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.     

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.     

Epidermal adhesion molecules and basement membrane components as target structures of autoimmunity

 Intraepidermal and dermal-epidermal cohesion are of paramount importance for the integrity of the skin. Some constituent molecules of keratinocyte adhesion complexes and basement membrane-associated structures are the targets of antibody-mediated autoimmune reactions that give rise to various (muco-)cutaneous blistering diseases. The current state of our knowledge about these molecules – along with the main clinical, histological, and immunohistochemical features of the corresponding autoimmune diseases and their pathogenetic mechanisms – comprise the subjects surveyed in this review. Among the desmosomal cadherins (desmogleins and desmocollins) that mediate epidermal cell–cell adhesion, it has been demonstrated that desmoglein 1 and desmoglein 3 are the autoantigens of pemphigus foliaceus and pemphigus vulgaris, respectively, both diseases that result in intraepidermal blistering. Further, desmocollin autoantibodies may be involved in IgA pemphigus. Paraneoplastic pemphigus is associated with autoantibodies directed against the desmosomal plaque protein, desmoplakin. Of the constituents of hemidesmosomes, the plaque protein, BP230 (BPAG1), and the collagen-like transmembrane protein, BP180 (BPAG2), are the autoantigens of bullous pemphigoid and pemphigoid gestationis, the manifestations of both of which include subepidermal blistering. Several diseases arise from autoimmune reactions against certain proteins associated with the basement membrane located beneath hemidesmosomes, for example laminin 5 (cicatricial pemphigoid), ladinin (LAD-1; linear IgA disease), uncein, and collagen VII (epidermolysis bullosa acquisita), the last of which is the constituent protein of the anchoring fibrils. Such recent advances in the elucidation of the molecular nature of autoantigens may serve as the basis for the development of novel molecule-based therapeutic strategies. Received: 12 September1997 / Accepted: 27 October 1997     

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.     

Detection and characterization of anti-envoplakin linker autoantibodies in paraneoplastic pemphigus using specific bead-based assay

Paraneoplastic pemphigus (PNP) is characterized by an autoantibody response directed against desmosomal antigens, desmogleins and plakin's proteins, i.e., periplakin and envoplakin. Notably, PNP antibodies were shown to recognize major epitopes located in the linker subdomain of human envoplakin and thus, may constitute a diagnostic marker of PNP. In this study, a recombinant envoplakin-linker subdomain (rENV-L) was produced and used to develop a specific bead-based assay to determine the prevalence and titers of anti-rENV-L antibodies in patients with different types of autoimmune bullous skin diseases. Sera from 33 PNP, 41 pemphigus vulgaris, 46 pemphigus foliaceus, 24 bullous pemphigoid and 74 normal subjects were analyzed by rENV-L bead-based assay: 23/33 (69.7%) PNP, 2/24 (8.3%) bullous pemphigoid and 4/74 (5.4%) healthy control sera showed IgG reactivity against rENV-L. For PNP, the sensitivity and specificity of the assay were 69.7% and 94.6%, respectively. Among the PNP sera reacting with envoplakin and periplakin by immunoblot analysis, 80.8% contained anti-rENV-L antibodies. Finally, we showed that an anti-rENV-L mAb that recognizes both envoplakin and periplakin gave the same fluorescence pattern on rat bladder sections than PNP sera. Thus, anti-rENV-L antibodies constitute a useful diagnostic marker of PNP and our bead-based assay, notably combined with other epidermal autoantigens, a useful tool to diagnose PNP.     

Anti-desmoglein 1 antibodies in Tunisian healthy subjects: arguments for the role of environmental factors in the occurrence of Tunisian pemphigus foliaceus

Pemphigus foliaceus is an autoimmune blistering skin disease mediated by autoantibodies directed against desmoglein 1 and occurs as a sporadic form throughout the world, or as an endemic form called fogo selvagem in Brazil. Healthy subjects living in Brazilian endemic areas produce antidesmoglein 1 antibodies, suggesting the role of environmental factors in the initiation of the autoimmune response. Tunisia was described recently as an endemic area where the disease is characterized by its high rate among young people, especially women. An enzyme-linked immunosorbent assay using recombinant desmoglein 1 as antigen was used to detect antibodies against desmoglein 1 and calibrated with sera from 67 French healthy blood donors, 20 French pemphigus foliaceus patients and patients with other bullous skin diseases. When sera from 179 healthy Tunisian blood donors were tested, 31 (17%) were found positive. The desmoglein 1 binding activity of these 31 sera was confirmed in 10 cases by indirect immunofluorescence analysis and/or immunoblotting using human epidermal extract. Subclass analysis of antidesmoglein 1 antibodies showed that they were almost exclusively of the IgG2 subclass in positive normal sera and of IgG4 subclass in patients with PF. Thus, antibodies against desmoglein 1 are prevalent in normal subjects living in Tunisia which, along with their IgG2 isotype, suggests the role of the environment in the pathogenesis of this endemic type of pemphigus foliaceus and the need for additional factors to switch from a subclinical to a clinical form of the disease.     

IgE-mediated mechanisms in bullous pemphigoid and other autoimmune bullous diseases

Autoimmune bullous diseases (AIBDs) are characterized by autoantibodies against structural proteins of the dermal–epidermal junction (in pemphigoid diseases) and the epidermal/ epithelial desmosomes (in pemphigus diseases). By far, the most common AIBD is bullous pemphigoid, which is immunopathologically characterized by autoantibodies against BP180 (type XVII collagen) and BP230. IgG and, to a lesser extent, IgA autoantibodies are the major autoantibody isotypes in these disorders. IgE autoantibodies are increasingly reported in particular in bullous pemphigoid. The development of specific and sensitive anti-BP180 IgE ELISA systems, the report of two experimental murine models employing IgE autoantibodies against BP180, and the successful treatment of bullous pemphigoid with the anti-IgE antibody omalizumab have raised interest in the role of IgE autoantibodies and the modulation of their production in AIBDs. Here, the relevance of IgE autoantibodies in the diagnosis, pathophysiology, and treatment decisions of AIBDs, with a focus on bullous pemphigoid, is reviewed.     

Profile of autoantibody to basement membrane zone proteins in patients with mucous membrane pemphigoid: long-term follow up and influence of therapy

Mucous membrane pemphigoid (MMP) is an autoimmune mucocutaneous blistering disease characterized by autoantibodies to components within the basement membrane zone. In this study, we report the titers of autoantibodies to antigens in the BMZ, in the sera of 13 patients, treated with intravenous immunoglobulin as monotherapy over a consecutive 18-month period. Using bovine gingiva lysate as substrate in an immunoblot assay, autoantibodies to human bullous pemphigoid antigens (BPAg1 and BPAg2), human beta4 integrin, and laminin 5 were measured. A statistically significant (P < 0.05) decline in the autoantibody titers to beta4-integrin was observed after 3.42 months of initiating the IVIg therapy. These titers were undetectable after 13 months of therapy. The titers of antibodies to BPAg1 and BPAg2 did not correlate with disease activity or response to therapy. Antibodies to laminins were not detected. In patients with MMP, autoantibody titers to beta4-integrin correlate with disease activity and response to therapy.     

IgG autoantibodies against desmocollin 3 in pemphigus sera induce loss of keratinocyte adhesion

Pemphigus is considered an autoimmune bullous skin disorder associated with IgG against the desmosomal components, desmoglein 3 (Dsg3) and desmoglein 1 (Dsg1). This concept is supported by the in vitro and in vivo pathogenicity of anti-Dsg3/Dsg1 IgG and the mucosal blistering phenotype of mice with a genetic deficiency of Dsg3. Mice deficient for another desmosomal adhesion molecule, desmocollin 3 (Dsc3), show a similar pemphigus phenotype, and we investigated the pathogenicity of Dsc3-reactive IgG autoantibodies that were identified previously in a subset of patients with atypical pemphigus. We here demonstrate that IgG against Dsc3 causes loss of adhesion of epidermal keratinocytes. Specifically, IgG against Dsc3 was purified from Dsc3-reactive pemphigus sera by affinity column chromatography using recombinant human Dsc3. Affinity purified IgG was functionally active and did not only react with recombinant Dsc3 but also with epidermis and cultured human keratinocytes. Moreover, Dsc3-reactive IgG induced loss of adhesion of epidermal keratinocytes in a dispase-based keratinocyte dissociation assay that was reversed on pre-adsorption with human Dsc3 but not Dsg3. These findings demonstrate that IgG autoantibodies against an additional component of the desmosomes, Dsc3, induce loss of keratinocyte adhesion and thus may contribute to blister formation in pemphigus.     

Pemphigus: A Complex T Cell-dependent Autoimmune Disorder Leading to Acantholysis

Pemphigus is a relatively rare autoimmune bullous disorder involving the skin and mucous epithelia. Clinically characterized by blisters and erosions, its histological hallmark is acantholysis induced by IgG antibodies (ab) against desmoglein 3 and/or desmoglein 1. The role of ab alone in inducing acantholysis is still a matter of debate as several mechanisms could be involved. Another intriguing area of research is the trigger factor inducing autoimmunity in pemphigus patients and the role of T and B cells in this process. This paper will review the data related to the mechanisms of acantholysis between keratinocytes and the role of T cell in this phenomenon.     

The role of T cells in cutaneous autoimmune disease

T cells assume a fundamental function in immunosurveillance and maintenance of the cutaneous immune barrier, yet derangement of their requisite role effects a range of cutaneous autoimmune diseases with significant associated morbidity. While blistering skin diseases, such as pemphigus vulgaris (PV), pemphigus foliaceus (PF) and bullous pemphigoid (BP) are mediated by antibodies directed against autoantigens found in the skin, recent evidence has shown that T cell activation is crucial for the initiation and coordination of this humoral response. Non-blistering skin diseases, such as alopecia areata (AA), vitiligo (VL) and psoriasis (PS) are increasingly believed to be directly mediated by the activities of autoreactive T cells. Here, we examine T lymphocyte control of antibody-mediated and cell-mediated processes involved in the pathoimmunology of the above mentioned skin diseases.     

The role of T cells in cutaneous autoimmune disease

T cells assume a fundamental function in immunosurveillance and maintenance of the cutaneous immune barrier, yet derangement of their requisite role effects a range of cutaneous autoimmune diseases with significant associated morbidity. While blistering skin diseases, such as pemphigus vulgaris (PV), pemphigus foliaceus (PF) and bullous pemphigoid (BP) are mediated by antibodies directed against autoantigens found in the skin, recent evidence has shown that T cell activation is crucial for the initiation and coordination of this humoral response. Non-blistering skin diseases, such as alopecia areata (AA), vitiligo (VL) and psoriasis (PS) are increasingly believed to be directly mediated by the activities of autoreactive T cells. Here, we examine T lymphocyte control of antibody-mediated and cell-mediated processes involved in the pathoimmunology of the above mentioned skin diseases.     

Autoimmunity in chronic bullous skin diseases. Immunofluorescent demonstration of three types of antibodies to skin in sera of patients with pemphigus, bullous pemphigoid and in other human sera

Antibodies to an intercellular substance of epidermis and other stratified squamous epithelia demonstrable by immunofluorescent staining have been reported to occur in the sera of patients with pemphigus vulgaris. Using sera of patients with bullous pemphigoid, antibodies to a component of the basement zone of skin had been found by the same technique. In the present study these observations have been confirmed and extended. In addition to the above mentioned immunofluorescent staining reactions, another type of antibody directed to a cytoplasmic or perinuclear component of epidermal cells was found in a variety of pathologic sera. This observation points to the need for differentiating other skin-reactive antibodies from those found only in pemphigus. Guinea-pig lip sections appeared to be satisfactory for these studies. Mixed immunofluorescence was found to yield staining reactions identical to those observed by the indirect staining method, and gave titres the same as or lower than those obtained by the indirect staining procedure. Using the indirect method with guinea-pig lip sections it was found that eight of ten sera of patients with pemphigus contained intercellular antibodies while 102 control sera failed to yield reactions of this type. The titres of antibodies appeared to be proportional to the severity of the disease process. Sera of two out of three patients suffering from bullous pemphigoid yielded immunofluorescence staining of the basement zone while 109 controls were negative for this antibody.     

Epidermolysis bullosa acquisita: Autoimmunity to anchoring fibril collagen

Epidermolysis bullosa acquisita (EBA) is a rare and acquired autoimmune subepidermal bullous disease of skin and mucosa. EBA includes various distinct clinical manifestations resembling genetic dystrophic epidermolysis bullosa (DEB), Bullous pemphigus, Brunsting–Perry pemphigoid, or cicatricial pemphigoid. These patients have autoantibodies against type VII collagen (C7), an integral component of anchoring fibrils (AFs), which are responsible for attaching the dermis to the epidermis. Destruction or perturbation of the normal functioning AFs clinically results in skin fragility, blisters, erosions, scars, milia, and nail loss, all features reminiscent of genetic dystrophic epidermolysis bullosa. These anti-C7 antibodies are “pathogenic” because when injected into a mouse, the mouse develops an EBA-like blistering disease. Currently, treatment is often unsatisfactory; however, some success has been achieved with colchicine, dapsone, photopheresis, plasmapheresis, infliximab, rituximab, and IVIG.     

C1q binding substances in pemphigus and bullous pemphigoid. Detection with a [131I] C1q binding assay.

A modification of the [125I]C1q binding assay was developed to allow the estimation of C1q binding activity (C1q BA) in pemphigus and bullous pemphigoid sera. The modifications include lower final concentration of PEG 6000 (1-5%) which permitted the use of sera that had been stored at -20 degrees C for extended periods of time; use of 131I instead of 125I and an [131I] C1q concentration of 5 microng/ml rather than 1 microng/ml. EDTA was used at a final concentration of 0-13 M to obviate the need for heat inactivation of sera. Sera from seventy-one patients with pemphigus and from 142 patients with bullous pemphigoid were tested for C1q BA. Of these 40% of the pemphigus and 20% of the bullous pemphigoid patients showed elevated C1q BA. A relationship between elevated C1q BA in serum and active disease was noted. Sequential samples from forty patients with pemphigus and thirty-seven patients with bullous pemphigoid demonstrated two different types of relationship between serum antibody titres to cutaneous antigens and C1a BA. In some patients serum antibody titres and C1q BA increased and decreased simultaneously; in others, increase of C1q BA followed increase of antibody titre and coincided with its decrease. The latter relationship supports the hypothesis that C1q BA may represent at least in part antigen-antibody complexes containing cutaneous antigens.     

Indirect cutaneous immunofluorescence : II Clinical significance

Sera of 532 patients with bullous diseases, connective tissue diseases and malignancies were tested for pemphigus epidermal intercellular fluorescence (ICF) and for the bullous pemphigoid ;tubular' band by the indirect fluorescent antibody technique. Human normal skin cryostat sections were used.The band and ICF were seen primarily only in bullous pemphigoid and pemphigus respectively, Some indirect band and ICF-negative patients demonstrated positive direct results in involved skin. suggesting that direct tests should be performed in indirect negative patients clinically thought to have pemphigus or bullous pemphigoid.No close correlation was found between disease activity and positive or negative indirect tests in bullous pemphigoid and pemphigus. Steroids did not interfere with positive results of this diagnostically extremely valuable test.     

Autoreactive cytotoxic T lymphocytes in pemphigus and pemphigoid

In order to know whether effector cells are capable of recognizing the epidermal targets in pemphigus vulgaris (PV) and bullous pemphigoid (BP), non-adherent cells (NAC) of peripheral blood from 27 primary PV, 19 BP patients and 12 healthy volunteers were used in cytotoxic tests. Autologous, allogenic and murine epidermal keratinocytes as well as autologous and allogenic fibroblasts were employed as targets. Total esterase activity of serine proteinases was measured in supernatants of samples and the results obtained were compared with the data received in 51Cr-release assay. These two techniques yielded similar results. The effector cells of PV and BP patients were found to lyse the epidermal target cells without presensitization in vitro. Epidermal cells from involved and uninvolved skin of PV patients, as well as from perilesional skin of BP patients, were highly sensitive to cytotoxic effect on the part of effector cells. The NAC activity in BP was higher than in PV. On the one hand, the obtained data suggest that PV and BP patients develop autoreactive cytotoxic T lymphocytes, sensitized to pemphigus and pemphigoid antigens, respectively, and on the other hand that epidermocytes in pemphigus and pemphigoid patients turned to be more fragile compared to normal human epidermal keratinocytes. It was supposed that cell-mediated cytotoxic reactions are probably related to enhanced proteolytic activity in the site of bullous eruption.     

A Korean case of anti-p200 pemphigoid

Anti-p200 pemphigoid is a newly defined autoimmune subepidermal blistering disease, which is characterized by the presence of IgG autoantibodies to the dermal side of 1 M NaCl split skin as well as by the reactivity of these antibodies to a novel 200-kDa antigen on immunoblot analysis of a dermal extract. We describe a 49-year-old Korean male who presented with a bullous eruption on the whole body, which clinically resembled bullous pemphigoid or epidermolysis bullosa acquisita. A histopathological examination of a lesional skin biopsy specimen showed an area of dermal-epidermal separation and mixed dermal inflammatory infiltrates consisting of lymphocytes, neutrophils, and eosinophils. Direct immunofluorescence showed a linear deposition of IgG and C3 along the basement membrane zone. Indirect immunofluorescence demonstrated circulating IgG autoantibodies directed against the dermal side of the 1 M NaCl split skin. Immunoblot analysis of dermal extracts revealed the patient's sera recognized the 200-kDa antigen. This is the first Korean case of an anti-p200 pemphigoid who showed good response to the treatment with systemic corticosteroids and dapsone.     

Characterization of BALB/c mice B lymphocyte autoimmune responses to skin basement membrane component type XVII collagen, the target antigen of autoimmune skin disease bullous pemphigoid

Bullous pemphigoid is an autoimmune blistering skin disease characterized by IgG autoantibodies targeting the skin basement membrane component type XVII collagen (BPAg2). To gain understanding of the disease's induction phase, we subcutaneously immunized adult BALB/c mice with peptides of human and/or the murine-equivalent BPAg2 pathogenic NC16A domain. Female mice were injected with peptides (human, murine, or combined human and murine), or PBS control emulsified in CFA, on a four-week interval. At the fourth and subsequent immunizations, all peptide-immunized mice were given murine peptides. Two weeks after the sixth immunization, ELISA detected IgG circulating autoantibodies against self peptides in 92% (47/51) of mice immunized with murine peptides; whereas none of the preimmune sera or the sera from PBS control-immunized mice reacted to the self peptides. In four mice their autoantibodies labeled mouse skin basement membrane. Breaking B-cell tolerance to BPAg2 sets the first step in dissecting the disease's induction phase.