Vitiligo, reactive oxygen species and T-cells...Vitiligo, reactive oxygen species and T-cells Steven...

Clinical Science (2011) 120, 99–120 (Printed in Great Britain) doi:10.1042/CS20090603 99

R E V I E W

Vitiligo, reactive oxygen species and T-cells

Steven J. GLASSMANDivision of Dermatology, Department of Medicine, University of Ottawa, 737 Parkdale Avenue, Ottawa, Ontario,Canada K1Y 1J8

A B S T R A C T

The acquired depigmenting disorder of vitiligo affects an estimated 1% of the world populationand constitutes one of the commonest dermatoses. Although essentially asymptomatic, thepsychosocial impact of vitiligo can be severe. The cause of vitiligo remains enigmatic, hamperingefforts at successful therapy. The underlying pathogenesis of the pigment loss has, however, beenclarified to some extent in recent years, offering the prospect of effective treatment, accurateprognosis and rational preventative strategies. Vitiligo occurs when functioning melanocytesdisappear from the epidermis. A single dominant pathway is unlikely to account for all casesof melanocyte loss in vitiligo; rather, it is the result of complex interactions of biochemical,environmental and immunological events, in a permissive genetic milieu. ROS (reactive oxygenspecies) and H2O2 in excess can damage biological processes, and this situation has beendocumented in active vitiligo skin. Tyrosinase activity is impaired by excess H2O2 through oxidationof methionine residues in this key melanogenic enzyme. Mechanisms for repairing this oxidantdamage are also damaged by H2O2, compounding the effect. Numerous proteins and peptides,in addition to tyrosinase, are similarly affected. It is possible that oxidant stress is the principalcause of vitiligo. However, there is also ample evidence of immunological phenomena in vitiligo,particularly in established chronic and progressive disease. Both innate and adaptive arms ofthe immune system are involved, with a dominant role for T-cells. Sensitized CD8+ T-cells aretargeted to melanocyte differentiation antigens and destroy melanocytes either as the primaryevent in vitiligo or as a secondary promotive consequence. There is speculation on the interplay,if any, between ROS and the immune system in the pathogenesis of vitiligo. The present reviewfocuses on the scientific evidence linking alterations in ROS and/or T-cells to vitiligo.

INTRODUCTION

Vitiligo is a common skin disorder characterizedby the acquired loss of constitutional pigmentationmanifesting as white macules and patches caused by a

loss of functioning epidermal melanocytes. Extent ofinvolvement is highly variable, ranging from focal togeneralized, and the onset can be abrupt or gradual.Although essentially asymptomatic, the psychosocialimpact of vitiligo can be devastating, and affected

Key words: immune system, melanocyte, reactive oxygen species (ROS), skin, T-cell, vitiligo.Abbreviations: α-MSH, α-melanocyte-stimulating hormone; 4-TBP, 4-tertiary butyl phenol; 6BH4, (6R)-l-erythro-5,6,7,8,-tetrahydrobiopterin; AchE, acetylcholinesterase; AIS, autoimmune susceptibility loci; BchE, butyrylcholinesterase; BH4,tetrahydrobiopterin; CLA, cutaneous lymphocyte-associated antigen; CTL, cytotoxic T-lymphocyte; CTLA-4, CTL antigen-4;CYP, cytochrome P450; DC, dendritic cell; ET-1, endothelin-1; FT, Fourier Transform; GSH-Px, glutathione peroxidase; GST,glutathione transferase; HLA, human leucocyte antigen; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin;iNOS, inducible NO synthase; MBEH, monobenzyl ether of hydroquinone; MDA, malondialdehyde; MITF-M, melanocyte-specific microphthalmia-associated transcription factor; MSR, methionine sulfoxide reductase; ONOO− •, peroxynitrite; POMC,pro-opiomelanocortin; ROS, reactive oxygen species; SCF, stem cell factor; SNP, single nucleotide polymorphism; SOD, superoxidedismutase; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α; TRP-1, tyrosinase-related protein-1; XO,xanthine oxidase.Correspondence: Dr Steven J. Glassman (email [email protected]).

C© The Authors Journal compilation C© 2011 Biochemical Society

http://dx.doi.org/10.1042/CS20090603

100 S.J. Glassman

persons are often desperate for effective therapy [1].As of 2010, this goal has not yet been reached, asthe underlying pathomechanisms in vitiligo are stillincompletely understood, despite intense scrutiny. Threerecurring themes emanate from clinical and scientificanalysis of melanocyte loss in vitiligo: genetic, immuneand biochemical. The dominant theorem describesan autoimmune process occurring in a geneticallysusceptible host and triggered by a variety of host andenvironmental factors [2]. The aim of the present reviewis to summarize the role of ROS (reactive oxygen species)and T-cells in the pathogenesis of vitiligo.

Vitiligo occurs worldwide, with an estimated preval-ence rate of about 1 %. A large population-based surveyfound a prevalence rate of 0.38 % in Denmark [3], anda clinic-based survey in Martinique found a prevalencerate of 0.34 % [4]. Other surveys have reported ratesfrom 0.15 % to 8.8 %, with the highest rates occurringin Indian locales [5]. Onset is before the age of 20 yearsin about half the cases, and three quarters have occurredby the age of 30 years. Sexes are equally affected, butthere might be a female preponderance owing to reportingbias [6]. Two major subtypes of vitiligo are described,segmental and non-segmental. Segmental vitiligo tends tohave an earlier onset, with the rapid evolution of unilateraldepigmentation in the distribution of a dermatome,suggesting neural involvement, or corresponding toBlaschko’s lines, suggesting a form of genetic mosaicism.Segmental vitiligo mostly affects the face and tends to bestable once fully developed. The area of depigmentationis sometimes not as well demarcated as in othertypes of vitiligo [7]. Non-segmental vitiligo is, by far, thecommoner type and is also known as generalized vitiligoor vitiligo vulgaris. Most work relating to pathogenesishas focused on this form of vitiligo exclusively. Onsetis usually gradual with depigmented macules evolvinginto well-demarcated patches with convex or scallopedborders, in a symmetrical distribution in sites ofpredilection. These include regions which are normallyslightly hyperpigmented, such as the face, groin, axillae,areolae and genitalia, or those prone to mechanicalinjury and repeated friction, such as the ankles, kneesand elbows. Macules can have indistinct borders,and other morphological variants include trichromevitiligo, where three shades in concentric zones arenoted: normal, intermediate depigmentation and fulldepigmentation, and inflammatory vitiligo, where thereis an erythematous, raised border to the lesion, withassociated itching. Ordinary vitiligo can also present withpruritus. Mixed patterns, including segmental and non-segmental types can occur. A hyperpigmented rim cansometimes be seen at the outer margin of a vitiligo lesion,especially after sun exposure. A loss of hair pigmentin affected areas is considered a late sign of vitiligo,with some prognostic significance. Therefore, it appearsthat epidermal melanocytes are lost before those in the

hair bulb [6]. Non-segmental vitiligo is a slow andprogressive disease, marked by occasional remissions andexacerbations, corresponding presumably to triggeringfactors. Full resolution of generalized vitiligo has neverbeen reported, emphasizing the relentless chronicityof the condition. Depigmented areas are surprisinglytolerant of sun exposure and relatively resistant tophotoaging and skin cancer. Up-regulation of wild-type p53 could be the reason [8]. The diagnosis isclinical, but occasionally, skin biopsy is required forconfirmation: melanocytes and melanin are absent.Examination of the affected areas with Wood’s light canhelp distinguish active vitiligo from several disorderscharacterized by varying degrees of hypopigmentationby showing fluorescence owing to the presence ofoxidized pteridines [9]. The degree of fluorescencedepends on the skin phototype, and fluorescence is notseen in postinflammatory hypopigmentation. The dif-ferential diagnosis of vitiligo includes piebaldism, nevusdepigmentosus, hypomelanosis of Ito, lichen sclerosus,pityriasis versicolor, idiopathic guttate hypomelanosis,progressive macular hypomelanosis, hypopigmentedmycosis fungoides, indeterminate leprosy, pityriasisalba and many other forms of postinflammatoryhypopigmentation, such as that following resolutionof psoriasis. Particularly challenging can be the totaldepigmentation which can follow repeated excoriationof the skin from atopic dermatitis and psychogenicexcoriations, and inflammation from connective tissuediseases like lupus erythematosus and systemic sclerosis.Depigmentation due to exposure to certain toxins canmimic vitiligo exactly and is termed chemical leukoderma.There can be shared pathomechanisms with ordinaryvitiligo. Depigmentation around melanocytic nevi (halonevi) can resemble vitiligo, and the two conditions canbe associated [10]. A regressing melanoma can alsodemonstrate vitiligo-like depigmentation. An accuratehistory is often the only way to distinguish pseudovitiligofrom the real entity. To improve the accuracy of diagnosisof vitiligo, and to monitor the response to treatment, theVitiligo European Task Force has created an assessmenttool, which incorporates a staging system [11]. Thetreatment of vitiligo encompasses preventative measures,camouflage makeup, medical and physical agents andsurgical procedures. Prevention focuses on avoidingtrauma, friction, sunburn and emotional stress. Thefriction from everyday activities like bathing and dryingthe skin has probably been overlooked. Tight clothingand sports apparel are also to be avoided when practical.Stable, inactive vitiligo, especially segmental vitiligoresponds quite well to surgical repigmentation [12].Evidence-based scrutiny supports treatment of vitiligowith UV light, especially narrow-band UVB, topicalcorticosteroids, topical calcineurin inhibitors and surgicaltechniques [13–15]. Repigmentation takes two mainforms, follicular and diffuse, and is usually incomplete. In


Vitiligo, ROS and T-cells 101

the former, tiny islands of pigmentation, correspondingto hair follicles, appear in the patch and graduallyincrease in diameter and coalesce. In the latter, there ishomogeneous repigmentation of the patch, starting witha pale hue and becoming darker. Certain sites, such as thehands and feet, are very resistant to therapy. Followingsuccessful treatment, relapse is very common at all sites.Very extensive vitiligo is sometimes best managed withdepigmentation of remaining islands of normal skin.Psychological interventions should be offered to helppatients with vitiligo cope with their disease. Patients canhave significant co-morbidities like autoimmune thyroiddisease, and ophthalmological and auditory effects fromthe loss of pigment have been reported [16]. The clinicalfeatures and management of vitiligo have been reviewedrecently [17–20].

MELANIN

It is interesting to reflect that one’s skin colour isnot a ‘permanent coat’ but the result of regularreplenishing or ‘repainting’ according to the turnoverof the epidermis. Melanin pigments in the form ofeumelanin (brown-black shades) and phaeomelanin (red-yellow shades) are transferred from melanocytes in thebasal layer to associated keratinocytes via the so-calledepidermal melanin unit. One melanocyte is in contactwith approximately 36 surrounding keratinocytes [21].Granules of melanin are transferred to keratinocytesvia melanosomes in a process of phagocytosis. Becausemelanocytes represent only 8–10 % of all epidermal cells[22], most of the skin’s pigmented colour is determinedby melanin in the keratinocytes, and only at localizedcollections of melanocytes, as occurs in melanocyticnevi or ‘moles’, is the colour due to the melanocytesthemselves. Dark skin has a higher content of eumelanin,with larger melanosomes, and lighter skin has a higherproportion of phaeomelanin, with smaller melanosomes.Skin colour is not determined by the number or sizeof melanocytes. Melanin is thought to filter out UVradiation and scavenge ROS, thereby limiting UV damageto other cutaneous cells. The supranuclear melanincap structure in keratinocytes presumably minimizesphotodamage to the nucleus [23,24]. Melanocytes arealso thought to play a role in the skin immune system,secreting a wide range of signal molecules and respondingto growth factors and cytokines. Melanocytes canphagocytize and eliminate exogenous antigens, whichhave penetrated the skin barrier [25], and they can processand present antigen in the form of peptides with HLA(human leucocyte antigen) class II molecules to T-cells,triggering an adaptive immune response. Melanosomalproteins are involved in this antigen processing [26].Activation of T-cells by melanocytes is shown bythe secretion of co-stimulatory molecules like ICAM

(intercellular adhesion molecule)-1 and LFA (leucocytefusion-associated molecule)-3 [25,27]. When melanocytesdie, migrate or stop functioning, skin reverts to itsunpigmented form. This default colour is best exemplifiedin the single gene disorder of albinism, where melaninproduction is severely curtailed or completely absent.An acquired, patchy form of melanocyte failure occursin vitiligo, with a fully depigmented area having thesame colour seen in albinism. Onset is usually gradual,as several epidermal turnovers are required before allremaining pigment in keratinocytes is lost in the processof keratinization and desquamation. It seems reasonableto conclude that the skin colour disappears in a processof fading, taking several weeks to months. The specificdynamics of this process are difficult to study in vivo.Melanocytes in hair follicles seem to be more robust, sothat normal hair colour often persists in the white patch[28]. Greying and eventual whitening of scalp and bodyhair in the condition of poliosis is not considered a form ofvitiligo, but there may be some shared pathomechanisms;early greying of the hair can be seen in some vitiligolesions [29]. New studies on the control of melanogenesisand constitutional skin colour point to roles for cytokineslike SCF (stem cell factor), ET-1 (endothelin-1) andgranulocyte macrophage colony stimulating factor [30].

VITILIGO AETIOPATHOGENESIS:HYPOTHESES

The subject of vitiligo aetiopathogenesis has beenreviewed by several groups recently [31–35], but despitetremendous progress in molecular biology and genetics,there is still no universally accepted hypothesis. Itcould well be that vitiligo represents a ‘syndrome’rather than one disease, with numerous different butnot mutually exclusive pathways leading to melanocytefailure or disappearance. Genetic factors probablydetermine which particular pathway predominates ina specific patient. This was first proposed in the‘convergence’ theory of Le Poole et al. [36] in 1993 andlater refined by Schallreuter et al. [34] in 2008. Oneproposed schema is as follows: increased endogenousor exogenous phenol/catechol concentrations aroundthe melanocyte compete with tyrosine for tyrosinasebinding sites in the melanocyte, generating aberrantsubstrates and curtailing melanin production. Theseaberrant substrates generate reactive quinones, especiallyin the presence of ROS and disturbed redox balance,which impair cell functions and bind covalently to thecatalytic centre of tyrosinase, impairing or inactivatingthe enzyme and further reducing melanogenesis [31].Similar detrimental effects from excessive ROS occurwith other peptides and enzymes. Another proposalemphasizes the immunogenicity of melanocytes andmelanosomal proteins, with aberrant T-cell attack


102 S.J. Glassman

resulting in melanocyte destruction by apoptosis. Vitiligorepresents an autoimmune disease under this proposal[24]. Within these polar views of vitiligo pathogenesis,there is speculation that ROS and the immune systemmight somehow interact synergistically, so that bothmechanisms might be relevant. Further hypothesesfocus on neural dysregulation, hormones, cytokines,infections, calcium imbalance and melanocytorrhagy inthe causation of vitiligo [37,38]. The molecular controlof melanogenesis has been more accurately definedin the past decade: new hypotheses regarding vitiligohave emerged from this work, in particular, the roleof SCF/KIT protein interactions, and the downstreameffector, MITF-M (melanocyte-specific microphthalmia-associated transcription factor) [30,39,40].

VITILIGO GENETICS

The genetics of generalized vitiligo was reviewed bySpritz in 2008 [41]. Genes are thought to play a role inall aspects of vitiligo pathogenesis. There is a positivefamily history in about 20 % of cases and similarconcordance in identical twins. Vitiligo is considereda complex trait, involving the interaction of multiplegenes with non-genetic factors, both environmentaland host. At present, there is strong support forrelatively few susceptibility genes, including certainHLA genes, PTPN22, NALP1 and perhaps CTLA-4[CTL (cytotoxic T-lymphocyte) antigen-4]. These areall associated with a tendency to autoimmunity. HLAmolecules present peptides to T-cells, and it has beenproposed that certain HLA haplotypes confer moreefficient presentation of cognate autoantigen, therebypredisposing to autoimmunity; an example is HLA-DQB1*0301 [28]. NALP1 is involved in the innateimmune response to pathogens. Recent fine-mappingstudies showed associations with chromosomes 7 and 9[42]. Numerous other candidate genes and susceptibilityloci bear ongoing scrutiny, including CAT, GST, COMT,ACE, mannose-binding lectin 2 and XBP1 [43–46]. Arecent genome-wide association study by Jin et al. [47]using European white subjects and controls showedsignificant associations of generalized vitiligo with thefollowing loci, which have been previously linked withautoimmune diseases: HLA class I and II molecules,PTPN22, LPP, IL2RA, UBASH3A and C1QTNF6. Twoadditional immune-related loci identified were REREand GZMB. The HLA class I association occurred inthe regions between HLA-A and HCG9, consistentwith previous reports of strong associations with theHLA-A*02 allele, and the HLA class II gene associationoccurred in the region between HLA-DRB1 and HLA-DQA1, in keeping with known associations to theHLA-DRB1*04 allele. With the exception of PTPN22,the associations were similar whether patients had

vitiligo alone or vitiligo as well as another autoimmunedisease. An important association with a non-immune-related gene was identified: SNPs (single nucleotidepolymorphisms) in the gene encoding tyrosinase, TYR.Tyrosinase is a melanocyte enzyme that catalyzes the rate-limiting step in melanin biosynthesis and is a putativetarget autoantigen in vitiligo. Interestingly, certain TYRSNPs are associated with melanoma risk, and some ofthese are in linkage disequilibrium with vitiligo TYRSNPs shown in this study. Vitiligo TYR SNPs couldbe more antigenic than melanoma TYR SNPs, therebyconferring protection from melanoma through immunesurveillance [47,48].

MELANOCYTE LOSS

It is generally accepted that vitiligo results from lossof melanocytes rather than reduced functioning alone.This is supported by histological, ultrastructural andimmunohistochemical techniques showing that vitiligoskin is devoid of melanocytes, and by the fact thatmelanocytes are almost never able to be culturedfrom affected skin. Whether melanocytes in vitiligoare eliminated by necrosis or apoptosis has beendebated. Histological and ultrastructural studies supportapoptosis by revealing nuclear shrinkage, vacuolization,loss of dendrites and detachment. Known cytotoxinslike phenolic compounds, which can induce leukodermasimilar to that seen in vitiligo, cause melanocyte loss withplasma membrane blebbing and DNA fragmentationsuggestive of apoptosis; similar mechanisms might occurin vitiligo. Cytokine changes and immune reactions caninitiate apoptosis too, and these are thought to be relevantin melanocyte loss in vitiligo [49].

ROS AND AUTOIMMUNITY

The potential role of oxygen free radicals in humanautoimmune disease was reviewed by Ahsan et al. [50]in 2003. Free radicals are atoms or molecules which canexist independently despite having one or more unpairedelectrons. This makes them more reactive than thecorresponding non-radical forms. Free radicals derivedfrom oxygen include four moieties: O2

• − (superoxideanion radical), 1O2 (singlet oxygen), •OH (hydroxylradical) and HO2

• (perhydroxyl radical). Together,these are termed ROS. ROS are routinely generatedduring many cellular biochemical and metabolic chemicalreactions. Superoxide anion is thought to be the first freeradical generated, primarily via the electron transportchain in mitochondria. Hydroxyl and perhydroxylradicals are formed directly from superoxide. H2O2 isnot as reactive as free radical-derived ROS, but it is animportant oxidant, which can cross biological membranesand generate highly reactive hydroxyl radicals throughan interaction with transition metal ions like Fe2+ and



Cu+ in the Fenton reaction. H2O2 can, therefore, beconsidered a marker of potential ROS. H2O2 is formedwhen superoxide anion undergoes a dismutation reactioncatalysed by SOD (superoxide dismutase). Hydroxylradical can also be generated from the interaction ofsuperoxide anion radical and hydrogen peroxide in theHaber–Weiss reaction. Hydroxyl radical seems to bethe most potent and damaging radical in biologicalsystems, capable of interacting with all macromoleculeslike lipids, proteins, nucleic acids and carbohydrates.Polyunsaturated fatty acids are particularly susceptibleto hydroxyl radicals, whereby removal of a hydrogenatom from the fatty acid starts the process of lipidperoxidation in a type of chain reaction. Aldehydesof lipid peroxidation can react with amino acids,altering their biological activity. Membrane structureand function are affected. Protein oxidation results incross-linking, fragmentation and the addition of aldehydegroups, all affecting structure and function of the protein.Nucleic acids are damaged by single-strand breaks, cross-linking and changes to individual nucleotide bases. DNAis damaged by single-strand breaks, base modifications,conformational changes and DNA–protein cross-links.Thymine and guanine are more susceptible to damagethan cytosine and adenine. The reaction product betweensuperoxide anion and NO is OONO− • (peroxynitrite),which is also a strong oxidant for proteins, lipids andnucleic acids, causing cell damage [51]. ROS can also begenerated by exogenous stimuli like UV radiation andenvironmental chemicals. ROS have beneficial effects too,notably in the ‘respiratory burst’ of neutrophils duringthe phagocytosis of bacteria. Host cells have developedprotective mechanisms to counter the deleterious effectsof ROS and other free radicals in the form of free radicalscavengers and antioxidant chemicals and reactions.An imbalance between production and removal ofROS leads to oxidant stress or redox imbalance, andthis phenomenon has been implicated in numerousdisease states as well as the process of normal aging.ROS can modulate the expression of a variety ofimmune and inflammatory molecules, leading to tissuedamage. Aberrant immune reactions have been related tooxidative imbalance, and antioxidant functions have beenlinked to anti-inflammatory and immunosuppressiveproperties. If ROS-induced damage to cells is severeenough, programmed cell death or apoptosis occurs.Apoptotic debris can be highly immunogenic, andefficient removal by proteasomal degradation is necessaryto prevent autoimmune reactions [52–56]. Antioxidantsystems have evolved to control the redox balance.In the skin, these include antioxidant enzymes likecatalase, thioredoxin reductase, glutathione peroxidaseand superoxide dismutase, and small molecules likemethionine, glutathione, vitamin C and vitamin E.Oxidatively damaged proteins are repaired by MSRs(methionine sulfoxide reductases) [57,58,122].

ROS IN VITILIGO

Human skin serves as an interface between theenvironment and the body. It is constantly exposedto a broad array of physical, chemical and biologicalagents, many of which are either inherent oxidants orcatalyse the generation of ROS. ROS can denatureproteins, alter apoptotic pathways, damage nuclearand mitochondrial DNA and mediate release ofproinflammatory cytokines [59,60]. ROS are believedto be involved in the pathogenesis of inflammatoryskin diseases, carcinogenesis, photoaging and hairgreying [61,62]. Mitochondria are the most importantendogenous source of ROS, but they are also a targetof ROS-mediated damage. Thus, ROS can lead tomitochondrial dysfunction, reduced efficiency and moreROS in a vicious cycle of oxidant imbalance [59]. Severalcompelling lines of research have shown evidence ofoxidative stress throughout the epidermis of patientswith vitiligo, attributed to massive amounts of H2O2

in the 10− 3-M range [63]. Impetus for this researchcame from the finding of low catalase levels in theepidermis of patients with vitiligo, first reported in1991 [64]. Catalase removes H2O2 by converting itinto water and oxygen. Generation of H2O2 is aphysiological reaction in all cells via several metabolicpathways. There are also numerous exogenous directand indirect sources of epidermal H2O2. While lowconcentrations, of the order of 10− 6 M, are necessaryfor cell signalling and transcription, high concentrationscan have deleterious effects. Ultrastructural changessuggestive of lipid peroxidation have been demonstratedin melanocytes, keratinocytes and Langerhans cells inthe skin of patients with vitiligo, both in affected andperilesional areas [65–68]. High levels of epidermal H2O2

as well as the methionine oxidation product, methioninesulfoxide, have been demonstrated in vivo in vitiligousing FT (Fourier Transform) Raman spectroscopy[34,63]. This augmented previous findings of increasedH2O2, which were in vitro, based on cell cultureand skin biopsies [63]. FT Raman spectroscopy alsorevealed oxidation of l-tryptophan in epidermal albumin,and HPLC showed the presence of allantoin in theepidermis, confirming the presence of oxidative stress invitiligo [69,70]. Oxidative destruction of polyunsaturatedfatty acids of phospholipids is referred to as lipidperoxidation. It is one of the hallmarks of oxidativestress. MDA (malondialdehyde) is an end-product oflipid peroxidation, and elevated serum levels of MDAhave been documented in patients with vitiligo [71–73].

Sources of epidermal H2O2There are numerous sources of H2O2 in the normalepidermis. NADPH oxidase activity in neutrophils andmacrophages generates H2O2 [74]. TNF-α (tumour nec-rosis factor-α) leads to the formation of H2O2 indirectly,


104 S.J. Glassman

by inducing manganese superoxide dismutase [39]. Othercytokines have been reported to generate H2O2: TGF-β(transforming growth factor-β), EGF (epidermal growthfactor) and PDGF (platelet-derived growth factor) [75].Monoamine oxidase A activity in the epidermis generatesH2O2 [76]. Nitric oxide synthases create H2O2 in anenvironment deficient in l-arginine [77]. XO (xanthineoxidase) catalyses the conversion of purine bases into uricacid, and generates H2O2 as a by-product [70]. Oxidationof aromatic phenols like 17β-oestradiol to catechols byan NADPH-dependent CYP (cytochrome P450) yieldssuperoxide anion, which disproportionates to H2O2;this diffuses into the epidermis [78]. Photo-oxidation ofepidermal 6-biopterin and sepiapterin yields H2O2 [80].The enzymes tyrosine hydroxylase and phenylalaninehydroxylase also produce H2O2 as by-products. Externalphenols, ortho- and para-quinols, UVA and UVB, andX-rays also generate H2O2 in the mM range [63,77].Metabolism of xenobiotics, principally by CYP enzymes,generates ROS and H2O2, through toxic quinone andsemiquinone intermediates [56].

Several of these epidermal sources of H2O2 areshown to be augmented in vitiligo, providing thepresumed source for the elevated levels, which havebeen documented, both in affected and normal skin inpatients with vitiligo (Table 1). Increased epidermal TNF-α levels have been shown [39]. Increased photo-oxidationof epidermal 6-biopterin and sepiapterin has been demon-strated [80]. Impaired recycling of the essential cofactor6BH4 [(6R)-l-erythro-5,6,7,8,-tetrahydrobiopterin] byelevated H2O2 causes accumulation of H2O2 in theepidermis and affects all cofactor-dependent mechanisms.6BH4 is an essential electron donor in the hydroxylationof the aromatic amino acids, l-phenylalanine, l-tyrosineand l-tryptophan. These amino acids are substratesfor melanogenesis, and thus, 6BH4 is an essentialcomponent of the pigmentary system [79]. Induciblenitric oxide synthase levels in vitiligo epidermis areelevated, producing both H2O2 and peroxynitrite [8].Homocysteine oxidation also causes elaboration of ROS,and elevated serum homocysteine levels have beenreported in vitiligo patients [81]. Increased monoamineoxidase A activity has been found in the epidermis ofpatients with vitiligo, elaborating H2O2 [76]. ElevatedSOD activity would seem a likely source of H2O2

in vitiligo, but results have been contradictory. Bothnormal and elevated serum and tissue SOD activityhas been shown [71,82–85]. XO catalyses the oxidativehydroxylation of hypoxanthine to xanthine, and xanthineto uric acid as part of purine degradation. These reactionsgenerate H2O2, and XO is considered a major biologicalsource of ROS, leading to oxidative stress in many organs.XO contains two non-haem iron atoms in its structure,which can react with H2O2 to produce OH− •, the mostpotent ROS with respect to DNA damage. Because H2O2

also oxidizes uric acid to allantoin, this metabolite is a

Table 1 ROS in vitiligo: sources and effects

Level Reference(s)

SourcesH2O2 ↑ [63]Peroxynitrite ↑ [8]NADPH oxidase ↑ [74]TNF-α ↑ [39]Oxidized pterins ↑ [80]6BH4 recycling ↓ [79,80]iNOS ↑ [8]Homocysteine ↑ [81]Monoamine oxidase A ↑ [76]SOD n/↑ [71,82–85]Thioredoxin reductase ↓ [106]Xanthine oxidase ↑ [70]Catecholamines ↑ [33]GTP-cyclohydrolase I ↑ [98,99]Catalase ↓ [64,85]GSH-Px n/↓ [73,83–85]Vitamin E ↓ [72,73]

EffectsTyrosinase ↓ [121]TRP-1 ↓ [122]MSR ↓ [57,125]Catalase ↓ [64,85,106]Thioredoxin reductase ↓ [106]Tyrosine hydroxylase ↓ [79,80]POMC peptides ↓ [109–111]L-Phenylalanine ↑ [103]Acetylcholine ↑ [106,107]6BH4 recycling ↓ [79,102]Calmodulin, furin ↓ [103,113,114]Albumin (epidermal) ↓ [112]Malondialdehyde ↑ [71–73]Methionine sulfoxide ↑ [34,57,125]Allantoin ↑ [70]

useful marker of oxidative stress. XO activity has beenshown in skin, and elevated plasma levels have beenmeasured in patients with vitiligo. Recently, XO activityin melanocytes and keratinocytes was confirmed, withH2O2 regulating enzyme activity in a concentration-dependent fashion: low levels (10− 6 M) up-regulatedactivity, whereas high levels were suppressive. Oxidationby H2O2 of tryptophan and methionine residues in XOis thought to be the mechanism for this effect. Allantoinwas detected in the epidermis of acute vitiligo but not incontrol skin, further supporting a role for ROS in vitiligo[70].

Exogenous ROSThe role of exogenous oxidants in vitiligo is highlightedby the conditions of chemical leukoderma and contactvitiligo, as shared mechanisms might elucidate trigger



factors and reasons for progression and chronicityin idiopathic vitiligo. Chemical leukoderma refers toacquired depigmentation at sites of contact with certainchemicals; contact vitiligo starts in the same manner, butdepigmentation then spreads to distant sites, in the sameway as generalized idiopathic vitiligo. Depigmentationin both cases occurs from loss of melanocytes in theepidermis. Chemicals involved are mostly phenolic andcatecholic derivatives, which resemble tyrosine and canoccupy the catalytic centre of tyrosinase as surrogatesubstrates in the melanin synthesis pathway [86]. Theyinclude hydroquinone, MBEH (monobenzyl ether ofhydroquinone) and 4-TBP (4-tertiary butyl phenol).These are oxidized by tyrosinase or tyrosinase-relatedprotein to more reactive o-quinones, with the generationof ROS, which contribute to oxidative stress [87]. Inthe presence of excess H2O2, this process is accelerated.Melanin synthesis is reduced because the intermediatedopaquinone is not synthesized, and melanocyte viabilitycan be compromised. 4-TBP, a potent cause of contactvitiligo, is cytotoxic to cultured human melanocytesin a dose-dependent manner, with melanocyte lossattributed to apoptosis. There is marked variation inindividual susceptibility to chemical leukoderma andcontact vitiligo, emphasizing the key role of geneticfactors in determining melanocyte sensitivity to thesestimuli. Highly reactive o-quinones can react withnucleophilic groups on proteins to create antigensand stimulate an immune response. Phenols in well-known contact allergens, like poison ivy, might also beoxidized by similar mechanisms to form antigens onkeratinocytes. This could be the link between ROS andan altered immune response in vitiligo. Interestingly,the clinical picture of occupational contact vitiligois similar to allergic contact dermatitis, with itching,redness and scaling. When MBEH is used to removeremaining pigment in patients with vitiligo universalis,a similar reaction is seen, but only in the pigmentedareas, suggesting involvement of melanocytes ratherthan keratinocytes. Patients with established generalizedidiopathic vitiligo can be exquisitely sensitive toexogenous phenols and catechols [38,88–91]. Generationof a reactive o-quinone from MBEH via tyrosinase wasconfirmed recently in vitro, with isolation of several by-products with potential relevance to melanocyte toxicity[92]. Cytotoxic experiments have also confirmed recentlythat both 4-TBP and MBEH induce melanocyte death,but by different pathways: 4-TBP activates the caspasecascade and causes DNA fragmentation with apoptosis,while MBEH induces release of Mobility Group Box-1protein, which causes necrosis rather. This is confirmedby ultrastructural studies of MBEH-treated melanocytes[93]. Many drugs are potential exogenous sources ofROS, especially when metabolized by CYP enzymes,as they produce reactive quinones and semiquinones[56]. Interestingly, proton pump inhibitors were recently

shown to reactivate vitiligo, and the mechanism mightalso be through generation of free radicals [94]; pHchanges relating to melanogenic enzymes could also playa role [95].

Endogenous ROS: sources and effects

CatecholsEndogenous catechols are a source of ROS: elevatedplasma and urine catecholamines like norepinephrine,epinephrine and dopamine and their metabolites havebeen documented in vitiligo patients [96]. Keratinocytespossess β-2 adrenoceptors and synthesize and degradecatecholamines, and melanocytes synthesize norepineph-rine. Patients with vitiligo have markedly elevated GTP-cyclohydrolase I activity, which leads to excessive de novoproduction of 6BH4, leading to increased synthesis ofcatecholamines in the epidermis [96–99]. Catecholaminesalso compete preferentially with tyrosine for tyrosinaseactive binding sites, becoming hydrolysed in the processand generating H2O2 [33]. Norepinephrine-inducedvasoconstriction in vitiligo skin could cause hypoxiaand predispose to oxidative stress by this mechanism[38]. These phenomena, among others, are the basis forthe ‘neural’ theory of vitiligo aetiopathogenesis. Patientsoften report increased emotional stress prior to onsetof vitiligo or concurrent with a flare of disease activity[100]. Estrogens and progesterone can generate H2O2 invitiligo, contributing to quinone-mediated DNA damagein peripheral blood lymphocytes [78].

BH4 (tetrahydrobiopterin)BH4 cofactor is essential for various enzyme activitiesand is present in probably all cells. Six and sevenisoforms of BH4 are synthesized de novo from GTP,but regeneration is crucial to adequate functioning,and requires two enzymes, pterin-4a-carbinolaminedehydratase and dihydropteridine reductase. The latterare deactivated by H2O2 by oxidation of active sitetryptophan and methionine residues, and H2O2 alsooxidizes both 6- and 7-BH4 to 6- and 7-biopterin.This is the reason for fluorescence, which can beseen in vitiligo patches under Wood’s light. Thus, thehomoeostasis of this important cofactor is compromised[79,101,102]. Some of the enzymes that depend on BH4

are phenylalanine hydroxylase, tyrosine hydroxylaseand tryptophan hydroxylase, as well as all nitricoxide synthase isoforms. Thus, BH4 deficiency affectsmelanin, catecholamine, serotonin and NO synthesis.l-phenylalanine levels would be expected to rise in thissetting, and in fact, increased epidermal phenylalaninelevels have been documented in patients with vitiligo byin vivo FT Raman spectroscopy [103]. Increased de novosynthesis of 6-BH4 in vitiligo contributes to elevatednorepinephrine levels and up-regulates monoamineoxidase A and catechol-O-methyl transferase in the


106 S.J. Glassman

epidermis. These result in increased epidermal H2O2

[74,104,105].

AcetylcholineHigh epidermal levels of acetylcholine have beenreported in vitiligo, and this is attributed to almostabsent epidermal AchE (acetylcholinesterase) and BchE(butyrylcholinesterase) activities due to the effect of highlevels of H2O2. While low levels of H2O2 (10− 6 M)activate AchE, high concentrations (10− 3 M) deactivatethe enzyme. This regulation of enzyme activity byH2O2 is seen with several other enzymes. Molecularmodelling of AchE suggests that the inhibition is due toH2O2-mediated oxidation of tryptophan and methionineresidues in the protein, causing disorientation of theactive-site histidine residue. The tetramerization domainand calcium-binding domains in BchE are also affectedby high levels of H2O2 [106–108].

POMC (pro-opiomelanocortin)Oxidative stress via H2O2 directly affects POMCpeptides in the epidermis of patients with vitiligo. POMCpeptides have a key role in melanogenesis. Prohormoneconvertases 1 and 2, which cleave POMC, are oxidized byH2O2, and POMC-derived peptides like ACTH, α-MSH(α-melanocyte-stimulating hormone) and β-endorphinare also altered directly by oxidation of their methionineresidues. Reduced epidermal levels of α-MSH andβ-endorphin have been demonstrated in vitiligo bothin vitro and in vivo. These changes are reversed bythe reduction of H2O2 levels with narrow-band UVB-activated pseudocatalase PC-KUS. Oxidation of α-MSHcan also be ameliorated in the presence of abundant6BH4; this cofactor becomes deficient in the presenceof abnormal levels of H2O2. Regulation of tyrosinase,the key enzyme for pigmentation, by α- and β-MSH via6BH4 and 7BH4 has also been proposed, another linkbetween ROS and dyspigmentation [109–111].

TryptophanEpidermal l-tryptophan is oxidized by H2O2 in vitiligo,as shown in vivo by FT Raman spectroscopy. Albumincontains a tryptophan residue in its sequence, andoxidation could explain low levels of epidermal albumin,which have been reported in vitiligo. Several otheramino acid residues in albumin, such as methionine, arealso prone to oxidation. Some oxidation products oftryptophan like 5-OH-Trp are generated from H2O2

by Fenton chemistry. Albumin plays a key role incalcium homoeostasis, and reduced albumin levels mightaccount, in part, for impaired calcium uptake, which hasbeen described in vitiligo. H2O2 also affects all fourcalcium EF-hand-binding domains of calmodulin, andcalmodulin-ATPase activity is low in vitiligo skin. Theuptake of l-phenylalanine, which is defective in vitiligo,is also a calcium-dependent process. Epidermal furin is a

calcium-dependent prohormone convertase, which playsa role in the cleavage of POMC. Loss of a calcium-bindingsite in furin because of oxidation from H2O2 has beenshown in progressive vitiligo skin [103,112–114].

CytokinesSCF, a paracrine cytokine produced by keratinocytes,has a major role in promoting melanogenesis and inmelanocyte survival. SCF interacts with its receptor onmelanocytes, KIT protein, produced by the c-kit gene.KIT protein interacts with MITF-M, which serves as atranscription factor regulating expression of tyrosinasemRNA. In response to SCF/KIT protein signalling,MITF-M is activated by phosphorylation through a MAPkinase, whereafter it is translocated to the nucleus toactivate several genes. MITF-M also interacts with Bcl-2 to prevent apoptosis of the melanocyte [115]. ET-1 isalso an important regulator of melanin production, via itsETBR receptor [116]. SCF and ET-1 were not shown tobe deficient in vitiligo lesions, suggesting that abnormalparacrine secretion by keratinocytes is not the cause of thehypopigmentation. At the edge of a vitiligo lesion, thereare still melanocytes expressing tyrosinase, ETBR andS100α, albeit at slightly lower levels than unaffected skin,but KIT protein and MITF-M are markedly reduced.Melanocytes expressing tyrosinase, S100α, ETBR, KITprotein and MITF-M protein were absent at the centre ofthe vitiligo lesion. This suggests that reduced expressionof KIT protein, and its downstream effectors like MITF-M, could explain melanocyte loss and/or dysfunction invitiligo [30]. It is interesting that oxidative stress likeexcessive H2O2 leads to down-regulation of MITF-Mexpression in cultured human melanocytes. Thus, ROScould be the cause of the cytokine abnormality seen invitiligo [117].

TraumaTrauma to the skin, UV radiation and other sourcesof inflammation probably contribute to the epidermalpool of H2O2 in a non-specific manner via NADPHoxidase stimulation. This could explain the prominentKoebner phenomenon seen in vitiligo, especially in activestages of the disease [118,119]. In particular, UVB inducesgeneration of free radicals, which damage DNA. Thiscan be ameliorated by vitamin D, which up-regulatesexpression of metallothionein, a free radical scavengerprotein with photoprotective properties [120].

TyrosinaseHigh levels of H2O2 (0.5–5.0 × 10− 3 M) have been shownto deactivate tyrosinase, and this effect is compounded byincreased 6BH4 [121]. Recent work on the mechanism ofsenile hair greying has provided a possible explanationfor the inhibition of tyrosinase by H2O2. It was shownthat methionine 374, at the enzyme active site, is oxidizedby H2O2, disrupting enzyme activity, especially when



methionine sulfoxide repair by MSRA and MSRB isalso affected by the same ROS. A similar scenarioevidently occurs in vitiligo [62]. Abnormal expression ofTRP-1 (tyrosinase-related protein-1) has been reportedfollowing oxidative stress to cultured melanocytes fromthe advancing border of vitiligo lesions. This leads to earlycell death, possibly through an interaction with calnexin[122].

PeroxinitriteRecently, significantly elevated levels of 8-oxoguaninewere reported in plasma and skin of patients withvitiligo, an indication of DNA damage. Together withthis, there was up-regulation of epidermal wild-typep53 and enhanced short-patch base-excision repair. Inaddition, high epidermal levels of iNOS (inducible nitricoxide synthase) were demonstrated, with correspondingelevations of 3-nitrotyrosine and nitrated p53. Thisimplies increased epidermal ONOO− •, a reactivenitrogen species, which can be added to the list ofradicals involved in the pathogenesis of vitiligo. H2O2

was shown to enhance the DNA binding capacity ofp53, while ONOO− • completely inhibited this binding.Interestingly, H2O2 at a concentration of 10− 3 Mabolished this deleterious effect of ONOO− •. Thus,H2O2 appears to be protective in the sense of improvingDNA repair via enhanced p53. This could partly explainthe relative absence of photoaging and skin cancer inchronic lesions of vitiligo [8].

AntioxidantsElevated levels of H2O2 and other oxidants wouldordinarily be countered by antioxidant defenses and freeradical scavengers. However, several of these processesare actually inhibited by H2O2 itself. H2O2 in the mMrange deactivates catalase by oxidizing its porphyrin ringand methionine and tryptophan residues in the structureof the enzyme-active site and the cofactor NADPH-binding site. This explains why catalase levels are lowin vitiligo, despite unchanged mRNA expression for theenzyme [106]. Low tissue levels of catalase in patientswith vitiligo were recently confirmed, with lower levelsin active rather than stable disease [85,123]. Severalheterogeneous SNPs in the catalase gene, observed invitiligo patients, might render these catalase variantsparticularly susceptible to deactivation by H2O2, therebypredisposing to vitiligo [43,124]. Another antioxidantenzyme system, thioredoxin reductase, is impaired byhigh H2O2 levels through oxidation of the enzyme-active site and the NADPH cofactor-binding site. Lowactivities of this enzyme have been shown in vitiligo[99]. Oxidation of MSRA and MSRB by H2O2 leadsto reduced functioning of this important protein-repairmechanism. Low levels of MSR have been shown invitiligo. Furthermore, in vitro down-regulation of MSRA

in normal cultured melanocytes rendered them verysusceptible to oxidative stress [57,125].

GSH-Px (glutathione peroxidase) converts H2O2 andother peroxides into water and oxygen, utilizing gluta-thione, glutathione reductase and NADPH. Seleniumis a cofactor for some isoforms of GSH-Px. Variablelevels of GSH-Px and selenium have been found in tissueand blood of patients with active vitiligo [73,83–85,126–128]. GST (glutathione transferase) is a superfamily ofbroadly expressed isoenzymes involved in defence againstoxidative stress. Polymorphisms in the GST gene havebeen identified, which confer risk of vitiligo [45]. VitaminE levels were reported as low in vitiligo patients, who alsoshowed elevated blood levels of MDA, as evidence of lipidperoxidation [72,73]. Total blood antioxidant status usingthe Randox kit was increased in vitiligo patients in onestudy [128].

Melatonin has a strong antioxidant effect, and anaberration of melatonin receptors was once proposedas a mechanism for the development of vitiligo[129]. Melatonin is synthesized in the skin yieldingpotent free radical scavengers like N1-acetyl-N2-formyl-5-methoxykynuramine, which also stabilize themitochondrial electron transport chain [130].

Antioxidants react in tandem with proteins like Bcl-2and caspases to regulate apoptosis, which can result fromoxidative challenge [131]. For example, Bcl-2 expressionprevents cell death from H2O2 and menadione [132]. Bcl-2 is expressed by melanocytes and keratinocytes and iscrucial to their survival. Melanocytes from individualswith vitiligo have lower levels of Bcl-2 than normal,presumably contributing to melanocyte fragility [91].Elevated levels of Bcl-2 have also been reported invitiligo [8], as well as normal levels [133], and the roleof melanocyte apoptosis in vitiligo is still unclear.

Further proof of the role of H2O2 and ROS requiresevidence of improvement in lesions with antioxidanttherapies. Topical pseudocatalase (PC-KUS) is a narrow-band UVB-activated bis-MnIII-(EDTA)2-(HCO3

− )2

complex that is applied to the entire skin as a lotion,followed by low-dose UVB exposure. It functions as asynthetic catalyst to oxidize H2O2 into O2 and H2O, thusmimicking natural catalase. This regimen causes rapidrepigmentation of even long-standing vitiligo [134,135].Consequent with this is a marked reduction in epidermalH2O2 as measured in vivo with FT Raman spectroscopy.Bathing in Dead Sea water also had a pseudocatalaseeffect, though milder, probably from its high content oftransition metals like manganese [135]. A randomizedcontrolled trial comparing narrow-band UVB withor without a pseudocatalase cream did not confirmadditional benefit from the cream, but the methodologywas different, and the pseudocatalase was not thesame as PC-KUS [136]. A catalase of vegetable origin,combined with SOD in a microsphere formulation,showed equivalent efficacy to topical betamethasone,


108 S.J. Glassman

Figure 1 Summary of sources, effects and interactions of ROS/H2O2 in vitiligo

when combined with sunlight exposure in the treatmentof vitiligo [137]. Oral vitamin E appeared to confersome additional benefit to narrow-band UVB therapyof vitiligo, with a corresponding decrease in plasmaMDA in the vitamin E group versus the control [138].ROS/H2O2 sources, effects and interactions in vitiligoare summarized in Figure 1.

VITILIGO AND AUTOIMMUNITY

Generalized vitiligo is widely considered an autoimmunedisease, with involvement of humoral and cellularcomponents of the innate and adaptive immune system.The belief is supported by the following lines ofepidemiological, clinical and investigational research: anassociation with other autoimmune disorders; chronicrelapsing and remitting course so typical of autoimmunedisorders; possible response to immunosuppressivetherapies like UV phototherapy, topical and oralcorticosteroids, and topical calcineurin inhibitors;circulating anti-melanocyte antibodies; T-cell infiltratesin perilesional skin; anti-melanocyte cytotoxic T-cells inthe skin and circulation and proinflammatory cytokinepatterns of a Th-1 type response. Autoimmunity mightbe the triggering event in vitiligo, but it could functioninstead as a promoter of disease progression andchronicity [26,139].

Autoimmune conditions associated with vitiligoinclude autoimmune polyendocrine syndrome types 1and 2, pernicious anaemia, Type 1 diabetes, Addison’sdisease, Graves’ disease, alopecia areata, systemic lupus

erythematosus, rheumatoid arthritis, psoriasis and myas-thenia gravis. A recent survey of 2600 vitiligo patientsshowed increased frequencies of autoimmune thyroiddisease, Addison’s disease, systemic lupus erythematosusand pernicious anaemia, with about 30 % of patientshaving at least one of these disorders. In addition,family members who did not have vitiligo still had atendency to the same autoimmune conditions, pointing toa genetic risk for a specific cluster of autoimmune diseases.Other studies report true associations only with thyroiddysfunction and thyroid antibodies, regarding the otherconditions as random concomitant events. Psoriasis orlichen planus occurring in vitiligo lesions has also beenreported, with a shared pathogenesis postulated. Organ-specific autoantibodies are recorded with increasedfrequency in vitiligo patients, often in the absence ofclinical symptoms. There is probably an increased riskof developing clinical or subclinical disease later [139–142]. Several immunogenetic factors predispose patientsto autoimmune diseases, and some of these are associatedwith vitiligo, adding to the evidence that vitiligo mayhave an autoimmune basis. Various HLA class II alleleshave been associated with vitiligo, in particular, HLA-DR4 [143]. The particular haplotype association variesaccording to ethnic origin. Genes involved in antigenpresentation and processing have been associated withautoimmune diseases and in some cases with vitiligo.These include LMP-1 and -7 (low-molecular-masspolypeptide-2 and -7) and TAP-1 and -2 (transporterassociated with antigen processing protein-1 and-2) [144]. Homozygous or heterozygous complement



2 and 4 deficiency is associated with autoimmunity, andthis has been described in vitiligo [145]. The CTLA-4gene product down-regulates T-cell activation andcontrols T-cell apoptosis. Certain CTLA-4 polymorph-isms predispose to vitiligo in patients who alreadyhave other autoimmune conditions [146]. Autoimmunepolyendocrine syndrome type 1, which often includesvitiligo, is due to mutations in the autoimmune regulatorgene, AIRE [147]. A missense mutation in the PTPN22gene, which encodes LYP (lymphoid protein tyrosinephosphatase), has been linked to several autoimmunediseases including vitiligo [148]. Loci on chromosomes1, 7 and 8 have been linked with autoimmune diseasesand termed AIS (autoimmune susceptibility loci) 1, 2 and3, respectively. AIS2 and -3, in particular, are associatedwith vitiligo. A locus designated SLEV1 on chromosome17p13 has also been linked to vitiligo [149].

Animal models of vitiligo show prominent roles foranti-melanocyte antibodies. Some of these cross-reactwith mammalian TRP-1 [150]. Antibodies to melanocyteshave been found in the circulation of patients with vitiligo[151]. These antibodies correlate with disease activityand extent [152]. Targets of these antibodies include avariety of melanocyte and melanosomal antigens. Thefrequency of each antibody in the vitiligo populationis relatively low, and a dominant antigen has notbeen found. Antibodies might trigger vitiligo as aprimary event, but they could also arise secondary tomelanocyte damage or serve to perpetuate the disease.Whatever their role in vitiligo, these antibodies have thecapacity to injure pigment cells in vivo and in vitro.Even if they are not pathogenic, study of melanocyteantibodies and target antigens might refine the diagnosticand prognostic testing of vitiligo, reveal putativeT-cell targets and add to the therapeutic armamentarium[153]. Circulating anti-parietal cell, thyroid and adrenalantibodies have been detected in vitiligo patients,as well as antinuclear antibodies and rheumatoidfactor, again suggesting an autoimmune pathomechanismfor the disease [151]. Autoimmune diseases are theresult of complex interactions between T and B cellsubpopulations. A recent flow cytometric study of thesecells in vitiligo did not show a pathological distributionof B cells, suggesting that T-cells might have a moredominant role. Circulating immune complexes were notelevated either [154]. Cases of generalized vitiligo inrecipients of bone marrow transplants from donors withvitiligo have been reported, illustrating the role of bonemarrow-derived cells in disease pathogenesis [155].

T-CELLS IN VITILIGO

Peripheral bloodLymphocyte analysis in peripheral blood of patients withvitiligo has yielded variable results (Table 2). Earlier

Table 2 T-cell changes in vitiligo

Level

PhenotypePeripheral [156–163]

Total T-cell n/↓CD4+/CD8+ n/↓/↑CD45RA+ ↓CD45RO+ ↑Total NK T-cell ↓

Tissue [179–193]Total T-cell ↑CD8+ ↑CD45RO+ ↑IL-2 receptor ↑CLA+ ↑

CytokinePeripheral [164–174]

IL-1 ↑IL-6 ↑IL-8 n/↑TNF-α n/↑/↓IFN-γ ↓TGF-β ↓IL-2 receptor ↑/↓TNF receptor n

Tissue [165–174]IL-2 receptor ↑IL-6 ↑TNF-α ↑IFN-γ ↑IL-10 ↑

studies found normal total levels of T-cells but a decreasedCD4+/CD8+ ratio and normal or increased total NKcells. There was no particular correlation noted withdisease activity [156–160]. Abdel-Naser et al. [161] foundnormal levels of total T-cells, CD4+ T-cells and totalNK cells, but a decreased proportion of CD45RA+

(naıve CD4+) T-cells and increased HLA-DR expression,suggesting the presence of activated T-cells. Mahmoud etal. [162] found decreased total T-cells, CD45RA+ cellsand NK T-cells, with increased CD45RO+ (memoryphenotype) T-cells in severe disease. Basak et al. [163]found normal total T-cells with a decreased CD4+/CD8+

ratio, as well as increased monocytes, CD45RO+ T-cells and total NK cells. Total T-cells were relativelyhigher in generalized than in acral/acrofacial vitiligo,and CD45RO+ T-cells were higher in acral/acrofacialdisease. Unlike former studies, Pichler et al. [154]found an increased CD4+/CD8+ ratio in peripheralblood, but noted that the ratio in tissues could bedifferent.


110 S.J. Glassman

CytokinesCytokine studies of peripheral blood and skin in patientswith vitiligo have also yielded variable results, butwith a trend to proinflammatory T-cell patterns. Earlierwork showed increased IL (interleukin)-6 and IL-8 butdecreased TNF-α and IFN (interferon)-γ in serum,with elevated soluble IL-2 receptor in blood and tissue[164,165]. Moretti et al. [166] found increased IL-6, TNF-α and minimal TGF-β in tissue, and Tu et al. [167]found increased IL-6 but normal IL-1β, IL-8 and TNF-α in blood. Franczuk et al. [168] reported decreasedsoluble IL-2 receptor in blood. Grimes et al. [169] notedincreased tissue TNF-α, IFN-γ and IL-10, and Zailaie[170] detected increased IL-1, IL-6, IL-8 and TNF-αin blood, while Birol et al. [171] found elevated tissueTNF-α but normal blood levels of this cytokine. Itwas recently noted that imiquimod often causes vitiligo-like depigmentation when used to treat superficial basalcell carcinoma. Imiquimod binds Toll-like receptors 7and 8 and evokes a Th-1 response with the productionof IFN-α, TNF-α and IL-12. Imiquimod also causesincreased IL-6, IL-8 and IL-10. Similar cytokines mightbe involved in vitiligo [172]. Taher et al. [173] notedthat topical tacrolimus, used successfully to treat vitiligo,increases tissue IL-10, which is an immunosuppressiveTh-2 cytokine. This suggests that vitiligo might be aTh-1 type of autoimmune disease. Pichler et al. [154]recently found normal blood levels of TNF receptor withslightly elevated IL-6, while Basak et al. [174] reportedsignificantly decreased serum levels of TGF-β in vitiligo,with potential inhibition of regulatory T-cell function.

InfiltratesEarlier histopathological studies of vitiligo focused ondegenerative changes in melanocytes and keratinocytes,particularly at the perilesional location. Vacuolaralteration of the basal layer was noted, with scatterednecrotic keratinocytes reminiscent of a lichenoid reaction.Electron microscopy showed intracellular oedema,cytoplasmic vacuolation and dilation of organelles withindegenerating melanocytes and keratinocytes in normal-appearing skin adjacent to amelanotic skin. In somecases, degeneration was limited to melanocytes, sparingkeratinocytes. Little attention was paid to mononuclearcell infiltrates, but these were, in fact, noted, being in aperivascular distribution and also within the epidermis.The infiltrate was considered to comprise lymphocytesand histiocytes, and while generally of a mild intensity,denser infiltrates were observed in vitiligo of morerecent onset and in active rather than stable disease[66,175–178]. Lichenoid-type inflammation implies a T-cell-mediated autoimmune reaction [179], and in fact,many of the mononuclear cells in the perilesional infiltrateof ordinary generalized vitiligo were shown to be T-cells by immunoperoxidase techniques. CD8+ T-cellspredominated in the epidermis, while in the dermis,

there was a normal ratio of CD4+/CD8+ T-cells [180].Activation of cell-mediated immunity was also suggestedby the early finding of abnormal expression of HLA classII molecules and ICAM-1 on perilesional melanocytes incases of vitiligo [181]. Highly significant overall increasesin CD3+, CD4+ and CD8+ T-cells were also notedby Badri et al. [182] in the margins of depigmentedskin. Many of the T-cells also expressed HLA class IImolecules and IFN-γ . CD45RO+ memory phenotypepredominated, with many T-cells expressing the skin-homing marker CLA (cutaneous lymphocyte-associatedantigen), which binds to E-selectin on dermal endothelialcells [182]. Mononuclear infiltrates in perilesional vitiligoskin were noted to be IL-2 receptor- and IFN-γ -positive,suggesting the presence of activated T-cells [178]. CLA+

T-cells clustered around disappearing melanocytes weredescribed by van den Wijngaard et al. [183]: mostof the T-cells were CD8+, and those interacting withmelanocytes were perforin and granzyme B-positive,suggesting a cytotoxic phenotype. There was focalincreased expression of HLA-DR and ICAM-1 in theepidermis at sites of interaction with T-cells [183].Melanocytes are sensitive to the granule exocytosispathway of apoptosis (perforin/granzyme) but resistantto the Fas ligand-mediated pathway [184]. Melanocytedeath in vitiligo has generally been attributed to apoptosisrather than necrosis, and cytokines like IL-1, IFN-γor TNF-α, released by lymphocytes, keratinocytes andmelanocytes, can initiate apoptosis of both melanocytesand keratinocytes. Fas/Fas ligand interactions may alsobe involved in the apoptosis of keratinocytes in vitiligo[185,186].

Cellular infiltrates were particularly noted in rare casesof ‘inflammatory’ vitiligo, characterized by elevated,reddish, scaly borders. Histopathological analysis of theborder often showed a lichenoid pattern of lymphocyticinfiltration, even a few centimeters away towards normalskin [187,188]. Lymphocytes were often juxtaposedto remaining melanocytes, suggesting a role in theirdestruction. Dense perivascular infiltrates were notedin the upper dermis, with exocytosis of lymphocytesinto the epidermis. Vacuolar degeneration of the basallayer, with necrotic keratinocytes and colloid bodies werenoted. Immunophenotyping of inflammatory vitiligoalso confirmed the presence of many T-cells, particularlywithin the epidermis. Epidermal T-cells were eitherCD8+ or CD4+, while dermal T-cells were mainly CD4+.T-cells showed a memory subtype, being CD45RO+.Keratinocytes from the inflamed border were HLA DR+,and ICAM-1 staining was increased in one study. T-cellsshowed increased IL-2 receptor expression, suggestingactivation, and many T-cells were CLA+ [189,190]. Thus,both ordinary and inflammatory vitiligo had similarpatterns of T-cell infiltration.

In vitro characteristics of infiltrating T-cells inordinary vitiligo were reported for the first time by



Wankowicz-Kalinska et al. [191] in 2003: biopsies ofperilesional skin in four patients with active and onewith stable vitiligo confirmed the presence of T-cellinfiltrates, mainly CD8+ T-cells, but with many CD4+

T-cells as well. These were often noted in appositionto residual melanocytes. Normal skin usually shows anequal distribution of CD4+ to CD8+ subsets, mostly in adermal perivascular location. The predominance of CD8+

over CD4+ T-cells in vitiligo, as well as their proximityto melanocytes, could represent the effector phase of ananti-melanocyte response, as seen in the Smyth chickenmodel of vitiligo [192]. CD8+ T-cells are known toinfiltrate organs like the pancreas and thyroid gland inother autoimmune disorders like diabetes mellitus andHashimoto’s thyroiditis, respectively [193,194]. A novelfinding by Wankowicz-Kalinska et al. [191] in the studywas the presence of T-cell infiltrates in normal-appearingskin remote from the site of vitiligo, with microscopic lossof melanocytes termed ‘microdepigmentation’. Thesecould well represent sites of future vitiligo. T-cells weregenerated by mitogenic expansion and tested for cytokineelaboration and antigen specificity. Phenotypes generatedwere similar to those observed in situ. In all four patientswith active vitiligo, a high proportion of both CD8+ andCD4+ cells produced IFN-γ and TNF-α, with relativelylittle IL-4, IL-5 and IL-13. This pattern was not seenin T-cells derived from normal skin in these patients.Thus, the T-cells in vitiligo are polarized to a Th-1-likeprofile. TNF-α, and IFN-γ , might act synergisticallyto up-regulate HLA Class II molecules and ICAM-1,as noted previously [191]. Melanocytes can process andpresent antigenic peptides to antigen-specific CD4+ cellsafter pretreatment with IFN-γ ; therefore, elevated IFN-γ and TNF-α could predispose melanocytes to immunesurveillance [195]. The patient with stable vitiligo had adifferent cytokine profile, more Th-2-like. It is interestingthat multiple sclerosis shows a Th-1 type response inactive disease and a Th-2-dominant response in quiescentstages; perhaps a similar phenomenon occurs in vitiligo[196]. Several CD8+ T-cell clones were able to lyseautologous cultured melanocytes in an HLA-restrictedmanner, the first time that T-cells from tissue rather thanblood of patients with vitiligo have shown this effect.Blocking with anti-HLA class I antibody preventedthe lysis, confirming a specific, HLA-restricted CD8+

cytotoxic T-cell response rather than an NK-like response[191].

Antigen-specific T-cellsThe study of antigen-specific T-cells in vitiligo benefitedfrom work on the cell-mediated immune response tomelanoma, shared melanocyte differentiation antigenswith melanoma and a simpler technique to detectantigen-specific T-cells, known as soluble tetramerstaining. Vitiligo is more frequent in patients withmetastatic melanoma than the general population [197–

200] and is associated with increased survival. Vitiligo-like depigmentation has been reported after successfulimmunotherapy of melanoma, including high dose IL-2therapy, infusions of peptide-pulsed DCs (dendritic cells)and Melan-A/MART-1-specific cytotoxic T-cell clones[201–206]. During adoptive transfer therapy, infusedmelanocyte-specific T-cells accumulated in the peripheryof sites of incipient depigmentation. Depigmentationwas not observed in non-responding patients orthose with unrelated malignancy [203]. Cytotoxic T-cells generated from melanoma recognize melanocytedifferentiation antigens expressed by normal melanocytesas well [203,207,208]. Clonally expanded T-cells withidentical T-cell receptor Vβ regions were simultaneouslydemonstrated in the depigmented halo around aregressing melanoma and within the melanoma [209].The possibility that melanocyte differentiation antigensrepresent the targets of cell-mediated autoimmunity invitiligo has been investigated recently. These antigensinclude melanosomal proteins like Melan-A/MART-1, tyrosinase and gp100, involved in the synthesisof melanin [197]. High numbers of Melan-A-specificcytotoxic T-cells have been observed in the peripheralblood of melanoma patients with concurrent vitiligo,but not in those without vitiligo [210]. Regression ofmetastatic melanoma and the simultaneous developmentof vitiligo have occurred after immunization with Melan-A peptide, associated with oligoclonal expansion ofMelan-A-reactive cytotoxic T-cells [211]. Utilizing thetechnique described by Altman et al. [212], multimericsoluble complexes of known peptides with HLA-A2molecules could be used to identify peptide–antigen-specific T-cells in HLA matched hosts. These peptide–HLA-A2 complexes bind more than one T-cell receptoron a specific T-cell, allowing a slower dissociation rateand enabling successful immunological staining [212].Utilizing this technique, Ogg et al. [213] showed forthe first time a high frequency of Melan-A-specificCD8+ T-cells in the circulation of HLA-A2+ patientswith vitiligo. These cells were able to lyse A2-matchedmelanoma cells in vitro. They were not observed inA2-negative vitiligo patients or in A2-positive controlpatients who did not have vitiligo. The T-cells alsoexpressed high levels of skin-homing CLA [213]. Lang etal. [214] confirmed these findings, with levels of Melan-A-specific CD8+ T-cells correlating with disease activity.Four peptides from Melan-A were used. Gp100 andtyrosinase-specific CD8+ T-cells were also demonstrated,using five peptides from gp100 and two from tyrosinase.Intracellular IFN-γ was elevated in the CD8+ T-cells,confirming the active state shown by the enzyme-linkedimmunospot technique [214]. Wankowicz-Kalinska etal. [191] also showed Melan-A specificity of clonedT-cells from perilesional skin by the tetramer technique,revealing a high proportion of Melan-A-specific CD8+

T-cells in two of five HLA-A2.01+ patients with vitiligo.


112 S.J. Glassman

Interestingly, these two patients failed to show similarCD8+ cells in the peripheral circulation, emphasizing theimportance of testing both peripheral and tissue sites,if possible [191]. Mandelcorn-Monson et al. [215] inan ex vivo study utilized peripheral blood of patientswith vitiligo to isolate antigen-specific CD8+ T-cells,without additional in vitro culture, and revealed a highproportion of gp100-positive CD8+ T-cells, unlike theother studies. Two epitopes of gp100 were used to increasesensitivity. Utilizing recognized epitopes from Melan-A and tyrosinase, specific T-cells were not isolated.One patient showed Melan-A and tyrosinase specificityafter in vitro re-stimulation [215]. Palermo et al. [216]utilized the tetramer technique with peripheral bloodof patients with vitiligo or melanoma and showed thepresence of Melan-A-specific cytotoxic T-cells at similarlevels in both HLA-A2-matched groups. However, onlyvitiligo cells showed T-cell-receptor down-regulationand IFN-γ production after exposure to HLA-matchedmelanoma cells, suggesting a qualitative difference inreceptor affinity in the two groups. Vitiligo has beenconsidered the effective variant of melanoma immunity[216,217]. Van den Boorn et al. [218] expanded onall these findings in 2009: their study showed aprominent role for T-cells in vitiligo pathogenesis,being causative rather than just a consequence of otherstimuli. Perilesional T-cells were easily identified in nineHLA-A2+ patients with progressive vitiligo, culturedand subjected to flow cytometric analysis using HLA-A2/peptide tetramers for tyrosinase-(369–377), gp100-(280–288), gp100-(209–217) and MART-1-(26–35). Acontrol antigen in the form of influenza virus-(58–66)was employed. Compared with healthy donor skin-residing cells, significantly increased levels of T-cellsrecognizing all the melanocyte antigens were found inthe perilesional T-cell population. Similar proportionsof these T-cells were also found in the peripheralblood, showing that the presence of melanocyte antigen-specific T-cells in the skin usually coincides with similarincreased levels of these cells in the blood. Functionalactivation of cultured perilesional T-cells upon exposureto melanocyte differentiation antigens was tested forthe first time. T-cells were stimulated with the poolof HLA-A2-binding peptides in five patients and threecontrols. In order to exclude bias from prolonged invitro culture, only those samples that grew sufficientcells within 14 days were analysed. Activation of T-cells was measured by CD-69 expression for earlyT-cell activation, CD137 for CD8+ T-cell activationand CD154 for CD4+ T-cell activation. Cytolyticaction was measured by granzyme-B production andcytotoxic degranulation by CD107a expression. IL-4, ahumoral response promoter, and the proinflammatorycytokines IL-17, TNF-α and IFN-γ were also measured.Results showed that after melanocyte antigen exposure,perilesional CD8+ T-cells became activated (CD69+,

CD137+, granzyme-B+ and CD107a+); they were notactivated by influenza controls. The magnitude of theCD8+ T-cell response was proportional to the extentof vitiligo or the presence of poliosis. PerilesionalCD4+ T-cells showed bystander activation, with CD69and CD154 expressed but not granzyme-B. Thisdemonstrates an antigen-independent activation of theseCD4+ T-cells in this HLA class-I-restricted stimulationsetup. There was a variable increase in TNF-α andIFN-γ by both perilesional CD4+ and CD8+ T-cells.One patient, who also had halo nevi, demonstratedelevated IL-17. Donor control skin-residing T-cells didnot show a cytotoxic response to the melanocyte antigens.Therefore, perilesional T-cells in vitiligo are primed toreact against melanocyte antigens unlike healthy skin-residing T-cells. To examine the functional capacityof these T-cells to kill melanocytes, a skin explantmodel, originally developed to predict GVHD (graftversus host disease) in bone marrow transplantation, wasused. Perilesional T-cells were co-cultured for 2 dayswith non-lesional, normally pigmented autologous skin;CD8+-enriched and -depleted populations were alsotested. Explants were subsequently analysed for thepresence of infiltrated T-cells, melanocytes and apoptosis.Results in all cases showed infiltration of explants, withinduction of apoptosis of melanocytes as indicated bycytoplasmic active caspase-3 staining. Other epidermalcells in the suprabasal layers, probably keratinocytes, alsounderwent apoptosis. CD8+-depleted samples showedinfiltration but minimal apoptosis, while CD8+ enrichedsamples had the most apoptosis of melanocytes andkeratinocytes, corresponding to sites of epidermalT-cell infiltration. Keratinocyte apoptosis did not occurin lesional skin devoid of melanocytes, and is thusconsidered a bystander effect from proinflammatorycytokines rather than a specific cytotoxic effect of T-cellson keratinocytes. Finally, the same explant experimentwas performed, but utilizing purified melanocyteantigen-specific T-cell populations. Gp100-(280–288)-specific CTLs were co-cultured with autologous skin anddemonstrated infiltration of the explant, with extensivedisruption of the skin tissue. CLSM (confocal laserscanning microscopy) showed CTLs infiltrating thedermis and epidermis, causing damage to the dermo-epidermal junction and basal melanocytes. When theseCTLs were stimulated in vitro with the same gp100-(280–288) antigen, they produced large amounts of IFN-γ .The same cells did not produce IFN-γ after stimulationwith irrelevant tyrosinase epitopes, or if there was noadditional peptide stimulation, indicating the antigen-specific nature of the activation. Tyrosinase-(369–377)-and MART-1-(26–35)-specific CD8+ T-cell clones werealso co-cultured with explant skin, also demonstratinginfiltration and apoptosis of melanocytes. Keratinocyteapoptosis was not observed in these samples. Noapoptosis was found when these T-cells were incubated



with lesional (melanocyte-free) skin explants, showingthat activation was antigen-specific and dependent on thepresence of melanocytes [218].

With respect to tyrosinase as a potential autoimmunetarget in vitiligo, it is interesting that the recentgenomewide association study confirmed a significantassociation with several TYR SNPs, in particularrs1393350. Significant epistasis was observed between thisSNP and the HLA-A SNP rs12206499, and it is postulatedthat this combination presents the TYR polypeptide moreefficiently to the immune system by HLA-A*02 [47].

Regulatory T-cellsAlthough T-cells are considered integral to the onsetof vitiligo, it is acknowledged that anti-melanocyteantibodies could play an important role in maintainingthe disease [219]. CTLs alone are probably necessarybut not sufficient for perpetuating generalized vitiligo,as regulatory mechanisms that prevent autoaggressionby the immune system need to be overcome first [220].Regulatory T-cells play a key role in removing self-reactive T-cells that have escaped the process of clonaldeletion by the thymus [221]. Some regulatory T-cellshave been shown in vitro to suppress activated T-cells in acontact-dependent process, utilizing CD25 and forkheadand winged-helix family factor FOXP3 (forkhead box P3)[33]. Regulatory T-cells may be stimulated by immatureDCs in a Tol–DC regulatory loop [222]. DCs alsohave the capacity to augment melanocyte apoptosis incertain circumstances: melanocytes exposed to 4-TBPgenerate HSP-70 (heat shock protein-70), which althoughpartly protective, also induces TRAIL (TNF-relatedapoptosis-inducing ligand) receptor expression on theircell surface. This activates DC effector functions andresults in melanocyte death [223]. Tumour and self-directed CD8+ T-cells are augmented by CD4+ T-cellsand suppressed by regulatory T-cells [224]. RegulatoryT-cells can inhibit CD8+ and CD4+ T-cells and NKcells, via the cytokine TGF-β [219]; decreased TGF-βlevels in peripheral blood of vitiligo patients were, in fact,recently reported, pointing towards impaired regulatoryT-cell functioning [174]. Regulatory T-cells also controlconversion of naıve CD4+ T-cells (CD45RA+) intoTh-17 T-cells, producing IL-17. In the absence of regulat-ory T-cells, naıve CD4+ T-cells become Th-1 cells [225].IL-17 is proinflammatory, acting synergistically with IL-6, IL-1β and TNF-α. IL-17 is increased in several chronicautoimmune diseases including psoriasis and systemicsclerosis [226]. Increased levels of IL-17-producing T-cells were reported in one patient with vitiligo [218],but serum IL-17 was not elevated in another studyof vitiligo patients [174]. Dysregulation of CTLA-4can also predispose to autoreactive T-cells, as thismolecule decreases T-cell responsiveness and raises thethreshold for T-cell activation. Increased CD8+ self-reactive function has been reported in the presence of

defective CTLA-4, and CD4+ T-cells are also involved inthis process [227]. CTLA-4 polymorphisms predispose tovitiligo in patients who already have other autoimmuneconditions [146].

CONCLUSIONS

A complex interplay of genetic, immunological, envir-onmental and biochemical factors occurs in the leadup to generalized vitiligo. The hierarchy of events isstill unknown, but evidence points to a major rolefor ROS and T-cells, probably acting in concert todestroy or incapacitate melanocytes in a vicious cycleof cellular fatigue, cytokine imbalance, immune attackand apoptosis. Melanocytes produce melanin, which hasantioxidant properties, but its synthesis predisposes toROS generation, and melanocytes, therefore, operate ina potentially hostile environment. Melanocytes seem tobe uniquely fragile in people with a tendency to vitiligo.Numerous endogenous and exogenous sources of H2O2

and other ROS have been described in vitiligo, which aredeleterious to a variety of melanocytic cellular processes,particularly in the context of impaired cellular antioxidantdefence. The resultant protein and lipid damage couldbe sufficient, on its own, to initiate melanocyte failure,but another effect of oxidization could be to initiatemelanocyte failure and apoptosis leading to uptake byLangerhans cells or DCs. If these Langerhans cells orDCs become activated, they may trigger a melanocyte-reactive immune response that can eradicate melanocytesin the skin, leading to depigmentation. This immuneresponse principally involves cytotoxic T-cells. Failureof regulatory T-cell mechanisms allows the processto continue indefinitely, in keeping with the chronic,relentless course of generalized vitiligo. Further researchinto the pathogenesis of this common and distressingdisorder will offer the potential for better therapiesand might even facilitate effective strategies for primaryprevention in genetically susceptible individuals.

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Received 23 November 2009/26 August 2010; accepted 26 August 2010Published on the Internet 20 October 2010, doi:10.1042/CS20090603


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