Beno Michel

Apoptotic mechanism in pemphigus autoimmunoglobulins-induced acantholysis—possible involvement of the EGF receptor

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.

An organ culture model for the study of pemphigus acantholysis

An in vitro model for the study of pemphigus acantholysis has been developed. The histological changes of pemphigus vulgaris were reproduced in vitro in organ culture by growing normal human skin in the presence of pemphigus vulgaris or pemphigus foliaceus sera. At 24 h a suprabasilar split was noted and at 72 h extensive suprabasilar acantholysis developed. Direct immunofluorescent tests demonstrated that pemphigus antibody became bound to the epidermal intercellular space antigen(s) during the first 6–12 h. As acantholysis increased the presence of tissue‐fixed antibody decreased. The fixation of the pemphigus antibody to the skin prior to the development of acantholysis provides strong evidence for the pathogenetic role of this antibody in the production of acantholysis. The data suggest that complement is not required in this model for the production of the acantholytic changes of pemphigus since heating the serum for 30 min at 56 C did not destroy the acantholytic activity and no complement (C3) could be detected by DIF of organ culture explants.

Corticosteroids, aurothioglucose and soybean trypsin inhibitor do not prevent pemphigus antibody-induced acantholysis in vitro

Hydrocortisone, triamcinolone acetonide, aurothioglucose and soybean trypsin inhibitor were added to normal human skin explants cultured with IgG from pemphigus serum to determine if acantholysis could be prevented. At the therapeutic concentrations used none of these compounds prevented binding of the autoantibody to the epidermal target cells, and none prevented acantholysis. These experiments support the concepts that the pemphigus antibody alone is responsible for producing the acantholytic lesions of pemphigus, that the therapeutic effectiveness of steroids and gold salts is probably due to their ability to reduce serum autoantibody titres and that pemphigus acantholysis is probably not caused by a serine proteinase.

ELEVATED LEVELS OE SERUM COMPLEMENT IN PATIENTS WITH PYODERMA GANGRENOSUM

ELEVATED levels of serum complement are seldom seen except in experimental and clinical inflammation (Fischel, 1963), myocardial infarction (Boltax and Fischel, 1956) or in connective tissue disorders such as rheumatoid arthritis, acute rheumatic fever and active dermatomyositis (Williams and Law, 1958), We wish to report 2 cases of pyoderma gangrenosum with elevated levels of serum complement. One patient also had rheumatoid arthritis, the other had a past history of chronic ulcerative colitis and rheumatic fever and the pyoderma was associated with active chronic ulcerative proctitis,

Epidermal nuclear immunofluorescence. Is it a specific marker for mixed connective tissue disease

In sixteen patients, the relationship of epidermal nuclear immunofluorescence to the spectrum of connective tissue diseases and to serum antibodies was investigated. The clinical diagnoses were mixed connective tissue disease (7 patients), systemic lupus erythematosus (5 patients), scleroderma (3 patients) and rheumatoid arthritis (1 patient). Detailed serologic evaluation in 13 of the 16 patients revealed positive rheumatoid factor (7 patients), antinuclear antibody (13 patients), anti‐DNA (2 patients), anti‐n‐RNP (11 patients), anti‐Sm (2 patients), anti‐SS‐A (3 patients) and anti‐RANA (5 patients). Anti‐SS‐B and anti‐Scl‐70 were absent in all. Epidermal nuclear immunofluorescence on skin biopsy correlated with serum anti‐n‐RNP and is a useful marker for mixed connective tissue disease. However, it did not appear to serve as a specific marker for any one connective tissue disease. We discuss the mechanism(s) for this immunofluorescent pattern and believe it is an in vivo phenomenon.