The epidermal basement membrane zone--structure, ontogeny, and role in disease

Since many dermatologic diseases affect the epidermal basement membrane zone, there has been intense investigation into the role of epidermal basement membrane zone constituents in various skin diseases, particularly subepidermal blistering skin diseases. The epidermal basement membrane zone consists of four major structural components--the basal cell plasma membrane, the lamina lucida, the lamina densa, and the sublamina densa zone, which contains anchoring fibrils. The lamina lucida is composed of laminin, bullous pemphigoid antigen (a disease-specific glycoprotein identified by antibodies circulating in patients' sera), and other as yet poorly defined antigens identified by in vivo bound and circulating antibodies in the sera of patients with herpes gestationis, scarring pemphigoid, and others. The lamina densa consists of type IV collagen and KF-1 antigen, which is noncollagenous and is identified by a skin-specific monoclonal antibody. The sublamina densa zone consists of AF-1 and AF-2 antigens and the epidermolysis bullosa acquisita antigen(s). Knowledge of the structure and chemical composition of the basement membrane zone is critical to an understanding of some of the genetic and immunologically mediated blistering skin diseases.     

Linear IgA Disease with Clinical and Immunopathological Features of Epidermolysis Bullosa Acquisita

Abstract: 10-year-old boy had a 3-month history of urticarial plaques and vesicles. Histologic and immunofluorescence testing confirmed the diagnosis of linear IgA disease. Immunoelectron microscopy revealed IgA deposits in the sublamina densa area similar to those seen in epidermolysis bullosa acquisita. Milia developed after resolution of the lesions, similar to lesions of epidermolysis bullosa acquisita.     

Location of basement membrane Type IV collagen beneath subepidermal bullous diseases

Using standard immunohistochemical methods, routinely processed sections, and a polyclonal antibody to Type IV collagen, we have determined the location of Type IV collagen, a substance located in the lamina densa of basement membrane, in a spectrum of acquired subepidermal bullous diseases. Type IV collagen was attached to the blister roof in five cases of well-established epidermolysis bullosa acquisita and to the blister base in 25 cases of bullous pemphigoid, four cases of dermatitis herpetiformis and 12 cases of porphyria cutanea tarda. Immunohistochemical localization of Type IV collagen in epidermal-dermal basement membrane is a simple, rapid and reliable technique which can be utilized to exclude and possibly to confirm the diagnosis of epidermolysis bullosa acquisita in routinely fixed paraffin-embedded tissues.     

KF-1 monoclonal antibody defines a specific basement membrane antigen defect in dystrophic forms of epidermolysis bullosa

The monoclonal antibody, KF-1, identifies a noncollagenous constituent of the lamina densa of the basement membrane zone (BMZ) of skin. In order to determine whether this BMZ constituent is affected in epidermolysis bullosa (EB), a mechanobullous skin disease often resulting in marked disfigurement, we have examined skin from patients with various forms of this disease for binding by KF-1 as well as for binding by polyclonal antibodies to laminin, type IV collagen, and bullous pemphigoid antigen, three other known BMZ components of normal skin. In all specimens from patients with simplex and junctional forms of EB, all four antibodies bound normally. In contrast, absent or diminished KF-1 binding was noted in all skin specimens from patients with dystrophic EB; antibodies directed against the other BMZ constituents, however, bound normally. This suggests that KF-1 may play a role in the structural integrity of normal skin and its absence or diminution may be important in the pathogenesis of lesion formation in dystrophic EB.     

Epidermolysis bullosa acquisita. Incidence in patients with basement membrane zone antibodies

We examined the incidence of epidermolysis bullosa acquisita in patients with circulating basement membrane zone antibodies. Serum samples from 100 sequential patients with basement membrane zone antibodies were tested by indirect immunofluorescence against 1 mol/L sodium chloride split skin and by Western immunoblot against epidermal and dermal extracts of skin. Ninety-two (92%) serum samples stained only the epidermal side of split skin, 5 (5%) stained only the dermal side, and 3 (3%) stained both sides. Four of the 5 serum samples with dermal staining but none of the serum samples with epidermal or combined staining reacted with the 290-kd epidermolysis bullosa acquisita antigen by Western immunoblot. These results indicate that approximately 5% of unselected patients with basement membrane zone antibodies have epidermolysis bullosa acquisita or bullous lupus erythematosus rather than bullous pemphigoid.     

Epidermal basement membrane alterations in epidermolysis bullosa acquisita

Immunopathological studies on a patient with clinical evidence of epidermolysis bullosa acquisita (EBA) revealed an increase in type IV collagen in the skin of the chronic scarred lesion, but not in the clinically normal skin. These findings, together with multiplication of the lamina densa of the chronic lesional skin demonstrated by electron microscopy, strongly suggest that repeated injury possibly stimulates epidermal cells to secrete type IV collagen, leading to multiplication of lamina densa of the basement membrane at the dermal-epidermal junction in EBA.     

Epidermolysis bullosa acquisita

Four patients with the clinical picture of epidermolysis bullosa acquisita were investigated. Biopsies were taken from the involved and uninvolved areas of the skin and the immunohistochemical and microscopic changes were studied. Direct immunofluorescence showed deposition of IgG and C3/4 in a linear or notched pattern along the epidermal basement membrane in both the involved and the uninvolved skin. In addition IgA (3/4), IgM (1/4), C4 (3/4) and properdin (3/4) could be detected. Indirect immunofluorescence revealed the presence of circulating antibodies against inter alia the epithelial basement membrane zone in one patient. Routine electron microscopy showed that the blister was situated in the dermis leaving the basal lamina in the roof of the blister. With immunoelectron microscopy using peroxidase-labelled antibody the in vivo deposition of IgG was observed just beneath the basal lamina in the dermis of both the perilesional and the uninvolved skin. These observations show that epidermolysis bullosa acquisita is a distinct entity, in which autoimmune mechanisms might possibly play a role.     

IgA-mediated epidermolysis bullosa acquisita: two cases and review of the literature

We describe 2 adult patients with a subepidermal bullous dermatosis with exclusively linear IgA depositions along the epidermal basement membrane zone that were deposited in the sublamina densa zone as witnessed by direct immunoelectron microscopy. Indirect immunofluorescence microscopy of patients' sera revealed circulating IgA autoantibodies that bound exclusively to the dermal site of salt-split skin substrate. Immunoblot analysis using dermal and keratinocyte extracts were negative. Indirect immunofluorescence microscopy using type VII collagen-deficient skin ("knockout" substrate) showed no IgA binding, whereas linear IgA binding was seen at the epidermal basement membrane zone in normal human skin. The autoantigen in the patients was thus type VII collagen. A diagnosis of IgA-mediated epidermolysis bullosa acquisita (IgA-EBA) was made. We systematically reviewed the literature of this subset of patients with linear IgA dermatosis on the basis of the following criteria: exclusive binding of serum-IgA to the dermal side of salt-split skin or IgA depositions in the sublamina densa zone by indirect or direct immunoelectron microscopy. We learned that IgA-EBA is clinically indistinguishable from the classic "lamina-lucida type" linear IgA dermatosis or from the inflammatory type of IgG-mediated epidermolysis bullosa acquisita (IgG-EBA). Only a minority of the patients with IgA-EBA showed milia or scarring or had therapy-resistant ocular symptoms as in the mechanobullous type of IgG-EBA. Most patients with IgA-EBA responded to dapsone therapy.     

Electron microscopy and morphometry enhances differentiation of epidermolysis bullosa subtypes. With normal values for 24 parameters in skin

Electron microscopy combined with morphometry was used to establish values for 24 parameters in normal skin. These results were compared with those similarly obtained from samples of epidermolysis bullosa with a view to facilitating classification of the disease. Six of the eight subtypes of epidermolysis bullosa investigated could be differentiated. Four subtypes showed values for structural components in intact skin which were outside the normal range: (1) epidermolysis bullosa simplex generalisata gravis (hemidesmosomes); (2) epidermolysis bullosa dystrophica Pasini and (3) Cockayne-Touraine (anchoring fibrils); and (4) epidermolysis bullosa acquisita (anchoring fibrils, hemidesmosomes, and lamina lucida and lamina densa aspects of the dermoepidermal junction). Two subtypes revealed specific features which could be assessed qualitatively: distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara and thick (30 nm diameter) cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Epidermolysis bullosa simplex Köbner and Weber-Cockayne forms could not be distinguished.     

Inflammatory epidermolysis bullosa acquisita with coexistent IgA antibodies to plectin

We present a case of inflammatory epidermolysis bullosa acquisita (EBA) with IgA antibodies to plectin. Analysis of lesional skin biopsies by electron microscopy revealed the split level to be in the sublamina densa zone, corresponding to the diagnosis of EBA. Direct immunofluorescence of perilesional skin demonstrated u-serrated depositions of IgG and IgA that under immunoelectron microscopy were shown to be located in the sublamina densa. In contrast, indirect immunofluorescence on salt-split skin revealed circulating IgA antibodies that stained the roof rather than the floor of the blister. Immunoblotting showed these serum antibodies to be directed to the cytoplasmic hemidesmosomal antigen plectin. The antiplectin specificity of these antibodies was confirmed by 'knockout' immunofluorescence analysis; the serum IgA did not bind to skin sections of a patient with plectin-deficient epidermolysis bullosa. To our knowledge, this case demonstrates for the first time the existence of IgA antibodies against plectin.     

The basement membrane and its significance in congenital and acquired diseases of the skin

Basal membranes are an ubiquitous component of all human organs and fulfil a large variety of functions. They separate epithelial from mesenchymal tissue and control the passage of substances, of inflammatory as well as tumor cells. They form the extracellular cytoskeleton, regulate growth processes, and play an important part in wound healing. Ultrastructurally, the lamina densa can be distinguished from the lamina lucida. Anchoring fibrils connect the basal membrane of the dermoepidermal junction with the underlying dermis, while anchoring plates connect it with the epidermal cells. During the last few years, various components of the basal membrane have been biochemically analyzed. Different proteins were characterized, and their corresponding morphological structure could be identified. The growing knowledge regarding the structure and composition of the dermo-epidermal junction has led to a better understanding of many diseases involving this structure, in particular inborn and acquired bullous diseases, but also that of the role of the basal membrane in metastasis and tumor invasions. The characterization of antigens in bullous diseases with circulating antibodies has led to exact diagnostic criteria concerning the often overlapping disorders and allows, e.g., the differentiation between bullous pemphigoid and epidermolysis bullosa acquisita.     

A mouse monoclonal antibody against a newly discovered basement membrane component, the epidermolysis bullosa acquisita antigen

A mouse monoclonal antibody, H3a, directed against the newly described epidermolysis bullosa acquisita (EBA) antigen was obtained using hybridoma techniques. The distribution of the monoclonal antibody is identical to that of the polyclonal serum antibody of patients with EBA. By immunofluorescence, a linear band is seen at the dermal-epidermal junction and, by immunoelectron microscopy, immune reaction products are present in the lamina densa and sublamina densa regions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot analysis shows that the monoclonal antibody recognizes 290 and 145 kilodalton proteins present in the immunizing junctional extract, identical with the newly discovered EBA antigen. This monoclonal antibody should be useful in the further isolation and characterization of the EBA antigen.     

Epidermolysis bullosa acquisita: an autoimmune disease with distinctive immunoultrastructural features

Epidermolysis bullosa acquisita is a subepidermal bullous disease of the skin with distinctive clinical, histologic, immunologic, and ultrastructural features. A case of epidermolysis bullosa acquisita is presented in which the onset of the disease was heralded by inflammatory blisters similar to herpes simplex or herpes zoster. The immunofluorescent findings were indistinguishable from bullous pemphigoid. Although some inflammatory blisters persisted throughout the evolution of the disease, the patient eventually developed noninflammatory blisters that healed with milia formation and scarring, lesions typical of classic epidermolysis bullosa acquisita. The diagnosis of epidermolysis bullosa acquisita was definitely made by electron and immunoelectron microscopy which showed a sub-lamina-densa blister cleavage plane and immune deposits beneath the lamina densa. This case illustrates that some cases of epidermolysis bullosa acquisita may have an inflammatory stage and immunofluorescent findings that make it difficult to distinguish from inflammatory bullous diseases. The value of electron microscopy and immunoelectron microscopy in making a firm diagnosis of epidermolysis bullosa acquisita is emphasized.     

Epidermolysis bullosa: variability of expression of cicatricial pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita antigens in clinically uninvolved skin

Indirect immunofluorescence was performed on skin from 13 patients with epidermolysis bullosa (EB) (simplex, 6; junctional, 2; dystrophic, 5) to compare the expression of 3 basement membrane components, bullous pemphigoid (BP) antigen, cicatricial pemphigoid (CP) antigen, and epidermolysis bullosa acquisita (EBA) antigen, in clinically uninvolved tissue. In addition, expression of laminin, type IV collagen, and KF-1 antigen was also evaluated. Whereas laminin, type IV collagen, and KF-1 antigen were each detectable in EB skin in a manner identical to that previously reported, marked variability was noted in the expression of BP, CP, and EBA antigens. However, no correlation was noted comparing lack of expression of any one of these latter antigens with either of the remaining two. Of these 3 antigens, BP antigen was the least often detectable, particularly in skin from patients with EB simplex. The lack of detectable BP antigen in EB simplex skin appeared to correlate with more extensive disease involvement and/or younger patient age. These findings may, therefore, limit the usefulness of BP serum in immunofluorescence mapping. Additionally, the disparity in expression of basement membrane antigens defined by BP and CP sera suggests that BP antigen and CP antigen are distinct entities.     

Immunoelectron microscopy in subepidermal autoimmune bullous diseases: a prospective study of IgG and C3 bound in vivo in 32 patients

Thirty-two patients suffering from subepidermal autoimmune bullous disease were studied prospectively by clinical examination and immunoelectron microscopy. Clinically, 1 patient had herpes gestationnis, 14 typical bullous pemphigoid (BP), 3 epidermolysis acquisita (EBA), 3 cicatricial pemphigoid (CP), and 11 patients overlapping clinical diseases. These 11 patients shared clinical features of BP, EBA, or CP and a clinical diagnosis could not be done safely. Immunoelectron microscopy revealed diaminobenzidine deposits in 20 patients on the epidermal side of dermo-epidermal junction in the lamina lucida as in BP. In 5 patients, deposits located mostly under the anchoring fibril zone, in the floor of a sublamina densa dermoepidermal separation for 2 of them, were consistent with a diagnosis of EBA. In 6 patients, deposits were located mostly in the lamina densa, in the floor of a dermoepidermal separation occurring in the lamina lucida for 3 of them. This suggests that some of these 6 patients had neither EBA or BP, but another autoimmune bullous disease again, an uncharacterized component of dermoepidermal junction located in the lamina densa. Finally, a correlation exists between the sites of IgG and/or C3 components on epidermal or dermal side of dermoepidermal junction and the presence or absence of characteristic clinical features such as scar, milia formation, or mucosal involvement.     

Epidermolysis bullosa acquisita antigen, a new major component of cutaneous basement membrane, is a glycoprotein with collagenous domains

The epidermolysis bullosa acquisita antigen is a major constituent of the basement membrane zone beneath stratified squamous epithelium. The antigen which is recognized in extracts of skin basement membrane by Western blot analysis with polyclonal or monoclonal antiepidermolysis bullosa acquisita antigen antibodies as 2 chains (a major chain of 290,000 and a minor chain of 145,000) has a native molecular weight over 800,000. Both epidermolysis bullosa acquisita antigen chains contain carbohydrate and the 290K chain is sensitive to collagenase.     

Bullous eruption of systemic lupus erythematosus: a clinicopathological study of four cases

We describe the clinical, histological and immunopathological features of four female patients with the bullous eruption of systemic lupus erythemutosus (bullous SLE). Three patients had circulating anti-basement membrane zone (BMZ) antibodies, and Western blot analysis in two cases revealed binding to type VII collagen. Immunoeiectron microscopy in one of these patients demonstrated deposition of antibody in the lamina densa and sublamina densa regions, thus sharing immunopatho-logical features with epidermolysis bullosa acquisita (KBA). The vesiculobullous lesions developed 8 months to 6 years after the initial symptoms of SLE, and cleared promptly with dapsone treatment.     

A Case of Nonscarring Inflammatory Epidermolysis Bullosa Acquisita: Characterization of IgG Autoantibodies by Immunofluorescence,Immunoblotting and Immunogold Electron Microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

Functional heterogeneity of immune complexes in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita is an inflammatory subepidermal bullous disease characterized by circulating and tissue-bound complement-binding anti-basement membrane zone autoantibodies to type VII procollagen. Lesions are characterized by neutrophil-predominant inflammation in some patients, but not in others. These features suggest complement activation and generation of complement-derived chemotactic factors for leukocytes by basement membrane zone immune complexes may contribute to inflammation, but that complexes may be heterogeneous in the ability to express that function. In this study, we measured the ability of basement membrane zone complexes from patients with (n = 4) and without (n = 6) neutrophil predominant inflammation to activate complement and generate complement-derived chemotactic activity using a complement-dependent neutrophil attachment assay. The results showed considerable heterogeneity in neutrophil attachment among EBA patients and that both the incidence (4/4 vs 2/6) and magnitude (81 +/- 34 vs 12 +/- 10 neutrophils/mm basement membrane zone) of attachment were greater in patients with neutrophil-predominant inflammation. Functional heterogeneity appeared to be due to differences in the amounts of complement-activating complexes formed at the basement membrane zone, which in turn appeared to be due to differences in the availability of circulating complement-binding anti-basement membrane zone antibodies. This was suggested by a positive correlation (r = 0.72, p less than 0.01) between neutrophil attachment and complement-binding anti-basement membrane zone antibody titers and the observation that high levels of neutrophil attachment could be generated in skin from patients with epidermolysis bullosa acquisita who did not have neutrophil-predominant inflammation by treating their skin in vitro with complement-binding anti-basement membrane zone antibodies. These results suggest tissue complexes in epidermolysis bullosa acquisita are heterogeneous in the ability to activate complement and generate complement-derived chemotactins (C5a, C5a des arg), and that functional heterogeneity contributes to histologic heterogeneity. The functional immunologic-pathologic correlations observed in this study suggest epidermolysis bullosa acquisita is an autoimmune "collagen" disease.     

A unique epithelial basement membrane antigen defined by a monoclonal antibody (KF-1)

The basement membrane zone (BMZ) of human skin is a complex structure which contains several well-defined components including bullous pemphigoid antigen, laminin, type IV collagen, and proteoglycan. Characterization of additional basement membrane (BM) constituents has been limited by their relative inaccessibility, insolubility, and low tissue concentration. We have produced a murine monoclonal antibody that has enabled us to define a unique constituent of the BMZ of human stratified squamous epithelia. The monoclonal antibody (KF-1) was raised by standard techniques using suction blister-derived trypsinized human epidermal cells as the antigen. Indirect immunofluorescence and immunoperoxidase staining of human and rhesus monkey tissues with KF-1 produced linear BMZ staining of stratified squamous epithelia. Glandular and vascular BMs were not stained. Immunoelectron microscopic studies of normal human skin and esophagus showed specific binding of KF-1 to the lamina densa of the BMZ, a localization identical to that of type IV collagen. However, unlike type IV collagen, which is not species specific and is found in all BMs, the antigen defined by KF-1 is collagenase-resistant and is specific for primate stratified squamous epithelia. These findings confirm the existence of regional variation in BM composition, and demonstrate for the first time that the lamina densa of stratified squamous epithelial BMs contains a constituent other than type IV collagen.