Studies on Cafe au lait Spots in Neurofibromatosis and Pigmented Macules of Nevus Spilus

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1 Tohoku J. exp. Med., 1976, 118, Studies on Cafe au lait Spots in Neurofibromatosis and Pigmented Macules of Nevus Spilus MASAAKI TAKAHASHI Department of Dermatology,* Tohoku University School of Medicine, Sendai TAKAHASHI, M. Studies on Cafe au lait Spots in Neurofibromatosis and Pigemented Macules of Nevus Spilus. Tohoku J. exp. Med., 1976, 118 (3), Cafe au lait spots from 14 Japanese patients with neurofibromatosis and nevus spilus from 9 Japanese patients were subjected to the studies on the differences in nature of their melanocytes. When the number of melanocytes of the pigmented lesions was compared with that of the surrounding normal skin, the former was always increased and that of cafe au lait spot was higher than that of nevus spilus. Giant pigment granules were recognized only in 6 patients out of 14 with neurofibromatosis but not in nevus spilus examined. 2 days after UV irradiation at 4 MED, the number of melanocytes was increased in both surrounding normal skin and pigmented lesion, and the rates of increase were lower in the pigmented lesion. Under the electron microscope, melanocytes in cafe au lait spots which received an ultraviolet light irradiation showed various changes in their cytoplasm ; a development of dendrites contain ing many mature melanosomes, an increased number of cytoplasmic vacuoles and mitochondria, a development of Golgi apparatus in their cytoplasm, appearances of some dense-bodies and of autophagosomal melanosome-complexes. In nevus spilus, the same kind of changes occurred, but they were moderate com pared with those developed in cafe au lait spots. Melanosomes in the keratinocytes of cafe au lait spots tended to come together around the nucleus and to form melanosome-complexes; while, melanosomes in the keratinocytes of nevus spilus seemed to be single-dispersed after irradiation. The causative factors of the hyperpigmentation and the different reactivity of melanocytes against UV irradiation in these two pigmented macules were discussed. cafe au lait spot; nevus spilus; melanocyte; keratinocyte; UV irradiation In clinical appearances, cafe an lait spots of the patients with neurofibromatosis are so similar to the pigmented macules of nevus spilus that it may be impossible to distinguish one from the other without finding of neurofibroma. It may be assumed, however, that the nevus spilus, which has been considered to be only a hyperpigmentation in the basal cell layer without malformation, is different in pathological changes of melanocytes and keratinocytes from cafe au lait spots which has been recognized as a part of the syndrome of neurofibromatosis. Johnson and Charneco (1970) revealed the differences in the number of melanocytes between cafe au lait spots in the patients with neurofibromatosis and the same sort of spots without neurofibromatosis. They concluded that an increased number of melanocytes and the presence of giant pigment granules were Received for publication, September 30, * Director: Prof. Makoto Seiji. 255

2 256 M. Takahashi invaluable aids in establishing the diagnosis of neurofibromatosis when only pigmented macules were present. According to the recent electron microscopic study by Jimbow et al. (1973), these giant pigment granules in neurofibromatosis had many similarities with the normal melanosomes and were called "macromelanosome". And Silvers et al. (1974) reported the cases of cafe au laic spots without giant pigment granules. Konrad et al. (1974) reported that the giant granules were also present in nevoid pigmented lesions. Thus, the giant pigment granules are not considered to be specific for cafe au laic spots of neurofibromatosis. The purpose of this study is to clarify the differences between cafe au lait spots in neurofibromatosis and the pigmented macules of nevus spilus. Patients MATERIAL AND METHODS 14 Japanese patients with neurofibromatosis and 9 Japanese patients with nevus spilus were selected. These patients fulfilled the diagnostic criteria for nevus spilus and neurofibromatosis in respects of clinical features and histopathological findings. And, 9 patients with nevus spilus had only one or two macules on their entire skin surface and no any other malformations. Preparation of the Dopa-whole mount specimen The skin specimens were taken through the biopsies with a surgical knife including surrounding normal skin. The following preparations were all carried out on both normal and pigmented lesions. After stripping off the horny layer with a scotch tape, skin biopsies were performed with a surgical knife under a local anesthesia of 1% xylocain solution. Separation of the epidermis from the dermis and Dopa-staining were performed with the methods described by Mizuno (1968b), or described originally by Staricco and Pinkus (1957). The specimens were placed in 2 N sodium bromide solution at 37 Ž for 3 1/2 hr. Then the epidermis was separated and washed in physiological saline for a few min. The epidermal sheets were then incubated in 0.1 % DL-Dopa in M/15 phosphate buffer solution, ph 7.3 at 37 C for 4 1/2 hr and during incubation the Dopa-solution was renewed a few times. The specimens were fixed in 10% neutral formaldehyde solution overnight, then washed in physiological saline and dehydrated in a series of ascending concentrations of alcohol. After cleaned in a xylene solution the specimens were mounted, the basal layer up, on glass slides. The counting of the number of melanocytes was made with the standard light micro scope at a magnification of 400 times. The width of microscopic field was measured with an objective micrometer and the number of melanocytes was estimated per mm2 of surface areas of the skin in plane projection. The calculations of Dopa-positive melanocytes were based on five readings, corresponding to mm2. The standard deviation of the mean is included in the Table. Effects of ultraviolet (UV) irradiation on the number of melanocytes in the normal and the pigmented lesions of cafe au lait spots and nevus spilus In order to examine the influence of UV irradiation on the melanocytes, the pigmented lesions and the normal skin areas were irradiated with the xenon lamp. The minimum erythema dose (MED) for each patient was estimated on the normal skin area as near as possible from the pigmented lesion. Effects of UV irradiation on the melanoeytes in the epidermis The following preliminary experiments were performed using the normal back skins

3 Melanocytes in Cafe au lait Spot and Nevus Spilus 257 of the four control persons in order to establish the irradiation procedures. a) The skin specimens were obtained from the irradiated and non-irradiated skin areas 2 days after UV irradiations of 1, 2, 3, 4, 5 and 6 MED. Melanocyte counts were carried out on the Dopa-whole mount specimens. b) On the normal back skin, an UV irradiation of 4 MED was carried out in each different part every day. Then, the skin specimens were taken from these irradiated skin at the end of 1, 2, 3, 4, 5 and 6 days after irradiations. Melanocyte counts were carried out as mentioned above. Comparative studies between cafe au lait spots and nevus spilus on the number of melanocytes after irradiation 7 patients with neurofibromatosis and 5 patients with nevus spilus were selected and they received an UV irradiation of 4 MED on their lesions and surrounding normal skins. The biopsies of the irradiated and non-irradiated lesions, and of the irradiated and non irradiated normal skins were performed 2 days after irradiation. Electron microscopic studies on the irradiated and non-irradiated cafe au lait spots and nevus spilus The specimens were taken from the lesions of 4 patients with neurofibromatosis and 3 patients with nevus spilus 2 days after 4 MED irradiation. Under a local anesthesia, 2 or 3 mm punch biopsies were carried out on the center of irradiated lesions and on the non-irradiated lesions. The tissues were fixed in 2.5% glutaraldehyde in phosphate buffer ph 7.2 for 2 hr at 4 C. Postfixation was carried out with 2 % osmium tetroxide in 0.1 M phosphate-buffered ph 7.4 solution at 4 Ž for 2 hr. After fixed, the tissues were dehydrated in ascending concentrations of ethanol and embedded in Epon 812. The sections were stained with uranyl acetate and lead citrate, and examined with a JEOL 100-B electron microscope. RESULTS Melanocyte counts in cafe au laic spots and nevus spilus Table 1 shows the melanocyte counts in the cafe au lait spots and surrounding normal skin. Table 2 shows those of nevus spilus. In all patients with TABLE 1. Melanocyte counts in cafe au lait spot and surrounding skin of the patients with neurofibromatosis normal Melanocyte counts are presented as an average number per mm2.

4 258 M. Takahashi TABLE 2. Melanocyte counts in pigmented macules and surrounding normal skins of the patients with nevus spilus Melanocyte counts are presented as an average number per mm2. Fig. 1. a: Dopa-stained-melanocytes in the epidermal preparation from the surrounding normal skin of cafe au lait spot. ( ~ 400) b: Melanocytes in the pigmented lesion of cafe au lait spot had many well-developed dendrites and were big in size, but were weaker in Dopa-reaction than in the surround ing normal skin. ( ~ 400) neurofibromatosis, the numbers of melanocytes in cafe au lait spots were more increased than those in the surrounding normal skins. On the other hand, in the patients with nevus spilus, the numbers of melanocytes in the pigmented lesions were almost the same as in the normal skin areas except three cases, which showed a significant increase; 30%, 30% and 15%.

5 Melanocytes in Cafe au lait Spot and Nevus Spilus 259 Fig. 2. a: Dopa-stained-melanocytes in the surrounding normal skin of nevus spilus. ( ~ 400) b : Melanocytes in the pigmented macule of nevus spilus were stronger in Dopa-reaction than in the surrounding normal skin, but they were not so big in size and had a few dendrites. ( ~ 400) Fig. 3. Giant pigment granules recognized in 6 patients with neurofibromatosis. Two giant pigment granules (arrow) are seen in a Dopa-positve cell. ( ~ 400) From the light microscopic observations, melanocytes seen in cafe au lait spots were big in size and possessed many well-developed dendrites, but the Dopa-reac tion was weaker than that of the normal skin (Fig. 1). On the other hand,

6 260 M. Takahashi Fig. 4. The relationship between melanocyte counts and the strength of UV (MED). Fig. 5. The time course of melanocyte counts after UV irradiation of 4 MED. melanocytes in pigmented lesions of nevus spilus were, in general, not so big in size and had a few dendrites, but their Dopa-reaction was stronger than that of the normal skin (Fig. 2). Although giant pigment granules in melanocytes and keratinocytes, as shown in Fig. 3, were found in 6 patients among 14 with neuro fibromatosis, they were never seen in the patients with nevus spilus. Effects of UV irradiation to the melanocytes in the normal skin The relationship between the strength of UV irradiation and the melanocyte counts is shown in Fig. 4. The number of melanocytes 2 days after irradiation rapidly increased up to 2 MED and then became stationary between 2 and 5 MED. The case received 6 MED irradiation formed a blister so that the melanocyte counts could not be made. The time course of melanocyte counts after UV irradiation is shown in Fig. 5. The number of Dopa-positive melanocytes did not change one day after irradia tion and it became 1.3 times as many as the original 3 days after, and thereafter it appeared to be stationary up to 6 days. The observation of Dopa-positive cells 2 days after irradiation indicated that they possessed many well-developed dendrites and became strongly positive in Dopa-reaction (Fig. 6). As the time passes after irradiation, the Dopa-reaction in

7 Melanocytes in Cafe au lait Spot and Nevus Spilus 261 Fig. 6. a: Melanocytes in the non-irradiated normal skin. ( ~ 400) b: Melanocytes 2 days after 4 MED irradiation. They have many well-developed dendrites and are stronger in Dopa-reaction than those of non-irradiated skin. ( ~ 400) melanocytes seems to become stronger. Comparative studies on the number of melanocytes between cafe au lait spot and nevus spilus after irradiation Table 3 shows the number of melanocytes in irradiated and non-irradiated cafe au lait spots and in those normal skins. Melanocyte counts increased in both pigmented lesions and surrounding normal skin after irradiation. The increasing rate in pigmented lesions was, however, smaller than that of surrounding normal skins except 1 case. Changes of the melanocyte counts in 6 patients with nevus spilus 2 days after 4 MED irradiation are shown in Table 4. The number of melanocytes in irradiated skins increased in both pigmented lesions and control normals. The increasing rates in pigmented lesions were, however, smaller than those of surrounding normal skins as seen in the cases of cafe au lait spots. The light microscopic changes of UV irradiated melanocytes in pigmented lesions were quite similar in both pigmented lesions of cafe au lait spots and of nevus spilus. UV irradiation caused an activation of Dopa-reaction and a develop ment of dendrites into the spinous cell layer. These changes were the same as seen in the normal skin.

8 262 M. Takahashi TABLE 3. Melanocyte counts in normal and cafe au lait spot before and after 4 MED UV Irradiation Melanocyte counts are presented as an average number per mm2. TABLE 4. Melanocyte counts in normal and nevus spilus before and after 4 MED UV irradation Melanocyte counts are presented as an average number per mm2. Electron microscopic observation In general, it was hard to recognize the ultrastructural differences between cafe au lait spots and nevus spilus, but there were some differences. Many mature melanosomes were seen in the cytoplasm of keratinocytes in both pigmented lesions, in nevus spilus they were mostly single-dispersed in the cytoplasm of keratinocytes; on the other hand, in cafe au lait spots, melanosomes tended to form complexes and many of them were dispersed in a form of membrane-bounded complex around

9 Melanocytes in Cafe au lait Spot and Nevus Spilus 263 the nucleus. There were well developed Golgi apparatus in the cytoplasm of melanocytes and many relatively electron dense particles, which seemed to be Golgi vesicles. These characteristics seemed to be almost the same in cafe au lait spot and nevus spilus. The giant pigment granules were also found in the electron micrographs of cafe au lait spots. They were recognized in both melanocytes and keratinocytes. As shown in Fig. 7, the giant pigment granule was seen as a round, electron-dense particle in the cytoplasm of a keratinocyte. It corresponded to the granule found in the Dopa-split specimen of cafe au lait spots in size and shape (Fig. 3). No granules like this were found in nevus spilus. Electron microscopic observation after irradiation When the epidermis of cafe au lait spots 2 days after UV irradiation were examined in the electron microscope, it was found that melanocytes and keratinocytes changed a great deal. In contrast to the non-irradiated lesions, there was an enlargement of intercellular space of adjacent spinous cells, where these cells had many cytoplasmic villi and processes. The clear cells thought to be melanocytes were more frequently encountered in the basal layer. They had many membrane-bounded vesicles, mitochondria and occasional irregular dense bodies in their cytoplasm. Melanosomes in melanocytes were single-dispersed but those in the keratinocytes came together around the nucleus and tended to form melanosome-complexes (Fig. 8). Melanocytes in the basal layer had many well-developed dendrites spreading into the intercellular spaces of the spinous cell layer. In the peripheral parts of these dendrites, there were many single mature melanosomes (Fig. 9). In some parts, there were seen some damaged melanocytes, in which the nuclei were shifted in the peripheral area and the cytoplasms contained many large lysosomal vesicles, mitochondria and autophagosomal melanosome-complexes (Fig. 10). These cells seemed to be in degeneration process due to the UV damage. In cases of nevus spilus, the electron micrography revealed that slight changes occurred after irradiation. In the spinous cell layer, keratinocytes had an increased number of melanosomes and intercellular spaces were enlarged and filled with many cytoplasmic villi and/or processes. Melanocytes in the basal cell layer had an increased number of Golgi vesicles, rough endoplasmic reticulum, premelanosomes and mitochondria (Figs. 11 and 12). Melanocytes had a few dendrites which were not so well developed. In the keratinocytes at basal and supra-basal layers, there were also an increased number of melanosomes and the distribution pattern of melanosomes was mainly single-dispersed (Figs. 11 and 12). As a whole, the changes in nevus spilus appeared to be not so dominant. DISCUSSION When the pigmentary disorders of the skin are seen, one must consider the following points as causative factors : 1. changes in the number of melanocytes, 2.

10 264 M. Takahashi alteration in production of melanosomes, 3. alteration in melanization of melano somes, 4. disturbance in melanosome transfer, 5. alteration in degradation process of melanosomes in melanocytes and keratinocytes, and 6. abnormal proliferation of melanocytes. At first, histochemical studies were carried out on the cafe au lait spots anc the nevus spilus in order to clarify the changes in the number of melanocytes. Although the number of nielanocytes was increased in both pigmented lesions, the increasing rate in cafe au lait spots was greater than that in nevus spilus. Johnson and Charneco (1970) revealed that the melanocyte counts were less in nevm spilus than in the normal skins and they were more in cafe au lait spots. Therefore, the results obtained were inconsistent with those of Johnson and Charneco with respect to melanocyte counts; they were not increased in 213 of the patients with nevus spilus. It is of interest that the hyperpigmentation in nevus spilus was not due to the increase of the melanocyte number in the basal layer. In fact, the Dopa-reaction was much stronger in nevus spilus than in the surrounding normal skins, while it was the same or weaker in cafe au lait spots than in the normal skins. Referring to these facts, it may be assumed that the increase of melanocytes in number is a main cause of brown hyperpigmentation of cafe au lait spots (Seiji et al. 1974), but not of nevus spilus. Therefore, some other mechanisms may be involved in nevus spilus. Szabo (1959) first observed the presence of giant pigment granules in the melanocytes and keratinocytes in the epidermis of the patients with neurofibro matosis. Benedict et al. (1968) reported that giant pigment granules were seen only in cafe au lait spots with neurofibromatosis, but not in the similar pigmented spots such as seen in Albright's syndrome. Johnson and Charneco (1970) also observed that these granules were found only in some but not in all of cafe an lait spots of neurofibromatosis, and never found in any of nevus spilus. Therefore, these granules were considered to be specific in cafe au lait spots of neurofibro niatosis. However, in 1974, Hirone et al. found these granules to be present in the keratinocytes of the lentigines lesion of the sole of the foot, and recently Konrad et al. (1974) also demonstrated these granules in so-called nevus spilus and called them "giant melanosomes". Therefore, giant pigment granules are no longer considered to be a specific granule for cafe au lait spots of neurofibromatosis. According to the results obtained here, giant pigment granules were found only in 6 patients among 14 with neurofibromatosis and never found in nevus spilus examined. Jimbow et al. (1973) studied these granules with the electron microscope and called them "macromelanosomes", because of many similarities with normal melanosomes. They presumed these granules were made in melanocytes and transferred to keratinocytes as a single unit after maturation. It is not known at present why they are not found in all cases of cafe an laic spots and never found in any cases of nevus spilus examined, the similar pigmented skin disorder. Further studies are needed as to how these granules are made in epidermal cells and as to what roles they play there.

11 Melanocvtes in Cafe au lait Snot and Nevis Snilus 2 When the normal skin was irradiated with UV light, melanogenesis of individual melanocytes was activated and the melanocytes increased in number. And also melanocytes became larger in size and to show well-developed dendrites, as have been observed (Pathak and Epstein 1971; Pathak et al. 1971; Mizuno 1968a, c). There are two explanations for the increase of Dopa-positive melanocytes after UV irradiation: the one is that melanocytes increased through the cell divis ion, and the other the Dopa-negative melanocytes became positive through the UV irradiation. It is not known at the present time which function is actually work ing (Seiji et al. 1973). IN irradiation performed to the cafe au lait spot and nevus spilus lesion was to elucidate the reactivities of melanocytes in these lesions against the UV irradiation. The preliminary experiments were carried out on the normal back skin to seek the most suitable conditions for studying the UV effects on melanocytes in pigmented skin lesions. The number of melanocytes increased in proportion to the strength of UV irradiated up to 2 MED level and thereafter it became stationary until 5 MED. The time course of the melanocyte counts after UV irradiation showed that Dopa-positive cells increased in number up to 2 or 3 days after UV irradiation and thereafter they stayed stationary until 5 days. Dopa-reaction in melanocytes gradually became stronger as the time passed after 2 days. Thus, it may be supposed that the first reaction of melanocytes against UV irradiation is the increase in number followed by the activation of the Dopa reaction. These results seem to be different a little from those of Mizuno (1968c). According to him, in Caucasians, the increase in melanocyte counts after irradiation occurred slightly later than 3 days. These preliminary experiments indicated that the best dose to be given was 4 MED and skin specimens were to be taken on 2 days after irradiation. Then, histochemical and electron microscopic studies were carried out on the lesions and the surrounding normal skins under these conditions determined above. Histochemical studies indicated that melanocyte counts increased by UV irradia tion in both pigmented lesions and the normal skin, but the rate of increase was smaller in the former than in the latter. Although the real reasons why the behaviors of melanocytes in these pigmented lesions differ from those of surrounding normal skin, are not known, the following two aspects may be considered: Minimum erythema dose would be higher in a pigmented lesions than in the normal skin area, because of the protective effects of melanin which is present in the pigmented lesions. The other would be that the threshold in the reactivity of melanocytes against UV irradiation is higher in the pigmented lesions than in the normal skin. The former, however, does not seem to play a major role, because the preliminary experiment showed that the increase rate of melanocyte number after irradiation was almost stationary from 2 to 5 MED (Fig. 4). Therefore, if 5 MED were given to the pigmented lesion the increase rate of melanocyt8 number would not be changed. Szabb (1966) examined the changes of melanocytes after a single and multiple exposures of UV and concluded that the changes occurred in melanocytes after a single exposure were not so significant as those in the multiple

12 266 M. Takahashi exposures, since there may be a drop or rise in melanocyte counts after a single exposure. It will be a prospective subject how melanocytes change in these pigmented lesions after receiving the multiple exposures of UV. These electron microscopic observations of non-irradiated lesions showed that it was hard to recognize differences in melanocytes between these two pigmented lesions, but in numbers and in the distribution pattern of melanosomes in keratinocytes, there was a little difference. Melanosomes in the keratinocytes of nevus spilus were mostly single-dispersed in the cytoplasm around the nucleus, while those of cafe au lait spots tended to form complexes in which the figures of degrading melanosomes were seen. These observations may suggest that the degradation of melanosomes in the keratinocytes of nevus spilus may be harder to take place than in the cafe au lait spots. Therefore, one of the causative factors of hyperpigmentation in nevus spilus is assumed to be the increased number of melanosomes in the keratinocytes due to the disturbance in degradation of melanosomes. Toda et al. (1972) demonstrated that the changes occurred in melanosomes of melanocytes and keratinocytes after a single exposure of long wave ultraviolet light irradiation ( nm) with the application of 4, 5', 8-trimethyl psoralen (TMP). Large melanosomes which are usually not seen in the Caucasoid skin were produced and they were transferred to the keratinocytes where they were single-dispersed. Wolff and Konrad (1972) indicated that the distribution pattern of melanosomes in the keratinocyte depends on the size of melanosomes. The results obtained here revealed that the distribution pattern of melanosomes changed after UV irradiation. Melanosomes in cafe au lait spots were mostly found to be melano some-complexes, but those in nevus spilus were frequently single-dispersed. How ever, the size of melanosomes did not seem to be so different in both pigmented lesions. The changes occurred in the normal skin after UV irradiation were almost the same as described by the previous authors (Nix 1967; Pathak and Epstein 1971; Pathak et al. 1971). Although many changes were observed in both pigmented lesions after W irradiation, the degree of changes in cafe au lait spots seemed to be greater than those in nevus spilus. It is supposed that the melanosomes are produced more abundantly in the melanocytes in cafe au lait spots and transferred into the keratinocytes where they are degraded after formed the melanosome complexes. On the other hand, melanosomes are not produced so many in the melanocytes of nevus spilus as in the cafe au lait spots, but the destruction of melanosomes does not appear to take place in the keratinocyte of nevus spilus. Jimbow and Fitzpatrick (1975) examined the changes of melanocytes and melanosomes after the irradiation of long-uv ( nm) plus visible light ( nm). The immediate tanning reaction of melanocytes after irradiation was a marked elongation and elaboration of the dendritic processes of the melanocytes, b ut 24 hr after irradiation the dendritic processes, which had been markedly elongated immediately after irradiation, became less developed and contracted.

13 Melanocytes in Cafe au lait Spot and Nevus Spilus days after irradiation may correspond to the beginning of the delayed tan ning stages. It is not known how the immediate tanning reactions affect on the melanocytes in these pigmented lesions. According to these results, it is assumed that the increased number of melanocytes may play an important role in the hyperpigmentation of cafe au laic spots, while the degradation of melanosomes in the keratinocytes may not be easier to take place in the pigmented lesions of nevus spilus. Acknowledgments This work was supported by the Cancer Research Grant (1974) from the Ministry of Education, Japan, and the Japan O'Leary Pigment Research Funds, Tokyo. References 1) Benedict, P.H., Szabo, G., Fitzpatrick, T.B. & Sinesi, S.S. (1968) Melanotic macules in Albright syndrome and in neurofibromatosis. J. Amer, med. Ass., 205, ) Hirone, T., Fukuda, S. & Fukushiro, R. (1974) Lentigines of palm and sole. Rinsho Derma (Jap.), 14, ) Jimbow, K. & Fitzpatrick, T.B. (1975) Changes in distribution pattern of cytoplasmic filaments in human melanocytes during ultraviolet-mediated melanin pigmentation. J. Cell Biol., 65, ) Jimbow, K., Szabb, G. & Fitzpatrick, T.B. (1973) Ultrastructure of giant granules (macromelanosomes) in the cutaneous pigmented macules of neurofibromatosis. J. invest. Derm., 61, ) Johnson, B.L. & Charneco, D.R. (1970) Cafe au lait spot in neurofibromatosis and in normal individuals. Arch. Derm., 102, ) Konrad, K., Wolff, K. & Honigsmann, H. (1974) The giant melanosome: A model of deranged melanosome-morphogenesis. J. Ultrastruct. Res., 48, ) Mizuno, N. (1968a) Epidermal changes after UV irradiation. Rinsho Derma (Jap.), 10, ) Mizuno, N. (1968b) A new method for Dopa whole mount specimen. Jap. J. clin. Derm. (Jap.), 22, ) Mizuno, N. (1968c) Behavior of melanocyte after single ultraviolet irradiation. Jap. J. elin. Derm. (Jap.), 22, ) Nix, T.E. (1967) Ultraviolet-induced changes in epidermis. In: Ultrastructure of Normal and Anbormal Skin, edited by A.V. Zelickson, Lea & Febiber Press, Philadelphia, pp ) Pathak, M.A. & Epstein, J.H. (1971) Normal and abnormal reactions of man to light. In: Dermatology in General Medicine, edited by T.B. Fitzpatrick et al., McGraw-Hill Book Company, New York, pp ) Pathak, M.A., Hori, Y., Szabo, G. & Fitzpatrick, T.B. (1971) The photobiology of melanin pigmentation in human skin. In: Biology of Normal and Abnormal Melanocytes, edited by T. Kawamura et al. Univ. Tokyo Press, Tokyo, pp ) Seiji, M., Takahashi, M. & Tanahashi, Y. (1973) The sun-protection mechanisms of human epidermis. In: Proceedings of the International Conference on Photosensitization and Photoprotection, edited by T. Kobori & T. Yasuda, Kanehara Shuppan, Tokyo, pp ) Seiji, M., Saito, N., Takahashi, M., Uzuka, M., Okazaki, K., Morikawa, F. & Toda, K. (1974) Recent problem on melanin metabolism. Korean J. Derm., 12, ) Silvers, D.N., Greenwood, R.S. & Helwig, E.B. (1974) Cafe au lait spots without giant pigment granules. Arch. Derm., 110, ) Staricco, R.J. & Pinkus, H. (1957) Quantitative and qualitative data on the pigment cells of adult human epidermis. J. invest. Derm., 28,

14 268 M. Takahashi 17) Szab6, G. (1959) Quantitative histological investigations on melanocytes system of human epidermis. In: Pigment Cell Biology, edited by M. Gordon, Academic Press, New York, pp ) Szab6, G. (1966) Photobiology of melanogenesis: Cytological aspects with special reference to differences in racial coloration. In: The Pigmentary System, edited by W. Montagna & Funan Hu, Pergamon Press, Oxford, pp ) Toda, K., Pathak, M.A., Parrish, J.A. & Fitzpatrick, T.B. (1972) Alteration of racial differences in melanosome distribution in human epidermis after exposure to ultraviolet light. Nature New Biol., 236, ) Wolff, K. & Konrad, K. (1972) Phagocytosis of latex beads by epidermal keratino cytes in vivo. J. Ultrastruct. Res., 39, Fig. 7. Giant pigment granule (G) in cafe au lait spot as revealed by electron microscopy. It looks like a round, electron-dense particle seen in the cytoplasm of keratinocyte (Kc). Me, melanocyte; BL, basal lamina. ( ~ 9,000) Fig. 8. Melanocyte in cafe au lait spot 2 days after 4 MED irradiation. Melanosomes in melanocyte were mostly single-dispersed, but those in keratinocyte come together and tend to form complexes. ( ~ 10,000)

15 Melanocytes in Cafe au lait Spot and Nevus Spilus 269

16 270 M. Takahashi Fig. 9. Melanocyte in the basal layer of cafe au lait spot 2 days after 4 MED irradiation. A well-developed dendrite extends into the intercellular space of keratinocytes. Many melanosomes are seen in the dendrite (arrow). ( ~ 10,000) Fig. 10. A melanocyte in the cafe au lait spot 2 days after 4 MED irradiation. The nucleus (N) is constricted and the cytoplasm includes many large lysosomal vacuoles, mitochondria and an autophagosomal melanosome-complex (Ag). This cell is recognized as a damaged cell. Kc, keratinocyte. ( ~ 10,000)

17 Melanocytes in Cafe au lait Spot and Nevus Spilus 271

18 272 M. Takahashi Fig. 11. Melanocyte (Me) and keratinocyte (Kc) in the pigmented macule of nevus spilus 2 days after 4 MED irradiation. There are a little increased number of mitochondria, melanosomes, rough endoplasmic reticulum and Golgi vesicles. But changes in the melanocyte seem to be not so dominant as those of cafe au lait spot. ( ~ 10,000) Fig. 12. Melanocyte (Me) and keratinocyte (Kc) in nevus spilus 2 days after 4 MED irradiation. Many melanosomes in keratinocyte are single-dispersed around the nucleus. ( ~ 9,500)

19 Melanocytes in Cafe au lait Spot and Nevus Spilus 273