Mechanisms Causing Acantholysis in Pemphigus-Lessons from Human Skin

Abstract

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease caused primarily by autoantibodies (PV-IgG) against the desmosomal adhesion proteins desmoglein (Dsg)1 and Dsg3. PV patient lesions are characterized by flaccid blisters and ultrastructurally by defined hallmarks including a reduction in desmosome number and size, formation of split desmosomes, as well as uncoupling of keratin filaments from desmosomes. The pathophysiology underlying the disease is known to involve several intracellular signaling pathways downstream of PV-IgG binding. Here, we summarize our studies in which we used transmission electron microscopy to characterize the roles of signaling pathways in the pathogenic effects of PV-IgG on desmosome ultrastructure in a human ex vivo skin model. Blister scores revealed inhibition of p38MAPK, ERK and PLC/Ca²⁺ to be protective in human epidermis. In contrast, inhibition of Src and PKC, which were shown to be protective in cell cultures and murine models, was not effective for human skin explants. The ultrastructural analysis revealed that for preventing skin blistering at least desmosome number (as modulated by ERK) or keratin filament insertion (as modulated by PLC/Ca²⁺) need to be ameliorated. Other pathways such as p38MAPK regulate desmosome number, size, and keratin insertion indicating that they control desmosome assembly and disassembly on different levels. Taken together, studies in human skin delineate target mechanisms for the treatment of pemphigus patients. In addition, ultrastructural analysis supports defining the specific role of a given signaling molecule in desmosome turnover at ultrastructural level.

Keywords: desmosomes; electron microscope; ex vivo skin model; pemphigus; signaling; ultrastructure.

Figure 1

(A) Electron micrograph showing single desmosome in a healthy skin with a superimposed schematic representation depicting molecular structure of a desmosome. Colour coding of each single molecule is the same as those represented in (B, C). A schematic representation of the distribution of desmosomal proteins along the different layers of (B) epidermis and (C) mucosa. Small schematic desmosomes are colour-coded the same as the bars representing the distribution.

Figure 2

Electron micrographs showing an overview (top) and zoomed in to a single desmosome (bottom) of (A) healthy skin injected with IgG from healthy volunteers. (B, C) skin injected with PV-IgG showing suprabasal blistering, interdesmosomal widening and altered desmosomes: (B) reduced keratin insertion (red arrow head) into a damaged plaque. Note that the distinction between the outer and inner desmosomal plaque is lost after incubation with PV-IgG; violet arrow heads indicate the basement membrane, (C) a split desmosome with half plaque (green arrow head), red asterisk indicates blister cavity. Scale bars: 2 µm (top) and 250 nm (bottom).

Figure 3

Ultrastructural quantification of desmosomes. (A) Desmosome density expressed in number of desmosomes per µm membrane length. Only desmosomes along clearly delineated cell borders of basal cells were considered. (B) Desmosome size measured along the linear length of the plaques and expressed in nm. (C) Percentage of split desmosomes both in acantholytic and non acantholytic areas. (D) Percentage of keratin dissociation from desmosomal plaques. Each data point represents individual desmosomes for (B), and the average per electron micrograph for (A, C and D) (n= 3-5 for each pathway. *p < 0.05 vs. control, #p < 0.05 vs. PV-IgG). Inhibitors used: SB202190 – p38MAPK inhibitor, Pp2 – Src inhibitor, Bim-X – PKC inhibitor, UO126 MEK (upstream of Erk1/2) inhibitor, U-73122 – PLC inhibitor, Xest (xestospongin) – IP3R inhibitor.