0022-202X/ 80/ 7405-0359$02.00/ 0 THE JOURNAL OF i NVESTIGATIVE D E RMATOLOGY, 74:359-362, 1980 Vol. 74, No. 5 Copyright © 1980 by The Williams & Wilkins Co. Printed in U.S.A . Pemphigus Acantholysis: A Unique Immunological Injury JOHN R. SCHILTZ, PH.D. Department of Dermatology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio, U.S.A. Pemphigus is a disease involving human stratified squamous in vitro as skin explants, intact epidermis or cell suspensions. epithelial tissues which is characterized histologically by th e From these studies a concept for pemphigus acantholysis has presence of intraepidermal blisters. These form above the basal been developed whereby t he living epidermal cell, without cell layer in pemphigus vulgaris and vegetans or through the participation of complement, responds to the pemphigus anti­ granular or upper malpighian layers in pemphigus erythema­ body by generation of an active "pemphigus acantholysis fac­ tosis and pemphigus foliaceous (including Brazilian pemphigus tor" (PAF). PAF appears to be a proteolytic enzyme which or "fogo selvagem"). The most distinguishing histologic featw-e originates from the epidermal cell and causes th e ch aracteristic of the pemphigus lesion is the distinctive acantholytic cell , lesion of the disease. A sin1ilar concept has been advanced by which is a physically separated epidermal cell containing a Farb, D ykes, and Lazarus [18]. Addition of pemphigus serum small pyknotic nucleus surrounded by a clear zone or "halo," to monolayer cultures of mouse epidermal cells resulted in an intensely eosinophilic cytoplasm and rounded borders [1]. antibody binding and reduced cellulru· adherence to the culture Diverse mechanisms have been proposed to explain the lesions vessel. Because the process was prevented by serine protease of pemphigus. Prominent theories have included bacterial and inhibitors, t hey postulated that loss of adhesion and pemphigus viral infections, electrolytic imbalances resulting from disturbed acantholysis was caused by a n epidermal serine protease. adrenocortical function, serum toxicity, and tissue and serum The techniques used to prepru·e human skin for explant [12] h ydrolytic e nzymes. Many of these notions have not sw-vived or cell suspension cultw-e [20] have been described. The skin is critical experimental protocols and some concepts involving usually obtained from nonpemphigus patients undergoing rad­ systemic or metabolic disorders have been shown to be second­ ical mastectomy. For explan t culture on floating lens paper ary consequences of the disease. rafts, the medium consists of Ham's F-10 to which h as been The discovery was made in 1964 that pemphigus patients added 10% heat-denatured fetal calf serum and IgG fractions have serum antibodies which bind to stratified squamous epi­ from human AB serum (controls) or pemphigus serum. For cell thelial cells [2,3]. Since these antibodies were unique to pem­ suspension cultw-es, fetal calf serum was omitted and a 3H­ phigus patients, were bound at the site of the lesion and the amino acid mixture was added to assess protein synthesis. All antibody titers correlated with disease activity [ 4], it was a incubations were at 37°C in a humid atmosphere containing 5% reasonable hypothesis that the antibodies were responsible for C02/95% air. producing the lesion. Direct experimental evidence for this hypothesis, however, was not presented until1972 when Wood, SKIN EXPLANT STUDIES Beutner, and Chorzelski [5] and Holubar et al [6] respectively Dming a 5-day period normal human skin explants cultured demonstrated that pemphigus antibody bound and acantholysis with pemphigus lgG underwent characteristic histological occw-red in monkey skin or oral epithelia following local injec­ changes. After 1 day extracellular spaces developed between tion of pemphigus serum. These animal experiments were re­ keratinocytes which were shown by electron microscopy to be garded with caution, however, because the repeated injections caused by dissolution of the cell sw-face glycocalyx in nondes­ which were required to produce acantholysis caused consider­ mosomal areas [16]. After 3 days extensive s uprabasilar acan­ able tissue trauma. In addition, it could not be demonstrated tholysis occurred which was indistinguishable fi-om that seen in that the pemphigus antibody was capable of fixing complement patient's lesions (Fig 1B). In mru·ked contrast, skin cultured 3 in indirect immunofluorescent tests [7,8] and precedent did not days with normal IgG displayed histological featm es chru·acter­ exist whereby an antibody could induce tissue damage without istic of explant cultures, including intercellulru· edema, para­ participation of complement and inflammatory cells. keratosis and epiboly, but acantholysis did not occur (Fig lA). In 1974, Michel and Ko [9,10] introduced a new approach to The extent and time of onset of acantholysis was dependent studying pemphigus by their demonstration that epidermal upon the antiepithelial antibody titer, and acanth olysis did not acantholysis occw-red in normal human skin explants cultured occur at titers less than 80. Furthermore, since the in vitro in vitro with whole pemphigus serum. Although Bellone and "lesions" resembled that seen in pemphigus vulgaris biopsies, Leone had reported in vitro experiments in 1956 which yielded even when th e IgG source was from patients with pemphigus similar results [ll], the work was not well publicized and the foliaceous or fogo selvagem, it may be that patients with differ­ significance of theix observations were overlooked. Schiltz and ent forms of the disease respond differently to a similru· autoan­ Michel [12] later showed that it was the IgG fraction which tibody. Autoradiographic studies demonstrated that in the pres­ caused the in vitro "lesion" and that complement was not ence of the a utoantibody t he capacity for the suprabasila1· required. Similar observations have since been made by others "tru·get cells" to accumulate RNA and proteins was reduced using human [13] or monkey skin.[14,15] and the ultrastructural with respect to that of basal cells and no accumulation of these changes which occw- in vitro are similar to those which occur macromolecules occw-red in the definitive acantholytic cell. in vivo [16,17]. Experiments were performed to determine if complement Ow· work will be reviewed which d eals with the interaction was required for acantholysis to occw· in vitro. When the of the pemphigus a ntibody with human epidermal cells cultured medium containing pemphigus IgG was heated at 58°C for l hr, acantholytic activity remained. When pemphigus lgG was digested with papain under controlled conditions so that the This work was supported by grant# AM 19115 from the National Fe Institutes of Health. portion of the molecule (which binds C3) was removed, the Reprint requests to: John R. Schiltz, Ph.D., Department of Derma­ resulting univalent Fab fragments produced acantholysis in to logy, University Hospi tals of Cleveland, Case Western Reserve Uni­ cultmed skin. These experiments provide strong evidence that versity, Cleveland, Ohio 44106. complement is not involved in the primary mechanism of Abbreviations: acantholysis. PAF: pemphigus acantholysis factor Explant cultures were employed to test selected compounds 359 360 SCHILTZ Vol. 74, No.5 for ability to prevent pemphigus IgG binding to the e pidermis and acantholysis [19]. At therapeutic concentrations, hydrocor­ 5 '<:t tisone and triamcinolone acetonide (both at 10- M) did not I • prevent IgG binding to the epidermis or acantholysis. Similarly, 0 2 5 • Normal IgG the t hioglucose salt of gold (10- M) or soybean trypsin inhibitor >< (100 llg/ ml), which is a serine protease inhibitor, did not prevent 2: binding or acantholysis. These experiments support the con­ a... 0 cepts that the t h erapeutic effectiveness of steroids and gold / salts is pro.bably due to their demonstrated ability to reduce 1) \ serum autoantibody titers and that pemphigu .D s acantholysis is ::I \0 probably not caused by a serine protease. / 0 "- Ill / Pemphigus IgG c EPIDERMAL CELL SUSPENSION STUDIES H .6 " 0 Although new general insights about pemphigus acantholysis / '--u--0 Q) had been gained u sing explant-cultured skin, that system had u limited utility for more detailed biochemical studies of the 0 mechanism. We th erefore studied effects of the pemphigus antibody on aspects of protein metabolism by freshly-prepared 0 2 3 4 human epidermal cells in short-term suspension culture [20]. Hours These studies have provided evidence that pemphigus acantho­ FIG 2. Effects of normal or pemphigus lgG on the accumulation of lysis is caused by autoantibody-induced synthesis or release of radioactive insoluble cellular proteins by suspensions of human epider­ an epidermal protease(s). mal cells. Cell suspension cultures (2 .1 x lOu cells in 0.6 ml F-10 Nearly 70% of epidermal cells prepared by trypsinization medium) were incubated with 11.4 mg/ ml normal or pemphigus IgG retained the capacity to bind pemphigus antibody to their (antiepithelial antibody titer = 150) + 5 /lCi per culture 3H-amino acid surface (immunofluorescence), and during 18-hr incubation pe­ mixture. At the indicated times the cellular-insoluble fraction was riods 75% of th ese cells were killed as compared to 14% in prepared (reference 20) and the radioactivity determined. control m edium containing normal IgG. When 3H-amino acids were included in the medium to assess protein synthesis during the water-insoluble cellular fraction. Pemphigus IgG caused a this period, accumulation of radioactive proteins was inhibited concentration-dependent shift from the insoluble fraction to about 57% by pemphigus IgG, relative to the normal IgG the medium. During the first 2 hr of culture, accumulation into controls. Kinetic studies sh owed that this inhibition was anti­ the cell-insoluble fraction was similar for cells c ultured with body concentration-dependent and time-dependent. Inhibition normal or pemphigus IgG(Fig 2). Beginning at 2 hr, however, did not occur at pemphigus antibody titers less than 50 and there was a rapid loss of insoluble material in pemphigus IgG­ began after 2 hr.