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.     

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     

Advances in pemphigus and its endemic pemphigus foliaceus (Fogo Selvagem) phenotype: a paradigm of human autoimmunity

Pemphigus encompasses a group of organ specific, antibody mediated autoimmune diseases of the skin characterized by keratinocyte detachment that leads to the development of blisters and erosions, which can become life-threatening. The pathogenic autoantibodies recognize desmogleins, which are members of the desmosomal cadherin family of cell adhesion molecules. Desmoglein 3 is targeted in pemphigus vulgaris while desmoglein 1 is targeted in pemphigus foliaceus and its endemic form, Fogo Selvagem. This review will briefly define the salient features of pemphigus and the proposed steps in pathogenesis. We will then summarize the most recent advances in three important areas of investigation: (i) epidemiologic, genetic, and immunologic features of Fogo Selvagem, (ii) molecular mechanisms of injury to the epidermis, and (iii) novel therapeutic strategies targeting specific steps in disease pathogenesis. The advances in each of these three seemingly separate areas contribute to the overall understanding of the pemphigus disease model. These recent advancements also underscore the dynamic interplay between the treatment of patients in a clinical setting and basic science research and have led to an integrative understanding of disease pathogenesis and treatment, allowing pemphigus to serve as a paradigm of human autoimmunity.     

A Perspective of Pemphigus from Bedside and Laboratory-Bench

Pemphigus represents a distinct organ-specific acquired autoimmune disease characterized by intra-epidermal blistering, which is induced by autoantibodies against desmosomal cadherins, desmoglein 1 (Dsg1), and Dsg3. Pemphigus is currently divided into three distinct varieties, i.e., pemphigus vulgaris (PV), pemphigus foliaceus (PF) and other variants of pemphigus (mostly associated with inflammation), depending on clinical features, the level of separation in the epidermis, and immunologic characteristics of auto-antigens. Blistering pathomechanisms differ for each of the types of pemphigus. Pemphigus, which results from autoantibodies against desmogleins and possibly to other proteins, binds to the cell surface antigens. This binding may cause steric hindrance to homophilic adhesion of desmogleins, and may, in turn, lead to internalization of desmogleins and inhibition of desmogleins' integration into desmosomes, resulting in the formation of Dsg3-depleted desmosomes in PV or Dsg1-depleted desmosomes in PF. Furthermore, PV-IgG activates an "outside-in" signaling pathway to induce disassembly of desmosomal components from the inside of the cells by phosphorylation of proteins, including Dsg3. On the other hand, Pemphigus-IgG-augmented signaling pathways may be linked to the secretion of cytokines such as in case of pemphigus herpetiformis and chemokines that initiate or activate inflammation. In this article, the classification of pemphigus and the characteristic pathomechanisms for acantholysis will be reviewed, with particular emphasis on the molecular and biochemical cell biology of these diseases.     

Pemphigus vulgaris immunoglobulin G can recognize a 130 000 MW antigen other than desmoglein 3 on peripheral blood mononuclear cell surface

Pemphigus vulgaris (PV) is considered to be an autoimmune disease affecting skin and mucous membranes. Traditionally, PV autoantibodies are thought to recognize antigens located in the intercellular substance (ICS) of keratinocytes; antigens represented mainly by the desmosomal cadherin desmoglein 3 (Dsg3). Accordingly, titres of anti-ICS and anti-Dsg3 immunoglobulin G (IgG) are considered to be major laboratory criteria when making a diagnosis of PV. In this paper, we demonstrated for the first time that PV IgG bind antigen(s) expressed on the surface of peripheral blood mononuclear cells (PBMC), as revealed by immunofluorescence studies. This novel autoantigen is immunoprecipitated by PV IgG as a 130 000 molecular weight protein. However, Western blot analysis of the immunocomplexes failed to show reactivity with anti-Dsg3 monoclonal and polyclonal antibodies. Taken together, our data provide strong evidence that PV autoimmunity targets a 130 000 antigen other than Dsg3 on PBMC. This shifting from epidermis to blood cells may open new perspectives for a better understanding of pemphigus autoimmunity and more rational approaches to its treatment.     

Paraneoplastic pemphigus is associated with the DRB1*03 allele

Pemphigus is a group of autoimmune blistering diseases caused by autoantibodies directed against keratinocyte adhesion molecules. Pemphigus vulgaris (PV) and pemphigus foliaceus (PF), in which autoantibodies bind, respectively, to desmoglein 3 and desmoglein 1, are strongly associated with HLA-class II DR4 and DR14 alleles. In paraneoplastic pemphigus (PNP), a rare variant associated with neoplasia, autoantibodies target proteins of the plakin family in addition to desmogleins 1 and 3. The presence of anti-desmoglein antibodies in all types of pemphigus raises the question of common molecular mechanisms of susceptibility, particularly similar MHC-class II allele associations, in the different forms of the disease. HLA-DRB1 typing was performed in 13 PNP patients and results were compared to those obtained from 84 healthy controls, 37 PV and 31 PF patients. Our data demonstrate a significant association of PNP with HLA-DRB1*03 allele which was found in 61.5% of the patients, whereas DRB1*04 and DRB1*14 appear not to be involved in PNP susceptibility. Therefore, the HLA-genetic background of PNP differs from that of other types of pemphigus, which suggests that distinct mechanism(s) initiate(s) the immunological response in this form of pemphigus.     

A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease, which is characterized by autoantibodies to a specific desmosomal constituent, i.e. desmoglein 3 (Dsg3). In this study, we analyzed phosphorylation of desmosomal proteins and their molecular interactions after PV-IgG binding to Dsg3 using DJM-1 cells, a squamous cell carcinoma cell line, and normal human keratinocytes. Cells were metabolically labeled with 32P inorganic phosphate, followed by stimulation with the IgG fractions from five PV patients or normal individuals for 20 min. Phosphorylation of specific desmosomal components and their molecular interactions were studied in immunoprecipitates using PV-IgG and anti-plakoglobin (PG) antibodies. PV-IgG binding alone induced the phosphorylation of Dsg3 at serine residues. Although Dsg3 and PG were coprecipitated by PV-IgG-immunoprecipitation when treated with normal IgG, PG was not coprecipitated with Dsg3 when stimulated with PV-IgG, suggesting that PV-IgG binding to Dsg3 caused the dissociation of Dsg3 from PG. These results demonstrate that the binding of pathogenic PV autoantibodies to the cell surface antigen Dsg3, which is an adhesion molecule categorized into desmosomal cadherins, caused particular phosphorylation of Dsg3 and its dissociation from PG.     

Immunoadsorption in pemphigus

The principle of extracorporal immunoadsorption (IA) is based on affinity adsorption of pathogenic (auto-)antibodies and circulating immune complexes (CIC) which reversibly bind to an immobilized ligand of the adsorber. In pemphigus, a blistering autoimmune disease affecting skin and mucous membranes, autoantibodies, mainly of the IgG subclass are directed against desmosomal adhesion molecules and other non-desmosomal antigens on the surface of epidermal keratinocytes, such as acetylcholine receptors. The pathogenicity of these autoantibodies has been shown in various in vitro and in vivo systems. Recently, IA was applied in severe pemphigus demonstrating that a rapid and dramatic decline in desmoglein (Dsg)-reactive autoantibodies is accompanied by clinical remission of mucocutaneous blisters and erosions. As an adjuvant treatment, IA was combined with systemic immunosuppressive medication and current protocols initially apply treatment cycles of 3-4 IAs on consecutive days followed by immunoapheresis once a week or repeating the initial cycle in 4 week intervals depending on the disease activity. IA in pemphigus is generally safe and well tolerated.     

Immunoadsorption in pemphigus

The principle of extracorporal immunoadsorption (IA) is based on affinity adsorption of pathogenic (auto-)antibodies and circulating immune complexes (CIC) which reversibly bind to an immobilized ligand of the adsorber. In pemphigus, a blistering autoimmune disease affecting skin and mucous membranes, autoantibodies, mainly of the IgG subclass are directed against desmosomal adhesion molecules and other non-desmosomal antigens on the surface of epidermal keratinocytes, such as acetylcholine receptors. The pathogenicity of these autoantibodies has been shown in various in vitro and in vivo systems. Recently, IA was applied in severe pemphigus demonstrating that a rapid and dramatic decline in desmoglein (Dsg)-reactive autoantibodies is accompanied by clinical remission of mucocutaneous blisters and erosions. As an adjuvant treatment, IA was combined with systemic immunosuppressive medication and current protocols initially apply treatment cycles of 3–4 IAs on consecutive days followed by immunoapheresis once a week or repeating the initial cycle in 4 week intervals depending on the disease activity. IA in pemphigus is generally safe and well tolerated.     

A Spontaneous Deletion within the Desmoglein 3 Extracellular Domain of Mice Results in Hypomorphic Protein Expression,Immunodeficiency, and a Wasting Disease Phenotype

Desmoglein 3 is a transmembrane component of desmosome complexes that mediate epidermal cell-to-cell adhesion and tissue integrity. Antibody blockade of desmoglein 3 function in pemphigus vulgaris patients leads to skin blistering (acantholysis) and oral mucosa lesions. Desmoglein 3 deficiency in mice leads to a phenotype characterized by cyclic alopecia in addition to the dramatic skin and mucocutaneous acantholysis observed in pemphigus patients. In this study, mice that developed an overt squeaky (sqk) phenotype were identified with obstructed airways, cyclic hair loss, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. Single-nucleotide polymorphism–based quantitative trait loci mapping revealed a genetic deletion that resulted in expression of a hypomorphic desmoglein 3 protein with a truncation of an extracellular cadherin domain. Because hypomorphic expression of a truncated desmoglein 3 protein led to a spectrum of severe pathology not observed in mice deficient in desmoglein 3, similar human genetic alterations may also disrupt desmosome function and induce a disease course distinct from pathogenesis of pemphigus vulgaris.Tissues experiencing mechanical stress are held together by supramolecular desmosome complexes composed of type I transmembrane glycoproteins from the desmoglein (Dsg) and desmocollin (Dsc) families of epithelial cadherins.¹ The extracellular protein domains of the desmogleins and desmocollins consist of four approximately 110–amino acid homologous cadherin domains (EC1 to EC4) and a proximal extracellular anchor domain. The desmosomal cadherins are differentially expressed in different cellular layers of select tissues. For example, desmoglein 1 (Dsg1) is expressed at high levels in the suprabasal outer layer of skin epidermis (stratified squamous epithelia) and thymus.^2,3 Desmoglein 2 (Dsg2) is expressed ubiquitously in the basal layers of the epidermis and desmosome-enriched cardiac tissues.^2,3 Desmoglein 3 (Dsg3) is primarily expressed by epidermal keratinocytes in the basal and immediate suprabasal layers of skin and in the basal layer of the mucosal epithelium of the mouth, eyes, and trachea.^2,3 Six Dsg and three Dsc genes are found in mice, with four Dsg and three Dsc genes in humans.⁴ Homophilic and heterophilic interactions between the Dsg and Dsc proteins lead to the formation of tightly packed desmosomal complexes.⁵The desmogleins are involved in human disease pathogenesis. Cleavage of the extracellular domain of Dsg1 by Staphylococcus aureus exfoliating toxin results in bullous impetigo in children, manifesting as skin blisters due to detachment (acantholysis) of the outer layer of epidermis.⁶ Inactivation of either the Dsg2 or Dsc3 gene is embryonic lethal.^7,8 The development of circulating IgG autoantibodies against Dsg1 or Dsg3 can result in the human autoimmune blistering disorders pemphigus foliaceus and pemphigus vulgaris due to reduced desmoglein expression on the cell surface.⁹ In patients with pemphigus foliaceus, acantholysis within the superficial layers of the epidermis results in clinical lesions that resemble those observed in lupus erythematosus and seborrheic dermatitis patients. Pemphigus foliaceus patients experience no oral involvement and have no associated mortality. By contrast, patients with pemphigus vulgaris experience acantholysis within the deep basilar and parabasilar portions of the epidermis, which results in lesions that may resemble toxic epidermal necrolysis. With pemphigus vulgaris, there is significant oral and skin involvement and untreated patients experience considerable mortality.Although mutations in the human Dsg3 gene have not been described, gene inactivation in Dsg3^−/− mice leads to fragility of the skin and oral mucous membranes, analogous to those found in pemphigus vulgaris patients,¹⁰ along with runting and progressive hair loss.¹¹ Two independent spontaneous mutations within mouse chromosome 18 affecting exons encoding the Dsg3 cytoplasmic domain also ablate protein expression and lead to a Dsg3^−/− phenotype.^10,12,13 Herein, a spontaneous gene mutation was identified in mice that develop an overt squeaky (sqk) phenotype with cyclic hair loss, obstructed airways, and severe immunodeficiency subsequent to the development of oral lesions and malnutrition. This phenotype was mapped to a partial exon deletion in the Dsg3 gene that results in hypomorphic expression of a truncated Dsg3 protein, which leads to a severe spectrum of pathology not observed in Dsg3^−/− mice.     

A complementarity-determining region peptide of an anti-desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity-determining region of the F12 heavy chain (VH-CDR3) was shown to share a four-amino-acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen-combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH-CDR3 inhibited its binding to target antigens while VH-CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self-antigen binding mechanism for some autoantibodies.     

The extent of desmoglein 3 depletion in pemphigus vulgaris is dependent on Ca(2+)-induced differentiation: a role in suprabasal epidermal skin splitting?

Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes and is characterized by development of autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 and formation of intraepidermal suprabasal blisters. Depletion of Dsg3 is a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer periods under high-Ca(2+) conditions, we hypothesized that Dsg depletion depends on Ca(2+)-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in skin of patients with PV and in an organotypic raft model of human epidermis incubated using IgG fractions from patients with PV. In addition, Dsg3 depletion and loss of Dsg3 staining were prominent in cultured primary keratinocytes and in HaCaT cells incubated in high-Ca(2+) medium for 3 days, but were less pronounced in HaCaT cultures after 8 days. These effects were dependent on protein kinase C signaling because inhibition of protein kinase C blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, protein kinase C inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Considered together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca(2+)-induced differentiation. Furthermore, prominent depletion in basal epidermal layers may contribute to the suprabasal cleavage plane observed in PV.     

Antibodies to desmogleins 1 and 3, but not to BP180, induce blisters in human skin grafted onto SCID mice

Pemphigus and bullous pemphigoid (BP) are blistering skin diseases associated with IgG autoantibodies to desmosomal and hemidesmosomal components. When autoantibodies to desmogleins 1 and 3 from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) or rabbit antibodies against the murine hemidesmosomal component BP180 are passively transferred into neonatal mice, they induce blisters in the skin of the mice. To develop an animal model that would duplicate the findings in the skin of the patients more closely, full-thickness human skin from healthy volunteers was grafted onto SCID mice. Injection of the purified IgG fraction from the serum of PF and PV patients led to subcorneal and suprabasal splits in the human grafts and human IgG was deposited intercellularly in the upper and lower layers of the epidermis, respectively. Interestingly, anti-BP180 autoantibodies purified from the serum of BP patients and from a rabbit immunized with recombinant human BP180 strongly bound to the basement membrane zone of the grafts (n=32), fixed murine complement, led to the recruitment of neutrophils to the upper dermis of the graft, but did not induce subepidermal blisters. We report a novel experimental model for PF and PV which should greatly facilitate further studies to dissect the immunopathological mechanisms in these diseases. Specifically, this model can be used to identify pathogenically relevant epitopes on human desmogleins 1 and 3 and to develop novel strategies for the treatment of pemphigus.     

Non-junctional human desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a pathway possibly involved in the pathogenesis of pemphigus vulgaris

E-cadherin, a classical cadherin, is an adhesion receptor in adherens junctions and has important functions in cell-cell adhesion and cell signalling. Recently we reported that a desmosomal cadherin, desmoglein 3 (Dsg3), an autoantigen in pemphigus vulgaris (PV), associates with E-cadherin and activates Src, which results in tyrosine phosphorylation of adherens junction proteins. However, the nature of such an interaction and its role in cell-cell adhesion remain unclear. In this report, we provide direct evidence that it is the detergent-soluble, non-desmosomal Dsg3 that regulates the activity of Src and its association with E-cadherin in adherens junction formation. Modulation of Dsg3 levels, either by Dsg3 silencing or over-expression, alters Src activity and its association with E-cadherin. Dsg3 silencing caused retardation of calcium-induced E-cadherin junction assembly and a reduction of desmosomal protein expression. Furthermore, we provide evidence that this signalling pathway is involved, at least in part, in the pathophysiology of pemphigus. Along with the reduced expression of Dsg3, loss and disruption of E-cadherin and a concomitant decreased pSrc signalling was identified in the basal keratinocytes surrounding the blisters in PV. These findings suggest a novel function for Dsg3 in the control of E-cadherin-Src signalling and cell-cell adhesion.     

Pemphigus antibody induced phosphorylation of keratinocyte proteins

The pemphigus family of autoimmune blistering diseases is characterized by an autoantibody response to desmosomal cadherins in epithelia. Autoantibodies against desmogleins, desmosome cell adhesion molecules, induce loss of cell-cell adhesion that is characterized clinically by blister formation. The mechanism by which these autoantibodies induce loss of cell-cell adhesion is under active investigation, but appears to involve a coordinated intracellular response including activation of intracellular signaling and phosphorylation of a number of proteins in the target keratinocyte. Activation of p38 mitogen activated protein kinase may have a critical role in the acantholytic mechanism as inhibitors of p38MAPK block the ability of pemphigus IgG to induce blistering in pemphigus animal models.     

Pemphigus antibody induced phosphorylation of keratinocyte proteins

The pemphigus family of autoimmune blistering diseases is characterized by an autoantibody response to desmosomal cadherins in epithelia. Autoantibodies against desmogleins, desmosome cell adhesion molecules, induce loss of cell–cell adhesion that is characterized clinically by blister formation. The mechanism by which these autoantibodies induce loss of cell–cell adhesion is under active investigation, but appears to involve a coordinated intracellular response including activation of intracellular signaling and phosphorylation of a number of proteins in the target keratinocyte. Activation of p38 mitogen activated protein kinase may have a critical role in the acantholytic mechanism as inhibitors of p38MAPK block the ability of pemphigus IgG to induce blistering in pemphigus animal models.     

Pemphigus IgG causes skin splitting in the presence of both desmoglein 1 and desmoglein 3

According to the desmoglein (Dsg) compensation concept, different epidermal cleavage planes observed in pemphigus vulgaris and pemphigus foliaceus have been proposed to be caused by different autoantibody profiles against the desmosomal proteins Dsg 1 and Dsg 3. According to this model, Dsg 1 autoantibodies would only lead to epidermal splitting in those epidermal layers in which no Dsg 3 is present to compensate for the functional loss of Dsg 1. We provide evidence that both pemphigus foliaceus-IgG containing Dsg 1- but not Dsg 3-specific antibodies and pemphigus vulgaris-IgG with antibodies to Dsg 1 and Dsg 3 were equally effective in causing epidermal splitting in human skin and keratinocyte dissociation in vitro. These effects were present where keratinocytes expressed both Dsg 1 and Dsg 3, demonstrating that Dsg 3 does not compensate for Dsg 1 inactivation. Rather, the cleavage plane in intact human skin caused by pemphigus autoantibodies was similar to the plane of keratinocyte dissociation in response to toxin B-mediated inactivation of Rho GTPases. Because we recently demonstrated that pemphigus-IgG causes epidermal splitting by inhibition of Rho A, we propose that Rho GTPase inactivation contributes to the mechanisms accounting for the cleavage plane in pemphigus skin splitting.     

Pathogenic relevance of IgG and IgM antibodies against desmoglein 3 in blister formation in pemphigus vulgaris

Pemphigus vulgaris is an autoimmune disease caused by IgG antibodies against desmoglein 3 (Dsg3). Previously, we isolated a pathogenic mAb against Dsg3, AK23 IgG, which induces a pemphigus vulgaris-like phenotype characterized by blister formation. In the present study, we generated a transgenic mouse expressing AK23 IgM to examine B-cell tolerance and the pathogenic role of IgM. Autoreactive transgenic B cells were found in the spleen and lymph nodes, whereas anti-Dsg3 AK23 IgM was detected in the cardiovascular circulation. The transgenic mice did not develop an obvious pemphigus vulgaris phenotype, however, even though an excess of AK23 IgM was passively transferred to neonatal mice. Similarly, when hybridoma cells producing AK23 IgM were inoculated into adult mice, no blistering was observed. Immunoelectron microscopy revealed IgM binding at the edges of desmosomes or interdesmosomal cell membranes, but not in the desmosome core, where AK23 IgG binding has been frequently detected. Furthermore, in an in vitro dissociation assay using cultured keratinocytes, AK23 IgG and AK23 IgM F(ab')(2) fragments, but not AK23 IgM, induced fragmentation of epidermal sheets. Together, these observations indicate that antibodies must gain access to Dsg3 integrated within desmosomes to induce the loss of keratinocyte cell-cell adhesion. These findings provide an important framework for improved understanding of B-cell tolerance and the pathophysiology of blister formation in pemphigus.     

Demonstration of an adhering-junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceus and pemphigus vulgaris

Pemphigus foliaceus and pemphigus vulgaris are skin diseases in which antibodies against the cell surface of keratinocytes destroy the adhesion between epidermal cells, producing blisters. Patients with pemphigus foliaceus have antibodies to a complex of three polypeptides of 260, 160, and 85 kd (the foliaceus complex), whereas patients with pemphigus vulgaris have antibodies to a complex of 210-kd, 130-kd, and 85-kd polypeptides (the vulgaris complex). The 160-kd polypeptide of the foliaceus complex has been identified as desmoglein, a desmosomal glycoprotein. We suspected that the 85-kd component in both these antigenic complexes might be plakoglobin, another molecule in the adhering junctions of cells. To characterize these antigenic complexes, we used the serum of five patients with pemphigus foliaceus, that of four patients with pemphigus vulgaris, and monoclonal antiplakoglobin antibodies. We found that monoclonal antibodies to plakoglobin immunoprecipitated the 85-kd polypeptide from the dissociated foliaceus and vulgaris complexes and precipitated both complexes from epidermal extracts. Serum from patients with pemphigus foliaceus or pemphigus vulgaris (but not from four normal controls) bound desmoglein and the 130-kd polypeptide, respectively, showing that these peptides (and not plakoglobin) are the antigenic binding sites in these disorders. We conclude that plakoglobin, a protein of the adhering junctions of epidermal cells, is the 85-kd molecule in the antigenic complexes found in both pemphigus foliaceus and pemphigus vulgaris, although it is not the binding site in either disorder.