Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line

Pemphigus vulgaris is an autoimmune blistering disease characterized by cell-cell detachment of epidermal cells. Autoantibody against desmoglein (Dsg) 3, a transmembrane glycoprotein that mediates the association of desmosomes, plays a major role in blistering in pemphigus vulgaris (PV). The mechanisms of autoantibody-induced acantholysis have not been clarified. We previously reported that PV-IgG induces phosphorylation of Dsg3, decreases Dsg3 on the cell surface and forms Dsg3-depleted desmosomes in cultured keratinocytes, and that cell treatment with a potent pathogenic monoclonal antibody against Dsg3 (AK23 mAb) decreases the amount of Dsg3 in cultured keratinocytes. Although the precise mechanisms remain unclear, we have proposed the involvement of intracellular signal transduction resulting from the binding of autoantibodies to Dsg3. In this study, we examined whether AK23 mAb augments phosphorylation of Dsg3 and p38 mitogen-activating protein kinase (MAPK) in a human squamous cell line, DJM-1 cells. AK23 mAb increased serine phosphorylation of Dsg3 and augmented activation levels of p38 MAPK. These results indicate that antibodies bind to Dsg3, but not other antigens, in the IgG fraction and can induce activation of signal transduction.     

Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line

Pemphigus vulgaris is an autoimmune blistering disease characterized by cell–cell detachment of epidermal cells. Autoantibody against desmoglein (Dsg) 3, a transmembrane glycoprotein that mediates the association of desmosomes, plays a major role in blistering in pemphigus vulgaris (PV). The mechanisms of autoantibody-induced acantholysis have not been clarified. We previously reported that PV-IgG induces phosphorylation of Dsg3, decreases Dsg3 on the cell surface and forms Dsg3-depleted desmosomes in cultured keratinocytes, and that cell treatment with a potent pathogenic monoclonal antibody against Dsg3 (AK23 mAb) decreases the amount of Dsg3 in cultured keratinocytes. Although the precise mechanisms remain unclear, we have proposed the involvement of intracellular signal transduction resulting from the binding of autoantibodies to Dsg3. In this study, we examined whether AK23 mAb augments phosphorylation of Dsg3 and p38 mitogen-activating protein kinase (MAPK) in a human squamous cell line, DJM-1 cells. AK23 mAb increased serine phosphorylation of Dsg3 and augmented activation levels of p38 MAPK. These results indicate that antibodies bind to Dsg3, but not other antigens, in the IgG fraction and can induce activation of signal transduction.     

Pathogenic autoantibody production requires loss of tolerance against desmoglein 3 in both T and B cells in experimental pemphigus vulgaris

Mechanisms of tolerance break against desmoglein 3 (Dsg3) in patients with pemphigus vulgaris (PV) producing pathogenic anti-Dsg3 IgG autoantibodies are unclear. In this study, using a novel PV mouse model involving Dsg3 knockout mice, we investigated the mechanisms leading to production of autoantibodies against Dsg3. Adoptive transfer of Dsg3(-/-) splenocytes immunized with recombinant mouse Dsg3 to Rag2(-/-) recipient mice expressing Dsg3 resulted in the stable production of anti-Dsg3 IgG and development of PV phenotypes including oral erosions with suprabasilar acantholysis. When purified T and B cells from Dsg3(-/-), Dsg3(+/-) or Dsg3(+/+) mice were mixed with various combinations and transferred to Rag2(-/-) mice, pathogenic anti-Dsg3 IgG production was observed only with a combination of Dsg3(-/-) T and Dsg3(-/-) B cells but not with the other combinations. These results suggest that loss of tolerance against Dsg3 in both B and T cells is important for the development of autoimmune state of PV.     

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.     

Transition from pemphigus foliaceus to pemphigus vulgaris: case report with literature review

The transition between the main subtypes of pemphigus, pemphigus vulgaris (PV), and pemphigus foliaceus (PF) has rarely been reported. Moreover, the development of PV in a patient with PF is much more unusual than that of PF in a patient with PV. We report a 48-year-old man who presented with cutaneous lesions showing the typical clinical and histological features of PF. Five years later, his skin lesions became extensive and he developed oral erosions. His condition did not respond well to steroids and azathioprine. Histological examination of a vesicle disclosed suprabasal acantholysis in contrast to the subcorneal acantholysis discovered upon initial histological evaluation. Indirect immunofluorescence revealed IgG antikeratinocyte cell surface antibodies at a titer of 1:640. The titer was 1:160 at initial diagnosis. Upon immunoblotting, the patient's serum reacted with 130 kiloDalton (kDa) and 160 kDa proteins, suggesting desmoglein (Dsg) 3 and 1, respectively. We herein report an unusual case of PV that developed from PF during the disease's flare-up.     

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.     

Pemphigus vulgaris autoimmune globulin induces Src-dependent tyrosine-phosphorylation of plakophilin 3 and its detachment from desmoglein 3

Abstract

The cell adhesion molecule plakophilin 3 (Pkp3) plays an essential role in the maintenance of skin integrity and is targeted in certain autoimmune conditions. In one example, we have shown that Pkp3 is instrumental in mediating the discohesive effects of sera from patients with pemphigus vulgaris (PV), a life-threatening autoimmune disease that targets intercellular adhesion in the epidermis. In the present study, we determine the effect of PV autoimmune globulin (PV IgG) on Pkp3 in an in-vitro model of PV. We demonstrate that Pkp3 becomes tyrosine phosphorylated as early as 30?min upon binding of PV IgG to keratinocyte surface and eventually detaches from its binding partner desmoglein 3 (Dsg3). In parallel, Pkp3 is depleted from the membrane (Triton X-soluble) fraction and accumulates in the cytoplasm within 240?min of incubation with PV IgG. Inhibition of Pkp3 phosphorylation by a Src inhibitor attenuates the discohesive effects of PV IgG. Taken together, the data demonstrate that activation of Src-kinase signalling is crucial for PV acantholysis and acts, at least in part, via phosphorylation of the adaptor protein Pkp3.     

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.     

Production of non-pathogenic human monoclonal antibodies to desmoglein 3 from pemphigus vulgaris patient

Pemphigus vulgaris is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3, a 130 kDa epidermal protein. To further characterize the epitope(s) of pemphigus vulgaris antigen we established two human-human hybridoma by fusion of the peripheral blood mononuclear cells with a human and mouse heterohybridoma. These hybridomas designated as MAb Dsg-3: 06 and MAb Dsg-3: 10 and stable in culture and demonstrated yield of monoclonal antibodies specific for pemphigus vulgaris. Immunofluorescence, immunoblot, ELISA assays demonstrated that both the monoclonal antibodies bind to the intercellular cement substance and to 130 kDa protein present in the skin and specifically binds to recombinant desmoglein 3 protein, but not to desmoglein 1 protein. The IgG subclass distribution study demonstrated that both the antibodies are of IgG1 subclass in nature. Both the antibodies were non-pathogenic as demonstrated in vitro by their inability to produce acantholysis in normal human skin in organ culture or in vivo by the induction of disease in neonatal BALB/c mice. The relevance and value of these monoclonal antibodies in the pathogenesis of pemphigus vulgaris is discussed.     

Production of Non-pathogenic Human Monoclonal Antibodies to Desmoglein 3 from Pemphigus Vulgaris Patient

Pemphigus vulgaris is a potentially fatal autoimmune mucocutaneous disease associated with production of IgG autoantibodies to desmoglein 3, a 130 kDa epidermal protein. To further characterize the epitope(s) of pemphigus vulgaris antigen we established two human-human hybridoma by fusion of the peripheral blood mononuclear cells with a human and mouse heterohybridoma. These hybridomas designated as MAb Dsg-3: 06 and MAb Dsg-3: 10 and stable in culture and demonstrated yield of monoclonal antibodies specific for pemphigus vulgaris. Immunofluorescence, immunoblot, ELISA assays demonstrated that both the monoclonal antibodies bind to the intercellular cement substance and to 130 kDa protein present in the skin and specifically binds to recombinant desmoglein 3 protein, but not to desmoglein 1 protein. The IgG subclass distribution study demonstrated that both the antibodies are of IgG1 subclass in nature. Both the antibodies were non-pathogenic as demonstrated in vitro by their inability to produce acantholysis in normal human skin in organ culture or in vivo by the induction of disease in neonatal BALB/c mice. The relevance and value of these monoclonal antibodies in the pathogenesis of pemphigus vulgaris is discussed.     

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.     

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.     

The specific proteolysis hypothesis of pemphigus: Does the song remain the same?

Within the last decade, a number of theories on the pathogenesis of pemphigus vulgaris (PV) have followed one another. Of these, plesminogen activation and desmoglein compensation hypotheses have been substantiated by a conspicuous body of evidence. A significant change of this scenario occurred with the discovery that autoimmunity in PV can target acetylcholine receptors and that PV serum elicits a pletora of intracellular signals. Since then, a myriad of explanations accounting for PV acantholysis have appeared in the literature. However, as revolutionary as they can be, the majority of organic theories seemed to be highly speculative. We have recently obtained evidence for a proteolytic cleavage of desmoglein 3 in an in vitro model of PV; furthermore, our previous findings suggested the possible involvement of proteases such as matrix metalloproteinase (MMP) 9 in PV acantholysis both in vitro and in vivo. Hence, in formulating the "specific proteolysis theory" we have kept the rationale and the well-established evidence of both plasminogen activation and desmoglein compensation hypotheses. However, the specific proteolysis theory proposed by us is not just a return to the past. On the basis of the current knowledge on MMP substrate specificity we propose that Dsg1 and Dsg3, along with other important cadherins which are likely to be proteolytically targeted in PV, could be cleaved by either ADAM or typical MMPs, respectively. Whether this view was confirmed by further investigations, these enzymes could be specifically targeted by selective drugs which would permit more rational approaches to the treatment of pemphigus.     

Induction of keratinocyte IL-8 expression and secretion by IgG autoantibodies as a novel mechanism of epidermal neutrophil recruitment in a pemphigus variant

A subset of pemphigus herpetiformis, a rare pemphigus variant, is characterized histopathologically by subcorneal acantholysis and neutrophilic infiltration. The mechanism of neutrophil infiltration is unknown, but chemokines such as IL-8 may play a role. We investigated the possible role of IL-8 in two such cases. Direct and indirect immunofluorescence studies demonstrated in vivo-bound and circulating IgG epithelial cell surface-binding autoantibodies, both predominated by IgG4 subclass. ELISA and immunoblotting studies revealed that the patients' IgG autoantibodies recognized recombinant desmoglein 1 but not desmoglein 3. Preadsorption of the patients' sera with recombinant desmoglein 1 completely removed the epidermal cell surface immunostaining. Significantly, immunohistochemistry demonstrated intense expression of IL-8, co-localized with in vivo-bound IgG, in the upper epidermis, where the acantholysis took place. Affinity-purified sera IgG from these two patients, a normal individual, and a pemphigus vulgaris patient containing desmoglein 1 autoantibodies, were incubated with normal human keratinocytes in vitro. Cells treated with these patients' IgG secreted a seven-to-nine-fold increase of IL-8 (30-37 pg/ml) compared with the controls (2-4 pg/ml) and expressed a higher intensity of cytoplasmic IL-8 staining. These data demonstrate a novel functional role for IL-8 in the pathogenesis of the neutrophil-dominant subset of pemphigus herpetiformis. The autoantibody-induced epidermal cell IL-8 expression may represent a novel mechanism of epidermal neutrophil recruitment.     

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 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.     

Modulation of desmoglein-3-specific T cell response and pathogenic antibody generation in mice

Pemphigus is a severe blistering disease of the skin and mucous membranes caused by pathogenic autoantibodies to desmosomal adhesion proteins desmoglein-3 (Dsg3) and desmoglein-1 (Dsg1). The antibody titer and the distinct isotype patterns correlated with the disease activity. To identify their functional properties and pathogenic potential, we immunized C57BL/6 and Balb/c mice with recombinant Dsg3 fusion protein plus complete Freund's adjuvant (CFA) or Aluminum Hydroxide hydrate (Alum). After immunization, the cytokine profiles on T cells, the antibody titers, and the isotypes were analyzed. The pathogenicity of different autoantibody isotypes was evaluated by antibody passive transfer approach. It was found that Th1 type cytokine interferon gamma (IFN gamma) was elevated in the CFA-treated group, while Th2 type cytokine interleukin-4 (IL-4) was increased in the Alum-treated group. IgG1 expression was persistent in the Alum group while IgG2a was predominant in the CFA group. Neonatal mice transferred with sera from the Alum group, but not the CFA group, developed skin lesions clinically and histologically with IgG deposition on the epidermal keratinocytes. Our findings suggest that distinct T cell responses could be switched after active immunization combined with different adjuvants, resulting in distinct anti-Dsg3 antibody isotypes with different pathogenic activities in disease development. These findings shed new light on the immunopathogenesis of PV and offer a new therapeutic strategy for this potentially fatal disorder.     

Detection of low avidity desmoglein 3-reactive T cells in pemphigus vulgaris using HLA-DR beta 1*0402 tetramers

In the present study, we developed a HLA class II tetramer-based detection system utilizing DRB1*0402 tetramers loaded with recently identified immunodominant peptides of desmoglein 3 (Dsg3), the major autoantigen of pemphigus vulgaris (PV). Initial experiments demonstrated staining of a Dsg3-reactive T cell hybridoma which was derived from HLA-DR0402-transgenic mice with loaded PE-labeled DRbeta1*0402 tetramers. However, staining of autoreactive T cell clones (TCC) derived from PV patients resulted only in positive staining by addition of exogenous peptides to the staining reactions. There was a dose-dependent specific binding of TCC to the tetramers with the agonistic Dsg3 peptide which was not altered by exogenous unrelated Dsg3 peptide. Noteworthy, the TCC did not stain with HLA-DR4 tetramers complexed with unrelated Dsg3 peptides. The findings of this study suggest that HLA class II tetramers may provide a highly specific approach to monitor ex vivo the T cellular autoimmune response against Dsg3 in patients with PV.     

Role of intramolecular epitope spreading in pemphigus vulgaris

Pemphigus vulgaris (PV) is an acquired immunobullous disorder. At the early stage of the disease (mucosal PV), patients display only autoimmunity to desmoglein (Dsg) 3 and develop mucosal blisters; while at the later stage of the disease (mucocutaneous PV), patients exhibit non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and acquire cutaneous as well as mucosal blisters. At these two disease stages, Dsg3 autoantibodies exhibit different tissue-binding patterns and pathogenic activities, suggesting that they may recognize distinct epitopes. To test this hypothesis and to investigate the mechanism underlying the disease transition, we studied Dsg3 autoantibody epitopes from mucosal PV patients and patients exhibiting disease transition to mucocutaneous PV. We demonstrated that autoantibodies from the majority of mucosal PV patients target epitopes at the COOH-terminal portion of the Dsg3 ectodomain. Interestingly, only autoantibodies against the Dsg3 NH2-terminal epitope(s) are able to bind human skin. Moreover, we discovered that the intramolecular epitope spreading from Dsg3(87-566) to Dsg3(1-88) is a critical step that precedes the intermolecular epitope spreading from Dsg3 to Dsg1. During disease transition, this mechanism dictates the development of Dsg3 autoantibodies that recognize human skin and lead to expression of cutaneous PV lesions.