Molecular diagnosis in autoimmune skin blistering conditions

Abstract

Blister formation in skin and mucous membranes results from a loss of cell-cell or cell-matrix adhesion and is a common outcome of pathological events in a variety of conditions, including autoimmune and genetic diseases, viral and bacterial infections, or injury by physical and chemical factors. Autoantibodies against structural components maintaining cell-cell and cell-matrix adhesion induce tissue damage in autoimmune blistering diseases. Detection of these autoantibodies either tissue-bound or circulating in serum is essential to diagnose the autoimmune nature of disease. Various immunofluorescence methods as well as molecular immunoassays, including enzyme-linked immunosorbent assay and immunoblotting, belong to the modern diagnostic algorithms for these disorders. There is still a considerable need to increase awareness of the rare autoimmune blistering diseases, which often show a severe, chronic-relapsing course, among physicians and the public. This review article describes the immunopathological features of autoimmune bullous diseases and the molecular immunoassays currently available for their diagnosis and monitoring.

Fig. (1)

Schematic representation of major skin autoantigens. The autoantigens shown here are molecules involved in maintaining the cell-cell and cell-matrix adhesion. The transmembrane desmosomal cadherins of neighboring cells, desmogleins and desmocollins, confer adhesion by homo- and heterophilic interaction in the extracellular space. On the cytoplasmic sides of the desmosome, the carboxy-terminal regions of cadherins are rooted in the desmosomal plaques composed of proteins belonging to the armadillo and plakins superfamilies, such as desmoplakin, which bind to the intermediate keratin filaments. The hemidesmosomes are important for the stable anchorage of basal keratinocytes to the underlying basement membrane. The intracellular portion of transmembrane hemidesmosomal proteins, incuding collagen XVII and integrin α6β4, binds to intracellular plaque proteins BP230 and plectin, which link hemidesmosomes to keratin intermediate filaments. Collagen XVII/BP180 and α6β4 integrin bind extracellularly to the extracellular matrix protein laminin (Ln) 332, a major component of the lamina densa. Laminin γ1 chain, present in the vessel walls and at the epidermal basement membrane as in laminin 511, may function as autoantigen. Finally, collagen VII is the main constituent of the anchoring fibrils, which connect lamina densa to the collagen fibers of the upper dermis.

Fig. (2)

Major clinical and laboratory findings in pemphigus vulgaris. (a) Extensive erosions with crusts and hyperpigmentation on the back of a 34-year old male patient with muco-cutaneous pemphigus vulgaris. (b) Histopathological examination reveals suprabasal acantholysis with modest inflammatory infiltrate. A single layer of basal keratinocytes remains attached to the basement membrane as a “row of tombstones”. (c) Direct immunofluorescence microscopy analysis of a perilesional skin biopsy shows deposits of IgG with an intercellular pattern in the epidermis. (d) Serum IgG autoantibodies binding with an intercellular pattern are detected by indirect immunofluorescence microscopy on monkey oesophagus (all magnification 200x).

Fig. (3)

Major clinical and laboratory findings in pemphigus foliaceus. (a) Erythema, blisters and erosions in a 61-year old male patient with pemphigus foliaceus. (b) Histopathological examination reveals sub-corneal acantholysis and an inflammatory infiltrate consisting mainly of neutrophil granulocytes. (c) Deposits of IgG with a cobblestone pattern within the epidermis by direct immunofluorescence microscopy analysis of a skin biopsy. (d) By indirect immunofluorescence microscopy on monkey oesophagus, serum IgG autoantibodies are detected, which bind with an intercellular pattern to epithelium (all magnification 200x).

Fig. (4)

Detection of desmoglein-specific autoantibodies by ELISA. (a) Pemphigus vulgaris (PV) may present with only mucosal (m) lesions and autoantibodies against desmoglein (Dsg) 3 or with mucocutaneous (m/c) involvement and autoantibodies to both Dsg 1 and 3. Pemphigus foliaceus (PF) patients show only skin lesions and autoantibodies exclusively directed to desmoglein (Dsg) 1. (b) Monitoring of Dsg1- and Dsg3-specific IgG autoantibodies during therapy with prednisolone, azathioprine (AZA), and mycophenolate mofetil (MMF). The gray area represents the disease activity as represented by the PDAI (pemphigus disease area index) scores [177].

Fig. (5)

Characteristic findings in paraneoplastic pemphigus. (a) Hemorrhagic erosions with crusts on the lips and oral cavity of a patient with non-Hodgkin lymphoma. (b) Interface dermatitis by histopathology (H&E staining). (c) Immunofluorescence (IF) microscopy analysis of a perilesional skin biopsy reveals deposits of IgG at the dermal-epidermal junction and at the intercellular spaces of keratinocytes. (d) Further serological testing by indirect IF microscopy shows binding of IgG autoantibodies to rat bladder urothelium (magnification, 200x). (e) Extract of cultured keratinocytes fractionated by 7.5% SDS-PAGE was transferred to nitrocellulose, and incubated with sera from patients with pemphigus vulgaris (lane 1), bullous pemphigoid (lane 2), paraneoplastic pemphigus serum (lane 3), and normal human serum (lane 4). PNP serum reacts specifically with envoplakin (210 kD, upper arrow) and periplakin (190 kD, lower arrow). The control serum shows no specific reactivity. Migration position for BP230, BP180, Desmoglein (Dsg) 1 and 3 are depicted on the left side of the panel.

Fig. (6)

IgA pemphigus. (a) Clinical picture of a 56-year old woman with IgA pemphigus showing pustules on the abdomen. Inset: close-up view showing pustules, blisters, erosions, and crusts on an erythematous background. (b) Histopathological examination reveals subcorneal acantholysis with an inflammatory infiltrate consisting mainly of neutrophils. (c) IgA autoantibody binding with an intercellular pattern on monkey esophagus by indirect immunofluorescence (IF) microscopy. (d) Indirect IF microscopy using COS-7 cells transfected with desmocollin 1-cDNA as substrate reveals autoantigen-specific IgA serum autoantibodies.

Fig. (7)

Bullous pemphigoid. (a) Blisters, erosions with crusts on an erythematous background in a 72-years old male patient with bullous pemphigoid. Inset: close-up view of blistering skin. (b) The histopathological examination reveals subepidermal cleavage with a inflammatory infiltrate consisting predominantly of eosinophils and neutrophils. (c) Direct immunofluorescence microscopy of perilesional skin shows C3c deposition at the dermo-epidermal junction of a patient with bullous pemphigoid. (d) Serum IgG autoantibodies from a bullous pemphigoid patient binding at the epidermal side of 1M NaCl-split skin by indirect immunofluorescence microscopy (all magnification 200x).

Fig. (8)

Molecular specificity of autoantibodies in bullous pemphigoid (BP). (a) Sera from patients with BP were tested by ELISA using a recombinant form of the 16th noncollagenous (NC16) A domain of the bullous pemphigoid (BP) antigen 180 and with recombinant BP230 as described [92, 93]. The dotted line represents the cut-off of the assay. (b) An extract of cultured keratinocytes was fractionated by 7.5% SDS-PAGE, transferred to nitrocellulose, and incubated with a BP180-specific monoclonal antibody (lane 1), serum from patients with BP (lanes 2–3), and serum from a healthy control (NHS; lane 4). The BP serum in lane 2 reacts with bullous pemphigoid antigen 180 (BP180, 180kDa, lower arrow) only. The BP serum depicted in lane 3 reacts with both bullous pemphigoid antigen 230 (BP230, 230 kDa, upper arrow) and BP180. Control serum shows no specific reactivity (lane 4).

Fig. (9)

Mucous membrane pemphigoid. (a) Buccal erosions in a 77-year old female with mucous membrane pemphigoid. (b) Histopathological examination of mucosa reveals a sub-epidermal blister and a mixed leukocytic infiltrate. (c) Direct immunofluorescence microscopy shows IgG deposits at the dermo-epidermal junction of a patient with mucous membrane pemphigoid. (d) Serum IgG autoantibodies binding to the epidermal side of 1M NaCL-split skin by indirect immunofluorescence microscopy (magnification 200x).

Fig. (10)

Linear IgA disease. (a) Erythema, blisters, erosions and crusts in a 4-year old child with linear IgA disease. (b) Histopathological examination reveals subepidermal cleavage and a rich inflammatory infiltrate dominated by neutrophils. (c) Direct immunofluorescence microscopy analysis of perilesional skin shows linear IgA deposition at the dermo-epidermal junction. (d) Serum IgA-autoantibodies bind to the epidermal side of 1M NaCl-split skin by indirect immunofluorescence microscopy (all magnification 200x).

Fig. (11)

Molecular specificity of IgA autoantibodies in pemphigoid diseases. (a) Spent medium of cultured keratinocytes was concentrated by ammonium sulfate precipitation, electrophoretically separated by 8% SDSPAGE, transferred on nitrocellulose and immunoblotted with serum from linear IgA disease (LAD) patients (lanes 1-3) and control serum (NHS) (lane 4). Bound autoantibodies were visualized using a peroxidase-labeled secondary antibody specific for human IgA. The shed ectodomain of BP180 of 120 kDa (LAD-1) and its 97 kDa degradation product (LABD97) are indicated by arrow and arrow head, respectively. (b) ELISA reactivity with the recombinant BP180 ectodomain of IgA autoantibodies from LAD, bullous pemphigoid (BP), dermatitis herpetiformis (DH) patients and age-matched, healthy donors (NHS). The cut-off of the assay is represented by a dashed line. Scatter plot adapted from [138].

Fig. (12)

Anti-p200 pemphigoid (a) Erythema, blisters, erosions and crusts in a 53-year old patient with anti-p200 pemphigoid. (b) Histopathological examination reveals subepidermal cleavage and a neutrophil-rich inflammatory infiltrate. (c) Direct immunofluorescence (IF) microscopy analysis of perilesional skin shows linear IgG deposition at the dermo-epidermal junction. (d) Serum IgG autoantibodies bind to the dermal side of 1M NaCl-split skin by indirect IF microscopy (all magnification 200x). (e) Dermal extracts were separated by 6% SDS-PAGE, transferred on nitrocellulose and immunoblotted with serum from patients with epidermolysis bullosa acquisita (EBA; lane 1), anti-p200 pemphigoid (p200; lane 2) and normal human serum (NHS, lane 3).

Fig. (13)

Diagnostic features of epidermolysis bullosa acquisita (EBA). (a) Clinical picture of a 61-year old female patient with the inflammatory form of EBA showing erythema, tense blisters, erosions and crusts on the lateral abdomen. (b) Histopathology analysis of the lesional skin shows dermal-epidermal separation and a neutrophil-rich inflammatory infiltrate. (c) Direct immunofluorescence microscopy of perilesional skin reveals deposits of IgG along the basement membrane zone. (d) Indirect immunofluorescence microscopy on 1M NaCl split-skin shows binding of IgG autoantibodies to the dermal side of the dermal-epidermal junction (magnification 200x).

Fig. (14)

Major clinical, histo- and immunopathological features of dermatitis herpetiformis. (a) Multiple excoriated papules, erosions and crusts on the elbow of a 44-year old patient with dermatitis herpetiformis. (b) Histopathological examination shows infiltration of neutrophils with incipient formation of papillary microabscesses and dermal-epidermal separation. (c) Direct immunofluorescence microscopy of a biopsy of non-affected skin reveals granular IgA deposits at the basement membrane. (d) By indirect immunofluorescence microscopy on monkey esophagus, anti-endomysial IgA antibodies are detected in the serum of a patient with dermatitis herpetiformis.

Fig. (15)

Diagnostic algorithm for autoimmune bullous diseases.