The Response of Mammalian Epidermal Melanocytes in Culture to Hormones*

\'l. 48, No. 4 Printed in U.S.A.

Toe JOURNAL OF INVESTIGATIVE DERMATOLOGY

Copyright

1967 by The Williams & Wilkins Co.

THE RESPONSE OF MAMMALIAN EPIDERMAL MELANOCYTES IN CULTURE TO HORMONES* SIDNEY N. KLAUS, M.D. AND RICHARD S. SNELL, M.D., Pu.D.

Changes in the skin color of mammals, ineluding man, have been brought about by the administration of certain hormones (1, 2, 3). The manner in which the hormones act at the cellular level is not yet clear. In this report, the effects of four hormones on normal adult guinea pig melanocytes in tissue culture are described.

The hormones are alpha melanocyte-stimulating hormone (a-MSH), acetylcholine, norepinephrine, and melatonin.

taming hormone. The melanocytes were observed and photographed for periods varying from 2 to more than 6 hours. Eight different cultures were used for each hormone. The concentrations of the various hormones ia the media are listed in Table I. Once a culture had been exposed to a hormone, it was not used again.

The melanin granule dispersion or aggregation

effect of each of the hormone-culture medium solu-

tions was tested by the in vitro frog skin technic (5). RESULTS

MATERIAL5 AND METHODs

Epidermal cells were cultivated from adult red

and brown guinea pig skin according to the

method of Cruickshank (4). Small pinch grafts were taken from the ears after they had been shaved and cleansed with 70 per cent alcohol. The grafts were floated, dermal surface downwards, on

Appeoronees of Cultures During the Control

Period After incubation for 24 hours, the cultures of epidermal cells seemed fairly uniform. Many cells had become attached to the coerglass and appeared flattened. The medium usually was renewed at this stage. During the changeover, cells that had not survived were washed out with the old medium. At 36—48 hours, the keratinocytes

a solution of 0.15 per cent trypsin (Difco 1:250) diluted in calcium- and magnesium-free Tyrode's solution, for 15—20 minutes at 37° C. The dermis was peeled from the grafts with forceps and the intact epidermis was transferred to a watch glass that contained a small amount of Eagle's Minimal underwent further flattening and their cytoEssential Medium with 10 per cent calf serum. The cells were dispersed with paired needles. Suf- plasm contained a number of highly refractile ficient medium then was added to produce a sus- bodies. The keratinocytes, at this stage, tended pension of approximately 200,000 cells/ml. Two- to be triangular and the cell membranes undutenths milliliters of the suspension was injected lated continuously (Fig. 1). During the next into each Cruickshank chamber, the aperture 3—4 Weeks the kcratinocytes enlarged and multisealed with beeswax, and the chamber incubated at 37° C., coverglass side downwards, for 24 hours. plied. As cell division continued, the small isThe chambers then were turned over and incu- lands of cells enlarged gradually and finally met bation continued with the coverglass uppermost. to form a continuous sheet of cells (Fig. 2). In this investigation, the ages of the cultures The melanocytes at 24-48 hours often had a ranged from 2 days to 29 days. The cells were observed by phase contrast mi- quadrilateral or fusiform shape (Fig. 3). Tocroscopy. A melanocyte was selected for observa- wards the end of this period they began to send tion, and its behavior was recorded with a time out multiple branching processes. Melanin granlapse movie unit on Kodak Plus-X Reversal film. ules were distributed throughout the cytoplasm Frames were exposed every eight seconds. During of the cell body of most of the melanocytes exthe first hour of observation, the normal range of movement of the melanin granules and cell amined. Sometimes the granules were more membranes in a hormone-free medium was estab- heavily concentrated around the periphery of lished. The medium then was aspirated from the the cytoplasm; at times, small areas of cytoculture chamber and replaced with medium con- plasm near the nucleus practically were devoid of granules. Time-lapse sequences indicated that * From the Departments of Medicine and Anatomy, Yale University School of Medicine, New the granules oscillated and cytoplasmic flow Haven, Connecticut. Received for pubhcation June 15, 1966.

This research was supported by the United

States Public Health Service grants Ca 04679-07 and AM 5264-06.

carried them along as the dendritic processes were extended or retracted. After only one or two days of cultivation, the melanocytes, With their pigment granules and multiple branching

352

353

EPIDERMAL MELANOCYTES IN CULTURE

processes, could be recognized scattered among the keratinocytes.

The cell membrane of the melanocyte was relatively passive compared with the membrane of the keratinocyte. Between 24 and 72 hours of cultivation, one and often two refractile bodies appeared in the melanocyte cytoplasm. If single, it was found near one pole of the nucleus and if there were two they were usually found at op-

TABLE I Concentrations of the various hormones used in the eultore media

a-MSH (Synthetic—CIBA Basle) posite poles of the nucleus. Towards the end Acetyleholine of the fourth week, the cultures became domi(Acetylcholine nated by coalescing sheets of keratinocytes. The Merck) time at which this occurred varied considerably Nnrepiciephrine

and depended to some extent on the frequency of the medium changes and the general over-all "fitness" of the cultures.

concentration of hormone in culture mediom

Hormone

Pharm.,

5 pg/mI 500 pg/mI

chloride—

(D. L.—Arterennl California Co., Biochemical Research) Melatonin

5 X l0 M 5 )< 101 M

5 X 10 M 5 X 10—2 M

0.1 mp/ml

The movements of the cells were dissimilar. Keratinocytes would move across the culture toward the more sedentary melanocytes and emit

(Synthetic—Upjohn Co.)

blunt, undulating, cytoplasmic processes towards

The ineffectiveness of hormones introduced

melanocyte then would seem actively drawn by the keratinoeyte. At this stage melanin granules were often transferred to the cytoplasm of the keratinocyte from the melanocyte (Fig. 4).

separating the physiology of amphibian melano-

10 mp/ml

the dendritic processes. The dendrite of the into the culture media emphasizes the wide gap

Appearances of Cultures Following the Introduction of Media Containing Hormones Cell morphology. Both the melanocytes and keratinocytes seemed unaffected by hormonal

phores and mammalian melanocytes. While rapid dispersion and aggregation of melanin granules in response to hormone is characteristic

of the amphibian dermal pigment cell, we saw

no indication of similar activity in the mammalian epidermal pigment cell. Because evidence

from other sources suggests that the lack of

hormonal effect may not be explained by a common factor, the results of each experiment will Melanin granules. The characteristic oscilla- be discussed separately. tion of the melanin granules within the cytoAlpha MSH. Darkening of human (1, 7, 5, 9) plasm of the melanocytes continued unchanged. and guinea pig (2, 3, 10) skin has been accomParticular attention was paid to possible altera- plished with a variety of natural and syntions in the streaming of melanin granules either thetic peptide hormones, including homogeneous peripherally or centrally within the cytoplasms. porcine a-MSH, /3-MSH, and ACTH. MicroNo definite changes occurred after the hormones scopic examination showed a marked increase in agents in the culture medium.

were introduced (Figs. 5—8).

the melanin content of the melanocytes which

Assay of hormones. To determine whether the appeared larger, as a result. The dendritic prochormones retained activity in the culture media, esses filled with melanin, looked longer, wider, assays of their lightening and darkening effects and more complex. The amount of free melanin

on frogs' skin were done at intervals follow- was increased (2, 3, 9). Of significance in the ing their introduction into the media. All prepa- above work was the absence of the immediate rations maintained appropriate activity for the darkening of the skin which occurs in amphibia. duration of the experiments. The results are It must be presumed that, in mammals, horsummarized in Table II.

mones affect the pigment cells shortly after administration but, so far, we have no evidence niscus sION that granule dispersion occurs at this time. The The general appearance of the melanocytes possibility still exists that conditions in vitro are and keratinocytes cultured by the Cruickshank sufficiently different from conditions in vivo technic have been described (4, 6). to preclude the dispersing effect. However,

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Fie. 1. Triangular keratinocyte in monolayer cell culture, three days after planting. Fia. 2. Confluent sheet of keratinocytes in monolayer cell culture, 17 days after planting. Fic. 3. Fusiform melanocyte in monolayer cell culture three days after planting, showing prominent polar bodies. Narrow membrane surrounds the cell. Fic. 4. Direct transfer of pigment granules from a melanocyte to a keratinocyte in monolayer cell culture 12 days after planting. 354

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FIG. 5. (a) Melanocyte in cell culture, five days. (b) No change in localization of melanin granules or in the cell size 20 minutes after adding MSH (500 /Lg/ml) to the medium. . . Fie. 6. (a) Melanocyte in cell culture, five days. (b) No significant change 20 minutes

after adding acetylchohne (5 X 10M) to the medium. 355

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Fie. 7. (a) Melanocyte in cell culture, five days. (b) No significant change 20 minutes

after adding norepinephrine (5 >< 102M) to the medium.

FIG. 8. (a) Melanocyte in cell culture, eight days. (b) No significant change 20 minutes after adding melatonin (10 g/ml) to the medium. 356

EPIDERMAL MELANOCYTES IN CULTURE

TABLE II Effect on frog skin met anophores of the culture media containing hormones Hormone resent in

a-MSH

Effect on frog skin melanophores

357

significantly inactivate the hormones used even after six hours.

The ability of norepinephrine to aggregate melanin granules rapidly in the pigment cells of lower animals prompted Stubblcfield et at. (17)

to add this hormone to cultures of Cloudman Very great darkening with melanoma, and to melanocytes derived from 11dispersion of melanin gran-

day-old mouse embryos. They reported a cesules in dermal pigment sation of cytoplasmic granule motion after the cells. Effect undiminished

hormone was added to the culture medium and Moderate lightening with ag- clumping of the melanin granules followed by gregation of melanin gran- a slow contraction of the whole cell. The pigules in dermal pigment mented mouse cells were reduced to 80 per cent cells. This effect was of their original area. However, aggregation of slightly reduced after six melanin granules was not described and no such hours of culture. observations were made in our cultures. Great lightening indicating Melatonin, which was first isolated from the aggregation of melanin pioeal glands of cows by Lerner et at., (18) does granules in dermal pigment during period of experiment.

Acetylcholine

Norepinephrine

cells. Effect unchanged during period of experiment. Melatonin

not affect skin pigmentation of man (19) or guinea pig (20). Therefore, it is unlikely that

nin granules in dermal

this compound plays a significant role in mammalian pigmentation. Hu (12) noted no "lightening" effect of melatonin on mouse melanoma cells in vitro at concentrations of 10 to 25 pg/mI.

experiment.

It is evident from our results that cultured melanocytes of guinea pig ear skin cannot re-

Very great lightening indicating aggregation of mela-

pigment cells. Effect unchanged during period of

spond to acetyleholine, norepinephrine, or melatonin in a manner similar to amphibian Novales (11) has reported that melanophores melanophores. The failure of these isolated cells from lower animals in tissue culture respond to

MSH as readily as in vivo. Hu (12) recently to react to ee-MSH although they respond in has introduced MSH to cultures of melanoma the intact animal and man suggests the followcells without dispersing melanin granules. It is ing possibilities. (1) In the mammal, the melanopossible that the methods used in the prepara- cytes may require exposure to the hormone for tion of the cultures damage the cell membranes several days before morphological changes beand prevent the hormone from reaching its come apparent. (2) The manipulation and exposure to trypsin associated with culturing the target. Trypsin, in particular, may damage the cells. However, both Cruickshank (4) and Cohen cells may damage the melanoeytes and prevent (13) have followed similar cultures of melano- the hormone's action. (3) The separation of the epidermal cells in the preparation of the culture cytes through several generations and have and the destruction of the melanocyte keratinofailed to note any abnormality. cyte relationship may have an adverse effect on Acetylcholine, norepinephrine, and melotonin the movement of the melanin granules. That an arc potent lightening agents of frogs' skin (14). The effect is due to the aggregation of melanin active part is played by the keratinocyte in the granules in the dermal pigment cells. Recently uptake of melanin from the melanocytes gen(15, 16) it has been observed that these hor- erally is acknowledged. It is possible that some mones affect the dcrmal pigment cells of the of the changes observed in melanocytes of intact frog, but do not affect the epidcrmal pigment skin in response to hormone therapy are due to cells. It is not surprising, therefore, that these a primary effect of the hormones on the hormones failed to cause aggregation of melanin keratinocytes (21).

SUMMARY granules in cultured guinea pig epidcrmal melanocytes. The various enzymes present in the Four hormones—aMSH, acetyleholine, norserum component of the culture medium did not epinephrine, and melatonin—have been added to

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

358

guinea pig epidermal melanocytes grown in cul-

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thectomy. Arch. Dermat., 94: 269, 1966. 10. Snell, R. S. and Lerncr, A. B.: The adrenocor-

ture. No changes in either the cell morpholwhen concentrations of the hormones which have

a known effect on the skin color of amphibians were used.

L. and McGuire, J. S. Vitiligo and sympa-

ticotrophic hormone and mammalian epi-

dermal melanocytes. Sixth International Pigment Cell Conference. Springer Verlag, New York, 1966.

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