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Chimeric Human Epidermal Reconstructs to Study the Role of Melanocytes and Keratinocytes in Pigmentation and Photoprotection

1998; Elsevier BV; Volume: 111; Issue: 6 Linguagem: Inglês

10.1046/j.1523-1747.1998.00405.x

1523-1747

S. Bessou‐Touya, Catherine Pain, Alain Taïeb, Mauro Picardo, Vittoria Maresca, Jean‐Étienne Surlève‐Bazeille,

Biochemical Analysis and Sensing Techniques

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. 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. catalase superoxide dismutase 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., 1979Fitzpatrick T.B. Szabò G. Seiji M. Quevedo W.C. Biology of the melanin pigmentary system.in: Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.W. Austin K.F. 2nd edn. Dermatology in General Medicine. McGraw-Hill, New York1979: 131-163Google Scholar). Cellular interactions between keratinocytes and melanocytes are important because keratinocytes act as paracrine effectors for melanocytic homeostasis (Halaban et al., 1988Halaban R. Langdon R. Birchall N. et al.Basic fibroblast growth factor from human keratinocytes is a natural mitogens for melanocytes.J Cell Biol. 1988; 107: 1611-1619Crossref PubMed Scopus (404) Google Scholar;Yaar and Gilchrest, 1991Yaar M. Gilchrest B.A. Human melanocyte growth and differentiation: a decade of new data.J Cell Biol. 1991; 115: 821-828Crossref PubMed Scopus (165) Google Scholar). 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 byFitzpatrick and Breathnach, 1963Fitzpatrick T.B. Breathnach A.S. Das epidermale Melanin-Einheit System.Dermatol Wochschr. 1963; 147: 481-489PubMed Google Scholar. 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, 1995Thody A.J. Epidermal melanocytes: Their regulation and role in skin pigmentation.Eur J Dermatol. 1995; 5: 558-565Google Scholar). 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, 1988Fitzpatrick T.B. The validity and practicality of sun-reactive skin type I through VI.Arch Dermatol. 1988; 124: 869-871Crossref PubMed Scopus (2674) Google Scholar). 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., 1997Bessou S. Gauthier Y. Surlève-Bazeille J.E. Pain C. Taïeb A. Epidermal reconstructs in vitiligo: an extrinsic factor is needed to trigger the disease.Br J Dermatol. 1997; 137: 890-897Crossref Scopus (20) Google Scholar). The peroxidation of the cell membranes may be the basis for conformational changes triggered by UV energy absorption, inducing signaling for melanogenesis and other photoprotective mechanisms (Rosette and Karin, 1996Rosette C. Karin M. Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factor and cytokine receptors.Science. 1996; 274: 1194-1197Crossref PubMed Scopus (926) Google Scholar). Using autologous reconstructed epidermis with keratinocytes and melanocytes plus UVB irradiation, we have already adequately reproduced tanning of various skin phototypesex vivo (Bessou et al., 1995Bessou S. Surlève-Bazeille J.E. Sorbier E. Taïeb A. Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB.Pigm Cell Res. 1995; 8: 241-249Crossref PubMed Scopus (59) Google Scholar,Bessou et al., 1996Bessou S. Surlève-Bazeille J.E. Pain C. Donatien P. Taïeb A. Ex-vivo study of skin phototypes.J Invest Dermatol. 1996; 107: 684-688Abstract Full Text PDF Scopus (42) Google Scholar). 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., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymatic and non-enzymatic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994 a; 102: 122-124Abstract Full Text PDF PubMed Google Scholar;Applegate and Frenk, 1995Applegate L.N. Frenk E. Cellular defense mechanism of the skin against oxidant stress and in particular UVA radiation.Eur J Dermatol. 1995; 5: 97-103Crossref Google Scholar), 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., 1996Picardo M. Grammatico P. Roccella F. Roccella M. Grandinetti M. Del Porto G. Passi S. Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma.J Invest Dermatol. 1996; 107: 322-326Crossref PubMed Scopus (94) Google Scholar). 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 toFitzpatrick, 1988Fitzpatrick T.B. The validity and practicality of sun-reactive skin type I through VI.Arch Dermatol. 1988; 124: 869-871Crossref PubMed Scopus (2674) Google Scholar from I to VI. According to a technique previously described (Bessou et al., 1995Bessou S. Surlève-Bazeille J.E. Sorbier E. Taïeb A. Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB.Pigm Cell Res. 1995; 8: 241-249Crossref PubMed Scopus (59) Google Scholar,Bessou et al., 1996Bessou S. Surlève-Bazeille J.E. Pain C. Donatien P. Taïeb A. Ex-vivo study of skin phototypes.J Invest Dermatol. 1996; 107: 684-688Abstract Full Text PDF Scopus (42) Google Scholar,Bessou et al., 1997Bessou S. Gauthier Y. Surlève-Bazeille J.E. Pain C. Taïeb A. Epidermal reconstructs in vitiligo: an extrinsic factor is needed to trigger the disease.Br J Dermatol. 1997; 137: 890-897Crossref Scopus (20) Google Scholar), 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, 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 2×105 cell per cm2 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 ofPruniéras et al., 1979Pruniéras M. Régnier M. Schlotterer M. Nouveau procédé de culture des cellules épidermiques humaines sur derme homologue ou hétérologue: préparation de greffons recombinés.Ann Chir Plast. 1979; 24: 357-362PubMed Google Scholar. 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 cm2 for 3 d, using a computerized irradiation programme and a Biotronic lamp (Vilbert Lourmat, Marne la Vallée, France). 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. Reconstructs were fixed in 10% formalin and embedded in paraffin. Melanocytes and melanin were observed in 5 μm sections after Fontana Masson staining (Gabe, 1968Gabe M. Techniques Histologiques. Paris, Masson1968Google Scholar). This technique was performed according toStaricco and Pinkus, 1957Staricco R.J. Pinkus H. Quantitative and qualitative data on the pigment cells of adult human epidermis.J Invest Dermatol. 1957; 28: 33-45Abstract Full Text PDF PubMed Scopus (183) Google Scholar. Briefly, reconstructs were separated from the dermis after a 2 h incubation at 37°C in a 2N bromide sodium solution. Reconstructs were then incubated in a 0.1% DOPA solution for 4 h at 37°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. 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). 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 fromRosenthal et al., 1973Rosenthal M.H. Kreider J.W. Shiman R. Quantitative assay of melanin in melanoma cells in culture and in tumors.Ann Biochem. 1973; 56: 91-96Crossref Scopus (54) Google Scholar andLogan and Weatherhead, 1978Logan A. Weatherhead B. Pelage color cycles and hair follicle tyrosinase activity in the siberian hamster.J Invest Dermatol. 1978; 71: 295-298Crossref PubMed Scopus (29) Google Scholar. The protein concentration was determined according toBradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205256) Google Scholar. 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., 1991Passi S. De Morrone C. Luca C. Ippolito F.L. Blood levels of vitamin E, polyunsaturated fatty acids of phospholipids, lipoperoxides and glutathione peroxidase in patients affected with seborrheic dermatitis.J Dermatol Sci. 1991; 2: 171-178Abstract Full Text PDF PubMed Scopus (55) Google Scholar). 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, 1985Clairborne A. Catalase activity.in: Greenwald R.A. Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton1985: 283-284Google Scholar), and SOD activity was evaluated as the inhibition of pyrogallol oxidation (Roth and Gilbert, 1987Roth E.F. Gilbert H.S. The pyrogallol assay for superoxide dismutase: absence of glutathione artefact.Anal Biochem. 1987; 137: 50-53Crossref Scopus (81) Google Scholar) and the data reported as U protein per mg. Vitamin E was analyzed by gas chromatography–mass spectrometry (Picardo et al., 1996Picardo M. Grammatico P. Roccella F. Roccella M. Grandinetti M. Del Porto G. Passi S. Imbalance in the antioxidant pool in melanoma cells and normal melanocytes from patients with melanoma.J Invest Dermatol. 1996; 107: 322-326Crossref PubMed Scopus (94) Google Scholar) and reported as ng per mg protein. 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 (Figure 1a). Heterologous reconstructs with type V melanocytes and Negroid or other Caucasoid keratinocytes produced a phototype similar to type V (Figure 1). 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 (Figure 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).Table 1UVB irradiation increases the number of DOPA positive melanocytes and melanin content in heterologous reconstructs (NIRE, non-irradiated reconstructed epidermis; IRE, irradiated reconstructed epidermis)Heterologous reconstructionsNIRE (melanocyte count)aCounts represent the mean ± SD of melanocytes per field. One field corresponds to 0.2 mm2. 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 ( Bessouet al. 1995).IRE (melanocyte count)aCounts represent the mean ± SD of melanocytes per field. One field corresponds to 0.2 mm2. 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 ( Bessouet al. 1995).NIRE (μg melanin per mg protein)IRE (μg melanin per mg protein)Melanocytes VI + keratinocytes III68.0 ± 7.492.2 ± 4.1cStudent's t test p < 0.001.14.1528.25Melanocytes VI + keratinocytes V88.25 ± 5.4128.0 ± 6.8cStudent's t test p < 0.001.21.7840.30Melanocytes V + keratinocytes III102.7 ± 27.7141.2 ± 21.4bStudent's t test p < 0.01.13.9927.87Melanocytes III + keratinocytes V53.75 ± 10.476.0 ± 9.3bStudent's t test p < 0.01.5.4512.38a Counts represent the mean ± SD of melanocytes per field. One field corresponds to 0.2 mm2. 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 ( Bessouet al. 1995).b Student's t test p < 0.01.c Student's t test p < 0.001. Open table in a new tab 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 (Figure 2a). Melanosomal transfer was increased after UVB irradiation (Figure 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 (Figure 2c). After UVB irradiation, those features were more pronounced (Figure 2d). The antioxidant pattern detected in each reconstructed epidermis before and 72 h after the last UVB treatment is reported inTable II. The sensitivity of cells to oxidative stress is better addressed by the fine balance between SOD and Cat activities (Shindo and Hashimoto, 1995Shindo Y. Hashimoto T. Antioxidant defence mechanism of the skin against UV irradiation: study of the role of catalase using acatalasemia fibroblasts.Arch Dermatol Res. 1995; 287: 747-753Crossref PubMed Scopus (20) Google Scholar), because if the activity of the two enzymes is disproportionate, hydrogen peroxide can accumulate within the cells (Halliwell and Gutteridge, 1989Halliwell B. Gutteridge Jmc Free Radicals in Biology and Medicine. Oxford University Press, New York1989Google Scholar). 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 were more susceptible to peroxidative damage (Figure 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.Table 2Antioxidant enzymatic activities and vitamin E levels before and after irradiation in autologous and heterologous epidermal reconstructsaNIRE, 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.U Cat per mgU SOD per U Catng vitamin E per mg proteinNIREIRENIREIRENIREIREK II + M II124 ± 794.8 ± 5 **p < 0.005.0.116 ± 0.0190.14 ± 0.0039 *p < 0.05;15.4 ± 317.5 ± 2K IV + M II134 ± 4120 ± 5 *p < 0.05;0.067 ± 0.00810.083 ± 0.006 **p < 0.005.11.66 ± 211.65 ± 3K V + M II124 ± 670 ± 4 *p < 0.05;0.058 ± 0.00690.14 ± 0.005 **p < 0.005.24.7 ± 433.28 ± 2K IV + M IV212 ± 5196 ± 7 *p < 0.05;0.037 ± 0.00780.046 ± 0.002318.7 ± 326 ± 3K II + M VI124 ± 6111.2 ± 5 *p < 0.05;0.077 ± 0.00920.048 ± 0.0031 *p < 0.05;19 ± 420.7 ± 3K IV + M VI186 ± 4174 ± 4 *p < 0.05;0.053 ± 0.0050.058 ± 0.01352.6 ± 759.44 ± 6a 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 < 0.005. Open table in a new tab 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 (Figure 4).Table 3Fatty acids determination before irradiation in epidermal reconstructsaNon-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.C 16: 0C 18: 0C 20: 0C 22: 0C 18: 1C 18: 2C 20: 4% unsK II + M II15.12 ± 365.36 ± 51.66 ± 0.31.86 ± 0.48.84 ± 0.56.06 ± 0.81.07 ± 0.0515.9 ± 2.16K IV + M II38.9 ± 432.96 ± 34.04 ± 0.73.19 ± 0.615.1 ± 3.4.87 ± 0.50.87 ± 0.820.8 ± 1.64K V + M II29.4 ± 3.46 ± 44.44 ± 0.62.37 ± 0.411.6 ± 2.5.6 ± 0.60.47 ± 0.0517.6 ± 2.02K IV + M IV59.4 ± 622.17 ± 32.46 ± 0.20.99 ± 0.211.93 ± 3.2.77 ± 0.20.17 ± 0.0314.8 ± 1.47K II + M VI70.9 ± 721.51 ± 21.99 ± 0.31.29 ± 0.33.92 ± 0.60.31 ± 0.080.03 ± 0.0064.2 ± 0.43K IV + M VI22.3 ± 365.6 ± 61.02 ± 0.31.57 ± 0.35.9 ± 0.53.25 ± 0.090.26 ± 0.059.4 ± 0.8a 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. Open table in a new tab 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 (Figure 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 (Figure 6).Figure 6SOD/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 < 0. 05.View Large Image Figure ViewerDownload (PPT) 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 (Wolff and Klaus, 1972Wolff K. Klaus K. Phagocytosis of latex beads by epidermal keratinocytes in vivo.J Ultrastruct Res. 1972; 39: 262-280Crossref PubMed Scopus (66) Google Scholar), has been demonstrated to be the regulating factor of the mode of uptake (Wolf et al., 1974Wolf K. Jimbow K. Fitzpatrick T.B. Experimental pigment donation in vivo.J Ultrastruct Res. 1974; 47: 400-419Crossref Scopus (29) Google Scholar). 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., 1974Wolf K. Jimbow K. Fitzpatrick T.B. Experimental pigment donation in vivo.J Ultrastruct Res. 1974; 47: 400-419Crossref Scopus (29) Google Scholar). 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., 1996Bessou S. Surlève-Bazeille J.E. Pain C. Donatien P. Taïeb A. Ex-vivo study of skin phototypes.J Invest Dermatol. 1996; 107: 684-688Abstract Full Text PDF Scopus (42) Google Scholar). 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., 1996Bessou S. Surlève-Bazeille J.E. Pain C. Donatien P. Taïeb A. Ex-vivo study of skin phototypes.J Invest Dermatol. 1996; 107: 684-688Abstract Full Text PDF Scopus (42) Google Scholar) 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 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., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymatic and non-enzymatic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994 a; 102: 122-124Abstract Full Text PDF PubMed Google Scholar), 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., 1991Yohn J. Norris D. Yrastorsa D. Bruno I. Leff J. Hake S. Repine J. Disparate antioxidant enzyme activities in cultured cutaneous fibroblasts, keratinocytes and melanocytes.J Invest Dermatol. 1991; 97: 405-410Abstract Full Text PDF PubMed Google Scholar). 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., 1993Moisan A. Marquis I. Gaboriau F. Santus R. Dubertret L. Morlière P. Ultraviolet A-induced lipid peroxidation and antioxidant defence system in cultured human skin fibroblasts.J Invest Dermatol. 1993; 100: 692-698Crossref Scopus (108) Google Scholar). 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, bothin vivo andin vitro (Shindo et al., 1993Shindo Y. Witt E. Packer L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.J Invest Dermatol. 1993; 100: 260-264Abstract Full Text PDF PubMed Google Scholar;Applegate and Frenk, 1995Applegate L.N. Frenk E. Cellular defense mechanism of the skin against oxidant stress and in particular UVA radiation.Eur J Dermatol. 1995; 5: 97-103Crossref Google Scholar). Its activity can be affected by high peroxide concentrations and by visible light (Demopoulos et al., 1980Demopoulos H.B. Pietroingrao D.D. Flamm E.S. Seligman M.L. The possible role of free radical reaction in carcinogenesis.J Environ Pathol Toxicol. 1980; 3: 273-303PubMed Google Scholar;Halliwell and Gutteridge, 1989Halliwell B. Gutteridge Jmc Free Radicals in Biology and Medicine. Oxford University Press, New York1989Google Scholar). Asin vivo (Shindo et al., 1994bShindo Y. Witt E. Han D. Tzeng B. Aziz T. Nguyen T. Packer L. Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure.Photodermatol Photoimmunol Photomed. 1994 b; 10: 183-191PubMed Google Scholar), 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., 1994bShindo Y. Witt E. Han D. Tzeng B. Aziz T. Nguyen T. Packer L. Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure.Photodermatol Photoimmunol Photomed. 1994 b; 10: 183-191PubMed Google Scholar), as noted, for example, in DNA repair disease xeroderma pigmentosum (Vuillaume et al., 1992Vuillaume M. Daya-Grosjean L. Vincens P. et al.Striking differences in cellular catalase activity between two DNA repair-deficient diseases: xeroderma pigmentosum and trichothiodystrophy.Carcinogenesis. 1992; 13: 321-328Crossref PubMed Scopus (78) Google Scholar). 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 UV-induced lipoperoxidation (Quiec et al., 1995Quiec D. Mazière C. Santus R. et al.Polynsaturated fatty acid enrichment increase ultraviolet A induced lipid peroxidation in NCTC 2544 human keratinocytes.J Invest Dermatol. 1995; 104: 964-969Crossref Scopus (16) Google Scholar). 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., 1995Archambault M. Yaar M. Gilchrest A. Keratinocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation.J Invest Dermatol. 1995; 104: 859-867Crossref PubMed Scopus (98) Google Scholar). 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., 1995Valverde P. Healy E. Jackson I. Rees J.L. Thody A.T. Variants of the melanocyte-stimulating hormone receptor gene are associated with red hair skin in human.Nature Genet. 1995; 11: 328-330Crossref PubMed Scopus (795) Google Scholar,Valverde et al., 1996Valverde P. Healy E. Sikkins S. et al.The Asp84Glu variant of the melanocortin 1 receptor (MC1R) is associated with melanoma.Hum Mol Genet. 1996; 5: 1663-1666Crossref PubMed Scopus (271) Google Scholar). 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, or H2O2 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., 1991Picardo M. De Zompetta C. Luca C. et al.Role of skin surface lipids in UV-induced epidermal cell changes.Arch Dermatol Res. 1991; 283: 191-197Crossref PubMed Scopus (48) Google Scholar). 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 H2O2 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.