisomerisation and high absorption of superficial stratum corneum proteins5 is that the action spectra for the production of cis-UCA in mouse and human skin in vivo are red-shifted from 656 | Photochem. Photobiol. Sci., 2008, 7, 655–667

the trans-UCA absorption spectrum (maximal at 268 nm) and have broad peaks of 280–310 nm.6,7 The trans-UCA triplet state has a triplet energy (E T ) of about 230 kJ mol−1 and a lifetime of approximately 10−7 s−1 and is capable of energy transfer to molecular (triplet state) oxygen to form singlet oxygen, O2 (1 Dg ).13,16,18–21 Photoacoustic spectroscopy has determined that this weak reaction occurs in the UVA region of 315–380 nm with peak efficiency at 340 nm16 which corresponds with a theoretical efficiency spectrum for trans-UCA intersystem crossing.18 Time-dependent density functional theory calculations support the existence of this transition at 340 nm.22,23 The shape of the action spectrum for trans-UCA photosensitized production of O2 (1 Dg ) mimics that for the production of sagging in mouse skin.24 It has therefore been suggested that trans-UCA may be a chromophore for O2 (1 Dg ) mediated skin aging.16,22 As O2 (1 Dg ) is short-lived and only reacts with adjacent molecules, it remains to be determined whether UCA photosensitized O2 (1 Dg ) formation in the terminally differentiated stratum corneum can influence cytokine and matrix metalloproteinase induction lower in the epidermis. There is evidence that trans-UCA photosensitized O2 (1 Dg ) generated during UVA irradiation can be scavenged inefficiently (kQ = 3.5 × 106 M−1 s−1 ) by ground state transUCA and form coloured oxidized UCA products that are more efficient than parent UCA at photosensitising O2 (1 Dg ) production and hence further UCA decomposition.18 These UCA oxidized products include longer lived hydroperoxides that are capable of cleaving plasmid DNA19 and inactivating viruses.20 In contrast to these type II reactions, UCA has been reported to act as a type I photosensitiser when exposed to UVA (355 nm) in the presence of methyl linoleate.25 When irradiated at 266 nm, both trans- and cisUCA also undergo photo-ionisation to form a long-lived radical that can efficiently react with oxygen (kQ = 1.3 × 109 M−1 s−1 ).26 In addition to its ability to photosensitise production of excited oxygen species, trans-UCA is an effective hydroxyl scavenger, although an inefficient peroxyl radical scavenger.27,28 In vitro studies using hydroxyl radical generating systems suggest that several UCA oxidation products may be produced from both transUCA and cis-UCA after UVB exposure, particularly imidazole4-carboxaldehyde (ImCHO), its oxidation product imidazole4-carboxylic acid (ImCOOH), imidazole-4-acetic acid (ImAc) and glyoxylic acid (GLX). Under more physiological conditions, ImCHO, ImAc and ImCOOH were shown to be formed in corneal scrapings of human skin after UVB, but not UVA irradiation.29 It is notable that certain of these UCA photoxidation products also possess immunosuppressive properties comparable to those of cis-UCA.30

Analysis of UCA isomers Methods There are various methods which can be used to measure UCA in biological tissues. The most common utilises high performance liquid chromatography (HPLC). A wide variety of columns and eluting buffers have been reported for the quantification of UCA isomers. The amount of free histidine is a major factor affecting the level of UCA in the skin. It is therefore useful to analyse skin histidine and UCA levels simultaneously. Tateda and colleagues31 have developed a HPLC method which detects UCA


through UVR absorption, and histidine by fluorescence following postcolumn derivatisation with o-phthalaldehyde. Hermann and Abeck32 also reported optimal separation of UCA isomers and histidine using a C8 column and a mobile phase consisting of 90% aqueous (0.01 M triethylammoniumphosphate with 5 mM sodium 1-octanesulfonate) and 10% acetonitrile. The same group also describe another technique which can detect UCA isomers and histidine using high-performance capillary electrophoresis (HPCE). UCA isomers are separated from histidine on a fusedsilica column with a mobile phase of 0.05M NaH2 PO4 buffer at a pH of 5.0.32 Levels of UCA are similar to those obtained by HPLC. In contrast to these extraction/chromatographic methods Caspers and co-workers33 have developed a confocal Raman microspectroscopy system that enables the levels of total UCA in skin to be measured non-invasively, although this apparatus cannot distinguish between the two UCA isomers. Concentration of urocanic acid in skin UCA is synthesised from histidine in a deamination reaction. Total UCA concentration appears to correlate with increasing dietary L-histidine levels so that, for instance, skin UCA content has been shown to rise in rats or mice fed diets rich in histidine.34,35 A study in mice examined the relationship between dietary histidine levels and UCA isomers in the skin after a single exposure to UVR at 312 nm.36 The group fed the histidine-rich diet had the same ratio of cis:trans-UCA after irradiation as the group fed the normal diet but showed a quicker return to baseline UCA isomer levels. Further interest in dietary constituents and UCA has focussed on malnutrition. For example De Fabo et al.35 found that low levels of dietary L-histidine resulted in an increase in skin trans-UCA in mice, consistent with the weight loss observed. This change might lead to increased immunosuppression following sunlight exposure because of the increased production of cis-UCA. Hug et al.37 have suggested that such a reduction in the immune response could be a factor contributing to the increased incidence of infectious diseases in malnourished children in many developing countries. In an interesting extension of this idea, Hug et al.38 speculate that the high levels of infection seen in astronauts, who are also known to suffer protein loss, may be related to enhanced UCA levels with UVR, either from the sun or spacecraft lighting, leading to the production of immunosuppressive cis-UCA. In a similar manner, travellers to high altitude can become malnourished, suffer protein catabolism, are exposed to enhanced solar UVB and often develop infectious diseases.39 These novel ideas have not yet been tested experimentally. The concentration of UCA in the skin of healthy individuals of different phototypes and at different body sites has been measured. It varied considerably between individuals at all body sites, with more than 10-fold differences being found.40,41 No correlation between UCA content and age, sex, minimal erythema dose (MED), phototype, pigment or stratum corneum thickness was apparent; any biological consequence of these large individual differences in UCA concentration remains unknown,41 although it has been suggested in a preliminary study that people with high UCA levels produce more DNA photoproducts, with a slower rate of repair of such lesions.42 Within each subject, there are only slight variation in UCA concentration between one body site and another, with higher levels being present in the buttock and arm

and lower levels in the forehead. A further study examined UCA isomers in the skin of healthy children.43 As is the case in adults, there was no correlation between total UCA concentration and MED. However the UCA content was significantly higher in the children than in adults at an unexposed site (buttock–median 22.2 vs. 13.6 nmol cm−2 ), but not different on a sun-exposed site (upper back), although a significantly higher percentage as cis-UCA was found in children compared with adults at this site (median 60.1 vs. 28.3%). This may reflect a higher degree of sun exposure in children than in adults in the weeks or days preceding the analysis. Alternatively, but less likely, is the possibility that children suffer a greater degree of immunosuppression following UVR exposure. In adults, the UCA content at both exposed and unexposed body sites decreased slightly in the summer months compared with the rest of the year (for example mean 7.2 nmol cm−2 in August vs. 9.0 nmol cm−2 in December on the forehead).44 This may be due to more extensive sweating and hence loss of UCA in the hotter temperatures of the summer, and argues against a role for UCA in natural photoprotection. Indeed it has been shown that UCA concentration in human skin does not correlate with photosensitivity.10 There are differences in UCA levels between species. While a median value of approximately 20 nmol cm−2 is present in human skin, the marsupial opossum, Monodelphis domestica, has about 12 nmol cm−2 and a range of Australian marsupials about 120 nmol cm−2 , most of which appeared to be present in the dermis, rather than in the epidermis, as is the case for human and mouse skin.45 One study of hairless mice reported a value of 5 nmol cm−2 .36 The skin concentration of UCA is known to vary considerably between strains of haired mice (A. Moodycliffe and M. Norval, unpublished). Photoisomerisation of urocanic acid Exposure to UVR induces a dose-dependent isomerisation from trans to cis-UCA, ultimately resulting in approximately 60-70% cis-UCA at the photostationary point, following four standard erythema doses of broadband UVB of human subjects.46 This amount of UVR would be expected to produce moderate erythema on unclimatised white skin but minimal or no erythema on previously exposed skin.47 The analysis of a group of subjects throughout a year revealed that, in the summer, cis-UCA levels are close to the maximum obtainable at all body sites tested (forehead, upper back, upper chest and outer and inner upper arm) except at a non-exposed site (buttock).41 In the winter months the UCA present as the cis-isomer fell to below 7% in all regions apart from the forehead where it remained at 18%. The relationship between the degree of pigmentation and the production of cis-UCA following UVR has been examined. Snellman et al. found no skin phototype-dependent difference in the ability to photoisomerise UCA.48 It should be noted that, in this study, the irradiations were performed with fractions of the MED calculated for each subject on an individual basis, implying that the darker-skinned individuals would receive higher UV doses that the fairer-skinned individuals. In contrast, de Fine Olivarius et al. demonstrated that the production of cisUCA was higher in subjects of skin types I and II compared with skin types III and IV following exposure to small doses of UVR.46 Thus, as the immunological activity of cis-UCA is



dose-dependent, at least in mice,34,49 low pigmentation may be considered as a risk factor for a higher degree of UV-induced immunosuppression. The increase in cis-UCA following UVR suggests that it may be possible to assess recent sun exposure by analysing the ratio of cis:trans-UCA in human urine. Sastry et al. tested this by measuring the concentration of UCA isomers in such samples before and following irradiation with 95% UVA/5% UVB to 90% of the body area, a protocol similar to that experienced in commercial suntanning parlours.50 A single exposure or repeated daily exposures of approximately 70% MED induced about a 5-fold increase in the ratio relative to baseline. Thus such an assay may be useful as a biomarker for recent sun exposure. Although photoisomerisation of UCA in human skin occurs optimally within the UVB region,6 both UVA-II and UVA-I have also been shown to induce cis-UCA production.6,8 Commercial sun beds emit mainly UVA wavelengths. Possible consequences of sunbed use on cis-UCA formation, immunosuppression and the induction of skin cancer remain unclear. Ruegemer et al.51 assessed UCA levels in volunteers before sunbed use and after twice weekly exposures for 6 weeks. They report an initial decrease in transUCA which then rose after the 12 sessions, and a significant increase in cis-UCA at most of the time points measured. These data suggest that consecutive sunbed use alters the total content and isomer ratio of UCA, indicating that a continuing detrimental effect on the immune system is possible during the development of a tan. As cis-UCA could be an important factor in cutaneous carcinogenesis (see ‘Role of cis-UCA in photocarcinogenesis’ section below), UCA isomer biomarkers may be useful in predicting the risk of developing skin cancer. This possibility has been tested by three groups. First de Fine Olivarius et al. assayed UCA isomers in UV-exposed and non-exposed body sites in patients with a past history of basal cell carcinoma (BCC) or cutaneous malignant melanoma (CMM) and in healthy controls.52 No differences were found between the groups in total UCA or in the percentage as the cis-isomer. The net production of cis-UCA (measured as a percentage of trans-UCA) following a single test UV dose was slightly higher in both cancer groups compared with the control group. However the biological significance of this change was thought doubtful as there was no difference between the groups in the absolute concentration of cis-UCA (measured as nmol cm−2 ) induced by the UVR. Similar results were recorded by Snellman et al. after analysis of patients with past histories of BCC or CMM and healthy controls.53 De Simone and colleagues assessed UCA isomers in the skin of patients with a past history of nonmelanoma skin cancer (NMSC, consisting of BCCs and squamous cell carcinomas) at different times of the year.54 No significant difference in total UCA or percentage as cis-UCA between patient and control groups was found in either photoexposed (outer arm) or non-photoexposed (buttock) sites in the winter period. In the summer months, the percentage of cis-UCA in the photoexposed site of the patients was considerably lower than in the controls (17% vs. 42%) although it did not differ in the non-photoexposed site. It is likely that this result does not reflect a risk factor for subjects to develop skin cancer but merely reflects the fact that people who have had skin cancer in the past are more likely to avoid direct sun exposure in the summer than people in the control group. 658 | Photochem. Photobiol. Sci., 2008, 7, 655–667

Thus the evidence to date indicates that people with a past history of either CMM or NMSC do not differ from healthy controls in their cutaneous UCA concentration, percentage as cis-UCA or rate of photoisomerisation.

Mechanism of action of cis-UCA as an initiator of UVR-induced immunosuppression Following UVR of the skin, suppression of cell-mediated immunity is induced. The major chromophores, present in the upper layers of the epidermis, that act as initiators of this process are DNA, trans-UCA and membrane components. The pathway leading from the absorption of UVR photons by the chromophores to the downregulation of T cell responses is complex and is shown in outline in Fig. 2. Readers are referred to the comprehensive reviews of Clydesdale et al.,55 Schwarz,56 Ullrich,57 Schade et al.,58 Hanneman et al.,59 and Norval60 for more details, and to Norval and El-Ghorr for a description of methods to determine the immunomodulatory effects of cis-UCA.61

Fig. 2 Outline of the pathway leading to suppression of cell-mediated immunity as a result of exposure to ultraviolet radiation (UVR).

At the outset, it is clear that the mechanism of action of cis-UCA as an immunosuppressive agent has not been fully explained. Currently there is evidence for more than one pathway and it is possible that cis-UCA may act on different cell types found in various body sites in different ways. After the UVRinduced isomerisation of trans to cis-UCA in the stratum corneum, cis-UCA persists in the epidermis of human subjects for at least 2 weeks following the exposure, gradually returning to the background level during that time.62 It is also found for several


weeks in suction blister fluid, presumably indicating that it is present in the dermis.63 Some is released systemically as it has been detected in serum for 1–2 days after UVR64 and is excreted in the urine for about 2 weeks.65 Thus there is the opportunity for cisUCA to affect immune cell populations, not only in the epidermis but also in the dermis, lymph nodes and even in the blood, spleen, thymus or bone marrow. To add to the complexity, not all types of antigens or immune responses may be affected by cis-UCA similarly. El-Ghorr and Norval66 irradiated mice with three UVR sources emitting various wavebands, followed by tests of contact hypersensitivity (CHS) and delayed type hypersensitivity (DTH) and by UCA analysis, and found that there was no correlation between the suppression of either hypersensitivity response and the concentration of cis-UCA in the skin. Furthermore Kim et al.67 showed that the viability of the antigen determined the differential contribution of cis-UCA or DNA as initiators of UVR-induced suppression of DTH. Here mice were UVR-irradiated and the importance of cis-UCA or DNA damage assessed by injection of a monoclonal antibody with specificity for cis-UCA or application of liposomes containing DNA repair enzymes to the skin. Subsequent DTH tests with live and either formalin-fixed or killed antigens revealed that cisUCA was the critical initiator molecule when live antigens were used, whereas damaged DNA was the critical initiator when dead antigens were used. The process by which cis-UCA affects the ability of various populations of antigen presenting cells to process live or dead antigens, together with their subsequent maturation and cytokine production is largely unknown. To provide clarity, the following sections deal separately with five ways in which cis-UCA has been proposed to down-regulate immunity. These are by occupying specific receptors, by altering cytokine production, by affecting antigen presentation, by functional effects on nerve/mast cells, and by inhibiting the generation of reactive oxygen species. Of course, in reality each of these mechanisms should not be considered singly, such as the occupation of a receptor by cis-UCA which could have significant consequences for the immune function of that cell type. A summary is provided in Table 1 of the current possibilities whereby cis-UCA might suppress cell-mediated immunity. It should also be noted that cis-UCA can be immunotoxic. A single administration of cisUCA leads to decreased splenocyte phagocytosis in C57BL/6N mice, while prolonged cis-UCA treatment (4 weeks) results in thymic atrophy and hypocellularity and in splenic and lymph node hypercellularity in both C57BL/6N and C3H/HeN strains of mice.68,69 The mechanisms for these changes and any resulting consequence for the immune response have not been investigated. Cis-UCA receptors Early experiments in mouse models demonstrated that histamine receptor antagonists/agonists could reverse the ability of cisUCA to down-regulate DTH responses. However attempts to show that cis-UCA acts through histamine-like receptors on keratinocytes, monocytes or Langerhans cells have failed. Laihia et al.70 analysed the binding of UCA isomers to histamine H1 , H2 and H3 receptors and, because of the structural similarities of UCA with c-aminobutyric acid (GABA), to the GABA receptors also, on rat cortex membranes. While competitive binding curves did not provide evidence that histamine receptors for cis-UCA were

Table 1 Summary of possible pathways by which cis-urocanic acid suppresses cell-mediated and innate immunity Mechanism

Details

Through receptors

GABAA on cortical cells.70,71 Serotonin (5-HT2A ) on unidentified cell type(s).74

Through cytokines and other immune mediators

Prostaglandin E2 production by keratinocytes in the presence of histamine.86 Cytokine production by human keratinocytes.85 IL-10 production by activated CD4+ T cells.87

Through antigen presenting cells

Impairment in antigen presentation by epidermal cells; reversal by IL-12.90

Through nerve/mast cells

Stimulation of neuropeptides (substance P, CGRP) from peripheral sensory nerves, degranulation of mast cells.73,92,93

Through neutrophils

Impairment in the generation of extracellular reactive oxygen species.96,97

present, both UCA isomers bound to GABAA receptors. Some stereospecificity was involved as cis-UCA displaced [3 H]-GABA by 48% and trans-UCA by 19%. Further experiments showed that both isomers were more potent at pH 5.5, the pH of the skin, than at pH 7.4, and that cis-UCA inhibited GABA responses, while trans-UCA slightly enhanced them.71 While both isomers acted with low potency on the GABAA receptors, it was noted that the UCA concentration in the skin is high, ranging from about 2 to 62 nmol cm−2 in human epidermis.41,72 This is similar to the range that was used experimentally. As various neuropeptides have been implicated as mediators in UVR-induced immunosuppression and as GABA is an inhibitory neurotransmitter, it was suggested that cis-UCA could bind to the GABAA receptor, thus encouraging the secretion of various cutaneous mediators, such as histamine and prostaglandins, that down-regulate local immune responses. However GABA receptors have not been located in the skin, although they are present on T cells, and they are not generally found on peripheral nerves. In addition Khalil et al.73 showed that cis-UCA could stimulate neuropeptide release from peripheral sensory nerves while GABA did not. Therefore, for all these reasons, it seems unlikely that cisUCA acts through the GABA receptors to initiate the suppression of cell-mediated immunity. Significant progress has been made recently with the discovery that cis-UCA may act via the serotonin, 5-hydroxytryptamine (5HT) receptor.74 The ring-like structure that cis-UCA forms in aqueous solution resembles the structure of 5-HT (see Fig. 3). A series of careful experiments showed first that cis-UCA bound to this receptor with relatively high affinity while trans-UCA did not. Treatment with a selective serotonin antagonist blocked the binding of cis-UCA, and 5-HT acted as a competitive inhibitor for the binding of cis-UCA. Secondly cis-UCA, but not trans-UCA, was demonstrated to mobilise intracellular calcium stores within cells expressing serotonin receptors, implying that it did this by engaging the serotonin receptor. Thirdly in vivo experiments were



Fig. 3 The structures of cis-urocanic acid and serotonin, illustrating their similarities.

set up in which mice were UVR-irradiated or were treated with cis-UCA or serotonin. They were then immunised with formalinfixed Candida albicans and the DTH assessed subsequently by intradermal injection of C. albicans. All these procedures resulted in suppression of the DTH response compared with untreated mice. However if the mice were injected with an anti-cis-UCA antibody or an anti-5-HT antibody prior to the UVR exposure, cis-UCA or 5-HT administration, the immunosuppression was blocked. Tests using a series of serotonin receptor agonists and antagonists with specificity for various 5-HT receptor subtypes revealed that cis-UCA activated the 5-HT2A receptor to suppress DTH. The cell target expressing the 5-HT2A receptors and its site in the body are not yet known. Such receptors are found on dendritic cells,75 on T and B cells,76 on nerve cells77 and on mast cells.78 Each of these cell types has been implicated in UVR-induced immunosuppression, as described in the sections below. Finally, it should be noted that results presented recently by Woodward et al.79 did not confirm that cis-UCA acts through the 5-HT2A receptor. Here human peripheral blood mononuclear cells were stimulated in vitro with lipopolysaccharide in the presence of cisUCA or serotonin, and the production of TNF-a monitored. Both cis-UCA and serotonin suppressed TNF-a release but the down-regulating effect of cis-UCA was abrogated by adding indomethacin (an inhibitor of prostaglandin production) to the cultures; in contrast, the down-regulating effect of serotonin was not abrogated. Similarly, when the production of prostaglandin E2 in the cultures was measured, cis-UCA induced it in a dose-dependent manner but serotonin was only effective at a supraphysiological concentration. The test system used here was an artificial one, relying on the in vitro response of the peripheral blood mononuclear cells alone, and as such is unlikely to mimic what happens in vivo, particularly locally in the skin. In addition the secretion of cytokines by monocytes is known to be suppressed by 5-HT2A receptor binding, rather than being activated.80 Cytokines and other immune mediators While broadband UVB is known to induce a variety of cytokines such as TNF-a and TGF-b, and other immune mediators such as platelet activating factor, prostaglandins and histamine, in the skin and systemically, the consequences of cis-UCA application are less clear. An early hypothesis, proposed by Kurimoto and Streilein,81 was that cis-UCA acts on keratinocytes to induce the production of TNF-a. However cis-UCA treatment of the murine keratinocyte cell line, PAM-212, failed to cause any detectable change in the mRNA or protein expression of the immunosuppressive cytokines TNF-a, IL-10 and TGF-b.82 In addition, studies in knock-out mice that were deficient in TNF receptor 1 and 2 showed that cis-UCA is still capable of suppressing both local and systemic 660 | Photochem. Photobiol. Sci., 2008, 7, 655–667

CHS, as it did in the wild-type mice.83,84 Thus these results indicate that TNF-a signalling is unlikely to play a major part in the mechanism of action of cis-UCA although it should be noted that the response of human keratinocytes may be different from that in mice. Kaneko et al.85 have demonstrated very recently that cis-UCA induces the production of several cytokine proteins, including TNF-a, in primary human keratinocytes. In addition prostaglandin-endoperoxide synthase 2 is highly up-regulated by cis-UCA, resulting in an enhanced secretion of prostaglandin E2 into the culture supernatant. Previously it has been shown that cis-UCA can act in synergy with histamine to induce the production of prostaglandin E2 from human keratinocytes in vitro.86 Whether these mechanisms involving immune mediators produced by keratinocytes are of importance in vivo remains to be established. An interesting report investigated the down-regulating effects of cis-UCA on the ability of mouse spleen cells to act as antigen presenting cells.87 It was shown that the reason for the suppression in antigen presentation was due to the stimulation of IL-10 production by activated CD4+ T cells. The main target for cis-UCA was shown to be this cell population and not the macrophages or any other cell type present in the spleen. The increase in IL-10 could then lead to inhibition of antigen presentation by dendritic cells, affect the production of T regulatory cells and down-regulate the T helper 1 response.87 These findings have not been corroborated, as far as we are aware, but it would be worthwhile to undertake further investigations using the reagents that are available currently to phenotype the CD4 cell population in the spleen that responds to cis-UCA by producing IL-10. In particular any interaction with T regulatory cells could be important in helping to explain how cis-UCA acts. Antigen presentation While it is an attractive concept that cis-UCA could affect the ability of Langerhans cells or dendritic cells to present antigen, such an effect has not been demonstrated in in vitro studies. For example, cis-UCA did not change the development of dendritic cells derived from bone marrow or their allo-antigen presenting capability.88 In addition cis-UCA does not alter the expression of MHC Class II or the co-stimulatory molecules required for effective antigen presentation.88,89 In contrast Beissert et al.90 have developed a mouse tumour model in which a suspension of epidermal cells (containing 5–15% Langerhans cells) were first pulsed ex vivo with a tumour-associated antigen, derived from a spindle cell line. The cells were then injected into the mice to immunise them, and the animals were challenged subsequently with the spindle cells to find out if they had been protected, as demonstrated by rejection of the tumour. Under normal circumstances, they were but, if the incubation of the epidermal cells with the tumour-associated antigen had taken place in the presence of cis-UCA, the protective effect was lost. It was concluded that cis-UCA had down-regulated the ability of the epidermal cells to present the tumour antigen, thus failing to immunise the mice. Further investigation revealed that, if the epidermal cells were incubated ex vivo with cis-UCA in the presence of IL-12, then the suppression in the protective effect was prevented.90 IL-12 treatment also abrogated the suppression in CHS responses induced by cis-UCA in mice. Finally incubation of the epidermal cells in the presence of cis-UCA with an antigen


that required processing before presentation, and another antigen that did not require processing before presentation, showed that cis-UCA acted mainly by impairing antigen presentation.90 These data indicate that cis-UCA may down-regulate antigen presentation and that IL-12 can protect against this effect, perhaps by inhibiting the apoptosis induced by T regulatory cells.91 It should be noted that the epidermal cell population used in the above experiments, although enriched for I-A+ cells, was not pure and an interaction of cis-UCA with other cell populations is certainly possible. Nerve/mast cells The first indication that cis-UCA might target mast cells came from studies in mast cell-deficient mice. It was shown that they were resistant to suppression of the CHS response, induced by cisUCA.92 If their dorsal skin was reconstituted with bone-marrow derived-mast cell precursors, the mice became susceptible to the immunomodulating effects of cis-UCA. In 1999 Wille et al.93 used human skin organ cultures treated with cis-UCA and showed that mast cell chymase was depleted and TNF-a was produced, demonstrating that cis-UCA had induced mast cell degranulation. More recently it has been revealed that the effect of cis-UCA on mast cells is more likely to be indirect, via the stimulation of neuropeptides from peripheral sensory nerves. Khalil et al.73 induced a blister on the hind footpad of rats and, following removal of the surface epithelium, perfused the area with cis-UCA, trans-UCA and other molecules. Cis-UCA, but not trans-UCA, increased the microvascular flow and this was due to neuropeptide release. Substance P and calcitonin gene related peptide (CGRP) were responsible for most of the cis-UCA-induced change in flow. Furthermore, in mice treated with capsaicin to deplete sensory neuropeptides, cis-UCA did not suppress CHS responses. It was concluded that cis-UCA acts first on the peripheral terminals of unmyelinated primary sensory nerves leading to production of neuropeptides, and that these, in turn, degranulate the mast cells. The products of the mast cells, particularly histamine, lead to stimulation of prostaglandin synthesis by keratinocytes and ultimately suppression of cell-mediated immunity by this route. The neuropeptides themselves can also be down-regulatory. For example, CGRP can bind to Langerhans cells and induce IL10 production,94 and substance P can bind to keratinocytes and stimulate IL-1 production.95 Equivalent studies to determine whether cis-UCA alters neuropeptide production in human skin have not been undertaken. Generation of reactive oxygen species by neutrophils Polymorphonuclear neutrophils provide an essential contribution to innate immunity. They are recruited to the site of an infection and, on activation, engulf and kill the microbial pathogens. This process is dependent on the production of microbicidal peptides, proteases and reactive oxygen species (ROS), also referred to as respiratory or oxidative burst activity. Kivisto et al.96 have shown in an in vitro study that the respiratory burst activity of human neutrophils can be inhibited by cis-UCA. Further investigation has revealed that cis-UCA suppresses the generation of extracellular superoxide by neutrophils while not affecting the generation of intracellular superoxide or other ROS.97 Phagocytic

and bactericidal activities were also not altered by cis-UCA. The mechanism involved and whether the interactions take place outside or inside the neutrophils have not been elucidated. These interesting results suggest that cis-UCA may have some therapeutic potential in situations where there is recruitment of large numbers of neutrophils as it could limit the production of extracellular ROS that damage the host tissues whilst preserving the activities required for microbial clearance.97

Role of cis-UCA in infectious diseases Exposure to UVB is known to suppress cell-mediated immune responses to a variety of infectious agents in mouse and rat models. Similar results have been obtained when the UVR is replaced by cis-UCA. For example, rats were infected orally with the parasitic worm, Trichinella spiralis, and then injected subcutaneously with a range of doses of cis or trans-UCA.98 The number of T. spiralis larvae in the muscle tissue was increased in the animals treated with cis-UCA compared with those treated with trans-UCA. In addition the DTH response to T. spiralis antigens was significantly suppressed by the cis-UCA treatment. If the rats were injected with an anti-cis-UCA antibody before being UVirradiated and infected with T. spiralis, the expected increase in larvae counts and downregulation in DTH were abrogated. Thus, in this model, cis-UCA is proved to be an important mediator of UVB-induced suppression of cell-mediated immunity. A study in a hairless guinea-pig model of Buruli ulcer disease reached the same conclusion.99 The disease, characterised by the development of chronic necrotising skin ulcers with an associated social stigma, is caused by an extracellular infection with Mycobacterium ulcerans. The animals were treated topically for 3 consecutive days with trans-UCA or a mixture of 1:9 cis:trans-UCA or vehicle. They were infected intradermally with M. ulcerans and the resultant skin lesions measured, together with the DTH response to M. ulcerans cell fragment antigens. Compared with the guinea-pigs treated with trans-UCA or vehicle, the animals treated with the UCA mixture developed more severe skin lesions and had suppressed DTH responses. These results indicate that cis-UCA enhances the ulcerative form of M. ulcerans disease. There is also evidence that cis-UCA can suppress the cellmediated immune response to viruses. Sleijffers et al.100 irradiated volunteers with one personal MED on each of 5 consecutive days. They were then vaccinated with hepatitis B surface antigen. The UCA isomer levels in the skin were assayed before and immediately after the UV exposure, and the in vitro lymphocyte response to various mitogens, recall antigens such as tetanus and diphtheria, and hepatitis B virus was also measured before and at several time points after UV exposure and vaccination. It was found that the subjects with a higher cis-UCA concentration after UV exposure had slightly lowered hepatitis B-specific lymphocyte responses. It is not known if this rather small (although statistically significant) difference in the T cell response due to cis-UCA content would lead to an actual reduced level of protection against a future hepatitis B infection, but this was thought unlikely by the authors. No such correlation was apparent between cis-UCA levels and the other lymphocyte responses tested, nor between cis-UCA levels and hepatitis B-specific antibody titres. While there is some evidence to support a role for UVA in UV-induced immunosuppression,101 Reeve et al.102 have shown



the reverse-that UVA can protect against UVB-induced immunosuppression. It has been suggested that this effect could be mediated through cis-UCA. One model of infection to support this view has been studied. Pre-exposure of mice to UVA before UVB irradiation or cis-UCA treatment and infection with Listeria monocytogenes resulted in a reversal of the expected down-regulation in the cell-mediated immune response to the bacterium.103 The reader is referred to the ‘Protection against the immunosuppressive effects of cis-UCA’ section below for further details. The elegant studies of Safer et al.104 demonstrate that UCA is a major chemoattractant for the skin-penetrating nematode, Strongyloides stercoralis. Although Safer et al. did not determine the isomer content of the UCA used in their experiments, it is likely to be trans-UCA. If the chemoattractant properties of UCA are isomer-specific, there may be some potential to reduce infection with S. stercoralis by exposing the skin to solar UVR so that about half of the UCA is converted to the cis-isomer. As S. stercoralis is a soil-dwelling parasite, most commonly infecting via the soles of the feet, such an activity may require an unusual sunbathing position!

Role of cis-UCA in photocarcinogenesis It is well established that topical application of UCA increases both the incidence and malignancy of UVR-induced skin cancers in a hairless mouse model.105 The hypothesis tested in this work was that increased levels of cutaneous UCA would lead to increased cis-UCA after UV exposure, thus enhancing photoimmunosuppression and increasing oncogenicity. This approach, however, does not mirror the natural situation as extra UCA was applied externally. In addition, as outlined in the ‘Photoisomerisation of UCA’ section above, there is no experimental evidence to date showing that individuals with a past history of skin cancer differ from healthy individuals in their total cutaneous UCA concentration or percentage as cis-UCA or rate of isomerisation from trans to cis-UCA on UV exposure.52–54 These studies contained subjects with phototype I-IV but none with phototype V or VI. An interesting suggestion in this context has been made by Hug106 that differing levels of epidermal UCA may explain the observation that black women with a previous NMSC are at more than three-fold increased relative risk of developing a further NMSC compared with white women.107 Quoting two reports in which the UCA content of black skin is found to be more than three times higher than in white skin, Hug argues that this may render black individuals more susceptible to increased photoimmunosuppression and thus to be at higher risk of NMSC development. The most convincing experimental evidence to date that endogenous cis-UCA plays an important role in photocarcinogenesis comes from the study of Beissert et al. in mice.80 The animals were irradiated with increasing doses of UVB over a period of 6 months and then observed for the development of skin tumours over the next 6 months. One group was treated with an anti-cis-UCA antibody, administered intraperitoneally before each exposure and two control groups contained mice that were only UV-irradiated, or were treated with an irrelevant isotype-matched antibody before each exposure. All the animals in both control groups developed tumours after 182 days but, in contrast, after 200 days, only 50% 662 | Photochem. Photobiol. Sci., 2008, 7, 655–667

of the animals in the anti-cis-UCA antibody treated group had developed tumours. Thus cis-UCA is involved in UV-induced skin cancer in mice and, as outlined in the ‘Antigen presentation’ section above, may act to inhibit antigen presentation, an effect that can be reversed by IL-12.80 It is not known at what point or points the down-regulation might happen. To try to address this issue, another study used a line of fibrosarcoma cells (FSA) which, when implanted subcutaneously into mice, grew into skin tumours.108 Cis-UCA applied topically or intradermally for three weeks before the injection of FSA cells had no effect on tumour outgrowth, while prior UVB irradiation enhanced the outgrowth considerably. In confirmation, treatment with an anti-cis-UCA antibody before each UVB exposure did not inhibit the increased growth of the tumours compared with unirradiated animals. This result was taken as evidence that UV-enhanced growth of skin tumours is not mediated by cis-UCA, and therefore that cis-UCA is more likely to act at an early stage of UV-induced carcinogenesis rather than at a later progression stage.

Protection against the immunosuppressive effects of cis-UCA UVR production of cis-UCA occurs via the singlet state of transUCA in the upper layers of the human epidermis. Strategies to protect against cis-UCA production are therefore very much restricted to limiting those UVR wavelengths responsible for maximum trans-UCA singlet formation reaching the skin. All sunscreens, if applied correctly, are likely to protect to some degree against both erythema and cis-UCA production.109 However, protection against these two UVR effects are highly wavelength dependent. As detailed above (see ‘Photochemistry of UCA’ section), the action spectrum for cis-UCA production is red-shifted from both the trans-UCA absorption spectrum and the UVR erythemal action spectrum in human skin.7 This explains why a predominantly UVB sunscreen might protect efficiently against erythema but be less protective against cis-UCA production.110 Also it may provide a reason for the discrepancy between the sun protection factor and the immune protection factor noted with UVB sunscreens which is not so apparent with broad spectrum (UVB and UVA) sunscreens.111,112 Interestingly, in mice, it has been shown that two UVB sunscreens, 2-ethylhexyl-p-methoxycinnamate (2-EHMC) and octyl-N-dimethyl-p-aminobenzoate (o-PABA), both fail to protect against UVR-induced cis-UCA production, but 2-EHMC is highly effective at protecting against UVR-induced suppression of CHS.113 Further study revealed that 2-EHMC also abrogates immunosuppression induced by topical cis-UCA in the absence of UVR, suggesting that a ‘neutralising’ chemical reaction may occur between 2-EHMC and cis-UCA.113 Paradoxically, the physical sunscreen, titanium dioxide, may reduce the production of cisUCA109 but can also photosensitise UVA-induced trans-UCA oxidation in vitro.114 The latter effect could result in increased levels of the immunosuppressive photooxidation products.30 Several studies in mice have investigated strategies to protect against the immunosuppressive effects of exogenously applied cisUCA. When applied topically, the omega-3 fatty acid, eicosapentaenoic acid (EPA), effectively inhibits UVB and topical cis-UCAinduced suppression of CHS responses.115 The mechanisms for this effect are not clear but are likely to be linked to either the


antioxidant properties of EPA or its ability to compete with omega-6 fatty acid cyclooxygenase substrates (e.g. arachadonic acid), thus reducing the production of immunosuppressive prostaglandin E2 .115 PycnogenolR , an antioxidant extracted from the bark of the French maritime pine, Pinus pinaster Ait., abrogates inhibition of CHS by UVR and by cis-UCA when applied to the skin immediately after either treatment.116 Significantly PycnogenolR also protects against photocarcinogenesis if applied after each daily irradiation, thus providing indirect evidence for the involvement of cis-UCA in this process.116 Certain isoflavonoids such as the genistein metabolite, [S]-4 7dihydroxyisoflavane (equol), extracted from red clover (Trifolium pratense) have radical scavenging properties, induce the cutaneous antioxidant metallothionein and are also phytoestrogens that bind efficiently to estrogen receptors (ER). In mice, topical equol partially reverses the suppression in CHS induced by UVR or by topically applied cis-UCA.117 Recent studies have indicated the mechanisms by which this may occur. Using metallothioneinknockout mice, it was clear that metallothionein protects against immunosuppression produced by either UVR or cis-UCA.118 In the same mouse model it was demonstrated that equol protection against both agents is also partially dependent on metallothionein production.119 Further studies using an ER antagonist suggest that the phytoestrogenic property of equol plays a role in reducing both UVR and cis-UCA-induced immunosuppression via an ER-dependent upregulation of metallothionein.120 Interestingly, 17-b-estradiol also abrogated both UVR and cis-UCA-induced suppression of CHS. This suggests a gender specific susceptibility that may relate to epidemiological and experimental results showing a lower risk of skin cancer in females compared with males.121,122 As mentioned above (see ‘Role of cis-UCA in infectious diseases’ section) prior or concurrent exposure of mouse skin to UVA is reported to reduce immunosuppression caused by either UVB or cis-UCA.102 This UVA protective effect is dependent on the induction of the cutaneous stress-protein, haem oxygenase-1 (HO-1) and can be reduced by tin protoporphyrin, a specific HO-1 inhibitor.123 As a product of its haem catabolic activity, HO-1 releases carbon monoxide (CO) and studies with specific CO generating systems suggest that it is CO generated by HO-1 activity that abrogates UVB and cis-UCA-induced immunosuppression.124 Further experiments have demonstrated that UVA immunoprotection is dependent on CO binding to cutaneous guanylyl cyclase (GC) and the consequent production of cyclic guanosine monophosphate (cGMP). Treatment of mice with sildenafil (ViagraR ) which inhibits cGMP degradation has the same effect as CO binding.125 Whilst the primary aim of the above studies has been to identify candidate targets to protect against photoimmunosuppression, they have also increased the understanding of how cis-UCA may mediate its immunomodulatory effects.

Conclusions Significant progress has been made in recent years towards a fuller knowledge of the photochemistry and photobiology of UCA, although much remains to be discovered. An understanding of the complex primary photoprocesses that contribute to the wavelength dependence of the quantum yield

for trans-UCA photoisomerisation has explained why the action spectrum for cis-UCA production is red-shifted from the transUCA absorption spectrum. The original argument linking transUCA photoisomerisation to photoimmunosuppression was based on the similarity of the trans-UCA absorption spectrum and the action spectrum for suppression of CHS. Now we know why the red-shift occurs, it is necessary to investigate other chromophores and photoproducts of trans-UCA to assess the relative contribution of cis-UCA production in photoimmunosuppression. From the studies discussed above, it is clear that the oxidative breakdown of trans-UCA may produce photoproducts that are as immunosuppressive as cis-UCA but whose photochemistry is largely unexplored. The photosensitising properties of the triplet state of trans-UCA and its interactions with biomolecules (particularly structural proteins which share a common location and are important in skin terminal differentiation) require further investigation. The contrasting abilities of trans-UCA to both photosensitise and scavenge ROS and other radical species also need to be explored more fully in relation to immunosuppression. The analyses of the concentration of UCA in human skin have revealed large differences between individuals that have failed to be related to any other biological parameter. It would be useful to measure histidine concentration as well as UCA isomers in future skin samples as the former may affect the latter. In addition the ability to assess histamine concentration in the skin of healthy subjects and those with various dermatoses would be interesting because histamine is formed in one step from histidine, as is trans-UCA, and it is an important cutaneous immune mediator following UVR. Although experiments in mice indicate that cisUCA suppresses hypersensitivity response dose-dependently, the range of cis-UCA doses used was limited. In humans, preliminary results demonstrate that fair-skinned people may be at relatively higher risk of immunosuppression following suberythemal UVB exposure, due to a higher rate of isomerisation to cis-UCA, compared with more pigmented people. In addition a high UCA level may lead to the production of more DNA photoproducts and an increased chance of a downregulated antigen-specific T cell response. However individuals with a history of skin cancer, where past exposure to solar UVR is recognised as a major risk factor, do not have a different cutaneous UCA content, percentage as cis-UCA or rate of isomerisation from trans to cis-UCA from individuals without a history of skin cancer. This argues against a critical role for cis-UCA in human photocarcinogenesis, although experiments in mice have shown that inhibiting the action of cisUCA provides partial protection against the generation of skin tumours. As the percentage of cis-UCA is at the maximum possible in most body sites during the summer months (at mid-latitudes), this means that there is no adaptation in UCA concentration or in the ratio of UCA isomers over an extended period of time as a result of repeated solar UVR. Cis-UCA is recognised as an important initiator of the complex cascade leading to suppression of cell-mediated immunity, but its mechanism of action is still not clear, despite many different approaches being taken. It is likely that it has more than one immunological effect, and may act not only locally in the skin where it is formed but also systemically. Recent results indicate that the 5-HT2A receptor is involved and it will be important to identify the cell target that expresses this receptor and where it is located. Evidence demonstrating that cis-UCA induces the production of



IL-10 by activated CD4+ T cells requires corroboration and also an identification of the subtype of CD4+ cell involved, particularly to find out if it could be classified as a T regulatory cell. Other data show that cis-UCA has an inhibitory effect on antigen presentation, a process that can be reversed by IL-12, and that it can indirectly target mast cells by stimulating the generation of neuropeptides from peripheral sensory nerves that then lead to the degranulation of the mast cells. The degree to which we wish to protect against UCA-mediated photoimmunosuppression needs to be debated fully. On the one hand reduced immunosuppression may reduce skin cancer risk but at the same time increase the incidence of immunological photodermatoses. In common with the majority of the research discussed in this review, the studies on protection against cis-UCAinduced immunosuppression have been conducted in mice and have revealed tantalising mechanistic pathways. Methodologies for studying clinical photoimmunology have improved dramatically over the last ten years and it is now time to explore these UCA pathways in human subjects.

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