Epidermal Melanin Units: Melanocyte-Keratinocyte Interactions

AM. ZOOLOCIST, 12:35-41 (1972). Epidermal Melanin Units: Melanocyte-Keratinocyte Interactions WALTER C. QUEVEDO, JR. Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912 SYNOPSIS. Current concepts of melanin pigmentation in mammals, with special reference to man, are explored centering on the notion that the multicellular epidermal melanin unit, rather than the melanocyte alone, serves as the focal point for melanin metabolism within mammalian epidermis. The structural and functional organization of epidermal melanin units is described with particular attention to the intrinsic and extrinsic mechanisms which regulate their activities. The significance of the epidermal melanin unit concept in the description and interpretation of skin color variations in man is evaluated in the light of new information. Melanin pigmentation of mammalian epidermis results from the interaction of melanocytes and keratinocytes in the synthesis, transfer, transport, and ultimate disposition of melanosomes, the specialized pigment-containing organelles elaborated by melanocytes (Pinkus et al., 1959; Fitzpatrick and Breathnach, 1963; Quevedo, 1969; Fitzpatrick and Quevedo, 1971; Quevedo, 1971). The epidermal melanin unit concept stresses that a structural and functional organization of melanocytes and keratinocytes exists at levels of biological organization that transcend those characterizing the individual component cells (Fitzpatrick and Breathnach, 1963; Hadley and Quevedo, 1966; Fitzpatrick and Quevedo, 1971). In this view, the basic mechanism for melanin pigmentation is multicellular in design, consisting of a melanocyte and an associated population of keratinocytes. It is now clear that melanosomes transferred from melanocytes to keratinocytes within epidermal melanin units may not be passively transported and shed along with the cornified cells at the epidermal surface; instead, in some cases, they may be catabolized en route (Olson, et al., 1970). Various phases of the author's work reported herein were supported in part by PHS Research Grant No. CA-06097 from the National Cancer Institute, United States Public Health Service. In man, where the most extensive studies of epidermal melanin units have been made thus far, there is evidence that active ("dopa-positive") melanocytes and "viable keratinocytes" (i.e., essentially cells of the malpighian layer) exist throughout the epidermis in a ratio of 1 to 36 (Frenk and Schellhorn, 1969). Since the number of epidermal melanocytes/mm 2 of the human epidermis varies significantly in different anatomical regions of the integument (Szab6, 1967), it follows that the monitoring mechanisms for melanocyte/keratinocyte proportions operate in the face of regionally varying populations of keratinocytes within the malpighian layer. It remains to be determined whether the fixed melanocyte/keratinocyte populations observed at the light microscope level are verifiable by quantitative electron microscopy. It is already clear that many more melanocytes exist in the epidermis than are resolvable by the lieht microscope (Mishima and Widlan, 1967; Zelickson and Mottaz, 1968). Conservatively, it would seem that some relationship exists between the numbers of "welldifferentiated" melanocytes and keratinocytes. By projection of available data (Frenk and Schellhorn, 1969), each epidermal melanin unit in man, on average, would consist of one melanocyte and 36 "viable keratinocytes" in various stages of progression toward the cornified layer. 35

36 WALTER C. QUEVEDO, JR. The numbers of epidermal melanocytes appear to be approximately the same in all races of man (Szab6, 1967). Accordingly, it would follow that human racial differences do not exist in the numbers of epidermal melanin units per unit area of skin. However, the precise ratio of keratinocytes to melanocytes among the various races remains to be determined. Racial variation in skin color results from differences in the properties and numbers of melanosomes synthesized by melanocytes and transferred to keratinocytes. Clinically, melanin pigmentation of the skin is determined by the amount of melanin within both types of cells (Gates and Zimmermann, 1953; Fitzpatrick and Quevedo, 1971). In view of the markedly greater population of keratinocytes relative to melanocytes, the numbers of melanosomes in transport within keratinocytes must make a major contribution to overall skin color (Fitzpatrick and Quevedo, 1971). Melanosomes are synthesized within melanocytes in accordance with a genetic program which specifies the detailed architecture of the melanosomes as well as the numbers in which they are produced (Markert and Silvers, 1956; Foster, 1965; Moyer, 1966; Rittenhouse, 1968a,&; Quevedo, 1969; Quevedo, 1971). Pigmentary genes may act directly by way of the genome of the melanocyte or indirectly through the tissue environment which surrounds it (Markert and Silvers, 1956; Mayer and Green, 1968). Melanin is ultimately derived from the amino acid tyrosine through a pathway, the initial steps of which are catalyzed by tyrosinase (Duchon et al., 1968). Melanin, a heteropolymer, is deposited within the membrane-limited melanosome where it coats the surface of the "sheet-like" protein matrix and occupies much of the inner space of the organelle (Duchon et al., 1968; Breathnach, 1969). Melanosomes are acquired by keratinocytes through a heterophagic process whereby they phagocytize bits of the melanosome-laden dendrites of the melanocytes (Mottaz and Zelickson, 1967). In man, the fate of melanosomes within keratinocytes is significantly influenced by the size of the individual melanosomes (Toda et al., 1971, 1972; Wolff and Konrad, 1971). It would appear that melanosomes which exceed approximately 0.8//, in greatest diameter will be distributed singly within membrane-limited vesicles of the keratinocytes (Toda et al., 1972). Melanosomes less than approximately 0.8ju in greatest diameter will be aggregated in groups of two or more to each membranelimited vesicle. The groups of melanosomes have been designated as "melanosome complexes" (Fitzpatrick and Quevedo, 1971). Electron cytochemistry has demonstrated that melanosomes within keratinocytes are associated with lysosomal enzymes in what are regarded as "lysosome-like vesicles" (Hori et al., 1968; Olson et al., 1970; Ohtaki and Seiji, 1971). Human racial differences (Fig. 1) exist in the manner in which melanosomes are distributed within keratinocytes of epidermal melanin units (Mitchell, 1968; Szab6 et al., 1969; Toda et al., 1971). In Negroids and Australoids, melanosomes exceed 0.8^ in greatest diameter and are distributed singly within keratinocytes (Toda et al., 1972). In Caucasoids and Mongoloids, melanosomes are less than 0.8^ in greatest diameter and form "melanosomecomplexes" within keratinocytes (Toda et al., 1972). Based on examinations with the electron microscope, there is the suggestion that the melanosomes of "melanosome complexes" may be more subject to degradation by lysosomal hydrolases than those distributed as single entities (Toda, personal communication). Whether this is the result of racial differences in the properties of individual melanosomes, such as the density of melanization, or in the amounts of lytic enzymes to which single and aggregated melanosomes are exposed, remains to be determined. The lysosomal hydrolases seem to attack the matrix of the melanosome but not its associated melanin (Ohtaki and Seiji, 1971). As a conse-

EPIDERMAL MELANIN UNITS 37 B 1? IG. 1. Diagrammatic representation o epidermal melanin units illustrating racial differences in the mode of transport of melanosomes within keratinocytes: (A) Caucasoids and Mongoloids Melanosomes form "melanosome complexes" within keratinocytes and there is evidence of melanoquence, melanin fragments may be released from degraded melanosomes within keratinocytes (Quevedo, 1971). The role of melanin fragments in the economy of the epidermis is not clear but they may account for melanoid pigment originally described and so interpreted by Edwards and Duntley (1939) based on reflectance spectrophotometry. If this relationship is correct, the demonstration that melanoid pigment occurs in all races would require that melanosome degradation is universal in the epidermal keratinocytes of man (Edwards and Duntley, 1939). Melanosomes have been observed in the cornified cells of all races examined (see Quevedo, 1969), but it remains to be shown whether some degradation within the lysosome-like complexes; (B) Negroids and Australoids Melanosomes are larger than those of Caucasoids and Mongoloids and arranged singly within keratinocytes. a greater proportion of the melanosomes synthesized by the melanocytes in Caucasoids and Mongoloids are degraded than is the case in Negroids and Australoids. The contributions that grouping of melanosomes within "melanosome complexes" and the degradation of melanosomes make to the overall color of the skin have not been studied quantitatively. Logically, the grouping of melanosomes would appear to "lighten" the skin. The possible influence of melanosome fragments on skin color permits a variety of interpretations. With respect to the significance of melanosome degradation in the epidermis, while recognizing the possibility of other

38 WALTER C. QUEVEDO, JR. ling evidence that environmental factors may alter the size of melanosomes synthesized and consequently the pattern of their transport within keratinocytes (Toda et al., 1971, 1972). Treatment of the skin of Caucasoids and Mongoloids with the photosensitizer, trimethyl psoralen, followed by exposure to UV results in an increase in the size of melanosomes synthesized by melanocytes and, consequently, in their dispersal singly rather than as "melanosome complexes" within keratinocytes. To date, it has not been established whether the single melanosomes are less prone to degradation than they would be if transported in "melanosome complexes." Based on reflectance spectrophotometry, the melanogenic activity of epidermal melanin units would appear to vary regionally in the normally clothed human integument (Edwards and Duntley, 1939). The skin is darkest in "fold regions" such as the axillae and perineum. The blueprint for the function of epidermal melanin units appears to be drawn in very fine detail. Whimster (1971) has submitted evidence that the skin of the adult human is a mosaic of many discrete unit areas, the properties of which are significantly maintained by the nervous system. He has illustrated how the "unit nature" and symmetry of many cutaneous diseases substantiate this principle. In vitiligo (Fig. 2), for example, there is occasionally a quite precise loss of melanocytes from the epidermis resulting in highly symmetrical patterns of cutaneous depigmentation (Breathnach, 1969; Whimster, 1971). Whimster's observations suggest that regional differentiation of the integument into a vast mosaic is achieved during embryonic development and maintained throughout life. The symmetrical loss of epidermal melanin unit function is possibly the result of melanocyte destruction by neurocytotoxic agents (Lerner, 1971). The region-specific loss of mechanisms protecting against the damaging action of neurocytotoxic agents would result in melanocyte death (Lerner, 1971). It would appear that region-specific programs determine the responsiveness of fc) FIG. 2- Vitiligo resulting in symmetrical cutaneous depigmentation in an African Negroid (from Whimster, I. W., Annali Italiani di Dermatologica Clinica e Sperimentale 16:357-384, 1961/62). conclusions (Quevedo, 1971), it would seem most likely that melanin fragments, particularly in the skin of Caucasoids, extend the protection that melanin provides against damaging action of solar UV. The presence of melanosome fragments may maximize the screening capacity provided by a limited number of melanosomes (Quevedo, 1971). Interaction of genes at three to four loci in man would seem sufficient to account for the world-wide variation in skin color (Harrison and Owen, 1964; Livingstone, 1969; Stern, 1970). The precise sites for differential gene action in determining racial differences in skin color have not been established. There is no doubt that genes in some way influence the numbers and general properties of the melanosomes synthesized (Quevedo, 1971). Although the size of melanosomes would appear to be genetically determined, there is compel-

EPIDERMAL MELANIN UNITS FIG. 3. Electropherograms demonstrating regional differences in activity patterns of lactate dehydrogenase isozymes in the epidermis of C57BL mice: (A) tail; (B) ear pinna; (C) dorsum of trunk; (D) foot. LDHS predominates in all regions; the ratio of LDH6 to LDHi varies (Quevedo, Bienieki, Holstein, and Dyer, unpublished data). epidermal melanin units to endogenous (e.g., hormones) or exogenous (e.g., UV) factors. Based on the patterns of depigmenting cutaneous disease (Whimster, 1971), it would appear that a limited number of epidermal melanin units is specified by a given program. Available information indicates that melanocyte function may be specified from within the epidermal melanin unit as well as by cues from the dermis (Quevedo, 1971). Anatomical regional differences have been demonstrated in the biochemistry of the epidermis (Fig. 3). Billingham and Silvers (1967) have provided experimental evidence in support of the view that the regional specificity of the epidermis depends on dermal regulation expressed throughout life. At present the mechanisms which regulate the structural and functional integrity of epidermal melanin units are not clearly 39 understood. The maintenance of a ratio of 36 "viable keratinocytes" to one melanocyte within an epidermal melanin unit would require a precise proportional control of the mitotic activity of melanocytes and keratinocytes. Bullough and Laurence (1961, 1964 1968; Bullough, 1971) have proposed that the mitotic activity of melanocytes and keratinocytes is regulated by tissue-specific inhibitory agents, possibly glycoproteins, which they have designated as chalones. They have submitted evidence that distinctly different chalones regulate the mitotic activity of melanocytes and keratinocytes (Bullough and Laurence, 1968). If the chalone concept is correct, the balance of melanocytes and keratinocytes in the epidermis would seem to depend on a close interaction of their chalone systems. For full effectiveness, chalones require the support of adrenaline and a glucocorticoid such as hydrocortisone (Bullough, 1971). Recently, the chalone concept of mitotic regulation in the epidermis has been challenged (Voorhees and Duell, 1971; Powell et al., 1971). Evidence has been submitted in favor of the view that mitotic activity in the epidermis is regulated by intracellular levels of cyclic AMP (Voorhees and Duell, 1971; Powell et al., 1971). It has been suggested that adrenaline, instead of strengthening the action of chalone, binds to cell surfaces at /}-adrenergic receptor sites and stimulates increased cyclic AMP production through the action of adenyl cyclase. In this view, chalones may simply be key ingredients of the cyclic AMP system. Iversen (1969) somewhat earlier proposed a merger of the chalone and cyclic AMP concepts of mitotic regulation. Current research should clarify this important issue. The epidermal melanin unit represents but one expression of complex cellular interactions which are initiated during embryonic development and maintained throughout life. A variety of evidence indicates that in the adult mammal, the tissue environment may act to specify melanocyte

40 WALTER C. QUEVEDO, JR. performance. In mice, a dramatic example is the occurrence of phaeomelanin (yellow melanin) synthesis only within the environment provided by the hair follicle (Markert and Silvers, 1956). Additionally, it is significant that UV stimulates melanin synthesis within melanocytes of the intact skin, but does not do so in cultures of melanoma cells (Kitano and Hu, 1969). It would seem that influences of keratinocytes may be necessary for radiationinduced tanning. At present little is known about the action of melanosomes within the keratinocytes of the epidermal melanin unit. It is possible that the arrival of melanosomes within keratinocytes stimulates metabolic changes such that pathways are activated which ultimately serve to regulate the melanogenic activity of melanocytes. It is conceivable that the degradation of melanosomes by lysosomal enzymes may yield small molecules which pass back by way of the keratinocytes or intercellular spaces to influence melanocyte performance through biological feedback. Finally, the mechanics of cell movements within the epidermis are now being clarified (Pinkus and Hunter, 1966; Penneys et al., 1970; Mackenzie, 1970; Karatschai et al., 1971; Menton and Eisen, 1971). The emerging impression is quite different from that entertained to date. The new information on the morphogenesis of the epidermis should significantly influence current understanding of the origin, distribution, and fate of melanosomes within epidermal melanin units. With this insight, the full biological significance of melanin pigmentation in man should be perceived more clearly. REFERENCES Billingham, R. E., and W. K. Silvers. 1967. Studies on the conservation of epidermal specificities of skin and certain mucosas in adult mammals. J. Exp. Med. 125:429-446. Birbeck, M. S. C. 1963. Electron microscopy of melanocvtes: the fine structure of hair-bulb premelanosomes. Ann. X. Y. Acad. Sci. 100:540-547. Breathnach, A. S. 1969. Normal and abnormal melanin pigmentation of the skin, p. 353-394. In M. Wolman [ed.]. Pigments in pathology. Academic Press, New York. Bullough, W. S. 1971. The actions of the chalones. Ag. Actions 2:1-7. Bullough, W. S., and E. B. Laurence. 1961. The study of mammalian epidermal mitosis in vitro. A critical analysis of technique. Exp. Cell Res. 24:289-297. Bullough, W. S., and E. B. Laurence. 1964. Mirotic control by internal secretion: the role of the chalone-adrenalin complex. Exp. Cell Res. 33:176-194. Bullough, W. S., and E. B. Laurence. 1968. Control of mitosis in mouse and hamster melanomata by means of the melanocyte chalone. Eur. J. Cancer 4:607-615. Duchon, J., T. B. Fitzpatrick, and M. Seiji. 1968. Melanin 1968: some definitions and problems, p. 6-33. A. W. Kopf and R. Andrade [ed.], In The 1967-68 year book of dermatology. Year Book Medical Publishers, Chicago. Edwards, E. A., and S. Q. Duntley. 1939. The pigments and color of living human skin. Amer. J. Anat. 65:1-33. Fitzpatrick, T. B., and A. S. Breathnach. 1963. Das epidermale Melanin-Einheit-System. Dermatol. Wochschr. 147:481-489. Fitzpatrick, T. B., and W. C. Quevedo, Jr. 1971. Biological processes underlying melanin pigmentation and pigmentary disorders. Mod. Trends Dermatol. 4:122-149. Foster, M. 1965. Mammalian pigment genetics. Advan. Genet. 13:311-339. Frenk, E., and J. P. Schellhorn. 1969. Zur Morphologie der epidermalen Melanineinheit. Dermatologica 139:271-277. Gates, R. R., and A. A. Zimmermann. 1953. Comparison of skin color with melanin content. J. Invest. Dermatol. 21:339-348. Hadley, M. E., and W. C. Quevedo, Jr. 1966. Vertebrate epidermal melanin unit. Nature 209:1334-1335. Harrison, G. A., and J. J. T. Owen. 1964. Studies on the inheritance of human skin colour. Ann. Hum. Genet. 28:27-37. Hori, Y., K. Toda, M. A. Pathak, W. H. Clark, Jr., and T. B. Fitzpatrick. 1968. A fine-structure study of the human epidermal melanosome complex and its acid phosphatase activity J. Ultrastruct. Res. 25:109-120. Iversen, O. H. 1969. Chalones of the skin, p. 29-53. In G. E. W. Wolstenholme and J. Knight [ed.], Ciba Foundation Symposium on Homeostatic Regulators. J. and A. Churchill Ltd., London. Karatschai, M., V. Kinzel, Kl. Goerttler, and R. Suss. 1971. "Geography" of mitoses and cell divisions in the basal cell layer of mouse epidermis. Z. Krebsforsch. 76:59-64. Kitano, Y., and F. Hu. 1969. The effects of ultraviolet light on mammalian pigment cells in vitro. J. Invest. Dermatol. 52:25-31. Lemer, A. B. 1971. On the etiology of vitiligo and gray hair. Amer. J. Med. 51:141-147.

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