Glucocorticosteroids are used to treat patients with pemphigus, but the mechanism of action is unknown. We studied the effect of methylprednisolone on acantholysis induced in vitro by incubation of normal skin with plasma from a patient with pemphigus. Normal human breast skin was maintained in organ cultures for several days in Ham F-10 medium. Plasma from a patient with active pemphigus vulgaris caused suprabasilar epidermal acantholysis when added to this culture system. In control cultures (F-10 medium and fetal bovine serum), no acantholysis occurred. Acantholysis was prevented when breast skin was preincubated for 24 h in a 0.25 mM solution of methylprednisolone in F-10 medium and fetal bovine serum, suggesting that methylprednisolone directly inhibits acantholysis. No suppression of acantholysis occurred when the methylprednisolone was added to the culture system simultaneously with the pemphigus plasma, suggesting a time requirement for alteration of cellular events. The inhibition of acantholysis was not caused by cell death since methylprednisolone did not alter keratinocyte viability as determined by exclusion of trypan blue dye when keratinocytes were exposed to pemphigus plasma. Similarly, the inhibition of acantholysis was not due to dissolution, alteration, or coating of pemphigus antigen on epidermal cells, since the intercellular antibodies in the plasma bound as well to methylprednisolone-treated epidermis as to untreated epidermis.
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Human skin was maintained in skin organ culture by a modification of the methods of Sarkany et a! . Acantholysis was induced in specimens by the method of Schiltz and Michel . Squares of normal human breast skin (4 X 4 X Y.! mm) were placed on sterile lens tissue. This tissue paper had waxed edges so that it and the skin specimen would float in various test media. Tissue, tissue paper, and media were placed in a watch glass and maintained in a sterile, covered culturedish containing wet gauze to insure high humidity. The system was Manuscript received June 22, 1981; accepted for publication April 13, 1983. This work was supported by grant T32AM07165 from the National Institutes of Health. Reprint requests to: Mark V. Dahl, M.D., Box 98 Mayo, University of Minnesota Medical Center, Minneapolis, Minnesota 55455. Abbreviations: FCS: fetal calf serum incubated in a humidifed 4% C02 atmosphere at 37°C. Specimens were incubated for 3 days. After incubation, organ culture specimens were removed from their paper rafts, fixed in formalin, sectioned, and stained with hematoxylin and eosin. Specimens were then coded and examined by light microscopy to determine whether acantholysis was present. Acantholysis was defined as the presence of a suprabasilar cleft. Specimens were read blindly by 3 histopathologists to eliminate any possible prejudice. To study the effects of methylprednisolone on inhibition of acantholysis, skin specimens were incubated in Ham F-10 medium  (1 ml) and fetal calf serum (FCS) (1 ml) with methylprednisolone (0.25 mM) (Upjohn Co., Kalamazoo, Michigan). Methylprednisolone at a 0.25 mM concentration approximates the distribution of 1 g of methylprednisolone in the extracellular fluid of a 60-kg woman. After a variable preincubation period, citrated plasma (1 ml) derived from a patient with active pemphigus was added to the medium (Table 1). All experiments were repeated at least 4 times. To induce acantholysis, plasma from a patient with active pemphigus with intercellular antibodies at 1:320 titer was added to the medium either initially or after a 24-h preincubation in FCS and Ham F-10 medium. As controls, organ culture specimens were incubated in Ham F -10 medium containing either FCS or normal human plasma either with or without 0.25% rnM methylprednisolone (Table II).