The location of binding sites of pemphigus vulgaris and pemphigus foliaceus autoantibodies: a post-embedding immunoelectron microscopic study

Pemphigus is a life-threatening autommune blistering discase of skin and mucous membranes that has two major subtypes based on clinical and histolgical features, pemphigus vulgaris (PV) and pemphigus foliaceus (PF). Autoantibodies against the PV antigen (desmoglein 3) and the PF antigen (desmoglein 1) are involved in the pathogenesis of blister formation. In the present study, the location of epitopes recognized by autoantibodies of patients with PV and PF was studied by postembedding immunogold electron microscopy. PV and PF autoantibodies were observed bound predominantly to the intercellular domains of desmosomes, but not to the non-desmosomal keratinocyte cell surface. The relationship between the location of PF antigen and other constitutive desmosomal proteins. desmocollin, desmoplakin and plakoglobin, in normal human skin was investigated using a double immunogold labelling techinique. It was observed that PF antigen and desmocollin co-localize within the intercellular domain of the desmosomes. In contrast, the antibodies against desmoplakin and plakoglobin bound predominantly to the intracellular desmosomal attachment plaque with the binding site of the antibody against plakoglobin closer to the desmosomal cell membrane than that of the antibody to desmoplakin. We show that the LR White postembedded immunogold electronmicroscopy technique is convenient and easily applied to studied to studies of autoimmune bullous skin diseases. We have used it to demonstrated the precise localization of the binding sites of PV and PF autoantibodies and their reltionship with other constitutive desmosomal proteins.     

Pemphigus group (vulgaris, vegetans, foliaceus, herpetiformis, brasiliensis)

Pemphigus is a rare autoimmune bullous disorder involving the skin and mucosa. The disease has a chronic course. It is characterized histologically by an intraepidermal cleavage and the production of pathogenic antibodies directed against different proteins of the desmosomes, which belong to the cadherin family. The diagnosis of the type of pemphigus is made on clinical features, the level of histologic cleavage, and the identification of the antigens recognized by circulating autoantibodies using immunoblot or ELISA analysis of serum. The epidemiology and clinical, histologic, and immunologic findings of pemphigus vulgaris, pemphigus foliaceus, pemphigus vegetans, and pemphigus herpetiformis are described.     

Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting

BACKGROUND: Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs. METHODS: We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid. RESULTS: The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens. CONCLUSIONS: Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.     

Immunoblot and immunoelectronmicroscopic analysis of endemic Tunisian pemphigus

Tunisian pemphigus is a newly described form of endemic pemphigus whose clinical, histological and epidemiological characteristics have recently been detailed. The objective of this study was to analyse the binding properties of autoantibodies present in sera from patients with endemic Tunisian pemphigus using immunoblotting and indirect immunoelectron microscopy (IEM). Thirty patients with pemphigus foliaceus (PF) and six with pemphigus vulgaris (PV) seen in the dermatology department of Tunis Hospital between 1992 and 1994 were selected for this study. Seven of 30 (23%) and six of 12 (50%) PF sera tested bound to the 160 kDa band of desmoglein 1 when tested on bovine tongue and human epidermal extracts, respectively. Two of six and two of three PV sera tested bound to the 130 kDa desmoglein 3 in these two extracts. Immunoblot and indirect IEM showed that 24 of 30 (80%) PF sera contained IgG1, IgG3 or IgG4 antibodies that bound to a 185-kDa polypeptide localized on the desmosomal plaque. This immunological analysis showed that most endemic Tunisian pemphigus sera correspond to PF sera and are characterized by a high frequency of autoantibodies directed against a recently identified 185-kDa antigen of the desmosomal plaque.     

Demonstration of antibodies to bovine desmocollin isoforms in certain pemphigus sera

Summary We have shown previously that IgG antibodies in certain pemphigus sera. particularly endemic Brazilian pemphigus foliaceus (BPF) sera. react with bovine desmocollins (Dsc). which are transmembranous glycoproteins of desmosome junctions. Desmocollins occur as three different isoforms (Dsc 1, 2 and 3). all of which are represented in the epidermis. In this study. we examined sera of various pemphigus types by immunoblotting purified bovine desmosomes and bovine Dsc l, 2 and 3 fusion proteins. expressed in pGEX expression vectors. Six of l5 (40.0%) BPF sera. two of 18 (11.1%) non-endemic pemphigus foliaceus sera. eight of 39 (20.5%) pemphigus vulgaris (PV) sera. and two of l l (18.2%) normal sera. showed reactivity with Dsc from desmosomes. Experiments with fusioni proteins showed that no Dsc isoform was specifically recognized by sera of any individual pemphigus type. Our results indicate that the pathogenesis of pemphigus might be more complex than previously believed.     

Autoantigens for IgA anti-intercellular antibodies of intercellular IgA vesiculopustular dermatosis

A new disease characterized by the presence of in vivo bound and/or circulating IgA anti-intercellular (IC) antibodies has recently been identified. We propose the term intercellular IgA vesiculopustular dermatosis (IAVPD) for this entity, which seems to be divided clinicopathologically into at least two distinct subtypes: intraepidermal neutrophilic IgA dermatosis (IEN type) and subcorneal pustular dermatosis-like cases (SPD type). Using immunoblot technique, we examined the antigen substances for the IgA anti-IC antibodies in the sera from one Japanese patient with IEN type of IAVPD and three Japanese patients with SPD type. A serum from a patient with IEN type reacted exclusively with a 120-kD protein in both the normal human skin extract and the bovine desmosome sample. Sera from three patients with SPD type reacted specifically with a doublet of 115-kD and 105-kD proteins, which appeared to be identical to desmocollins I and II, well known desmosomal core proteins, in the bovine desmosome sample. IgA antibody from our patients with IAVPD bound to neither pemphigus vulgaris antigen nor pemphigus foliaceus antigen. From these results, we suggest that IAVPD is different from pemphigus and is heterogeneous in terms of the antigens to which IgA autoantibodies bind.     

An immunohistological study of desmosomal components in pemphigus

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune diseases in which there is loss of cohesion between keratinocytes (acantholysis) and blistering within the epidermis. PV is characterized by acantholysis predominantly between the epidermal basal cells and suprabasal layers, whereas in PF intraepidermal cleavage is higher in the epidermis. Adhesion between keratinocytes is dependent on the function of transmembrane glycoproteins of the cadherin family present in specialized adhesion junctions, the desmosomes. The pathogenesis of acantholysis In pemphigus is uncertain, but the pemphigus autoantibodies bind to epithelial cadherins. We have used monoclonal antibodies to desmosomal components to investigate their distribution in different forms of pemphigus. Our results show that the localization of desmosomal components is abnormal in intact perilesional epidermis, intact epidermis above the blisters in PV and intact epidermis below the blisters in PF. We suggest that autoantibody binding may have a direct effect on the function of specific epithelial cadherins, but will only cause cell separation where the antigen is the principal adhesion molecule.     

Ultrastructural binding site of pemphigus foliaceus autoantibodies: comparison with pemphigus vulgaris

We studied in vivo binding sites of pemphigus foliaceus (PF) auto-antibodies by immuno-gold labelling technique, and compared them with those of pemphigus vulgaris (PV). In early acantholytic lesions of PF, the bound antibodies indicated by 5 nm protein A-colloiclal gold particles were observed on the surface of keratinocytes, with particular affinity for desmosomes and separated attachment plaques. Nondesmosomal cell surfaces were sparsely labeled with the gold particles. A similar binding pattern was seen in the epidermal sheets obtained from a PV patient utilizing the Nikolsky phenomenon. These findings indicate that both PF and PV antigen-antibody complexes are densely located on the desmosomal areas in early pemphigus lesions, suggesting the pathogenic importance of functional impairment of desmosomes by the autoantibodies.     

Ultrastructural localization of cell junctional components (desmoglein, plakoglobin, E-cadherin, and β-catenin) in Hailey-Hailey disease, Darier's disease, and pemphigus vulgaris

The distribution of desmoglein, plakoglobin, E-cadherin, and β-catenin in the peri-lesional and lesional skin of Hailey-Hailey disease, Darier's disease, and pemphigus vulgaris was examined by immunoelectron microscopy. In the peri-lesional skin, the immunolabeling of these desmosomal components was localized to desmosomes. Adherens junction-associated E-cadherin and β-catenin were at the cell periphery, excluding desmosomes. The labeling pattern was similar among these diseases, but the labeling intensity particularly that of plakoglobin in Hailey-Hailey disease and Darier's disease, was less than that of normal controls, suggesting that these glycoproteins are quantitatively less concentrated in the normal epidermis of these inherited diseases. In the acantholytic cells of Hailey-Hailey disease and Darier's disease the immunolabeling of the components of desmosomes was diffusely distributed in the cytoplasms, whereas that of adherens junction was mostly at the cell periphery and partly diffusely in the cytoplasm. In contrast, desmosomes of detaching keratinocytes in pemphigus vulgaris still showed the labeling of desmoglein and plakoglobin. These findings suggest that the inherited acantholytic diseases, i.e., Hailey-Hailey disease and Darier's disease have a different pathogenesis from that of autoimmune acantholysis in pemphigus vulgaris: The intracellular components of desmosomes may primarily be disrupted in the genetic acantholytic diseases in the initial stages of acantholysis. Several unsolved questions in the previous light microscopic immunofluorescence sttidies using the same antibodies are now answered: 1) the diffusion of desmosomal proteins is not due to the internalization of desmosomes, 2) intracellular components of adherens junction are also finally dissolved, 3) diffuse cytoplasmic immunofluorescence patterns of desmosomal components could be explained by immunoelectron microscopy as those attached to cell membrane and trapped in tonofilament aggregates.     

Childhood Pemphigus Foliaceus with Exclusive Immunoglobulin G Autoantibodies to Desmocollins

Pemphigus refers to a group of potentially fatal blistering skin diseases that are often due to the deleterious effects of autoantibodies directed against desmosomal antigens. Although desmogleins have been mainly implicated as autoantigens in pemphigus, a steadily growing body of evidence suggests that other desmosomal proteins may be causally involved as well. Antibodies directed against desmocollin‐3 have been shown to play a direct role in the pathogenesis of several types of pemphigus. Here we describe the case of a child with localized pemphigus foliaceus and immunoglobulin G (IgG) reactivity exclusively directed to desmocollins. The present report suggests that autoantibodies against nondesmoglein antigens may play a role in the pathogenesis of superficial pemphigus, in addition to pemphigus vulgaris, paraneoplastic pemphigus, and IgA pemphigus.     

Desmosomes and their autoimmune pathologies

Desmosomes guarantee the integrity of the epidermis, by functioning both as an adhesive complex and as a cell-surface attachment site for the keratin intermediate filaments of the cytoskeleton. Considerable progress has been made in our knowledge of desmosomes and their components. The structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have been elucidated. Desmosomal proteins are major antigens in pemphigus. Each type of pemphigus has its own antigenic targets, but in the last few years it has been shown that certain autoantibody populations are not restricted to just one form of pemphigus. The production of autoantibodies against multiple intracellular and extracellular desmosomal proteins, whose pathogenic role remains to be elucidated, suggests an overlapping distribution of antibody specificities among different forms of pemphigus.     

Autoantibody formation against a 190-kDa antigen of the desmosomal plaque in pemphigus foliaceus

Summary We report changes in the antigen recognition pattern of sera from two pemphigus foliaceus patients with a long-term follow-up. The patients' sera were analysed by immunoblotting using different antigenic sources: cultured human keratinocytes, bovine tongue epithelium and a recombinant protein corresponding to the C-terminal end of the 230-kDa bullous pemphigoid antigen. While initial serum samples reacted exclusively with the 160-kDa desmoglein 1, the later sera reacted both with desmoglein 1 and a 190-kDa antigen immunolocalized to the desmosomal plaque, previously demonstrated to be recognized by sera of some patients with paraneoplastic pemphigus. lgG subclass analysis further showed that antidesmoglein 1 antibodies were of lgGl and/or IgG4 subclasses, while anti-190-kDa antibodies were lgG3. The patients were free of malignancy.     

Desmosomal dissolution in Grover's disease, Hailey-Hailey's disease and Darier's disease

Proteins involved in the formation of desmosomes and simpler adherens junctions were studied in three types of non-immune acantholytic diseases; specifically, four cases of Grover's disease (GD), one case of Hailey-Hailey's disease (HMD) and one case of Darier's disease (DD), and these were compared to two cases of immune-mediated acantholytic disease pemphigus vulgaris (PV). The proteins studied included: 1. The intracellular desmosomal proteins, desmoplakin I and II and plakoglobin; 2. The intercellular desmosomal proteins, desmoglein and CD44; and 3. vinculin, which is a major intracellular protein of the simpler aherens junctions. In GD, HHD and DD, immunostaining showed a loss of desmoplakin I and II and plakoglobin from the desmosomes, and a diffuse staining in the cytoplasm. In contrast, in pemphigus vulgaris, these proteins seemed intact and were localized to dot-like spots on the cell surface. Also, desmoglein, and CD44 were slightly affected in GD, and moderately affected in HHD and DD. Absence of desmosomal attachment plaques, the lack of labeling with desmoglein in the affected desmosomes and a diffusion of the labels into cytoplasm were demonstrated with electron microscopy using an immunogold technique. In PV, desmoglein III is one of the target antigens for the autoantibodies in this disease and was only partially preserved in a small number of lesional cells, while CD44 was mostly preserved. Vinculin was intact in GD, HHD and DD, but was lost in PV. This study, our previous work, and that of others, suggest that: 1. In GD, HHD and DD, the proteins of the desmosomal attachment plaque are primarily affected; 2. In PV, the intercellular glycoproteins are primarily involved; and 3. Simple adherens junctions are intact in GD, HHD and DD, but are damaged in PV.     

Specific detection of anti-cell surface antibodies in herpes gestationis sera

Abstract We examined 42 herpes gestationis sera with immunofluorescence of normal human skin sections, and found that anti-keratinocyte cell surface antibodies were detected specifically in 10 herpes gestationis sera. The diagnosis of these herpes gestationis cases was confirmed by detecting antibodies against the 180 kD bullous pemphigoid antigen with immunoblotting of its fusion protein. The results of immunoadsorption assay using baculoproteins of both pemphigus vulgaris and pemphigus foliaceus antigens indicated that the herpes gestalionis sera did not recognize common pemphigus antigens. Immunoblotting of human epidermal extracts and immunofluorescence of various tissues also suggested that the sera did not recognize any other desmosomal components or paraneoplastic pemphigus antigens. The significance of this reactivity is unclear. However, because no control bullous pemphigoid sera showed this reactivity, it may suggest a different pathophysiology between herpes gestationis and bullous pemphigoid.     

Beyond steric hindrance: the role of adhesion signaling pathways in the pathogenesis of pemphigus

Epidermal cell adhesion depends on the intercellular interactions of transmembrane cadherin glycoproteins, which form the basis of adherens junctions and desmosomes. Pemphigus is a blistering disease of the skin and mucous membranes characterized by autoantibodies against the cell surface desmosomal cadherins, desmoglein (Dsg) 3 and Dsg1. An unanswered question in pemphigus pathophysiology is the mechanism of acantholysis, or loss of keratinocyte cell adhesion. One longstanding theory for pemphigus pathogenesis is the concept of steric hindrance, in which pathogenic pemphigus autoantibodies cause loss of intercellular adhesion by directly interfering with desmosomal cadherin trans-interactions. However, several recent studies have demonstrated that modulation of p38MAPK, Rho family GTPase, c-myc, protein kinase C, and phospholipase C signaling pathways prevents keratinocyte dissociation induced by pemphigus autoantibodies. As it is unlikely that desmosomal signaling would occur only in response to pemphigus autoantibodies, these studies suggest that numerous different signaling molecules may play a role in desmosomal homeostasis. Many of these same signaling pathways regulate classical cadherins in adherens junctions. Given the recent discovery of bidirectional crosstalk between adherens junctions and desmosomes, it would be valuable to understand how signaling pathways implicated in pemphigus pathogenesis may be involved in more general mechanisms of desmosome and adherens junction regulation. In this review, we will summarize the evidence supporting a role for steric hindrance and signaling mechanisms in the pathogenesis of pemphigus acantholysis and discuss potential analogues in the classical cadherin literature.     

Pemphigus foliaceus antibodies and a monoclonal antibody to desmoglein I demonstrate stratified squamous epithelial-specific epitopes of desmosomes

Pemphigus foliaceus (PF) is a human autoimmune disease in which autoantibodies are directed against the cell surface of epidermal cells. Using an immunoblotting technique, we recently demonstrated that a subgroup of PF patients have autoantibodies to the desmosomal core glycoprotein, desmoglein I (DGI). There are desmosomes in all epithelia and in heart, yet PF affects only stratified squamous epithelia. One explanation for this finding might be that there are tissue-specific differences in desmosomes. Thus, to determine whether certain epitopes of DGI are tissue-restricted, we performed immunofluorescence studies on various monkey tissues with the following antibodies: a rabbit polyclonal antiserum against whole desmosomes, which demonstrated the desmosomes in all tissues tested; a mouse monoclonal antibody against DGI, MmDGI-1; and PF sera that bound DGI on immunoblotting (PF IB+). In addition, we tested the tissues with PF sera that did not bind DGI by immunoblotting (PF IB-) to determine if these sera were different from PF IB+ sera in their tissue specificity. PF IB+, PF IB-, and MmDGI-1 antibodies stained all stratified squamous epithelia tested, including skin, tongue, upper esophagus, conjunctiva, and cornea; however, they did not stain heart or any nonstratified squamous epithelia, including gall bladder, small intestine, liver, ureter, and bladder. These results indicate that there is tissue heterogeneity of desmosomes, and that epitopes on DGI defined by both PF antibodies and a monoclonal antibody are present only in stratified squamous epithelia. In addition, PF IB- sera had the same tissue specificity as PF IB+ sera. These results may partially explain why PF involves only stratified squamous epithelia.     

Immunomorphologic and biochemical identification of the pemphigus foliaceous autoantigen within desmosomes.

Desmosomes are specialized domains of the plasma membrane that play a fundamental role in intercellular adhesion. This adhesive function is mediated at least in part by the cadherin homologous cell adhesion molecule (CAM) desmoglein (dg). Autoantibodies (aab) from patients with pemphigus foliaceous (pf), a blistering disease of the epidermis, have been shown by immunochemical methods to bind to desmoglein. However, the molecular localization of the binding sites of these antibodies, especially as it relates to the ultrastructure of the desmosomes, has not been definitively characterized. We therefore performed pre-embedding direct immunoelectron microscopy (IEM) on perilesional skin of patients with pf and post-embedding indirect IEM using sera from five patients with pf. We first confirmed by immunoprecipitation and immunoblotting that these sera bound dg. Both IEM methods showed that pf-aab exclusively bind to desmosomes. Double-labeling IEM of several other constitutive desmosomal proteins further suggests that most likely pf-aab bind to an extracellular domain of the transmembrane CAM dg. Our studies suggest one possible pathophysiologic mechanism for the clinical manifestations of pf: namely, that the binding of aab to an extracellular epitope of desmoglein might impair the adhesive properties of desmosomes mediated by dg and result in the loss of cell adhesion leading to acantholysis and blister formation.     

Detection of pemphigus vulgaris and pemphigus foliaceus antigens by immunoblot analysis using different antigen sources

In an immunoblot analysis with human epidermal extract as a source of antigens, all (28/28) pemphigus vulgaris (Pv) sera showed a specific reactivity with a 130-kD protein. Several, but not all, Pv sera reacted with similar antigens in both a bovine muzzle desmosome preparation and extract of cultured human squamous carcinoma cells. On the other hand, some pemphigus foliaceus (Pf) sera exhibited reactivity with a 150-kD protein, which is most likely desmoglein I, in both the human epidermal extract and the bovine desmosome preparation, but no Pf serum reacted with this antigen in the squamous carcinoma cell extract. Furthermore, 4/16 Pv sera also reacted with a 150-kD protein in the desmosome preparation, which seemed to be the same as Pf antigen. These results show a relationship between antigens of both Pf and Pv and desmosomes, as well as heterogeneities of both Pv and Pf antigens in terms of antigenic molecules or epitopes. Furthermore, this study presents the possibility that immunoblot analysis can be routinely used for differentiation of Pv and Pf antibodies.     

天疱疮抗体结合靶抗原的定位研究

目的 研究天疱疮抗体铺皮细胞间抗原在超微结构水平的部位。方法 采用ＬＲＷｈｉｔｅ树脂为包埋剂,用金标记包一直接和间接免疫电镜技术,观察天疱疮患者皮损中ＩｇＧ的沉积部位和患者血清中ＩｇＧ型自身抗体结构正常人皮肤的部位。结果 寻常型天疱疮和落叶型天疱疮的直接和间接免疫电镜均在表皮细胞间的桥粒部位觅金颗粒沉积,在非桥数部位的角质形成细胞间未金颗粒沉积。结论寻常型天疱疮和落叶型天疱疮的靶抗原均是桥粒成分,     

A nonfatal case and 2 fatal cases of paraneoplastic pemphigus: can a complement indirect immunofluorescent test help to identify fatal "group A" paraneoplastic pemphigus cases?

We studied 3 recent cases of paraneoplastic pemphigus (PNP) in detail. Two patients died despite concerted management efforts. One patient received no treatment after the appearance of PNP and recovered completely from both PNP and lymphoma. Multiple serum studies of these 3 patients plus 9 other proven PNP cases revealed that 8 of 9 fatal PNP cases (referred to here as "group A") had distinctive cell surface antibodies detected by complement indirect immunofluorescent (CIIF) tests on monkey esophagus sections. By contrast, none of the sera from 3 patients with PNP who experienced long-term survival (referred to here as "group B") and none of 20 pemphigus vulgaris or 10 pemphigus foliaceus control sera revealed similar beaded cell surface CIIF reaction patterns, a difference that is statistically significant (P <.0001). Cell surface CIIF reaction patterns of group A PNP antibodies resemble the pattern of pemphigus antibody reactions in indirect immunofluorescent tests on the same substrate; however, the latter tend to be thinner and more linear, whereas the cell surface CIIF pattern tends to be more beaded, suggesting possible desmosomal reactions. We believe this test is useful in identifying an aggressive group A form of PNP.