Differences in skin color between humans are related to

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1 Chimeric Human Epidermal Reconstructs to Study the Role of Melanocytes and Keratinocytes in Pigmentation and Photoprotection Sandrine Bessou-Touya, Mauro Picardo,* Vittoria Maresca,* Jean-Etienne Surlève-Bazeille, Catherine Pain, and Alain Taïeb Dermatology Laboratory, University Victor Ségalen Bordeaux II, France; *Pathophysiology Laboratory, San Gallicano Dermatology Institute, Rome, Italy; Cell Regulation and Defence Mechanisms Laboratory, University of Bordeaux I, France Chimeric epidermal reconstructs made with Negroid melanocytes and Caucasoid keratinocytes (or vice versa) were studied before and after UVB irradiation to understand the respective roles of these cells in tanning and photoprotection, especially lipoperoxidation and enzymatic defences against free radicals. Using this approach, we have confirmed overall the theory of the epidermal melanin unit. We have also shown that melanocytes of poorly tanning Caucasoids, which have a comparatively higher content of unsaturated fatty acids in their cell membrane, are more prone to the peroxidative effects of UV light, and that keratinocytes participate in photoprotection via phototype-dependent antioxidant enzyme activities, especially for catalase. Key words: catalase/epidermal lipids/melanin/superoxide dismutase. J Invest Dermatol 111: , 1998 Differences in skin color between humans are related to the number, size, type, distribution, and degradation of melanosomes and to tyrosinase activity (Fitzpatrick et al, 1979). Cellular interactions between keratinocytes and melanocytes are important because keratinocytes act as paracrine effectors for melanocytic homeostasis (Halaban et al, 1988; Yaar and Gilchrest, 1991). Cooperation between keratinocytes and melanocytes is already obvious as far as melanosomial donation at the ultrastructural level is concerned. One melanocyte should theoretically be able to interact with 36 keratinocytes in the epidermal melanin unit defined by Fitzpatrick and Breathnach (1963). This model supposes collaboration between melanocytes that produce melanosomes and keratinocytes that receive, disperse, and degrade them. The regulation of UV-mediated melanogenesis and the importance of melanins in photoprotection in the human epidermis are not yet fully understood. The activation of protein kinases, the production of diacylglycerol, and the secretion of mitogens and melanogenic factors from keratinocytes have been considered as major physiologic events following UV exposure (Thody, 1995). At the clinical level, skin phototypes (skin types) are classified according to the susceptibility of the skin to burn or to tan after sun exposure (Fitzpatrick, 1988). There is a coarse correlation between skin phototypes, eumelanogenesis, and photoprotection. Besides eumelanin contained in melanosomes, other antioxidant defences, especially enzymes such as superoxide dismutase (SOD), catalase (Cat), and glutathione peroxidase, are crucial to protect the epidermis from reactive oxygen species. Cellular interactions in antioxidant defences may pertain to some diseases with a loss of melanocytes, such as vitiligo (Bessou et al, 1997). The peroxidation of the cell membranes may be the basis for conformational changes Manuscript received October 8, 1997; revised August 3, 1998; accepted for publication August 9, Reprint requests to: Prof. A Taïeb, Unité de Dermatologie Pédiatrique, Hoˆpital Pellegrin-Enfants, Bordeaux, France. Abbreviations: Cat, catalase; SOD, superoxide dismutase. triggered by UV energy absorption, inducing signaling for melanogenesis and other photoprotective mechanisms (Rosette and Karin, 1996). Using autologous reconstructed epidermis with keratinocytes and melanocytes plus UVB irradiation, we have already adequately reproduced tanning of various skin phototypes ex vivo (Bessou et al, 1995, 1996). To investigate the epidermal melanin unit with this model, we have made heterologous chimeric epidermal reconstructs with keratinocytes and melanocytes of healthy donors of Caucasoid (II to V) and Negroid (VI) skin phototypes. For that purpose, we studied the reconstructs at the morphologic level, especially for melanosomial donation within the epidermis with respect to the original phototype of keratinocytes and melanocytes. To understand further the respective role of melanocytes and keratinocytes in photoprotection, we studied biochemical changes following UVB irradiation in non-irradiated and irradiated reconstructs. Among the possible antioxidants, we evaluated those known to be modified following acute and chronic UV irradiation (Shindo et al, 1994a; Applegate and Frenk, 1995), namely vitamin E (Vit E), SOD, and Cat activities. The fatty acid pattern of cell membrane phospholipids was evaluated as a target of free radical-mediated damage (Picardo et al, 1996). MATERIALS AND METHODS Sources of skin and skin phototype determination Skin samples were obtained from non-photoexposed skin from adult donors undergoing plastic surgery (breast reconstruction and abdominoplasty) and foreskins of children submitted to circumcision. The phototype was determined according to Fitzpatrick (1988) from I to VI. Epidermal reconstruction ex vivo According to a technique previously described (Bessou et al, 1995, 1996, 1997), chimeric combinations of keratinocytes and melanocytes of caucasoid and negroid skin phototypes were studied: keratinocytes II melanocytes VI, keratinocytes III melanocytes V, keratinocytes III melanocytes VI, keratinocytes IV melanocytes VI, keratinocytes V melanocytes III, keratinocytes V melanocytes VI, keratinocytes VI melanocytes II, keratinocytes VI melanocytes III, keratinocytes VI melanocytes V. In parallel experiments, two types of autologous reconstructs were studied: keratinocytes II melanocytes II, X/98/$10.50 Copyright 1998 by The Society for Investigative Dermatology, Inc. 1103

2 1104 BESSOU-TOUYA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY keratinocytes IV melanocytes IV. For each cellular combination a minimum of 12 reconstructs was studied. According to the number of reconstructs produced, morphology alone, or morphology plus biochemisty (melanin content and/or lipids and enzyme determinations) was performed. Briefly, keratinocytes and melanocytes cultured in MCDB 153 were seeded at cell per cm 2 into a stainless steel ring deposited on the surface of a dead de-epidermized dermis at a 1:20 melanocyte/keratinocyte ratio. Acellular killed dermis was prepared according to the technique of Pruniéras et al (1979). After cell adhesion culture chambers were removed and the system was covered with complete medium. After 72 h of culture, the system was lifted at the air liquid interface for 15 d with three media changes per week. The same batch of fetal calf serum was used for all cultures so that the undefined source of essential elements, fatty acids, and vitamins in the medium was identical for all specimens biochemically studied. Reconstructs were irradiated after 8 d at the air liquid interface at 312 nm, 0.15 J per cm 2 for 3 d, using a computerized irradiation programme and a Biotronic lamp (Vilbert Lourmat, Marne la Vallée, France). Morphologic studies Cultures were stopped after 15 d at the air liquid interface and reconstructs were prepared for histologic studies, DOPA reaction on split-thickness skin, and electron microscopy. Histology Reconstructs were fixed in 10% formalin and embedded in paraffin. Melanocytes and melanin were observed in 5 µm sections after Fontana Masson staining (Gabe, 1968). Dopa reaction This technique was performed according to Staricco and Pinkus (1957). Briefly, reconstructs were separated from the dermis after a2h incubation at 37 C in a 2N bromide sodium solution. Reconstructs were then incubated in a 0.1% DOPA solution for 4hat37 C. After incubation, reconstructs were fixed in 10% formol for 10 min and then mounted (dermal side up) in a glycerol/phosphate buffer for examination. The number of DOPA positive melanocytes was estimated by counting at least five fields at 100 magnification. Ultrastructure Biopsies were fixed in 2.5% glutaraldehyde buffered with 0.1 M cacodylate at ph 7.4 for 60 min at room temperature. Samples were post-fixed for 1 h in 2% osmium tetroxide in a cacodylate buffer and then dehydrated in a graded series of ethanol (5mn each) and embedded in epon. Sections were cut using a diamond knife with a Reichert ultramicrotome (Nussloch, Germany) and stained with uranyl acetate and lead citrate. Ultrathin sections were examined at 60 kv under a JEOL 100S electron microscope (Tokyo, Japan). Melanin quantitation The melanin content was evaluated after enzymatic digestion of the reconstructs with proteinase K at 45 for 48 h, extraction in chloroform-methanol (2:1), and spectrophotometric analysis at 436 nm using a technique modified from Rosenthal et al (1973) and Logan and Weatherhead (1978). The protein concentration was determined according to Bradford (1976). Fatty acid pattern of membrane phospholipids and antioxidant measurements The fatty acid pattern and antioxidant levels were studied before and after UVB irradiation in each series of reconstructs. All quantitations were done blindly on coded samples after 15 d of culture at the air liquid interface, and, for irradiated reconstructs, 3 d following the last irradiation. From two to four samples of each heterologous/autologous condition were analyzed and each determination was repeated twice. Reconstructed epidermis were separated from the dermis with trypsin-ethylenediamine tetraacetic acid for 24 h at 4 C. Epidermis was homogenized in phosphate-buffered saline, centrifuged at 800 g, and the protein concentration of the supernatant determined by Bio-Rad assay. All samples were diluted in phosphate-buffered saline at the same concentration. The fatty acid pattern of membrane phospholipids was trans-methylated from the purified phospholipid fraction, and the fatty acid methyl esters were analyzed by a combined gas chromatography mass spectrometry method (Passi et al, 1991). After time integration of the chromatogram and final processing of the peak areas, data were reported as the percentage of each fatty acid with respect to the total fatty acids analyzed. The degree of lipoperoxidation was evaluated as the modification of the percentage of unsaturated fatty acids in irradiated as against to nonirradiated samples. Cat activity was determined as the disappearance of hydrogen peroxide (Clairborne, 1985), and SOD activity was evaluated as the inhibition of pyrogallol oxidation (Roth and Gilbert, 1987) and the data reported as U protein per mg. Vitamin E was analyzed by gas chromatography mass spectrometry (Picardo et al, 1996) and reported as ng per mg protein. RESULTS Epidermal pigmentation is determined by the melanocyte phototype In all heterologous reconstructions, we obtained, macroscopically and microscopically, the phototype determined by melanocyte origin. Pigmentation increased progressively during the 1 wk irradiation time course. All heterologous reconstructions with Negroid melanocytes, independent of keratinocyte origin, resulted in a Negroid epidermis. Melanin transfer increased after UV irradiation (Fig 1a). Heterologous reconstructs with type V melanocytes and Negroid or other Caucasoid keratinocytes produced a phototype similar to type V (Fig 1b). Heterologous reconstructs containing type III melanocytes with Negroid or Caucasoid keratinocytes resulted in a phototype equivalent to phototype III: the reconstructs were lightly pigmented, and pigmentation was increased after UVB irradiation. Microscopically, the melanocytes were moderately pigmented and there was a melanin transfer up to the stratum corneum that increased after UV exposure (Fig 1c). With melanocytes originating from type III and V phototypes, significant differences in epidermal color were found after UVB irradiation. In all heterologous epidermal reconstructs UVB irradiation induced an increase of DOPA positive melanocytes and an increase in melanin content (Table I). To examine the size and transfer of melanosomes in heterologous reconstructs, an electron microscopy study was performed. In reconstructs containing type VI melanocytes, and irrespective of keratinocyte origin, isolated melanosomes were transferred up to the stratum corneum, a typical feature of Negroid epidermis (Fig 2a). Melanosomal transfer was increased after UVB irradiation (Fig 2b). In heterologous reconstructions with type II, III, or V melanocytes and Caucasoid or Negroid keratinocytes, melanosomal complexes and a few isolated melanosomes were transferred up to the stratum corneum, and, without irradiation, this transfer was greater with type V melanocytes than with type II or III melanocytes (Fig 2c). After UVB irradiation, those features were more pronounced (Fig 2d). The keratinocyte phototype influences epidermal antioxidant enzyme activities The antioxidant pattern detected in each reconstructed epidermis before and 72 h after the last UVB treatment is Figure 1. Correlation macroscopymicroscopy for chimeric reconstructs of various combined phototypes. For each part of the figure, the macroscopy before (NIRE) and after (IRE) irradiation is in part (A). An area of epidermis corresponds to 0.64 cm 2. The corresponding microscopy is shown below (B, NIRE; C, IRE); Fontana- Masson stain, 5 µm sections, original magnification 200. (a) Negroid melanocytes (VI) with Caucasoid keratinocytes type II; (b) melanocytes type V with Caucasoid type III keratinocytes; (c) melanocytes type III with Caucasoid type II keratinocytes. See comments in text.

3 VOL. 111, NO. 6 DECEMBER 1998 PIGMENTATION AND PHOTOPROTECTION EX VIVO 1105 Figure 2. Electron microscopy of heterologous reconstructed phototypes. (a) Isolated melanosomes in stratum corneum in melanocytes type VI/caucasoid keratinocytes type II reconstruction. (b) Same type of construct after UVB irradiation: augmentation of isolated melanosome transfer. (c) Melanocytes type III/keratinocytes type VI construct, after UVB irradiation: melanosomal complexes and isolated melanosomes (arrows) in the spinous layer. (d) Melanocytes type V/ keratinocytes type VI construct, after UVB irradiation: isolated melanosomes in the granular layer and stratum corneum. Scale bars: 4µm. Table I. UVB irradiation increases the number of DOPA positive melanocytes and melanin content in heterologous reconstructs (NIRE, non-irradiated reconstructed epidermis; IRE, irradiated reconstructed epidermis) Heterologous reconstructions NIRE IRE NIRE IRE (melanocyte count) a (melanocyte count) a (µg melanin per mg protein) (µg melanin per mg protein) Melanocytes VI keratinocytes III c Melanocytes VI keratinocytes V c Melanocytes V keratinocytes III b Melanocytes III keratinocytes V b a Counts represent the mean SD of melanocytes per field. One field corresponds to 0.2 mm 2. Due to the clustering of melanocytes after irradiation, an understimate of DOPA positive cells counted in split-irradiated reconstructs is probable. Melanin content was evaluated after enzymatic digestion of reconstructs and spectrophotometric analysis as previously reported (Bessou et al, 1995). b Student s t test p c Student s t test p Table II. Antioxidant enzymatic activities and vitamin E levels before and after irradiation in autologous and heterologous epidermal reconstructs a U Cat per mg U SOD per U Cat ng vitamin E per mg protein NIRE IRE NIRE IRE NIRE IRE KII M II ** * KIV M II * ** KV M II ** ** KIV M IV * KII M VI * * KIV M VI * a NIRE, non-irradiated reconstructed epidermis; IRE, irradiated reconstructed epidermis. Samples were irradiated with UVB as reported in Materials and Methods and analysed 3 d after the last irradiation. Enzymatic activities superoxide dismutase (SOD) and catalase (CAT) were determined by spectrophotometry and expressed as U protein per mg. Each result represents the mean SD of at least triplicate analyses. *p 0.05; **p reported in Table II. The sensitivity of cells to oxidative stress is better addressed by the fine balance between SOD and Cat activities (Shindo and Hashimoto, 1995), because if the activity of the two enzymes is disproportionate, hydrogen peroxide can accumulate within the cells (Halliwel and Gutteridge, 1989). Therefore this ratio has been evaluated in each sample. There was a correlation between the SOD/CAT ratio and the phototype of the keratinocytes. The highest ratios corresponded to the lowest keratinocyte phototypes, suggesting that these reconstructs

4 1106 BESSOU-TOUYA ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 3. SOD/CAT ratio correlates with keratinocyte phototype. Correlation between keratinocyte/melanocyte phototype and the ratio SOD/ Cat in non-irradiated autologous and heterologous reconstructs after 15 d of culture at the air liquid interface. The enzymatic activities were determined by a spectrophotometer in cell lysates and reported as U protein per mg. Each result represents the mean SD of at least triplicate analyses. Solid symbols, melanocytes; open symbols, keratinocytes. R 0.75; p with respect to keratinocyte phototype. R 0.32 with respect to melanocyte phototype Figure 4. Percent of unsaturated fatty acids correlates with melanocyte phototype in non-irradiated reconstructs. Correlation between melanocyte/keratinocyte phototype and the percentage of unsaturated fatty acids (C18:1 C18:2 C20:4/total fatty acids analyzed) in non-irradiated autologous and heterologous reconstructs after 1 d of culture at the air liquid interface. Each result represents the mean SD of at least triplicates analyses. Solid symbols, melanocytes; open symbols, keratinocytes. R 0.91; p with respect to melanocyte phototype; R 0.5 with respect to keratinocyte phototype. Figure 5. UVB-induced lipoperoxidation of unsaturated fatty acids varies according to cell phototype in reconstructs. Percentage of unsaturated fatty acids with respect to the total fatty acids analyzed in reconstructs before and 3 d after last UVB irradiation. Each result represents the mean SD of at least triplicate analyses. Figure 6. SOD/CAT ratio correlates with degree of unsaturated fatty acid peroxidation. Correlation between the modification of the ratio SOD/ Cat and the modification of the percentage of unsaturated fatty acids (C18:1 C18:2 C20:4/total fatty acids) 3 d after the last irradiation. R 0.53; p were more susceptible to peroxidative damage (Fig 3). Prior to or 72 h following UV treatment, no significant difference in SOD activity and Vit E concentration were observed in all the samples (Table II). On the contrary, a significant decrease in Cat activity (p 0.05; p 0.005) was detected in all heterologous and autologous reconstructs 72 h after the last irradiation. The melanocyte phototype determines epidermal unsaturated lipid content and influences UVB-induced epidermal lipid peroxidation The non-irradiated samples evidenced variations in the percentage of unsaturated fatty acids among the different reconstructs (Table III) and a correlation was found with the melanocyte phototype (Fig 4). The degree of lipoperoxidation following irradiation was determined as the difference in the percentage of unsaturated fatty acids analyzed, i.e., C18: 1n9, C18: 2 n6, C20: 4 n6, with respect to the total fatty acids analyzed (Fig 5). In reconstructs made with Negroid melanocytes, there was an increase in the unsaturated fatty acids content 72 h following the last irradiation, whereas the SOD/Cat ratio remained dependent upon the keratinocyte phototype. Overall, the alteration of the SOD/Cat ratio was correlated with the degree of unsaturated fatty acid peroxidation (Fig 6). DISCUSSION Pigment transfer in mammalian skin is considered to be a heterophagic process involving melanosomes. The size of the melanosomes, like experimentally that of latex beads (Wolf and Konrad, 1972), has been demonstrated to be the regulating factor of the mode of uptake (Wolf et al, 1974). As a result, large melanosomes, as found in Negroid melanocytes, should be ingested singly, whereas for smaller Caucasoid, Mongoloid, and American Indian melanosomes collective uptake should take place (Wolf et al, 1974). Those principles of pigment donation were respected in the reconstructs studied ultrastructurally in this study. Basically, our findings concerning pigmentation in chimeric human epidermal reconstructs parallel those noted previously in autologous reconstructs of the corresponding melanocytic phototype (Bessou et al, 1996). Caucasoid melanosomes were transferred as melanosomal complexes and Negroid melanosomes in isolation. UV stimulated donation but not its intrinsic pattern. All these experiments indicate that epidermal pigmentation is under strict melanocytic control, as expected in the theory of the epidermal melanin unit. Overall, the size of the melanosomes determined their subsequent cellular handling. The presence of a few isolated melanosomes in keratinocytes has already been noted in Caucasoid autologous reconstructs (Bessou et al, 1996) and thus should not be regarded as an exception to this rule. This finding was more marked even without irradiation with donor melanocytes of the higher phototypes (V) that produced melanosomes

5 VOL. 111, NO. 6 DECEMBER 1998 PIGMENTATION AND PHOTOPROTECTION EX VIVO 1107 Table III. Fatty acids determination before irradiation in epidermal reconstructs a C16:0 C18:0 C20:0 C22:0 C18:1 C18:2 C20:4 %uns KII M II KIV M II KV M II KIV M IV KII M VI KIV M VI a Non-irradiated epidermal reconstructs were analysed for the fatty acid pattern of membrane phospholipids by GC-MS as reported in Materials and Methods. The values are expressed as a percentage of each fatty acid respect to the total fatty acids analysed, and represent the mean SD of three determinations. of Caucasoid size, suggesting that single dispersion can occur even without UV irradiation as a physiologic event in culture. The determinations of antioxidant enzymatic and non-enzymatic activities in the reconstructs were not significantly different from those previously reported in normal human skin (Shindo et al, 1994a), further supporting the reliability of our model. It has been demonstrated in isolated cell cultures that the antioxidant enzyme concentrations of keratinocytes are higher than those of melanocytes (Yohn et al, 1991). Therefore, in our system, keratinocytes may be expected to contribute most to the enzymatic activities. Interestingly, a significant correlation between the SOD/Cat ratio and the keratinocytic phototype was found, with an increased ratio for lower phototypes, suggesting that these cells are more susceptible to peroxidative damage. The SOD/ Cat ratio has been evidenced to be a suitable marker of cell sensitivity to UV exposure (Moisan et al, 1993). Moreover, antioxidant systems interact in a complex fashion, so that changes in activity or concentration in one component can affect the whole system. Cat, however, is the enzyme most susceptible to the effect of UV irradiation in the epidermis and its recovery is slower than that of other enzymes, both in vivo and in vitro (Shindo et al, 1993; Applegate and Frenk, 1995). Its activity can be affected by high peroxide concentrations and by visible light (Demopoulos et al, 1980; Halliwell and Gutteridge, 1989). As in vivo (Shindo et al, 1994b), the recovery capacity of the reconstructs was suggested because SOD activities and vitamin E concentations were similar to those observed in non-irradiated samples 3 d after the last irradiation. Conversely, the significant decrease in Cat activity in all heterogeneous and autologous reconstructs confirms that Cat activity is altered in UV-induced free radical-mediated damage (Shindo et al, 1994b), as noted, for example, in DNA repair disease xeroderma pigmentosum (Vuillaume et al, 1992). The relationship between the melanocytic phototype and the percentage of unsaturated fatty acids prior to irradiation suggests that the latter essentially arise from the presence of melanocytes and that melanocytes of poorly tanning individuals are more prone to UV light peroxidative damage. It has already been evidenced that the enrichment of cell membranes with polyunsaturated fatty acids increases UVinduced lipoperoxidation (Quiec et al, 1995). Ex vivo, the presence of keratinocytes appears to protect melanocytes from UV-induced damage and death. This phenomenon has been linked to the production of growth factors and cytokines by keratinocytes (Archambault et al, 1995). Our data provide a complementary explanation for the photoprotective effect of keratinocytes. After UV irradiation, the alteration of the SOD/Cat ratio was correlated to the level of peroxidation of polyunsaturated fatty acids, suggesting a link between a mostly keratinocyte-dependent trait (SOD-Cat equipment) and a mostly melanocytic target (membrane unsaturated lipids). Thus, the higher the protective capability due to antioxidant enzymes, the lower the peroxidation of the unsaturated components of cell membranes. Considering that the generation of cutaneous UV-induced free radicals has been associated with the occurrence of skin cancer, our data suggest that epidermal cells from fair skinned individuals with low tanning ability may be more susceptible to free radical-mediated damage due to (i) a keratinocyte-associated genetic background for antioxidant enzyme activities, and (ii) a higher content in polyunsaturated fatty acids in melanocyte membranes. This particular susceptibility to oxidative stress can probably be interpreted as an intrinsic part of the clinical concept of phototype, independent of the production of pheomelanin, the pigment found in Caucasians with pale skin, red or light hair, freckling, and an inability to tan, which is associated with a genetic variant of the melanocortin receptor MCR-1 and an increased risk of melanoma (Valverde et al, 1995, 1996). Based on our data, a speculative link between melanogenesis and antioxidant enzymatic defences in photoprotection could be theorized as follows: antioxidant enzyme activities, especially Cat, which are mostly dependent upon the keratinocyte phototype, protect epidermal cells to maintain a continuous flow of pigment in the epidermal melanin unit. Following UV irradiation, the intracellular reactive oxygen species generated must be eliminated. If the level of antioxidant enzymes is constitutionally low, as in the keratinocytes of low phototype individuals, hydrogen peroxide accumulates and has to be scavenged by melanin provided by the melanocytes. Depending on the melanin concentration in the keratinocytes, hydrogen peroxide can reach melanocytic membranes to produce lipoperoxidative damage. Thus, the response of the melanocytes may depend on several factors produced by the keratinocytes, such as TNFα, orh 2 O 2 production, and may be regulated by the phototype-linked percentage of polyunsaturated fatty acids in cell membranes. It has been shown that low concentrations of reactive oxygen species and lipoperoxides can stimulate cell proliferation and melanin synthesis in the melanocytes, whereas higher concentrations are toxic (Picardo et al, 1991). This complex interaction could explain a differential effect of reactive oxygen species according to the melanocytic phototype. In particular, the increase in polyunsaturated fatty acids in reconstructs made with Negroid melanocytes may be linked to the stimulatory properties of H 2 O 2 on melanocytic function. Overall, our work confirms the theory of the epidermal melanin unit for pigmentation, in which the melanocytes are the key actors for melanin production and distribution; however, keratinocytes are functionally important in photoprotection, due to their antioxidant content that interacts in a complex fashion with neighboring melanocytes. This study was supported by a grant of the Conseil Régional d Aquitaine to AT and a grant of the Italian Ministry of Health to MP. We thank the plastic surgery and pediatric surgery departments for providing skin specimens. REFERENCES Applegate LN, Frenk E: Cellular defense mechanism of the skin against oxidant stress and in particular UVA radiation. Eur J Dermatol 5:97 103, 1995 Archambault M, Yaar M, Gilchrest A: Keratinocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation. 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