Epidermolysis Bullosa Acquisita Antigen Research Articles

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

Fluorescence overlay antigen mapping using laser scanning confocal microscopy differentiates linear IgA bullous dermatosis from epidermolysis bullosa acquisita mediated by IgA

Linear IgA bullous dermatosis (LABD) and epidermolysis bullosa acquisita (EBA) mediated by IgA antibodies belong to the group of autoimmune subepidermal bullous diseases mediated by IgA autoantibodies. Early and correct diagnosis is crucial because the management and prognosis of the diseases are different. To determine whether fluorescence overlay antigen mapping using laser scanning confocal microscopy (FOAM-LSCM) is helpful in the differentiation between these diseases. FOAM-LSCM and immunoblot studies were performed in 19 patients with disseminated tense blisters who presented with in vivo bound and circulating IgA antibasement membrane zone (BMZ) antibodies on immunofluorescence. Using FOAM-LSCM, in vivo bound IgA above type IV collagen, which is characteristic for LABD, was seen in 14 of the 19 cases, whereas five of the 19 cases had IgA deposits below type IV collagen, typical for EBA. Immunoblot studies showed that IgA antibodies in 11 of the 14 patients with deposits above type IV collagen reacted with different epitopes on BP180, mainly with LAD-1, which is a target antigen in LABD. Among the five patients with deposits below type IV collagen, one showed IgA antibodies to the 200-kDa laminin γ-1 and one had antibodies to the 290-kDa type VII collagen, EBA antigen. Additionally, enzyme-linked immunosorbent assay with recombinant type VII collagen was positive in three of the five cases who presented with IgA deposits below type IV collagen on FOAM-LSCM. The results using FOAM-LSCM were consistent with those obtained on immunoblotting. FOAM-LSCM is useful in routine diagnostics in cases with undetectable circulating anti-BMZ antibodies, and can differentiate LABD from IgA-EBA, the former with in vivo bound IgA above type IV collagen and the latter with IgA deposits below type IV collagen.

Accuracy of indirect immunofluorescence on sodium chloride-split skin in the differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita

Previous reports have shown that indirect immunofluorescence (IIF) performed on sodium chloride-split skin (SSS) is helpful to differentiate epidermolysis bullosa acquisita (EBA) from bullous pemphigoid (BP). Antibodies of BP may bind to the epidermal side of SSS, while antibodies of EBA bind to the dermal side. To determine the accuracy of IIF-SSS in the differential diagnosis of EBA and BP utilizing immunoblotting (IB) analysis. Sera from 78 patients, diagnosed with BP by clinical features, histopathology, and direct immunofluorescence (DIF), were assayed using IIF-SSS and IB. Of the 43 serum samples with an epidermal reaction to IIF-SSS assay, 42 were recognized with BP antigens (180 kDa or 230 kDa). Of the 11 serum samples with a dermal reaction pattern, 7 were recognized with the 290 kDa antigen of EBA and 3 with sera bound BP antigens. Seven serum samples with epidermal and dermal combined staining, of which 5 of them reacted with BP antigens, 1 reacted with both BP and EBA antigens. One serum sample from each group showed a negative result by IB. Approximately 9.0% (7/78) of patients diagnosed with BP using regular methods were actually EBA. Epidermal reaction using the IIF-SSS assay highly correlated with the diagnosis of BP. However, dermal reactions correlated poorly with EBA, with some serum samples from BP patients binding to dermal-side antigens. In both epidermal and dermal stained sera using IIF-SSS, there was a possibility of BP and EBA. Differential diagnosis should be confirmed using IB, especially in cases of dermal and double staining patterns assayed using IIF-SSS.

Generation of Antibodies of Distinct Subclasses and Specificity Is Linked to H2s in an Active Mouse Model of Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease, characterized by antibodies to type VII collagen (COL7). EBA can be induced in mice by immunization with a fragment of the non-collagenous 1 domain of murine COL7. Contrary to other autoimmune diseases, e.g., rheumatoid arthritis, little is known about the genetic susceptibility for EBA. We therefore used the EBA mouse model to address the hypothesis that disease induction depends on the major histocompatibility complex (MHC) haplotype. Mice from different inbred strains were immunized with recombinant murine COL7. Five distinct responses were observed: induction of (i) severe disease in SJL/J (H2s) and female MRL/MpJ (H2k), (ii) mild and transient disease in C57Bl/10.s (H2s), (iii) microscopic blistering in DBA/1J (H2q), (iv) only presence of non-pathogenic autoantibodies in C57Bl/6J (H2b), NZM2410/J (H2z), BXD2 (H2b), and male MRL/MpJ, and (v) complete resistance to EBA in NOD/ShiLtJ (H2g7) and C57Bl/10.q (H2q) mice. Overall, susceptibility to EBA was strongly associated with H2s. In addition, the diseased phenotype was associated with autoantibodies to specific regions of COL7. Our findings show that induction of antibodies with a distinct specificity is linked to the MHC haplotype in experimental EBA. Furthermore, our data are the basis for future studies with the goal of identifying non-MHC EBA susceptibility genes.

Epidermolysis bullosa acquisita: concise review and practical considerations

Introduction Epidermolysis bullosa acquisita (EBA) is a cutaneous subepi-dermal autoimmune blistering disorder (CSEAIBD) that resultsfrom the effects of IgG autoantibodies directed against the145-kDa noncollagenous amino-terminal (NC-1) domain ofcollagen VII, a major component of anchoring fibrils (Fig. 1).

Potential of Fibroblast Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin-blistering disorder caused by mutations in the COL7A1 gene that lead to reduced type-VII collagen and defective anchoring fibrils at the dermal-epidermal junction (DEJ). Presently there are no effective treatments for this disorder. Recent mouse studies have shown that intradermal injections of normal human fibroblasts can generate new human type-VII collagen and anchoring fibrils at the DEJ. To assess potential clinical benefits in humans, we gave single intradermal injections of allogeneic fibroblasts to five subjects with RDEB. We noted increased type-VII collagen at the DEJ at 2 weeks and at 3 months following injection and increased anchoring fibrils, although none of these had normal morphology. No adverse effects, clinical or immunopathologic, were noted. We believe the major effect of allogeneic fibroblasts is to increase the recipients' own COL7A1 mRNA levels with greater deposition of mutant type-VII collagen at the DEJ and formation of additional rudimentary anchoring fibrils. Nevertheless, this mutant protein may be partially functional and capable of increasing adhesion at the DEJ. This is the first study demonstrating that intradermal injections of allogeneic fibroblasts have therapeutic potential in human subjects with RDEB.

The Epidermolysis Bullosa Acquisita Antigen (Type VII Collagen) is Present in Human Colon and Patients with Crohn's Disease have Autoantibodies to Type VII Collagen

Epidermolysis bullosa acquisita is an autoimmune blistering disease of the skin characterized by IgG autoantibodies against type VII collagen. Systemic diseases are often associated with epidermolysis bullosa acquisita, Crohn's disease being the most frequent. This study sought to determine if type VII collagen, the epidermolysis bullosa acquisita autoantigen, was present in normal human colon by western blotting and immunofluorescence. The 290 kDa type VII collagen alpha chain was demonstrated by western blotting in four normal intraoperative colon specimens. Antibodies to type VII collagen labeled the junction between the intestinal epithelium and the lamina propria. We also used an enzyme-linked immunosorbent assay to test sera from patients with Crohn's disease (n = 19), ulcerative colitis (n = 31), celiac disease (n = 17), rheumatoid arthritis (n = 15), and normal controls (n = 16). It was found that 13 of 19 patients with Crohn's disease and four of 31 patients with ulcerative colitis demonstrated reactivity to type VII collagen. Sera from control subjects, patients with celiac disease or rheumatoid arthritis were negative. The sera from Crohn's disease patients also reacted with type VII collagen by immunoblot analysis. It was concluded that patients with inflammatory bowel disease may have IgG autoantibodies to type VII collagen, which exists in both the skin and the gut.

Psoriasis vulgaris coexistent with epidermolysis bullosa acquisita

Autoimmune bullous diseases, such as bullous pemphigoid or pemphigus vulgaris, occasionally develop in psoriatic patients. In addition, a novel subepidermal bullous disease with autoantibodies against a lower lamina lucida antigen of 200 kDa has recently been reported in association with psoriasis. We describe here a patient with psoriasis vulgaris who developed epidermolysis bullosa acquisita (EBA). Direct immunofluorescence revealed linear deposition of IgG and C3 at the basement membrane zone. The patient's serum bound to the dermal side of salt-split normal human skin. However, immunoblot analysis demonstrated that the patient's serum reacted with an EBA antigen of 290 kDa. EBA should be included in the list of autoimmune diseases associated with psoriasis vulgaris.

Diagnostic Value of Indirect Immunofluorescence on Sodium Chloride-Split Skin in Differential Diagnosis of Subepidermal Autoimmune Bullous Dermatoses

To determine the diagnostic value of indirect immunofluorescence on sodium chloride-split skin (SSS) in differentiating the pemphigoid group of subepidermal autoimmune bullous dermatoses, including bullous pemphigoid (BP), cicatricial pemphigoid, and pemphigoid gestationis, from epidermolysis bullosa acquisita (EBA). Serum samples were tested using immunofluorescence on SSS and immunoblot assay on epidermal and dermal extracts, a recombinant protein corresponding to the C-terminal end of the 230-kd BP antigen, and purified laminin-5. An immunodermatology laboratory. One hundred forty-two serum samples from patients with BP (n = 98), cicatricial pemphigoid (n = 23), pemphigoid gestationis (n = 10), EBA (n = 10), and anti-type IV collagen (n = 1). Binding sites of serum to the epidermal and/or dermal sides of SSS were correlated with their antigenic specificities. Epidermal staining on SSS was highly specific for pemphigoid. Alternatively, a poor correlation was found for the dermal-reacting serum samples and the diagnosis of EBA; of the 19 serum samples with dermal staining on SSS, only 10 reacted with the EBA antigen. The remaining serum samples were from patients with cicatricial pemphigoid having antibodies to the alpha 3 or beta 3 chains of laminin-5 (n = 5) or patients with BP having antibodies to the 180-kd BP antigen (n = 2). One sample recognized exclusively a 185-kd dermal antigen corresponding to type IV collagen. One more BP serum sample with dermal staining did not recognize any dermal or epidermal antigen. In case of immunofluorescent dermal staining, the precise diagnosis should be confirmed by identification of the involved antigen, since it may reveal antibodies to laminin-5 or type XVII or IV collagen, in addition to the EBA antigen.

The presence of anti-basement membrane zone antibodies in the sera of patients with non-bullous lupus erythematosus

We performed indirect immunofluorescence (IF) studies using 1 mol/l sodium chloride split skin to determine whether or not a positive IF is specific to patients with bullous lupus erythematosus (LE). We examined the sera from 21 patients with systemic LE (SLE), three of which were obtained from two SLE patients and one subacute cutaneous LE (SCLE) patient with bullous eruptions. As a comparison, we also studied the sera from patients with discoid LE (DLE, n = 7), SCLE (n = 1), systemic sclerosis (SSc, n = 20), bullous pemphigoid (n = 2) and normal individuals (n = 10). Sera from 16 SLE, four DLE and two SSc revealed a linear deposition of IgG isotype antibody at the epidermal side and/or the dermal side on indirect IF of split skin. The sera from three patients with bullous eruption and from 12 patients of SLE, SCLE, DLE without bullous eruption or SSc were further analysed by immunoblotting using five defined antigens, i.e. dermal extract, epidermal extract, three fusion proteins of 230 kDa bullous pemphigoid antigen (BPAG). 180 kDa BPAG, and human epidermolysis bullosa acquisita (EBA) antigen. Two SLE sera as well as one of the SCLE and the DLE serum reacted with 230 kDa BPAG in epidermal extract, and one of the SCLE and the DLE serum also reacted with the fusion protein of 180 kDa BPAG. No serum reacted with the dermal extract or the fusion protein of 230 kDa BPAG or EBA antigen. There was no consistent correlation between split-skin IF results and immunoblotting results. These results may suggest that even non-bullous LE patients often have autoantibodies to the basement membrane zone antigens, most of which are less pathogenic. Although we rarely examine the sera from non-bullous LE patients, we should keep this phenomenon in mind to avoid overestimating the results of split-skin test and immunoblotting.

Human skin basement membrane in health and in autoimmune diseases.

Skin basement membrane zone (BMZ) is an ultrastructurally defined area situated between the outer layer of skin, the epidermis, and the inner layer of skin, the dermis. The major function of skin BMZ is to serve as an adherent connection between the epidermis and the dermis. Heritable skin diseases characterized by genetic mutations that result in defective BMZ protein production, such as junctional and dystrophic epidermolysis bullosa, manifest clinically as skin fragility and subepidermal blister formation inducible by minor trauma. Many distinct, yet interconnected BMZ components, have been identified and studied extensively. Some of these essential components, interestingly, were discovered because they were targeted by an autoimmune reaction. As a result of studying the autoimmune diseases, these BMZ components involved in autoimmune reaction, or autoantigens, were isolated by molecular biology techniques. The isolation of these autoantigens, in turn, facilitates our studies of the normal structures and functions of skin BMZ. The skin BMZ can be divided into four ultrastructurally distinct areas: the hemidesmosome/upper lamina lucida, the lower lamina lucida, the lamina densa, and the sub-lamina densa. Well-characterized BMZ components include the hemidesmosome/upper lamina lucida-located bullous pemphigoid antigens (BP230 and BP180), a 6b4 integrin, and plectin; the lower lamina lucida-located laminin-1, laminin-5 (previously named kalinin, epiligrin, nicein, BM600), laminin-6 (previously named k-laminin), p105, and entactin/nidogen; the lamina densa-located type IV collagen and perlecan; and the sub-lamina densa-located type VII collagen (epidermolysis bullosa acquisita antigen). Mucosal BMZ also contain identical components as skin BMZ. Autoantibodies targeting skin and/or mucosal BMZ components, like that of genetic mutation of BMZ components, result in a histopathologically defined subepidermal blistering disease, that is, a blister which occurs just below the epidermis/epithelium. Well-characterized autoimmune subepidermal blistering diseases include bullous pemphigoid (BP), linear IgA bullous dermatosis, cicatricial pemphigoid (with subsets of patients characterized by autoantibodies targeting either BP antigens or laminins), anti-p105 pemphigoid, and epidermolysis bullosa acquisita. Using a simple method of direct or indirect immunofluorescence testing on a skin section chemically separated at the middle portion of lamina lucida, in combination with immunoelectron microscopy and target antigen determination, accurate diagnoses for various autoimmune subepidermal blistering diseases can be obtained.

Acquired subepidermal bullous diseases associated with psoriasis: a clinical, immunopathological and immunogenetic study

Clinical, immunopathological and immunogenetic studies of four patients with a subepidermal bullous disease associated with psoriasis were carried out to determine the true nature of the blistering disease and to investigate further the relationship between psoriasis and acquired subepidermal bullous diseases. Autoantibodies in all four patients bound to the epidermal side of salt-split skin by indirect immunofluorescence and detected the major bullous pemphigoid (BP) antigens by immunoblotting. One had additional IgA autoantibodies binding an epidermal polypeptide of 270/280 kDa and another had circulating IgG autoantibodies which detected both BP and epidermolysis bullosa acquisita (EBA) antigens. All patients had active psoriasis at the onset of the bullous disease. Three patients were being treated with tar when blisters developed; one patient also received UVB radiation and experienced a relapse after exposure to sunlight. HLA phenotypes in three patients were determined. All the patients responded well to methotrexate. These findings demonstrate that BP is the subepidermal bullous disease most associated with psoriasis. Changes at the basement membrane zone in psoriasis may be responsible for the heterogeneous antibody response and may trigger the bullous disease, as may antipsoriatic treatment including tar and UV radiation. However, common immunogenetic mechanisms may play a crucial part in this disease association.

Autoantibodies to bullous pemphigoid and epidermolysis bullosa acquisita antigens in an infant

We describe a 1-year-old boy with multiple tense blisters on the skin, who showed circulating autoantibodies directed to both bullous pemphigoid and epidermolysis bullosa acquisita antigens. The patient's serum IgG antibodies bound to the 290-kDa epidermolysis bullosa acquisita antigen with immunoblot analysis of human dermal extracts. Immunoblot analysis also demonstrated that the patient's serum autoantibodies were reactive with recombinant NC16a domain of the 180-kDa bullous pemphigoid antigen. This study confirmed the presence of circulating autoantibodies directed to both bullous pemphigoid antigen and epidermolysis bullosa acquisita antigen.

The 105-kDa Basement Membrane Autoantigen p105 Is N-Terminally Homologous to a Tumor-Associated Antigen

Certain constitutive skin basement membrane components, such as bullous pemphigoid antigens and epidermolysis bullosa acquisita antigen, were discovered because they were targeted by an autoimmune reaction. We aimed to purify and characterize a 105-kDa skin basement membrane protein termed p105 recognized by autoantibodies (anti-p105) from patients with a unique immune-mediated subepidermal blistering skin disease. A simian virus 40-transformed human fibroblast cell line that synthesizes and secretes p105 was utilized as the protein source. p105 was partially purified by salt-gradient fractionation of serum-free conditioned medium through a Mono Q anion-exchange column and by examining each fraction with protein staining and immunoblotting against anti-p105. p105 was isolated from polyacrylamide gel electrophoresis gels, blotted onto polyvinylidene difluoride membrane, and subjected to protein microsequencing. The 20 microsequenced N-terminal amino acids exhibited no homology to known basement membrane proteins but exhibited a 70% homology to a 90-kDa tumor-associated antigen. Antibodies raised against a peptide generated from these amino acid sequences reacted to a 105-kDa western-blotted keratinocyte and fibroblast protein and a basement membrane component. p105 resisted digestion by glycosidases chondroitinase ABC, neuraminidase, and N-glycosidase F but was cleaved by protease V8 to antigenic fragments of 22 kDa and 14 kDa. The synthesis of p105 was inhibited by cycloheximide. We conclude that p105 is a unique basement membrane component produced by both keratinocytes and fibroblasts.

A case of linear IgA buUous dermatosis with IgA anti-type VII collagen autoantibodies

In this study we present a patient with the sublamina densa type of linear IgA bullous dermatosis (LABD), with IgA autoantibodies reactive with the 290-kDa type VII collagen (the epidermolysis bullosa acquisita (EBA) antigen) and with immunoblotting of normal human dermal extracts. The clinical and histological features of the present case were compatible with those of LABD but quite different from those of EBA. Although EBA sera reacted with the bacterial fusion protein of the N-terminal globular (NC1) domain of type VII collagen, this patient's serum did not show reactivity. Furthermore, ultrastructural localization of target epitopes on the anchoring fibrils in this patient was considerably different from EBA. These results indicate that, whereas EBA antibodies react with the NC1 domain of type VII collagen, the epitope in this case is different from that of EBA (and is most likely on the central triple helical domain). This difference may be responsible for the clinical presentation in this patient being distinct from that of EBA.

A case of generalized pustular psoriasis followed by bullous disease: and atypical case of bullous pemphigoid or a novel bullous disease?

We describe a 31-year-old Japanese woman with generalized pustular psoriasis treated with PUVA who subsequently developed a bullous disease. Throughout the disease course, there was no phase of psoriasis vulgaris. Although several reports describe coexistence of psoriasis vulgaris and bullous disease such as bullous periphigoid, coexistence of generalized pustular psoriasis without any phase of psoriasis vulgaris and bullous disease is rare. As for the bullous disease, direct immunofluorescence study showed IgG and C3 deposition along the basement membrane zone. Indirect immunofluorescence disclosed IgG antibasement membrane zone antibodies. Indirect immunofluorescence on 1 mol/l sodium chloride-split skin demonstrated linear IgG staining almost exclusively on the dermal side of the split. Western immunoblot analysis revealed that the antibody was directed to neither epidermolysis bullosa acquisita antigen nor bullous pemphigoid antigens. Considering the unusual clinical course, we suspect the possibility of a novel autoimmune blistering disease.

Epidermolysis bullosa acquisita: report of a case with comparison of immunogold electron microscopy using pre- and postembedding labelling

A patient with epidermolysis bullosa acquisita (EBA), who has been diagnosed as having bullous pemphigoid for 7 years, is reported. By immunoblotting, both the latest serum and a 4-year-stored serum sample of the patient, were shown to react with the 290-kDa EBA antigen or type VII collagen, but not with bullous pemphigoid antigens. Pre-embedding immunogold electron microscopy demonstrated that the serum bound to the 'anchoring plaque' and to both ends of the anchoring fibrils in the fashion reported previously. In contrast, postembedding immunoelectron microscopy showed binding mainly to the lamina densa. These results indicate that EBA antigens are localized mainly at the lamina densa. Further studies are necessary for confirmation.

Use of Animal Skin Substrates for Indirect Immunofluorescence Diagnosis of Subepidermal Autoimmune Bullous Diseases

We read the article by Pang et al¹concerning the use of frog skin for the differentiation of bullous pemphigoid (BP) from epidermolysis bullosa acquisita (EBA). Based on sporadic observations of a floor-pattern indirect immunofluorescence on 1 mol/L of sodium chloride—split skin (SSS) obtained from BP sera, these authors used skin from a toad to establish a differential diagnosis between BP and EBA. Their rationale was the fact that, as suggested by earlier studies, the BP antigen is present in the skin of some animals,²from which the EBA antigen—being phylogenetically more restricted—is absent.³Since this procedure seems attractive, we undertook a retrospective comparative study to better delineate the usefulness of animal skin substrates for the diagnosis, using indirect immunofluorescence, of subepidermal autoimmune bullous diseases. During the last 3 years, we screened over 1500 human serum samples sent to us under the suspicion of a subepidermal autoimmune

Acquired bullous diseases of childhood: re-evaluation of diagnosis by indirect immunofluorescence examination on 1 M NaCl split skin and immunoblotting.

Acquired autoimmune bullous diseases of childhood are rare, and can be difficult to distinguish clinically. We have studied 12 children, with an initial diagnosis of bullous pemphigoid (BP) in eight patients, cicatricial pemphigoid (CP) in one, chronic bullous disease of childhood (CBDC) in one, and epidermolysis bullosa acquisita (EBA) in two. All patients had positive indirect immunofluorescence (IIF) of the BMZ with IgG. Using 1 M NaCl split skin, six patients showed epidermal binding of IgG, with additional IgA in three cases, and in five patients IgG antibodies bound a dermal protein. Immunoblotting studies revealed an antibody to type VII collagen (EBA antigen) in three patients who had a dermal pattern on IIF. Six sera reacted with an epidermal protein of 180 and/or 220 kDa, characteristic of BP and CP. One of the three IgA-positive sera detected 220- and 180-kDa epidermal proteins using anti-IgA antibody. Following these studies the diagnosis was changed in three of the children. The diagnosis of CBDC was changed to either BP or EBA because of the presence of circulating IgG autoantibodies. In two children with an initial diagnosis of BP the diagnosis was changed to EBA. We conclude that the clinical picture in bullous disorders of childhood shows considerable overlap, and is often misleading. Additional circulating IgA autoantibodies seem to be more common in BP than has been recognized previously. Indirect immunofluorescence investigation on 1 M NaCl split skin may be helpful in differentiating between BP and EBA, but does not replace immunoblotting studies. EBA is apparently more common in children than in adults. No difference was found between the children with BP and EBA with regard to the duration of disease. The long-term outlook is good, although the course may be protracted.

Identification of a Novel Basement Membrane Antigen (p84) Defined by Sera with Antibodies to Both the Epidermal and Dermal Side of Split Skin

The diagnosis of autoimmune blistering skin disease is increasingly based on the skin antigen(s) that is the target of the autoimmune process. Some patients with bullous diseases have unusual basement membrane autoantibodies that react to antigens present in both the epidermal and dermal sides of skin split with 1 M NaCl. This combined staining pattern is due to antibodies directed in part to a 160-kilodalton (kD) basement membrane antigen present on the epidermal side of the split skin and to different antigen(s) present on the dermal side. The identity of the current antigen(s), and the significance of this unusual type of combined staining antibody response is not known. Sera of five patients with combined staining antibodies, 19 patients with other types of basement membrane antibodies (15 directed only to the epidermal and four only to the dermal side of split skin), and 23 patients without basement membrane antibodies were tested by Western immunoblot for reactivity to 4% sodium dodecyl sulfate+2 M urea extracts of dermal and epidermal side of salt-split skin. Three (60%) of the combined staining sera had antibodies to an 82-84-kD antigen (p84) reproducibly present in dermal, but not epidermal, extracts of normal human skin. Antibodies to this antigen were absent in the 42 control sera. Western blot affinity-purified antibody to p84 bound only to the basement membrane, on the dermal side of 1 molar salt-split skin. By co-migration experiments, p84 differed from other basement membrane antigens including the major and minor bullous pemphigoid, cicatrizing pemphigoid, and epidermolysis bullosa acquisita antigens, types III and IV collagen, laminin, and epiligrin. Thus, combined staining antibodies are directed in part to an 82-84-kD antigen (p84), a normal but previously underscribed component of the basement membrane of human skin. This antigen differs from the basement membrane antigens recognized by autoantibodies in other subepidermal bullous diseases. The antibody response to p84 provides a specific marker for a novel autoimmune subepidermal blistering skin disease.