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Porokeratoses: Immunohistochemical, light and electron microscopic evaluation W. Jurecka, MD, R. A. Neumann, MD, and R. M. Knobler, M D Vienna, Austria Punch biopsy specimens of 14 patients with porokeratosis of Mibelli (n = 1), disseminated superficial porokeratosis (n = 6), disseminated superficial actinic porokeratosis (n = 4), porokeratosis plantaris, palmaris et disseminata (n = 2), and punctate porokeratosis (n = 1) were obtained for light and electron microscopy for evaluation of possible differences between these clinical variants. Langerhans cells in close contact with early degenerating keratinoeytes could be observed in the epidermis. To study the cellular composition of the epidermal inflammatory infiltrate immunohistochemistrywas performed. These studies demonstrated that the predominant cell type in these infiltrates are helper T ceils, intermingled with Leu-6+ Langerhans cells. Despite the clinical variation and possible different etiologic or triggering mechanisms, the immunohistochemicaland morphologic changes in all types of porokeratosis are the same and seem to represent a uniform reaction pattern. (J AM ACAD DERMATOL 1991;24:96-101 .)

Under the assumption that typical lesions had their origin in the orifices of sweat ducts and/or hair follicles Mibelli t in 1893 coined the term porokeratosis for a previously unknown dermatosis. In the same year Respighi 2 described a more disseminated superficial variant (DSP). Subsequently several other clinical variants of porokeratosis have been described: disseminated superficial actinic porokeratosis (DSAP), 3 porokeratosis plantaris, palmaris et disseminata (PPPD), 4 linear porokeratosis, 5 and porokeratosis punctata. 6 Reed and Leone 7 proposed that porokeratosis is an epidermal disease in which a mutant clone of epidermal cells expands peripherally. This leads to formation of the cornoid lamella, the histologic hallmark of all variants, at the boundary between the clonal population and normal epidermis. For DSAP an autosomal dominant mode of inheritance with variable expression has been assumed. 8 In many reports induction of porokeratosis has been associated with exposure to UV light9, 10 and immunosuppression.11.12 A limited number of electron microscopic investigations in porokeratosis have been performed. No significant differences between porokeratosis of

From the Department of Dermatology II, University of Vienna. Reprint requests: Wolfgang Jureeka, MD, Dept. Dermatol. II, University of Vienna, Alserstrasse 4, A-1090 Vienna, Austria.

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Fig. 1. Light microscopy of typical porokeratotic lesion with cornoid lamella and vacuolated and dyskeratotie keratinocytes. (Hematoxylin-eosin stain; •

Mibelli (PM) and DSAP were found. 13-16 We observed 14 patients with different variants of porokeratosis from whom material for light microscopy, electron microscopy, and immunohistochemistry could be obtained. The purpose of the study was to

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Fig. 2. In semithin sections, in addition to dyskeratotic and vacuolated keratinocytes, dendritic cells with clear cytoplasm (arrow)can be seen below cornoid lamella. (Toluidine blue stain; •

Fig. 3. Electron microscopy reveals close cell-to-cell contact of Langerhans cells (LC) to early degenerating keratinocyte characterized by thick tonofilaments (arrowheads) and granular material (arrows)in cytoplasm. (Uranyl acetate and lead citrate stain; X23,000.)

elucidate possible ultrastructural differences in the epidermis among clinical variants. In addition, we studied dermal changes and the inflammatory infiltrate below the cornoid lamella.

PATIENTS AND M E T H O D S Fourteen patients (eight women, six men; age range from 36 to 75 years) with P M (n = 1), PPPD (n = 2), punctate porokeratosis (n = 1), DSAP (n = 4), DSP af-

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Fig. 4. In dermal infiltrate numerous Langerhans cells (LC) surrounded by lymphocytes (arrowheads) can be observed. These lymphocytes show close cell-to-cell contact to Langerhans cells. (Uranyl acetate and lead citrate stain; X6000.)

Fig. 5. Higher magnification of Langerhans cell shown in Fig. 4. Note typical Birbeck granules (arrow and inset) and close relation to lymphocytes (12).(Uranyl acetate and lead citrate stain; X 15,000.)

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ter immunosuppressivetherapy (n = 2), and DSP with no known triggering mechanism (n = 4) were studied. In all patients several 4 mm punch biopsy specimens from representative peripheral ridges were obtained with patients under local anesthesia. For light microscopy the specimens were fixed in buffered formaldehyde. Paraffin sections were stained with hematoxylin and eosin. For electron microscopy biopsy specimens were fixed in 2% buffered glutaraldehyde, postfixed in osmium tetroxide, and embedded in Epon. Semithin sections were stained with toluidine blue and ultrathin sections with uranyl acetate and lead citrate. Immunohistochernistry was performed either on frozen or paraffin-embedded material, with anti-UCHL-1, anti-MB-2, anti-Leu-3a, antiLeu-6, anti-Leu-12, and anti--S-100 antibodies.

RESULTS Light microscopy The diagnosis of porokeratosis was confirmed in hematoxylin-eosin-stained paraffin sections (Fig. 1 ). A moderately dense, chronic inflammatory infiltrate was seen in the dermis. These histologic findings were similar in all cases and differences in the clinical variants were only quantitative. In semithin sections cells with a clear cytoplasm and a lobulated nucleus can be observed in the epidermis (Fig. 2). These cells are located in the basal cell layer and to a larger amount in the prickle cell layer and were characterized as Langerhans cells by electron microscopy.

Electron microscopy The epidermis below the cornoid lameUa showed severe intraepidermal edema. The keratinocytes were separated by broad intercellular clefts. A striking feature in the epidermis below the cornoid lameila was the presence of vacuolated keratinocytes with a more or less pronounced perinuclear edema, accumulation of autophagic vacuoles and heterolysosomes, and peripheral dislocation of tonofilaments. In addition, isolated keratinocytes displayed dyskeratotic changes with individual cell keratinization. These morphologic changes were essentially the same in all types of porokeratosis. Within the epidermis we observed many Langerhans cells. A number of these cells revealed close cell-to-cell contact with early degenerating (Fig, 3) or vacuolized keratinocytes. At the junctional zone between epidermis and dermis a considerable duplication of the basal lamina was present.

Fig. 6. Immunohistochemical staining of dermal infiltrate below the cornoid lamella with Leu-3a antibody reveals dense reaction product at surface of most of the inflammatory cells, characterizing them as helper T cells.

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In the papillary dermis multiple ectatic capillaries, surrounded by inflammatory cells, were seen. The infiltrate was composed of lymph~cytes and histiocytic cells, intermingled with some macrophages. In addition, cells with long cytoplasmic protrusions, clear cytoplasm, few organelles, and with lobulated nuclei were observed. In these cells Bitbeck organelles were occasionally detected, characterizing them as dermal Langerhans cells (Figs. 4 and 5). Each dermal Langerhans cell was surrounded by lymphocytic ceils with which they frequently were in close cell-to-cell contact (Figs. 4 and

5). Immunohistochemistry The majority ofthelymphocytic cells did not stain with MB-2 and Leu-12 antibodies but were positive for U C H L - I and Leu-3a (Fig. 6). This indicates that they are helper T cells. Several cells between the lymphocytes expressed the CD 1 antigen as demonstrated by positivity for Leu-6 (Fig. 7). Most &these cells also revealed positivity for S-100 protein.

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Fig. 7. Staining with Leu-6 antibody shows Langerhans cells in dermal infiltrate and epidermis (arrow). (•

DISCUSSION Ultrastructural studies have been performed on five cases of P M and two cases of DSAP.13-16 These investigations were restricted to the epidermis below the cornoid lamella and revealed comparable changes in all cases: severe intraepidermal edema, vacuolization, and dyskeratosis of keratinocytes. These results were confirmed by our ultrastructural studies. This uniform epidermal reaction pattern suggests that all types of porokeratosis m a y be regarded as variants of the same entity. Immunohistochemical and electron microscopic investigations reveal that the inflammatory infiltrate is mainly composed of helper T cells intermingled with closely attached Langerhans cells. It is generally accepted that the major function of Langerhans cells is to provide the sensitizing signal in skin-induced i m m u n e response by modifying or processing an antigen in such a way that immunologically relevant determinants are presented to the T lymphoeytes. Contact between antigen-presenting cells and T lymphocytes is required for effective T-cell activation (e.g., by migration of antigen-laden Langerhans cells across the dermoepidermal junction initiating T-cell sensitization in the dermis 17-19).

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In this sense the electron microscopic and immunohistochemical observations in porokeratotic lesions suggest that similar mechanisms m a y take place in their development. Reed and Leone 7 suggested that in porokeratosis there is a mutant clone of epidermal cells tha~ expands peripherally. Autotransplantation experiments 3 and detection of abnormal D N A ploidy in porokeratotic epidermis 2~support this hypothesis. Under certain circumstances this genetically aberrant cell population might be activated and become clinically overt. Although there is evidence that immunosuppression in various forms (e.g., by UV light 9, to or by drugs 12) can trigger this disease, many patients lack any apparent history. It has also been suggested that an infective agent (e.g., a virus) might induce expression of this disease in immunosuppressed patients. 21, 22 Whether classical immune mechanisms are involved in the development of porokeratotic lesions is not yet known, but McMillan and Roberts 21 suggested that the inflammatory infiltrate found beneath the cornoid lameUa participates in their establishment. The presence of Langerhans cells in the epidermis in relation to early degenerating keratinocytes as well as to helper T cells in the dermis is strong evidence for immunologic mechanisms induced by antigen presentation. It is possible that one of the immune-modulating or suppressive mechanisms described earlier directly affects the contribution of Langerhans cells to the induction or suppression of porokeratotic lesions. Future sequential studies of developing lesions should help clarify the role Langerhans cells play in the pathogenesis of porokeratosis. REFERENCES 1. Mibelli V. Porokeratosis. Contributo allo studio della ipercheretosi dei aenali sudoriferi (porokeratosis). G Ital Mal Venereol 1893;28:313-55. 2. RespighiE. Di una ipercheraosinon ancora descritta. G Ital Mal Ven Pell 1893;28:356-86. 3. Chernosky ME, Freeman RG. Disseminated superficial actinic porokeratosis (DSAP). Arch Dermatol 1967; 96:611-24. 4. Guss SB, Raymond AO, Marvin AL. Porokeratosis plantaris, palmaris et disseminata. Arch Dermatol 1971; 104:366-73. 5. Rahbari H, Cordero AA, Mehregan AH. Linear porokeratosis: a distinct clinical variant of porokeratosisof Mibelli. Arch Dermatol 1974;109:526-8. 6. Chernosky ME. Porokeratosis. Arch DermatoI 1986; 122:869-70. 7. Reed R J, Leone P. Porokeratosis: a mutant clonal keratosis of the epidermis.Arch Dermatol 1970;101:340-7.

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8. Anderson DE, Chernosky ME. Disseminated superficial actinic porokeratosis: genetic aspects. Arch Dermatol 1969; 99:408-12. 9. Chernosky ME, Anderson DE. Disseminated superficial actinic porokeratosis: clinical studies and experimental prodUction of lesions. Arch Derrnatol 1969;99:401-7. 10. Neumann RA, Knobler RM, Jurecka W. Disseminated superficial porokeratosis: experimental induction and exacerbation of skin lesions. J AM ACAD DERMATOL 1989; 21:1182-8. 11. Lederman JS, Sober A J, Lederman GS. Immunosuppression: A cause of porokeratosis? J AM ACAD DERMATOL 1985;13:75-9. 12. Neumann RA, Jurecka W, Knobler R, et al. Disseminated superficial porokeratosis and immunosuppression. Br J Dermatol 1988; 119:375-80. 13. Sato A, Anton-Lamprecht I, Schnyder UW. Ultrastructure of inborn errors of keratinization. VII. Porokeratosis Mibelli and disseminated superficial actinic porokeratosis. Arch Dermatol Res 1976;255:271-84. 14. Mann PR, Cort DF, Fairburn EA, et al. Ultrastructural studies on two cases of porokeratosis of Mibelli. Br J Dermatol 1974;90:607-17. 15. Marghescu S, Anton-Lamprecht I, Melz-Rothfuss B. Dis-

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16. 17.

18. 19. 20. 21. 22.

seminated bilateral hyperkeratotic variant of porokeratosis Mibelli. Arch Dermatol Res 1987;279:39-47. Besenhard HM, Korting HC, Stolz W, et al. Disseminierte superfizielle ak-tinische Porokeratose (DSAP) mit Morbus Bowen. Hautarzt 1988;39:286-90. Stingl G, Wolff K. Langerhans cells and their relation to other dendritic cells and mononuclear phagocytes. In: Fitzpatrick TB, Eisen AZ, Wolff K, et al, eds. Dermatology in general medicine. 3rd ed; vol. 1. New York: McGraw-Hill, 1987:410-26. Shevach EM. Macrophages and other accessory ceils. In: Paul WE, ed. Fundamental immunology. New York: Raven Press, 1984:71-5. Silberberg-Sinakin I, Thorbecke G J, Baer RL, et al. Antigen-bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell Immunol 1976;25:137-51. Otsuka F, Chi HI, Shima A, et al. Cytological demonstration of abnormal DNA ploidy in the epidermis of porokeratosis. Arch Dermatol Re,s 1988;280:61-3. Macmillan AL, Roberts SOB. Porokeratosis of Mibelli after renal transplantation. Br J Dermatol 1974;90:45-51. Foulds IS, Slater DN. Porokeratosis of Mibelli and immune complex glomerulonephritJs. Clin Exp Dermatol 1983 ;8:6975.

Porokeratoses: immunohistochemical, light and electron microscopic evaluation.

Punch biopsy specimens of 14 patients with porokeratosis of Mibelli (n = 1), disseminated superficial porokeratosis (n = 6), disseminated superficial ...
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