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The pemphigoids, autoimmune blistering diseases of the skin and mucosae: immunopathogenic mechanisms, prognostic and diagnostic markers (PEMPHIGOIDS & IMMUNITY)


The pemphigoids (comprising bullous pemphigoid, gestational pemphigoid and mucous membrane pemphigoid) are a group of autoimmune bullous disorders of the skin and mucous membranes characterised by defective epithelial adhesion and by circulating and tissue-bound autoantibodies against components of the cutaneous basement membrane zone. Pemphigoid patient autoantibodies preferentially target two components of the epithelial adhesion structures called hemidesmosomes: the BP180 antigen (or collagen XVII), a type II collagenous transmembrane protein, and the cytoplasmic BP230 antigen. In particular, the majority of patients present autoantibodies against the NC16A subdomain of BP180 ectodomain. The third major antigenic target in pemphigoid patients is the epithelial adhesion ligand laminin 5. In this project, we have characterized the humoral immune response in the pemphigoids and developed novel diagnostic ELISAs. Specifically, we have obtained: %1) nine ELISAs based on different regions of BP180 (five different regions of the ectodomain and also the entire ectodomain) and BP230 (three regions spanning almost the entire antigen), produced as baculovirus-derived recombinant proteins, which were validated testing large cohorts of bullous pemphigoid patients sera. Overall these ELISAs displayed a high sensitivity and specificity. However and in keeping with current concepts on disease pathogenesis, the global diagnostic properties of the BP180–ELISA outperformed those of the BP230-ELISA. These ELISAs also allowed to correlate antigen reactivity and clinical phenotypes and to delineate markers for disease activity and course; 2) six ELISAs based on BP180 regions produced as prokaryotic recombinant proteins that were used to detect intramolecular epitope spreading events during the disease course and to improve the diagnostic performance of the commercially available ELISA based on NC16A alone. These ELISAs were based on epitopes selected by screening a BP180 random epitope lambda library with bullous pemphigoid patient’s sera. 3) an ELISA based on native laminin 5 purified from the conditioned medium of a squamous carcinoma cell line. When used to screen a large cohort of pemphigoid patients sera, our ELISA detected anti-laminin 5 reactivity in the majority of mucous membrane pemphigoid sera and also in a part of bullous pemphigoid sera tested. Moreover, the titers of laminin 5-specific IgG correlated with clinical severity of mucous membrane pemphigoid. Collectively our findings show that these novel ELISAs represent a highly sensitive and specific assay for the rapid diagnosis of the pemphigoids and may provide predictive parameters for the management of pemphigoid patients.
Our studies have further our understanding of the molecular organization of hemidesmosomes and of their linkage with the cytoskeleton and the basement membrane. Hemidesmosomes are junctional adhesion complexes promoting epithelial-stromal cohesion in stratified epithelia, such as skin. Acquired and inherited (gene mutations) defects of components of hemidesmosomes and of their associated structures cause a number of life-threatening skin diseases, such as the autoimmune blistering diseases called pemphigoids and the inherited diseases known as epidermolysis bullosa. Our results have characterized the interactions of the hemidesmosomal components BP180 (BPAG2) and BP230 (BPAG1e) with either the cytokeratins K5/14 intermediate filament system or extracellular matrix protein laminin-5. The results indicate that sequences within the C-terminal domain of BP230 are critical for its association with K5/K14 keratins and that BP180 is able to directly bind to laminin-5. New knowledge about processes of basic biological importance, such as cell adhesion, maintenance of cytoarchitecture and cytoskeleton organization has been gained. This information contributes to a better understanding of the physiopathology of a group of acquired and inherited disorders of the skin characterized by skin blistering and fragility. Clinically, it will be possible to better explain to patients affected by these rare diseases the mechanisms leading to skin blistering and fragility with failure of the skin to remains “sticked” together. Finally, it is expected that the acquired new knowledge will facilitate the development of better tailored therapies for a group of patients with acquired and congenital skin blistering disorders.
The major autoantigen of bullous pemphigoid, BP180 or collagen XVII, has two forms, a transmembrane and a short, soluble form, which results from shedding of the ectodomain from the keratinocyte surface. The major objective here was to identify the molecular mechanisms of shedding. Using a broad panel of proteinase inhibitors, recombinant enzymes and substrates, and enzyme-deficient cells from knockout mice we demonstrated that collagen XVII is shed by ADAMs (a disintegrin and metalloproteinase) proteinases. The prototype sheddase TACE (TNF-alpha converting enzyme, or ADAM-17), ADAM-10 and ADAM-9 contribute to shedding of collagen XVII. This was the first description of shedding of a transmembrane collagen by ADAMs, which possibly release also other transmembrane proteins from the keratinocyte surface. In order to define the cleavage region and to assess its structure and requirements for shedding, deletion mutants of the NC16A domain were expressed in COS-7 cells. Analysis of the deletions pointed to the stretch of amino acids 528-547 as important for sheddase recognition and cleavage. Secondary protein structure predictions showed that deletion of this stretch resulted in conformational changes in the collagen XVII homotrimer, indicating that the conformation of the NC16A domain and steric availability of the cleavage site influence shedding and are important for folding of collagen XVII. Monoclonal antibodies recognizing the proteolytically released ectodomain of collagen XVII were also produced and characterized. As a development of studies on the regulation of BP180 shedding, we investigated the role of plasma membrane microenvironment in BP180 shedding. ADAMs-mediated BP180 shedding was induced by disintegration of lipid rafts and by low-cholesterol membrane levels, and confocal microscopy analysis indicated that BP180 is incorporated in lipid rafts. These data represent the first evidence of the role of plasma membrane lipid organization in the regulation of BP180 shedding and therefore of keratinocyte migration and differentiation.New highly relevant knowledge has been obtained about basic aspects of keratinocyte biology, such as cell adhesion and migration. Besides, the data on shedding are important for understanding the generation of autoantigen epitopes in bullous pemphigoid and for design of biologically valid novel therapeutic strategies. Finally, the monoclonal antibodies to the shedded ectodomain of collagen XVII can contribute both to the research reagent repertoire for study of hemidesmosomes and to the development of clinical diagnostic tests (a patent application has been submitted by the involved participant, University of Freiburg).

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