Journal of Dermatological Science 66 (2012) 160–168

Letter to the Editor

Contents lists available at SciVerse ScienceDirect

Journal of Dermatological Science

jou r nal h o mep ag e: w ww .e lsev ier . co m / jds

Migration of human melanocytes into keratinocyte monolayersin vitro

Keywords:

Melanocyte; Migration; Dendrites; Co-

culture; Conditioned medium

The repigmentation of adult skin after wound healing, or loss ofmelanocytes (e.g. vitiligo), poses a particular and unique challengeto the invading population of melanocytes. The melanocytes, ortheir precursors, resident in adjacent skin or in nearby niches, mustbe activated to proliferate, and then negotiate their way through atightly held epidermal barrier. The mechanisms whereby mela-nocytes negotiate their way through the epidermis to achieve thisrepigmentation have not been described in any detail and mayoccur trans-epidermally, and/or along the basement membraneand/or in the dermis. While there have been many studies onmigratory cells during embryonic development and in particularphysiological processes [1,2], these situations differ from the adultskin epidermal environment. To migrate through the tight spacesbetween keratinocytes, either junctions must be broken and spacesthus created, or alternatively, quite extreme cellular contortionswould need to occur to allow the cell to move through the narrowparacellular spaces. Further knowledge of these processes willcertainly contribute towards elucidating the mechanisms ofrepigmentation in pigmentary disorders.

In this study, through the use of both lateral and vertical/transwell migration assays, we investigate ways in whichmelanocytes might move through layers of keratinocytes usingislands of HaCaT keratinocytes [3] and melanocytes (Hermes 4acells [4] or primary M9602 cells [5]). These cells were cultured, inclose proximity to each other, in FETI medium [5], and assessed formelanocyte migration. Over a 9 day culture period the melano-cytes left their seeding boundaries and translocated into the spacesbetween the keratinocyte sheets (Fig. 1a and b). Interestingly, asthe melanocytes neared the keratinocyte population, they becamebipolar and orientated perpendicular to the keratinocyte edge withtheir dendrites protruding into the spaces (Fig. 1b). In the controlexperiment, where melanocytes only were seeded a distance fromeach other, those melanocytes leaving the seeding boundary,migrated in a random fashion, suggesting that in the absence ofkeratinocyte-derived factors, directed migration does not occur. Inaddition, it suggests that the melanocyte dendrites may function aspathfinding and anchorage structures during the migrationprocess.

To simulate whether melanocytes are able to migrate verticallythrough the keratinocyte sub-basal area into the epidermis,melanocytes (Hermes or M9602) were seeded onto the underside,

0923-1811/$36.00 � 2012 Japanese Society for Investigative Dermatology. Published b

while HaCaT keratinocytes were seeded onto the upper surface ofTranswell Millipore culture inserts (0.45 mm). After 14 days,melanocyte cell bodies and dendrites (Fig. 1c) (red, insert)migrated through the filter membrane and into the spacesbetween the confluent HaCaT keratinocyte monolayer (green)(Fig. 1c). The melanocytes on the side of seeding appeared to bemorphologically normal and displayed a bipolar phenotypeindicative of cell proliferation or were bluntly terminated, wherepart of their dendrites were not visible (Fig. 1d, arrows). Theseproved to be melanocyte nuclei as they were also seen inexperiments using keratinocyte-conditioned medium only(Fig. 1e). As there are distinct areas of membrane-bound stemcell factor (SCF) signal on the basal surfaces of keratinocytes [6], wesuggest that this may provide both a directive and proliferativesignal to melanocytes. This is in agreement with others who showthat melanocytes are attracted to and migrate towards keratino-cytes or keratinocyte secreted factors [6,7].

The intercellular spaces between keratinocytes range between0.08 and 0.19 mm in culture and 0.3–0.7 mm in vivo [8]. Scanningelectron microscopy performed on our membrane assays demon-strated that the melanocyte, including its nucleus, was able tomigrate through a 0.45 mm Millipore filter pore (Fig. 1f–h). In someinstances the melanocytes had either not completely migratedthrough the membrane (i.e. only part of their dendrites hadmigrated through the pores (Fig. 1f, arrow), similar to that seen inFig. 1d) or only parts of the cell cytoplasm had penetrated themembrane. At higher magnification the dendrites or portionsthereof could be seen invading the membrane surface andanchoring them into spaces in the membrane (Fig. 1g, yellowarrows). Furthermore, dendrite tips appeared on the uppermembrane surface containing conditioned medium (Fig. 1h,yellow boxes), suggesting that the migration of melanocytesthrough small spaces may be facilitated by both extension andanchorage of its dendrites. Since the melanocyte nucleus isapproximately 1.5–7 mm in diameter [9], the results suggest thatthese nuclei are highly malleable and are able to negotiate throughsmall spaces during migration. Of particular relevance here,Pajerowski et al. [10] elegantly demonstrated the plasticity of astem cell nucleus by revealing that these structures are highlydeformable and that their rigidity increased with differentiation.This ability to deform was attributed to the lack of laminin A/Cexpression as well as changes in chromatin density. Our resultswould suggest the possibility of a similar reorganisation in themelanocyte’s cytoplasm in order to regulate the diameter of thenucleus to increase its ‘fluidity’, as well as via regulation ofadhesion molecules such as laminins and integrins on themelanocyte dendrite. Overall, despite a reduced pore size, themelanocytes in this study, under the influence of both keratino-cytes and keratinocytes-derived growth factors, seemed to be ableto display a novel fluidity and malleability that allowed them tomigrate through the membrane and settle in amongst thekeratinocytes.

y Elsevier Ireland Ltd. All rights reserved.

Fig. 1. (a and b) Droplet assay to assess the vertical migration of melanocytes through a keratinocyte monolayer. (a) Day 5 (Mc, Hermes melanocytes and Kc, HaCaT

keratinocytes). (b) Using fluorescent antibodies, groups of melanocytes (red) could be seen interspersed between the keratinocytes (green), nuclei (blue). The melanocytes

were found in the available spaces lying perpendicular to the leading edge of the keratinocytes (arrow). (c and d) Membrane assay to assess the migration of melanocytes

through the basal surfaces of a keratinocyte monolayer. Immortalised Hermes melanocytes (S100, red), keratinocytes (E-cadherin, green) and nuclei (DAPI, blue).

Melanocytes and keratinocytes were fluorescently labelled with S-100 (1:200, Dakopatts, Glostrup, Denmark) and E-cadherin (1:500, Santa Cruz Biotechnology, CA, USA)

antibodies, respectively. Fluorescently conjugated secondary antibodies, donkey-anti rabbit-Cy3 (Biorad, CA, USA) and goat-anti mouse-Alexa (Biorad, CA, USA) were used. (c)

Monolayer of keratinocytes containing melanocyte dendrites (red dots, arrow head) and melanocyte cell bodies (arrow). The inserted image (yellow box) clearly

demonstrates the association between the melanocyte dendrites and the keratinocytes. (d) A bluntly terminated melanocyte was identified on the lower surface. (e) Analysis

of the affect of conditioned medium on stimulating primary melanocyte migration. Primary melanocytes seeded on the lower surface of the Millipore membrane migrated

towards the keratinocyte conditioned medium added to the upper surface. (f–h) Scanning electron microscopy images of primary melanocytes cultured on Millipore

membrane. (f) M9602 melanocytes on the lower surface of the membrane with part of its dendrite having already passed through the membrane pores (arrow). (g)

Protrusions off the surface of the dendrite into the membrane surface (arrows). (h) Tips of the dendrites protruding out of the membrane (yellow boxes). (a) 100 mm; (b)

50 mm. (c–e) 20 mm; (f) 10 mm; (g) 2 mm and (h) 1 mm. Images were viewed using the Zeiss Axiovert 200 M fluorescent microscope and captured using the Zeiss AxioCam HR

Monochrome camera with associated Axiovision software (versions 4.5 and 4.7). SEM samples were prepared according to instructions outlined by the membrane

manufacturers (Millipore, MA, USA) and viewed using a S440 scanning electron microscope (Leo, Leica).

Letters to the Editor / Journal of Dermatological Science 66 (2012) 160–168 161

Fig. 2. Proposed model for mode of melanocyte migration. Upon stimulation by migratory cues e.g. SCF/c-KIT signalling (a), the melanocyte dendrite extends into the spaces

between the keratinocytes (b). The dendrite functions as an anchor by adhering to the keratinocytes via various adhesion molecules e.g. E-cadherin (c). Anchorage of the

melanocyte dendrite to the keratinocytes allows for the melanocytes to be pulled through the small spaces between the keratinocytes (d). Once the entire cell has migrated to

its destination, new intercellular connections can be established and pigment production and transfer can resume (e).

Letters to the Editor / Journal of Dermatological Science 66 (2012) 160–168162

Using the novel results presented here, we propose a possiblemodel that may describe the route of melanocyte migration(Fig. 2). We propose that upon stimulation by keratinocyte factors,melanocytes adopt an elongated and bipolar morphology. Thepolarised melanocytes extend their dendrites at the cell front intointercellular spaces between the keratinocytes. These dendritesand tiny protrusions on the dendrites aid in attachment to thekeratinocytes for anchorage. The cell body and malleable nucleusare then pulled through and enter between the keratinocytes. Oncethe melanocyte has reached its destination, it becomes moredendritic and re-establishes connections possibly by upregulatingE-cadherin, and other adhesion molecules. This stable interactionbetween the melanocyte and surrounding keratinocytes inducesthe production of keratinocyte-derived factors and adhesionbetween the two cell types that upregulates melanogenesis andhence repigmentation of the skin.

Acknowledgements

The authors would like to thank Professor Dorothy Bennett forkindly contributing the Hermes 4a cells and Dr Nico Smit for the

M9602 melanocytes. Confocal images were captured with the helpof Dr Elizabeth van der Merwe. Scanning electron micrographimages were captured with the help of Dr Miranda Waldron. Thisresearch was funded by the National Research Foundation (SHK),Medical Research Council (LMD) and the University ResearchCouncil at the University of Cape Town (DK).

References

[1] Aubin-Houzelstein G, Bernex F, Elbaz C, Panthier JJ. Survival of patchworkmelanoblasts is dependent upon their number in the hair follicle at the end ofembryogenesis. Dev Biol 1998;198:266–76.

[2] Harris ML, Hall R, Erickson CA. Directing pathfinding along the dorsolateralpath – the role of EDNRB2 and EphB2 in overcoming inhibition. Development2008;135:4113–22.

[3] Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, FusenigNE. Normal keratinization in a spontaneously immortalized aneuploid humankeratinocyte cell line. J Cell Biol 1988;106:761–71.

[4] Gray-Schopfer VC, Cheong SC, Chong H, Chow J, Moss T, Abdel-Malek ZA, et al.Cellular senescence in naevi and immortalisation in melanoma: a role for p16?Br J Cancer 2006;95:496–505.

[5] Wenczl E, Van der Schans GP, Roza L, Kolb RM, Timmerman AJ, Smit NPM, et al.(Pheo)melanin photosensitizes UVA-induced DNA damage in cultured humanmelanocytes. J Invest Dermatol 1998;111:678.

Letters to the Editor / Journal of Dermatological Science 66 (2012) 160–168 163

[6] Wehrle-Haller B, Weston JA. Altered cell-surface targeting of stem cell factorcauses loss of melanocyte precursors in Steel17H mutant mice. Dev Biol1999;210:71–86.

[7] Kunisada T, Yoshida H, Yamazaki H, Miyamoto A, Hemmi H, Nishimura E, et al.Transgene expression of steel factor in the basal layer of epidermis promotessurvival, proliferation, differentiation and migration of melanocyte precursors.Development 1998;125:2915–23.

[8] Cozzani E, Cacciapuoti M, Parodi A. Adhesion molecules in keratinocyte. ClinDermatol 2001;19:544–50.

[9] Zhang R, Zhu W, Xia M, Feng Y. Morphology of cultured human epidermalmelanocytes observed by atomic force microscopy. Pigment Cell Res 2004;17:62–5.

[10] Pajerowski JD, Dahl KN, Zhong FL, Sammak PJ, Discher DE. Physical plasticity ofthe nucleus in stem cell differentiation. Proc Natl Acad Sci U S A 2007;104:15619–24.

Letter to the Editor

Fig. 1. (a) Immunostaining of DKK1 in non-lesional and lesional skin in a 20 year-old male

old female patient. Lesional dermis shows higher number of DKK1-positive cells i

bars = 200 mm. (c) Mean expression grade of DKK1 in non-lesional and lesional skins in i

between non-lesional and lesional skins (*p = 0.018). (e) The relative ratio of beta-cate

Dheshnie Keswell, Lester M. Davids*, Susan H. KidsonUniversity of Cape Town, Cape Town, South Africa

*Corresponding author at: University of Cape Town,Faculty of Health Sciences, Anzio Road, Cape Town 7925,South Africa.

Tel.: +27 21 4066787; fax: +27 21 4066228E-mail address: Lester.Davids@uct.ac.za (L.M. Davids)

23 June 2011

doi:10.1016/j.jdermsci.2012.01.005

DKK1 is highly expressed in the dermis of vitiligo lesion: Is thereassociation between DKK1 and vitiligo?

Dickkopf1 (DKK1), which is secreted by fibroblasts is aninhibitor of the Wnt signaling [1]. Recently, it has been reportedthat DKK1 is highly expressed in palmoplantar fibroblasts at themRNA and protein levels and is responsible for thickened andhypopigmented palmoplantar epidermis [2,3]. DKK1 inhibits thefunction and proliferation of melanocytes by suppressing the Wnt/beta-catenin/Microphthalmia-associated transcription factor(MITF)-signaling pathway [4]. In this study, to confirm ourhypothesis which DKK1 could be involved in the pathogenesis

of vitiligo as one of dermal factor, immunohistochemistry and real-time RT-PCR for DKK1, beta-catenin and PAR-2 were performedusing vitiligo lesional and non-lesional skin specimens.

Fifteen patients (M:F = 1:2) with vitiligo vulgaris were enrolledin this study. This study was approved by the Institutional ReviewBoard of Yonsei University Severance Hospital. Four-mm punchbiopsy was taken at the lesional skins and sites at least 2 cm awayfrom stable lesional areas in each patient. For immunohistochem-istry, rabbit polyclonal anti-human PAR-2 (1:100 dilution; SantaCruz, CA, USA) and rabbit polyclonal anti-human DKK1 (1:200dilution; Abcam, Cambridge, UK) were used. Color reaction wasperformed with diaminobenzidine. For immunofluorescence

patient. (b) Immunostaining of DKK1 in non-lesional and lesional skin in an 80 year-

n lesional skins than in non-lesional skins. Original magnification 100�. Scale

mmunohistochemistry (*p = 0.007). (d) The relative ratio of DKK1 mRNA expression

nin mRNA expression between non-lesional and lesional skins (*p = 0.043).