Acantholysis In Pemphigus Vulgaris Research Articles

The Role of Non-Neuronal Acetylcholine in the Autoimmune Blistering Disease Pemphigus Vulgaris

The importance of acetylcholine (ACh) in keratinocyte adhesion and acantholysis has been investigated over the last three decades, particularly in the pathophysiology of autoimmune blistering dermatoses. Pemphigus vulgaris (PV) is an autoimmune blistering skin disease where autoantibody-mediated suprabasilar intraepidermal splitting causes flaccid blisters and non-healing erosions of the oral mucosa and sometimes also of the skin. Historically, acantholysis in PV was thought to be driven by anti-desmoglein (Dsg) antibodies. Herein, we describe the role of autoantibodies against keratinocyte muscarinic and nicotinic acetylcholine receptors, as well as the annexin-like molecule pemphaxin that also binds ACh, in the immunopathogenesis of PV. The identification of targets in this disease is important, as they may lead to novel diagnostic and therapeutic options in the future for this potentially deadly disease.

Innate immune activation as cofactor in pemphigus disease manifestation.

Molecular mechanisms underlying auto-antibody-induced acantholysis in pemphigus vulgaris are subject of current research to date. To decipher the discrepancy between ubiquitous antibody binding to the epidermal desmosomes, but discontinuous disease manifestation, we were able to identify Ultraviolet A (UVA) as a cofactor for acantholysis. UVA induces interleukin (IL)-1 secretion in keratinocytes, mirroring innate immune system activation. In an in vitro keratinocyte dissociation assay increased fragmentation was observed when UVA was added to anti-Desmoglein 3 Immunoglobulins (anti-Dsg3 IgG). These results were confirmed in skin explants where UVA enhanced anti-Dsg3-mediated loss of epidermal adhesion. The UVA-mediated effect was blocked in vitro by the pan-caspase-inhibitor zVAD-fmk. Thus, we introduce UVA as a caspase-dependent exogenous cofactor for acantholysis which suggests that local innate immune responses largely contribute to overt clinical blister formation upon autoantibody binding to epidermal cells in pemphigus vulgaris.

What protein kinases are crucial for acantholysis and blister formation in pemphigus vulgaris? A systematic review.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by cell–cell detachment (or acantholysis) and blister formation. While the signaling mechanisms that associate with skin/mucosal blistering are being elucidated, specific treatment strategies targeting PV‐specific pathomechanisms, particularly kinase signaling, have yet to be established. Hence, the aim of this review was to systematically evaluate molecules in the class of kinases that are essential for acantholysis and blister formation and are therefore candidates for targeted therapy. English articles from PubMed and Scopus databases were searched, and included in vitro, in vivo, and human studies that investigated the role of kinases in PV. We selected studies, extracted data and assessed risk of bias in duplicates and the results were reported according to the methodology outlined by the Preferred Reporting Items for Systematic Reviews and Meta‐analyses (PRISMA). The risk of bias assessment was performed on in vivo studies utilizing SYRCLE's risk of bias tool. Thirty‐five studies were included that satisfied the pathogenicity criterion of kinases in PV, the vast majority being experimental models that used PV sera (n = 13) and PV‐IgG (n = 22). Inhibition of kinase activity (p38MAPK, PKC, TK, c‐Src, EGFR, ERK, mTOR, BTK, and CDK2) was achieved mostly by pharmacological means. Overall, we found substantial evidence that kinase inhibition reduced PV‐associated phosphorylation events and keratinocyte disassociation, prevented acantholysis, and blocked blister formation. However, the scarce adherence to standardized reporting systems and the experimental protocols/models used did limit the internal and external validity of these studies. In summary, this systematic review highlighted the pathogenic intracellular events mediated by kinases in PV acantholysis and presented kinase signaling as a promising avenue for translational research. In particular, the molecules identified and discussed in this study represent potential candidates for the development of mechanism‐based interventions in PV.

Caspase Inhibition as a Possible Therapeutic Strategy for Pemphigus Vulgaris: A Systematic Review of Current Evidence.

Simple SummaryPemphigus vulgaris is a potentially fatal disease characterised by blister formation affecting the skin and mouth. The mechanisms of blister formation may involve a biological process called apoptosis—a type of cell death—and some death-associated molecules known as caspases. Our review of the existing literature shows that caspase inhibitors exhibit an inhibitory effect on PV-induced apoptosis formation in vitro. In particular, activity of caspase 1 and caspase 3 is essential for the development of PV in vitro and in vivo. However, a majority of in vivo studies assessing caspase inhibition in PV models have a high risk of bias.Background: Pemphigus vulgaris (PV) is an IgG-mediated autoimmune disease characterised by epithelial cell–cell detachment (acantholysis) resulting in mucocutaneous blistering. The exact pathogenesis of blister formation is unknown and this has hampered the development of non-steroidal, mechanism-based treatments for this autoimmune disease. This systematic review aims to investigate the role of caspases in the pathogenesis of PV to inform the choice of more targeted therapeutic agents. Methods: A systematic search of MEDLINE/PubMed and Scopus databases was conducted to identify eligible studies. Multiple phases of inclusion and exclusion of the primary articles were conducted in pairs, and studies were recorded and analysed according to the latest version of the preferred reporting items for systematic reviews and meta-analyses (PRISMA). Risk of bias assessment was conducted for extracted in vivo animal intervention studies using SYRCLE’s risk of bias tool. Results: Eight articles from a total of 2338 in vitro, in vivo, and human studies met the inclusion criteria, with a high degree of inter-rater reliability. By and large, the results show that caspase activation was pathogenic in experimental PV because pan-caspase inhibitors could block or reduce PV acantholysis and blistering in vitro and in vivo, respectively. The pathogenic pathways identified involved caspase-1 and caspase-3. One study failed to show any improvement in the PV model with a caspase inhibitor. The majority of animal studies had high or unclear risk of bias. Conclusion: There are consistent data pointing towards a pathogenic role of caspase activation in PV acantholysis. However, high-quality evidence to confirm that caspase inhibition can prevent PV-induced blistering in vivo is limited. Therefore, further research is required to test the preclinical efficacy of caspase inhibitors in PV.

Mechanism-based therapeutic targets of pemphigus vulgaris: A scoping review of pathogenic molecular pathways.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterised by cell-cell detachment or acantholysis. The mechanisms which follow antibody (Ab) binding and culminate in acantholytic changes and skin/mucosal blistering have not been fully clarified. Current treatment strategies are not specific to PV pathophysiology and although life-saving, harbour considerable side effects. We aimed to systematically assess the molecules amenable to targeted treatments that follow Ab binding and are associated with PV acantholysis. The resulting scoping review was conducted under PRISMA-ScR guidelines with clear inclusion and exclusion criteria and focused specifically on kinases, caspases, proteases, hydrolytic enzymes and other molecules of interest postulated to take part in the pathophysiology of PV. The review process resulted in the identification of 882 articles, of which 56 were eligible for qualitative synthesis. From the included articles, the majority (n=42) used PV-IgG as the pathogenic agent, mainly via in vitro (n=16) and in vivo (n=10) models. Twenty-five molecules were found to play a pathogenic role in PV, including uPA, ADAM10, EGFR, Src, PKC, cdk2, ERK, PLC, calmodulin, NOS, p38MAPK and caspase-3. Selective inhibition of these molecules resulted in varying degrees of reduction in acantholysis and blistering. The pathogenic molecules identified in this review represent potential candidates for clinical translation.

Modulation of Mechanical Stress Mitigates Anti-Dsg3 Antibody-Induced Dissociation of Cell-Cell Adhesion.

It is becoming increasingly clear that mechanical stress in adhesive junctions plays a significant role in dictating the fate of cell-cell attachment under physiological conditions. Targeted disruption of cell-cell junctions leads to multiple pathological conditions, among them the life-threatening autoimmune blistering disease pemphigus vulgaris (PV). The dissociation of cell-cell junctions by autoantibodies is the hallmark of PV, however, the detailed mechanisms that result in tissue destruction remain unclear. Thus far, research and therapy in PV have focused primarily on immune mechanisms upstream of autoantibody binding, while the biophysical aspects of the cell-cell dissociation process leading to acantholysis are less well studied. In work aimed at illuminating the cellular consequences of autoantibody attachment, it is reported that externally applied mechanical stress mitigates antibody-induced monolayer fragmentation and inhibits p38 MAPK phosphorylation activated by anti-Dsg3 antibody. Further, it is demonstrated that mechanical stress applied externally to cell monolayers enhances cell contractility via RhoA activation and promotes the strengthening of cortical actin, which ultimately mitigates antibody-induced cell-cell dissociation. The study elevates understanding of the mechanism of acantholysis in PV and shifts the paradigm of PV disease development from a focus solely on immune pathways to highlight the key role of physical transformations at the target cell.

Study of Various Vesiculobullous Lesions Using Tzanck Smear

Background: The vesiculobullous reaction pattern is characterized by the presence of vesicles or bullae within the epidermis or at the dermoepidermal junction. Despite some having characteristic presentations, it’s difficult to make a definite diagnosis clinically. Hence, cytological evaluation is required for reliable and early diagnosis. Objectives of the study are to determine the incidence of various vesiculobullous lesions and evaluate cytology as a tool for early diagnosis of vesiculobullous lesions.
 Methods: For Tzanck smears fresh vesicle or bulla was selected and incised with scalpel, reflecting the roof of bulla. Base of the blister scraped gently and material spread on a glass slide. These smears were stained with MGG (air dried), Pap and H&E stains (fixed).Skin punch biopsies from the bullae were subjected to routine fixation, processing, sectioning and staining.
 Results: A total of 62 Tzanck smears were done for vesiculobullous lesions of skin of which 29 had histopathological correlation. Herpes constituted the most common vesiculobullous disorder (42%) followed by bullous pemphigoid (27.4%) and pemphigus vulgaris (19.3%). Most patients were in the age group 61- 70 years. The M:F ratio of 1:1.38 showing female preponderance. Tzanck smears showed acantholytic cells in pemphigus group, eosinophils in bullous pemphigoid and multinucleate giant cells in viral blisters. Histopathology showed intraepidermal acantholysis in pemphigus vulgaris, subcorneal blister in pemphigus foliaceus and subepidermal in bullous pemphigoid.
 Conclusion: Cytohistopathological correlation showed an overall sensitivity of 79%. Tzanck smear showed 96% sensitivity for viral infections. Tzanck smear is a quick, non-invasive method for the early diagnosis of vesiculobullous disorders.

The involvement of ADAM10 in acantholysis in mucocutaneous pemphigus vulgaris depends on the autoantibody profile of each patient.

Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). We used three PV-IgG fractions from different patients containing high or low levels of anti-Dsg1 and anti-Dsg3 antibodies, and the presence or not of anti-desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. Although all of the PV-IgG fractions produced suprabasal acantholysis, only those containing anti-Dsg1/3, but not anti-Dsc2/3 antibodies, induced ADAM10 activation in a Src-dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti-Dsc2/3 antibodies, in addition to anti-Dsg1/3, triggered earlier and ADAM10-independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. All PV-IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti-Dsg antibodies or the presence of non-Dsg antibodies, such as anti-Dsc, more severe cell-cell epidermal detachment will occur at different times, and in an ADAM10-dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non-Dsg proteins, and therefore more specific therapeutic approaches in PV should be used. What's already known about this topic? Suprabasal acantholysis in pemphigus vulgaris (PV) may be triggered by both desmoglein (Dsg) and non-Dsg autoantibodies. The autoantibody profile of each patient is associated with a distinct intracellular signalling pattern. What does this study add? In patients with PV with anti-Dsg3 and anti-Dsg1, but not anti-desmocollin (Dsc)3 antibodies, ADAM10 activation is induced in an Src-dependent way, together with an increase in the epidermal growth factor receptor (EGFR) ligands EGF and betacellulin. The presence of anti-Dsc3 antibodies triggers an earlier and ADAM10-independent acantholysis, without increasing EGFR ligands, and is associated with more severe epidermal detachment. Lower levels of anti-Dsc3 antibodies are associated with less severe acantholysis. What is the translational message? In some patients with PV, the severity and the timing for cell-cell detachment seem to depend on the level of anti-Dsg1/3 antibodies, although other as yet uncharacterized antibodies may also participate. These patients with PV would exhibit inhibition of acantholysis by Src, ADAM10, EGF and EGFR inhibitors. In other patients, the presence of non-Dsg antibodies, such as anti-Dsc2/3, would produce an earlier and more severe ADAM10-independent suprabasal acantholysis.

Acantholysis may precede elevation of circulating anti-desmoglein 3 antibody levels in pemphigus vulgaris presenting with desquamative gingivitis.

Pemphigus vulgaris (PV) is an autoimmune, blistering disease that affects the mucosa and skin. The current theory favors the concept that anti‐desmoglein (Dsg) 3 autoimmunity is the only pathogenic event needed to induce acantholysis. However, a few cases of active PV in the oral cavity had no detectable anti‐Dsg 3 antibody. The aim of this study was to evaluate the differences in clinical and laboratory findings, whether or not the anti‐Dsg 3 antibodies were present. This study was based on a retrospective review of 10 PV cases. The evaluation of the circulating autoantibody titers to Dsg 3 was conducted by using enzyme‐linked immunosorbent assay (ELISA). An index value of 20 or more was used as the cutoff for a positive reaction. Only five of the 10 PV cases had a positive Dsg 3 ELISA. There were no differences in clinical, cytological, histopathological, and direct immunofluorescence findings, whether or not the anti‐Dsg 3 antibodies were present. Of the five patients with a negative reaction at the time of diagnosis, the Dsg 3 ELISA became positive in the follow‐up period in three cases. In the remaining two cases, the Dsg 3 ELISA was consistently negative for 18 months. Dsg 3 ELISA was negative early in some PV cases. Therefore, PV acantholysis may precede the elevation of circulating anti‐Dsg 3 antibody levels. The diagnosis of PV should be considered based on comprehensive clinical, histopathological, and immunofluorescent criteria.

Locations of acantholysis in pemphigus vulgaris and pemphigus foliaceus.

Acantholysis in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) occurs predominantly in the suprabasal area and the granular layer, respectively. However, acantholysis can occasionally be observed in unusual locations. We retrospectively studied the acantholysis locations in 35 PV and 27 PF cases, and analyzed them using the index value of Desmoglein (Dsg) 1 and Dsg3 by enzyme-linked immunosorbent assay, clinical data, and inflammatory cells. We also analyzed the relationship between clinical subtype and various parameters. In PV, acantholysis was noted in the suprabasal area in 3 cases, in the lower half of the epidermis in 19 cases, and throughout the epidermis in 13 cases. In PF, acantholysis was observed in the granular layer in 6 cases, in the upper half of the epidermis in 14 cases, and throughout the epidermis in 7 cases. Mean index value of Dsg1 in PV patients with acantholysis throughout the epidermis was 2-fold higher than other PV patients. Neutrophils tended to infiltrate the dermis and epidermis more in PF than in PV. Higher Dsg1 index values seem to correlate with acantholysis in the upper part of the epidermis in PV. Neutrophils may play some role in unusual acantholysis locations in PF.

An Adjuvant Capsule in the Treatment of Pemphigus Vulgaris: A Novel Hypothesis

Pemphigus vulgaris (PV) is one of the most common and studied autoimmune disorders of skin. The knock down of desmogleins in the skin by IgG antibodies will eventually leads to mucocutaneous problems in both genders. Targeting the cardinal molecules involved in the pathways of immune response is the main goal of given therapy. With regard to the side effects of long term use of immunosuppressive treatments, patients may use various adjuvant drug combinations. Sulfasalazine (SSZ) and Pentoxifylline (PTX) are two low-cost anti-TNF medications that can halt the acantholysis in PV. Tetracycline is an effective antibiotic with immunomdulatory properties on down-regulation of pro-inflammatory molecules such as NO, IL-1β. Moreover, the anti-inflammatory effects of Omega-3 take this supplement into account as a potential addition to anti-PV armamentarium. Thus, this complex could be utilized as a safe and effective adjuvant capsule against PV.

Signalling pathways in pemphigus vulgaris

Acantholysis in pemphigus vulgaris is induced by binding of autoantibodies to desmoglein 3 (Dsg3). The roles of signalling pathways on development of acantholysis have recently been extensively studied. In the study by Sayar et al., recently published in Exp Dermatol, epidermal growth factor receptor (EGFR) signalling was activated in both in vivo and in vitro pemphigus vulgaris experimental models. However, while EGFR inhibitors suppressed activity of p38 mitogen-activated protein kinase (p38MAPK) linearly, they suppressed activity of c-Myc and acantholysis in a non-linear, V-shaped relationship. These findings indicated complicated interactions among EGFR, p38MAPK and c-Myc in pemphigus vulgaris pathology.

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

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.

Neural nitric oxide synthase participates in pemphigus vulgaris acantholysis through upregulation of Rous sarcoma, mammalian target of rapamycin and focal adhesion kinase

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis produced as a consequence of desmoglein (Dsg) and non-Dsg autoantibodies binding to several targeting molecules localized on the membrane of keratinocytes. Nitric oxide (NO) may exert a pathogenic function in several immunological processes. We have previously demonstrated that neural nitric oxide synthase (nNOS) plays part in PV acantholysis. Also, our group has described a relevant role for HER [human epidermal growth factor receptor (EGFR) related] isoforms and several kinases such as Src (Rous sarcoma), mammalian target of rapamycin (mTOR) and focal adhesion kinase (FAK), as well as caspases in PV development. Using a passive transfer mouse model of PV, we aimed to investigate the relationship between the increase in nNOS and EGFR, Src, mTOR and FAK kinase upregulation observed in PV lesions. Our results revealed a new function for nNOS, which contributes to EGFR-mediated PV acantholysis through the upregulation of Src, mTOR and FAK. In addition, we found that nNOS participates actively in PV at least in part by increasing caspase-9 and caspase-3 activities. These findings underline the important issue that in PV acantholysis, caspase activation is a nNOS-linked process downstream of Src, mTOR and FAK kinase upregulation.

A Possible Role for CD8+ T Lymphocytes in the Cell-Mediated Pathogenesis of Pemphigus Vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering disease whose pathogenesis involves both humoral and cell-mediated immune response. Though the pathogenetic role of autoantibodies directed against desmoglein 3 is certain, a number of other factors have been suggested to determine acantholysis in PV. In this study we examined the possible role of CD8+ T cells in the development of acantholysis by a passive transfer of PV autoantibodies using CD8 deficient mice, and we also studied the inflammatory infiltrate of PV skin lesions by immunohistochemical staining. The results of the immunohistochemical staining to study the expression of CD3, CD4, and CD8 in PV skin lesions showed that CD4+ are more expressed than CD8+ in the inflammatory infiltrate of PV lesions, confirming the data of the previous literature. The passive transfer study showed a lower incidence of pemphigus in the group of CD8 deficient mice compared to the control one of wild-type mice. These results suggest that CD8+ T cells may play a role in the pathogenesis of PV, perhaps through the Fas/FasL pathway.

P38 MAPK Signaling in Pemphigus: Implications for Skin Autoimmunity

p38 mitogen activated protein kinase (p38 MAPK) signaling plays a major role in the modulation of immune-mediated inflammatory responses and therefore has been linked with several autoimmune diseases. The extent of the involvement of p38 MAPK in the pathogenesis of autoimmune blistering diseases has started to emerge, but whether it pays a critical role is a matter of debate. The activity of p38 MAPK has been studied in great detail during the loss of keratinocyte cell-cell adhesions and the development of pemphigus vulgaris (PV) and pemphigus foliaceus (PF). These diseases are characterised by autoantibodies targeting desmogleins (Dsg). Whether autoantibody-antigen interactions can trigger signaling pathways (such as p38 MAPK) that are tightly linked to the secretion of inflammatory mediators which may perpetuate inflammation and tissue damage in pemphigus remains unclear. Yet, the ability of p38 MAPK inhibitors to block activation of the proapoptotic proteinase caspase-3 suggests that the induction of apoptosis may be a consequence of p38 MAPK activation during acantholysis in PV. This review discusses the current evidence for the role of p38 MAPK in the pathogenesis of pemphigus. We will also present data relating to the targeting of these cascades as a means of therapeutic intervention.

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.

Deregulation of PERK in the autoimmune disease pemphigus vulgaris occurs via IgG-independent mechanisms

Serum and IgG isolated from patients with the autoimmune blistering disease pemphigus vulgaris (PV) trigger complex intracellular pathways in keratinocytes, including alterations of the cell cycle and metabolism, which ultimately lead to cell-cell detachment (acantholysis). We have shown previously that one of the earliest pathogenic events in PV is the activation of protein kinases, including the PKR-like endoplasmic reticulum (ER) kinase PERK. In the present study we investigated in more detail the role of PERK in the pathogenesis of PV. PERK levels were assessed by Western blotting and in-cell enzyme-linked immunosorbent assay, and PERK expression was silenced by siRNA technology. The effects of PV sera/IgG on keratinocyte cultures were investigated by flow cytometry, MTT and adhesion assays. We show that PERK is activated in keratinocytes exposed to PV serum, as demonstrated by an increase in phosphorylated PERK levels and phosphorylation of eIF2α. Decreased expression of PERK by siRNA reduced the effects of PV serum on the cell cycle and keratinocyte viability, two key events in PV pathophysiology. As impairment of metabolic activity in PV is partially due to non-IgG serum factors, we then investigated the activation of PERK in keratinocytes incubated with whole PV serum, purified PV IgG and IgG-depleted PV serum. The data demonstrated that PV sera depleted of IgG, but not PV IgG, triggered PERK phosphorylation and this correlated with a marked reduction of metabolic activity in keratinocytes exposed to IgG-free serum. Knockdown of PERK by siRNA abrogated the changes in the cell cycle and apoptosis induced by IgG-depleted PV serum. Finally, the reduction of metabolic activity observed in keratinocytes exposed to IgG-depleted PV serum was almost absent in PERK-deficient cells. Taken together, the results demonstrate that activation of PERK participates in the reduction of metabolic activity and cell viability seen in PV and that this phenomenon depends on non-IgG factors. PERK activation may represent a novel signalling mechanism linking ER stress and acantholysis in PV.

Vitamin D inhibits captopril-induced cell detachment and apoptosis in keratinocytes

Captopril, an angiotensin I-converting enzyme inhibitor, is a commonly prescribed antihypertensive drug. Its cutaneous side-effects include pemphigus vulgaris acantholysis and bullous pemphigoid-like cell-matrix detachment. This medication also triggers apoptosis in human keratinocytes. Calcitriol, the hormonally active vitamin D metabolite, protects keratinocytes from programmed cell death induced by various noxious stimuli. To examine if calcitriol protects proliferating keratinocytes from the damage inflicted by captopril. Autonomously proliferating HaCaT keratinocytes, used as a model for basal layer keratinocytes, were exposed to captopril. Cell detachment was examined visually by light microscopy. Cytotoxicity was assessed by Hoechst 33342 staining and lactate dehydrogenase release. Apoptotic death was assessed by monitoring caspase 3-like activity. Cells exposed to captopril detached and became round. This process was accompanied by programmed cell death. From time-dependent monitoring of cell detachment and apoptosis, and examination of pan-caspase inhibitor effects on cell detachment we concluded that cell death is the consequence of cell detachment from the culture plate and not vice versa. Pretreatment with calcitriol significantly attenuated these events. The effects of calcitriol were already evident at 1 nmol L(-1) concentration of the hormone. The results of this study show that calcitriol protects keratinocytes from captopril-induced cell detachment and apoptosis.

Controversial Role of Antibodies against Linear Epitopes of Desmoglein 3 in Pemphigus Vulgaris, as Revealed by Semiquantitative Living Cell Immunofluorescence Microscopy and in-Cell Elisa

A novel explanation of pemphigus vulgaris (PV) pathogenesis suggests that serum autoantibodies may affect desmoglein 3 (Dsg3)-mediated adhesion by triggering depletion of Dsg3 from desmosomes. Furthermore, abrogation of Dsg3 from the cell seems to depend on anti-Dsg3 pemphigus IgG. In this study we sought to gain more insights into the role of PV IgG recognizing non-conformational epitopes of Dsg3 (anti-Dsg3-L IgG) by semi-quantitative living cell immunofluorescence (LCIF) microscopy, in-cell ELISA and morphometric analysis of acantholysis. Our data demonstrate that PV serum and PV IgG can induce acantholysis and reduce the total amount of Dsg3 in cultured keratinocytes, whereas anti-Dsg3-L IgG fail to do so when administered at concentrations comparable to those present in pathogenic PV sera. However, the Dsg3-depleting activity of such polyclonal anti-Dsg3 IgG was acquired when used at 1 microg/ml. Interestingly, both PV sera and IgG, including anti-Dsg3-L IgG, caused early depletion of surface Dsg3 while slightly affecting the total cell content of Dsg3 until late acantholysis. This raises a possibility that depletion of Dsg3 from cell membrane and reduction of the total cellular levels of Dsg3 represent distinct phenomena in PV acantholysis. Taken together, our data demonstrate that anti-Dsg3 PV IgG against linear epitopes of Dsg3 can induce acantholytic changes of keratinocytes in a dose- and time-dependent manner. Specifically, both morphological and biochemical changes suggestive of acantholysis are seen only at high IgG concentrations. We conclude that anti-Dsg3L IgG play a minor role in experimental PV under physiologic conditions.