Electron Microscopy of Melanocytes in Human Piebaldism* Aodan S

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ELECTRON MICROSCOPY OF MELANOCYTES IN HUMAN PIEBALDISM* AODAN S. BREATHNACH, M.D.,** T. B. FITZPATRICK, MD.5"' AND LUCILE M.-A. WYLLIE** Application of the modern technics of cell Piebaldism may be defined as a localized, biology to the study of melanin pigmentationcongenital hypomelanosis of the skin and hair in has led to significant advances during the lastman, that is usually inherited as an autosomal decade. Electron microscopy has made possibledominant trait, Its clinical manifestations are the certain identification of unmelanized mela-uniform (7) and the presence of a bilaterally nocytes, and has revealed in melanocytessymmetrical, triangular white forelock is char- specialized eytoplasmic organelles that cannotacteristic. Kugelman and Lerner (5), employing be seen by light microscopy (1). Furthermore,light microscopy, found "clear cells" in the isolation of these organelles by ultracentrifuga-basal layer of skin from a patient with pie- tion has permitted their biochemical analysisbaldism; they concluded that the lesions in this (2). As a result, the various steps in the bio-skin were histologically indistinguishable from synthesis of melanin and the morphogenesis ofthose of oculo-cutaneous albinism, and that, the melanin granule are now reasonably wellcontrary to generally expressed opinion, pie- understood (3—5). baldism in man is not equivalent to the "white These advances provide a sound basis forspotting" of experimental animals. "White investigation of the influence of genes at differ-spotting" in animals is usually attributed to ent stages of the process of melanization, andtotal absence of melanocytes. Using light mi- for examination of some of the heritable dis-croscopy, a number of workers (9, 10) have orders of pigmentation in man. For example,failed to detect "clear cells" in the hair follicles light microscopy revealed that, in both oculo-or the basal layer of epidermis from the white cutaneous albinism and vitiligo, "clear cells"skin of spotted mice and guinea pigs. It has are present in the basal layer of the epidermis;seemed desirable to amplify such findings by electron microscopy, however, disclosed that inelectron microscopy so that the possible mor- these two disorders the "clear cells" are struc-phological defects of hypomelanotic skin from turally different. The hypomelanosis of oculo-piebald subjects may be compared with the cutaneous albinism, a recessive trait, is due todefects seen in the skin of subjects with oculo- decrease or absence of melanin biosynthesis; incutaneous albinism and vitiligo. This report is this disorder, unmelanized organelles, premcla-concerned with the ultrastructure of melano- nosomcs, are present within the melanocyte,cytes in the hypomelanotic skin of piebald sub- which appears to be otherwise normal. Injects. vitiligo, an acquired hypomelanosis with a Before describing the findings in piebald familial tendency, normal melanocytes are re-skin, the ultrastructural characteristics of the placed in the basal layer by Langerhans cellsthree types of "clear" cells will be briefly sum- that contain characteristic disc-like cytoplasmicmarized. All three types (Table I) can be dis- organelles but no premelanosomes (6). Thetinguished from adjacent keratinocytes by their hypomelanosis associated with these two condi-relatively "clear" cytoplasm devoid of tono- tions therefore results from totally differentfilaments and by the absence of desmosomes defects. from their plasma membranes. The cytoplasm Supported by grants from the Medical Re-of normal melanocytes contains melanosomes in search Council and the Wellcome Trust, and invarious stages of melanization (11, 12), whereas part by U.S. Public Health Service Research Grant CA-SOlO-OS, National Cancer Institute. albino melanocytes, in mice, contain largely Received for pubhcation July 17, 1964. unmelanized organelles or premelanosomes (13) * From the Department of Anatomy, St. Mary'sand, in man, a few partially melanized organelles Hospital Medical School,** London W.2., England, the Department of Dermatology, Harvard Medical(melanosomes). The cytoplasm of Langerhans School*** and the Dermatological Service, Mas-cells contains no premelanosomes or melano- sachusetts General Hospital Boston, Massachusetts 02114. somes, but it does contain distinctive disc-like 25 MELANOCYTES IN HUMAN PIEBALDISM 29 granules which have rod-shaped profiles when TABLE I sectioned; the nucleus is characteristically in- The ultrostructure of "clear" *cells dented (6). Tn normal epidermis, melanocytes and rare Langerhans cells are found in the basal Location in EpidermisSpecialized Organdies layer; Langerhans cells are also present in the supra-basal layers. The exact nature of theNormal mela-Basal layer In various nocyte stages of mel- Langerhans cells is not fully understood, but anization there is evidence that it is related to and very (pre-melan- probably derived from the melanocyte. osomes and melano- MATERIALS AND METHODS somes) Theskin studied was obtained by biopsy re-Langerhans Supra-basal Disc-like forms movel from a 21-year-old mant with classical cell layers and presenting piebaldism (cutaneous albinism). Specimens were occasionally rod-shaped taken from two hypomelanotic areas, the white basal layer profiles on forelock region of the scalp and the flexor aspect in normal section of the forearm, and from one apparently normal area (control specimens) on the dorsal aspect of epidermis. the forearm. Immediately after excision, the skin Sole type in was fixed for two hours in buffered osmium vitiligo tetroxide at 4° C, then dehydrated in graded alco-Albino melano-Basal layer Unmelanized hols and finally embedded in Araldite. Some speci- cyte (oeulo- (pre-mela- mens were stained with 1 per cent phosphotungstic cutaneous nosomes); acid in 90 per cent alcohol before being embedded; albinism) rare melano- others were stained with lead hydroxide (Karnov- somes sky) on the grid after being embedded and sectioned. Thin sections cut on a Huxley ultra- *Maybe distinguished from keratinocytes by microtome were examined in a Siemens Elmi-their "clear" cytoplasm which contains no tone- skop-I. filaments. RESULTS Control Specimens (Normolly Pigmented Skin "Normal" skin from the piebald subject from the Piebald Subject) definitely contained more basal Langerhans cells than are found in epidermis from normal sub- In control specimens from this piebald subjects. Langcrhans cells were present in the supra- ject, the majority of melanocytes appeared tobasal layers. be essentially normal although they differed in several respects from melanocytes found in the Hypomelanotic Epidermis from the Forearm epidermis of normal subjects: the degree of nuclear indentation was greater than normal; Hypomclanotic epidermis from the forearm irregular vesicles were present in the cytoplasmcan best be discussed in terms of the abnor- and there was a tendency to produce unusualmally numerous "clear" cells present in the basal melanosomes. These variations were particu-layer, many of which seem to have shrunk larly evident in cells with dark-staining granu-away from adjacent keratinocytes. While this lar cytoplasm that had usually retracted fromshrinkage can be regarded as the result of the keratinocytes that flanked them (Fig. 1).processing, the fact that it was seen almost Melanoeytes of similar appearance have beenexclusively in "clear" cells suggests that it seen in the heavily pigmented epidermis over-may have wide significance as evidence of lying an intradermal nevus (14) and are occa-inherent cellular abnormality. On the basis of sionally encountered in normal epidermis, al-morphology, the "clear" cells seen in the basal though much less frequently than in this skinlayer may be divided into three groups. from the piebald subject. Fig. 2 shows two Group 1 includes cells with indented nuclei morphologically irregular melanosomes in theand diffusely granulated cytoplasm that con- cytoplasm of a cell of this type. tains a few partially melanized melanosomes and peculiar rounded granules. These granules *Patientof Dr. L. Fisch, to whom we are grate- ful for the opportunity to carry out this investiga-are almost invariably surrounded by a space, tion. and their internal structure is ill defined (Fig. .rC .11 ft.. -• -,

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Fio. 3. Melanocyte from hypomelanotic skin of the forearm. PTA stain, 16,000 X. The cytoplasmic granules (m) surrounded by a space are probably abnormal melanosomes. me = melanosomesof normal appearance.

3). As seen with the electron-microscope they rounded granules are almost certainly not mito- are very similar in size and shape to the gran- ehondria, which in these cells are very sparse ules in normal pigmented skin that are thought and of smaller size. Fig. 4 shows another cell of to be irregular melanosomes (Fig. 2). The this group in which the cytoplasm contains no

FIG. 1. Melanocyte of pigmented skin from the forearm of a piebald subject. Lead hydroxide stain, 24,000 x. Note the indented nucleus and the relatively electron-deose, granular cytoplasm containing somewhat irregular melanosomes (m). The cell has shrunk away from flanking keratinocytes (K). Arrows point to the basement membrane. Fio. 2. A portion of the cytoplasm of a melanocyte of pigmented skin from the forearm of a piebald subject. Lead hydroxide stain, 51,000 X. g =melaningranule of normal morphology; m =melanosomesof apparently irregular form; mi =mitochondria;v =irregular vesicles. ?- '4 I U 0: I e 'a (I IA C.&.S.' '% •4nr'.-

FIG. 4. Melanocyte from hypomelanotic skin of the forearm. PTA stain, 9,600 X. The cell has shrunk away from flanking keratinocytes (k). Except for an occasional granule (m) of the type illustrated in Fig. 3, few cytoplasmic details are apparent. FIG. 5. A Langerhans cell (L) in the basal layer of hypomelanotic skin of the forearm. Lead hydroxide stain, 6,000 X. K =keratinocyte.Arrows point to the basement membrane. A portion of the cytoplasm of this cell is shown in greater detail in Fig. 6. 32 MELANOCYTES IN HUMAN PIEBALDISM 33 organdies except one or two of the rounded Langerhans cells were also present in the granules; the cell itself seems to be disintegrat-supra-basal layers of this epidermis. In one of ing. The cells in Figs. 3 and 4 were both stainedthese cells there was a granule similar to those by phosphotungstie acid; cells stained on theseen in abnormal melanocytes of the basal layer. grid with lead hydroxide had similar characteristics, and their cytoplasm was coarsely Hypomelanotic Epidermis from the granular like that of the darker-staining mela- White Forelock Region nocytes of pigmented skin (Figs. 1 and 2). Neither normal melanocytes, nor abnormal Group 2 includes Langerhans cells that ap-melanocytes of the type found in hypomelanotic pear essentially normal. They were compara-epidermis from the forearm were scdll in skin tively rare. from the region of the white forelock. There Group 3 contains Langerhans cells whichwere present in the basal layer, however, (a) an differ from the normal in exhibiting a tendencyabnormally large number of Langerhans cells of toward nuclear-cytoplasmic separation (Fig.completely typical appearance (Fig. 7), and (b) 5). In lead-stained sections of these cells, theclear cells with indented nuclei and all the cytoplasm of the perinuclear region (Fig. 6)cytoplasmic characteristics of Langerhans cells had the dark-staining, granular appearance ofexcept the rod-shaped profiles (Figs. S & 9). the irregular melanocytes of pigmented skin.Even in normal epidermis, however, so few It seems likely that this granularity is due torod-shaped profiles are present in the Langer- an abundance of RNP particles. The number ofhans cells of the basal layer that they may not Langerhans cells, of whatever type seen, in theappear in all sections cut from any given cell basal layer was greater than normal. (15). Since nothing in any way resembling the

ttt a' Fm. 6. A portion of the cytoplasm of the Langerhans cell shown in Fig. 5. 34,000X. Note the tendency toward nuclear-cytoplasmic separation and the granularity of the perinoclear cytoplasm. n nucleus; p1 =plasmamembrane of Langerhans cell beyond which are desmosomes associated with adjacent keratinocytes (K); r =characteristicrod-shaped profiles. 34 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

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FIG. 7. A Langerhans cell in the basal layer of epidermis from the white forelock. PTA stain, 15,000 X.Kkeratinocyte; rrod-shaped profiles. FIG. 8. Two clear cells (L) in the basal layer of epidermis from the white forelock. PTA stain, 8,000 x.Allthe characteristics of Langerhans cells are present except for the absence of rod-shaped profiles. Arrows point to the basement membrane. MELANOCYTES IN HUMAN PIEBALDISM 35 1 C) — —a.

FIG. 9. A more highly magnified section through one of the clear cells shown in Fig. 8. PTA stain, 21,000 X. The cytoplasm is typically that of a Langerhans cell, hut no rod-shaped profiles are present. K —keratinocyte;mi =mitochondria. premelanosomes of albino melanocytes was seenIt also has revealed that there are differences in clear basal cells from the white forelock, it isbetween melanocytes from the region of the concluded that the clear cells found in thiswhite forelock and those from other hypopig- hypomelanotic area are in fact Langerhans cellsmented areas in the same piebald subject. These and that all clear cells present in the basalfindings are of interest from various points of layer of skin from the white forelock are of thisview. type. In epidermis from the white forelock, it ap- In the supra-basal layers of epidermis frompears that melanocytes are replaced by Langer- the white forelock, Langerhans cells seemed tohans cells, a phenomenon identical with that be abnormally numerous. found in vitiligo(6). This histopathologic similarity may seem surprising, because it is nIsCUssION customary to differentiate these two disorders In this study, electron microscopy has re-clearly on both etiological and clinical grounds. vealed that the melanocytes of hypomelanoticIt is not uncommon, however, for totally "piebald" skin differ in certain respects fromdisparate etiological factors to evoke similar, those found in the hypomelanotic skin of sub-nonspecific alterations in cellular material. The jects with oculc-cutaneous albinism, a distinc-replacement of melanocytes by Langerhans cells tion not made evident by light microscopy (8).in vitiligo can be explained by the recently 36 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY postulated (16) existence of a developmental The findings reported here are not incom- relationship between these two types of cells.patible with the concept that there are histologi- Whether or not the changes found in cells fromcal differences between the hypopigmentation of the white forelock can be explained in the samepicbaldism and that of white-spotted animals. way is not yet known. All that can be said atOn the basis of the present findings, one could the moment is that the observations reportedagree with Kugelmann and Lerner's (8) con- here tend to support the concept that theclusion that piebaldism in man and "white- Langerhans cell is an immature melanocyte inspotting" in animals are histologically different, an arrested phase of development. A study byprovided, that is, that one is prepared to accept electron microscopy of the melanoblasts andwhite-spotting as being due to a total absence of melanocytes in fetal skin might throw somemelanocytes. This interpretation is based en- light on this question. tirely upon failure to demonstrate "clear cells" Perhaps more surprising than the findings inby light microscopy in hair, and in the basal skin from the white forelock arc those in thelayer of the epidermis of white spots. This type hypomelanotic skin from the forearm of piebaldof evidence can no longer be considered ade- subjects. ]\/Ielanocytes from the forearm, likequate, and white-spotting requires re-examina- those from subjects with oculo-cutancous al-tion with electron microscopy. In fact, clear binism, contain a few premelanosomes and ancells that are almost certainly Langerhans cells occasional partially melanized melanosome.have recently been demonstrated by this means They differ from the melanocytes of oculo-in the basal layer of the surface epidermis and cutaneous albinism in that they also (a) containof the outer root sheath of the hair follicle, in what appear to be morphologically abnormalthe white skin of recessively spotted guinea pigs melanosomes; (6) have an empty appearance(18). This finding confirms Silver's observation, of cytoplasm; and (c) tend to become dis-which seems to have been overlooked in later sociated from the keratinocytes that flank them.discussions of the nature of white-spotting (17, Some of these characteristics are manifested in19), that "gold-positive dcndritic cells were very minor degree by the melanocytes of apparentlyprominent in both basal and superficial epi- normal pigmented skin. The fact that the mela-dermis of white spots" (20). It would seem, nocytes of piebald subjects have undergonetherefore, that white spotting may be due not to more profound change than those of subjectsabsence of epidermal melanocytcs, but to an with oculo-cutaneous albinism can be explainedalteration within the melanocytes of the epi- by postulating that oculo-cutaneous albinismdermis similar to that found in the hypopig- results from the effect of a single gene thatmerited skin of vitiligo and of the white fore- blocks the biosynthesis of tyrosinase, whereaslock of piebald subjects. Whether or not piebaldism results from the action of severalcomparably altered cells will be found in the genes. Studies of the genetics of skin and hairbulbs of white hairs remains to be seen. pigmentation in experimental animals (17) have shown that genes from a number of loci SUMMARY are concerned, either alone or in combination, Melanocytes of bypopigmented epidermis with the determination of pigmentary patternsfrom the general body surface of a piebald hu- and defects. Specific genes arc known to influ-man subject showed a much greater tendency ence melanoblast differentiation, melanocytethan normal to be dissociated from flanking morphology, and the basic protein structure ofkeratinocytes, and the cytoplasm of many the melanosomc, all of which appear to beexhibited a characteristically "empty" appear- affected in the hypomclanotic skin of subjectsance. In addition to non-melanized premelano- with piebaldism. The abnormally large numbersomes of normal structure, premelanosomes and of Langerhans cells present in hypomelanoticmelanosomes of frankly abnormal morphology skin from the forearm, and the fact that thesewere present in these cells. Melanocytes of the Langcrhans cells resemble melanocytes in certainapparently normal pigmented epidermis re- ways is not incompatible with this interpreta-vealed similar features but to a much lesser tion and favors the view that the two cellsdegree. In the white forelock region of the described are developmentally related. scalp, the basal epidermal melanocytes were MELANOCYTES IN HUMAN PIEBALDISM 37 replaced by Langerhans cells, a situation of human piebaldism and white forelock. Irish J. Med. Sci., *'398:86,1959. identical with that found in vitiligo, and one 8. Kugelman, T. P. and Lerner, A. B.: Albinism, which strongly reinforces the suggestion that partial nlbinism, and vitiligo. Yale J. Biol. the two cell types are related. Med., 33:407,1961. 9. Silvers, W. K.: Pigment cells: occurrence in The above findings show that the melanocytcs hair follicles. J. Morph., 99: 41, 1956. in piebaldism are more profoundly affected than10. Quevedo, W. C., Jr.: Loss of clear cells in hair in oculo-cutaneous (total) albinism, a situation follicles of X-irradiated albino mice. Anat. Rec., 127:725,1957. which light microscopy has failed to reveal.11. Barnicot, N. A. and Birbeck, M. S. C.: The The plurality of defects in piebaldism involving electron microscopy of human melanocytes and melanin granules. In The Biology of such features as melanocyte morphology and Hair Growth, edited by W. Montagna and the basic protein structure of the melanosome R. A. Ellis. New York, Academic Press Inc., indicates that a number of different gene loci 1958. 12. Drochmans, P.: Electron microscope studies of are concerned in determining this condition. epidermal melanocytes nnd the fine structure of melanin granules. J. Biophys. Biochem. REFERENCES Cytol., 8:165,1960. 13. Birbeck, M. S. C. and Barnicot, N. A.: Elec- 1. Birbcck, M. S. C.: Electron microscopy of tron microscope studies on pigment forma- melanocytes: the fine structure of hair-bulb tion in human hair follicles. In Pigment Cell premelanosomes. Ann. N. Y. Acad. Sci., 100: Biology, edited by M. Gordon. New York, Part2: 540, 1963. Academic Press Inc., 1959. 2. Seiji, M., Shimao, K., Birbeck, M. S. C. and14. Breathnnch, A. S.: Electron microscopy of a Fitzpatrick, T. B.: Subcellular localization of small pigmented cutaneous lesion. J. Invest. melanin biosynthesis. Ann. N. Y. Acad. Sci., Berm., 42:21,1964. 100:Part2: 497, 1963. 15. Breathnach, A. S.: Observations on cytoplasmic 3. Moyer, F. H.: Genetic effects of melanosome organelles in Langerhans cells of human fine structure and ontogeny in normal and epidermis. J. Anat., London, 98:265,1964. malignant cells. Ann. N. Y. Acad. Sd., 100: 16. Breathnach, A. S.: A new concept of the re- Part2: 584, 1963. lation between the Langerhans cell and the 4. Fitzpatirck, T. B., Seiji, M. and McGugan, melnnocyte. J. Invest. Berm., 40:279,1963. A. D.: Melanin pigmentation. New Eng. J.17. Sdvers, W. K.: Genes and the pigment cells Med., 265:328;374, 430, 1961. of mammals. Science, 134:368,1961. 5. Rdcy, V. and Fortner, J. G., editors: The18. Breathnach, A. S. and Goodwin, D.: Unpub- Pigment Cell: Molecular, Biological, and lished, 1964. Clinical Aspects. New York, New York19. Billingham, H. E. and Silvers, W. K.: The Academy of Sciences, 1963. melanocytcs of mammals. Quart. Rev. Biol., 6. Birbeck, M. S. C., Breathnach, A. S. and 35:1,1960. Everall, J. D.: An electron microscope study20. Silvers, W. K.: An histological and experi- of basal melanocytes and high-level clear mentnl approach to determine the relation- cells (Langerhans cells) in vitiligo. J. Invest. ship between gold-impregnated dcndritic Berm., 37:51,1961. cells and malanocytes. Amer. J. Anat., 100: 7. Froggntt, P.: An outline, with bibliography, 225,1957.