melanocytes are not absent in lesional skin of long duration vitiligo

Original Paper

Melanocytes are not absent in lesional skin of longduration vitiligo

Desmond J. Tobin1, Nelle N. Swanson2, Mark R. Pittelkow2, Eva M. Peters1 and Karin U. Schallreuter1,3*1 Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford, UK2 Department of Dermatology, Mayo Clinic, Minnesota, USA3 Institute for Pigmentary Disorders e.V. in Association with the Ernst-Moritz-Arndt University Greifswald Biotechnikum, Walter-Rathenau-Str. 49a,17489 Greifswald, Germany

*Correspondence to:Professor K. U. Schallreuter,Clinical and ExperimentalDermatology, Department ofBiomedical Sciences,University of Bradford, Bradford,West Yorkshire BD7 1DP, UK.E-mail: [email protected]

Received: 6 July 1999

Revised: 29 November 1999

Accepted: 21 March 2000

Published online: 8 June 2000

Abstract

This paper provides evidence that melanocytes are still present in the depigmented epidermis of

patients with vitiligo even after stable disease of 25 years' duration. Melanocyte cultures were

successfully established from depigmented epidermal suction blister tissue of all 12 randomly

selected patients and these cells produced melanin. Even under in vitro conditions, vacuolation of

melanocytes was demonstrated in ®ve patients with active disease, which was reversible upon

exogenous addition of bovine catalase to the culture medium. Full skin biopsies from 17 patients

with vitiligo, obtained from depigmented and normally pigmented areas, con®rmed the

involvement of melanocytes, keratinocytes, and Langerhans cells in this disorder. In addition,

the presence of clustered and single pre-melanosomes in basal and supra-basal keratinocytes of

lesional and normal epidermis, as well as the retention of single melanocytes in lesional epidermis,

was demonstrated by light and electron microscopy. Upon topical application of a narrow band

UVB-activated pseudocatalase, vacuolation, granulation, and dilatation of the endoplasmic

reticulum completely recovered, but the ectopic pre-melanosome shedding remained. Taken

together, these observations indicate that melanocytes are never completely absent in the

depigmented epidermis and that these melanocytes can recover their functionality in vivo and invitro upon the removal of hydrogen peroxide. Furthermore, this study supports the concept that

vitiligo involves the entire epidermal unit in both depigmented and `normal' pigmented skin.

Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: pre-melanosome; oxidative stress; vacuolation; epidermal melanin unit; electron-

microscopy; cell culture

Introduction

Vitiligo is an acquired idiopathic and, in the majorityof cases, a progressive, unpredictable disorder of theskin [1]. The family history is positive in approximately30±40% of cases and there is no gender or racial bias[1]. The onset is mostly early in life and it has anestimated worldwide incidence of 0.5±4%. While theaetiology of vitiligo is as yet unknown, severalhypotheses have been proposed for the loss offunctioning melanocytes in the skin of these patients.These include the presence of autoantibodies againstvarious tissues [2,3]; cytotoxic T-cells [4]; auto-destruction of melanocytes by intermediates of themelanogenesis pathway [5]; the presence of intrinsic/extrinsic metabolic defects in the melanocytes them-selves or in the epidermal melanin unit, leading tooxidative stress [6±8]; and the neural hypothesis [1].Recently, a `convergence theory' has been suggested,combining all hypotheses of this disease [9]. While theliterature is replete with structural and morphologicalstudies of vitiligo, many reports repeatedly describesome or all features, such as absence of melanin andmelanocytes from affected epidermis, degeneration ofmelanocytes and/or basal/supra-basal keratinocytes,

extracellular granular material, a dermal lymphohisto-cytic in®ltrate at the border between lesional and`normal' pigmented skin, the relocation of Langerhanscells to the basal layer and an increase in their number,and a dilated endoplasmic reticulum [10±23]. In thispaper, we de®ne `normal' skin in vitiligo as the fullypigmented epidermis. However, it should be recognizedthat in addition to vacuolation and the above-described abnormalities, a number of biochemicalchanges have also been found in this so-called`normal' epidermis [6±8].

Most of these early studies concluded that fullywhite/lesional vitiligo skin is characterized microscopi-cally by the complete absence of melanocytes. Therecent extensive study by Le Poole et al. concluded thatthere was loss of melanocytes in lesional skin ofvitiligo, using a panel of 16 monoclonal antibodies[13]. However, there are also some con¯icting reportsindicating that vitiligo lesions are not totally devoid ofmelanocytes [24]. Signi®cantly, residual amounts ofthe melanocyte-speci®c enzyme tyrosinase have beendetected in lesional vitiligo skin, providing unam-biguous evidence for the presence of these cells[24] (Schallreuter unpublished data). These authorsshowed that enzymatic hydroxylation of tyrosine to

Journal of PathologyJ Pathol 2000; 191: 407±416.DOI: 10.1002 /1096-9896(2000)9999 :9999<: :AID-PATH659>3.0.CO;2-D

Copyright # 2000 John Wiley & Sons, Ltd.

dopa in epidermal homogenates of vitiligo skin wasdue to tyrosinase [24]. A recent study reported thepresence of low amounts of melanin in white/lesionalskin [25]. These melanosomes were in late-stagematuration (stage III/IV) and were commonly clumpedas melanin granules within basal keratinocytes, even inlong-lasting disease. Since the depigmentation processcan be reversible, either spontaneously or by usingvarious therapeutic approaches, it seemed justi®able toreaddress the issue and to re-explore a possiblereservoir of melanocytes.

In this context, it has been shown that patients withvitiligo overproduce and accumulate (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) as well as the non-enzymatically generated 7-isomer (7BH4) in theirepidermis [26,27]. Consequently, the characteristic¯uorescence of the white/lesional skin upon Wood'slight examination (UVA 351 nm) was attributed to thepresence of oxidized pterins [26,27]. Furthermore, itwas demonstrated that the redox status of these pterinscan be signi®cantly in¯uenced by epidermal hydrogenperoxide (H2O2) production in both melanocytes andkeratinocytes [28,29]. Using Fourier-transform Ramanspectroscopy, it was possible to con®rm epidermalH2O2 accumulation in vivo in these patients [28,29].Furthermore, it has been shown that H2O2 canef®ciently oxidize 6BH4 as well as 7BH4 to 6- and 7-biopterin, respectively [29]. Here, it is noteworthy thatmicromolar amounts of 6-biopterin can be cytotoxic tonormal human melanocytes [30]. Under these in vitroconditions, the melanocytes retracted their dendritesand also lost the expression of melanocyte-speci®cmarkers such as NKI/beteb, HMB-45, and dopaoxidase. This process was reversible upon removal ofthe high concentrations of 6-biopterin from the culturemedium. These results suggested that the epidermalbiopterin redox status can signi®cantly affect melano-cyte homeostasis/function [30].

Considering these newer observations, we wishedto re-examine the ultrastructure and immunohisto-chemistry of white/lesional and `normal' pigmentedskin of patients with vitiligo by focusing particularlyon the presence/absence of functioning melanocytesand on the melanocyte±keratinocyte interaction. Tosubstantiate further the presence/absence of epidermalmelanocytes in white/lesional skin, we also aimed toestablish melanocyte cultures from suction blistertissue obtained from both white/lesional and `normal'pigmented epidermis from 12 randomly selectedpatients with different disease durations.

Materials and methods

Patients

Twenty-seven patients with the clinical subtype vitiligovulgaris (15 males/12 females) and a mean age of 41years (range 15±63 years) were included in this study.The mean duration of the disease for the group was 8years (range 1±25 years). The photo skin types

(Fitzpatrick classi®cation) were type II (n=2), typeIII (n=19), type IV (n=2), type V (n=1), and type VI(n=3). Seven of the patients had no treatment prior tothis study, whereas 20 patients were treated with atopical narrow band UVB-activated pseudocatalaseover 6±10 months [31]. This pseudocatalase has beenshown not to cause additional morphological changes,except recovery from the cellular vacuolation. Tenhealthy age- and photo skin type-matched probandsserved as controls. All patients were otherwise healthy.Patient consent was obtained prior to the study, whichwas approved by the local Ethics Committee.

Cell cultures

Epidermal melanocyte and keratinocyte cultures wereestablished in MCDB-153 medium, according to themethod of Pittelkow and Shipley, from suction blistertissue of 1±2 blisters with 1 cm in diameter of bothwhite/lesional and `normal' pigmented epidermis of 12randomly selected patients (eight male/four female, age14±48 years, mean 37 years) with active vitiligo andfrom eight healthy controls [32]. From this group, ®vepatients were pretreated with pseudocatalase, whereasseven patients had no treatment prior to the procedure.It is noteworthy that epidermal suction blister roofs donot contain any parts of hair follicles; hence themelanocytes could only originate from the epidermalblister roofs. The numbers of seeded melanocytespresent in suction blister material from the depigmen-ted epidermis of patients with vitiligo depend on thesize of each suction blister and vary from 10 to 50viable melanocytes per T25 ¯ask, compared with anorder of magnitude more cells per T25 ¯ask from`normal' pigmented skin and healthy controls. Mela-nocyte identity was con®rmed by morphologicalcriteria and by the expression of melanocyte-speci®cmarkers. The ability of cultured melanocytes toproduce and secrete melanin was followed by bright®eld microscopy. In addition, shed material wascollected from the culture medium and processed forconventional transmission electron microscopy.

Light and transmission electron microscopy

The specimens from 27 patients (seven untreated, tentreated, and ten controls) were prepared for standardlight and transmission electron microscopy andimmunohistochemistry as previously described [33,34].Brie¯y, patient and control tissue samples for light andelectron microscopic analysis were ®xed in half-strength Karnovsky's ®xative [35], post-®xed in 2%osmium tetroxide and uranyl acetate, and embedded inaraldite resin [33,34]. Semi-thin resin sections (1±2 mm)of `normal' pigmented and white/lesional skin wereobtained from each patient sample and from normalcontrols, using a Reichart±Jung microtome. The semi-thin sections were examined by bright ®eld lightmicroscopy, either stained with the metachromatictoluidine blue/borax stain or unstained for improvedobservation of melanin granules, followed by photo-

408 D. J. Tobin et al.

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 407±416.

graphic documentation. The occurrence of intracellularvacuoles was assessed in full-length 1 mm resin sectionsof 3 mm punch biopsies from clinically normal andlesional vitiligo skin, as well as from normal healthycontrols. Arbitrary units were ascribed to each patientsample, using 0 for absence of vacuolation, 1 for a fewaffected cells, and 2 for many affected cells. Numberswere pooled and statistical signi®cance was calculatedusing the t-test for unpaired samples. Ultrathinsections (50±100 nm) were stained with uranyl acetateand lead citrate and examined using a Jeol 100CXtransmission electron microscope. In order to demon-strate dopa-oxidase activity, ®xed skin specimens werealso incubated in 0.1% dopa for 4 h at 37uCand thereafter processed conventionally. The dopa-incubated tissue was examined by both light andtransmission electron microscopy.

Immunohistochemistry

Five-micrometre cryostat sections were cut from eachskin specimen and air-dried, followed by acetone®xation. Sections were incubated with the antibodiesNKI/beteb for the detection of pre-melanosomes andHMB-45 for the detection of pre- and more maturemelanosomes, using the Vectastain Elite ABC mouseIgG kit (Vector Laboratories). In addition, sectionswere stained with Fontana-Masson to visualize mela-nin. Dopa incubation was employed to detect tyrosi-nase activity [34].

Results

This study in untreated patients with vitiligo con®rmedpreviously reported morphological features, includingvacuolation/degeneration of basal keratinocytes, mela-nocytes and Langerhans cells; increased presence ofLangerhans cells in the basal layer of the epidermis inclose proximity to affected melanocytes; ®brillar/fattydegeneration and intracellular granular debris; anddilated endoplasmic reticulum (data not shown). Thequanti®cation of epidermal vacuolation of full skinbiopsies is summarized in Figure 1, which shows asigni®cant difference in both lesional and `normal' cellscompared with healthy controls. Although these dataonly con®rm previous results obtained by otherinvestigators, we can now unambiguously showthat these morphological changes are most likelythe consequence of H2O2-derived oxidative stress[10±12,29]. More importantly, additional observationsshow that melanocytes were never completely absentfrom white/lesional vitiligo epidermis and that thesecells retain the ability to become functionally active,even after long-lasting disease.

In vitro evidence for melanocytes in white/lesionalepidermis in vitiligo

Suction blister tissue was obtained from white/lesionaland `normal' pigmented skin from 12 randomly

selected patients with vitiligo. This technique allowedthe establishment of cells from pure epidermal origin.Five patients had no treatment, whereas seven weretopically treated with UVB-activated pseudocatalase[31]. Figures 2a±2d demonstrate a typical example ofboth white/lesional and `normal' pigmented epidermalsuction blister tissue used for this study. Notably, nocontaminating hair follicle material was seen in any ofthe samples used for cell culture. In all 12 cases, eighthealthy controls (Figures 2a and 2b) as well asmelanocyte and keratinocyte cell cultures were estab-lished from white/lesional and `normal' pigmentedsuction blister tissue. After the establishment of thesecell cultures, two morphologically distinct types ofmelanocytes were always observed using the antibodyNKI/beteb for melanosome identi®cation: a highlydendritic cell with multiple melanosomes and asecond cell type which showed only occasional mela-nosome formation (Figure 3c). The presence of shedmelanosomes in the culture medium was shown byelectron microscopic examination. Evaluation of thisshed material con®rmed the presence of some unat-tached melanocytes containing melanosomes at differ-ent stages of maturation and of free melanosomes inthe culture medium (data not shown). In our hands,after an initial lag period for both sources, melanocytesderived from `normal' pigmented skin presentedsigni®cantly slower growth than melanocytes fromwhite/lesional skin (data not shown). Surprisingly,melanocytes and keratinocytes originating frompatients with extremely progressive vitiligo (n=5)exhibited extensive vacuolation followed by sequentialdendrite retraction, even under culture conditions.Temporal changes as observed in Figure 3 werefollowed for ®ve independent melanocyte cell linesestablished from depigmented epidermal suction blis-ters from these patients. The exposure of normalmelanocytes (n=5) to micromolar concentrations ofH2O2 caused neither vacuolation nor dendrite retrac-tion, as observed with the establishment of thesepigment cells from patients with vitiligo. Upon addi-

Figure 1. Melanocyte and keratinocyte vacuolation in `normal'and lesional vitiligo epidermis. Intracellular epidermal vacuolationin full skin biopsies from patients with untreated vitiligo (n=7)compared with controls (n=10). Statistical signi®cance wascalculated using the t-test for unpaired samples

Melanocytes in lesional vitiligo skin 409


tion of exogenous bovine catalase to the culture

medium, this process was reversible in all cases

(Figures 4a±4c). These in vitro results were supported

in vivo by FT-Raman spectroscopy, where only the 12

patients with vitiligo demonstrated epidermal H2O2

production in the micromolar range, compared with

healthy controls [28,29].

Figure 2. Suction blister tissue used for the isolation andculture of melanocytes. Melanocytes were obtained from`normal' pigmented (a, b) and white/lesional (c, d) epidermis ina 47-year-old male with vitiligo of 25 years' duration. Melanin isdetectable in `normal' pigmented, but not white/lesional epider-mis. Note that hair follicle material did not contaminate theseblister specimens. V=vacuoles, M=melanin, SC=stratum cor-neum. (a) r1620; (b) r450; (c) r1620; (d) r450

Figure 3. Evidence for melanocytes in white/lesional skin.Primary cultures of melanocytes (MC) and keratinocytes (KC)derived from the suction blisters of `normal' pigmented (a) andwhite/lesional epidermis (b) obtained from a 22-year-oldmale with stable vitiligo of 20 years' duration. (a, b) r607.5.(c) Primary melanocyte culture derived from a suction blister ofwhite/lesional skin obtained from a 22-year-old male with stablevitiligo of 20 years' duration. Note that the differentiatedpigmented cell (MM) is NKI/beteb-positive throughout the cell,while the amelanotic melanocyte (AM) displays only punctatestaining (PM). r817.5



In vivo evidence for melanocytes in depigmentedepidermis

Direct evidence of melanocytes in white/lesional skin ofpatients with vitiligo was provided by the observationof rare clear cells in the epidermis of these patients,even after 25 years' disease duration (Figure 5a). Onelectron microscopy, these cells were typically smalland amelanotic (Figure 5b), although some containedabnormal melanosomes (Figure 5c). They were clearlydistinguishable from Langerhans cells by their lack of;characteristic granules, indented nuclei, and plentifulcytoplasm containing lysosomes, and from keratino-cytes by the absence of desmosomal junctions ortono®laments. De®nitive proof of a melanocyticidentity for at least some of these cells was providedby their expression of tyrosinase as determined by apositive dopa reaction (Figures 5d and 5e). Nocontaminating hair follicle material was observed inthe blister specimens. While these rare melanocytes hadno consistent pattern of location, they were mostcommonly widely separated and sometimes situatedclose to rete ridges. The isolation and establishment ofmelanocyte cultures from epidermal suction blistertissue, even from blisters of amelanotic skin in vitiligoof long-standing disease, highlight the presence of thesecells in lesional white skin of patients with vitiligo,although in signi®cantly lower numbers.

Ultrastructural evidence for melanin granules indepigmented epidermis

Circumstantial evidence in support of the retention ofmelanocytes in white/lesional epidermis was providedby the repeated observation of mature stage IVmelanin granules in the cytoplasm of basal and supra-basal keratinocytes in the depigmented lesional epider-mis. This ®nding was present in 10 of 12 vitiligopatients and surprisingly even in the amelanotic skinof patients with 25 years' disease duration. Thisfeature was independent of treatment with a UVB-activated pseudocatalase. These rare melanin-containing cells were typically distributed in groups of

one to three (Figures 5f and 5g). Given that melano-cytes are presumably absent in white/lesional epider-mis, the origin of this melanin is unclear. Bartosik et al.ascribed these melanin granules to functioning mela-nocytes in the perilesional, rather than the amelanoticepidermis [25]. Since we have demonstrated thepresence of residual melanocytes in vivo and in vitro,we conclude that melanin granules could originatefrom perturbed functioning melanocytes in white/lesional skin.

Impaired melanocyte±keratinocyte interaction in`normal' pigmented and white/lesional epidermis

To date, there is accumulating evidence that vitiligomay be a disorder of the entire epidermal melanin unit[26,27,36±38]. To examine this view, we reassessed theinteraction between the melanocyte and keratinocyte invitiligo skin. In this context, we observed that `normal'pigmented and white/lesional skin of vitiligo patientscould demonstrate evidence of defective transfer ofmelanosomes from donor melanocytes to recipientbasal and supra-basal keratinocytes. Extracellularmelanin granules were detected in the `normal' pig-mented epidermis in 6 of 12 patients, regardless oftreatment. These granules were detected in the basallayer, where most were `free' in the interstitial space,although they were also occasionally associated withmelanocyte fragmentation (Figures 6a±6c). Interest-ingly, some indication of melanosome movement wasevidenced by the apparent uptake of mature melaningranules into Langerhans cells (Figures 6a and 6c), inaddition to melanin granule uptake by presumptivemelanocyte dendrites/fragments, as evidenced by theirassociation with coated pits (Figure 6b). Langerhanscells also showed phagolysosomes containing multiplemelanin granules (Figure 6c). The transfer of melano-somes was not restricted to mature stage IV granules.Melanosomes in all stages of maturation were foundwithin keratinocytes, either within lysosomes or `free'in the cytoplasm in both white/lesional and `normal'pigmented epidermis (Figures 7a±7d). One possible

Figure 4. Effect of exogenous catalase (30 mg/ml) on melanocyte vacuolation and loss of dendricity. (a) Vacuolation in themelanocyte after day 19 in culture. Dendrites were lost by day 25 in culture; (b) 6 days after addition of catalase; (c) completerecovery of cell after 13 days. V=vacuolation. (a±c) r2040



mechanism of pre-melanosome entry into keratinocytesis suggested by the direct fusion of melanocyte and

keratinocyte plasma membranes (Figures 7c and 7d).Pre-melanosomes were detected close to the fusionzone, a region associated with apparently heterotypiccytoplasmic continuity (Figure 7d). Further evidence

for a dysfunctional epidermal melanin unit could bethe observation of focal clustering of mature granules

in the mid-epidermis and above (Figures 8a and 8b).After treatment with UVB-activated pseudocatalase,the entire epidermis recovered from vacuolation in allten treated patients.

Figure 5. Retention of melanocytes in white skin in vitiligo. (a) Melanocyte vacuolation (V) in the white/lesional epidermis ofa 35-year-old male with vitiligo of 2 years' duration. MC=melanocytes, D=dermis. (b) Poorly melanized melanocyte in theepidermis of white lesional skin in a 35-year-old female with vitiligo of 5 years' duration. M=melanin granule in keratinocyte;PM=premelanosome, D=dermis. (c) Bizarre melanosomes in a rare metabolically active melanocyte in the white/lesional epidermisof a 22-year-old male with vitiligo of 8 years' duration. Note the extensive Golgi and associated vesicles (G). (d) Presence of a singledopa-positive melanocyte (MC) in the white lesional epidermis of a 35-year-old female with vitiligo of 21 years' duration. Note thepresence of melanin (M) in the keratinocyte above the dopa-positive melanocyte. (e) High magni®cation of dopa-positive melanocyte(MC). (f) Melanin (M) restricted to a small number of adjacent basal cells in the white/lesional skin of a 35-year-old male with vitiligoof 2 years' duration. (g) Melanin (MC) in a single vacuolated (V) basal keratinocyte (KC) in the white/lesional skin of a 47-year-oldwoman with vitiligo of 6.5 years' duration. D=dermis. (a) r3366; (b) r7055; (c) r17 000; (d) r1190; (e) r2337.5; (f) r2932.5;(g) r8627.5



Immunohistochemical analysis

Abnormalities were observed in the entire epidermalmelanin unit, even in the `normal' skin. Clusters of pre-melanosomes (stage I/II) were detected in the mid-

layers of the epidermis using the melanocyte-speci®cantibody NKI/beteb, which usually only stains mela-nocytes in the basal layer of normal healthy epidermis.Here, focal staining of the `normal' pigmented mela-nocytes and of focal keratinocytes was evident. This®nding was supported by the identical staining patternobtained using the antibody HMB-45, which alsoreacts intensely with premelanosomes, suggesting thatthe target antigen (pre-melanosomes) is located in bothmelanocytes and keratinocytes (Figure 8c). It should benoted that under normal healthy conditions this cell isnot a recipient of immature melanosomes. Additionalanalysis with Fontana-Masson did not show anypositivity in lesional skin, whereas the staining patternwas completely unremarkable in `normal' pigmentedepidermis. These ®ndings are in agreement with LePoole et al. [13]. However, the observation of ectopicpre-melanosomes would suggest defective melanosomematuration and transfer in vitiligo skin.

Discussion

The results of this study have con®rmed earlierobservations of melanocyte degeneration, includingvacuolation, the presence of a dilated endoplasmicreticulum, and granular deposits. In addition, we alsocould not demonstrate immunohistochemically anyintact melanocytes in the white/lesional epidermisemploying some antibodies identical to those usedby Le Poole et al. [13]. However, to our surprise,melanocytes could be isolated and established in vitrofrom all randomly selected patient samples of white/lesional and `normal' pigmented skin, independent ofdisease duration and independent of treatment. Somemorphological changes such as vacuolation and den-drite retraction in melanocytes were even observedunder in vitro conditions, in cells derived from patientswith aggressive progressive vitiligo; these in vitrochanges could be reversed by the addition of exogen-ous bovine catalase to the culture medium (Figures4a±4c). Small amounts of mature melanin granuleswere present in the amelanotic skin of vitiligo patients,even up to 25 years of disease duration (Figures 5f and5g). This observation suggests that some partiallyfunctioning melanocytes must be retained in thisdisorder, as it is highly unlikely that this melanincould be transferred from outside the lesion. Theretention of rare intact melanocytes in clearly inter-follicular lesional skin of vitiligo patients supports theview that a subpopulation of `resistant' epidermalmelanocytes could well be a feature of this disorder.While these rare vitiligo melanocytes were usuallyamelanotic, some contained poorly melanized granules(Figure 5). Further support for defective melanocyte±keratinocyte interaction in the entire epidermis couldbe gained from the presence of extracellular melaningranules within both amelanotic and `normal' epider-mis of these patients. Since these granules were notalways associated with melanocyte cytoplasm or

Figure 6. Melanin incontinence in vitiligo. (a) Uptake of melaningranules (M) released from a melanocyte (MC) into basalLangerhans cells (LC) in the white/lesional skin of a 22-year-oldmale with vitiligo of 20 years' duration. LG=Langerhans granule.(b) Presumptive melanocyte dendrites/fragments (MF) in the`normal' pigmented skin of a 47-year-old female with vitiligo of6.5 years' duration. Note the apparent discharge of a melaningranule (M) into extracellular space. Inset: high-power view of acoated pit (CP) containing a melanin granule (M). (c) Langerhanscell in the `normal' pigmented epidermis of a 34-year-old femalewith vitiligo of 1 years' duration. Note the presence of melaningranules within a phagolysosome (PL). Inset: high-power view of atypical Langerhans granule (LG). EM=melanin, ER=endoplasmicreticulum, T=tono®laments; KC=keratinocyte. (a) r29 325;(b) r23 970 (inset: r47 889); (c) r3595.5 (inset: r5950)



melanocyte dendrites, they could possibly be releasedby degenerative or partially functioning melanocytes.The ectopic distribution of pre-melanosomes withinkeratinocytes of the basal and supra-basal layers ofboth amelanotic and `normal' pigmented epidermis, asobserved in this study, suggests a premature delivery ofthis organelle from melanocytes to keratinocytes, oringestion of immature melanosomes by keratinocytesafter fragmentation/degeneration of melanocytes. Thislatter mechanism could be a result of melanocyte deathby apoptosis, but at this time, in the absence ofadditional data, this can only be speculative. Thepositive staining for clustered pre-melanosomes usingthe antibodies NKI/beteb and HMB-45 in the absenceof fully stained cells in the basal layer and negativestaining with Fontana-Masson could indicate a defectin melanosome maturation and transfer. These arenew ®ndings which are currently under furtherinvestigation.

How can we reconcile these observations? Takinginto consideration that the entire epidermis of vitiligo

patients seems to have low catalase levels in associationwith varying degrees of H2O2 accumulation, and thatthe epidermal vacuolation recovers after treatment/substitution with a topical application of a narrowband UVB-activated pseudocatalase complex, we con-clude that the decrease of epidermal H2O2 certainlyfosters normal epidermal homeostasis [6,29,37,38]. Thequestion arises: why do melanocytes from healthyindividuals not respond with vacuolation upon exo-genous H2O2 exposure? The explanation is most likelybased on suf®cient H2O2 removal in these cells,whereas catalase activity is impaired in vitiligo skin[6]. Consequently, H2O2 accumulates as a result ofvarious identi®ed mechanisms [7,8,36±38]. Hence, asshown earlier [39], the active site of catalase can bedestroyed by its own substrate (H2O2), probablycaused by constant H2O2 stress and a build-up as aresult of various identi®ed mechanisms [6±8,31,36±38].

At the present time, it remains unclear what comes®rst, but clearly H2O2 removal is important for the cellintegrity of melanocytes and keratinocytes. It is

Figure 7. Ectopic distribution of pre-melanosomes in keratinocytes in patients with vitiligo. (a) Supra-basal keratinocytes containingmelanosomes at different stages of maturation in the `normal' pigmented epidermis of a 50-year-old male with vitiligo of 2 years'duration. Note that pre-melanosomes (PM) are located in the keratinocyte cytoplasm and also in the intercellular space.DS=desmosome, T=tono®laments. (b) Basal keratinocytes containing stage I and II pre-melanosomes (PM) in the white/lesionalskin of a 35-year-old male with vitiligo of 2 years' duration. T=tono®laments, D=dermis. (c, d) Entry of pre-melanosomes into abasal keratinocyte by fusion of melanocyte (MC) and keratinocyte plasma membranes (F) in the clinically normal epidermis of a35-year-old male with vitiligo of 2 years' duration. T=tono®laments. (a) r35 360; (b) r33 150; (c) r9265; (d) r28 050



tempting to speculate that the high epidermal 6BH4

levels observed in this disorder will be oxidized byH2O2 to 6-biopterin and that this oxidation productcould be one culprit to blame for melanocyte degen-eration as observed under in vitro conditions [30]. Thisspeculation is substantiated by the observation ofrepigmentation/melanocyte repopulation after H2O2

removal in approximately 65% of a large patientgroup (n=320) (KUS, unpublished data).

Acknowledgements

This research was generously supported by Stiefel International

Laboratory with a grant to KUS. We are indebted to the

patients and to Mrs Angela Panske from the Institute for

Pigmentary Disorders e.V. in association with the Ernst-Moritz-

Arndt University Greifswald in Germany for her constant

efforts. Miss Helen Bartle typed the manuscript.

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Figure 8. Defective melanocyte±keratinocyte interaction in the epidermis of vitiligo patients. (a) Presence of melanin in the basal,supra-basal, and upper layers of the clinically normal epidermis of a 22-year-old male with vitiligo of 8 years' duration. Note thepresence of keratinocyte degenerative change below melanin clumping in the mid-epidermis (EM). D=dermis. (b) Clumping ofmature melanin granules in the upper `normal' pigmented epidermis of a 34-year-old woman with vitiligo of 1 years' duration. Notethe giant complex of mature melanin granules (GM) within a vacuolated (V) keratinocyte. KC=keratinocyte. (c) Presence of NKI/beteb-reactive melanosomes in the `normal' pigmented epidermis of a 47-year-old male with vitiligo of 25 years' duration. Note thepresence of ectopic melanosomes (EM) in the basal, supra-basal and mid-epidermis. D=dermis. (a) r969; (b) r6790 (inset:r13 175); (c) r947.75



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melanocytes are not absent in lesional skin of long duration vitiligo

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