The use of ultraviolet light to induce melanogenesis in the epidermis of the rhesus monkeyAn ultrastructural and biochemical study.код для вставкиСкачать
The Use of Ultraviolet Light to Induce Melanogenesis in the Epidermis of the Rhesus Monkey: An Ultrastructural and Biochemical Study ' f 2 KENT L. ERICKSON3 Department of Cutaneous Biology, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, Oregon 97005 ABSTRACT The general body epidermis of the rhesus monkey (Macaca mulatta) contains no discernible melanocytes, but after repeated ultraviolet irradiation DOPA-positive melanocytes appear and increase numerically up to 30 exposures. With continued irradiation, however, the number again declines. Experiments to determine how melanogenic activity, assayed by the incorporation of labeled DOPA or tyrosine, is related to DOPA positivity indicated that biochemical activity corresponded to the histochemical pattern. Ultrastructural studies demonstrated that after the exposure to ultraviolet light, a pool of indeterminate cells in the skin of rhesus monkeys developed into melanocytes. The melanosomes formed by these cells, however, differed from the eumelanin melanosomes described in other species; they had no internal filamentous matrix with periodicity but appeared similar to phaeomelanin melanosomes. Long term ultraviolet light irradiation may damage keratinocytes and render them incapable of phagocytizing melanosomes. The pigmentary reaction to chronic ultraviolet exposure has already been described in the rhesus monkey (Macaca mulatta) by Erickson and Montagna ('75). They have shown that although the general body epidermis of the rhesus monkey has no discernible melanocytes, histochemically demonstrable DOPA-positive melanocytes appeared after sequential ultraviolet (UV) irradiation, increased to peak numbers after 30 exposures, then steadily declined to basal level. The experiments reported here were designed to determine how ultrastructural features and melanogenic activity in skin sequentially irradiated with UV light are related to DOPA positivity. mals received no irradiation. The spectral region of the lamp was 185 nm to 313 nm. Before irradiation, biopsies were removed from all animals, and 14 times during the exposure period nine biopsies were removed from three randomly selected animals (Erickson and Montagna, '75). MATERIALS AND METHODS Electron microscopy Blocks of tissue, 1 to 2 mm2, were fixed directly at 4°C in buffered 1% OsOc (pH 7.2) (Palade, '52) or 2.0% paraformaldehyde plus 2.5% glutaraldehyde solution in 0.03 M Millonig's phosphate buffer (pH 7.2) (Russell, '72), washed in 0.16 M phosphate buffer then postfixed in 1% Os04 in 0.08 M phosphate buffer. Tissues were dehydrated through graded ethanol to propylene oxide and embedded (Spun, Irradiation methods These studies were conducted on eight adult male rhesus monkeys (Macaca mulatta). After the hair had been removed, the thorax and abdomen of six animals were exposed five times per week to 7.0 X lo5 pw/cm2 of UV light (Hanovia Aero kromayer UV light) for 83 doses; two ani- Received July 7, '75. Accepted Oct. 7, '75. 1 Publication No. 818 of the Oregon Regional Primate Research Center supported in part by Public Health Service, National Institutes of Health Grants RR00163 of the Animal Resources Branch, and AM 08445 of the National Institute of Arthritis and Metabolic Diseases. ZPresented. in uart. at the 87th Annual Session of the American Association of Anatomists, Cleveland, Ohio, 1974. (Anat. Rec., 178: 351, 1974). 3 Present address: Department of Human Anatomy, University of California. School of Medicine, Davis, California 95616. ANAT. REC., 184: 637-646. 637 638 KENT L. ERICKSON '69). One-micron sections were stained with toluidine blue. Sections for electron microscopy were stained with lead citrate and uranyl acetate and viewed with a Philips 200 electron microscope operating at 60 KV. Biochemical studies After 10, 13, 18, 23, 28, 32, 38, 43 53, and 68 exposures, four 1 x 3 cm elliptical biopsies were taken from two randomly selected animals, one of the two nonirradiated controls and one of the six irradiated animals, Tyrosinase activity was measured with a modified technique described by Kitano and Hu ('71) for determining melanin synthesis. Epidermal sheets were prepared by incubating full-thickness biopsies in 2 N NaBr for one and one-half hours at 37°C and separating the epidermis from the dermis. The sheets were blotted dry, weighed, and incubated in TC Hanks solution (Difco Labs) for 30 minutes at 37°C. For a control, M 1-phenyl-2-thiourea was used. This compound inhibits the deposition of melanin on the internal matrix of the melanosome by complexing with the copper present in tyrosinase. To the solution, 0.5 &i/ml, DL-3, 4-dihydroxyphenylalanine-2-C" (DOPA) (New England Nuclear, spec act 9.31 mCi/mM) was added for two hours at 37"C, after which the samples were incubated in a 0.1% DOPA solution for 15 minutes. The samples labeled with c'"tyrosine were incubated with 0.5 pCi/ml L-tyrosine-l-Ci4 (New England Nuclear, spec act 53.5 mCi/mM) for two hours at 37"C, then postincubated in 0.1% aqueous solution of tyrosine. For a control, 1-phenyl-2-thiourea and/or 100 nig/ml puromycin were added. (The latter was used to inhibit gener a1 protein synthesis. ) The epidermal sheets were homogenized in a blender and trichloracetic acid (TCA) was added to the homogenate for a final TCA concentration of 5% ; samples were placed in an ice bath overnight. The resulting precipitates were collected on millipore filters (HAWP25) and washed first with cold 5% TCA and then absolute isopropanol. Dry filters were placed in glass scintillation vials containing toluene and omnifluor (New England Nuclear) and counted in a liquid scintillation spectrom- eter. The dermis was processed exactly like the epidermis except that before homogenation it was frozen in liquid nitrogen and pulverized in a cold stainless-steel cylinder, The rate of incorporation of C14-DOPA or Ci4-tyrosine puromycin into ultraviolet-stimulated epidermis was compared with that of controls (the above labeled compounds plus phenylthiourea) . The difference is reported as the rate of melanin synthesis. + RESULTS Ultrastructural study Ultrastructural investigations confirmed the earlier histological observations (Erickson and Montagna, '75) that no melanocytes were present in the general body epidermis of the nonirradiated adult rhesus. Most epidermal cells were ker atinocytes , but two types of dendritic cells were also present, Langerhans cells and indeterminate cells. The former were found in all strata of the Malpighian layer but most frequently in the spinous layer. These cells contained microfilaments and had irregular, indented nuclei and characteristic Langerhans granules which, like the electron-opaque bodies, were randomly dispersed (fig. 1). Since acid phosphatase activity has been demonstrated in these latter structures (Rowden, '67; Wolff, '67), they may be lysosomes. Like other dendritic cells, the indeterminate cells possessed similar features, e.g., microfilaments which do not aggregate into bundles but lack the defining cytoplasmic organelles, Langerhans granules or melanosomes, and desmosomes. These cells usually had indented nuclei but lacked the typical electron-opaque bodies seen in the Langerhans cells. Vesicles were sometimes Fig. 1 A Langerhans cell in the epidermis of nonirradiated skin. The surrounding keratinocytes contain tonofibrils whereas the Langerhans cell has distinct microfilaments (MF). Characteristic of this cell is the Langerhans granules (LG). Numerous lysosomes ( L ) or electron opaque bodies are present. x 30,000. Fig. 2 The indeterminate cell in the epidermis of nonirradiated skin. Neither Langerhans granules nor melanosomes are present. This cell lacks the typical electron opaque bodies shown in the previous figure but contains well-defined microfilaments (arrows 1. x 19,000. MELANOGENESIS IN THE RHESUS MONKEY 639 640 K E N T L. ERICKSON observed in the cytoplasm, more often in the basal layer, only occasionally in the spinous layer. Though less common than Langerhans cells, these cells constituted a sizable component of the normal epidermis in the rhesus monkey (fig. 2). After four irradiations, a few active melanocytes with all stages of melanosome formation were visualized. Most developing melanosomes (stages I and 11) lacked a n organized internal matrix, but a few had a filamentous network (fig. 3). Melanization seemed to derive from the deposition of a central core of flocculent material which gradually filled the membrane-limited vesicle until it became uniformly electron opaque (fig. 3 ) . After four irradiations, a few fully developed melanosomes (stage IV) were seen. During this period, very few single melanosomes and no membrane-bounded complexes were transferred to the surrounding keratinocytes. Stimulated melanocytes had an active Golgi apparatus and an extensive rough endoplasmic reticulum, Numerous small vesicles (50-80 n m ) were also dispersed throughout the cytoplasm. Mitotic figures were observed in the keratinocytes of the basal layer but not in the developing melanocytes or indeterminate cells. After 15 exposures, the increased population of melanocytes contained all stages of melanosome formation, but at this stage developing melanosomes were less numerous than after four exposures. A small number of indeterminate cells had active Golgi zones and many small vesicles (40150 nm). The stimulated melanocytes occasionally contained partially melanized melanosomes or clumps of smaller particles which resembled melanosomes and were similar to phaeomelanin (fig. 4). After 25 exposures, a few melanosomes were transferred to the surrounding keratinocytes, the rest remaining near the periphery of the melanocytes. At peak activity (30 exposures), the melanocytes contained fully developed melanosomes (stage I V ) but no clearly discernible early-stage melanosomes (stages I and 11). The rough endoplasmic reticulum was not as active as before; often very few or no Golgiassociated vesicles could be seen (fig. 5). After 37 exposures, the melanocytes contained only a few melanosomes (fig. 6) and showed no signs of extensive activity; however, no signs of damage were apparent, In specimens which had received additional irradiation, the population of indeterminate cells seemed to be increasing. These cells were quite similar to those in untreated epidermis except that they had more microfilamen t s. Biochemical study In general, the uptake of C'*-DOPA or C'*-tyrosine plus puromycin by melanosomes was similar (fig. 7): with sequential irradiation, the incorporation of melanin precursor rose to a peak; with continued irradiation, it decreased to a basal level, The maximum uptake and the length of time it took to incorporate them differed for both compounds, but both had a single peak. In addition, phenylthiourea did not completely inhibit melanin synthesis. In figure 8, only the rate of melanin synthesis (i.e., the rate of incorporation of C"-DOPA, minus the rate of CI4-DOPAplus phenylthiourea) is given. It indicates a pattern similar to that shown by the histochemical data, i.e., an increase to Deak activity followed by an equally rapid decrease. DISCUSSION The technique used for determining the rate of melanin synthesis is probably sensitive enough to demonstrate melanogenic activity when small numbers of melanocytes are present. But one problem with this technique is that phenylthiourea does not completely inhibit tyrosinase activity even though Lerner and his co-workers ('50) reported it to be the most effective inhibitor available. Kitano and Hu ('71) also reported that it is virtually impossible to abolish tyrosinase activity with inhibitors without simultaneously imparing protein synthesis. Protein synthesis, but apparently not melanoprotein synthesis, is inhibited by puromycin. Moreover, the cessation of protein synthesis does not affect Fig. 3 A dendrite of a melanocyte. Several stages of melanosomes (11-IV) are present. The early stage melanosomes generally lack an organized internal matrix although occasional filamentous networks are seen (single arrow). Melanization appears to be a deposition of a flocculent material (double arrows). A few completely developed melanosomes ( I V ) are found within the melanocyte. x 35,000. MELANOGENESIS I N THE RHESUS MONKEY Figure 3 64 1 642 KENT L. ERICKSON Fig. 4 A n epidermal melanocyte after 21 exposures. Some melanosomes (arrow), which appear morphologically similar to phaemelanin, exhibit incomplete melanization. x 14,000. Insert x 36,000. tyrosinase activity (Kitano and Hu, '71). In my experiments, the level of inhibition by phenylthiourea, which was of the same magnitude as that reported by Kitano and Hu ('71), varied according to the number of ultraviolet exposures, but the activity was never completely inhibited. The pattern of biochemical activity parallels that of histochemical data. Why the peak incorporation of C14-DOPA or of C"-DOPA plus inhibitors occurs at 33 days instead of at 28 days when the difference between the two is maximal is not known. This part of the study demonstrates that there is no compensation on the part of enzyme activity, i.e., when the number of morphological units decreases, so does the total amount of enzyme activity. There is no consensus about the origin of DOPA-positive cells after UV irradiation. After irradiating several species of vertebrates, a number of investigators (Sato and Kawanda, '72a, '72b; Snell, '63; Wolff and Winkelmann, '67) concluded that the DOPA-negative dendritic cells at the dermo-epidermal junction are amelanotic melanocytes and that the increase in DOPA-positivity is partly due to the activation of these normally inactive cells. Sat0 and Kawanda ('72a) based their conclusion on an experiment in which 1.1% of the DOPA-positive cells were labeled with H3-thymidineduring ultraviolet irradiation. On the basis of mitotic figures and incorporation of H3-thymidine within the melanocytes, Quevedo et al. ('63), however, concluded that division accounts for at least part of the population increase in the melanocytes of irradiated pedal skin. Mishima and Widlan ('67) substantiated the latter hypothesis. In my experiments, exposure to ultraviolet light stimulated a pool of indeterminate cells in the epidermis of the hairy skin to form melanocytes. This conclusion is based on the observation that significant numbers of indeterminate cells are found in nonirradiated skin and in skin biopsied MELANOGENESIS I N THE RHESUS MONKEY 643 Fig. 5 A melanocyte with the surrounding keratinocytes after 30 exposures. The melanosomes are fully developed. Although two stage I11 melanosomes are present (arrows), no earlier forms are evident. x 24,000. 644 KENT L. ERICKSON Fig. 6 A melanocyte in the basal layer of the epidermis after 37 exposures. Only a few melanosomes are present (arrow). x 19,000. after peak melanogenic activity, but virtually none at the peak of melanogenic activity. Other authors have arrived at similar conclusions. Zelickson and Mottaz ('68) suggested that indeterminate cells represent either a form of premelanocyte in which melanin synthesis can be induced or an effete melanocyte which has ceased to function. If the latter, then indeterminate cell should also be found in the upper layers of the epidermis; but as I have shown in these studies, they axe predominantly in the lower layers. Zelickson and Mottaz ('68) also thought that indeterminate cells are undifferentiated cells that give rise to Langerhans cells, melanocytes, or completely unrelated cells. Later they reported that a decrease in indeterminate cells as well as an increase in the mitotic activity of existing melanocytes accounted in their study for the increase in melanocytes (Zelickson and Mottaz, '70). After two weeks of daily ultraviolet irradiation, no Langerhans or indeterminate cells were observed. Another group of investigators (Tsuji et al., '69) hypothesized that indeterminate cells are undifferentiated cells which may give rise to another epidermal dendritic type cell or that melanocytes and Langerhans cells can transform into each other through indeterminate cells. The indeterminate cell may also represent a form of premelanocyte in which melanin synthesis can be induced, but whether any Langerhans cell-melanocyte transformation can occur is doubtful. On the basis of morphological and experimental data, the latter two are probably separate cell populations in the epidermis (Breathnach et al., '68); therefore, the transformation hypothesis is not cogent. Although these experiments do not explain what happens to the formed melanosomes and why the melanocytes suddenly stop forming them, some conclusions can be drawn. Melanocytes, melanosomes, and keratinocytes are closely related (Fitzpatrick and Breathnach, '63; Hadley and Quevedo, '66, ' 6 7 ) , and the latter probably MELANOGENESIS IN THE RHESUS MONKEY 645 Melanin Synthesis in Melanocytes of Irradiated Skin 1000- -- 900- 000 C'*-DOPA -___-_C'*- DOPA + Ph. - - C''-Tyrosine+Pu. I 700 - -.-.-. C'*-Tyrosine+Pu.+Ph i 2 600- g 500- a, 1 -2 % 400- 300 200 - 100- 10 20 30 40 50 60 Number of Ultraviolet Light Irradiations 70 Fig. 7 Uptake of labeled melanin precursors in the epidermis after exposure to 7.0 x 105 pw/cm2 uv light. Bmchets indicate standard error of the mean. Ph., phenylthiourea, h, Puromycin. Melanin Synthesis in Melanocytes of Irradiated Skin (C'=DOPA)-(C"-DOPA + Ph.) 250 r l 10 , l , 20 Number of l . 30 l 40 , l 50 , l , 60 l 70 Ultraviolet Light Irradiations Fig. 8 Rate of melanin synthesis after sequential daily irradiation. Data are extrapolated from figure 7. phagocytize portions of the melanin-laden dendrites of melanocytes (Fitzpatrick and Breathnach, '63; Cruickshank and Harcourt, '64). Thus, the rate of melanin synthesis within melanocytes may be regu- lated by a feedback mechanism which depends upon the rate at which melanosomes are removed by keratinocytes. This mechanism may function in man, but probably not in the rhesus monkey in which melanosomes are not rapidly transferred to surrounding keratinocytes. In irradiated rhesus skin, no melanosomes or melanocytes pass into the dermis and few or none remain in the epidermal melanocytes. Actually, melanosomes are transferred to keratinocytes but probably at such a slow rate that any new melanosome synthesis is inhibited. This may be a defect in the ability of the keratinocytes to take up the melanosomes. Several other investigators have reported the inability of keratinocytes to take up melanosomes under various pathological conditions. Mitchell ('63) reported that after prolonged solar irradiation of Caucasian skin, melanocytes seemed unable to transmit pigment to the keratinocytes, but very dark skin, like that of aborigines, did not appear to be damaged even by years 646 KENT L. ERICKSON of exposure to the sun (Mitchell, '68). Solar degeneration in xeroderma pigmentosum also prevents pigment transfer from occurring normally (Olson et al., '70). In this condition, some keratinocytes contained little melanin but others showed higher than normal amounts. Likewise, the passage of melanosomes to keratinocytes was inhibited after minor trauma, which had caused intercellular edema. Mottaz et al. ('71) thought that this condition resembles atopic dermatitis or that it is another example of the inability of melanosomes to be transferred to keratinocytes. As I have reported earlier (Erickson and Montagna, ' 7 5 ) , UV light may cause some physical-chemical changes in the epidermis such as cell injury since after successive periods of irradiation and nonirradiation, reirradiation causes active melanocytes to reappear. The exact nature of this injury is difficult to determine until better techniques have been developed. ACKNOWLEDGMENTS I would like to express my gratitude to Drs. William Montagna and Mary Bell for their generous assistance and advice, and to Drs. Funan Hu and W. H. Fahrenbach for their helpful suggestions. LITERATURE CITED Breathnach, A. S., W. K. Silvers, J. Smith and S. Heyer 1968 Langerhans cell in mouse skin experimentally deprived of its neural crest component. J. Invest. Dermatol., 50: 147-160. Cruickshank, C . N. D., and S. A. Harcourt 1964 Pigment donation in vitro. J. Invest. Dermatol., 42: 183-184. Erickson, K. L., and W. Montagna 1975 The induction of melanogenesis by ultraviolet light i n the pigmentary system of rhesus monkeys. J. Invest. Dermatol., 65: 279-284. Fitzpatrick, T. B., and A. S. Breathnach 1963 Das epidermale Melanin-Einheit-System. Dermatol. Wchschr., 147: 4 8 1 4 8 9 . Hadley, M. E., and W. C. Quevedo, Jr. 1966 Vertebrate epidermal melanin unit. Nature, 209: 1334-1335. 1967 The role of epidermal melanocytes in adaptive color change in amphibians. In: Advances in Biology of Skin. Vol. 8. The Pigmentary System. W. 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