No abstract

Pemphigus is an autoimmune cutaneous disease characterized by circulating autoantibodies that cause blistering and erosions on skin and mucous membranes. Circulating autoantibodies bind to epidermal cell membrane and cause cell-cell detachment (acantholysis), leading to epidermal tissue damage and cell death. The principal target of pemphigus vulgaris autoantibodies (PV-IgG) is desmosomal cadherin desmoglein 3 (Dsg3), a constituent of desmosomes, mediating cell-cell adhesion. Several hypotheses for the mechanisms of acantholysis induction by PV-IgG exist, but the actual mechanism is not clear as yet. We have previously reported on apoptosis induction in PV-IgG-mediated epidermal tissue and cell damage as a possible mechanism of acantholysis and cell death (Wang et al. 2004, Apoptosis, 9:131-143). In this study we investigated the involvement of the EGFR and intracellular signal transduction pathways in the PV-IgG-induced apoptosis. We show here that PV-IgG induced activation/autophosphorylation of EGFR in cultured keratinocytes in vitro. The specific tyrosine kinase inhibitor AG1478 abrogated EGFR autophosphorylation, cell death, FasL appearance and acantholysis, all induced by PV-IgG, in parallel, confirming the involvement of EGFR in this Fas apoptotic cascade. Activation of EGFR was followed by phosphorylation of its downstream substrates, MAP kinase ERK and transcription factor c-Jun, and internalization of EGFR. Pharmacological inactivation of the EGFR and ERK kinase activities, by use of specific inhibitors AG1478 and PD98059 respectively, blocked PV-IgG-induced phosphorylation of EGFR, ERK and c-Jun and cellular apoptosis, measured by flow cytometry and caspase 3 activity. Prolonged activation of EGFR by PV-IgG led to dramatic internalization of this receptor, possibly reducing the ability of the cell to perform survival signals. This suggests that activation of EGFR, followed by its internalization, is pivotal for intracellular apoptotic signal transduction via ERK/c-Jun pathways, leading to acantholysis. Our experimental data indicate that the EGFR is instrumental in transducing apoptotic/acantholytic signals in keratinocytes cultures in response to PV-IgG treatment. The acantholytic effect caused by PV-IgG binding to cell surface receptors begins with and depends on cell surface receptor (EGFR) activation of intracellular signaling pathways (ERK pathway) and apoptosis induction (FasR pathway), which later lead to major cell-cell separation (acantholysis) and cell death.

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.