AN ELECTRONMICROSCOPIC STUDY OF ACANTHOLYSIS AND DYSKERATOSIS IN HAlLEY AND HAlLEY'S DISEASE* GEORGE F. WILGRAM, M.D., PH.D., JAMES B. CAULFIELD, M.D. AND WALTER F. LEVER, M.D.
Hailey and Hailey's disease (benign familialand acantholysis in Hailey and Hailey's disease chronic pemphigus) is a rare hereditary skinprompted us to investigate this disease by elec- disorder characterized by recurrent eruptionstron microscopy. of vesicles and bullae involving predominantly In this publication the electron microscopic the neck, groins, and axillae (1). On histologicchanges of acantholysis in Hailey and Hailey's examination, this disease shows numerousdisease will be described. They will be compared acantholytic cells and the so-called suprabasalwith the lesions seen in pemphigus vulgaris. type of blister formation. Although the aca.n-The emphasis will center around the epidermal tholytic cells have lost their desmosomes (inter-tonofilaments which are generally regarded as cellular bridges), many of them still maintain aone of the precursors of keratin (7, 8, 9, 10). loose coherence. Such clusters of loosely coheringA comparison with the electron microscopic cells may float into the bulla, but often they stillchanges seen in Darier's disease will be under- retain adherence to the epidermis. Their presencetaken in a subsequent paper. gives the epidermis the appearance of a "tumbling brick wall." (Fig. 1). MATERIALS AND METHODS Under the light microscope, the acantholytic Two patients with Hailey and Hailey's disease cells in Hailey and Hailey's disease are roundedwere examined. One patient was a 60 year old and have a homogeneous eosinophilic cytoplasmCaucasian male; the other a 22 year old Oriental suggesting abnormal keratinization (2). Severalmale. In each case a family history of a similar skin disease was obtained, but the relatives were observers have described grains and corps rondsnot available for examination. Biopsy examina- similar to those found in Darier's disease (3, 4).tions were performed on both patients during an As a matter of fact, chronic benign familialexacerbation of their eruption. Only small blisters pemphigus has been considered by some authorsmeasuring not more than 2 mm. in diameter were taken by punch biopsy under minimal novocain as a bullous variant of Darier's disease (3, 4, 5).anesthesia from the region of the lower posterior Our interest in Hailey and Hailey's diseaseaspect of the neck. An attempt was made to select stems from previous electron microscopic studieslesions which (with reasonable certainty) were of acantholysis in pemphigus vulgaris (6). Itof recent origin. Both patients had been without was observed that in pemphigus vulgaris acan-therapy during the six months preceding the biopsy examination. tholysis was initiated by a destruction of the The biopsy specimens were fixed in buffered tonofilaments within epidermal cells. Subse-1% osmium tetroxide (11, 12) with sucrose for one quently, the desmosomes began to disappear.and one-half hours. They were embedded in Eventually, with the disappearance of the des-prepolymerized n-butyl methacrylate with 1% mosomes, acantholysis set in and bullae formed.Luperco (13). The sections were cut on a Porter- Blum microtome with diamond knives and were Dyskeratosis was not seen in pemphigus vul-mounted on carbon-coated grids (14, 15, 16). The garis. The observation under the light micro-pictures were taken with either an RCA EMU 3B scope of an associated occurrence of dyskeratosisor a Siemens Elmiscope. *Fromthe Department of Dermatology, Tufts University; and Department of Pathology, Massa- RESULTS chusetts General Hospital, Harvard University, Boston, Mass. In the two cases of benign familial pemphigus This investigation was supported by U.S.P.H.the epidermis was found to be predominantly National Institutes of Health Grants RG 9726,involved. The only obvious changes within the C-4955 and 2A-5220. The material in this manuscript was part of adermis were a marked increase in the number presentation given before the Twenty-secondof lymphocytes and monocytes and the presence Annual Meeting of the Society for Investigativeof an occasional neutrophil. The collagen, elastic Dermatology, New York City, June, 1961. Received for publication March 21, 1962. tissue, vessels, and nerves appeared normal. 373 374 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY
a
Abbreviations:BM—Basement membrane C—Collagen D—Desmosome M—Mitochondrion N—Nucleus T—Tonofil ament Fic. 1. This light microscopic photograph of a section of methacrylate-embedded material from a patient with Hailey-ilailey's disease is a serial section close to that shown in Figure 6. The basal cells in the center of the picture have no visible desmosomes. The prickle cells in the upper part of the picture show to a varying degree a decrease in the number of desmosomes. (1< 740)
There was no observable alteration in the base- In normal skin the basal cells contain through- ment membrane. out their cytoplasm numerous strands of tono- In the epidermis, changes were present in bothfilaments (17). Although the exact synthesis of basal cells and malpighian cells. The melanocyteskeratin is not known, it is likely that the tono- did not appear disturbed. The alterations werefilaments are precursors of keratin (7, 8, 9, 10). focal. In each case areas of normal epidermisThe tonofilaments insert at highly organized were interspersed with altered epidermal cells.points along the cell surface. The resultant com- The degree of involvement did not depend on theplex has a counterpart in the adjacent cells. stratum in which the epidermal cell was located.The combination of these two opposed highly Severely altered cells were seen within the basalorganized regions is a desmosome. The desmo- layer, as well as at various levels of the mal-somes are the points of coherence of neighboring pighian layer. cells throughout the epidermis. Desmosomes
FIG. 2. Normal stratum corneum. The desmosomes differ in appearance from those seen in the prickle cell layer. There are no visible subcellular elements, such as the nucleus, mitochondria or vesicles, but only a fine fibrillar matrix. Some of these fibrils are attached to the desmosomes in the same manner as seen in the prickle layer. (X 74,000) Fio. 3. Dermal-epidermal junction of normal skin. Numerous desmosomes are present at the base of a basal cell (arrows). They vary from the desmosomes in other locations in that only one cell contributes to the complex. The basement membrane (BM) is modified in the region of the desmosome, being slightly denser and in closer proximity to the basal cell than in areas without desmosomes. Tonofilaments (T) are seen to be inserting in the region of the desmosome. M =mitochondria;N =nucleus;C =collagen. (X 28,000) ACANTHOLYSIS AND DYSKERATOSIS IN HAlLEY—HAlLEY'S DISEASE 375
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FIGs.2—3 376 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY
may even be seen in the cornified layer and The clumping of tonofilamcnts following their disappear only in the final stages of keratiniza-separation from the dcsmosomes was accentuated tion (Figure 2). In the upper portion of thein many cells by the accumulation of large malpighian cell layer, the tonofilaments areamounts of disorganized filamcntous material associated with large, dense granules, presum-(Figure 7). No kcratohyalinc granules were ably representing the keratohyalinc granulespresent in cells containing these disorganized seen with the light microscopy (9, 17, 19). tonofilaments. As the filamcntous material in- The coherence of the basal cells and the base-creased in amount and aggregated, a large dense ment membrane is different from the coherencebody formed within the cell. These large dense between cpidcrmal cells. The dcsmosomcs ofintracellular bodies closely resembled the ones basal cells opposite the basement membraneseen in electron microscopic sections of Darier's differ from desmosomes elsewhere in the epi-disease and probably corresponds to the "corps dermis in that only one cell contributes to theirronds" seen with the bght microscope (3, 4, 5). structure (Figure 3). They are referred to asThe size and density of these bodies varied in junction granules (17). In addition, the base-the two cases, but their basic filamcntous struc- ment membrane is modified in the region of theture was the same (Figure 8). The cpidcrmal cells desmosomc (18). showing these marked abnormalities in their The two examples of Hailey and Hailcy'stonofilamcnt -dcsmosome-complexappeared disease which were studied deviated from thisotherwise normal. In particular, the mito- general scheme. In the least involved epidermalchondria, the various vesicular and mcmbranous cells, the tonofilaments were separated from somecomponents and the nucleus appeared undis- of the dcsmosomcs. The separation resulted in aturbed. In the lower epidermis normal appearing retraction of the tonofilamcnts from the plasmamelanin granules were present within melan- membrane and in a thickening of the strands ofocytes and epidermal cells. filaments (Figure 4). In areas where separation of tonofilaments had taken place over wide areas, DISCUSSION the desmosomes had disappeared and cellular Hailey and Hailey's disease, like pemphigus cohesion was lost (Figure 5). When this lossvulgaris, is primarily a disorder of the epidermis. of cohesion involved large areas of adjoining cellsThe electron microscopic findings in Hailey acantholysis occurred and lacunac were formed.and Hailcy's disease bear out the view gained Many basal cells showed loss of desmosomes onby Dupont (20) on examination with the light all surfaces except their basal surface (Figuresmicroscope that in this disease there is a selective 1 and 6). The presence of relatively normal loss of intercellular bridges. dcsmosomes (junction granules) adjacent to the The earliest change seen by electron micros- basement membrane with total loss on all otherscopy is a separation of the tonofilaments from surfaces is similar to the situation in pcmphigusthe dcsmosomes. Following this, the dcsmosomcs vulgaris. This preservation of coherence betweenof the affected cell disappear. Adjacent cpidcrmal basal cells and basement membrane resulted incells from which an altered cell separates fre- the suprabasal location of the lacunac and bullac.quently show normal desmosomes and tonofila- Many of the cells seen within the cavity of thements. Occasionally, ameboid leukocytes mi- bullae remained loosely adherent to one another,grating from the dermis into the epidermis resulting in clusters of acantholytic cells. separate the desmosomes of adjoining epidermal Polymorphonuclear leukocytcs and monocytescells; but this separation does not cause any were observed on occasion among relativelyalteration in the appearance of the tonofila- normal cpidermal cells, as well as within lacunaements and dcsmosomes. Thus, the alteration of and bullac. the tonofilaments seen in Hailey-Hailey's disease
FIG. 4. Cells from the malpighian layer of a case of Hailey and Hailey's disease. The cells show some loss of desmosomes and consequent separation from one another. The tonofilaments (arrow) in one of the cells show early retraction and clumping. The remainder of the cell components are normal. (X 13,000) FIG. 5. A cell from the malpighian layer of a case of Hailey and Hailey's disease. This cell shows more complete loss of desmosomes than the cells in Figure 4. The tonofilaments (T) are more densely aggregated around the nucleus. As in the less involved cells the other subeellular components show no morphological alterations. (X 18,000) ACANTHOLYSIS AND DYSKERATOSIS IN HAlLEY—HAlLEY'S DISEASE 377 a __
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FIGS.4—5 378 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY most likely derives from an abnormal intracellularand Hailey's disease the cellular structures of status. It is not clear whether the basic defect inthe acantholytic cells other than the tonofila- Hailey and Hailey's disease consists of a dis-ments and desmosomes remain intact. The cells integration of the whole tonofilament-desmosomeinvolved in pemphigus usually undergo necrotic complex or whether simple separation of thechanges which prevent the formation of dys- tonofilaments from the desmosome is sufficientkeratotic masses seen in Hailey and Hailey's to cause the disappearance of this highly special-disease. ized structure. Since desmosomes persist during No obvious reason can be given for the per- the process of normal keratinization up to thesistence of the desmosomes located adjacent to cornified layer (Figure 2), their disappearancethe basement membrane, the so-called junction at the level of the basal or malpighian layer isgranules, even after all demonstrable desmo- definitely a pathological event. somes on the other cell surfaces have disap- Retraction and clumping of the tonofilamentspeared. It is possible that the normal base- is most pronounced in cells which have lost allment membrane has a "protective" effect on their desmosomes. It is seen less in cells with athe junction granules. few remaining desmosomes and is least apparent Since neither the dermis nor the basement when the majority of desmosomes are present.membrane is damaged in Hailey and Hailey's The clumping of tonofilaments does not seem todisease, the basal cells continue to adhere and occur as long as the strands of filaments aresuprabasal blister formation results when acan- seen to insert in a desmosome. These considera-tholysis sets in. In pemphigus, too, the cohesion tions lead to the following hypothesis: Separationbetween basement membrane and basal cells is of the tonofilaments from the desmosomes resultspreserved, even though the basal cells appear in contraction of the filaments, which is as-quite degenerate. In contrast to Hailey-Hailey's sociated with a concentration of the tonofilamentsdisease and pemphigus vulgaris, the dermis and around the nucleus. Since in many cells there isthe basement membrane are altered in bullous an accumulation far greater than could beerythema multiforme. These dermal alterations accounted for by simple concentration, excessivetogether with focal epidermal cell damage lead or faulty synthesis of tonofilaments must taketo a loss of dermo-epidermal cohesion and to place. This excess production of tonofilamentoussubepidermal blister formation (21). material produces the "corps ronds" described The selective change in the tonofilament- by some authors on examination with the lightdesmosome-complex observed in Hailey and microscope (3, 4). The fact that the accumulationHailey's disease is consistent with the hypothesis of tonofilaments in the malpighian cells is notthat acantholysis in Hailey and Hailey's disease associated with keratohyaline granules suggestsis based on a hereditary error causing a faulty that these masses of tonofilaments representsynthesis or faulty maturation of the tonofila- not only immature, but also abnormal keratinments with subsequent degeneration of the tono- so that it is justified to speak of dyskeratosis.filament-desmosome-complex. On the other hand, In pemphigus vulgaris the early events arein pemphigus vulgaris, where there is degenera- similar to those observed in Hailey and Hailey'stion of the entire epidermal cell, we may assume disease. In both diseases the earliest change is athat acantholysis is brought about by a destruc- disintegration of the tonofilament-desmosome-tion of tonofilaments, rather than by faulty complex with its sequellae, namely: (1) clumpingsynthesis or faulty maturation. of tonofilaments; (2) dissolution of the des- mosomes; and (3) acantholytic separation of SUMMARY epidermal cells. However, there is a marked Two eases of Hailey and Hailey's disease have difference in the subsequent changes. In Haileybeen studied by electron microscopy. The
FIG. 6. This section is a serial section close to that shown in Figure 1. The basal cell depicted is de- void of desmosomes except those adjacent to the basement membrane, which are shown here. These desmosomes (arrows) are normal in appearance. On the cytoplasmic side tonofilaments arc inserting. Adjacent to the cellular component of the desmosome the basement membrane is more dense and fre- quently in closer apposition to the cell than elsewhere. (x25,000) Fro. 7. The tonofilaments of this cell are more severely altered than those of Figure 5. The filamentous component is not as clearly seen, although the magnification is comparable. Even at this state of dense aggregation of the tonofilaments no keratohyaline granules are visible. (X 17,000) ACANTHOLYSIS AND DYSKERATOSIS IN HAlLEY—HAlLEY'S DISEASE 379
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Fio. 8. Acantholytic cell from the malpighian layer of a case of Hailey and Hailey's disease. The plasma membrane at the top of the picture is totally free of desmosomes as was the entire surface visible in the section. No other cells were in contact with this cell. The cytoplasm visible in the picture con- tains a large quantity of loosely aggregated filamentous material. This material was present through- out the remainder of the cytoplasm not included in the picture. (X 22,800) 380 ACANTHOLYSIS AND DYSKERATOSIS IN HAlLEY—HAlLEY'S DISEASE 381 dermis and the basement membrane did not 203, 1957. Part II. The hair cuticle. J. Biophys. Biochem. Cytol., 3: 215, 1957. show any striking abnormalities. The earliest 8. MERcEE, E. H.: The electron microscopy of change appeared to be a separation of the keratinized tissues. In The Biology of Flair tonofilaments from the desmosomes with subse- Growth, p. 91. Academic Press, Inc., N. Y., 1958. quent degeneration of the desmosomes and loss 9. BEODY, I.: An ultrastructural study on the of cohesion of the involved cells. Dyskeratosis role of the keratohyaline granules in the was observed. This appeared to be the result of keratinization process. J. Ultrastruct. Res., 3:84, 1959. alterations in the tonofilaments in still viable 10.BEODY, I.:The ultrastructure ofthe tonofila- epidermal cells. The dyskeratotic material is ments in the keratinization of normal human epidermis. J. Ultrastruct. Res., 4: 264, 1960. formed by a condensation of tonofilaments in11. PALADE, C. E.: A study of fixation for electron the absence of keratohyaline granules. microscopy. J. Exp. Med., 95: 285, 1952. 12. CAULFIELD, J. B.: Effects of varying the Although the early changes in Hailey and vehicle for 0S04intissue fixation. J. Bio- Hailey's disease are similar to those in pemphigus phys. Biochem. Cytol., 3: 827, 1957. vulgaris, the later changes differ in that the13. B0RY5K0, E.: A study of the polymerization damage phenomenon by phase contrast acantholytic cells in pemphigus become necrotic microscopy. J. Biophys. Biochem. Cytol., and thus do not form dyskeratotic material. 2: 3, 1956 Suppl. No. 1. It is possible that the primary defect in Hailey14. PORTER, K. R. AND BLUM, J.: A study in the microtomy for electron microscopy. Anat. and Hailey's disease consists of an inherited Rec., 117: 685, 1953. faulty synthesis or maturation of the tonofila-15. FEaNANDEz-MORAN, H.: Applications of a diamond knife for ultrathin sectioning to the ment-desmosome-complex. study of the fine structure of biological tis- REFERENCES sues and metals. J. Biopbys. Biochem. 1. HAlLEY, H. AND HAlLEY, H.: Familial benign Cytol., 2: 29, 1956. Suppl. No. 1. chronic pemphigus. Arch. Dermat., & Syph.,16. WATSON, M. L.: The use of carbon films to 39: 679, 1939. support tissue sections for electron micros- 2. LEvEE, W. F.: Histopcthology of the Skin. 3rd copy. J. Biophys. Biochem. Cytol., 1: 183, Edition, p. 61. Philadelphia, J. B. Lippin- 1955. cott Co., 1961. 17. SELBY, C. C.: An electron microscopic study 3. ELLIS, F. A.: Vesicular Darier's disease. Arch. of the epidermis of mammalian skin in thin Dermat. & Syph., 61: 715, 1950. sections. J. Biophys. Biochem. Cytol., 1: 4. FINNEEUD, C. W. AND SZYMAN5KI, F. J.: 429, 1955. Chronic benign familial pemphigus, a pos-18. TAKAsE, K.: An electron microscopic study sible vesicular variant of keratosis I ollicu- on the dermu-epidermal junction. Acta laris. Arch. 1)ermat. & Sypb., 61: 737, 1950. Dermat. vener., 41: 481, 1961. 5. WINEE, L. H. AND LEEB, A. J.: Benign familial 19. LADEN, E. L., GETRNER, P. AND EmeRsoN, pemphigus. Arch. Dermat. & Syph., 67: J.0.:Electron microscopic study of kerato- 77, 1953. hyaline in the formation of keratin. J. 6. WILGEAM, G. F., CAULFIELD, J. B. AND LEVEE, Invest. Derm., 28: 325, 1957. W. F.: An electron microscope study of20. DUPONT, A.: Note sur l'histologie du pemphi- acantholysis in pemphigus vulgaris. J. gus familial héréditaire bénin. Ann. Derm. Invest. Dermat., 36: 373, 1961. Syph. (Paris) 78: 703, 1951. 7. BIEBEcK, M. S. C. AND MERcER, E. H.: The 21. CAULFJELD, I. B. AND WILGEAM, G. F.: An electron microscopy of the human hair electron microscopic study of blister forma- follicle. Part I. Introduction and the hair tion in erythema multiforme. J. Invest. cortex. J. Biophys. Biochem. Cytol., 3: Derm., (in press).