Pathogenesis Of Pemphigus Vulgaris Research Articles

Population-Specific Association between a Polymorphic Variant in ST18, Encoding a Pro-Apoptotic Molecule, and Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease caused by anti-epithelial antibodies, leading to disruption of cell-cell adhesion. Although the disease is exceedingly rare worldwide, it is known to be relatively prevalent in Jewish populations. The low prevalence of the disease represents a significant obstacle to a genome-wide approach to the mapping of susceptibility genes. We reasoned that the study of a genetically homogeneous cohort characterized by a high prevalence of PV may help exposing associated signals while reducing spurious results due to population sub-structure. We performed a genome-wide association study using 300K single-nucleotide polymorphisms (SNPs) in a case-control study of 100 PV patients of Jewish descent and 397 matched control individuals, followed by replication of significantly associated SNPs in three additional cohorts of Jewish, Egyptian, and German origin. In addition to the major histocompatibility complex locus, a genomic segment on 8q11.23 that spans the ST18 gene was also found to be significantly associated with PV. This association was confirmed in the Jewish and Egyptian replication sets but not in the German sample, suggesting that ST18-associated variants may predispose to PV in a population-specific manner. ST18 regulates apoptosis and inflammation, two processes of direct relevance to the pathogenesis of PV. Further supporting the relevance of ST18 to PV, we found this gene to be overexpressed in the skin of PV patients as compared with healthy individuals.

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.

Urban legends: pemphigus vulgaris

Pemphigus vulgaris (PV) is the most common type of pemphigus. PV pathogenesis is still debated, and treatment remains challenging. We investigated five controversial topics: (1) What are the target antigens in PV? (2) Do desmogleins adequately address PV pathophysiology? (3) How does acantholysis occur in PV? (4) Is PV still a lethal disease? (5) What is the role of rituximab (RTX) in PV treatment? Results from extensive literature searches suggested the following: (1) Target antigens of PV include a variety of molecules and receptors that are not physically compartmentalized within the epidermis. (2) PV is caused by a variety of autoantibodies to keratinocyte self-antigens, which concur to cause blistering by acting synergistically. (3) The concept of apoptolysis distinguishes the unique mechanism of autoantibody-induced keratinocyte damage in PV from other known forms of cell death. (4) PV remains potentially life-threatening largely because of treatment side effects, but it is uncertain which therapies carry the highest likelihood of lethal risk. (5) RTX is a very promising treatment option in patients with widespread recalcitrant or life-threatening PV. RTX's cost is an issue, its long-term side effects are still unknown, and randomized controlled trials are needed to establish the optimal dosing regimen.

The pathogenic role of autoantibodies in pemphigus vulgaris

Pemphigus vulgaris (PV) is a severe autoimmune bullous disease involving both the skin and mucosal areas, and characterized by intraepithelial flaccid blisters and erosions. The pathogenesis of this disease is not yet completely understood, but novel insights into desmoglein biology and autoantibody pathogenesis have recently been published. Acantholysis in PV seems to result from a collective action of autoantibodies against various keratinocyte self antigens, of which desmogleins 1 and 3 are the most important. Additional antigens including desmocollins and nondesmosome components, such as the mitochondrion, might take part in disease activation. Recently, apoptosis was reported as a possible underlying mechanism of acantholysis. Furthermore, apoptolysis is believed to be the link between suprabasal acantholytic and cell-death pathways. We review the possible hypotheses of the pathogenesis of PV: the desmoglein compensation theory, the antibody-induced apoptosis theory, the basal-cell shrinkage hypothesis and the newly published apoptolysis theory.

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.

Soluble Fas in pemphigus vulgaris.

The Fas/Fas ligand (FasL) system has been recognized as an important pathway for apoptosis induction in cells and tissues. It has recently been shown that skin lesions of pemphigus vulgaris are associated with Fas mediated apoptosis. The aim of this study was to evaluate the level of serum soluble Fas of ten newly diagnosed patients with pemphigus vulgaris. Sera were collected from ten patients with pemphigus vulgaris. Commercial sandwich enzyme-linked immunosorbent assay (ELISA) for quantitative detection of soluble Fas was applied. Patients with mucosal skin involvement had higher median values in contrast to patients with cutaneous involvement. Elevation of soluble Fas in our study may give insights for the pathogenesis of pemphigus vulgaris. Suppression of this underlying mechanism may be an important target for novel therapies and relapse prevention.

The extend of desmoglein 3 depletion in pemphigus vulgaris is dependent on Ca2+‐induced differentiation – a role in suprabasal epidermal skin splitting?

Pemphigus vulgaris (PV) is an autoimmune disease of skin and mucous membranes characterized by formation of intraepidermal blisters. Patients develop autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and 1, leading to suprabasal keratinocyte detachment. Depletion of Dsg3 was reported to be a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer time periods in high Ca2+ conditions, we hypothesized that Dsg depletion was dependent on Ca2+-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in PV patient skin and an organotypic raft model of human epidermis. Also, depletion was prominent in cultured primary keratinocytes as well as in HaCaT cells incubated in high Ca2+ media for 3d but is less pronounced in HaCaT cultures after 8d. These effects were dependent on protein kinase C alpha (PKCα) signaling because inhibition of PKCα blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, PKCα inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Taken together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca2+-induced differentiation, which may contribute to the suprabasal cleavage plane observed in PV.

Loss of the Desmosomal Protein Perp Enhances the Phenotypic Effects of Pemphigus Vulgaris Autoantibodies

Pemphigus vulgaris (PV) is an autoimmune bullous disease in which autoantibodies against proteins of the desmosomal adhesion complex perturb desmosomal function, leading to intercellular adhesion defects in the oral mucosa and skin. Previous studies have demonstrated a central role for downregulation of the desmosomal cadherin desmoglein 3 (DSG3) in the pathogenesis of PV. However, the effects of non-cadherin desmosomal proteins in modulating the cellular manifestations of PV remain poorly understood. Here, we characterize the expression and functional importance of Perp, a newly discovered tetraspan desmosomal protein, in PV. Our data demonstrate that PV autoantibodies disrupt Perp expression at the membrane and trigger its internalization along with DSG3 into the endosomal pathway, where it is ultimately targeted to the lysosome for degradation. We further show that Perp deficiency exacerbates the pathogenic effects of PV autoantibodies on keratinocytes by enhancing both the depletion of desmosomal DSG3 and intercellular adhesion defects. Together, our findings highlight the importance of non-cadherin desmosomal proteins in modulating PV phenotypes and provide new insight into Perp's role in the desmosome.

Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus

Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment.

Apoptosis is not required for acantholysis in pemphigus vulgaris

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory proteins FLIP(L) and FLIP(S) to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.

Pemphigus Vulgaris IgG Directly Inhibit Desmoglein 3-Mediated Transinteraction

The autoimmune blistering skin disease pemphigus is caused by autoantibodies against keratinocyte surface Ags. In pemphigus vulgaris (PV), autoantibodies are primarily directed against desmosomal cadherins desmoglein (Dsg) 3 and Dsg 1, whereas pemphigus foliaceus (PF) patients only have Abs against Dsg 1. At present, it is unclear whether Dsg autoantibodies contribute to pemphigus pathogenesis by direct inhibition of Dsg transinteraction. Using atomic force microscopy, we provide evidence that PV-IgG directly interfere with homophilic Dsg 3 but, similar to PF-IgG, not with homophilic Dsg 1 transinteraction, indicating that the molecular mechanisms in PV and PF pathogenesis substantially differ. PV-IgG (containing Dsg 3 or Dsg 1 and Dsg 3 autoantibodies) as well as PV-IgG Fab reduced binding activity of Dsg 3 by approximately 60%, comparable to Ca(2+) depletion. Similarly, the mouse monoclonal PV Ab AK 23 targeting the N-terminal Dsg 3 domain and AK 23 Fab reduced Dsg 3 transinteraction. In contrast, neither PV-IgG nor PF-IgG blocked Dsg 1 transinteraction. In HaCaT monolayers, however, both PV- and PF-IgG caused keratinocyte dissociation as well as loss of Dsg 1 and Dsg 3 transinteraction as revealed by laser tweezer assay. These data demonstrate that PV-IgG and PF-IgG reduce Dsg transinteraction by cell-dependent mechanisms and suggest that in addition, Abs to Dsg 3 contribute to PV by direct inhibition of Dsg transinteraction.

Evidence of Key Role of Cdk2 Overexpression in Pemphigus Vulgaris

The pathogenesis of pemphigus vulgaris (PV) is still poorly understood. Autoantibodies present in PV patients can promote detrimental effects by triggering altered transduction of signals, which results in a final acantholysis. To investigate mechanisms involved in PV, cultured keratinocytes were treated with PV serum. PV sera were able to promote the cell cycle progression, inducing the accumulation of cyclin-dependent kinase 2 (Cdk2). Microarray analysis on keratinocytes detected that PV serum induced important changes in genes coding for one and the same proteins with known biological functions involved in PV disease (560 differentially expressed genes were identified). Then, we used two different approaches to investigate the role of Cdk2. First, small interfering RNA depletion of Cdk2 prevented cell-cell detachment induced by PV sera. Second, pharmacological inhibition of Cdk2 activity through roscovitine prevented blister formation and acantholysis in the mouse model of the disease. In vivo PV serum was found to alter multiple different pathways by microarray analysis (1463 differentially expressed genes were identified). Major changes in gene expression induced by roscovitine were studied through comparison of effects of PV serum alone and in association with roscovitine. The most significantly enriched pathways were cell communication, gap junction, focal adhesion, adherens junction, and tight junction. Our data indicate that major Cdk2-dependent multiple gene regulatory events are present in PV. This alteration may influence the evolution of PV and its therapy.

A Case of Pemphigus Vulgaris with Serum Autoantibodies Binding Blood Cell Antigen(s) on Immunofluorescence: A Case Report

Patients with pemphigus vulgaris (PV) may develop autoimmunity against an array of non-desmoglein antigens, including keratinocyte acetylcholine receptors. The latter, along with yet unknown autoantigens, could participate to PV pathogenesis. Here, we report a case of PV developing autoimmunity against peripheral blood mononuclear cell (PBMC) antigens. The patient was poorly responsive to steroidal and immunosuppressive drugs. Although the role for this PBMC antigen has not been established, it could affect the behaviour of immune cells that regulate autoantibody production in PV. Deregulation of PBMC activity may therefore influence patient's response to pharmacological therapy.

Oral mucosal disease: Pemphigus

Pemphigus defines a group of rare mucocutaneous autoimmune diseases of which pemphigus vulgaris (PV) is the most common. The aetiology and pathogenesis of PV are not completely clear, but there is a fairly strong genetic background: ethnic groups such as Ashkenazi Jews and people of Mediterranean and Indian origin are particularly susceptible and there is a link to HLA class II alleles. The initiating event in PV is not clear, but circulating IgG autoantibodies develop, directed particularly against the intercellular cadherin desmoglein 3 (Dsg3) in desmosomes of stratified squamous epithelium. Oral lesions often herald the disease and are initially vesiculobullous, but they rupture readily to leave ulcers. Involvement of other mucosa and skin is almost inevitable and PV is potentially life threatening. The diagnosis is confirmed by biopsy with histological examination and immunostaining. Management is largely by systemic immunosuppression with corticosteroids, usually azathioprine or other agents, but newer treatments with potentially fewer adverse effects look promising.

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.

Plakoglobin‐dependent disruption of the desmosomal plaque in pemphigus vulgaris

We recently reported that the pathogenesis of pemphigus vulgaris (PV), an autoimmune blistering skin disorder, is driven by the accumulation of c-Myc secondary to abrogation of plakoglobin (PG)-mediated transcriptional c-Myc suppression. PG knock-out mouse keratinocytes express high levels of c-Myc and resemble PVIgG-treated wild-type keratinocytes in most respects. However, they fail to accumulate nuclear c-Myc and loose intercellular adhesion in response to PVIgG-treatment like wild-type keratinocytes. This suggested that PG is also required for propagation of the PVIgG-induced events between augmented c-Myc expression and acantholysis. Here, we addressed this possibility by comparing PVIgG-induced changes in the desmosomal organization between wild-type and PG knock-out keratinocytes. We found that either bivalent PVIgG or monovalent PV-Fab (known to trigger blister formation in vivo) disrupt the linear organization of all major desmosomal components along cell borders in wild-type keratinocytes, simultaneously with a reduction in intercellular adhesive strength. In contrast, PV-Fab failed to affect PG knock-out keratinocytes while PVIgG cross-linked their desmosomal cadherins without significantly affecting desmoplakin. These results identify PG as a principle effector of the PVIgG-induced signals downstream of c-Myc that disrupt the desmosomal plaque at the plasma membrane.

CD4+CD25high Regulatory T Cells Are Markedly Decreased in Blood of Patients with Pemphigus Vulgaris

Background: It remains to be determined whether pemphigus vulgaris (PV), an autoimmune blistering disease, has a reduction and/or dysfunction of CD4⁺CD25^high regulatory T (Treg) cells. Objectives: To evaluate the frequency and phenotypes of Treg cells in blood of patients with PV. Methods: Peripheral blood mononuclear cells were prepared from PV patients as well as normal and disease control volunteers, and the frequency and phenotypes of Treg cells were determined by flow cytometry. CD4⁺CD25⁺ and CD4⁺CD25^– T cells isolated from peripheral blood mononuclear cells of PV patients and normal controls were subjected to real-time semiquantitative RT-PCR for the expression of Foxp3 gene. Results: The proportion of Treg cells in all PV patients was severely reduced, approximately ten times less than controls. These observations were further confirmed by both diminished gene and protein expression of Foxp3 in the CD4⁺CD25⁺ T cell population in PV patients. Conclusions: Numerical impairment of Treg cells may be involved in the pathogenesis of PV.

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Pemphigus foliaceus treated with etanercept

To the Editor: The role of tumor necrosis factor alpha (TNF-α) in the pathogenesis of pemphigus vulgaris (PV) has been demonstrated by several studies.1,2 Recently, successful treatments with TNF-α antagonists have been reported in bullous autoimmune disorders.3-5

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