Mapping of epitopes on the BP180 ectodomain targeted by IgA and IgG autoantibodies in patients with the lamina lucida-type of linear IgA disease

Abstract Linear IgA disease (LAD) is an autoimmune subepidermal blistering skin disease characterized by the linear deposition of IgA at the dermoepidermal junction. Serum from patients with LAD most commonly contains autoantibodies that are directed against the hemidesmosomal transmembrane glycoprotein BP180 (type XVII collagen). Various antigenic sites on the extracellular domain of this anchoring filament protein have been shown to be targeted by autoantibodies in different autoimmune bullous skin diseases, including bullous pemphigoid and cicatricial pemphigoid (CP). However, little is known about epitopes on BP180 recognized by autoantibodies in LAD. In this study, we used three recombinant GST fusion proteins, together roughly covering the entire BP180 ectodomain, to characterize the autoimmune response in serum from patients with LAD. Interestingly, we found both IgA and IgG reactivity to all three portions of the BP180 ectodomain. The strongest reactivity was observed with the C-terminal portion of BP180. This is also the major region recognized by autoantibodies in patients with CP. This finding correlates with the observation that there may be significant overlap of the clinical and immunopathological findings in LAD and CP. Received: 21 August 2000 / Revised: 8 November 2000 / Accepted: 13 December 2000     

A child with localized vulval pemphigoid and IgG autoantibodies targeting the C-terminus of collagen XVII/BP180

Localized vulval pemphigoid of childhood (LVPC) has previously been reported in six girls. Clinical features and immunopathological data have suggested it to be a morphological variant of bullous pemphigoid. Epitope targets of the autoantibodies of these patients have not been defined in detail. We describe a 9-year-old girl with possible cicatricial LVPC and circulating IgG antibodies directed against native collagen XVII/BP180, its 120-kDa soluble ectodomain and against the C-terminus of collagen XVII/BP180. No reactivity was detected towards the NC16A domain of collagen XVII/BP180. Linear IgG and C3 deposits were found along the cutaneous basement membrane zone. On 1 mol/L salt-split skin, IgG autoantibodies were shown to bind to the epidermis, and the HLA type II allele DQB1*0301, a marker with significantly increased occurrence in patients with ocular and oral cicatricial pemphigoid, was identified in this patient. Our data suggest that LVPC is a variant of bullous pemphigoid in which direct immunofluorescence microscopy combined with immunoblot analysis can deliver valuable diagnostic information for differential diagnosis. However, differentiation between the scarring and non-scarring course of the disease cannot be made with the present diagnostic markers and therefore careful follow-up of patients with LVPC is required.     

IgG, IgA and IgE autoantibodies against the ectodomain of BP180 in patients with bullous and cicatricial pemphigoid and linear IgA bullous dermatosis

BACKGROUND: Bullous pemphigoid (BP), linear IgA bullous dermatosis (LABD) and cicatricial pemphigoid (CP) are clinically distinct autoimmune bullous skin diseases characterized by autoantibodies against components of the epidermal basement membrane. Like most patients with BP, a significant subgroup of patients with CP has circulating IgG specific for BP180, a transmembraneous protein of hemidesmosomes. Moreover, sera of patients with LABD contain IgA autoantibodies reactive with a 97/120-kDa protein, LABD antigen 1, which is highly homologous to the extracellular portion of BP180. OBJECTIVES: We aimed to determine whether, in these diseases, autoantibody reactivity to BP180 is restricted to distinct immunoglobulin subtypes. METHODS: Utilizing a baculovirus-encoded form of the ectodomain of BP180, sera from patients with BP (n = 10), CP (n = 9), LABD (n = 10) and normal human control sera (n = 10) were analysed by immunoblot for IgG, IgA and IgE reactivity against BP180. RESULTS: All of 10 BP sera displayed IgG, IgA and IgE reactivity with BP180. Six and seven of nine CP sera, respectively, contained IgG and IgA autoantibodies reactive with BP180, but none of nine sera contained BP180-specific IgE. Nine of 10 LABD sera contained IgA, and six of 10 IgG, which was reactive with BP180, but none of 10 sera showed IgE reactivity to BP180. CONCLUSIONS: The presence of IgG and IgA autoantibody responses to BP180 in patients with three clinically distinct autoimmune bullous diseases indicates that an autoimmune response to the same distinct adhesion protein may lead to different clinical manifestations. It is therefore conceivable that variable epitopes of BP180 are targeted by the different autoantibody isotypes, resulting in the distinct clinical pictures.     

Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP1801

Linear IgA disease is an autoimmune subepidermal blistering disease characterized by IgA deposits at the cutaneous basement membrane zone. IgA antibodies from linear IgA disease sera react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), both of which appear to be fragments of the extracellular domain of bullous pemphigoid 180 (type XVII collagen). The aim of this study was to determine whether linear IgA disease sera react with the immunodominant region of BP180 (NC16A domain), which is a major target of IgG autoantibodies produced by patients with bullous pemphigoid. Indeed, 11 of 50 linear IgA disease sera were found to contain IgA autoantibodies that recognized a recombinant form of NC16A by immunoblotting. The same sera also reacted with NC16A by enzyme-linked immunosorbent assay. An epitope mapping analysis uncovered four linear IgA disease-associated epitopes located within the 45 amino acid N-terminal stretch of NC16A, all of which were previously identified as antigenic sites targeted by bullous pemphigoid autoantibodies. Eight of the linear IgA disease sera that were reactive with NC16A also recognized LAD-1 secreted by the SCC-25 cell line, and five sera recognized BP180 extracted from keratinocytes. Linear IgA disease sera depleted of reactivity to NC16A by immunoadsorption continued to react with both the LAD-1 antigen and BP180 by immunoblotting and with the basement membrane zone by indirect immunofluorescence microscopy. Our results demonstrate that IgA autoantibodies from a subset of linear IgA disease patients react with the same sites on BP180 that are targeted by IgG autoantibodies in bullous pemphigoid.     

Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230

Cicatricial pemphigoid is a heterogeneous group of autoimmune subepidermal blistering diseases associated most commonly with autoantibodies to bullous pemphigoid (BP)180 and less frequently with those to laminin 5 or type VII collagen. In addition, a few cases have been described with autoantibodies to the beta4 subunit of alpha6beta4 integrin. We describe a patient with extensive disease of ocular, oral, pharyngeal, laryngeal and genital mucous membranes that healed with scarring of conjunctivae. IgG autoantibodies bound to the dermal-epidermal junction on direct immunofluorescence (IF) microscopy and to the epidermal side of 1 mol L(-1) NaCl-split skin on indirect IF microscopy. Our patient's circulating IgG recognized a 205-kDa protein in extracts of 293T cells transfected with the beta4 subunit of alpha6beta4 integrin and in the cell extract of DJM-1 cells. Our patient's IgG and IgA autoantibodies also reacted with full-length BP180 derived from epidermal extracts and the ectodomain of BP180 (LAD-1) derived from culture supernatant of keratinocytes. In addition, a weak IgG reaction with BP230 was noted. The disease rapidly responded to dexamethasone-cyclophosphamide pulse therapy, and immunoblot reactivity to both beta4 integrin and BP180 decreased according to disease activity.     

Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity

BACKGROUND: Mucous membrane pemphigoid (MMP), a chronic autoimmune subepithelial blistering disease, is associated with circulating IgG and/or IgA autoantibodies against several basement membrane zone antigens. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. OBJECTIVES: To analyse serum autoantibody profile and HLA class II alleles in MMP patients and to correlate this with the clinical presentation of disease. METHODS: Well-defined subgroups consisting of 124 patients with MMP were examined for IgG and IgA reactivity with immunoblotting using human epidermal, dermal and placental amnion proteins. The results were further analysed on the basis of detailed clinical (sites of involvement and disease severity) and immunopathological criteria (immunofluorescence study and HLA class II alleles). RESULTS: Immunoblot assay revealed that the majority of MMP patients had IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to BP180 (including its soluble ectodomains, 120-kDa LAD-1 and 97-kDa LABD97 antigens). Other antigens targeted predominantly by IgG autoantibodies included: BP230 in 34 (27%), beta4 integrin in 26 (21%), and laminin 5 in three (2%). All the BP230+ sera and 23 (88%) beta4 integrin+ sera also reacted with at least one of the BP180 antigens. Over 85% of patients with reactivity to beta4 integrin had ocular involvement. In most cases of MMP, more severe clinical features were associated with antibody reactivity to multiple basement membrane zone antigens, as well as reactivity to multiple BP180 component antigens. Dual BP180/LAD-1 reactivity with IgG and IgA was associated with a more severe phenotype. In addition, the subset-dependent autoantibody reactivity correlated well with specific HLA class II alleles, DQB1*0301, DRB1*04 and DRB1*11. CONCLUSIONS: Our results confirmed that BP180 is a major autoantigen targeted by the sera of patients with MMP. The disease-prevalent HLA class II alleles and humoral autoimmune response against the particular subsets of antigenic epitope(s) within BP180 ectodomain may contribute to the clinicopathological significance and disease severity of MMP.     

IgA autoantibodies in the pemphigoids and linear IgA bullous dermatosis

Please cite this paper as: IgA autoantibodies in the pemphigoids and linear IgA bullous dermatosis. Experimental Dermatology 2010; 19: 648–653. Background: Patients with bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and pemphigoid gestationis (PG) have IgG antibodies against BP180 and BP230, components of the hemidesmosomes. Patients with linear IgA bullous dermatosis (LABD) have IgA autoantibodies against a 97/120‐kDa protein which is highly homologous to a shedded fragment of the BP180‐ectodomain. Objectives: The aim of our study was to determine the incidence of IgA autoantibodies directed against BP180/BP230 in the pemphigoids and LABD and to determine the antigenic regions that are targeted by IgA autoantibodies. Methods: Utilizing baculovirus‐expressed recombinant BP180 and BP230 proteins, we performed immunoblot analyses for IgA reactivity of sera from patients with BP (n = 30), MMP (n = 10), PG (n = 6), LABD (n = 6) and from control patients with non‐related pruritic dermatoses (n = 8). Results: IgA reactivity against BP180 and/or BP230 was detected in 19/30 of the BP, in 7/10 of the MMP, in 6/6 of the LABD and in 3/6 of the PG sera, respectively, but not in the control group. In all subgroups, the major antigenic site recognized by IgA antibodies was located within the NH₂‐terminus of the BP180‐ectodomain, but only a minority of the sera showed also IgA reactivity against the BP180‐NC16a‐domain. IgA reactivity against the central domain of BP180 was more frequently seen than against its COOH‐terminus. IgA against the COOH‐ and NH₂‐terminus of BP230, respectively, was detected in 6/30 of the BP, 1/10 of the MMP, 1/6 of the LABD and 0/8 control sera. Conclusion: IgA reactivity against BP180 and/or BP230 is a common finding in the pemphigoids.     

Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on both intra- and extracellular domains of bullous pemphigoid antigen 180

BACKGROUND: Cicatricial pemphigoid (CP) is an autoimmune subepidermal blistering disease where autoantibodies target various components of the dermal-epidermal junction, including the bullous pemphigoid antigen 180 (BP180). OBJECTIVE: We determined the exact specificity of circulating IgG and IgA autoantibodies to BP180 in a large number of CP patients. METHODS: Twenty-six consecutive CP sera were analysed by Western blotting using a panel of cell-derived and recombinant proteins covering the entire BP180 molecule. RESULTS: Circulating autoantibodies were detected in all CP sera. Seven sera reacting with laminin-5 were excluded from further analyses; the remaining 19 sera recognized BP180, including six sera (32%) that showed only IgA reactivity to this protein. With the combined use of the soluble BP180 ectodomain (LAD-1) and recombinant BP180 NC16A, 16 of these 19 CP sera (84%) targeted BP180. IgG reactivity was preferentially found against NC16A, whereas IgA antibodies predominantly recognized LAD-1. Thirty-two per cent of the BP180-reative sera revealed reactivity with the intracellular domain of this protein. CONCLUSIONS: Our findings demonstrate that autoantibodies in CP target epitopes on both extra- and intracellular domains of BP180 and highlight the importance of testing for both IgG and IgA reactivity in these patients' sera.     

Mapping the binding sites of anti-BP180 immunoglobulin E autoantibodies in bullous pemphigoid

Bullous pemphigoid (BP) is a subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal protein BP180 (BPAg2, type XVII collagen). NC16A, a non-collagenous stretch of the BP180 ectodomain, is the primary target of pathogenic immunoglobulin (Ig)G autoantibodies and IgE class autoantibodies. This study further characterized the IgE-reactive regions of BP180. Of the ten sera from untreated BP patients, eight contained IgE reactive with the entire BP180 ectodomain. The IgE in four of these eight sera reacted with NC16A, whereas in the remaining four sera IgE immunoreactivity was restricted to sites downstream of NC16A. In contrast, IgG reactivity to NC16A was detected in nine of the ten BP sera, and in the remaining serum, IgG, as well as IgE, reacted exclusively with non-NC16A sites on the BP180 ectodomain. Fine mapping of the antigenic sites within NC16A revealed very similar reactivity patterns for IgE and IgG, with NC16A subregion-2 being the major site recognized by both isotypes. Eight of the untreated BP patients were tested for histamine release from their basophils in response to NC16A. Antigen-specific histamine release was observed only in those patients with detectable circulating IgE directed against NC16A (three of eight). Future studies will investigate the pathogenic relevance of anti-BP180 IgE.     

Linear IgA disease: the IgA and IgG response to the epidermal antigens demonstrates that intermolecular epitope spreading is associated with IgA rather than IgG antibodies, and is more common in adults

BACKGROUND: Linear IgA disease (LAD; adult and childhood) is a dapsone-responsive, acquired immunobullous disorder mediated by IgA antibodies directed at target antigens within the epithelial basement membrane. These antigens have not been completely characterized. OBJECTIVES: To identify the target antigens in LAD, and to correlate these with the antibody isotype. METHODS: We used 101 LAD sera without IgG antibodies detected by indirect immunofluorescence. The sera were analysed by immunoblotting for IgA (65 adults and 36 children) and IgG (61 adults and 34 children) autoantibodies, on salt-split, urea-extracted epidermal tissue extracts. RESULTS: Antigens were targeted in LAD by IgA antibodies (54 adults and 23 children), IgG antibodies (34 adults and 19 children), and both isotypes (30 adults and 16 children). Three major antigens were recognized by IgA antibodies: LAD285 (22 adults and three children), BP230 (30 adults and eight children) and BP180 (collagen XVII), including the 97-kDa ectodomain (52 adults and 20 children). Seven 'minor' antigens were occasionally detected (18 adults and 13 children). IgA antibodies bound multiple antigens (33 adults and nine children) more frequently than single antigens (21 adults and 14 children), but the binding to multiple antigens was more restricted in children than in adults. IgG antibodies mainly bound a single antigen (29 adults and 16 children), predominantly BP180. CONCLUSIONS: There was variation in the autoantibody response within the disease and the patient, with regard to target molecules and autoantibody class. The finding that IgG as well as IgA autoantibodies predominantly target BP180 supports a pivotal role for collagen XVII in adult and childhood LAD. The IgG response was very restricted compared with IgA autoantibodies (P < 0.01). Autoantibodies from children had a more restricted antigen repertoire than from adults (P < 0.05). Epitope spreading is common in LAD and is affected by the class of autoantibody and age of the patient.     

Cicatricial pemphigoid differs from bullous pemphigoid and pemphigoid gestationis regarding the fine specificity of autoantibodies to the BP180 NC16A domain

Bullous pemphigoid (BP), pemphigoid (herpes) gestationis (PG), cicatricial pemphigoid (CP), and lichen planus pemphigoides (LPP) are autoimmune subepidermal bullous diseases that are characterized by circulating autoantibodies to the transmembrane hemidesmosomal protein BP180/type XVII collagen. Previous studies demonstrated that the majority of patients with BP, PG, and LPP show antibodies to an immunodominant, membrane-proximal non-collagenous domain (NC16A) on the extracellular portion of BP180. By the use of non-overlapping peptides of the NC16A domain, we previously demonstrated that autoantibodies from BP and PG patients mainly react with epitopes clustered within the N-terminus of this immunodominant site of BP180; antibodies from patients with LPP also recognized the C-terminal portion of NC16A. However, some of these results had been obtained indirectly by preadsorption studies. The aim of the present study was to analyze the fine specificity of IgG autoantibodies to NC16A in sera from patients with CP and to compare their reactivity with antibodies from BP, PG, and LPP patients using a series of new overlapping fragments covering the entire NC16A domain. We confirm that BP and PG sera mainly react with N-terminal epitopes of NC16A, whereas sera from patients with LPP also bind to C-terminal portions, of this domain. Interestingly, out of ten patients with CP, the sera of seven reacted with NC16A; within NC16A, these sera bound to both C-terminal fragments and an N-terminal epitope right next to the cell membrane. Our data demonstrate a heterogeneous binding pattern of autoantibodies to BP180 NC16A in patients with CP.     

Mucous membrane pemphigoid with immunoglobulin G autoantibodies against full-length and 120-kDa ectodomain of BP180

Mucous membrane pemphigoid (MMP) is a rare autoimmune, subepidermal, bullous disease characterized by erosive lesions on the mucous membranes and skin. MMP reacts with various target antigens including BP180, laminin-332, β4 integrin, α6 integrin or type VII collagen. We present a 67-year-old male MMP patient who had lesions on the oral and ocular mucous membranes and facial skin. By immunoblot analyses, immunoglobulin G autoantibodies in the patient's sera reacted with full-length BP180 and the 120-kDa ectodomain of BP180 (LAD-1).     

Omalizumab as an alternative therapeutic tool in the treatment of bullous pemphigoid: A case report

Bullous pemphigoid (BP) is an acquired autoimmune bullous disease characterized by autoantibodies against the hemidesmosomal proteins found in the basal keratinocytes of the basement membrane zone (BMZ): a 180 kDa protein (type XVII collagen) mainly and the 230 kDa antigen. There is such evidence that the antibodies against the BMZ components are not only of IgG type, but also this bullous disease may have IgE antibodies directed to the BMZ that contribute to the pathogenesis of the disorder. IgE is not only thought to contribute to the pathogenesis of BP, it has also been suggested that eosinophils play a role in the development of the first signs associated with BP. A humanized monoclonal antibody directed to IgE, omalizumab, is approved for the treatment of severe asthma and chronic spontaneous urticaria, and it may be useful in the treatment of BP in the first stages of the disease.     

Plectin, an unusual target antigen in bullous pemphigoid

BACKGROUND: Bullous pemphigoid (BP) is a blistering disease associated with autoantibodies directed against two components of hemidesmosomes, BP180 and BP230. OBJECTIVES: To assess whether BP patients have autoantibodies targeting plectin, another hemidesmosomal component showing extensive homology to BP230. METHODS: Examination of sera from 16 patients with BP, using immunoprecipitation studies followed by immunoblotting. RESULTS: Serum of one of the 16 (6%) patients with BP contain autoantibodies binding to plectin, while no reactivity was found with sera from three control subjects. Sera from all 16 BP patients immunoprecipitated BP230 from extracts of biosynthetically radiolabelled human keratinocytes. CONCLUSIONS: Our results indicate that sera from BP patients might contain autoantibodies binding to plectin. Although this protein and BP230 are closely sequence-related, the occurrence of autoantibodies binding to plectin is a rare phenomenon in BP.     

IgE autoantibodies against the intracellular domain of BP180

Background  Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease characterized by autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. BP not only involves IgG-mediated neutrophil activation, leading to blistering, but also IgE-dependent activation of mast cells and basophils. While IgG and IgE autoantibodies target the extracellular noncollagenous (NC) 16A domain of BP180, little is known whether other BP180 regions are targeted by these antibody classes.
Objectives  To characterize IgE and IgG autoantibody binding to antigenic sites on the intracellular domain (ICD) of BP180 compared with BP180 NC16A.
Methods  IgE/IgG autoreactivity against recombinant BP180 ICD and NC16A was determined by immunoblotting of sera from 18 patients with BP and 10 controls.
Results  Total serum IgE was elevated in 16 of 18 BP sera. Most BP sera tested positive (15 of 18) to NC16A with both immunoglobulin classes. Additionally, 14 of 18 sera showed IgE reactivity with an epitope mapped to the ICD of BP180 (amino acid residues 103–266). Mapping of ICD antigenic sites revealed similar IgE and IgG reactivities for most regions except for greater IgE reactivity to amino acid residues 234–398 (11 of 18 BP sera) than IgG (five of 18). Control sera failed to display IgE reactivity to these antigens.
Conclusions  The results indicate that BP180 NC16A is not the only antigenic determinant of IgE autoantibodies in BP and that additional, novel epitopes exist on different regions of the ICD of BP180. The heterogeneous autoimmune response against BP180 suggests intramolecular epitope spreading during disease progression.     

A Case of Chronic Bullous Disease of Childhood That Was Reactive to the Antigen of 120 kDa (LAD-1)

Chronic bullous disease of childhood (CBDC) is an autoimmune blistering disease that is characterized by Immunoglobulin A (IgA) deposits at the basement membrane zone. IgA autoantibodies (aAbs) from the serum of patients with CBDC react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), and both of which are fragments of the extracellular domain of bullous pemphigoid 180 (BP180, type XVII collagen). The CBDC sera reacts with the immunodominant NC16a domain of BP180, which is the major region recognized by IgG aAbs in patients with bullous pemphigoid. A five-year-old boy presented with multiple pruritic tense blisters on the umbilical and inguinal areas for six weeks. The direct immunofluorescence of the perilesional area demonstrated linear deposits of IgA at the basement membrane zone. Using immunoblotting and an enzyme linked immunosorbent assay (ELISA), we identified the IgA aAbs reactive to antigens with a molecular weight of 120 kDa (LAD-1), which is a fragment of the extracellular domain of BP180.     

Refractory bullous pemphigoid leaving numerous milia during recovery

Recovery with milia may occur in bullous pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA). Scarring commonly occurs in MMP and EBA. Here, we report a 62‐year‐old man patient with BP, who was left with numerous milia during recovery. The patient had immunoglobulin (Ig)G autoantibodies to the recombinant protein of the BP180‐NC16a domain and the soluble 120‐kDa ectodomain of BP180 (linear IgA bullous dermatosis [LAD]‐1). There are cases of BP with IgG autoantibodies to LAD‐1 and/or the recombinant protein of BP180 C‐terminal domain. Extensive milia formation during recovery may be associated with immunological predisposition and/or improper interaction between hemidesmosomes and the extracellular matrix components.     

Linear IgA dermatosis with IgA and IgG autoantibodies to the 180 kDa bullous pemphigoid antigen (BP180): evidence for a distinct subtype

BACKGROUND: Autoantibodies in linear immunoglobulin A (IgA) disease (LAD) are reported to be of IgA class and directed against a 97-120 kDa epidermal antigen. METHODS: We report a 39-year-old woman with clinical features of LAD and with circulating IgA and IgG autoantibodies to the 180 kDa bullous pemphigoid antigen (BP180). RESULTS: Histopathology of lesional skin revealed a subepidermal blister with mixed inflammatory cell infiltrate. Direct immunofluorescence of perilesional skin showed linear deposits of IgA along the dermal-epidermal junction. The antigen specificity of the patient's circulating antibodies was determined by Western blotting and enzyme-linked immunoabsorbent assay (ELISA) using various antigen sources, including cultured human keratinocytes, dermal protein lysates, and purified laminin-5, as well as proteins corresponding to BP180, the 230 kDa bullous pemphigoid antigen (BP230), laminin-5 subunits, and collagen IV alpha1-alpha6 chains. IgA and IgG antibodies in the patient's serum were directed against BP180, and no IgA or IgG reactivity was found against the other skin antigens. CONCLUSIONS: These data provide evidence for the presence of a subtype of LAD with dual IgA and IgG autoimmune response to BP180.     

Assessment of skin basement membrane zone antibodies in the urine of patients with acquired subepidermal immunobullous diseases

BACKGROUND: In bullous pemphigoid (BP), cicatricial pemphigoid (CP) and linear IgA disease (LAD), autoantibodies to the basement membrane zone (BMZ) are found in skin and mucosa, blood and blister fluid. OBJECTIVES: To assess whether BMZ antibodies might also be detected in urine. METHODS: Urine and serum samples from 62 patients (32 with BP, 17 with CP and 13 with LAD) were analysed for antibody isotypes and subclasses by indirect immunofluorescence, and urine and serum samples from 40 patients (25 with BP, eight with CP and seven with LAD) were screened for target antigens using immunoblotting. RESULTS: Fourteen of 32 patients with BP had detectable levels of IgG BMZ autoantibodies in their urine, and all 32 had positive sera. Of these 14 BP patients, 13 had epidermal-binding serum autoantibodies at a titre > 1 : 160, and one had dermal-binding serum antibodies at a titre of 1 : 40. BMZ autoantibodies were not detected in the urine of the CP or LAD patients, but the corresponding sera were of low titre or negative. IgG subclasses (IgG1-4) were less frequently detected in urine than in serum. IgG4 was the predominant subgroup found (10 urine samples and all 14 sera), followed by IgG1 (two urine samples and 12 sera); IgG2 was detected in a single urine sample and three sera, and IgG3 was not detected. Eight of 25 BP and one of eight CP urine samples were positive on immunoblotting, and bound BP230 and/or BP180 with IgA and/or IgG autoantibodies. IgA autoantibodies were not detected in the urine of the seven LAD patients. The corresponding sera were often more positive, with 21 of 25 BP, five of eight CP and six of seven LAD sera immunoblotting the major BP antigens. CONCLUSIONS: The detection of IgG autoantibodies from urine samples using indirect immunofluorescence correlated with a high titre of IgG autoantibodies in the serum. IgG and IgA autoantibodies in the urine were detected by immunoblotting, although less frequently than in serum. The finding of BMZ antibodies in the urine of many BP patients may have clinical relevance, and may have a restricted application in the diagnosis of immunobullous disease.     

Production of the entire extracellular domain of BP180 (type XVII collagen) by baculovirus expression

Bullous pemphigoid (BP) is an acquired autoimmune skin disease, and its target antigens are a 230 kDa plaque protein (BP230) and a 180 kDa transmembrane protein with interrupted collagenous domains (BP180, type XVII collagen), which localize at the hemidesmosome. In this study we have attempted to express the entire extracellular domain of BP180 (rBP180EC) as a secreted protein by baculovirus expression. Seventy out of 83 BP sera (84.4%) showed positive reactivity against rBP180EC by immunoblot analysis, and 56 out of 83 BP sera (67.5%) were positive against rBP180EC by ELISA. These figures were comparable with those when a bacterial recombinant protein encoding the NC16a domain of BP180 (rNC16a) was used as an antigen source. Reactivity of BP sera against rBP180EC by ELISA was completely abolished or significantly reduced by immunocompetition with rNC16a in 11 out of 14 BP sera tested, while the reactivity was not altered in the rest of the three sera. These findings indicate that the NC16a domain represents the major epitopes on the extracellular domain of BP180, although there are some other minor epitopes outside of NC16a which are uniquely expressed by rBP180EC. rBP180EC will be useful to develop a diagnostic tool for BP as well as to dissect a molecular role for BP180 in interactions of keratinocytes with epidermal basement membrane.