melanosome capping of keratinocytes in pigmented reconstructed epidermis – effect of ultraviolet...

PIGMENT CELL RES 13: 458–466. 2000 Copyright © Pigment Cell Res 2000ISSN 0893-5785Printed in Ireland—all rights reser6ed

Original Research Article

Melanosome Capping of Keratinocytes in Pigmented ReconstructedEpidermis – Effect of Ultraviolet Radiation and3-Isobutyl-1-Methyl-Xanthine on Melanogenesis

SUSAN GIBBS, SEEMA MURLI, GERRIT DE BOER, AAT MULDER, A. MIEKE MOMMAAS and MARIA PONEC

Department of Dermatology, Leiden Uni6ersity Medical Centre, The Netherlands*Address reprint requests to Susan Gibbs, Ph.D., Department of Dermatology, Leiden Uni6ersity Medical Centre, Wassenaarseweg 72, Bld3, Syl6ius Laboratory, 2333 AL Leiden, The Netherlands. E-mail: [email protected]

Received 12 August 2000; in final form 2 September 2000

crease in pigmentation was entirely due to the increase inReconstructed pigmented epidermis was established by co-melanocyte activity as observed by increased dendrite forma-seeding autologous melanocytes and keratinocytes onto a

dermal substrate and culturing for up to 6 weeks at the tion, melanin production and transport to the keratinocytesair–liquid interface. Inspection of the tissue architecture re- and was not due to an increase in melanocyte proliferation. In

contrast, after UV irradiation, the increase in pigmentationvealed that melanocytes are regularly interspersed only in thebasal layer and transfer melanosomes to the keratinocytes. was also accompanied with an increase in melanocyte prolifer-We report for the first time, the in vitro formation of supranu- ation as well as an increase in melanocyte activity.clear melanin caps above the keratinocyte nuclei. The forma- In conclusion, we describe the establishment of pigmented

reconstructed epidermis with autologous keratinocytes andtion and abundance of these melanin caps could be enhancedmelanocytes that can be kept in culture for a period of at leastby pigment modifiers such as ultraviolet light and 3-isobutyl-1-

methyl-xanthine (IBMX). In untreated cultures, the capping 6 weeks. The complete program of melanogenesis occurs:melanosome synthesis, melanosome transport to kerati-was observed in the spinous layers after 6 weeks of culture,

whereas after irradiation or supplementation of the culture nocytes, supranuclear capping of keratinocyte nuclei and tan-ning of the epidermis. This enables sustained application ofmedium with IBMX, the capping occurred already in the basalpigment stimulators over a prolonged period of time and alsolayer 2 weeks after initiation of the stimulus.repeated application of pigment stimulators to be studied.In this study, we show that IBMX and ultraviolet irradia-

tion stimulate pigmentation via different mechanisms. AfterKey words: Melanocyte, Keratinocyte, Autologous, Co-culturesupplementation of the culture medium with IBMX the in-

result that the healed area is often hyper- or hypo-pigmented.

For these reasons, science and industry are now demand-ing models that closely resemble living skin as not allresearch can be performed in vivo on human volunteers.Many models have been proposed varying in their complex-ity such as epidermal cell cultures grown in plastic culturedishes (4–6) and organ cultures (7). However, cells grownon plastic are not spatially related to one another as they are

INTRODUCTIONThe main function of the epidermal melanocyte is to pro-duce melanin and transport it to neighbouring keratinocyteswhere it protects the cell against possible assault fromdamaging radiation. This process of melanogenesis involvesmany complex regulatory mechanisms (1, 2). Defects inmelanogenesis are clearly apparent in pigmentary disorderssuch as vitiligo where normal pigmentation of the skin isimpaired. Furthermore, during wound healing, a high de-gree of variability in repigmentation occurs (3) with the

Abbre6iations – BL, basal layer; IBMX, 3-isobutyl-1-methyl-xanthine; SB,suprabasal; UV, ultraviolet

Pigment Cell Res. 13, 2000458

in the epidermis so that the normal functional propertieswhich are derived from an ordered morphology are limited.Organ cultures provide the complexity and organisationtypical of the in vivo situation. However, these cultures havean extremely limited life span of only a few days. Animalmodels have also been proposed (8). The problem withanimal models is that the morphology and many of the cellregulatory mechanisms differ between epidermal cells ofanimal and human origin. In order to overcome theseproblems, various pigmented skin equivalent models havebeen developed of entirely human origin (9–12). In thesemodels, the co-culture of keratinocytes and melanocytes isgrown at the air–liquid interface and all nutrients reach theliving cell layers by diffusion from the culture medium viathe dermal substrate. However, most of these skin modelshave a limited life span, which prevents the study of long-term effects of pigmentation modifiers on melanogenesis andalso prevents the study of the effects of repeated application.Furthermore, little is known about the main protectivefunction of melanin – the induction of supranuclear cappingof keratinocyte nuclei (13). Skin equivalent models havebeen used in our laboratory to investigate the regulation ofepidermal proliferation and differentiation (14–16), epider-mal lipogenesis (17, 18), barrier function (19), irritation (20),

wound healing (21) and several aspects of melanoma devel-opment (22).

In this communication, we describe the establishment ofpigmented reconstructed epidermis, which can be kept inculture for at least 6 weeks. The melanocytes maintain theirability to function as pigment suppliers to keratinocytes andrespond to external physiological stimuli.

MATERIALS AND METHODSCell Culture

Cultures of normal human epidermal cells (obtained fromhealthy patients undergoing surgical corrections: skin typesII or III) were established essentially as described earlier (23,24). Briefly, the epidermis was trypsinised in 0.25% trypsinand the single cell suspension of keratinocytes andmelanocytes was co-cultured in DMEM/Hams F12 3:1(Greiner, Alphen a/d Rijn, Netherlands) supplemented with5% Hyclone bovine serum, 1 mM hydrocortisone, 1 mMisoproterenol, 0.1 mM insulin. De-epidermised dermis wasalso prepared as described earlier (25). Human skin equiva-lents were generated as follows: secondary cultures of adulthuman keratinocytes and melanocytes were seeded into ametal ring with an area of 0.8 cm2 placed on de-epidermiseddermis (2 cm2). The cultures were grown submerged in amedium consisting of a mixture of 3:1 DMEM and Ham’sF12 medium supplemented with 5% Hyclone bovine serum,1 mM hydrocortisone, 1 mM isoproterenol, 0.1 mM insulin,1.0×10−5 M L-carnitine, 1.0×10−2 M L-serine, 1 mMdl-a-tocopherol-acetate and enriched with a lipid supple-ment as described by Boyce and Williams (26) (containing2.5×10−5 M palmitic acid, 1.5×10−5 M linoleic acid,0.7×10−5 M arachidonic acid, and 2.4×10−5 M BSA[essential fatty acid free, as carrier]). After 2 days, theconcentration of serum in the medium was decreased to 1%.After an additional 2 days, 5 ng/ml KGF was added to themedium. Three days thereafter, cultures were lifted to theair–liquid interface and the same culture medium was used,except that serum was omitted and 50 mg/ml ascorbic acidwas added (17). In some experiments IBMX was also addedto the medium when the cultures became air-exposed. Alladditives were purchased from Sigma (St. Louis, MO). Themedium was refreshed three times per week and all experi-ments were performed in triplicate.

UV Irradiation

Irradiation was performed using a UV solar simulator(Schoeffel Instrument Div. Of Kratos, Inc.), containing 95%UVA, 5% UVB and 0% UVC. After 2 weeks of air-expo-sure, cultures were irradiated in phenol red free Hank’sbalanced salt solution (Gibco BRL, Gaithersburg, MD,USA) on ice. Cultures were irradiated three times a week for2 weeks with 700 mJ/cm2 of total UV (equivalent to 1 MEDfor skin type II/III). The output from the lamp was 0.6mW/cm2 and the duration of irradiation was 20 min. Adigital thermometer with K type thermocouple (AldrichChemical Company Inc., Milwaukee, USA) was used tocontrol that the temperature of the reconstructed epidermisdid not rise above 10°C during irradiation. Cultures wereharvested 3 days after the last irradiation.

Fig. 1. An autologous co-culture of keratinocytes and melanocyteswas established under submerged culture conditions. Keratinocytesare shown as round or polygonal cells growing in clones andmelanocytes are shown as individual dendritic cells. The culture isP=0, 5 days after plating.

Pigment Cell Res. 13, 2000 459

Fig. 2. Melanosome capping ofkeratinocyte nuclei in reconstructedpigmented epidermis. Transmissionelectron micrographs show pigmentedreconstructed epidermis cultured for 2 (A),4 (B) and 6 (C) weeks at the air–liquidinterface. Columnar shaped keratinocytesin the BL of a 6 week air-exposed cultureare shown in D. Small arrowheads:melanosomes in keratinocytes; largearrowheads: melanosomes cappingkeratinocyte nuclei; small arrows:dendrites containing melanosomes; largearrows: melanocytes. Scale bars, 10 mm.

Morphology and Immunohistochemistry

Cultures were harvested and washed in PBS, fixed in4% formaldehyde and processed for conventional paraffinembedment. For morphological observations, 5-mmsections were cut, deparaffinised and rehydrated and thenstained with haematoxylin and eosin. In order to identifymelanocytes, immunohistochemical analysis of mela-noma associated antigen (100/7) (clone NKI/beteb pur-chased from Sanbio, Uden, The Netherlands) was per-formed on 5-mm frozen sections (antibody was diluted1:20). The avidin biotin peroxidase complex methodwas used essentially as described by the suppliers(Streptavidin-biotinylated horseradish peroxidase com-plex [Amersham Life Science, RPN 1051, Uppsala, Swe-den]).

Estimation of the Melanocyte-Keratinocyte Ratio in the BasalLayer

The proportion of melanocytes present in the culture wasassessed by determination of the ratio of NKI/BTEB stainedcells to the total number of basal cells. In each section, threedifferent regions containing a minimum of 100 basal cellswere counted at 200×magnification and a mean value wasdetermined. This value was determined for three indepen-dent experiments, each of which was performed in duplicate.The resulting value is expressed as mean with standard errorof the mean (SEM) in the results.

Colorimetric Determination

The extent of pigmentation was quantified using a colorime-ter (Minolta Chroma Meter CR-300 using the Probe Holder


CR-A12 and the data processor DP-301 [Minolta CameraBenelux BV, Maarssen, Netherlands]) and measuring theL�a�b� colour space as described by Bessou et al. (27). Inthis colour space, L� represents luminance (L�=0, absoluteblack; L�=100, absolute white – thus values are inverselyproportional to pigmentation) and a� and b� are chromatic-ity coordinates (+a� is the red direction and +b� is theyellow direction). The absolute colour difference betweentreated and non-treated cultures can be expressed as a singlenumerical value, DE�ab. DE�ab is defined by the followingequation:

DE�ab=(DL�)2+ (Da�)2+ (Db�)2.

Electron Microscopy

Specimens were processed for electron microscopy as previ-ously described (19). Briefly, cultures were fixed in 2%paraformaldehyde/2% glutaraldehyde and then post-fixed in1% osmium tetroxide. After fixation, the specimens weredehydrated in graded ethanol up to 70% and embedding inEpon 812. Ultrathin sections were stained with uranyl ace-tate and lead hydroxide.

ResultsAutologous Keratinocyte and Melanocyte Culture

An autologous co-culture of keratinocytes and melanocyteswas established under submerged culture conditions (Fig. 1).Keratinocytes can be identified as round or polygonal cellsgrowing in clones and melanocytes can be seen as individualdendritic cells often making contact with neighbouringkeratinocytes.

Tissue Architecture of Pigmented Skin Equivalents

Reconstructed pigmented epidermis was established by co-seeding autologous melanocytes and keratinocytes onto adermal substrate and culturing it for up to 6 weeks at theair–liquid interface (Fig. 2). Melanocytes can be recognised

as dendritic cells, with a lighter coloured cytoplasm contain-ing many melanosomes. In 2-week-old cultures, themelanocytes are evenly distributed throughout the basal celllayer forming close contacts with the surrounding kerati-nocytes (Fig. 2A). No melanocytes are found in SB layers.After 4 weeks of culture, melanosomes are distributedevenly within the keratinocytes of the lower epidermal layers(Fig. 2B). After 6 weeks of culture, the melanosomes arecapping the keratinocyte nuclei in the SB cell layers (Fig.2C). It should be noted that in 4- and 6-week air-exposedcultures BL keratinocytes have a columnar shape very simi-lar to that found in vivo, in contrast to 2-week-old cultureswhere the BL cells are more rounded (Fig. 2D).

Using an antibody directed against melanoma, associatedantigen melanocytes have been detected in the BL of thereconstructed epidermis. In 2-week air-exposed cultures,6.691.1% of the cells present in the BL are melanocytes(Fig. 3). This percentage is comparable to that found innative epidermis (4.492.0%). The relative number ofmelanocytes present in the reconstructed epidermis does notsignificantly change during an 8-week culture period. After 8weeks culture, the number of melanocytes in the basal layeris 7.294.67%.

IBMX Increases Pigmentation by Stimulating MelanocyteActivity Rather than Melanocyte Proliferation in SkinEquivalents

Supplementation of the culture medium with IBMX resultsin a marked increase in pigmentation (Fig. 4A vs. B). Themaximum increase in pigmentation was observed at IBMXconcentration of 0.05 mM: DE=13.595.2 (Fig. 5A) andslightly decreases when the concentration was increased to0.2 mM (DE=10.191.7). IBMX added at 0.4 mM exertstoxic effects (data not shown). The increase in absolutecolour difference (DE) can be attributed to a decrease inluminescence (L�) (which corresponds to an increase indarkness): control cultures have an L� value of 68.792.7compared to cultures supplemented with 0.05 mM IBMXwhich have an L� value of 55.998.1 (Fig. 5B). Increasingthe concentration of IBMX in the culture medium above0.05 mM results in an increase in L� value. A slight butsignificant increase in a� is observed when the IBMX con-centration in the medium is increased to 0.2 mM (control=5.691.9; 0.2 mM IBMX=10.091.5) indicating that thecolour is changing in the red direction (Fig. 5C), whereas nochange in b� is observed (Fig. 5D).

Morphological analysis of the cultures reveals that IBMXstimulates pigmentation by stimulating melanocyte activity.Supplementation with IBMX stimulates melanocyte den-drite formation (Fig. 4C vs. D): dendrites can be seenextending into the fourth SB cell layer compared to un-treated cultures where dendrites rarely extend as far as thesecond SB cell layer. Melanosome transfer to the kerati-nocytes is stimulated and melanosomes can be seen cappingthe keratinocyte nuclei in the basal layer already after 2weeks of air-exposure compared to 6 weeks in untreatedcultures (compare Fig. 2 to Fig. 4E and F).

In order to determine whether the increase in pigmenta-tion observed after addition of IBMX is also due to an

Fig. 3. The relative number of melanocytes present remains con-stant over an 8-week culture period. Reconstructed epidermis wasgrown for 2, 4, 6 or 8 weeks at the air–liquid interface. Thepercentage of melanocytes present was determined immunohisto-chemically from the number of NKI/BTEB positively stained cellspresent in the BL as compared to the number of negatively stainedBL keratinocytes (see Materials and Methods). The values indicatedare the means and SEM of three independent experiments eachperformed in duplicate.


Fig. 4. IBMX induces melanosome cap-ping of keratinocyte nuclei in BL. Cul-tures were grown at the air–liquidinterface for 2 weeks in the absence (A,C and E) or presence (B, D and F) of0.05 mM IBMX. Upper panels (A andB) show stereo photographs of the cul-tures at the time of harvesting; panels Cand D (magnification 800× ) and panelsE and F (magnification 2000× ) showhaematoxylin and eosin staining of 5-mmsections.

increase in the number of melanocytes, the percentage ofmelanocytes in the BL was determined (Fig. 6). Increasingthe concentration of IBMX in the culture medium from 0 to0.2 mM IBMX did not result in a significant change in thenumber of melanocytes in the BL (control: 6.792.8%, 0.05mM IBMX=8.191.4%; 0.2 mM IBMX=7.593.0%.).

Solar Simulator Irradiation Increases Pigmentation byStimulating Melanocyte Proliferation as well as MelanocyteActivity in Skin Equivalents

To examine the effects of UV irradiation on pigmentation,cultures were first grown under air-exposed conditions for aperiod of 2 weeks to allow the development of a competentbarrier function. The cultures were then irradiated three

times a week (700 mJ/cm2 total UV) for a period of 2 weeks(thus a total of six irradiations). Cultures were harvested 3days after the last irradiation. UV irradiation resulted in amarked increase in pigmentation of the cultures (Fig. 7A vs.B). The increase in pigmentation only occurred within theinitial seeding area of the cultures – the region of the culturethat had expanded outside this area remained unpigmentedin both controls and irradiated cultures. Furthermore, thetissue architecture of the expanded region of the irradiatedculture showed signs of severe burning due to the presenceof an immature stratum corneum (data not shown). Theregion of the culture within the initial seeding area where thestratum corneum had developed a fully competent barrierfunction at the time of irradiation did not show signs ofburning (Fig. 7D vs. F). The absolute colour difference


Fig. 5. IBMX results in an increase in absolute colour difference (DE) which can be attributed to a decrease in luminescence (L�). The extentof pigmentation after supplementation of the culture medium with IBMX was quantified using a colorimeter and by measuring the L�a�b�colour space as described in Materials and Methods. Cultures were grown in the absence or presence of IBMX for 4 weeks. DE representsthe absolute colour difference (A); L� represents luminance (B); a� and b� represent chromaticity coordinates (C and D, respectively).

within the initial seeding area (DE=7.090.2; Fig. 8A) canbe attributed to a decrease in luminescence of the cultures(unirradiated: L�=67.193.7; irradiated: L�=60.492.7)(Fig. 8B) and is not due to changes in the chromaticitycoordinates a� and b (Fig. 8C and D).

As with supplementation of the culture medium withIBMX, UV irradiation stimulated dendrite formation (Fig.7C vs. D) and also melanosome transport and capping ofthe keratinocyte nuclei in the basal layer (Fig. 7E vs. F).Furthermore an increase in melanocyte proliferation wasobserved: the percentage of melanocytes in the BL of non-ir-radiated cultures is 9.591.3% whereas in irradiated culturesit is 15.591.5% (Fig. 9).

DISCUSSIONIn this study we show that during autologous co-culture ofmelanocytes and keratinocytes at the air–liquid interfaceover a period of 6 weeks, melanocytes maintain their abilityto function as pigment suppliers to keratinocytes and torespond to external physiological stimuli. The ratio ofmelanocytes to keratinocytes remains constant during theentire culture period and is similar to that found in nativeskin suggesting that cell division is regulated by paracrineinteractions between the two cell types. Similar observationshave also been made by both De Luca et al. (28) andStaiano-Coico et al. (6) in melanocyte–keratinocyte co-cul-tures grown for three subpassages under submerged cultureconditions. However, Staiano-Coico et al. found that during

an initial culture period of 7 days the total number ofmelanocytes in epidermal cultures decreased to 10% of thenumber present in normal skin and then the keratinocyte tomelanocyte ratio was preserved during further primary cul-ture and passaging. De Luca did not make such a compari-son to the in vivo situation. Scott and Haake (29), bycomparing human foetal and neonatal skin equivalents,showed that keratinocytes regulate melanocyte proliferationand the major growth factor involved in this process isprobably basic fibroblast growth factor (30).

Fig. 6. IBMX does not alter the relative number of melanocytespresent. The percentage of melanocytes in the BL was determinedafter supplementation of the culture medium with IBMX for 4weeks. Melanocytes were detected immunohistochemically usingNKI/bteb antibody as described in Materials and Methods. Thevalues indicated are the means and SEM of three independentexperiments each performed in duplicate.


Fig. 7. UV irradiation inducesmelanosome capping of keratinocytenuclei in the BL. Cultures wereirradiated with 700 mJ/cm2 of UVderived from a solar simulator asdescribed in Materials and Methods.Unirradiated cultures are represented inA), C) and E) and irradiated culturesare represented in B), D) and F).Upper panels (A and B) show stereophotographs of the cultures at the timeof harvesting. Panels C and D(magnification 800× ) and panels Eand F (magnification 2000× ) showhaematoxylin and eosin staining of5-mm sections.

Inspection of the tissue architecture revealed that in pig-mented reconstructed epidermis cultured at the air–liquidinterface over a 6-week period the melanocytes are regularlyinterspersed in the BL and transfer melanosomes to kerati-nocytes. Similar to normal skin of type II/III, nomelanosomes are found in the stratum corneum. We reportfor the first time the in vitro formation of supranuclearmelanin caps above the keratinocyte nuclei. The formationand abundance of these melanin caps could be furtherenhanced by pigment modifiers such as UV light andIBMX. In untreated cultures, the capping can be observedin the spinous layers after 6 weeks of air-exposed culture,whereas after irradiation or supplementation of the culturemedium with IBMX, the capping occurs already in the basallayer 2 weeks after the initiation of the stimulus. Theproliferating cell population is present in the BL of theepidermis and it has been shown that supranuclear melanincaps reduce DNA damage induced by UV radiation (13).Therefore, we can conclude that the in vitro reconstructed

pigmented epidermis is able to respond to pigment stimula-tors in such a way as to protect the epidermis againstpossible assault from UV irradiation. The fact that otherinvestigators have not observed supranuclear melanin cap-ping of keratinocytes in the absence (31–33) or presence ofIBMX (27) or after UV irradiation (11, 12) could be due tothe limited duration of their experiments or due to the factthat pigmented epidermis was not reconstructed with au-tologous melanocytes and keratinocytes.

In this study, we show that IBMX and UV irradiationstimulate pigmentation via different mechanisms. After sup-plementation of the culture medium with IBMX, the in-crease in pigmentation was entirely due to the increase inmelanocyte activity as observed by increased dendrite for-mation, melanin production and transport to the kerati-nocytes and is not due to an increase in melanocyteproliferation. In contrast, after UV irradiation, the increasein pigmentation was also accompanied with an increase inmelanocyte proliferation as well as an increase in


melanocyte activity. Our finding that UV radiation increasesmelanocyte proliferation and dendricity is in accordancewith that of other laboratories despite a different radiationsource and application – a higher dose (UVB only) onconsecutive days (34, 35). To ensure the full recovery of thekeratinocytes from DNA damage caused by UV irradiation,we have chosen to irradiate three times per week for 2consecutive weeks with a low physiological dose of UVAand UVB. This approach was based on the findings fromclinical studies that have shown that more frequent irradia-

tion results in accumulation of DNA damage (De Winter etal., submitted). In addition, heat alone (42°C) can inducemost of the effects observed after UV irradiation such as anincreased number of Dopa positive cells, increased dendric-ity, and larger cell bodies (36). Therefore, during all irradia-tions (and sham irradiations) the cultures were kept on iceand the temperature of the epidermis was regularly checkedwith a digital thermometer.

In conclusion, in the present study we describe the estab-lishment of pigmented reconstructed epidermis with au-tologous keratinocytes and melanocytes that can be kept inculture for a period of at least 6 weeks. The completeprogram of melanogenesis occurs: melanosome synthesis,melanosome transport to keratinocytes, supranuclear cap-ping of keratinocyte nuclei and tanning of the epidermis.This enables sustained application of pigment stimulatorsover a prolonged period of time and also repeated applica-tion of pigment stimulators or UV radiation to be studied.

Acknowledgements – The authors would like to thank JohannaKempenaar and Hans van der Meulen for their skilful technicalassistance. Henkel KGaA Research Technology, Dusseldorf, Ger-many, partially financially supported this study.

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melanosome capping of keratinocytes in pigmented reconstructed epidermis – effect of ultraviolet...

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