This article is published as part of a themed issue of Photochemical & Photobiological Sciences on

Topical and systemic photoprotection

Published in issue 4, 2010

Behaviour and measurement

Chronic sun damage and the perception of age, health and attractiveness, P. J. Matts and B. Fink, Photochem. Photobiol. Sci., 2010, 9, 421

Knowledge, motivation, and behavior patterns of the general public towards sun protection, J. M. Goulart and S. Q. Wang, Photochem. Photobiol. Sci., 2010, 9, 432

Molecular modifications of dermal and epidermal biomarkers following UVA exposures on reconstructed full-thickness human skin, M. Meloni et al., Photochem. Photobiol. Sci., 2010, 9, 439

In vitro tools for photobiological testing: molecular responses to simulated solar UV of keratinocytes growing as monolayers or as part of reconstructed skin, L. Marrot et al., Photochem. Photobiol. Sci., 2010, 9, 448

Comparison between UV index measurements performed by research-grade and consumer-products instruments, M. P. Corrêa et al., Photochem. Photobiol. Sci., 2010, 9, 459

Topical photoprotection

Ultraviolet filters, N. A. Shaath, Photochem. Photobiol. Sci., 2010, 9, 464

The long way towards the ideal sunscreen—where we stand and what still needs to be done, U. Osterwalder and B. Herzog, Photochem. Photobiol. Sci., 2010, 9, 470

Percutaneous absorption with emphasis on sunscreens, H. Gonzalez, Photochem. Photobiol. Sci., 2010, 9, 482

In vitro measurements of sunscreen protection, J. Stanfield et al., Photochem. Photobiol. Sci., 2010, 9, 489

Human safety review of “nano” titanium dioxide and zinc oxide, K. Schilling et al., Photochem. Photobiol. Sci., 2010, 9, 495

Sunscreens and occupation: the Austrian experience, H. Maier and A. W. Schmalwieser, Photochem. Photobiol. Sci., 2010, 9, 510

UVA protection labeling and in vitro testing methods, D. Moyal, Photochem. Photobiol. Sci., 2010, 9, 516

Sunscreens: the impervious path from theory to practice, P. U. Giacomoni et al., Photochem. Photobiol. Sci., 2010, 9, 524

Dose-dependent progressive sunscreens. A new strategy for photoprotection?, A. Gallardo et al., Photochem. Photobiol. Sci., 2010, 9, 530

The FDA proposed solar simulator versus sunlight, R. M. Sayre and J. C. Dowdy, Photochem. Photobiol. Sci., 2010, 9, 535

How a calculated model of sunscreen film geometry can explain in vitro and in vivo SPF variation, L. Ferrero et al., Photochem. Photobiol. Sci., 2010, 9, 540

Filter–filter interactions. Photostabilization, triplet quenching and reactivity with singlet oxygen, V. Lhiaubet-Vallet et al., Photochem. Photobiol. Sci., 2010, 9, 552

Systemic photoprotection

Mechanistic insights in the use of a Polypodium leucotomos extract as an oral and topical photoprotective agent, S. Gonzalez et al., Photochem. Photobiol. Sci., 2010, 9, 559

Vitamin D-fence, K. M. Dixon et al., Photochem. Photobiol. Sci., 2010, 9, 564

UV exposure and protection against allergic airways disease, S. Gorman et al., Photochem. Photobiol. Sci., 2010, 9, 571

Photoprotective effects of nicotinamide, D. L. Damian, Photochem. Photobiol. Sci., 2010, 9, 578

The two faces of metallothionein in carcinogenesis: photoprotection against UVR-induced cancer and promotion of tumour survival, H. M. McGee et al., Photochem. Photobiol. Sci., 2010, 9, 586

Dietary glucoraphanin-rich broccoli sprout extracts protect against UV radiation-induced skin carcinogenesis in SKH-1 hairless mice, A. T. Dinkova-Kostova et al., Photochem. Photobiol. Sci., 2010, 9, 597

Mice drinking goji berry juice (Lycium barbarum) are protected from UV radiation-induced skin damage via antioxidant pathways, V. E. Reeve et al., Photochem. Photobiol. Sci., 2010, 9, 601

Oestrogen receptor-β signalling protects against transplanted skin tumour growth in the mouse, J.-L. Cho et al., Photochem. Photobiol. Sci., 2010, 9, 608

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

KView Article Online / Journal Homepage / Table of Contents for this issue

PERSPECTIVE www.rsc.org/pps | Photochemical & Photobiological Sciences

Vitamin D-fence

Katie M. Dixon,*a Vanessa B. Sequeira,a Aaron J. Campa,b and Rebecca S. Masona

Received 2nd December 2009, Accepted 8th February 2010First published as an Advance Article on the web 3rd March 2010DOI: 10.1039/b9pp00184k

The role of vitamin D in the immune system is complex. Vitamin D is produced in the skin followingexposure to ultraviolet radiation. There is compelling evidence that vitamin D compounds protectagainst ultraviolet radiation-induced DNA damage and immune suppression, suggesting it may bebeneficial as a skin cancer preventive agent. However, vitamin D has many modulatory effects on theimmune system and it has in fact been suggested that the immune suppression generally attributed tothe UVB portion of sunlight is mediated through vitamin D. Here we describe the role of vitamin Dcompounds as “defence” molecules against UVR-induced damage, and investigate both sides of the“fence” regarding the effects of vitamin D compounds and the immune system.

1. Introduction

Vitamin D is the collective term for both vitamin D2 and vitaminD3. Whilst it is referred to as a vitamin, it is in fact a steroidhormone precursor, as it can be synthesized endogenously in thepresence of UV light. Vitamin D2 (ergocalciferol) is obtained fromplants. Dietary sources such as oily fish account for less than10% of total vitamin D for most people. The major source ofvitamin D is that produced in the skin from sunlight exposure.Pre-vitamin D3 is formed when 7-dehydrocholesterol in the skinis exposed to ultraviolet radiation (UVR) in the UVB range (290–320 nm)1 and undergoes a rapid rearrangement of its three doublebonds to form vitamin D3 (cholecalciferol).2 Prolonged sunlightexposure cannot result in excessive production of vitamin D3. Thisis because during sun exposure, both the previtamin D3 and thethermal isomerization product vitamin D3 (in the skin) absorb

aDepartment of Physiology and Bosch Institute, University of Sydney, NSW,Australia 2006. E-mail: katied@physiol.usyd.edu.au; Fax: +61 2 9351 2510;Tel: +61 2 9351 6526bDepartment of Anatomy, School of Biomedical Sciences and Pharmacy,University of Newcastle, NSW, Australia

Katie M. Dixon

Dr Katie M. Dixon PhD is aResearch Fellow in the BoschInstitute, University of Sydney,Australia. Her major research in-terests are vitamin D, photobiol-ogy, photoimmunology and can-cer cell biology.

Vanessa B. Sequeira

Vanessa B. Sequeira is a PhDcandidate at the University ofSydney and a member of theYoung Investigators Committeeof the Bosch Institute. Vanessahas an interest in the mechanismsof the photoprotective effects ofvitamin D analogs.

the solar UVR and are converted to several “over-irradiation”photoproducts including lumisterol and tachysterol.3 Vitamin Ditself is biologically inactive; it is transported in the blood streamvia the vitamin D-binding protein and converted to its active formin two sequential hydroxylation reactions, the first in the liverat the 25 position of vitamin D, and the second in the kidneysat the 1a position. These hydroxylation reactions result in theformation of 25-hydroxyvitamin D3 (25OHD3) and the activemetabolite 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol),respectively.4 Vitamin D2 compounds are similarly hydroxylated.Importantly, there is recent evidence that the enzymes required forboth hydroxylation steps are present in epidermal keratinocytes,and that they produce active 1,25(OH)2D3 in the 2–5 nM range.5–8

Dendritic cells are also capable of metabolizing vitamin D3 tothe active form.9 The two vitamin D activation steps have alsobeen shown to occur in several other tissues. Activity of 25-hydroxylase has been observed in the intestine, adrenal gland,lung, kidney and bone, whilst 1a-hydroxylation of 25(OH)D3 toform 1,25(OH)2D3 also occurs in many tissues, including bone,placenta, testes, prostate, brain, intestine, adrenal medulla10 andgranulomatous lymph nodes.11

564 | Photochem. Photobiol. Sci., 2010, 9, 564–570 This journal is © The Royal Society of Chemistry and Owner Societies 2010

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

2. Vitamin D signal transduction pathways

The active compound, 1,25(OH)2D3, binds to the vitamin Dreceptor (VDR), in one of its many target tissues, throughwhich it mediates genomic biological responses. In the skinthese cells include keratinocytes, Langerhans cells, melanocytes,CD11b+ macrophages, CD3+ T-lymphocytes, dermal fibroblasts,and epithelial cells of the epidermal appendages.12,13 This steroidhormone/receptor complex then interacts with vitamin D re-sponse elements within the promoter region of target genes, allow-ing regulation of transcription. Such genomic processes usuallyrequire several hours or days. However, vitamin D compoundscan generate a variety of non-genomic responses requiring amuch shorter time frame, in the order of seconds to minutes.The first vitamin D-mediated rapid response to be observed wastranscaltachia in chick intestinal cells.14

The ability to utilise multiple signal transduction pathwaysis conferred, at least in part, by the fact that the 1,25(OH)2D3

molecule is highly conformationally flexible. This renders themolecule capable of undergoing very rapid shape changes, and thusforming a variety of different ligand conformations.15 The natureof the receptor involved in vitamin D non-genomic responsesremains controversial. It has been suggested to be a membranereceptor (VDRmem) or an alternate ligand-binding domain withinthe classical VDR,16 or an interaction between the two. It is known,however, that the molecular shape preferred for non-genomicresponses is the 6-s-cis steroid-like conformation.15 Fig. 1 outlinesboth the non-genomic and genomic vitamin D signal transductionpathways that lead to biological responses.

3. Ultraviolet radiation

Only a small portion of wavelengths below 320 nm reachthe dermis, but the biological effects of these wavelengths aresignificant even in small doses. Exposure to UVB radiationfrom sunlight can result in pathological responses in the skinincluding sunburn (erythema), oedema, melanin pigmentation,actinic keratosis, photoaging, cataracts, immunosuppression andskin cancer.17–19 Despite this, UVB is beneficial in that it providesthe energy for formation of vitamin D. It is now clear that UVAis not innocuous and can produce many of the harmful effects

Fig. 1 Vitamin D signal transduction pathways. The 1,25(OH)2D3

molecule is highly conformationally flexible and is capable of undergoingrapid conformational changes to form different ligand shapes. 1. Thenature of the receptor involved in vitamin D non-genomic responsesremains controversial. Suggestions of a separate membrane receptor(VDRmem) or an alternate ligand binding domain within the classicalVDR located at the membrane have been proposed. The molecularshape preferred for non-genomic responses is the 6-s-cis conformation.2. The active compound, 1,25(OH)2D3, binds to the vitamin D receptor(VDR), through which it mediates genomic biological responses. Genomicprocesses usually require a longer time frame than non-genomic responses,though the timeline is not exact.

induced by UVB, including cell damage, DNA damage, actinicelastosis, alteration of immunologic functions of epidermal cellsand increased melanin production,20 although some have reportedbeneficial effects of the UVA spectrum.21,22

Both UVA and UVB are carcinogenic, though UVA is lesseffective in producing skin carcinoma. UVA has been implicatedin melanoma induction,23 though this was not the case in amore recent study. In a murine study in which several differentUVR sources were used, De Fabo et al. showed that only UVB-containing sources initiated melanoma. Furthermore, they showedthat in mice irradiated with an isolated UVA waveband, or alight source in which more than 96% of UVB was removed,the response was similar to unirradiated control animals. They

Aaron J. Camp

Dr Aaron J. Camp PhD is aResearch Fellow at the Universityof Newcastle, Australia, and anhonorary associate of the Depart-ment of Physiology, Universityof Sydney, Australia. His majorresearch focus is in neurobiologyof the special senses.

Rebecca S. Mason

Prof. Rebecca S. Mason MBBSPhD leads the Vitamin D, Boneand Skin Research Laboratory,part of the Bosch Institute and theDepartment of Physiology at theUniversity of Sydney. Her majorresearch interests are vitamin D,photoprotection and bone physi-ology.

This journal is © The Royal Society of Chemistry and Owner Societies 2010 Photochem. Photobiol. Sci., 2010, 9, 564–570 | 565

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

concluded that UVB was the wavelength responsible for develop-ment of cutaneous malignant melanoma, whereas UVA, even atenvironmentally relevant doses, was ineffective.24,25

4. UVR-induced immunosuppression

The UVR present in sunlight is immune suppressive.26 Theseimmunosuppressive effects facilitate skin carcinogenesis by de-pressing cell-mediated immune reactions that are normally in placeto destroy developing skin tumours.27

4.1 The skin immune system

The skin immune system consists of multiple cell types withinthe epidermis and dermis, and cells connecting the skin with theblood or lymph. The major cells involved in skin immunity areLangerhans cells, keratinocytes, mast cells and T cells. In theepidermis, Langerhans cells form a dendritic cell network thatfunctions to capture invading microorganisms, ingest them, andthen process their antigens into a form that can be recognizedby T cells. They then migrate to the skin-draining lymph nodeswhere they present antigen to naı̈ve T cells to initiate a protectiveimmune response.28 Mast cells are traditionally known for theirproinflammatory functions, but more recently mast cells and mastcell-derived interleukin-10 (IL-10) have been shown to limit theskin pathology associated with immunological responses thatcan damage the skin, such as contact dermatitis and chronicexposure to ultraviolet radiation.29 Keratinocytes also play animportant role in the skin immune system. They produce severalcytokines, including IL-1, IL-6, IL-7, IL-8, IL-10, IFNg, TGFband TNFa, that also contribute to the overall immune responsein the skin.30 UVR has a direct effect on immune cells in theskin. It causes a marked reduction in Langerhans cells31 andimpairs the antigen-presenting ability of those cells remaining.32,33

In addition, UVR promotes the release from keratinocytes ofsoluble mediators which enter the bloodstream and have systemiceffects.34

4.2 Immunosuppression: UVA vs. UVB

UVB is well known to cause immunosuppression, but the role ofUVA in UVR-induced immunosuppression is controversial. Thisdiversity may be accounted for by differences between animalsand humans, differences in irradiation sources and/or protocols,and/or differences in the immune end-points studied.35 Some stud-ies have shown no involvement of UVA in UVR-induced immuno-suppression. In one study, UVA was shown to not cause significantimmunosuppression by itself in human subjects, but it augmentedsolar-simulated radiation-induced immunosuppression.35 In con-trast, studies by Reeve et al. demonstrated a protective effect ofUVA on UVB-induced immunosuppression and inflammatoryoedema in hairless mice21,22 and further showed involvement ofinterferon-g and heme oxygenase induction in UVA-mediatedimmune protection.36 Other studies have reported failure of UVAto induce immunosuppression in hairless mice at doses consideredto be environmentally relevant for sun exposure in humans [fora review see Tyrrell and Reeve, 200637]. It has also been shownthat immunosuppression by UVA was dose- and time-dependent

in human subjects, requiring 48 h from UVR, and that there isinteraction between UVA and UVB in immunosuppressive effects72 h post-UVR.38

4.3 Immunosuppression models: CHS vs. DTH

UVR has been shown to suppress different immune responses,including contact hypersensitivity reactions (CHS)39 and delayed-type hypersensitivity reactions (DTH) to viral,40 bacterial41 andfungal antigens.42 Both CHS and DTH are the main experimentalmodels used to study UVR-induced immunosuppression. CHSis initiated with the application of a contact sensitizer appliedepicutaneously in the induction phase, followed by a furtherapplication several days later to induce the elicitation phase,resulting in an inflammatory response. In DTH the same protocolis followed except that the antigen is more complex and isusually injected intradermally or subcutaneously so that antigenpresenting cells other than those in the epidermis are involved.43

UVR-induced immunosuppression is described in terms of localimmune suppression and systemic immune suppression. Theseterms refer to the site of irradiation with respect to antigenexposure and are not used to describe whether the immuneresponse generated is local or systemic. Local immune suppressionoccurs when the immune response to antigen applied to the UV-irradiated site is reduced. Systemic immune suppression occurswhen the immune response to antigen applied at a non-exposedsite, distant from the irradiated site, is impaired.

4.4 Immunosuppression and DNA damage

UVR also induces DNA damage, which has been shown toplay a role in immunosuppression. The most common form ofUVR DNA photoproducts are cyclobutane pyrimidine dimers(CPD), the major subset of which are thymine dimers.44 Ina study by Applegate et al.,45 UVR-induced suppression ofCHS in the South American opossum, whose DNA damage isrepaired by a visible light-activated photoreactivating enzyme,was completely prevented when the opossum skin was exposedto visible light immediately after UVB irradiation. In a differentstudy, when UVB-irradiated mice were treated topically withT4N5-containing liposomes [bacteriophage T4 endonuclease V,an excision repair enzyme for CPDs], the number of CPDs wasreduced, and suppression of CHS and DTH and induction ofsuppressor T cells were blocked.46 Furthermore, IL-10, whichis capable of suppressing antigen presenting cell function,47 issecreted by UV-irradiated keratinocytes, but not by keratinocytestreated with T4N5 prior to UVR. This suggests that DNA damagemay activate keratinocytes to produce this immune regulatorycytokine.48

4.5 Immunosuppression and skin carcinogenesis

One of the first studies that suggested a role for UVR-inducedimmunosuppression in skin carcinogenesis was by the Kripkegroup in 1976.26 Skin tumours were induced in mice by chronicUVR exposure. The tumours were then excised and transplantedinto normal immune-competent non-irradiated mice, in whichthey were consistently rejected due to their highly antigenicnature. If the recipient mice were UV-irradiated before the

566 | Photochem. Photobiol. Sci., 2010, 9, 564–570 This journal is © The Royal Society of Chemistry and Owner Societies 2010

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

transplantation, the tumours were not rejected and grewprogressively.26 The importance of immunological factors in UVR-induced skin carcinogenesis is highlighted in renal transplantationpatients, who were shown to have an 82-fold increase in riskof invasive squamous cell carcinoma compared with the non-transplanted population.49 Patients undergoing immunosuppres-sive chemotherapy also appear to be at increased risk of skincancer.50

5. Immunomodulatory effects of vitamin D

5.1 Basal effects of 1,25(OH)2D3 and analogs in the absence ofUVR

Vitamin D compounds are well known for their ability tomodulate immune function in the skin. They inhibit maturationand function of antigen presenting cells, induce regulatory Tcell activation, suppress T-helper type 1 (Th1) T-cell responsesand modify cytokine patterns within the skin.51 Furthermore,binding of vitamin D compounds to the classical VDR suppressesrelB, a NF-kB family member, which plays an important rolein the differentiation and maturation of dendritic cells.52,53 TheVDR is present in several immune cells, including monocytes,macrophages and activated T and B cells.54 Thus, it been suggestedthat local production of active 1,25(OH)2D3 in skin cells may beinvolved in UVB-mediated immune suppression.55 Furthermore,1,25(OH)2D3 was reported to increase IL-10 production in mastcells.56

Whilst high concentrations of vitamin D have been shownto be immunosuppressive,57 vitamin D deficiency also causesimmunosuppression.58 There are also distinct differences withrespect to murine and human studies of vitamin D and im-munosuppression. In human subjects, the vitamin D analogcalcipotriene suppressed CHS responses to dinitrochlorobenzeneby 64%, a level similar to that caused by solar simulated UVR.59 Astudy by Gorman et al. showed increased suppressive capacityof CD4+CD25+ regulatory T cells in the skin-draining lymphnodes of BALB/c mice following a single topical applicationof 1,25(OH)2D3.60 A similar effect of topical calcipotriol wasreported by Ghoreishi et al. in C57 BL/6 mice.61 A more recentstudy by Damian et al. in human subjects showed that, in theabsence of UVR, 1,25(OH)2D3 was immunosuppressive in arecall DTH (Mantoux) model when total 1,25(OH)2D3 doses of1 mg or higher were applied topically. Furthermore, there werereductions between the initial Mantoux reactions and the post-treatment reaction at the vehicle-treated site, raising the possibilitythat the application of 1,25(OH)2D3 to nearby sites was causingeither systemic or para-local immunosuppression. Indeed, in theabsence of 1,25(OH)2D3 treatment there was no suppression of theMantoux reaction at vehicle-treated sites.51

In the study by Gorman et al., purified CD4+ cells from theskin draining lymph nodes of 1,25(OH)2D3-treated or UVB-irradiated BALB/c mice suppressed CHS to dinitrofluorobenzenewhen cells were injected into naı̈ve BALB/c mice, comparedto mice who received equal numbers of these cells from un-treated control mice.60 Conversely, other studies in mice haveshown that 1,25(OH)2D3 and analogs caused no direct changein immune responses in the absence of UVR.62,63 Possible reasons

Fig. 2 Summary of 1,25(OH)2D3 immunomodulation and protectionfrom UVR-induced immunosuppression in different models. The activecompound 1,25(OH)2D3 has been shown to cause immunosuppressionin the BALB/c mouse in a CHS model, but studies in Skh:hr1 mice infact showed the opposite effect; that is, 1,25(OH)2D3 and analogs JNand QW caused no basal immune suppression but conferred protectionagainst UVR-induced suppression of CHS. Both 1,25(OH)2D3 and analogcalcipotriol caused basal suppression of DTH and CHS responses inhuman subjects. When applied topically to human subjects, 1,25(OH)2D3at doses low enough to avoid basal immunosuppression failed to provideprotection against UVR-induced suppression of DTH. Whilst this isnot a comprehensive list of studies, it highlights the differences betweenspecies, mouse strains, and experimental protocols that currently limit ourunderstanding of the role of vitamin D compounds in immunosuppression.* Since 1,25(OH)2D3 caused basal immunosuppression in this study,the dose for the UVR protection study was lowered considerably toprevent basal immunosuppression. For this reason the results of the UVRprotection study are uninformative.

for discrepancies between these studies (Fig. 2) are proposed insection 5.3.

5.2 Effects of vitamin D compounds on UVR-inducedimmunosuppression

Topical 1,25(OH)2D3 has conferred protection against UVR-induced immunosuppression in mice in numerous studies.62–64 InSkh:hr-1 hairless mice, topical 1,25(OH)2D3 provided significantprotection against UVR-induced suppression of CHS to oxa-zolone. Mice were exposed to a 3¥ minimal erythemal dose andsubsequently treated on the dorsal surface with 1,25(OH)2D3.The dose of vitamin D used in this study was 1.65 mM,equivalent to 22.8 mmol cm-2 or 0.01 mg cm-2 (total dose0.07 mg in a 25 g mouse). Furthermore, both 1,25(OH)2D3 and

This journal is © The Royal Society of Chemistry and Owner Societies 2010 Photochem. Photobiol. Sci., 2010, 9, 564–570 | 567

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

a low-calcemic analog, 1a-hydroxymethyl-16-ene-24,24-difluoro-25-hydroxy-26,27-bis-homovitamin D3 (QW), protected againstUVR-induced systemic immunosuppression using the same CHSto oxazolone protocol,63 and both compounds caused no directchange in immune responses in the absence of UVR. Thesame group confirmed that a cis-locked analog, 1a,25-dihydroxy-lumisterol (JN), which can only activate the non-genomicpathway,65 provided similar protection to 1,25(OH)2D3 againstUVR-induced immunosuppression using the same protocol as theinitial study62 and again caused no direct immune suppression inthe absence of UVR in this model (Fig. 2).

In the study by Damian et al. outlined in section 5.1, theeffects of 1,25(OH)2D3 on UVR-induced immunosuppression inhuman subjects using a DTH model were also tested. In thisstudy a 0.01mg cm-2 dose of 1,25(OH)2D3 (total dose 0.56mg),considerably lower than the dose that caused immunosuppressionby itself, caused no suppression at the vehicle-treated sites butwas also ineffective in preventing UVR-induced suppression ofMantoux responses.51 However, the results were uninformativesince in order to investigate any protective effects, the dose of1,25(OH)2D3 had to be lowered to the point where it caused nobasal immunosuppression by itself. Intriguingly, the same studyshowed a reduction in UVR-induced DNA damage in the formof thymine dimers and in apoptotic sunburn cells with 0.1mg cm-2

1,25(OH)2D3 treatment.51 This pattern of partial photoprotectionis consistent with other studies in which sunscreens were shownto protect from genetic damage66 but failed to protect the immunesystem.67,68

5.3 Vitamin D and immunosuppression: both sides of the fence

The differences between studies showing immunosuppressiveeffects of vitamin D compounds and those showing protectionmay be explained by a number of factors. For example, in thestudy by Gorman et al.,60 a 1,25(OH)2D3 dose of 3 mM wasused—this is almost twice the dose that showed protection inother studies.62 This is consistent with higher doses of 1,25(OH)2D3

being immune suppressive,51 although a dose similar to that usedin the Gorman study was effective in producing photoprotectionin Skh:hr1 mice (unpublished data). It should be noted that the1,25(OH)2D3 dose of 0.01mg cm-2 that failed to prevent UVR-induced suppression of DTH in human subjects in the studyby Damian et al. was about 2000-fold less than the equivalentamount given per kilogram in mice. It was necessary in thehuman study to use low enough doses so as not to cause basalimmunosuppression. The authors suggested that this differencein dose relative to body weight and/or the greater absorptionthrough thin mouse skin may account for the protection fromUVR-induced immunosuppression in mice but not humans.51 Asnoted in an earlier publication, concentrations of 1,25(OH)2D3 of10-10–10-9 M in vitro could reasonably be justified by studies of1,25(OH)2D3 production in human skin.6,8,64 As far as possible,it was intended to mimic these concentrations in studies usingtopical applications.62,63

In relation to differences observed between murine studies,genetic differences in mouse strains have been reported to resultin different levels of immune responses to UVR.69 This raises thequestion as to whether the direct effect of 1,25(OH)2D3 to cause im-mune suppression in the absence of UVR60 is species specific, even

within strains of murine species. Differences between the BALB/cand Skh:hr1 strains in terms of UVR-induced immunosuppressionremain to be investigated. Interestingly, Skh:hr-1 hairless micewere shown to express estrogen receptor-b but not -a.70 This issignificant since an earlier study by Widyarini et al. revealed theinvolvement of estrogen receptor signalling in the regulation ofimmune suppression by UVB.71 In a preliminary communicationby Malley et al., dietary vitamin D3 was shown to preventUVB-induced suppression of CHS in C57BL/6 mice but not inBALB/c mice. Offspring from vitamin D deficient C57BL/6 miceshowed significantly higher levels of immunosuppression whencompared with their replete counterparts.72 This indicates thatprotection against UVR-induced immunosuppression by vitaminD compounds may be mouse strain dependent.

Differences in immune responses between mouse strains, orbetween mice and human subjects could also be explained bydifferences in the experimental protocols. Studies showing protec-tion against UVR-induced immunosuppression by 1,25(OH)2D3

utilised the CHS protocol, whereas studies in human subjects werecarried out using the DTH method. DTH studies in mice arerequired to fully elucidate the role of 1,25(OH)2D3 in protectionagainst UVR-induced immunosuppression.

Furthermore, studies in Skh:hr-1 mice showed that a cis-lockedanalog of 1,25(OH)2D3 that is only capable of activating the non-genomic pathway, JN, was as effective as 1,25(OH)2D3 in pro-tecting against immunosuppression.62 Both QW and 1,25(OH)2D3

are capable of generating both genomic and non-genomic re-sponses, so it is possible that they mediate their protective effectson UVR-induced immunosuppression through the non-genomicpathway.63 The biological activity of the analog calcipotriol hasbeen attributed to its binding to the VDR, however whetherit is capable of eliciting non-genomic responses is unclear.73 Itwas suggested by Kuritzky et al. that the opposing effects of1,25(OH)2D3 may be explained if the non-genomic pathway isresponsible for photoprotection, and the genomic pathway isresponsible for immunosuppression.74

6. Conclusion

While it is clear that vitamin D compounds modulate the immunesystem, our understanding of their precise role remains limited.Further studies are required to elucidate the role for vitamin Din local and systemic immunosuppression. Since vitamin D com-pounds have been shown to provide photoprotection, and sinceprotection against UVR-induced immunosuppression is key topreventing skin carcinogenesis, further studies in human subjectsusing cis-locked low-calcemic analogs are critical in determiningthe mechanisms of vitamin D compounds in immunosuppression.

Acknowledgements

This research was supported by the National Health and MedicalResearch Council of Australia. K. M. Dixon was the recipient ofa Cancer Institute NSW Research Scholar Award.

References

1 M. F. Holick, J. A. MacLaughlin, M. B. Clark, S. A. Holick, J. T. Potts,Jr., R. R. Anderson, I. H. Blank, J. A. Parrish and P. Elias, Science,1980, 210, 203–205.

568 | Photochem. Photobiol. Sci., 2010, 9, 564–570 This journal is © The Royal Society of Chemistry and Owner Societies 2010

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

2 X. Q. Tian, T. C. Chen, Z. Lu, Q. Shao and M. F. Holick, Endocrinology,1994, 135, 655–661.

3 M. F. Holick, Am. J. Clin. Nutr., 2004, 80, 1678S–1688S.4 P. J. Malloy, J. W. Pike and D. Feldman, Endocr. Rev., 1999, 20, 156–188.5 B. Lehmann, O. Tiebal and M. Meurer, Arch. Dermatol. Res., 1999,

291, 507–510.6 B. Lehmann, P. Knuschke and M. Meurer, Photochem. Photobiol.,

2000, 72, 803–809.7 D. D. Bikle, M. K. Nemanic, J. O. Whitney and P. W. Elias,

Biochemistry, 1986, 25, 1545–1548.8 B. Lehmann, T. Genehr, P. Knuschke, J. Pietzsch and M. Meurer,

J. Invest. Dermatol., 2001, 117, 1179–1185.9 H. Sigmundsdottir, J. Pan, G. F. Debes, C. Alt, A. Habtezion, D. Soler,

E. C. Butcher, H. Sigmundsdottir, J. Pan, G. F. Debes, C. Alt, A.Habtezion, D. Soler and E. C. Butcher, Nat. Immunol., 2007, 8, 285–293.

10 K. Vantieghem, L. Overbergh, G. Carmeliet, P. De Haes, R. Bouillonand S. Segaert, J. Cell. Biochem., 2006, 99, 229–240.

11 R. S. Mason, T. Frankel, Y. L. Chan, D. Lissner and S. Posen, Ann.Intern. Med., 1984, 100, 59–61.

12 J. Reichrath, U. G. Classen, V. Meineke, H. DeLuca, W. Tilgen, A.Kerber and M. F. Holick, Histochem. J., 2000, 32, 625–629.

13 P. Milde, U. Hauser, T. Simon, G. Mall, V. Ernst, M. R. Haussler, P.Frosch and E. W. Rauterberg, J. Invest. Dermatol., 1991, 97, 230–239.

14 I. Nemere, Y. Yoshimoto and A. W. Norman, Endocrinology, 1984, 115,1476–1483.

15 A. W. Norman, W. H. Okamura, M. W. Hammond, J. E. Bishop, M. C.Dormanen, R. Bouillon, H. van Baelen, A. L. Ridall, E. Daane, R.Khoury and M. C. Farach-Carson, Mol. Endocrinol., 1997, 11, 1518–1531.

16 M. T. Mizwicki, D. Keidel, C. M. Bula, J. E. Bishop, L. P. Zanello, J. M.Wurtz, D. Moras and A. W. Norman, Proc. Natl. Acad. Sci. U. S. A.,2004, 101, 12876–12881.

17 I. E. Kochevar, M. A. Pathak and J. A. Parrish, in Fitzpatrick’sDermatology in General Medicine, ed. I. M. Freedberg, A. Eisen,K. Wolff, K. F. Austen, L. Goldsmith, S. Katz and T. B. Fitzpatrick,McGraw-Hill, New York, 1999, pp. 220–230.

18 J. M. McGregor and J. L. M. Hawk, in Fitzpatrick’s Dermatology inGeneral Medicine, ed. I. M. Freedberg, A. Eisen, K. Wolff, K. F. Austen,L. Goldsmith, S. Katz and T. B. Fitzpatrick, McGraw-Hill, 1999, pp.1555–1561.

19 M. A. Pathak, P. Ngheim and T. B. Fitzpatrick, in Fitzpatrick’sDermatology in General Medicine, ed. I. M. Freedberg, A. Eisen,K. Wolff, K. F. Austen, L. Goldsmith, S. Katz and T. B. Fitzpatrick,McGraw-Hill, 1999, pp. 1598–1607.

20 F. R. de Gruijl, Methods Enzymol., 2000, 319, 359–366.21 V. E. Reeve, M. Bosnic, C. Boehm-Wilcox, N. Nishimura and R. D.

Ley, Int. Arch. Allergy Immunol., 1998, 115, 316–322.22 M. Allanson, D. Domanski and V. E. Reeve, J. Invest. Dermatol., 2006,

126, 191–197.23 S. Q. Wang, R. Setlow, M. Berwick, D. Polsky, A. A. Marghoob, A. W.

Kopf and R. S. Bart, J. Am. Acad. Dermatol., 2001, 44, 837–846.24 E. C. De Fabo, F. P. Noonan, T. Fears and G. Merlino, Cancer Res.,

2004, 64, 6372–6376.25 F. P. Noonan and E. C. De Fabo, J. Invest. Dermatol., 2009, 129, 1608–

1610.26 M. L. Kripke and M. S. Fisher, J. Natl. Cancer Inst., 1976, 57, 211–215.27 D. X. Nghiem, N. Kazimi, D. L. Mitchell, A. A. Vink, H. N.

Ananthaswamy, M. L. Kripke and S. E. Ullrich, J. Invest. Dermatol.,2002, 119, 600–608.

28 M. L. Kripke, C. G. Munn, A. Jeevan, J. M. Tang and C. Bucana,J. Immunol., 1990, 145, 2833–2838.

29 M. A. Grimbaldeston, S. Nakae, J. Kalesnikoff, M. Tsai and S. J. Galli,Nat. Immunol., 2007, 8, 1095–1104.

30 Y. Matsumura and H. N. Ananthaswamy, Toxicol. Appl. Pharmacol.,2004, 195, 298–308.

31 G. B. Toews, P. R. Bergstresser and J. W. Streilein, J. Immunol., 1980,124, 445–453.

32 J. C. Simon, P. D. Cruz, Jr., R. E. Tigelaar, R. D. Sontheimer and P. R.Bergstresser, J. Invest. Dermatol., 1991, 96, 148–151.

33 A. Tang, M. C. Udey, A. Tang and M. C. Udey, J. Immunol., 1991, 146,3347–3355.

34 A. Schwarz and T. Schwarz, Exp. Dermatol., 2002, 11(s1), 9–12.35 J. M. Kuchel, R. S. Barnetson and G. M. Halliday, J. Invest. Dermatol.,

2002, 118, 1032–1037.

36 V. E. Reeve and R. M. Tyrrell, Proc. Natl. Acad. Sci. U. S. A., 1999, 96,9317–9321.

37 R. M. Tyrrell and V. E. Reeve, Prog. Biophys. Mol. Biol., 2006, 92,86–91.

38 T. S. Poon, R. S. Barnetson and G. M. Halliday, J. Invest. Dermatol.,2005, 125, 840–846.

39 J. M. Jessup, N. Hanna, E. Palaszynski and M. L. Kripke, Cell.Immunol., 1978, 38, 105–115.

40 S. Howie, M. Norval and J. Maingay, J. Invest. Dermatol., 1986, 86,125–128.

41 A. Jeevan and M. L. Kripke, J. Immunol., 1989, 143, 2837–2843.42 Y. Denkins, I. J. Fidler and M. L. Kripke, Photochem. Photobiol., 1989,

49, 615–619 .43 M. Norval, Prog. Biophys. Mol. Biol., 2006, 92, 108–118.44 P. J. Rochette, J. P. Therrien, R. Drouin, D. Perdiz, N. Bastien, E. A.

Drobetsky and E. Sage, Nucleic Acids Res., 2003, 31, 2786–2794.45 L. A. Applegate, R. D. Ley, J. Alcalay and M. L. Kripke, J. Exp. Med.,

1989, 170, 1117–1131.46 M. L. Kripke, P. A. Cox, L. G. Alas and D. B. Yarosh, Proc. Natl. Acad.

Sci. U. S. A., 1992, 89, 7516–7520.47 K. W. Moore, R. de Waal Malefyt, R. L. Coffman and A. O’Garra,

Annu. Rev. Immunol., 2001, 19, 683–765.48 C. Nishigori, D. B. Yarosh, S. E. Ullrich, A. A. Vink, C. D. Bucana,

L. Roza and M. L. Kripke, Proc. Natl. Acad. Sci. U. S. A., 1996, 93,10354–10359.

49 F. J. Moloney, H. Comber, P. O’Lorcain, P. O’Kelly, P. J. Conlon andG. M. Murphy, Br. J. Dermatol., 2006, 154, 498–504.

50 B. H. Hill, Australas. J. Dermatol., 1976, 17, 46–48.51 D. L. Damian, Y. J. Kim, K. M. Dixon, G. M. Halliday, A.

Javeri and R. S. Mason, Exp. Dermatol., 2009, DOI: 10.1111/j.1600-0625.2009.00955.x.

52 X. Dong, W. Lutz, T. M. Schroeder, L. A. Bachman, J. J. Westendorf,R. Kumar and M. D. Griffin, Proc. Natl. Acad. Sci. U. S. A., 2005, 102,16007–16012.

53 M. D. Griffin, X. Dong, R. Kumar, M. D. Griffin, X. Dong and R.Kumar, Arch. Biochem. Biophys., 2007, 460, 218–226.

54 V. Matheu, O. Back, E. Mondoc, S. Issazadeh-Navikas, V. Matheu, O.Back, E. Mondoc and S. Issazadeh-Navikas, J. Allergy Clin. Immunol.,2003, 112, 585–592.

55 J. Reichrath and G. Rappl, J. Cell. Biochem., 2003, 89, 6–8.56 L. Biggs, C. Yu, B. Fedoric, A. F. Lopez, S. J. Galli and M. A.

Grimbaldeston, J. Exp. Med., 2010, DOI: 10.1084/jem.20091725.57 S. Yang, C. Smith and H. F. DeLuca, Biochim. Biophys. Acta, Gen.

Subj., 1993, 1158, 279–286.58 S. Yang, C. Smith, J. M. Prahl, X. Luo and H. F. DeLuca, Arch.

Biochem. Biophys., 1993, 303, 98–106.59 K. K. Hanneman, H. M. Scull, K. D. Cooper and E. D. Baron, Arch.

Dermatol., 2006, 142, 1332–1334.60 S. Gorman, L. A. Kuritzky, M. A. Judge, K. M. Dixon, J. P. McGlade,

R. S. Mason, J. J. Finlay-Jones and P. H. Hart, J. Immunol., 2007, 179,6273–6283.

61 M. Ghoreishi, P. Bach, J. Obst, M. Komba, J. C. Fleet, J. P. Dutz, M.Ghoreishi, P. Bach, J. Obst, M. Komba, J. C. Fleet and J. P. Dutz,J. Immunol., 2009, 182, 6071–6078.

62 K. M. Dixon, S. S. Deo, A. W. Norman, J. E. Bishop, G. M. Halliday,V. E. Reeve and R. S. Mason, J. Steroid Biochem. Mol. Biol., 2007, 103,451–456.

63 K. M. Dixon, S. S. Deo, G. Wong, M. Slater, A. W. Norman, J. E.Bishop, G. H. Posner, S. Ishizuka, G. M. Halliday, V. E. Reeveand R. S. Mason, J. Steroid Biochem. Mol. Biol., 2005, 97, 137–143.

64 R. Gupta, K. M. Dixon, S. S. Deo, C. J. Holliday, M. Slater, G. M.Halliday, V. E. Reeve and R. S. Mason, J. Invest. Dermatol., 2007, 127,707–715.

65 L. P. Zanello and A. W. Norman, J. Biol. Chem., 1997, 272, 22617–22622.

66 M. C. van Praag, L. Roza, B. W. Boom, C. Out-Luijting, J. B.Henegouwen, B. J. Vermeer and A. M. Mommaas, J. Photochem.Photobiol., B, 1993, 19, 129–134.

67 P. Hersey, M. MacDonald, C. Burns, S. Schibeci, H. Matthews and F. J.Wilkinson, J. Invest. Dermatol., 1987, 88, 271–276.

68 M. C. van Praag, C. Out-Luyting, F. H. Claas, B. J. Vermeer and A. M.Mommaas, J. Invest. Dermatol., 1991, 97, 629–633.

69 R. Bestak, R. S. Barnetson, M. R. Nearn and G. M. Halliday, J. Invest.Dermatol., 1995, 105, 345–351.

This journal is © The Royal Society of Chemistry and Owner Societies 2010 Photochem. Photobiol. Sci., 2010, 9, 564–570 | 569

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online

70 J. L. Cho, M. Allanson, D. Domanski, S. J. Arun and V. E. Reeve,Photochemical & Photobiological Sciences, 2008, 7, 120–125.

71 S. Widyarini, D. Domanski, N. Painter and V. E. Reeve, Proc. Natl.Acad. Sci. U. S. A., 2006, 103, 12837–12842.

72 R. C. Malley, H. K. Muller and G. M. Woods, in 5th Joint Meetingof The Societies for Free Radical Australasia and Japan & Mutagenesis

and Experimental Pathology Society of Australasia, Sydney, Australia,2009, p. 87.

73 J. P. Berg, K. M. Liane, S. B. Bjorhovde, T. Bjoro, P. A. Torjesen and E.Haug, J. Steroid Biochem. Mol. Biol., 1994, 50, 145–150.

74 L. A. Kuritzky, J. J. Finlay-Jones and P. H. Hart, Clin. Exp. Dermatol.,2008, 33, 167–170.

570 | Photochem. Photobiol. Sci., 2010, 9, 564–570 This journal is © The Royal Society of Chemistry and Owner Societies 2010

Dow

nloa

ded

by L

aure

ntia

n U

nive

rsity

on

09/0

4/20

13 1

2:56

:37.

Pu

blis

hed

on 0

3 M

arch

201

0 on

http

://pu

bs.r

sc.o

rg |

doi:1

0.10

39/B

9PP0

0184

K

View Article Online