Immunopathology and molecular diagnosis of autoimmune bullous diseases

Abstract

Autoimmune bullous diseases are associated with autoimmunity against structural components maintaining cell-cell and cell matrix adhesion in the skin and mucous membranes. Pemphigus diseases are characterized by autoantibodies against the intercellular junctions and intraepithelial blisters. In pemphigoid diseases and epidermolysis bullosa acquisita, sub-epidermal blistering is associated with autoantibodies targeting proteins of the hemidesmosomal anchoring complex. The autoantigens in autoimmune blistering diseases have been extensively characterized over the past three decades. In general, the pathogenicity of autoantibodies, already suggested by clinical observations, has been conclusively demonstrated experimentally. Detection of tissue-bound and circulating serum autoantibodies and characterization of their molecular specificity is mandatory for the diagnosis of autoimmune blistering diseases. For this purpose, various immunofluorescence methods as well as immunoassays, including immunoblotting, enzyme-linked immunosorbent assay and immunoprecipitation have been developed. This review article describes the immunopathological features of autoimmune bullous diseases and the immunological and molecular tests used for their diagnosis and monitoring.

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Schematic diagram of the desmosome and the dermal-epidermal junction. Here are represented only structural proteins that function as autoantigens in autoimmune bullous skin diseases. Neighbouring keratinocytes are associated via the extracellular portions of desmosomal cadherins. As examples, homophilic interactions between desmoglein 1, desmoglein 3 and desmocollin 1 are depicted. Their intracellular portions bind to desmosomal plaque proteins that mediate the interaction of desmosomes with keratin filaments. Keratin filaments also bind to bullous pemphigoid antigen 230 (BP230) and plectin, the main intracellular constituents of the hemidesmosomes. BP230 and plectin function as ligands for transmembrane hemidesomosomal proteins, type XVII collagen (BP180) and α₆β₄ integrin. These may connect the hemidesmosomes to laminin 5, which in addition to type IV collagen, is a major component of the lamina densa. Laminin 5 is a known ligand for type VII collagen, the major constituent of the anchoring fibrils, which connect lamina densa to the collagen bundles of the upper dermis.

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Serological tests used for the diagnosis of pemphigus. Circulating autoantibodies show an intercellular binding pattern by indirect immunofluorescence microscopy on monkey esophagus (magnification, 3200).

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Serological tests used for the diagnosis of paraneoplatic pemphigus. (A) Serum autoantibodies from a patient with paraneoplastic pemphigus bind to rat bladder urothelium, as revealed by indirect immunofluorescence microscopy (magnification, 200). (B) An extract of cultured keratinocytes was fractionated by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with paraneoplastic pemphigus serum (lane 1) and with normal human serum (lane 2). Paraneoplastic pemphigus serum reacts with envoplakin (210 kD; upper arrow) and periplakin (190 kD;lower arrow), while control serum shows no specific reactivity. Molecular weight markers are depicted on the right side of the panel.

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IF microscopy analysis in bullous pemphigoid and pemphigoid gestationis. (A) Direct immunofluorescence microscopy of perilesional skin reveals deposition of complement C3 at the dermal-epidermal junction of a patient with bullous pemphigoid (magnification, ×100). (B) Serum bullous pemphigoid IgG autoantibodies bind to the epidermal side of 1M NaCl-split skin, by indirect immunofluorescence microscopy (magnification, ×100). Cryosections of normal human skin were incubated with (C) pemphigoid gestationis serum or (D) normal human serum, and, subsequently, with fresh serum from a healthy control, used as a source of complement. Staining with an FITC-labeled antibody to human C3 reveals linear complement deposits at the dermal-epidermal junction in the case of the section incubated with the pemphigoid gestationis serum, but not with normal human serum (magnification, ×200).

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Serological tests used for the diagnosis of bullous pemphigoid and pemhigoid gestationis.(A) An extract of cultured keratinocytes was fractionated by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with serum from patients with bullous pemphigoid (BP;lanes 1–2), pemphigoid gestationis (PG;lane 3) and a healthy control (NHS;lane 4). The bullous pemphigoid serum depicted in lane 1 reacts with both bullous pemphigoid antigen 230 (BP230, 230 kD, upper arrow) and bullous pemphigoid antigen 180 (BP180, 180kD, lower arrow), while the bullous pemphigoid serum shown in lane 2 and the pemphigoid gestationis serum shown in lane 3 react with BP180 only. Control serum shows no specific reactivity (lane 4). Molecular weight markers are depicted on the right side of the panel. (B) The ELISA using a recombinant form of the 16th non-collagenous A domain of the bullous pemphigoid antigen 180 is a specific and sensitive tool for the diagnosis of bullous pemphigoid and pemphigoid gestationis. Sera from patients with bullous pemphigoid (BP), pemphigoid gestationis (PG), and normal healthy controls (NHS), were tested in triplicate with immobilized recombinant protein. The dotted line represents the cut-off of the assay (modified from [78]).

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Circulating autoantibodies from patients with linear IgA disease react with LAD-1 and LABD97. Concentrated supernatant of cultured keratinocytes was separated by 10% SDS-PAGE, transferred to nitrocellulose, and incubated with sera from patients with linear IgA disease (LAD;lanes 1–2), bullous pemphigoid (BP;lane 3), and healthy controls (NHS; lane 4). Subsequently, the nitrocellulose strips were incubated with HRP-labeled secondary antibodies to human IgA. Autoantibodies from patients with linear IgA disease react with both proteolytic fragments of BP180, LAD-1 (120 kD; lanes 1–2, upper arrow) and LABD97 (97 kD; lane 1, lower arrow). Serum autoantibodies from a patient with bullous pemphigoid recognize LAD-1 (lane 3). Control serum shows no specific reactivity (lane 4). Molecular weight markers are depicted on the right side of the panel.

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Serological tests used for the diagnosis of mucous membrane pemhigoid. (A) Serum autoantibodies from most patients with mucous membrane pemhigoid bind to the epidermal side of the salt-split skin by indirect immunofluorescence microscopy (magnification, ×100). (B) In a subgroup of patients with mucous membrane pemhigoid, circulating autoantibodies show a dermal binding pattern by indirect immunofluorescence microscopy (magnification, ×100). (C) Extracellular matrix of cultured keratinocytes was separated by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with mucous membrane pemhigoid and control sera [160]. Autoantibodies from mucous membrane pemhigoid patients react with non-processed (200 kD; lane 1, upper closed circle) and processed (165 kD;lane 1, middle closed circle, and lane 2, upper closed circles) forms of the 3-chain of laminin 5, as well as with the 2-chain (155 kD;lane 1 and 2, lower closed circles) of laminin 5. Control serum shows no specific reactivity (lane 3). Molecular weight markers are depicted on the left side of the panel.

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Serum autoantibodies from patients with epidermolysis bullosa acquisita react with both full-length type VII collagen and with its immunodominant domain. Dermal extracts (DE) and a recombinant form of the non-collagenous (NC) domain 1 of type VII collagen were separated by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with sera from patients with epidermolysis bullosa acquisita (lanes 1 and 3) or healthy controls (lanes 2 and 4). Sera from patients with epidermolysis bullosa acquisita react with both full-length type VII collagen (290 kD;upper arrow) and with its immunodominant NC1 domain (145 kD;lower arrow), extracted from dermis (lane 1). The same serum binds also to the recombinant form of NC1 (lane 3, arrowhead). Control serum did not react with any of the molecules (lane 2, 4). Molecular weight markers are depicted on the left side of the figure.

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IF microscopy analysis in dermatitis herpetiformis. (A) Direct immunofluorescence microscopy of a perilesional skin biopsy reveals granular IgA deposits in the papillary dermis (magnification, ×400). (B) By indirect immunofluorescence microscopy on monkey esophagus, anti-endomysial IgA antibodies are detected in serum of a patient with dermatitis herpetiformis (magnification, 200).