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Keratinocytes Play a Role in Regulating Distribution Patterns of Recipient Melanosomes In Vitro

2001; Elsevier BV; Volume: 117; Issue: 2 Linguagem: Inglês

10.1046/j.0022-202x.2001.01411.x

1523-1747

Ljiljana Minwalla, Yang Zhao, Raymond E. Boissy, I. Caroline Le Poole, R. Randall Wickett,

Circadian rhythm and melatonin

Melanosomes in keratinocytes of Black skin are larger and distributed individually whereas those within keratinocytes of Caucasian skin are smaller and distributed in clusters. This disparity contributes to differences in skin pigmentation and photoprotection, but the control of these innate distribution patterns is poorly understood. To investigate this process, cocultures were established using melanocytes and keratinocytes derived from different racial backgrounds and were examined by electron microscopy. Melanosomes transferred to keratinocytes were categorized as individual or in various clusters. Melanosome size was also determined for individual and clustered melanosomes. Results indicate that, in our model system, melanosomes in keratinocytes from different racial backgrounds show a combination of clustered and individual melanosomes. When keratinocytes from dark skin were cocultured with melanocytes from (i) dark skin or (ii) light skin, however, recipient melanosomes were individual versus clustered in (i) 77% vs 23% and (ii) 64% vs 36%, respectively. In contrast, when keratinocytes from light skin were cocultured with melanocytes from (iii) dark skin or (iv) light skin, recipient melanosomes were individual versus clustered in (iii) 34% vs 66% and (iv) 39% vs 61%, respectively. These results indicate that recipient melanosomes, regardless of origin, are predominantly distributed individually by keratinocytes from dark skin, and in membrane-bound clusters by those from light skin. There were also differences in melanosome size from dark or light donor melanocytes. Melanosome size was not related to whether the melanosomes were distributed individually or clustered, however, in cocultures. These results suggest that regulatory factor(s) within the keratinocyte determine recipient melanosome distribution patterns. Melanosomes in keratinocytes of Black skin are larger and distributed individually whereas those within keratinocytes of Caucasian skin are smaller and distributed in clusters. This disparity contributes to differences in skin pigmentation and photoprotection, but the control of these innate distribution patterns is poorly understood. To investigate this process, cocultures were established using melanocytes and keratinocytes derived from different racial backgrounds and were examined by electron microscopy. Melanosomes transferred to keratinocytes were categorized as individual or in various clusters. Melanosome size was also determined for individual and clustered melanosomes. Results indicate that, in our model system, melanosomes in keratinocytes from different racial backgrounds show a combination of clustered and individual melanosomes. When keratinocytes from dark skin were cocultured with melanocytes from (i) dark skin or (ii) light skin, however, recipient melanosomes were individual versus clustered in (i) 77% vs 23% and (ii) 64% vs 36%, respectively. In contrast, when keratinocytes from light skin were cocultured with melanocytes from (iii) dark skin or (iv) light skin, recipient melanosomes were individual versus clustered in (iii) 34% vs 66% and (iv) 39% vs 61%, respectively. These results indicate that recipient melanosomes, regardless of origin, are predominantly distributed individually by keratinocytes from dark skin, and in membrane-bound clusters by those from light skin. There were also differences in melanosome size from dark or light donor melanocytes. Melanosome size was not related to whether the melanosomes were distributed individually or clustered, however, in cocultures. These results suggest that regulatory factor(s) within the keratinocyte determine recipient melanosome distribution patterns. Melanosomes transferred within keratinocytes of dark skin individuals are predominantly distributed as individual organelles throughout the cytoplasm, with an increased density apically over the nucleus, of both sun-exposed and nonexposed skin. In contrast, three to eight melanosomes clustered into membrane-bound groups predominate within keratinocytes of Caucasians (Szabo, 1969Szabo G. Racial differences in the fate of melanosomes in human epidermis.Nature. 1969; 222: 1081Crossref PubMed Scopus (189) Google Scholar;Konrad and Wolff, 1973Konrad K. Wolff K. Hyperpigmentation, melanosome size, and distribution patterns of melanosomes.Arch Dermatol. 1973; 107: 853-860Crossref PubMed Scopus (64) Google Scholar;Jimbow et al., 1998Jimbow K. Sugiyama S. Melanosomal translocation and transfer.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J. The Pigmentary System Physiology and Pathophysiology. Oxford University Press, New York, Oxford1998: 107-114Google Scholar). These clusters also accumulate in the supranuclear position in light skin keratinocytes. The distribution patterns of melanosomes within keratinocytes are one determining factor in skin color. The distribution of single melanosomes increases light absorption exhibited physiologically by a darker skin color. More importantly, this distribution plays an important role in photoprotection. In fact, it is widely agreed that melanin and the distribution of melanosomes in the epidermis are the most important factors in the protection of human skin from the detrimental effects of ultraviolet (UV) light. Much of this protection is offered by the fact that melanin can absorb effectively throughout the visible and UV electromagnetic spectrum. Various specific mechanisms have been proposed for melanin-mediated photoprotection. They include the filtering and attenuation of impinging radiation by scattering, absorption, and subsequent dissipation (as heat), absorption accompanied by redox reactions, and absorption accompanied by electron transfer processes (Kollias et al., 1991Kollias N. Sayre R.M. Zeise L. Chedekel M.R. New trends in photobiology (Invited review).J Photochem Photobiol. 1991; 9: 135-160Crossref PubMed Scopus (337) Google Scholar). Regardless of mechanisms by which melanin attenuates the effects of UV radiation, the melanosomes shield the keratinocytes' nuclear DNA from these damaging rays by forming a protective "cap" over the nuclei. It was postulated that clustered melanosomes in Caucasian keratinocytes redistribute into single entities within the keratinocytes upon UV exposure to more efficiently shield the nuclei (Toda et al., 1972Toda K. Pathak M.A. Parrish J.A. Fitzpatrick T.B. Quevedo Jr, W.C. Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light.Nature. 1972; 236: 143-145Crossref Scopus (115) Google Scholar;Jimbow et al., 1991Jimbow K. Fitzpatrick T.B. Wick M.M. Biochemistry and physiology of melanin pigmentation.in: Goldsmith S.A. 2nd edn. Physiology, Biochemistry, and Molecular Biology of the Skin. Vol. II. Oxford University Press, New York, Oxford1991: 873-909Google Scholar). Thus, the constitutive and facultative distribution of melanosomes within keratinocytes is of primary concern with regard to the damaging effects of radiation, and this differs widely between different skin colors. The mechanism regulating the constitutive distribution of melanosomes within keratinocytes has not been elucidated, however. Whereas the mechanism of melanosome transfer from melanocytes to keratinocytes is not fully understood, some studies indicate that the mode of transfer of melanosomes, either singly or as clusters, is a melanocytic-dependent process and is responsible for constitutive differences in distribution patterns of melanosomes. Recently,Bessou-Touya et al., 1998Bessou-Touya S. Picardo M. Maresca V. Surleve-Bazeille J. Pain C. Taieb A. Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection.J Inv Dermatol. 1998; 111: 1103-1108Crossref PubMed Scopus (56) Google Scholar established heterologous melanocyte-keratinocyte reconstructions and examined them macroscopically and microscopically. Their conclusions suggested that epidermal pigmentation is determined by the melanocyte phototype. Other studies indicate a correlation between melanosome size and the distribution pattern of melanosomes within secondary lysosomes of keratinocytes. Melanosomes larger than 1 µm are singly distributed, whereas those smaller than 1 µm in long axis are aggregated to form melanosome complexes (Szabo, 1969Szabo G. Racial differences in the fate of melanosomes in human epidermis.Nature. 1969; 222: 1081Crossref PubMed Scopus (189) Google Scholar;Toda et al., 1972Toda K. Pathak M.A. Parrish J.A. Fitzpatrick T.B. Quevedo Jr, W.C. Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light.Nature. 1972; 236: 143-145Crossref Scopus (115) Google Scholar;Wolff et al., 1974Wolff K. Jimbow K. Fitzpatrick T.B. Experimental pigment donation in vivo.J Ultrastruct Res. 1974; 47: 400-419Crossref PubMed Scopus (29) Google Scholar;Yamamoto and Bhawan, 1994Yamamoto O. Bhawan J. Three modes of melanosome transfers in Caucasian facial skin: hypothesis based on an ultrastructural study.Pigment Cell Res. 1994; 7: 158-169Crossref PubMed Scopus (69) Google Scholar;Jimbow et al., 1998Jimbow K. Sugiyama S. Melanosomal translocation and transfer.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J. The Pigmentary System Physiology and Pathophysiology. Oxford University Press, New York, Oxford1998: 107-114Google Scholar). These observations have led investigators to suggest that a melanosome-associated signal regulates its distribution pattern in the recipient keratinocyte (Wolff and Konrad, 1971Wolff K. Konrad K. Melanin pigmentation: an in vivo model for studies of melanosome kinetics within keratinocytes.Science. 1971; 174: 1034-1035Crossref PubMed Scopus (39) Google Scholar;Jimbow et al., 1998Jimbow K. Sugiyama S. Melanosomal translocation and transfer.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J. The Pigmentary System Physiology and Pathophysiology. Oxford University Press, New York, Oxford1998: 107-114Google Scholar). This hypothesis was tested and supported experimentally by observing phagocytosis by keratinocytes of either isolated melanosomes or latex beads of different particle sizes (Korn and Weisman, 1967Korn E.D. Weisman R.A. Phagocytosis of latex beads by acanthamoeba. II. Electron microscopic study of the initial events.J Cell Biol. 1967; 34: 219-227Crossref PubMed Scopus (107) Google Scholar;Wolff and Konrad, 1971Wolff K. Konrad K. Melanin pigmentation: an in vivo model for studies of melanosome kinetics within keratinocytes.Science. 1971; 174: 1034-1035Crossref PubMed Scopus (39) Google Scholar;Wolff and Konrad, 1972Wolff K. Konrad K. Phagocytosis of latex beads by epidermal keratinocytes in vivo.J Ultrastruc Res. 1972; 39: 262-280Crossref PubMed Scopus (66) Google Scholar). In contrast, there have been reports demonstrating that melanosome size does not correlate with their distribution pattern within keratinocytes. In some pigmentary disorders and after certain chemical treatments, the melanosome distribution in secondary lysosomes is not consistent with their size (Jimbow et al., 1998Jimbow K. Sugiyama S. Melanosomal translocation and transfer.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J. The Pigmentary System Physiology and Pathophysiology. Oxford University Press, New York, Oxford1998: 107-114Google Scholar). In Caucasians, for example, nitrogen mustard treatment of skin produces a condition in which almost all of the small melanosomes are singly distributed rather than forming melanosome complexes in keratinocytes as expected (Flaxman et al., 1973Flaxman B. Sosis A. Van Scott E. Changes in melanosome distribution in caucasoid skin following topical application of nitrogen mustard.J Invest Dermatol. 1973; 60: 321-326Crossref PubMed Scopus (41) Google Scholar). Because melanosome size has not been altered, some other controlling property might have changed. Furthermore,Bhawan et al., 1976Bhawan J. Purtilo D.T. Riordan J.A. Saxena V.K. Edelstein L. Giant and 'granular melanosomes' in Leopard syndrome. An ultrastructural study.J Cutan Pathol. 1976; 3: 207-216Crossref PubMed Scopus (43) Google Scholar conducted an ultrastructural study of pigmentary abnormalities including melanosome size, shape, and distribution in patients with Leopard syndrome. They reported that individual melanosomes smaller than 0.8 μm were found easily in keratinocytes. And, even though melanosome complexes were composed of small melanosomes, melanosomes larger than 0.35 μm were commonly found in complexes. In fact, they reported that even giant melanosomes were occasionally seen in melanosome complexes. The authors suggest that their studies indicate that, in the Leopard syndrome, distribution of melanosomes in keratinocytes is not dependent on either race or melanosome size, but probably on a defect at the genetic level. For our study, we utilized cocultures of mixed melanocytes and keratinocytes established from light and dark skinned individuals to assess the pattern of melanosome distribution within keratinocytes and correlate with cell donor type and melanosome size. Our results indicate that the recipient keratinocytes, rather than the size of the melanosome, dictates the ultimate distribution pattern of transferred pigment granules. Cultures of normal human melanocytes and keratinocytes were established from individual neonatal foreskins (from dark and light skin infants) that were obtained from the nursery of University Hospital after routine circumcision. Skins were incubated in 0.25% trypsin for 2 h at 37°C. The tissue was gently vortexed for 30 s to separate the dermis from the epidermal cell suspension. The dermis was removed and then the epidermal cells were pelleted by centrifugation and resuspended in either melanocyte or keratinocyte growth medium for seeding in 25 cm2 tissue culture flasks. Melanocytes were maintained in M154 basal medium (Cascade Biologicals, Portland, OR) supplemented with 4% heat-inactivated fetal bovine serum, 1% antibiotic/antimycotic solution (Gibco BRL, Grand Island, NY), 1 µg per ml transferrin, 1 µg per ml vitamin E, 5 µg per ml insulin, 0.6 ng per ml human recombinant basic fibroblast growth factor, 10-8 M α-melanocyte stimulating hormone, and 10-9 M endothelin-1. The growth medium for normal human keratinocyte cultures consisted of M154 basal medium (Cascade Biologicals) supplemented with human keratinocyte growth supplements (Cascade Biologicals) and 1% antibiotic/antimycotic solution (Gibco BRL). We have previously demonstrated that keratinocytes in culture no longer contain residual melanocytes by passage 1 (Minwalla et al., 2001Minwalla L. Zhao Y. Cornelius J. Babcock G.F. Wickett R.R. Le Poole I.C. Boissy R.E. Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system.Pigment Cell Res. 2001; 14: 185-194Crossref PubMed Scopus (62) Google Scholar). All of the above reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise stated. All cultures were maintained in a tissue culture incubator at 37°C with 5% CO2. Growth medium was routinely changed twice a week. For ultrastructure of the skin, a 3 mm diameter punch biopsy was obtained from the forearm of an African American and a Caucasian adult with informed consent and fixed with half-strength Karnovsky's fixative overnight at 4°C as previously described (Boissy et al., 1991Boissy R.E. Liu Y.Y. Medrano E.E. Nordlund J.J. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients.J Invest Dermatol. 1991; 97: 395-404Abstract Full Text PDF PubMed Google Scholar). The tissue was then cut into quarters and processed for routine electron microscopy as described below. For the ultrastructural study of cultured cells, primary melanocytes were cocultured for 4 d with primary keratinocytes at a ratio of 1:2, respectively, and maintained in 1:2 normal melanocyte growth medium to normal keratinocyte growth medium, respectively. Cells were seeded into one-well Laboratory-Tek chamber slides (Nunc, Naperville, IL). On the fifth day, adherent cells were processed for electron microscopy as previously described (Boissy et al., 1991Boissy R.E. Liu Y.Y. Medrano E.E. Nordlund J.J. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients.J Invest Dermatol. 1991; 97: 395-404Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were fixed in wells with half-strength Karnovsky's fixative in 0.2 M sodium cacodylate buffer at pH 7.2 for 30 min at room temperature. When using melanocytes derived from light skin, it was necessary to stain the melanosomes for improved visualization by incubation in l-dihydroxyphenylalanine (DOPA), which reacts with tyrosinase to yield a brown/black polymerous end product, both in situ and postfixation. For in situ DOPA treatment, 250 µM L-DOPA was added to the medium on days 1 and 2 for 5 h each day. For postfixation DOPA histochemistry, fixed cells were incubated in a 0.1% solution of L-DOPA twice for 2.5 h (Boissy et al., 1991Boissy R.E. Liu Y.Y. Medrano E.E. Nordlund J.J. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients.J Invest Dermatol. 1991; 97: 395-404Abstract Full Text PDF PubMed Google Scholar). All cells were then washed three times in buffer and treated with 1% osmium tetroxide containing 1.5% potassium ferrocyanide for 30 min. The cells were washed, stained en bloc with 0.5% uranyl acetate for 30 min, dehydrated, and embedded in Eponate 12. For one sample of each type of coculture, 10 areas of each coculture were cut out of the Epon cast, mounted on Epon pegs, and sectioned on an RMC MT 6000-XL ultramicrotome. Ultrathin sections were then stained with aqueous solutions of uranyl acetate (2%) and lead citrate (0.3%) for 15 min each, and viewed and photographed in a JEOL JEM-100CX transmission electron microscope. (All tissue processing supplies were purchased from Ted Pella, Tustin, CA.) Cocultures of six combinations of racially different melanocytes and keratinocytes (at passages 1–3) were established and processed for electron microscopy as described in the previous section. These groups consisted of (1) dark skin derived melanocytes cocultured with dark skin derived keratinocytes; (2) dark skin derived melanocytes cocultured with light skin derived keratinocytes; (3) light skin derived melanocytes cocultured with dark skin derived keratinocytes; (4) light skin derived melanocytes cocultured with light skin derived keratinocytes; (5) light skin and dark skin derived melanocytes combined at 1:1 and cocultured with dark skin derived keratinocytes; and (6) light skin and dark skin derived melanocytes combined at 1:1 and cocultured with light skin derived keratinocytes. For one sample of each of the cocultures, 10 areas were sectioned and 10 micrographs of keratinocytes or areas of keratinocytes from each section were obtained for analysis as described below. For melanosome distribution, micrographs of groups (1)–(4) (described above) were subsequently analyzed for number of melanosomes per keratinocyte within all keratinocytes in each field, and grouped as being either individually distributed or in membrane-bound clusters of two to three, four to six, or greater than six by two independent investigators who were blinded to the source of the micrograph. For melanosome size, melanosomes within keratinocytes were assessed for size (i.e., area) from groups (2), (4), (5), and (6) (described above) using a Zidas Image Analyzer and image analysis software (Carl Zeiss, Thornwood, NY). First, from each group, the area of transferred melanosomes (µm2) was determined for 200 random (indiscriminately measured individual and clustered) unclassified melanosomes within all melanocytes in each field. Second, the areas (µm2) for 100 individually distributed melanosomes and the areas (µm2) for 100 clustered melanosomes were determined within all keratinocytes in each field. Statistical analysis was performed to determine that (i) the distribution patterns observed in the coculture of dark skin derived melanocytes and dark skin derived keratinocytes were significantly different from those observed in the coculture of dark skin derived melanocytes and light skin derived keratinocytes, and (ii) the distribution patterns observed in the coculture of light skin derived melanocytes and dark skin derived keratinocytes were significantly different from those observed in the coculture of light skin derived melanocytes and light skin derived keratinocytes. The parameters of interest tested were the population mean and variance. A 100(1 - α)% confidence interval for the mean was constructed. The relevant test statistic was Z. To confirm an expected difference between the two investigative groups (individual means compared and clustered means compared for each type of melanocyte), the p-values for the Z statistic of the one-tailed test should be less than α, 0.05. All calculations were performed using Microsoft Excel 2000. Statistical parameters were calculated for individual, clustered, and random (indiscriminately chosen individual and clustered) populations for melanosomes in light skin derived keratinocytes cocultured with dark skin derived melanocytes (groups 2 and 4) and in light skin derived keratinocytes cocultured with a 1:1 mixture of dark and light skin derived melanocytes (groups 5 and 6), to test whether the sizes of melanosomes that were distributed individually or in clusters differed. The parameters of interest were the population mean and variance. A 100(1 - α)% confidence interval for the mean was constructed. The relevant test statistic was obtained by using a two-tailed paired t test. To statistically assess differences between the average size of individual and clustered melanosomes from each of dark and light skin derived melanocytes, the p-value for the t statistic should be less than or equal to α, 0.05. All calculations were performed using Microsoft Excel 2000. Figure 1 exhibits the typical differences in melanosome distribution patterns that exist between Black and Caucasian skin. Melanosomes within Black keratinocytes Figure 1a are distributed individually throughout the cytoplasm, being predominantly localized apically over the nucleus. In contrast, melanosomes within Caucasian keratinocytes Figure 1b are almost exclusively in membrane-bound clusters that are also predominantly localized over the nucleus. This is an important feature that correlates with differences in complexion coloration. When melanosomes are larger and more spread out, they can absorb more incoming light so that less is refracted. This gives skin a darker coloration. When the melanosomes are smaller and distributed in clusters, they absorb less light and more is refracted, thus giving skin a lighter coloration. In cell culture the differences in melanosome distribution between Black and Caucasian skin were not absolutely recapitulated. Keratinocytes used in experiments did not contain any residual melanosomes within them Figure 2c. In keratinocyte-melanocyte cocultures derived from either dark or light skins, keratinocytes contained recipient melanosomes that were both individual and clustered. This difference observed between in vitro and in vivo conditions might reflect differences in cell behavior due to culture conditions or, as the exact race of donor individuals could not be confirmed, it possibly might reflect racial differences. Keratinocytes in cocultures derived from dark skin and light skin, however, do appear to contain melanosomes that are predominantly individual and clustered, respectively Figure 2. Heterologous cocultures using melanocytes and keratinocytes derived from dark and light foreskins were established in all four possible permutations as described in Materials and Methods. Subsequently, melanosomes were assessed as being either individually distributed or in membrane-bound clusters of two to three, four to six, or greater than six by two independent investigators who were blinded to the heterologous combinations Table I. In dark skin derived keratinocytes cocultured with dark skin derived melanocytes, 77% of melanosomes were individually distributed and 23% were clustered; in dark skin derived keratinocytes cocultured with light skin derived melanocytes, 64% of melanosomes were individual and 36% were clustered. In contrast, in light skin derived keratinocytes cocultured with light skin derived melanocytes, 34% of melanosomes were individually distributed and 66% were clustered, and in light skin derived keratinocytes cocultured with dark skin derived melanocytes, 39% of melanosomes were individual and 61% were clustered. This clearly seems to indicate that, regardless of the donor melanocyte, dark skin derived keratinocytes will predominantly distribute recipient melanosomes individually, and light skin derived keratinocytes will predominantly distribute recipient melanosomes in membrane-bound clusters.Table IDistribution of melanosomes within keratinocytes from heterologous cocultures established from melanocytes and keratinocytes derived from dark or light foreskinaResults are given as percentages of total melanosomes counted. 1–10 = Values between these numerical groups exhibited p(Z) ≤ α= 0.05.Distribution (percentages)Clusters categorizedGroupDescription of cocultured groupIndividualClusters combined2–34–6> 61Dark melanocytes + dark keratinocytes7712333 5271892Dark melanocytes + light keratinocytes39161385874593Light melanocytes + dark keratinocytes642364562829104Light melanocytes + light keratinocytes34266426386110a Results are given as percentages of total melanosomes counted. 1–10 = Values between these numerical groups exhibited p(Z) ≤ α= 0.05. Open table in a new tab The sizes of individual and clustered melanosomes were measured as described in Materials and Methods. Briefly, the Zidas Image Analyzer and software allows the calculation of the area within a circled region using a stylus. Here the outer membrane of the melanosome defined that region. In the light skin derived keratinocytes cocultured with either dark or light skin derived melanocytes (i.e., groups 2 and 4, respectively, of Table I) the areas of 200 random (individual and clustered) melanosomes per group were determined (Table II, column A) as described in Materials and Methods. The average size of melanosomes from dark skin derived melanocytes (2.00 × 10-2 µm2) was significantly larger than that of melanosomes from light skin derived melanocytes (0.63 × 10-2 µm2).Table IISizes of melanosomes donated either from dark or light skin derived melanocytes within keratinocytesaSizes of individually distributed melanosomes can be compared with sizes of clustered melanosomes. 1 = Values between these numerical groups exhibited p(t) ≤ α = 0.05. 2–5 = Values between these numerical groups exhibited p(t) ≥ α = 0.05.Average melanosome size (µm2) × 10-2Type of coculturedA = random melanosomesB = individual melanosomesC = clustered melanosomesDark melanocytes + light keratinocytes2.00 ± 0.081,21.70 ± 0.0721.64 ± 0.072Light melanocytes + light keratinocytes0.63 ± 0.031,30.78 ± 0.0330.86 ± 0.043Light and dark melanocytes + dark keratinocytes0.78 ± 0.00140.62 ± 0.0340.63 ± 0.034Light and dark melanocytes + light keratinocytes0.78 ± 0.00150.60 ± 0.0350.76 ± 0.025a Sizes of individually distributed melanosomes can be compared with sizes of clustered melanosomes. 1 = Values between these numerical groups exhibited p(t) ≤ α = 0.05. 2–5 = Values between these numerical groups exhibited p(t) ≥ α = 0.05. Open table in a new tab From the same sets of cocultures, the sizes of 100 melanosomes that were individual and 100 melanosomes within keratinocytes that were clustered were subsequently determined (Table II, columns B and C, respectively). In cocultures of dark skin derived melanocytes, the average size of melanosomes that were distributed individually (1.70 × 10-2 µm2) was not significantly different from the average size of melanosomes that were distributed in clusters (1.64 × 10-2 µm2). Similarly, in cocultures of light skin derived melanocytes, the average size of melanosomes that were distributed individually (0.78 × 10-2 µm2) was not significantly different from the average size of melanosomes that were distributed in clusters (0.86 × 10-2 µm2). In the second series of experiments, cocultures of light and dark skin derived keratinocytes were separately established with a mixture of both light and dark skin derived melanocytes Figure 3. Again, the areas of 200 random melanosomes per group were determined initially (Table II, column A) and subsequently the areas of 100 individual and 100 clustered melanosomes per group were determined (Table II, columns B and C, respectively). The average size of melanosomes from dark skin derived melanocytes (1.025 × 10-2 µm2) was significantly larger than that from light skin derived melanocytes (0.5293 × 10-2 µm2). Within either the dark or light skin derived keratinocytes cocultured with a mixture of dark and light skin derived melanocytes, the size of melanosomes distributed individually versus those distributed in clusters did not differ significantly when assessed either randomly within melanocytes (column A) or categorized within keratinocytes (columns B and C). Specifically, in cocultures of combined dark and light skin derived melanocytes with dark skin derived keratinocytes (i.e., group 5), the average size of individual and clustered melanosomes was 0.62 × 10-2 µm2 and 0.63 × 10-2 µm2, respectively. Similarly, in cocultures of combined dark and light skin derived melanocytes with light skin derived keratinocytes (i.e., group 6), the average size of individual and clustered melanosomes was 0.60 × 10-2 µm2 and 0.76 × 10-2 µm2, respectively. In addition, the melanosomes transferred to keratinocytes in groups (5) and (6) were predominantly individual and clustered, respectively, consistent with data presented in Table I. These results demonstrate that, in our model system, the size of the donated melanosomes did not affect their distribution pattern within keratinocytes, suggesting that some regulatory factor attributed to the keratinocyte is responsible for this arrangement. When melanosomes are larger and individually dispersed throughout the cytosol of the keratinocyte, they can absorb more incoming light and refract less, and give skin a darker coloration. Conversely, when the melanosomes are smaller and aggregated in clusters, less light is absorbed and more light is refracted, thus giving skin a lighter coloration. Therefore, melanosome distribution is an important feature that contributes to differences in complexion coloration and photoprotection. In spite of this distinction, analysis of cellular mechanisms governing this distribution of melanosomes within keratinocytes has been minimal to date. The use of tissue culture affords a research opportunity to further analyze regulatory mechanisms governing melanosome distribution in keratinocytes. In 1990, the development of an epidermal reconstruction model demonstrated that melanosomes were transferred to keratinocytes via an apparently phagocytic process and the transferred melanosomes could be retained within a membrane-bound cluster (Valyi-Nagy et al., 1990Valyi-Nagy I.T. Murphy G.F. Mancianti M. Whitaker D. Herlyn M. Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model.Lab Invest. 1990; 62: 314-324PubMed Google Scholar). Precise complexion coloration of the donors of the cell type was not mentioned in this paper, however. Recently, analysis of an epidermal reconstruction model utilizing phototype-identified donors of melanocytes and keratinocytes in various combinations concluded that epidermal pigmentation is under strict melanocyte control (Bessou-Touya et al., 1998Bessou-Touya S. Picardo M. Maresca V. Surleve-Bazeille J. Pain C. Taieb A. Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection.J Inv Dermatol. 1998; 111: 1103-1108Crossref PubMed Scopus (56) Google Scholar), but analytic confirmation of this conclusion was not provided. We report the quantitation of melanosome distribution in heterologous cocultures of melanocytes and keratinocytes derived from light and dark skin donors. Our results demonstrate that the keratinocyte predominantly governs the distribution patterns of melanosomes transferred to keratinocytes. This contrasts with the results described above that indicate that the melanocyte regulates the subsequent distribution pattern of recipient melanosomes. Our in vitro model system allowed us to mix and match melanocytes and keratinocytes from different skin colored donors. This experimental design permitted us to determine the cell type responsible for the differential melanosome distribution noted above. Our model system does not completely recapitulate the in vivo situation regarding racial differences, however. Both clustered and individual melanosomes were observed in both dark and light skin derived keratinocyte-melanocyte cocultures Figure 2. Reliable quantitative analysis could be obtained by using the Zidas Image Analyzer. Other procedures are available for morphometric analysis of melanosomal quantitation and distribution patterns. In particular a method was used byHönigsmann et al., 1986Hönigsmann H. Schuler G. Aberer W. Romani N. Wolff K. Immediate pigment darkening phenomenon. A reevaluation of its mechanisms.J Invest Dermatol. 1986; 87: 648-652Crossref PubMed Scopus (52) Google Scholar in which micrographs are processed digitally by a high resolution TV camera and then subjected to a cluster recognition method by means of the partition procedure using the mean spatial distribution of melanosomes, and, in addition, local dilatation of single particles is carried out after transformation of the picture into a binary-valued image. This allowed recognition and quantification of melanosomes and melanosome complexes. There, however, only fully melanized (stage IV) melanosomes were evaluated. These techniques were not available to us, and furthermore we hoped to analyze melanosomes in keratinocytes at all stages of development that were found within keratinocytes. Use of the Zidas Image Analyzer also provided for quantification of melanosomes at all stages of development, and did not include other electron-dense particles. Quantification of melanosome distribution in our various cocultured combinations Table I demonstrates that, regardless of the donor melanocyte, dark skin derived keratinocytes predominantly distributed recipient melanosomes individually, and light skin derived keratinocytes predominantly distributed recipient melanosomes in membrane-bound clusters. Therefore, we propose that the keratinocyte governs the distribution pattern of the recipient melanosomes. Much has been offered to explain the different distribution of melanosomes in light and dark skin. It remains unknown whether melanosomes are transferred from melanocytes to keratinocytes individually or in membrane-bound clusters. There has been much speculation that melanosome transfer is the consequence of phagocytosis of melanocytic dendrites by recipient cells (Birbeck et al., 1956Birbeck M.S.C. Mercer E.H. Barnicot N.A. The structure and formation of pigment granules in human hair.Exp Cell Res. 1956; 10: 505-514Crossref PubMed Scopus (42) Google Scholar;Valyi-Nagy et al., 1990Valyi-Nagy I.T. Murphy G.F. Mancianti M. Whitaker D. Herlyn M. Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model.Lab Invest. 1990; 62: 314-324PubMed Google Scholar;Jimbow et al., 1998Jimbow K. Sugiyama S. Melanosomal translocation and transfer.in: Nordlund J.J. Boissy R.E. Hearing V.J. King R.A. Ortonne J. The Pigmentary System Physiology and Pathophysiology. Oxford University Press, New York, Oxford1998: 107-114Google Scholar). Melanosomes, particularly clustered melanosomes, within keratinocytes are packaged within secondary lysosomes that contain acid phosphatase (Hori et al., 1968Hori Y. Toda K. Pathak M.A. Clark Jr, W.H. Fitzpatrick T.B. A fine-structure study of the human epidermal melanosome complex and its acid phosphatase activity.J Ultrastruc Res. 1968; 25: 109-120Crossref PubMed Scopus (68) Google Scholar). It is possible that these clusters of melanosomes are not broken down and released as efficiently by acid phosphatase activity in light skin derived keratinocytes compared with dark skin derived keratinocytes where the melanosomes are subsequently distributed individually. There is strong evidence that microtubules regulate melanosome distribution in melanocytes (Byers et al., 2000Byers H.R. Yaar M. Eller M.S. Jalbert N.L. Gilchrest B.A. Role of cytoplasmic dynein in melanosome transport in human melanocytes.J Invest Dermatol. 2000; 114: 990-997https://doi.org/10.1046/j.1523-1747.2000.00957.xCrossref PubMed Scopus (53) Google Scholar). Studies also indicate that the distribution of melanosomes within keratinocytes may be regulated by microtubules via dynein as well (Byers and Maheshwary, 2000Byers H. Maheshwary S. Cytoplasmic dynein expression in human keratinocytes: role in perinuclear aggregation of phagocytosed melanosomes.J Invest Dermatol. 2000; 114: 856Google Scholar). Individual melanosomes may be shuttled by various motor proteins and this may explain their distribution in keratinocytes derived from dark skin. Phagocytosed clusters of melanosomes also tend to migrate toward the nucleus in keratinocytes derived from light skin, however, and it is unclear whether and how microtubules accomplish regulation of this process as well. These data indicate that melanocytes play less of a role in melanosome distribution patterns than previously predicted (Bessou-Touya et al., 1998Bessou-Touya S. Picardo M. Maresca V. Surleve-Bazeille J. Pain C. Taieb A. Chimeric human epidermal reconstructs to study the role of melanocytes and keratinocytes in pigmentation and photoprotection.J Inv Dermatol. 1998; 111: 1103-1108Crossref PubMed Scopus (56) Google Scholar). As noted above, however, one important indicator of melanosome distribution has been proposed as the actual size of the melanosome itself. Experimental studies conducted by Konrad and Wolff (Wolff and Konrad, 1971Wolff K. Konrad K. Melanin pigmentation: an in vivo model for studies of melanosome kinetics within keratinocytes.Science. 1971; 174: 1034-1035Crossref PubMed Scopus (39) Google Scholar,Wolff and Konrad, 1972Wolff K. Konrad K. Phagocytosis of latex beads by epidermal keratinocytes in vivo.J Ultrastruc Res. 1972; 39: 262-280Crossref PubMed Scopus (66) Google Scholar;Konrad and Wolff, 1973Konrad K. Wolff K. Hyperpigmentation, melanosome size, and distribution patterns of melanosomes.Arch Dermatol. 1973; 107: 853-860Crossref PubMed Scopus (64) Google Scholar) on the phagocytosis of latex beads by epidermal keratinocytes of guinea pigs show that the mode of uptake of these melanosome-like particles is size dependent. Large latex beads were incorporated singly into cells whereas small particles were taken up in groups. In addition, others have investigated the uptake of mouse melanosomes by guinea pig keratinocytes induced experimentally in vivo without the action of melanocytes. In this system, large melanosomes were taken up individually by keratinocytes and dispersed singly within their cytoplasm whereas small melanosomes were incorporated as aggregates into the keratinocytes, where they maintained their aggregated form. As this model system bypasses the melanocyte, the authors suggest that the size of the individual melanosome appears to be the decisive factor that determines the distribution of pigment organelles (Wolff et al., 1974Wolff K. Jimbow K. Fitzpatrick T.B. Experimental pigment donation in vivo.J Ultrastruct Res. 1974; 47: 400-419Crossref PubMed Scopus (29) Google Scholar). In our model system, we demonstrated that melanosomes derived from dark skin derived melanocytes are indeed larger than those from light skin derived melanocytes Table II. This is consistent with the literature (Toda et al., 1972Toda K. Pathak M.A. Parrish J.A. Fitzpatrick T.B. Quevedo Jr, W.C. Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light.Nature. 1972; 236: 143-145Crossref Scopus (115) Google Scholar;Konrad and Wolff, 1973Konrad K. Wolff K. Hyperpigmentation, melanosome size, and distribution patterns of melanosomes.Arch Dermatol. 1973; 107: 853-860Crossref PubMed Scopus (64) Google Scholar;Okazaki et al., 1976Okazaki K. Uzuka M. Toda K. Seiji M. Transfer mechanism of melanosomes in epidermal cell culture.J Invest Dermatol. 1976; 67: 541-547Crossref PubMed Scopus (76) Google Scholar;Jimbow et al., 1991Jimbow K. Fitzpatrick T.B. Wick M.M. Biochemistry and physiology of melanin pigmentation.in: Goldsmith S.A. 2nd edn. Physiology, Biochemistry, and Molecular Biology of the Skin. Vol. II. Oxford University Press, New York, Oxford1991: 873-909Google Scholar). In contrast to the results described in the previous paragraph, however, melanosome size does not correlate with the ultimate pattern of distribution within the keratinocyte. When quantitating the size of melanosomes that were clustered as opposed to those that were individual, in our heterologous cocultures, we found that the sizes of clustered and individually distributed melanosomes from either dark or light skin derived melanocytes were not significantly different from each other within each type of recipient keratinocyte Table II. These results indicated that, in our model system, the size of the melanosome did not play a role in how it is distributed within the keratinocyte. In addition, we designed cocultures using a mixture of dark and light skin derived melanocytes so that both large dark skin derived melanosomes and small light skin derived melanosomes could be simultaneously presented to, and distributed within, the keratinocyte. Results demonstrated that the keratinocyte organized these different melanosomes indiscriminately. The heterogenous donated melanosomes predominantly were distributed individually in dark skin derived keratinocytes, and in clusters in light skin derived keratinocytes. In both cases, however, distribution as individual or clustered melanosomes was not significantly related to size Table II. These results clearly demonstrate that melanosome size does not correlate with status of distribution in our model system using human derived skin cells. Future directions should focus on the molecular mechanism(s) regulating melanosome transfer, uptake, and distribution within keratinocytes. Our results clearly suggest that the keratinocytes play a dominant role in distributing melanosomes in the distinctive patterns represented physiologically in different skin types. It should be noted, however, that these findings were limited to cells isolated from a few donor populations, subjectively identified as dark and light skin, to represent each skin type group albeit from the same gender and age. Therefore more expansive studies may be required to confirm the conclusions presented. In addition, our model system was somewhat limited in that it was a monolayer cocultured system. This may account for some of the differences in distribution patterns of melanosomes in keratinocytes compared with what is observed in vivo. Therefore, the next approach might be to develop a skin equivalent system to more closely recapitulate the in vivo system (Valyi-Nagy et al., 1990Valyi-Nagy I.T. Murphy G.F. Mancianti M. Whitaker D. Herlyn M. Phenotypes and interactions of human melanocytes and keratinocytes in an epidermal reconstruction model.Lab Invest. 1990; 62: 314-324PubMed Google Scholar;Gibbs et al., 2000Gibbs S. Murli S. De Boer G. Mulder Aat. Mommaas A.M. Ponec M. Melanosome capping of keratinocytes in pigmented reconstructed epidermis – effect of ultraviolet radiation and 3-isobutyl-1-methyl-xanthine on melanogenesis.Pigment Cell Res. 2000; 13: 458-466https://doi.org/10.1034/j.1600-0749.2000.130608.xCrossref PubMed Scopus (60) Google Scholar). Nevertheless, our results provide evidence that suggests a more proactive role for keratinocytes in regulating distribution patterns of recipient melanosomes. Interestingly, Scott and Haake had demonstrated in 1991 that keratinocytes also regulate the density of melanocytes during fetal and neonatal developmental times. This work was supported by the Proctor and Gamble Company and the Society of Cosmetic Chemists. We thank Dr. Rangaprasad Sarangarajan and Emma Lou Cardell for helpful discussions.