Redox Enzymes of the Thioredoxin Family as Potential and Novel Markers in Pemphigus.

Pemphigus vulgaris (PV) is a severe autoimmune blistering disease affecting both skin and mucous membranes. Its pathogenesis is related to IgG autoantibodies primarily targeting the cellular adhesion protein desmoglein (Dsg) 3, one of the major desmosome components. Impaired redox regulation is considered a major player in the pathogenesis of autoimmune diseases such as pemphigus by enhancing inflammation and breakdown of immunological tolerance by structural protein modifications. Despite many recent advances, local and systemic redox profiles that characterize the immune response in pemphigus are virtually unknown but potentially crucial in further advancing our understanding of redox-dependent modifications that eventually lead to clinical manifestation. Here, we have analyzed the individual expression pattern of four major redox enzymes that are members of the thioredoxin (Trx) fold superfamily (peroxiredoxins (Prxs) 1 and 4, glutaredoxin (Grx) 2, and Trx1) in serum and PBMCs as well as their distribution in the skin of pemphigus patients compared to healthy controls. We show that in groups of five pemphigus patients, Prx1 is upregulated in both serum and PBMCs, while its epithelial distribution remains within the spinous epithelial layer. Expression of Grx2 and Prx4 is both reduced in serum and PBMCs, while their distinct and similar expression in the skin changes from an even distribution throughout the basal layer (healthy) to ubiquitous nuclear localization in pemphigus patients. In PV patients, Trx1 is secreted into serum, and cellular distribution appears membrane-bound and cytosolic compared to healthy controls. We furthermore showed that a 3D ex vivo human skin model can indeed be used to reproduce similar changes in the protein levels and distribution of redox enzymes by application of cold atmospheric plasma. Deciphering the relationship between redox enzyme expression and autoimmunity in the context of pemphigus could be critical in elucidating key pathogenic mechanisms and developing novel interventions for clinical management.

Evaluation of antioxidant enzyme activity and antioxidant capacity in patients with newly diagnosed pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucosa. Increased levels of reactive oxygen species (ROS) were previously reported in PV. AIM: Because oxidative stress has an important role in the inflammatory process, we designed this study to evaluate the antioxidant status in patients with PV and to compare it with that of healthy controls (HCs). METHODS: In this case-control study, 43 newly diagnosed patients with PV were compared with 58 HCs. The severity of the disease was estimated according to Harman scores. Erythrocyte glutathione peroxidase (GPx), superoxide dismutase (SOD), CAT and serum malondialdehyde (MDA) activities and total antioxidant capacity were measured. Data were analyzed by independent t-test. RESULTS: Both groups were similar in sex, age and body mass index. Mean duration of disease was 5.6 months. Mean oral and skin severities were 1.79 and 2.3 respectively, based on Harman scores. SOD activity was not significantly different between groups (1003.30 ± 39.96 vs. 1009.76 ± 32.68 U/gHb). Levels were noticeably higher in patients with PV than in HCs for both GPx (52.13 ± 2.85 vs. 36.63 ± 1.49 U/gHb, respectively; P < 0.001) and CAT (205.69 ± 8.10 vs. 130.26 ± 6.80 kU/gHb, respectively; P < 0.001) activities, and CAT activity correlated with disease severity. In addition, patients had lower total antioxidant capacity than controls (3.39 ± 0.06 vs. 3.72 ± 0.09 mmol/L, P = 0.006). There was no noticeable difference in serum MDA between the two groups (P = 0.45). CONCLUSIONS: Patients with PV have significantly higher antioxidant enzyme activities and lower total antioxidant capacity compared with HCs. These data indicate the importance of improving antioxidant level in patients with pemphigus.

Overexpression of interleukin-23 and interleukin-17 in the lesion of pemphigus vulgaris: a preliminary study.

IL-23/IL-17 axis has been identified as major factor involved in the pathogenesis of several autoimmune diseases; yet its pathogenetic role in pemphigus vulgaris (PV) remains controversial. The aim of this research was to investigate the potential role of IL-23/IL-17 axis in the immunopathogenesis of PV, and correlation between IL-23+ cells and IL-17+ cells was also evaluated. For this purpose, ten patients with PV, three patients with pemphigus foliaceus (PF), and six healthy individuals were allocated to this research. The lesional skin biopsy specimens were obtained before treatment. Then immunofluorescence staining was performed to analyze the expression of IL-23 and IL-17 in the PV/PF patients and the healthy individuals. The results showed that the numbers of IL-23+ and IL-17+ cells were significantly higher in PV lesions, compared to PF lesions and normal control skins, respectively (all P < 0.05). Moreover, the correlation between IL-23+ cells and IL-17+ cells was significant (r = 0.7546; P < 0.05). Taken together, our results provided evidence that the IL-23/IL-17 axis may play a crucial role in the immunopathogenesis of PV and may serve as novel therapeutic target for PV.

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.

Biomarkers of oxidative stress in epidermis of Tunisian pemphigus foliaceus patients.

BACKGROUND: Reactive oxygen species play a key role in the development of many dermatological disorders. OBJECTIVE: The purpose of this study is to examine the lipid peroxidation, protein oxidation and antioxidative profile in Tunisian pemphigus foliaceus (PF) patients. METHODS: Malondialdehyde (MDA), conjugated dienes (CD), protein thiol levels, catalase (CAT) and superoxide dismutase (SOD) activities were evaluated in skin biopsies of 13 patients compared to biopsies of 7 healthy controls. RESULTS: Oxidative stress was confirmed in these three types of patient biopsies as compared to controls. Thus, MDA, CD levels and catalase CAT and SOD activities were significantly increased in lesional, perilesional and normal biopsies of PF patients than in those of control subjects. Protein oxidative was confirmed by lower levels of protein thiols in lesional, perilesional and normal biopsies than in control's biopsies. Otherwise, in patients, a significant rise of these biomarkers was observed in lesional and perilesional biopsies compared with normal biopsies. CONCLUSION: This study shows that oxidative stress could be involved in the pathogenesis of PF by the spread of skin lesions and/or by the increase in auto-antibodies' reactivity.

p38 mitogen-activated protein kinase (p38 MAPK)-mediated autoimmunity: lessons to learn from ANCA vasculitis and pemphigus vulgaris.

Evidence is beginning to accumulate that p38 mitogen activated protein kinase (p38 MAPK) signaling pathway plays an important role in the regulation of cellular and humoral autoimmune responses. The exact mechanisms and the degree by which the p38 MAPK pathway participates in the immune-mediated induction of diseases have started to emerge. This review discusses the recent advances in the molecular dissection of the p38 MAPK pathway and the findings generated by reports investigating its role in the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and autoimmune hepatitis. Application of newly-developed protocols based on sensitive flow cytometric detection has proven to be a useful tool in the investigation of the phosphorylation of p38 MAPK within different peripheral blood mononuclear cell populations and may help us to better understand the enigmatic role of this signaling cascade in the induction of autoimmunity as well as its role in immunosuppressive-induced remission. Special attention is paid to reported data proposing a specific role for autoantibody-induced activation of p38 MAPK-mediated immunopathology in the pathogenesis of autoimmune blistering diseases and anti-neutrophilic antibody-mediated vasculitides.

Varicella-zoster virus (VZV) and alpha 1 antitrypsin: a fatal outcome in a patient affected by endemic pemphigus foliaceus.

BACKGROUND: Herpes virus infections are well known infectious complications of pemphigus and bullous pemphigoid. We describe pathologic findings utilizing autopsy tissue from several organs from a patient affected by a new variant of endemic pemphigus in El Bagre, Colombia, South America. CASE REPORT: We describe a patient by a new variant of endemic pemphigus foliaceus from El Bagre that was receiving high-dosage immunosuppressants when hospitalized and died suddenly following contact with a second patient affected by chicken pox. MATERIALS AND METHODS: We performed studies utilizing hematoxylin and eosin, immunohistochemistry, and direct immunofluorescence techniques on tissues from several organs. RESULTS: We detected the presence of varicella zoster virus, as well as strong positivity for α-1 antitrypsin in the heart, kidneys, spleen, liver, skin, brain, lungs, pancreas, small and large intestines, and skeletal muscle. In regard to structural damage in the kidney and heart, we believe the observed damage is associated with the presence of autoantibodies to these organs, since both of them are rich in plakins and El Bagre-EPF patients present significant antibodies to plakin molecules. CONCLUSION: In patients with endemic pemphigus foliaceus, we recommend complete isolation of the patient when receiving high dosages of systemic immunosuppressive agents. We further suggest the clinical possibility of a synergistic, fatal interaction between active pemphigus foliaceus, varicella zoster virus, herpes simplex virus, immunosuppressive agents, and a systemic activation of α-1 antitrypsin. Thus, we suggest adequate bed spacing, barrier nursing, and preventative testing for α-1 antitrypsin activation are warranted in these patients to address these complications.

Inhibition of FAK prevents blister formation in the neonatal mouse model of pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FI before PV-IgG injection prevented the changes in both Bax and Bcl-2 expression and caspase-9 and caspase-3 activities induced by PV-IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases.

Hepatotoxicity and liver enzyme alteration in patients with immunobullous diseases receiving immunosuppressive therapy.

To avoid complications of high dose corticosteroid, pemphigus patients are usually co-treated with other immunosuppressive agents. Liver enzyme abnormality occurs commonly during treatment and occasionally causes discontinuation of drugs. To assess the rate of therapy-induced hepatotoxicity in patients with immunobullous diseases, we conducted a study of 250 pemphigus patients under immunosuppressive therapy prospectively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) plasma levels were recorded before the start of treatment and every week under treatment (up to 3 weeks). Hepatotoxicity was defined as the rise in the ALT plasma levels to greater than twice the upper normal limit. Approximately 81% of patients received prednisolone and azathioprine. Approximately 12% received only prednisolone. Hepatotoxicity occurred in 2.9% (n = 8) of patients after 1 week, in 7.8% (n = 20) after 2 weeks and in 11.5% (n = 29) after 3 weeks. No patient had jaundice or other clinical manifestations of hepatitis. The mean values of ALT and AST before the start of treatment were 20.7 ± 13.7 and 17.6 ± 10.8 U/L, respectively that grew to 47.5 ± 28.5 and 26.8 ± 14.5 U/L, 3 weeks after the initiation of treatment. Distribution of changes was not significantly different among groups of age, sex, immunosuppressive drugs and isoniazid consumption. Under usual treatment of pemphigus, hepatotoxicity occurs in 10% of patients during the first 3 weeks of therapy that does not seem to be associated with azathioprine or mycophenolate mofetil exclusively. High doses of prednisolone may play a role.

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.

PTPN22 1858T is not a risk factor for North American pemphigus vulgaris.

Alterations in the protein tyrosine phosphatase N22 (PTPN22) gene affect the threshold for lymphocyte activation. The PTPN22 1858T polymorphism leads to uninhibited T-cell receptor cascade propagation. An elevated PTPN22 1858C/T genotype frequency has been correlated with several autoimmune disorders which have T-cell and humoral components. However, a recent Tunisian report demonstrated no association between PTPN22 1858T and patients with Pemphigus vulgaris (PV), an autoantibody-associated blistering disorder. Because PTPN22 1858T allele frequency is known to vary across ethnic populations, we conducted a case-control study investigating the relationship between PTPN22 1858T and PV in North American patients of either Ashkenazi Jewish or Caucasian (non-Ashkenazi) decent. Participant genotype was determined in 102 PV patients and 102 healthy controls by restriction fragment length polymorphism-polymerase chain reaction genotyping. Relationships were calculated using Fisher's exact tests and chi-squared tests. We report that the PTPN22 1858C/T genotype is not significantly associated with PV in either Caucasians (P = 0.83) or Ashkenazi Jews (P = 0.60). Further stratification of the patient population by gender, age of disease onset, HLA-type, family history of autoimmune disease, history of anti-desmoglein (anti-Dsg) 3 or anti-Dsg1 antibody response, history of lesion morphology, and disease duration did not uncover significant associations between the PTPN22 1858T allele and PV subgroups. Our data indicate that the PTPN22 1858T mutation is not associated with PV in the North American population. We do observe an elevation of PTPN22 1858C/T genotype frequency in male PV patients. Further investigation will be required to determine if this trend reaches significance in larger studies.

Role of Rho GTPases in desmosomal adhesion and pemphigus pathogenesis.

Desmosomes are distinct intercellular contacts essential to the integrity of epithelial tissues and the heart muscle. This function is impaired in the disease pemphigus, in which patients develop autoantibodies against the cadherin-type desmosomal core proteins desmogleins. Autoantibody binding induces loss of cell-cell adhesion leading to blisters within the epidermis and mucous membranes. Despite the relevance of desmosomes for integrity of such essential organs as the skin, data on the regulation of desmosome assembly and maintenance and desmosome-mediated adhesion are only slowly emerging. Small guanosine triphosphatases (GTPases) of the Rho family have long been established as regulators of other cell junctions such as adherens junctions, but also have been implicated in participating in the formation of desmosomes. In this short review we summarize two papers from our group dealing with the role of Rho family GTPases for desmosomal adhesion and pemphigus and discuss these data integrating novel work recently published.

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

BACKGROUND: 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. OBJECTIVES: In the present study we investigated in more detail the role of PERK in the pathogenesis of PV. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Thioguanine nucleotides and thiopurine methyltransferase in immunobullous diseases: optimal levels as adjunctive tools for azathioprine monitoring.

OBJECTIVE: To prospectively determine optimal levels of 6-thioguanine nucleotide for disease remission in patients with immunobullous disease treated with azathioprine. DESIGN: Prospective, longitudinal study. Laboratory tests and clinical evaluations were performed monthly for 6 months and then every 2 to 3 months (median follow-up, 13.4 months). SETTING: Tertiary care medical center. PATIENTS: Twenty-seven patients with immunobullous disease treated with azathioprine were enrolled during a 2-year period. Twelve met the criteria for evaluation of optimal levels of 6-thioguanine nucleotide. MAIN OUTCOME MEASURES: Blood levels of 6-thioguanine nucleotide, 6-methylmercaptopurine, and thiopurine methyltransferase by polymerase chain reaction and enzyme activity were measured longitudinally during treatment. RESULTS: The range of 6-thioguanine nucleotide was 48 to 457 pmol/8 x 10(8) red blood cells (RBCs), with an average optimal level of 190.7 pmol/8 x 10(8) RBCs for all patients. The mean optimal levels were 179.4 and 205.6 pmol/8 x 10(8) RBCs for pemphigus and pemphigoid, respectively. Limited disease required less 6-thioguanine, with a mean of 145.3 pmol/8 x 10(8) RBCs. Longitudinal induction of thiopurine methyltransferase activity was observed during treatment. Patients with recalcitrant disease showed higher induction of enzyme activity (with an increase of 9.1 to 23.6 U/mL of RBCs above baseline) than did those with responsive disease. CONCLUSIONS: Optimal levels of 6-thioguanine nucleotide metabolites for disease remission in dermatology patients are 150 to 300 pmol/8 x 10(8) RBCs. High levels of the inactive metabolite 6-methylmercaptopurine and induction of thiopurine methyltransferase are associated with recalcitrant disease.

Biphasic activation of p38MAPK suggests that apoptosis is a downstream event in pemphigus acantholysis.

In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.

Involvement of the apoptotic mechanism in pemphigus foliaceus autoimmune injury of the skin.

Pemphigus foliaceus (PF) is an organ-specific autoimmune skin disease characterized by subcorneal epidermal cell detachment (acantholysis) and pathogenic autoantibodies against desmoglein 1. The mechanism responsible for pemphigus autoantibody-induced epidermal injury is not fully understood. In this study, we used the IgG passive transfer mouse model of PF to investigate the relevance of the apoptotic mechanism in pemphigus pathogenesis. TUNEL-positive epidermal cells and increased oligonucleosomes in the epidermal cytosolic fractions were detected in the diseased mice. Time course study reveals that TUNEL-positive epidermal cells appear before intraepidermal blisters. Moreover, the proapoptotic factor Bax was up-regulated at the earlier time points (2 and 4 h), whereas the antiapoptotic factor Bcl-x(L) was down-regulated at the later time points (6, 8, and 20 h) post-PF IgG injection by Western blot analysis. The active forms of caspase-3 and -6 were detected at the later time period (6, 8, and 20 h). Administration of Ac-DEVD-cmk, a peptide-based caspase-3/7 inhibitor, protected mice from developing intraepidermal blisters and clinical disease induced by PF IgG. The same protective effect was also observed using a broad-spectrum caspase inhibitor, Bok-D-fmk. Collectively, these findings show that biochemical events of apoptosis are provoked in the epidermis of mice injected with PF autoantibodies. Caspase activation may contribute to acantholytic blister formation in PF.

The role of nitric oxide synthases in pemphigus vulgaris in a mouse model.

BACKGROUND: Pemphigus vulgaris (PV) is a blistering autoimmune disease characterized by IgG autoantibodies against desmoglein 3. Nitric oxide synthases (NOS) may contribute to the increase of inflammation in tissues by the generation of nitrotyrosine residues (NTR). OBJECTIVES: To investigate whether the production of NTR mediated by NOS may participate in the development of inflammation and acantholysis in PV. METHODS: Mice were pretreated or not with NOS, tyrosine-kinase (TK) or nuclear factor (NF)-kappaB inhibitors, and then injected with PV-IgG. PV manifestations were examined in all mice. The expression of NTR, constitutive NOS (cNOS) [endothelial NOS (eNOS) and neuronal NOS (nNOS)], inducible NOS (iNOS) and NF-kappaB factor were studied in epidermis of mice using immunohistochemical techniques. RESULTS: After PV-IgG injection, expressions of NTR, iNOS, eNOS and nNOS increased in acantholytic cells, as did nuclear translocation of NF-kappaB in the basal cells of the epidermis. Pretreatment of mice with inhibitors of TK, nNOS and nonselective NOS, completely prevented NTR expression and the clinical and histological findings of PV in mice. TK inhibitor genistein inhibited both nNOS and iNOS expression on the membrane of basal keratinocytes, and nuclear translocation of NF-kappaB. CONCLUSIONS: Upregulation of cNOS and iNOS, NTR generation and nuclear translocation of NF-kappaB may contribute to increased inflammation and tissue damage in PV lesions. The absence of the clinical and histological findings of PV and NTR expression in mice injected with PV-IgG, through pretreatment with TK and nNOS inhibitors, provides compelling evidence that these signalling molecules should be considered as potential therapeutic targets in PV.

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.

Inhibition of Rho A activity causes pemphigus skin blistering.

The autoimmune blistering skin diseases pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are mainly caused by autoantibodies against desmosomal cadherins. In this study, we provide evidence that PV-immunoglobulin G (IgG) and PF-IgG induce skin blistering by interference with Rho A signaling. In vitro, pemphigus IgG caused typical hallmarks of pemphigus pathogenesis such as epidermal blistering in human skin, cell dissociation, and loss of desmoglein 1 (Dsg 1)-mediated binding probed by laser tweezers. These changes were accompanied by interference with Rho A activation and reduction of Rho A activity. Pemphigus IgG-triggered keratinocyte dissociation and Rho A inactivation were p38 mitogen-activated protein kinase dependent. Specific activation of Rho A by cytotoxic necrotizing factor-y abolished all pemphigus-triggered effects, including keratin retraction and release of Dsg 3 from the cytoskeleton. These data demonstrate that Rho A is involved in the regulation of desmosomal adhesion, at least in part by maintaining the cytoskeletal anchorage of desmosomal proteins. This may open the possibility of pemphigus treatment with the epidermal application of Rho A agonists.

p38MAPK inhibition prevents disease in pemphigus vulgaris mice.

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering skin disease characterized by detachment of keratinocytes (acantholysis). It has been proposed that PV IgG might trigger signaling and that this process may lead to acantholysis. Indeed, we recently identified a rapid and dose-dependent phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and heat shock protein (HSP) 27 after binding of PV antibodies to cultured keratinocytes. In human keratinocyte cultures, inhibitors of p38MAPK prevented PV IgG-induced phosphorylation of HSP27 and, more importantly, prevented the early cytoskeletal changes associated with loss of cell-cell adhesion. This study was undertaken to (i) determine whether p38MAPK and HSP25, the murine HSP27 homolog, were similarly phosphorylated in an in vivo model of PV and (ii) investigate the potential therapeutic use of p38MAPK inhibition to block blister formation in an animal model of PV. We now report that p38MAPK inhibitors prevented PV blistering disease in vivo. Targeting the end-organ by inhibiting keratinocyte desmosome signaling may be effective for treating desmosome autoimmune blistering disorders.