Anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita: Differentiation by use of indirect immunofluorescence microscopy

doi:10.1067/mjd.2003.99

Journal of the American Academy of Dermatology

Volume 48, Issue 4, April 2003, Pages 542-547

https://doi.org/10.1067/mjd.2003.99 Get rights and content

Abstract

Binding of autoantibodies to laminin 5 and type VII collagen causes anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita, respectively. Differentiation between these two dermal-binding autoimmune bullous dermatoses is not yet possible by indirect immunofluorescence microscopy. In this study we tested whether two recently described immunofluorescence techniques, “knockout” skin substrate and fluorescent overlay antigen mapping, can differentiate between anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita. A total of 10 sera were tested: 4 with antilaminin 5, and 6 with antitype VII collagen autoantibodies, as characterized by either immunoblot or immunoprecipitation analysis. Differentiation between anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita was possible in all 10 sera by indirect immunofluorescence using either knockout skin substrate or fluorescent overlay antigen mapping technique. (J Am Acad Dermatol 2003;48:542-7.)

Section snippets

Serum characterization

Serum samples were collected from 4 patients with AECP and 6 patients with mechanobullous IgG-mediated EBA. The sera had been characterized by immunoblotting, immunoprecipitation, or both as described previously.8, 12 The AECP sera reacted with the α₃ subunit of laminin 5 in immunoblotting. The EBA sera reacted in immunoblot using dermal extract with a 290-kd antigen corresponding with type VII collagen. By IIF, using 1-mol/L salt-split skin,¹³ all 10 sera showed binding of IgG4 antibodies

Indirect immunofluorescence microscopy on knockout skin

IIF with AECP sera showed negative or faint staining on JEB-H skin (the faint staining being a result of cross-reactivity of the autoantibodies to laminin 6 of the epidermal BMZ in laminin 5-deficient skin) (Fig 1, A).

. IIF staining for serum IgG on knockout skin substrates lacking either laminin 5 (A and C) or type VII collagen (B and D). Antiepiligrin cicatricial pemphigoid serum shows faint (or absent) IgG binding in laminin 5-deficient skin (A), but clearly positive linear IgG binding to

Discussion

In this study we describe two IIF methods that differentiate AECP and EBA, subepidermal bullous dermatoses characterized by dermal-binding IgG anti-BMZ autoantibodies directed against the dermal side of 1 mol/L salt-split skin. The first IIF method using knockout skin differentiated between AECP and EBA by determining loss of IgG binding to laminin 5- or type VII collagen-deficient skin substrates. Laminin 5-negative skin was obtained from a patient with JEB-H. The loss of laminin 5 in such

References (26)

Z Lazarova et al.
Reactivity of autoantibodies from patients with defined subepidermal bullous diseases against 1 mol/L salt-split skin. Specificity, sensitivity, and practical considerations
J Am Acad Dermatol
(1996)
CA Egan et al.
Anti-epiligrin cicatricial pemphigoid and relative risk for cancer
Lancet
(2001)
P Verrando et al.
Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmosomes
Exp Cell Res
(1987)
LS Chan et al.
Laminin-6 and laminin-5 are recognized by autoantibodies in a subset of cicatricial pemphigoid
J Invest Dermatol
(1997)
N Domloge-Hultsch et al.
Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease
J Clin Invest
(1992)
N Domloge-Hultsch et al.
Antiepiligrin cicatricial pemphigoid: a subepithelial bullous disorder
Arch Dermatol
(1994)
M Leverkus et al.
Antiepiligrin cicatricial pemphigoid: an underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous diseases?
Arch Dermatol
(1999)
P Bernard et al.
Incidence and distribution of subepidermal autoimmune bullous skin diseases in three French regions: bullous diseases French study group
Arch Dermatol
(1995)
D Zillikens et al.
Incidence of autoimmune subepidermal blistering dermatoses in a region of central Germany
Arch Dermatol
(1995)
Z Lazarova et al.
Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice
J Clin Invest
(1996)

RF Ghohestani et al.

A and b subunits of laminin-5 are target antigens in a subset of patients with cicatricial pemphigoid

J Invest Dermatol

(1996)

C Nieboer et al.

Epidermolysis bullosa acquisita; immunofluorescence, electron microscopic and immunoelectron microscopic studies in four patients

Br J Dermatol

(1980)

RM Vodegel et al.

The use of skin substrates deficient in basement membrane molecules for the diagnosis of autoimmune bullous disease

Eur J Dermatol

(1998)

Cited by (25)

Epidermolysis bullosa acquisita
2022, Anais Brasileiros de Dermatologia
Citation Excerpt :
The sensitivity varies from 20% to 80%, according to the COLVII source and the type of NC domain used.44 False-negative results may occur due to conformational changes in epitopes during the preparation of the recombinant proteins.51 Circulating anti-COLVII IgG can be detected after incubation of the serum with human cells transfected with the NC1 domain.
Epidermolysis bullosa acquisita is a rare autoimmune disease, characterized by the synthesis of anti-collagen VII autoantibodies, the main component of hemidesmosome anchoring fibrils. The antigen-antibody binding elicits a complex inflammatory response, which culminates in the loss of dermo-epidermal adhesion of the skin and/or mucous membranes. Skin fragility with bullae, erosions, and milia in areas of trauma characterizes the mechanobullous form of the disease. In the inflammatory form of epidermolysis bullosa acquisita, urticarial inflammatory plaques with tense bullae, similar to bullous pemphigoid, or mucosal lesions can determine permanent scars and loss of functionality in the ocular, oral, esophageal, and urogenital regions. Due to the similarity of the clinical findings of epidermolysis bullosa acquisita with other diseases of the pemphigoid group and with porphyria cutanea tarda, the diagnosis is currently confirmed mainly based on the clinical correlation with histopathological findings (pauci-inflammatory subepidermal cleavage or with a neutrophilic infiltrate) and the demonstration of the presence of anti-collagen VII IgG in situ by direct immunofluorescence, or circulating anti-collagen VII IgG through indirect immunofluorescence and/or ELISA. There is no specific therapy for epidermolysis bullosa acquisita and the response to treatment is variable, usually with complete remission in children and a worse prognosis in adults with mucosal involvement. Systemic corticosteroids and immunomodulators (colchicine and dapsone) are alternatives for the treatment of mild forms of the disease, while severe forms require the use of corticosteroid therapy associated with immunosuppressants, intravenous immunoglobulin, and rituximab.
Mucous membrane pemphigoid
2022, Autoimmunity Reviews
Citation Excerpt :
Several other in-house tests are in use in specialized laboratories [55,88,183]. Indirect IF microscopy on laminin 332-deficient skin is another elegant method for the detection of anti-laminin 332 antibodies [184]. This method is, however limited to sera reactive with human skin and those that do not contain antibodies against another antigen of the BMZ.
Mucous membrane pemphigoid (MMP) is a clinically and immunopathologically heterogenous disease with an incidence of about 2/million inhabitants/year in central Europe. Pemphigoid diseases are characterized by autoantibodies against structural proteins of the epidermis and/or surface-close epithelia. MMP has been defined as pemphigoid disease with predominant mucosal lesions. Most frequently, the oral cavity and the conjunctivae are affected. Lesions outside the mouth tend to heal with scarring leading to visual impairment and finally blindness, as well as, more rarely, impairment of breathing and food intake. Autoantibodies target BP180 (collagen type XVII), laminin 332, BP230 (nearly always in conjunction with other antigens), and type VII collagen in about 75%, 10–20%, 10–30%, and <5% of MMP patients, respectively. While the main autoantibody isotype is IgG, additional, and less frequently exclusive, IgA autoantibodies can be detected in the majority of patients. Assaying for anti-laminin 332 reactivity is pivotal, since in about a quarter of patients with anti-laminin 332 MMP, a malignancy, mainly solid cancers, is associated. The pathophysiology of MMP is yet incompletely understood. A recent mouse model of anti-laminin 332 MMP replicating characteristic clinical and immunopathological findings of the human disease may be helpful to close this knowledge gap. Diagnosis is established by the clinical picture with predominant mucosal lesions and visualization of tissue-bound anti-basement membrane zone antibodies by direct immunofluorescence microscopy. In recent S3 guidelines initiated by the European Academy of Dermatology and Venereology, the clinical spectrum and diagnostic strategies are detailed. In addition, treatment regimens for different clinical situations including patients with exclusive oral or ocular involvement are outlined. Future studies are needed to better understand the clinical complexity and associations as well as to establish widely available diagnostic assays and evidence-based therapeutic strategies.
Epidermolysis bullosa acquisita: A comprehensive review
2019, Autoimmunity Reviews
Citation Excerpt :
Using IF and perilesional skin from patients with EBA, digitized color images are formed by staining lamina lucida IgG deposits one color, against collagen VII, a different color. In patients with EBA there is an overlap of the fluorescence whereas in patients with BP, for example, the two stains are seen to be separate [80,92]. With immunoelectron microscopy (IEM), deposition of IgG beneath the basal lamina in the dermis of perilesional skin is visualized [7].
Epidermolysis bullosa acquisita is a rare autoimmune blistering disease which results in vesicle and bullae formation on the skin and erosions on the mucous membranes. EBA is mediated by autoantibodies to collagen VII. Clinically, it can present with numerous phenotypes, though the most common are the mechanobullous and inflammatory variants. Patients with mechanobullous EBA develop non-inflammatory bullae and erosions at sites of trauma while patients with the non-mechanobullous type develop inflammatory lesions which often mimic other blistering conditions including bullous pemphigoid, linear IgA bullous disease, and mucous membrane pemphigoid. Diagnosis is established by having a consistent clinical presentation, DIF, and autoantibodies against collagen VII. In apparent “seronegative” patients, the diagnosis is challenging due to the need for confirmatory tests which are often not routinely accessible outside of the specialized center. In light of EBA's rarity, and lack of any randomized controlled trials, treatment guidelines rely on the small case series presented in the literature. There has been variable success utilizing the arsenal of immunosuppressants and biologics. Development of experimental murine models has facilitated a deeper understanding of EBA's pathogenesis and allows for preclinical testing of numerous novel drug targets predominantly targeting inhibition of neutrophil activation. We herein review the presentation, diagnosis, treatments, and future avenues of research in EBA.
Detection of collagen VII autoantibodies to NC1 and NC2 domains of collagen VII by ELISA in suspected epidermolysis bullosa acquisita and bullous lupus erythematosus patients
2012, Journal of Dermatological Science
Differentiating antiepiligrin cicatricial pemphigoid from epidermolysis bullosa acquisita by indirect immunofluorescence of skin substrates lacking Type VII collagen or laminin 332: A case report and review of literature
2011, Dermatologica Sinica
Citation Excerpt :
However, these are often time consuming and expensive. Diagnostic indirect IF on antigen-deficient substrates2,3 of Type VII collagen-deficient or laminin 332-deficient skin from patients with inherited dystrophic and junctional epidermolysis bullosa, respectively, can provide a rapid and simple test to help clinicians differentiate between AECP and EBA. If indirect IF on antigen-deficient skin substrates show positive reaction on Type VII collagen-deficient skin and negative finding on laminin 332-deficient skin, this demonstrates that the patient’s serum contains antilaminin 332 autoantibodies instead of anti-Type VII collagen autoantibodies.
Antiepiligrin cicatricial pemphigoid (AECP) is a chronic autoimmune subepidermal blistering disease characterized by clinical features of cicatricial pemphigoid and circulating IgG antibasement membrane autoantibodies directed against laminin 332. There is growing evidence of an increased relative risk for solid cancers and lymphomas in AECP patients, especially in the 1^st year after the onset of blisters. However, it is difficult to distinguish patients with initially skin-predominant AECP from similar findings of epidermolysis bullosa acquisita merely based on clinical, histopathologic, and immuno-pathologic examinations. This is a report on a case of AECP confirmed by indirect immunofluorescence of type VII collagen- and laminin 332-deficient skin as substrates to differentiate it from epidermolysis bullosa acquisita.
The vesiculobullous reaction pattern
2009, Weedon's Skin Pathology: Third Edition

View all citing articles on Scopus

^☆: Funding sources: Zon/MW.

^☆☆: Conflict of interest: None identified.

^★: Reprints not available from authors.

^★★: Correspondence: Prof Dr Marcel F. Jonkman, MD, Department of Dermatology, Groningen University Hospital, PO Box 30.001, 9700 RB Groningen, the Netherlands. E-mail:[email protected].

^♢: 0190-9622/2003/$30.00 + 0

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ReportsAnti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita: Differentiation by use of indirect immunofluorescence microscopy☆,☆☆,★,★★,♢

Abstract

Section snippets

Serum characterization

Indirect immunofluorescence microscopy on knockout skin

Discussion

J Am Acad Dermatol

Lancet

Exp Cell Res

J Invest Dermatol

Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease

J Clin Invest

Antiepiligrin cicatricial pemphigoid: a subepithelial bullous disorder

Arch Dermatol

Antiepiligrin cicatricial pemphigoid: an underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous diseases?

Arch Dermatol

Incidence and distribution of subepidermal autoimmune bullous skin diseases in three French regions: bullous diseases French study group

Arch Dermatol

Incidence of autoimmune subepidermal blistering dermatoses in a region of central Germany

Arch Dermatol

Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice

J Clin Invest

A and b subunits of laminin-5 are target antigens in a subset of patients with cicatricial pemphigoid

J Invest Dermatol

Epidermolysis bullosa acquisita; immunofluorescence, electron microscopic and immunoelectron microscopic studies in four patients

Br J Dermatol

The use of skin substrates deficient in basement membrane molecules for the diagnosis of autoimmune bullous disease

Eur J Dermatol

Reports
Anti-epiligrin cicatricial pemphigoid and epidermolysis bullosa acquisita: Differentiation by use of indirect immunofluorescence microscopy☆,☆☆,★,★★,♢