The Ichthyosiform Dermatoses II: Autoradiographic Studies of Epidermal Proliferation

Clinically evident thickening of the stratum corneum is characteristic of all the ichthyosi-form dermatoses. Although these disorders are grouped together because they have this important feature in common, the patho-physiologi-cal basis for the presence of excess scale on the body surface may be quite different in each disorder. On the basis of anatomical and cellular kinetic observations, Frost and Van Scott have separated the major ichthyosiform dermatoses into four groups: ichthyosis vulgaris; lamellar ichthyosis; epidermolytic hyperkeratosis; and psoriasiform erythroderma (1). Other names by which these conditions have been known are listed in Table I. Their salient clinical features are summarized in Table II.Our previous studies have shown that mitotic activity was greatly increased above normal control levels in all of the above categories except ichthyosis vulgaris (1). Hyperprolifera-tion of epidermal cells was, therefore, suggested as a major factor leading to the appearance of excess scale in lamellar ichthyosis, epidermolytic hyperkeratosis, and psoriasiform erythroderma. The present study presents additional data obtained with autoradiographic technics on the epidermal cellular kinetics of these disorders.Although some patients with psoriasiform erythroderma have erroneously been described under the title of congenital ichthyosiform erythroderma, they are actually instances of extensive psoriasis beginning in childhood (1). Since similar kinetic studies of psoriasis havebeen reported previously (2), these patients were omitted from the present investigation.METHODS AND MATERIALSSelection of patientsFour patients with ichthyosis vulgaris, three with lamellar ichthyosis, three with epidermolytic hyperkeratosis, and 18 normal controls were selected for study. Because the method involved the in vivo use of radioactive isotopes, only patients at least 40 years old and not planning to have more children were investigated.Autoradiographic technicFive ne of tritiated thymidine (sp. act. = 1.9 C/mmole) or tritiated glycine (sp. act. = 1.0 C/mmole) were injected intradermally into each of several sites. Sequential biopsy specimens were obtained after 1 hour and at intervals of 2-3 days. The biopsy specimens were processed for autoradiographic analysis according to established procedure (2, 3, 4). Slides with 6fi thick histologic sections were dipped into Kodak NTB-3 liquid emulsion and exposed in light-tight boxes for one to four weeks. The slides were developed and stained with hematoxylin and eosin.Tritiated thymidine is incorporated into cells which are in the DNA synthesis period at the time of injection. After mitosis, each daughter cell retains half of the original labelled DNA which permits its identification as it subsequently ascends through the stratum malpighii. Tritiated glycine is incorporated into epidermal-cell proteins and becomes an autoradiographic marker as these cells become a part of the horny layer (3).Numerical dataThe number of cells labelled with tritiated thymidine in the specimens obtained one hour after injection is a measure of the proliferative activity of that tissue since these cells will all go into mitosis. The number of labelled cells is expressed per unit basal line length and per unit surface length for each specimen. These lengths were determined with a map-measure on projected images of histologic sections, as described previously (1). The average distance a 0-particle from tritiated thymidine will travel through histologic sections to reach the photographic emulsion above is less than 2 microns. A certain number of visible nuclei which have incorporated tritiated thymidine will not appear labelled since they may be 2fi or more from the surface and their emis-561562THE JOURNAL OF INVESTIGATIVE DERMATOLOGYTABLE I1. Ichthyosis vulgaris-ichthyosis simplex2. Lamellar ichthyosis-ichthyosis vulgaris; dryform of congenital ichthyosiform erythroderma3. Epidermolytic hyperkeratosis-bullous form ofcongenital ichthyosiform erythroderma; ichthyosis hystrix; bullous ichthyosis4. Psoriasiform erythroderma-congenital ichthy-osiform erythrodermasions will not reach the emulsion. Therefore, the number of labelled cells counted was multiplied by a calculated correction factor of 1.4 to obtain the number of cells actually containing tritiated thymidine (5).The transit time is defined as the minimal time necessary for a labelled basal cell to reach the top of the stratum granulosum, or the top of stratum malpighii, if there is no granular layer, as in the case* of ichthyosis vulgaris (Figures 1, 2).RESULTSThe data obtained on labelled cells per unit length of surface and basal lines are presented in Table III. The transit time was 10-14 days in ichthyosis vulgaris (Figures 1, 2), 4r-5 days in lamellar ichthyosis (Figures 3, 4) and 4 days in epidermolytic hyperkeratosis (Figures 5, 6). The transit time through normal epidermis was previously reported as 12-14 days (2).A kinetic study of the stratum corneum with the use of tritiated glycine poses certain problems. First, although one can follow a front of labelled cells as it ascends to the skin surface, the thickness of the stratum corneum is so variable due to differences among individuals and histological processing, that a transit time through this horny layer is of limited signifi-cance. However, the enormously thickened stratum corneum in epidermolytic hyperkeratosis permitted certain novel observations. Auto-radiographs of one-hour specimens revealed heavy label throughout the epidermis, as anticipated (Figure 7). The three-day specimen showed a sharply demarcated front of grains in the stratum corneum and a relative decrease in grain density over the viable epidermis, which represents the movement of most of the epidermal cells into the stratum corneum during the preceding three days (Figure 8). In a specimen taken seven days after glycine injection, the band of radioactivity had moved to the mid-stratum corneum with a relatively clear horny layer and epidermis below (Figure 9). This clear horny layer beneath the radioactive band represents the upward migration of an additional population of epidermal cells generated after the time of initial labelling.DISCUSSIONOne purpose of this investigation was to obtain more information regarding the pathophysiology of excess scale formation in the ichthyosiform dermatoses. The present studies lend further support to the concept that hyper-proliferation is a contributing factor in the production of the thickened horny layer in lamellar ichthyosis and epidermolytic hyperkeratosis, but not in ichthyosis vulgaris. The average number of labelled cells per cm surface line was approximately three times the control levels in lamellar ichthyosis, and approximately four times the control counts in epidermolytic hyperkeratosis, while the ichthyosis vulgaris counts fell within the control range.As a reference unit, the number of labelledTABLE IIClinical SummaryDisorder Gross features Histologic features Inheritance Ichthyosis vulgaris Fine scales; spares flexural Hyperkeratosis ; absent gran- Autosomal dom- creases ular layer inant Lamellar ichthyosis Large, coarse scales Hyperkeratosis ; prominent Autosomal re- granular layer cessive (?) Epidermolytic hyper- Verrucous hyperkeratosis ; Hyperkeratosis; reticular Autosomal dom- keratosis flexural creases involved; bullae spaces in epidermis inant Psoriasiform erythro- Universal erythema and Parakeratosis; acanthosis, Autosomal dom- derma scaling clubbing of rete ridges inant ICHTHYOSIFORM DERMATOSES563** • #' Jl'-**m mm L, #*4 /R2'S ' P-. * -?r/Fig. 1. Ichthyosis vulgaris. Specimen of skin taken 1 hour after injection of tritiated thymidine. Note absent granular layer. X 480.Fig. 2. Ichthyosis vulgaris. Specimen of skin taken 10 days after injection of tritiated thymidine. Labelled nuclei (arrows) present at top of stratum malpighii. X 450.cells per cm surface line is most useful since it indicates the number of cells which will eventually reach a specified surface area in a given time. The same count expressed per unit basal line is of limited significance, since with increasing papillomatosis this value may remain constant, although many more cells will reach a given surface area per unit time. Other authors have made use of a labelling index, i.e. the number of labelled cells per 100 or 1000 germinative cells for the purpose of computing the cell cycle time (6-9). The present study, however, measures the turnover of an entire population of cells, ie., the epidermis. In the controls and in ichthyosis vulgaris labelled cells were seen only in the basal layer. Labelled cells were noted in the lowest 2-3 rows in lamellar ichthyosis and in epidermolytic hyperkeratosis. The exactTABLE IIISummary of kineticsCondition Labelled cells per cm basal line ±S* Labelled cells per cm surface line ±S* Cell transit time through epidermis (days) Normal 68.9 ± 17 86.2 ± 21 12-14 controls Ichthyosis 49.3 ± 3.5 69.6 ± 7.2 10-14 vulgaris Lamellar 134 ± 15 231 ± 36 4-5 ichthyosis Epidermo- 251 ± 104 294 ± 85 4 lytic hyper- keratosis * S = one standard deviation from the mean.564THE JOURNAL OF INVESTIGATIVE DERMATOLOGYtt/ *- Art ;<'*V , 'A:m/ gj•/ ;' ' * • *W3*cs -,*vFig. 3. Lamellar ichthyosis. Specimen of skin taken 1 hour after injection of tritiated thymidine. Labelled nuclei present in lower cell layers of epidermis. Note presence of granular layer. X 480.Fig. 4. Lamellar ichthyosis. Specimen of skin taken 4 days after injection of tritiated thymidine. Labelled nuclei (arrow) are present at top of stratum granulosum. X 370.size of the germinative populations in these conditions is, therefore, difficult to ascertain precisely.It has been suggested REFERENCES