Normal human skin was maintained in organ cultures for several days in Ham's F-10 medium with good preservation of the epidermal cells. When the partially purified IgG fraction from the pooled sera of patients with pemphigus vulgaris or pemphigus foliaceous was added to this culture system, after 24 hr some evidence of epidermal acantholysis was seen. By 72 hr, extensive suprabasilar epidermal acantholysis had occurred in which the acantholytic cells were indistinguishable histologically from the acantholytic cells in biopsies from skin lesions of patients with pemphigus vulgaris. In the control cultures (i.e., F-10 medium or F-10 medium + normal human serum IgG), none of these changes was seen. Direct immunofluorescent staining of these explants using fluorescein-labeled goat antihuman IgG showed that by 6 hr binding of the pemphigus IgG had occurred in the intercellular cement substance of the epidermis. The staining intensity was maximal by 18 to 20 hr. When the pemphigus serum was fractionated by DEAE-cellulose column chromatography, three major IgG-containing peaks (presumably IgG) were eluted which bound to the epidermoid intercellular substance and caused acantholysis in culture. The complement system did not play a role in the antibody-induced acantholysis since complement was not included in this system and heating the reconstituted F-10 + pemphigus IgG for 1 hr at 58 degrees C did not destroy the acantholytic activity. Autoradiographic experiments showed that after about 2 days in culture the rates of incorporation of RNA and protein precursors in the suprabasilar cells in the presence of pemphigus IgG were reduced to less than 10% of the normal IgG controls, whereas these synthetic activities of the basal cells were only slightly affected. These observations lead to the proposal that it is the interaction of the pemphigus autoantibody(s) with the suprabasilar epidermal cell which initiates and possibly substains the process(es) of acantholysis.

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A B S T R A C T The mechanism of pemphigus acantholysis has been studied with an in vitro system. Freshly prepared human skin epidermal cells were incubated in F-10 medium which contained the immunoglobulin G fraction from either pemphigus serum or normal human serum. During 18-h incubation periods, the pemphigus antibody became bound to the surface ofthe epidermal cells, caused the destruction of 75% of the viable cells as compared to only 14% in the normal immunoglobulin G controls (trypan blue exclusion), prevented the accumulation of newly synthesized proteins by nearly 60% as determined by radioactive tracer studies, and caused a dramatic shift in distribution of the newly synthesized proteins from an insoluble cell-associated fraction to an extracellular soluble fraction. These effects on the accumulation and partitioning of newly synthesized proteins were antibody concentration-dependent. Kinetic studies showed that at a fixed pemphigus antibody concentration the inhibition of protein accumulation preceded solubilization by about 1 h, at which time rapid solubilization of up to 70% of the insoluble cellular material occurred. Several lines of evidence suggested that this phenomenon was caused by enzymatic activity. Epidermal extracts solubilized a prepared substrate of radioactively labeled insoluble epidermal cell material. This activity was heat labile and pH dependent, with pH optima ranging from 4.5 to 6.5. Enzymes with pH optima between 6 and 6.5 were recovered in the culture medium after a 2-day incubation of pure, intact epidermis with the pemphigus antibody.We propose the following hypothesis to account for pemphigus acantholysis. The pemphigus antibody reacts with the epidermal cell surface and produces such a severe disturbance that the integrity of the cell