Autoradiographic Analysis of Turnover Times of Normal and Psoriatic Epidermis*

Vol. 45, No. 4

Tac JOURNAL OF INVESTIOATFVE DERMATOLOOY

Copyright

Printed in U.S.A.

1965 by The Williams & Wilkins Co.

AUTORADIOGRAPHIC ANALYSIS OF TURNOVER TIMES OF NORMAL AND PSORIATIC EPIDERMIS* GERALD D. WEINSTEIN, M.D.t AND EUGENE J. VAN SCOTT, M.D.

The epidermis is a constantly proliferating thymidine labels the nuclei of epidermal cells synthesizing DNA in a premitotic phase, and cells are equal. Early studies of mammalian serial biopsy specimens at intervals after tissue epidermal turnover time, reviewed by Lehlond labeling permits following the outward progresand Walker (1), found ranges of 13—100 days. sion of labeled cells (3), direct information on In a recent study in humans using I.V. injected the kinetics of human epidermal cell populations tissue in Which the birth and death of epidermal

glycine-C'4 followed by biochemical analysis of labeled proteins reaching the epidermal surface, Rothberg, et al. determined a turnover time of 26—28 days for normal human epidermis (2). A turnover time of approximately 30 days of pig epidermis was recently found by Weinstein (3)

in an autoradiographie study utilizing both tritiated thymidine and glycine-C" as nuclear and protein labels respectively. The study was

was sought. MATERIALS AND METHOD5

Normol Epidermis. Uninvolved skin areas of seven patients (2 psoriasis, 4 mycosis fungoides, 1

basal cell carcinoma) were used for this part of the study. Ten sites in normal appearing skin on the trunk were marked and injected sub-epidermally with 5 jzc (0.1 ml) of tritiated thymidine (specific activity = 1.9 C/mM) in water. Sequential 4 mm punch biopsy specimens were ob-

prompted because of morphologic similarities of tained from injected sites at 1, 12, 24 hours, and at pig and human epidermis. 2, 4, 8, 10, 12, 14, 16 days. In comparison to normal epidermis, the lesion Psoriesis. Lesioos of four patients with active, of psoriasis is characterized by hyperplasia of untreated psoriasis were likewise injected with epidermal cells and an increase in number of tritiated thymidine and serial biopsy specimens from injected sites at 1, 3, 5, 12, 18, 24, 36, mitoses. Two studies to date have agreed on the obtaioed 48, 72 and 96 hours. kinetics of accelerated epidermal turnover rates The preparation of autoradiographs is based on found in psoriasis. Using the isotope assay the procedure of Joftes (6). Speeimeos were fixed method mentioned above Rothberg, et al. (2) in 10% formalin, embedded in Paraplast and unfound the turnover time of psoriatie epidermis to stained histological sections prepared. The slides were dipped in Kodak NTB-3 liquid emulsion, be 3—4 days. In a histogeometrie study of normal dried and exposed in light-tight boxes for one to and psoriatie epidermis Van Scott and Ekel (4) four weeks. The slides were developed and immeconfirmed this finding, and found as well that the diately stained with hematoxylin and fast green. major change contributing to increased turnover RESULTS

time in psoriasis is an increase in germinative

Normal Epidermis. Labeled nuclei were seen in the epidermal basal cell layer in all specimens raphy have provided teehnies to derive direct taken at 1 hour post-injection (Fig. 1). A nucleus information on the kinetics of selected epidermal was considered labeled if it contained 5 or more cell population. Recent advances of high resolution autoradiog-

cell populations (5). The purpose of this study grains within its nuclear boundaries. The grain was to determine whether confirmatory values count was always substantially above the of turnover times for normal and psoriatic minimal background, which was usually lower than 1 grain per cell. An occasional nucleus was epidermis could be obtained by this technic. Since sub-epidermally injected tritiated labeled in cells immediately distal to the basal cell layer in the early specimens. Labeled nuclei Presented at the Twenty-sixth Annual Meeting were scattered randomly in the basal cell layer. of The Society for Investigative Dermatology, Labeled mitoses were not seen in appreciable Inc., NeW York, N. Y., June 20, 1965. * From the Dermatology Branch, National Can- numbers until 12 hours post-injection. After 12 cer Institute, National Institutes of Health, Bethesda, Maryland. Public Health Service, U. S. De- hours, pairs of labeled cells were often seen, partment of Health, Education and Welfare. signifying post-mitotie daughter cells.

t Present address: Department of Dermatology, With passing days the labeled nuclei were seen University of Miami School of Medicine, Miami, more distally in the stratum malpighii. Between Florida. 257

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T4 FIG. 1. Autoradiograph of normal human epidermis one hour after local intradermal injection of tritiated thymidine. Epidermal cells labeled in basal cell layer only. FIG. 2. Autoradiograph of normal epidermis six days after tritiated thymidine injection. Labeled epidermal cells have reached mid-stratum malpighii. Labeled pair of cells may be post-mitotic daughter cells moving outward together. 258

NORMAL AND PSORIATIC EPIDERMIS

4 and 8 days the labeled nuclei were in the midstratum malpighii (Fig. 2). In 4 patients, labeled cells were seen in the stratum granulosum by the 12th or 14th day (Fig. 3). In the remaining three patients, no labeled cells had reached the stratum granulosum by the 8th and 12th day. Specimens on later dates were of low technical quality and

259

ing the process of becoming a stratum corneum

cell, tritiated thymidine can be used as a marker only while the nucleus is intact, i.e.

while the cell traverses the stratum malpighii. The epidermal cell in psoriasis, on the other hand, retains part or all of its nucleus while traversing the stratum corneum (parakeratosis), prevented a finite analysis in these patients. thus labeled cells are seen in the stratum corHowever, the movement of labeled nuclei in neum. Therefore, while in normal epidermis the these patients appeared to be progressing at the technic permits measuring turnover time of same rate as in the first four patients. As only the viable epidermal cell population (i.e. expected, no cells in the stratum corneum were cells of the stratum malpighii), in psoriasis deterlabeled, inasmuch as nuclei are normally absent mination of the turnover time of the entire in this layer. epidermis is possible. Psoriasis. Biopsy specimens removed at 1 and The data of this study indicate that the transit 3 hours after injection contained labeled cells in time of cells through the normal stratum the lowest three epidermal cell layers (Fig. 4). malpighii is approximately 13 days. The transit A few labeled cells seeming to be in the mid- time through the same cell compartment in the stratum malpighii were found to be adjacent to hyperplastic psoriatic epidermis, however, is only dermal papillae visualized in neighboring sec- 48 hours. tions. The number of labeled cells was markedly Studying incorporation of systemically-admingreater than found in normal epidermis. Labeled istered glycine-C'4 into epidermal proteins, Roth-

mitoses were seen in appreciable numbers as berg, et al. (2) demonstrated that the labeled early as three hours post-injection, as contrasted

to their appearance considerably later in nor-

proteins reached the epidermal surface at 13—14 days, an interval indicative of the turnover time

mal epidermis. It was not possible to determine the appearance of pairs of post-mitotic daughter

of the stratum corneum. When the turnover

was much more rapid than in normal epidermis and the mid-stratum malpighii was reached in

study (13—14 days), a value of 26—27 days is

time of the normal stratum malpighii determined

cells, since many labeled cells were initially in this study (13 days) is added to the turnover side by side. Distal movement of labeled cells time of the stratum corneum from the Rothberg

24 hours. In all four patients labeled cells

found for the turnover time of the entire epidermis. In psoriasis, the results of the present

reached the outer limits of the stratum malpighii

study show that the transit time of labeled cells

at 48 hours (Fig. 5). In two patients labeled through the parakeratotic stratum corneum is nuclei were seen in the outermost layers of the two days, thereby indicating a total turnover parakeratotic stratum corneum at 96 hours. DIsCUssION

Several autoradiographic studies in animals confirm the observation that epidermal basal cells incorporate tritiated thymidine into DNA synthesized in the pre-mitotic period (3, 5, 7, 5). Following mitosis, labeled cells randomly leave the basal layer and migrate to the surface.

time of 4 days for the entire psoriatic epidermis.

It is interesting to note that in psoriasis the turnover time of the parakeratotic stratum corneum cells equals that of the stratum malpighii.

In normal human and pig epidermis (3), the turnover times of these strata are also equal, although the total turnover time is many times longer.

In a recent publication, Kaku, et at., reported

In this study normal and psoriatic epidermis their observations on the cytokinetics of normal were exposed in vivo to minute quantities of and psoriatic human skin by autoradiographic tritiated thymidine to analyze the turnover times technics similar to those employed by us (9). of these tissues. Initial labeling was mainly con- They found the turnover time of the stratum fined to basal cells (one cell layer in normal malpighii in normal epidermis to be 21 days and epidermis and about 3 layers in psoriasis) with subsequent movement of the labeled cells distally. Since the squamous cell in normal epidermis loses its nucleus or nuclear contents dur-

in psoriasis to be 5 days. These results are quite

different from our findings of 13 and 2 days respectively for the same cell compartments. The reason for the difference in results of the

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! a Fw. 3. Autoradiograph of normal epidermis 12 days after injection of tritiated thymidine. Labeled epidermal cell is now at outer limits of stratum malpighii. Fro. 4. Autoradiograph of psoriatic epidermis 1 hour after local intradermal injection of

tritiated thymidine. Nuclear labeling is seen primarily in the first three cell layers of epidermis which constitute the germinative zone.

260

NORMAL AND PSORIATIC EPIDERMIS

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5. Autoradiograph of psoriatic epidermis 48 hours after isotope injection. Labeled

cells have reached the outer limits of the stratum malpighii, in areas of thick epidermis overlying rete ridges as well as over the dermal papillac.

t\vo studies is not entirely clear but may be due

to the small number of specimens studied by Kaku and coworkers. Moreover, they examined biopsy specimens only at 1 hour, 7, 14 and 21

days after isotope injection in normal skin,

and at 1 hour and 5 days in psoriatic skin. Our early observations suggest that in psoriasis significant percentages of mitoscs arc labeled

several hours earlier than in normal epidermis (10). This may indicate a difference in the DNA synthesis period (S phase) and/or the "prcmitotic rest period" (G2 phase) between normal and psoriatic cpidermal germinative cells. If this

proves to be the case it would mean that in addition to an enlarged germinative cell population in psoriasis there is a biochemical pathway enabling the individual psoriatic epidermal cell

to reproduce more rapidly than the normal cpidcrmal cell. SUMMARY

The turnover (transit) times of cpidcrmal ccli populations in normal and psoriatic buman skin were measured by autoradiographic technics. Tritiatcd thymidinc was injected sub-cpidermally into normal and psoriatic skin in vivo. Autoradiographs of biopsicd skin specimens from injected sites reveal that the outward migration of labeled basal cells through the stratum malpighii of normal epidermis is approximately 13 days. Further analyses reveal that in psoriasis the transit time through the stratum malpighii and stratum corncum are equal, i.e., two days each. A similar equal relationship exists in nor-

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

262

mal epidermis even though the absolute times are longer.

Billingham, R. E. (eds.): Advances in Biol-

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