Fc-binding proteins enhance autoantibody-induced BP180 depletion in pemphigoid

Immunoglobulins (Igs) consist of two antigen-binding regions (Fab) and one constant region (Fc). Protein A and protein G are bacterial proteins used for the purification of IgG by virtue of their high affinities for the Fc fragment. Rheumatoid factors are autoantibodies against IgG Fc fragments, which are present in the body under physiological conditions. Little is known about the influence of Fc-binding proteins on the pathogenicity of antibody-induced autoimmune diseases. Pemphigoid diseases are a group of autoimmune subepidermal blistering disorders that includes bullous pemphigoid and mucous membrane pemphigoid. IgGs targeting the non-collagenous NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) are known to induce skin fragility in mice and the depletion of BP180 in keratinocytes. In this study, mAb against NC16A in combination with Fc-binding proteins was found to enhance BP180 depletion. Although mAb against the C-terminus of BP180 does not show pathogenicity in vivo or in vitro, mAb treatment with Fc-binding proteins clearly induced skin fragility in mice and BP180 depletion in keratinocytes. Anti-BP180 mAbs and Fc-binding proteins were colocalized in the cytoplasm and at the basement membrane zone. Cell adhesion strengths were decreased in parallel with BP180 amounts. Clinically, bullous pemphigoid patients had higher rheumatoid factor titers than controls. Anti-BP180 mAb in combination with high-titer rheumatoid factor serum was found to enhance BP180 depletion. Furthermore, saliva from mucous membrane pemphigoid patients contained larger quantities of bacteria and Fc-binding proteins than controls. Our results suggest that Fc-binding proteins (rheumatoid factor or protein G) may enhance the pathogenicity of autoantibodies in pemphigoid diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Autoimmune responses in patients with linear IgA bullous dermatosis: both autoantibodies and T lymphocytes recognize the NC16A domain of the BP180 molecule

Linear IgA bullous disease (LABD) is an autoimmune skin disease characterized by subepidermal blisters and IgA autoantibodies directed against the epidermal basement membrane zone (BMZ) of the skin. Various antigens have been identified as targets of IgA autoantibodies including BP180, a type II glycoprotein that spans the BMZ and lamina lucida. Previously, we have identified a subset of LABD patients whose sera contained IgA antibodies against the 16th noncollagenous (NC16A) domain of BP180. NC16A was previously shown to harbor epitopes that are recognized by both autoantibodies and T cells from patients with bullous pemphigoid and herpes gestationis and is thought to be associated with the development of these immunobullous diseases. The aim of this study was to determine whether T lymphocytes from LABD patients with anti-NC16A IgA autoantibodies respond to epitopes in the same region of the BP180 protein. Indeed, of the four LABD patients in our study, all had T cells that specifically proliferated in response to NC16A. Moreover, two subfragments of NC16A were identified as the predominant targets of LABD T cells. Further analysis of T cell lines and clones derived from these patients revealed that these cells express a CD4 memory T cell phenotype and secrete a Th1/Th2 mixed-cytokine profile, characteristics similar to those of T cells in bullous pemphigoid patients. Our data suggest that the BP180 protein, typically the NC16A region, is the common target of both cellular and humoral immune responses in some LABD patients. This information helps to further elucidate the autoimmune mechanisms in this disease.     

Recent advances on the 180-kDa epidermal antigen in autoimmune subepidermal bullous skin diseases

Summary The blistering skin disease bullous pemphigoid (BP) is an example of how autoantibodies from patients can be used as tools to improve our knowledge of the molecular structure of normal skin. BP autoantibodies bind to the basement membrane zone of normal human skin and usually react with two main epidermal antigens of approximately 230-kDa and 180-kDa. From recent studies, it is now established that the 180-kDa polypeptide is distinct from the 230-kDa BP antigen and is a major component of hemidesmosome. In this article, the recent data regarding the localization and structure of the epidermal 180-kDa BP antigen are reviewed in detail, together with its clinical significance, since it appears to be also a target antigen for pemphigoid (herpes) gestationis and, perhaps, cicatricial pemphigoid autoantibodies.     

IgG antibodies against immunodominant C-terminal epitopes of BP230 do not induce skin blistering in mice

Bullous pemphigoid, the most common autoimmune blistering disease in Western Europe and the USA is characterized by the presence of circulating and tissue-bound autoantibodies against the hemidesmosomal proteins BP230 and BP180/collagen XVII. After binding to their target antigens at the basement membrane of the dermal–epidermal junction these autoantibodies are thought to trigger an inflammatory cascade comprising complement- and granulocyte-dependent reactions that result in tissue damage. Whereas the role of anti-BP180 antibodies has been extensively characterized, few and conflicting data is available on the contribution of anti-BP230 antibodies to bullous pemphigoid pathogenesis. Therefore, we addressed in the present study the role of autoantibodies to BP230 in experimental bullous pemphigoid. Rabbit polyclonal antibodies generated against epitopes of the C-terminal fragment of murine BP230 bound to the basement membrane and activated the complement system ex vivo. Affinity-purified antibodies were subsequently subcutaneously transferred into neonatal and adult BALB/c mice. In vivo, we observed a dose-dependent binding of transferred antibodies in the murine skin; however, there was no complement activation and these mice showed no clinical or histological signs of inflammatory disease, in contrast to mice receiving anti-BP180 antibodies. We further conducted ex vivo experiments and demonstrated that rabbit IgG anti-BP230-specific antibodies, in contrast to antibodies from bullous pemphigoid patients or rabbit IgG anti-BP180 antibodies used as positive controls, did not activate human granulocytes to induce dermal–epidermal separation in skin cryosections. Our present findings demonstrate that antibodies against BP230 are non-pathogenic in experimental models of bullous pemphigoid and suggest that proper activation of the complement and granulocytes represent prerequisites for conferring bullous pemphigoid autoantibodies their tissue destructive potential.     

IgE recognition of bullous pemphigoid (BP)180 and BP230 in BP patients and elderly individuals with pruritic dermatoses

Bullous pemphigoid (BP) is the most common autoimmune bullous disease of the elderly and is associated with IgG and IgE autoantibodies against the hemidesmosomal proteins, BP180 and BP230. The purpose of this study was to characterize the epitope specificity of IgE against defined regions of BP180 and BP230 in 32 BP patients and 21 elderly patients with pruritic disorders who did not yet fulfill all the criteria of BP by immunoblot (IB), ELISA and indirect immunofluorescence microscopy. Our findings show that IgE from BP sera preferentially targets the COOH-terminus of BP230 (IB: 16/32, ELISA: 12/32) and, to a lesser extent, the BP180-NC16A domain (IB: 11/32, ELISA: 9/32). Noteworthy, a subgroup of elderly patients with pruritic dermatoses also showed IgE recognition of BP180-NC16A (IB: 1/21, ELISA: 4/21) and less frequently of BP230 (IB: 2/21, ELISA: 2/21). Thus, IgE recognition of the BP autoantigens is presumably an early pathogenetic event in BP.     

Evaluation of recombinant antigen-based assays for diagnosis of bullous autoimmune diseases

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.     

Multicenter prospective study of the humoral autoimmune response in bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous disease, associated with autoantibodies directed against the hemidesmosomal components BP180 and BP230. In this study for the first time different laboratories have analyzed the autoantibody profile in the same group of 49 prospectively recruited BP patients. The results show that: 1) disease severity and activity correlated with levels of IgG against the BP180-NC16A domain, but also against a COOH-terminal epitope of BP180, 2) distinct epitopes of the BP180 ectodomain other than BP180-NC16A were recognized by 96% of the BP sera; and 3) the combined use of BP180 and BP230 ELISA led to the detection of IgG autoantibodies in all the BP sera. These results demonstrate the usefulness of the combined ELISAs based on various BP180 and BP230 fragments in establishing the diagnosis of BP and support the concept that BP180 is the major autoantigen of BP.     

T cell receptor gene usage of BP180-specific T lymphocytes from patients with bullous pemphigoid and pemphigoid gestationis

BP180 is the autoantigen of different immunobullous diseases, including bullous pemphigoid (BP) and pemphigoid gestationis (PG). Previously, we demonstrated that the NC16A domain of this autoantigen harbors key epitopes of autoantibodies and T cells, indicating that it plays an essential role in the pathogenesis of diseases. Moreover, NC16A-specific T cell clones derived from these patients were shown to express a CD4+ memory T cell phenotype and secrete cytokines that may promote autoantibody production. In this study, we further characterize the properties of these T cells by analyzing their epitope specificity and T cell receptor (TCR) gene usage. We discovered that 83% of T cell clones obtained from BP patients preferentially express TCRBV13, while clones derived from a PG patient express the TCRBV3 gene. However, no preferential TCRBJ gene usage was identified. In conclusion, our results provide an advanced understanding of the characteristics of autoimmune T cells in immunobullous diseases.     

Humoral and cellular autoimmunity in autoimmune bullous skin disorders

Autoimmune bullous skin diseases, such as pemphigus vulgaris (PV) and bullous pemphigoid (BP), are severe, frequently life-threatening skin disorders. Immunologically, they are characterized by the presence of serum autoantibodies (auto-Ab) targeting distinct adhesion molecules of the epidermis or dermoepidermal basement membrane zone. Antibody (Ab) binding interferes with the adhesive function of these molecules, leading to detachment and subsequently blister formation. PV is the classical example of an Ab-mediated autoimmune disease affecting epidermal adhesion. Auto-Ab against the desmosomal adhesion molecule, desmoglein 3 (Dsg3), are critical in the pathogenesis of this disease, since the transfer of serum IgG Ab reactive with Dsg3 into newborn mice induces a bullous skin disease resembling PV. Autoreactive T cell responses to Dsg3 may be critical in the pathogenesis of PV because: (1) Ab production generally requires T cell help; (2) the involvement of CD4+ T lymphocytes in PV has been suggested by the strong association with distinct HLA class II alleles, and (3) T cell recognition of epitopes of Dsg3 may be crucial for the initiation and perpetuation of the production of Dsg3-specific auto-Ab by B cells. In PV and BP, autoreactive CD4+ T cells recognize distinct epitopes of the extracellular portions of Dsg3 and BP180 [BP antigen 2 (BPAG2) or type XVII collagen], respectively, and produce preferentially T helper type 2 (TH2) cytokines. Auto-Ab of the TH2-dependent IgG4 subtype are preferentially seen in the active stages of both PV and BP, while auto-Ab of the TH1-dependent IgG1 subclass are predominant during the chronic course of these disorders. These observations suggest that autoreactive TH2 cells may provide targets to specifically modulate the T cell-dependent production of pathogenic auto-Ab in these disorders.     

Granulocyte-derived elastase and gelatinase B are required for dermal-epidermal separation induced by autoantibodies from patients with epidermolysis bullosa acquisita and bullous pemphigoid

Epidermolysis bullosa acquisita (EBA) and bullous pemphigoid (BP) are two clinically and immunologically distinct autoimmune subepidermal blistering skin diseases associated with IgG autoantibodies against the dermal-epidermal junction. BP antibodies are directed against the hemidesmosomal antigens BP180 and BP230, and those in patients with EBA target type VII collagen, a major component of anchoring fibrils. While the pathogenetic mechanisms of subepidermal blistering in BP have been previously studied using a passive transfer mouse model, the effector pathways of blister formation in EBA are largely unknown. Autoantibodies to type VII collagen and BP180 have recently been shown to induce leucocyte-mediated subepidermal cleavage in cryosections of human skin. The aim of the present study was to identify human leucocyte protease(s) instrumental in dermal-epidermal separation induced by autoantibodies to type VII collagen and BP180. When incubated with cryosections of human skin pretreated with IgG from patients with EBA or BP but not from patients with anti-laminin 5 mucous membrane pemphigoid or healthy controls, granulocytes were recruited to the dermal-epidermal junction and induced subepidermal splits. A combination of broad-range protease inhibitors as well as inhibitors of serine and matrix metalloproteases completely abolished dermal-epidermal separation induced by EBA or BP autoantibodies. When characterizing the proteases involved more specifically, selective inhibition of human leucocyte elastase or gelatinase B/MMP-9 was also found to result in suppression of blistering. These findings strongly suggest that elastase and gelatinase B are essential for granulocyte-mediated proteolysis resulting in dermal-epidermal separation in EBA and BP patients' skin.     

T cell participation in autoreactivity to NC16a epitopes in bullous pemphigoid

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies against the NC16a domain of bullous pemphigoid 180. This study was performed to characterize and map the fine specificity of T cell responses to NC16a. Peripheral blood mononuclear cells (PBMC) from a total of 28 bullous pemphigoid patients and 14 matched controls were tested for proliferative and cytokine responses to recombinant NC16a and a complete panel of 21 overlapping peptides spanning this region of BP180. Proliferative responses to NC16A and the peptide panel in the patients with active disease were similar in frequency and magnitude to those in healthy donors, and included late responses typical of naive cells in approximately 60% of each group. Interleukin (IL)-4 responses were slightly stronger for six peptides, and significantly stronger for Nc16a, in patients than in controls. Factor analysis identified factors that separate responses to the peptide panel discretely into IL-4, T helper type 2 (Th2) pattern, interferon (IFN)-γ, Th1 pattern and IL-10 or transforming growth factor [TGF-β, regulatory T cell (T_reg)] pattern. Factors segregating IL-10 versus IFN-γ were predicted by active blistering or remission, and TGF-β or IL-10 versus IFN-γ by age. Finally, we confirmed a significant up-regulation of IgE responses to BP180 in the patients with pemphigoid. This shows the complexity of T cell phenotype and fine autoreactive specificity in responses to NC16A, in patients and in normal controls. Important disease-associated factors determine the balance of cytokine responses. Of these, specific IL-4 and IgE responses show the strongest associations with pemphigoid, pointing to an important contribution by Th2 cytokines to pathogenesis.     

Patients with bullous pemphigoid and linear IgA disease show a dual IgA and IgG autoimmune response to BP180

Bullous pemphigoid (BP) and linear IgA disease (LAD) are autoimmune subepidermal blistering skin diseases associated with autoantibodies against the transmembrane hemidesmosomal protein BP180/type XVII collagen. It has been demonstrated previously that BP is characterized predominantly by IgG autoantibodies, while autoantibodies in LAD mainly belong to the IgA isotype. The aim of the present study was to investigate the hypothesis that there is a significant overlap in the autoantibody isotype profiles associated with these two diseases. Several new recombinant forms of BP180 were generated in the baculovirus expression system, including the full-length protein. IgG autoantibodies to BP 180 were detectable in 39 of 40 (98%) of BP sera; interestingly, 88% of BP sera also contained IgA anti-BP180 autoantibodies. Similarly, anti-BP180 reactivity in LAD sera (n=22) was also attributed to both an IgA (68%) and an IgG (76%) autoantibody response. IgA and IgG autoantibodies to the intracellular portion of BP180 were found in 14% and 28% of BP sera, respectively, and in 8% of LAD sera (same percentage for both isotypes). Our findings clearly demonstrate that both BP and LAD patients have a dual IgA and IgG autoimmune response to BP180 which is directed not only to the ectodomain, but also to the intracellular portion of this protein.     

IgE Antibodies in Sera from Patients with Bullous Pemphigoid Are Autoantibodies Preferentially Directed Against the 230-kDa Epidermal Antigen (BP230)

Bullous pemphigoid (BP) is unique among autoimmune skin diseases in which a high serum IgE level has been detected. We sought to determine the antigenic specificity of these IgE antibodies in 39 BP sera by immunofluorescence microscopy, immunoblot, and ELISA. The patient's sera contained IgG antibodies to 230-kDa (BP230) (n = 20), 180-kDa (BP180) (n = 9), and both BP230 and BP180 (n = 10) antigens. Serum IgE levels varied from 29 to 5000 kIU/L (mean ± SD, 856 ± 1426 kIU/L), among which sera containing IgG antibodies to BP230 had an IgE level on average 4.3 times higher than anti-BP180 sera. IgE antibodies in 18 sera were found to be autoantibodies reactive either with an epidermal component of basement membrane zone by immunofluorescence microscopy on 1 M NaCl-split skin or with a 230-kDa antigen by immunoblots of cultured human keratinocytes.⁷ The 230-kDa epidermal antigen recognized by IgE antibodies comigrated with the BP230 as labeled by a specific human monoclonal antibody. IgE anti-BP230 antibodies in patients' sera were always associated with IgG autoantibodies. No sera contained IgE antibodies to BP180 or to any other epidermal or dermal antigens as verified by immunoblot and ELISA. A good correlation was found between the presence of IgE circulating autoantibodies and the level of serum IgE (P < 0.004). IgE antibodies to BP230, like IgG autoantibodies, were mapped primarily to the C-terminal end of the protein, as they labeled rBP55, a BP230 recombinant protein encoded by a cDNA for the C-terminal end of BP230.     

IgG4 autoantibodies are inhibitory in the autoimmune disease bullous pemphigoid

The IgG4 subclass of antibodies exhibits unique characteristics that suggest it may function in an immunoregulatory capacity. The inhibitory function of IgG4 has been well documented in allergic disease by the demonstration of IgG4 blocking antibodies, but similar functions have not been explored in autoimmune disease. Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease characterized by autoantibodies directed against BP180 and an inflammatory infiltrate including eosinophils and neutrophils. Animal models have revealed that the NC16A region within BP180 harbors the critical epitopes necessary for autoantibody mediated disease induction. BP180 NC16A-specific IgG belong to the IgG1, IgG3, and IgG4 subclasses. The purpose of this study was to determine effector functions of different IgG subclasses of NC16A-specific autoantibodies in BP. We find that IgG4 anti-NC16A autoantibodies inhibit the binding of IgG1 and IgG3 autoantibodies to the NC16A region. Moreover, IgG4 anti-NC16A blocks IgG1 and IgG3 induced complement fixation, neutrophil infiltration, and blister formation clinically and histologically in a dose-dependent manner following passive transfer to humanized BP180-NC16A mice. These findings highlight the inhibitory role of IgG4 in autoimmune disease and have important implications for the treatment of BP as well as other antibody mediated inflammatory and autoimmune diseases.     

Epstein-Barr virus-transformed lymphocytes from patients with bullous diseases produce autoantibodies to cytokeratins

Human cell lines obtained after Epstein-Barr virus transformation of lymphocytes from seven patients with bullous diseases (Bullous pemphigoid, Pemphigus) and five controls were screened for the production of autoantibodies against skin antigens. In five out of seven patients, the culture supernatants tested by indirect immunofluorescence on frozen sections of normal human skin and rabbit lip showed the production of autoantibodies with different specificities: basal epidermal cells, whole epidermis, Merkel cells, fibroblasts endothelial cells, etc. All autoantibodies were of IgM class and reacted with intracellular structures. Some of them were further tested by immunoblotting against epidermal keratins and were found to react with the main human epidermal keratins (56 to 67 kDa). In contrast, even when patients had circulating autoantibodies, no supernatant showed any reactivity against the antigens usually involved in these diseases, i.e., the dermoepidermal junction or the intercellular spaces of epidermis. Supernatants from controls did not show any reactivity by immunofluorescence. The results demonstrated that human lymphoid cell lines obtained from patients with bullous diseases elicited the production of anti-intermediate filament autoantibodies known to occur spontaneously in normal patients. It is suggested that this phenomenon may be linked to the blistering conditions that provoke tissue destruction.     

Oral pemphigoid autoantibodies preferentially target BP180 ectodomain

Mucous membrane pemphigoid (MMP) comprises a heterogenous group of autoimmune subepithelial bullous diseases very frequently having oral involvement. Very few studies have investigated the immunological status of a subset of MMP, termed oral pemphigoid (OP), presenting with exclusive oral lesions. In this study we show that 75% of 20 OP patients without scarring phenotype possessed circulating autoantibodies against the BP180 molecule, indicating a prominent role of this protein as a target antigen in OP. Of note, the frequency of reactivity against BP180 ectodomain epitopes in OP was similar to that previously reported for MMP with cicatricial phenotype, while the lack of significant recognition of BP180 intracellular domain appears to characterize OP with respect to other diseases of the pemphigoid group. Finally, the combined use of sensitive techniques allowed the detection of circulating autoantibodies in 90% of OP patients, supporting the usefulness of this approach in the diagnosis of MMP disease.     

The Pathophysiology of Bullous Pemphigoid

Bullous pemphigoid (BP) is a blistering skin disease characterized by an autoimmune response to 2 hemidesmosomal proteins within the dermal–epidermal junction, designated BP180 and BP230. While BP230 localizes intracellularly and associates with the hemidesmosomal plaque, BP180 is a transmembrane glycoprotein with an extracellular domain. Most BP patients have autoantibodies binding to an immunodominant region of BP180, the noncollagenous 16A domain (NC16A), which is located extracellularly close to the transmembrane domain of the protein. Autoreactive T and B cell responses to BP180 have been found in patients with BP. Passive transfer of antibodies to the murine BP180 ectodomain triggers a blistering skin disease in mice that closely mimics human BP. Lesion formation in this animal model depends upon complement activation, mast cell degranulation and accumulation of neutrophils and eosinophils. Patients’ autoantibodies to BP180 induce dermal–epidermal separation in cryosections of human skin when co-incubated with leukocytes. The loss of cell-matrix adhesion is mediated by proteinases released by granulocytes. The increased knowledge of the pathophysiology of BP should facilitate the development of novel therapeutic strategies for this disease.     

Diagnosis and classification of pemphigus and bullous pemphigoid

Pemphigus and bullous pemphigoid represent the two major groups of autoimmune blistering diseases. Pemphigus has three major variants: pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus and is characterized by autoantibodies directed against the cell surface of keratinocytes, producing acantholysis that in turn leads to intraepithelial blisters in the skin and/or mucous membranes. In bullous pemphigoid, the autoantibodies are present at the dermo-epidermal junction and attack the hemidesmosomes, causing subepidermal blister formation. The classification of the major variants of both the pemphigus group and bullous pemphigoid can be based on the combination of clinical, histopathological and immunopathological criteria. Many tools are available for the diagnosis of these entities including biopsy, direct and indirect immunofluorescence, immunoprecipitation, immunoblotting and ELISA. However, currently there are no generally accepted criteria for the diagnosis of these disorders. The present review provides a proposal for diagnostic criteria.     

A highly sensitive and simple assay for the detection of circulating autoantibodies against full-length bullous pemphigoid antigen 180

Bullous pemphigoid antigen 180 (BP180) is the target of autoantibodies in various subepidermal blistering diseases. The most common one is bullous pemphigoid (BP). The pathological importance of anti-BP180 antibodies has been demonstrated in a passive transfer mouse model. However, sensitive assays for routinely detecting circulating antibodies directed against both intra- and extracellular domains of BP180 are only available in specialized laboratories. In addition, most current assays use prokaryotic recombinant fragments of BP180 that lack conformation-dependent epitopes. A simple and very sensitive immunofluorescence (IF) assay based on eukaryotic cells is described here. Sf21 insect cells were transfected with full-length (FL) BP180. As revealed by FACS and confocal laser scanning microscopy the protein was expressed as type II transmembrane protein as in human keratinocytes. By testing serial dilutions of BP180-specific mouse monoclonal antibodies, the eukaryotic IF assay was demonstrated to be more sensitive compared to conventional assays including (1) indirect IF microscopy of human salt-split skin, (2) Western blotting (WB) of the keratinocyte-derived BP180 ectodomain, (3) WB of recombinant BP180 NC16A, and (4) WB of FL-BP180 extracted from Sf21 insect cells. When applied to sera from patients with BP (n = 65), pemphigoid gestationis (n = 16), and cicatricial pemphigoid (n = 7), the novel assay revealed that 58 (89%), 13 (81%), and 6 (84%), respectively, were positive. In contrast, all control sera (pemphigus, n = 20; epidermolysis bullosa acquisita, n = 5; anti-laminin 5 cicatricial pemphigoid, n = 5; systemic lupus erythematosus, n = 5; atopic dermatitis, n = 7; contact dermatitis, n = 3; normal human sera, n = 30) were negative indicating that the assay is highly specific. In addition, reactivity of the assay was conserved to a large extent when the cells had been stored at -20 degrees C for 3 months. Thus, this assay meets the demands of a simple and effective diagnostic tool for detecting circulating antibodies against FL-BP180 and may also be used in laboratories without access to molecular biological technology.     

Recent advancement on autoantigens,autoantibodies and inflammatory cells in subepidermal autoimmune bullous diseases

Subepidermal autoimmune bullous diseases (SABD) are some autoimmune skin diseases that can present in a variety of forms and can be a challenging disease to treat. An overview of the different forms of SABD are discussed including bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), cicatricial pemphigoid (CP), bullous systemic lupus erythematosus (BSLE), and Anti-p200 pemphigoid. Emphasis on recent advancement is presented. In recent years, improved Knowledge of the mechanisms of intercellular and cell-matrix adhesion has led to better understanding of the blistering process in some SABD. Defects of such structures cause the subepidermal bullous diseases and have also led to the discovery of new diseases (e.g. anti-p200-pemphigoid). Recent studies have outlined the important role of autoantibodies, mast cell lymphocytes and their cytokines in pathogenesis of SABD.