Ca2+ signalling is critical for autoantibody-induced blistering of human epidermis in pemphigus.

BACKGROUND: Pemphigus is a severe bullous autoimmune skin disease. Pemphigus foliaceus (PF) is characterized by antidesmoglein (Dsg) 1 IgG causing epidermal blistering; mucosal pemphigus vulgaris (mPV) by anti-Dsg3 IgG inducing erosions in the mucosa; and mucocutaneous pemphigus vulgaris (PV) by affecting both, with autoantibodies targeting Dsg1 and Dsg3. OBJECTIVES: To characterize the Ca2+ flux pathway and delineate its importance in pemphigus pathogenesis and clinical phenotypes caused by different antibody profiles. METHODS: Immunoprecipitation, Ca2+ flux analysis, Western blotting, immunofluorescence staining, dissociation assays and a human skin ex vivo model were used. RESULTS: PV IgG and PF IgG, but neither Dsg3-specific monoclonal antibody (AK23) nor mPV IgG, caused Ca2+ influx in primary human keratinocytes. Phosphatidylinositol 4-kinase α interacts with Dsg1 but not with Dsg3. Its downstream target - phospholipase-C-γ1 (PLC) - was activated by PV IgG and PF IgG but not AK23 or mPV IgG. PLC releases inositol 1,4,5-trisphosphate (IP3) causing IP3 receptor (IP3R) activation and Ca2+ flux from the endoplasmic reticulum into the cytosol, which stimulates Ca2+ release-activated channels (CRAC)-mediated Ca2+ influx. Inhibitors against PLC, IP3R and CRAC effectively blocked PV IgG and PF IgG-induced Ca2+ influx; ameliorated alterations of Dsg1 and Dsg3 localization, and reorganization of keratin and actin filaments; and inhibited loss of cell adhesion in vitro. Finally, inhibiting PLC or IP3R was protective against PV IgG-induced blister formation and redistribution of Dsg1 and Dsg3 in human skin ex vivo. CONCLUSIONS: Ca2+ -mediated signalling is important for epidermal blistering and dependent on the autoantibody profile, which indicates different roles for signalling complexes organized by Dsg1 and Dsg3. Interfering with PLC and Ca2+ signalling may be a promising approach to treat epidermal manifestations of pemphigus.

A new ex vivo human oral mucosa model reveals that p38MAPK inhibition is not effective in preventing autoantibody-induced mucosal blistering in pemphigus.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by blister formation in the epidermis and oral mucosa due to loss of keratinocyte cohesion. Autoantibodies present in patients with PV (PV-IgG) are known to primarily target desmoglein (Dsg)1 and Dsg3 in desmosomes. The mucosal-dominant subtype of PV (mdPV) is caused by PV-IgG autoantibodies against the cadherin-type adhesion molecule Dsg3. p38 mitogen-activated protein kinase (p38MAPK) signalling has been characterized as an important pathway downstream of PV-IgG binding and its inhibition is protective in ex vivo human skin. However, the role of p38MAPK signalling in mdPV is unknown as no experimental model has been available. OBJECTIVES: To establish a human ex vivo oral mucosa culture, and evaluate the p38MAPK dependency of blister formation and of ultrastructural alterations of desmosomes induced by mdPV-IgG. METHODS: Human labial mucosa was injected with mdPV-IgG as well as AK23, a pathogenic mouse monoclonal Dsg3 antibody, in the presence or absence of p38MAPK inhibitors. Viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay. Blister score was determined following haematoxylin and eosin staining and Dsg3 distribution by immunostaining. Samples were processed for transmission electron microscopy to analyse desmosome ultrastructure. RESULTS: Both AK23 and mdPV-IgG induced blisters and caused reduction in desmosome size and number in labial mucosa. Inhibition of p38MAPK was not effective in preventing these alterations. CONCLUSIONS: In contrast with human epidermis, PV-IgG and AK23 induce blisters and desmosome ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK. What's already known about this topic? Pemphigus vulgaris IgG (PV-IgG) induces blistering as well as a reduction in desmosome number and size mediated by p38 mitogen-activated protein kinase (p38MAPK) signalling in ex vivo human skin. What does this study add? This study establishes a new human ex vivo mucosa model to test pathomechanisms mediated by PV-IgG. The study demonstrates that both AK23 and mucosal-dominant PV induce blisters and associated ultrastructural changes in labial mucosa via a mechanism not dependent on p38MAPK signalling. What is the translational message? This study highlights the respective tissue-specific responses of oral mucosa and skin related to PV pathogenesis, similar to the patient situation.

Inhibition of p38MAPK signalling prevents epidermal blistering and alterations of desmosome structure induced by pemphigus autoantibodies in human epidermis.

BACKGROUND: Pemphigus vulgaris (PV) is a skin blistering disease caused by autoantibodies targeting the desmosomal adhesion proteins desmoglein (Dsg) 3 and 1. The mechanisms underlying pemphigus skin blistering are not fully elucidated but p38 mitogen-activated protein kinase (p38MAPK) activation is one of the signalling events necessary for full loss of cell cohesion. However, it is unclear whether ultrastructural hallmarks of desmosome morphology as observed in patients' lesions are mediated by p38MAPK signalling. OBJECTIVES: In this study, we tested the relevance of p38MAPK for blister formation and the ultrastructural changes induced by PV autoantibodies in human skin. METHODS: Human skin samples were injected with IgG fractions of one patient suffering from mucocutaneous PV (mcPV-IgG), one from mucosal-dominant PV (mdPV-IgG) or AK23, a pathogenic monoclonal Dsg3 antibody derived from a pemphigus mouse model. Samples were processed for histological and electron microscopy analyses. RESULTS: mcPV-IgG and AK23 but not mdPV-IgG reduced desmosome size, caused interdesmosomal widening and formation of split desmosomes, and altered keratin filament insertion. In contrast, full epidermal blister formation and lower desmosome number were evident in tissue samples exposed to mcPV-IgG only. Pharmacological inhibition of p38MAPK blunted the reduction of desmosome number and size, ameliorated interdesmosomal widening and loss of keratin insertion and prevented mcPV-IgG-induced blister formation. CONCLUSIONS: Our data demonstrate that blistering can be prevented by inhibition of p38MAPK in the human epidermis. Moreover, typical morphological alterations induced by mcPV-IgG such as interdesmosomal widening and the reduction of desmosome size at least in part require p38MAPK signalling.