In vitro C3 mRNA expression in Pemphigus vulgaris: complement activation is increased by IL-1alpha and TNF-alpha

BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. OBJECTIVE: In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. METHODS: Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1alpha and TNF-alpha are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1alpha and TNF-alpha. RESULTS: Incubation of NHuK with PV sera caused their detachment from the plates after 20-30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1alpha and TNF-alpha in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression.CONCLUSION: This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1alpha and TNF-alpha.     

Desmoglein-3 is a target autoantigen in spontaneous canine pemphigus vulgaris

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease of humans and companion animals. In human patients, PV is associated with the production of IgG autoantibodies specific for keratinocyte desmosomal glycoproteins of the cadherin family. The purpose of this study was to determine whether antikeratinocyte IgG autoantibodies were present in the skin and serum of dogs with PV, and also to identify the canine PV autoantigen(s) targeted by circulating autoantibodies. Eleven dogs were selected because of the microscopic demonstration of suprabasal epithelial acantholysis. Direct immunofluorescence revealed the presence of IgG autoantibodies bound to the membrane of keratinocytes in skin biopsy specimens of 8/9 dogs (89%). Using indirect immunofluorescence, serum-circulating IgG autoantibodies were found in 10/11 (91%) and 5/11 (45%) dogs, using normal canine gingiva and cultured canine oral keratinocytes, respectively. By immunoblotting using cultured canine oral keratinocyte protein lysates, IgG autoantibodies from 7/9 (78%) tested dogs recognized a 130-kDa antigen that comigrated with that identified by rabbit polyclonal antibodies raised against desmoglein-3. This 130 kDa antigen was confirmed to represent the canine equivalent of human desmoglein-3 by immunoprecipitation-immunoblotting. The results of these studies provide evidence that the canine desmoglein-3 homologue is a major autoantigen in dogs with PV. These observations further establish spontaneous canine PV as a natural model for research on pathogenesis, etiology and novel therapeutic approaches for this disease of humans.     

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.     

Clues to diagnosis for unusual mucosal pemphigus demonstrating undetectable anti‐desmoglein 3 serum antibodies by routine tests

Pemphigus is an autoimmune blistering disease caused by immunoglobulin (Ig)G autoantibodies against desmogleins (Dsg). In mucosal‐dominant pemphigus vulgaris (PV), anti‐Dsg3 antibodies play a critical role in acantholysis. We followed two mucosal‐dominant PV cases who suffered from refractory oral mucosal erosions. In these cases, anti‐Dsg3 serum antibodies were not detected by indirect immunofluorescence and enzyme‐linked immunosorbent assay (ELISA). However, direct immunofluorescence showed the intercellular IgG deposition in the epidermis and histopathological findings revealed suprabasal acantholysis. In order to analyze the pathomechanisms in these cases, we first examined the Dsg3 expression patterns in lesional sites and compared them with those of typical mucosal‐dominant PV cases. In typical PV cases, the alteration of Dsg3 distribution was observed in lesional sites by immunostaining. The aggregation of Dsg3, which is the characteristic change in PV mucosal lesions, was observed as the initial change prior to acantholysis. In our cases, a clustering of Dsg3 was observed at mucosal lesions, and the expression levels of Dsg3 in acantholytic lesions were decreased, as observed in typical mucosal‐dominant PV cases. Although anti‐Dsg3 serum antibodies could not be detected by routine tests, anti‐Dsg3 serum antibodies were detected by Dsg3 ELISA using 10‐times more concentrated sera (highly sensitive ELISA). Moreover, purified and concentrated PV IgG showed high pathogenicity when examined by dissociation assay. In conclusion, the detection of morphological changes in Dsg3 distribution and highly sensitive ELISA method could be useful for the early diagnosis of PV recurrence.     

Is Grover's disease an autoimmune dermatosis?

Grover's disease (GD) is a transient or persistent, monomorphous, papulovesicular, asymptomatic or pruritic eruption classified as non‐familial acantholytic disorder. Contribution of autoimmune mechanisms to GD pathogenesis remains controversial. The purpose of this study was to investigate antibody‐mediated autoimmunity in 11 patients with GD, 4 of which were positive for IgA and/or IgG antikeratinocyte antibodies by indirect immunofluorescence. We used the most sensitive proteomic technique for an unbiased analysis of IgA‐ and IgG‐autoantibody reactivities. Multiplex analysis of autoantibody responses revealed autoreactivity of all 11 GD patients with cellular proteins involved in the signal transduction events regulating cell development, activation, growth, death, adhesion and motility. Semiquantitative fluorescence analysis of cultured keratinocytes pretreated with sera from each patient demonstrated decreased intensity of staining for desmoglein 1 and/or 3 and PCNA, whereas 4 of 10 GD sera induced BAD expression, indicating that binding of autoantibodies to keratinocytes alters expression/function of their adhesion molecules and activates apoptosis. We also tested the ability of GD sera to induce visible alterations of keratinocyte shape and motility in vitro but found no specific changes. Thus, our results demonstrated that humoral autoimmunity in GD can be mediated by both IgA and IgG autoantibodies. At this point, however, it is impossible to conclude whether these autoantibodies cause or are caused by the disease. Antidesmoglein antibodies may be triggered by exposure to immune system of sequestered antigens due to disintegration of desmosomes during primary acantholysis. Clarifying aetiology of GD will help improve treatment, which currently is symptomatic and of marginal effectiveness.     

Searching for experimental models of <Emphasis Type="Italic">Pemphigus vulgaris</Emphasis>

The current knowledge on Pemphigus vulgaris (PV) pathophysiology suggests that blister formation relies on both PV IgG and non-IgG serum factors activity. PV autoimmunity seems to develop against both desmoglein 1/3 and acetylcholine receptors leading to transduction of signals to the cell mediated by phosphorilation events. Serum factors other than IgG also participate to PV acantholysis through apoptotic or cytokine-mediated mechanisms. Apart from the role played by each actor within the acantholysis, however, the current scenario arises important methodological issues. For example, the use of PV IgG or monoclonal anti-Dsg3 antibodies to experimentally reproduce the disease appears inadequate, as it does not take into account the role of non-IgG factors. On the basis of the above observations and those from our laboratories, here we propose that using whole sera from PV patients with active disease represents the most faithful manner to mimic the disease.     

Pemphigus vulgaris acantholysis ameliorated by cholinergic agonists

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune, IgG autoantibody-mediated disease of skin and mucosa leading to progressive blistering and nonhealing erosions. Patients develop autoantibodies to adhesion molecules mediating intercellular adhesion and to keratinocyte cholinergic receptors regulating cell adhesion. OBSERVATIONS: To determine whether a cholinergic agonist can abolish PV IgG-induced acantholysis, litter mates of neonatal athymic nude mice were injected with PV IgG together with carbachol (0.04 micro g/g body weight). None of these mice developed skin lesions. Through in vitro experiments, we measured the expression of adhesion molecules in monolayers of normal human keratinocytes incubated overnight in the presence of 0.25mM carbachol using semiquantitative Western blot and immunofluorescence. Carbachol caused an elevation of the relative amount of E-cadherin in keratinocytes (P<.05) without changing that of plakoglobin (P>.05). The phosphorylation level of E-cadherin and plakoglobin was increased by PV IgG, whereas this effect of PV IgG was attenuated in the presence of 0.5mM carbachol. Pyridostigmine bromide, an acetylcholinesterase inhibitor, produced effects similar to those of carbachol, which helps explain its clinical efficacy in a patient with active PV that was resistant to treatment with systemic glucocorticosteroids. Treatment with pyridostigmine bromide (360 mg/d) in a patient with PV allowed to keep his disease under control at a lower dose of prednisone than that used before starting pyridostigmine bromide treatment.Conclusion Elucidation of the cholinergic control of keratinocyte adhesion merits further consideration because of a potential for the development of novel antiacantholytic therapies using cholinergic drugs.     

Herpetiform pemphigus with characteristic transmission electron microscopic findings of various-sized ballooning vacuoles in keratinocytes without acantholysis

We report a unique case of a Japanese woman with herpetiform pemphigus (HP) who had IgG autoantibodies reactive with nondesmosomal sites of keratinocytes and presented characteristic transmission electron microscopic (TEM) findings of various-sized vacuoles in keratinocytes without acantholysis. The patient presented with pruritic annular oedematous erythemas with small blisters lining the margins on the trunk and extremities. Histopathological examinations showed intraepidermal blisters with prominent infiltrations of eosinophils. Direct and indirect immunofluorescence tests revealed the presence of in vivo bound and circulating IgG autoantibodies to the keratinocyte cell surfaces. However, enzyme-linked immunosorbent assays for desmoglein (Dsg) 1, Dsg3 and desmocollins 1–3 showed negative results. Immunoblotting using the full-length human Dsg1 recombinant protein showed a positive band. TEM examination showed various-sized vacuoles squashing the nuclei in many keratinocytes, resulting in rupture of the cells. Immunoelectron microscopic examination revealed IgG deposition over the entire keratinocyte cell surfaces, which spared the desmosomes. IgG antibodies were also present on the inside walls of the vacuoles around the nuclei of keratinocytes and on the cell surfaces of infiltrating eosinophils. This patient also had marked eosinophilia and high levels of thymus and activation-regulated chemokine and interleukin-5 in the serum. These results indicated a novel autoantigen on the nondesmosomal keratinocyte cell surfaces and the pathogenesis of bullous spongiotic change with inflammation in HP.     

寻常型天疱疮患者血清对HaCaT细胞Dsg、MMP-9表达的影响

 目的:探讨寻常型天疱疮（PV）患者血清对HaCaT细胞桥粒芯蛋白（Dsg）1、3和基质金属蛋白酶（MMP）9表达的影响及其参与PV棘层松解的可能机制。方法:用含5%PV血清的高糖DMEM培养基孵育HaCaT细胞,以正常培养组、健康血清组以及抗Dsg3抗体组（阳性对照组）作为对照。刮取细胞提取总RNA及蛋白质,Q PCR检测Dsg1、Dsg3和MMP 9 mRNA转录水平,Western Blot检测Dsg1、Dsg3和MMP 9表达,荧光单克隆Dsg1/3抗体分别检测细胞表面Dsg1、Dsg3表达。 结果:Q PCR结果显示：与正常培养组比较,含5% PV血清培养组HaCaT细胞Dsg1、Dsg3、MMP 9基因的mRNA相对表达量分别上升约432%、402%、402%,而抗Dsg3抗体组分别上升约495%、488%、604%,PV血清组与正常培养组间有明显差异（P值均<0.05）。 Western Blot结果显示：与正常培养组相比,PV血清组HaCaT细胞表面Dsg1、Dsg3、MMP 9蛋白表达升高。Dsg1、Dsg3荧光单克隆抗体检测显示：PV血清组HaCaT表面Dsg1线状荧光消失,替代为颗粒状、团块状荧光颗粒分散于细胞表面和胞浆区域,而细胞表面Dsg3线状荧光仍然存在,但强度减弱。 结论:PV血清能促进Dsg1、Dsg3和MMP 9转录及表达,Dsg3抗体可通过诱导角质形成细胞Dsg1蛋白内吞,削弱抗体补偿作用参与棘层松解。     

IgG binds to desmoglein 3 in desmosomes and causes a desmosomal split without keratin retraction in a pemphigus mouse model

Pemphigus vulgaris (PV) is an autoimmune blistering disease caused by IgG autoantibodies against desmoglein 3 (Dsg3). In this study, we characterized the ultrastructural localization of in vivo-bound IgG, Dsg3, and desmoplakin during the process of acantholysis in an active mouse PV model, using post-embedding immunoelectron microscopy. In non-acantholytic areas of keratinocyte contact, IgG labeling was restricted to the extracellular part of desmosomes, and was evenly distributed throughout the entire length of the desmosome. The distribution of in vivo IgG was similar to that of anti-Dsg3 labeling in the control mouse. Within the acantholytic areas, there were abundant split-desmosomes with keratin filaments inserted into the desmosomal attachment plaques. These split-desmosome extracellular regions were also decorated with anti-Dsg3 IgG and were associated with desmoplakin staining in their cytoplasmic attachment plaques. No apparent split-desmosomes, free of IgG-labeling were observed, suggesting that Dsg3 was not depleted from the desmosome before the start of acantholysis in vivo. Desmosome-like structures (without keratin insertion) were found only on the lateral surfaces of basal cells, but not on the apical surfaces at the site of acantholytic splits. These findings indicate that anti-Dsg3 IgG antibodies can directly access Dsg3 present in desmosomes in vivo and cause the subsequent desmosome separation that leads to blister formation in PV.     

Small-interfering RNA targeted at antiapoptotic mRNA increases keratinocyte sensitivity to apoptosis

Background Gene silencing RNA interference technology concentrates on downregulation of gene expression using specific double‐stranded RNA molecules (small‐interfering RNA, siRNA), which induce the degradation of complementary mRNA. SiRNA therapeutics is currently being tested in several clinical trials. Skin disorders are an attractive target for siRNA‐based technology because effective agents can be delivered by topical application. In several inflammatory skin disorders, the barrier function of the skin is markedly impaired and this may increase the delivery efficiency. Psoriasis is a very common skin disorder. The characteristics of this disorder are abnormal keratinocyte proliferation and inflammation. Keratinocytes from psoriatic epidermis express much higher levels of the antiapoptotic protein, Bcl‐xL, compared with normal keratinocytes. Insulin‐like growth factor 1 receptor (IGF‐1R) plays a major role in cell growth, differentiation and apoptosis in many cell types, including keratinocytes and IGF‐1R activation plays an important role in the pathogenesis of psoriasis. Keratinocytes from patients with psoriasis are more susceptible to IGF‐1‐stimulated proliferation compared with normal keratinocytes. IGF‐1R is expressed by proliferating basal and suprabasal keratinocytes and is more abundant in psoriatic lesions. Objectives To prove the validity of IGF‐1R and Bcl‐xL as useful targets for siRNA‐based therapeutics and to deliver siRNA selectively and efficiently to primary human keratinocytes; this is a primary essential step in the development of siRNA. Methods Primary normal human keratinocytes were transfected with various siRNA molecules, and transfection efficiency was monitored by fluorescent labelling. Cell growth and apoptosis induction were evaluated in the transfected cells. Results We were able to deliver efficiently siRNA targeting Bcl‐xL or IGF‐1R to primary human keratinocyte cultures. We also showed that siRNAs targeting Bcl‐xL and IGF‐1R induce growth inhibition, apoptosis and increased sensitivity to ultraviolet B in keratinocytes. Conclusions The present findings demonstrate that Bcl‐xL and IGF‐1R are valid, important targets for siRNA‐based technology directed at the suppression of keratinocyte hyperproliferation.     

In vivo blockade of pemphigus vulgaris acantholysis by inhibition of intracellular signal transduction cascades

BACKGROUND: Pemphigus vulgaris (PV) is an autoimmune disease characterized by mucocutaneous intraepithelial blisters and pathogenic autoantibodies against desmoglein 3. The mechanism of blister formation in pemphigus has not been defined; however, in vitro data suggest a role for activation of intracellular signalling cascades. OBJECTIVES: To investigate the contribution of these signalling pathways to the mechanism of PV IgG-induced acantholysis in vivo. METHODS: We used the passive transfer mouse model. Mice were injected with IgG fractions of sera from a patient with PV, with or without pretreatment with inhibitors of proteins that mediate intracellular signalling cascades. RESULTS: Inhibitors of tyrosine kinases, phospholipase C, calmodulin and the serine/threonine kinase protein kinase C prevented PV IgG-induced acantholysis in vivo. CONCLUSIONS: These observations strongly support the role of intracellular signalling cascades in the molecular mechanism of PV IgG-induced acantholysis.     

UVB与钙离子对体外培养人角质形成细胞表达天疱疮抗原的影响

目的 研究中波紫外线(UVB)照射以及不同钙离子浓度对角质形成细胞表达天疱疮抗原的影响.方法 通过改变培养基中的钙离子浓度以及采用不同剂量UVB照射体外培养的人角质形成细胞,在不同时间段以寻常型天疱疮(PV)或落叶型天疱疮(PF)血清作为一抗进行免疫荧光检测,观察寻常型天疱疮抗原(PVA)和落叶型天疱疮抗原(PFA)的表达情况;提取细胞或皮肤表皮组织蛋白质,用PV和PF血清进行免疫印迹检测.结果 无论是否增加钙离子浓度,体外培养人角质形成细胞间隙均可以检测到PV血清特异性着色.只有增加培养基中钙离子浓度后,形成的复层化角质形成细胞间隙才可以检测到PF血清特异性荧光着色.不同剂量UVB照射后的角质形成细胞均不产生PF血清的特异性着色.PF血清与160000条带反应,PV血清可与130000和160000两条带反应.结论 体外培养的单层或复层角质形成细胞均可以表达PVA,提高培养基中钙离子浓度可以诱导培养人角质形成细胞复层化并表达PFA,而UVB照射不能促使人角质形成细胞在体外培养条件下表达PFA.     

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.     

Differences in the expression of pemphigus antigens during epidermal differentiation

Epidermal desmogleins with molecular weights of 130/140kDa (Dsg3 or PVA) and 150/160 kDa (Dsg1 or DGI) are recognized by autoantibodies from patients with pemphigus vulgaris (PV) and pemphigus foliaceus (PF), respectively. In order to understand the histogenesis of both types of pemphigus, we studied the expression patterns of Dsgl and Dsg3 during stratification of cultured keratinocytes. Monolayers of cultured normal human keratinocytes demonstrated uniform inter cellular staining with PV sera. The staining pattern was distinct from the focal staining with PF sera observed only in the stratified areas. Both Dsgl and Dsg3 proteins and their mRNA were expressed by the monolayers, whereas no production of Dsg2 (HDGC) mRNA was found. The relative ratio of Dsg3 to the total desmogleins, as determined by density on immunoblotting, decreased as the cultured keratinocytes stratified. In the completely stratified keratinocytes cultured on collagen membrane, Dsgl became predominant, with subsequent reduction of PV antigen expression. The relative decrease of Dsg3 (PVA) during epidermal differentiation might be responsible for the induction of suprabasal acantholysis in PV.     

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

Background 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. Objectives To examine if calcitriol protects proliferating keratinocytes from the damage inflicted by captopril. Methods 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. Results 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. Conclusions The results of this study show that calcitriol protects keratinocytes from captopril‐induced cell detachment and apoptosis.     

microRNA-mediated keratinocyte hyperproliferation in psoriasis vulgaris

Background Psoriasis is a chronic inflammatory skin disease characterized by intense proliferation and abnormal differentiation of keratinocytes, although the pathogenesis is still not completely clarified. Objectives We investigated the mechanism of keratinocyte proliferation seen in psoriasis, focusing on microRNA (miRNA). Materials and methods miRNAs were extracted from tissues and sera of psoriasis, atopic dermatitis and healthy control. To determine pathogenic miRNAs, we performed miRNA polymerase chain reaction (PCR) array analysis. The results were confirmed with quantitative real‐time PCR, in situ hybridization, immunohistochemistry, transient transfection of siRNA and inhibitor in cultured keratinocytes and Western blotting. Results PCR array analysis using tissue miRNA demonstrated miR‐424 level was markedly decreased in psoriasis skin in vivo. Protein expression of mitogen‐activated protein kinase kinase 1 (MEK1) or cyclin E1, predicted target genes of miR‐424, was increased in psoriatic skin, although their mRNA levels were not. The transfection of specific inhibitor of miR‐424 in normal human keratinocytes led to upregulation of MEK1 or cyclin E1 protein, and resulted in increased cell proliferation. On the other hand, cell number was significantly decreased when cells were transfected with siRNA for MEK1 or cyclin E1. Furthermore, we first investigated serum miRNA levels in psoriasis. Although not significant, serum miR‐424 concentration tended to be decreased in patients with psoriasis compared with healthy controls. Conclusions Decreased miR‐424 expression and subsequently increased MEK1 or cyclin E1 may play a key role in the pathogenesis of psoriasis. Investigation of the regulatory mechanisms of keratinocyte proliferation by miRNA may lead to new treatments and a disease activity marker.     

Specific immunoadsorption of pathogenic autoantibodies in pemphigus requires the entire ectodomains of desmogleins

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are life‐threatening autoimmune blistering skin diseases. They are characterized by circulating autoantibodies which bind to the ectodomains of desmoglein (Dsg) 1 and Dsg3. These antibodies induce acantholysis in skin and mucous membranes. In severe cases of pemphigus, immunoadsorption is applied to remove total IgG from patient plasma using protein A or other ligands. To develop a specific adsorber for anti‐Dsg antibodies, epitope mapping studies of Dsg1 and Dsg3 ectodomains were conducted. Dsg variants were expressed on the surface of HEK‐293 cells and analysed for reactivity with pemphigus and control sera by indirect immunofluorescence technique. For Dsg1, a construct consisting of domain 1 directly fused to domain 5, seemed to be suitable for specific immunoadsorption of anti‐Dsg1 antibodies. The recognized epitopes were mainly conformation‐dependent. However, adsorption of pemphigus foliaceus IgG using this protein coupled to a Sepharose matrix did not completely remove pathogenicity from the sera, as proven by a keratinocyte dissociation assay. In contrast, full‐length Dsg1 and Dsg3 ectodomains were able to specifically adsorb anti‐Dsg antibodies and to efficiently eliminate pathogenicity. Therefore, the complete and correctly folded ectodomains of both desmogleins are required for therapeutic immunoadsorption.     

Apoptolysis: a novel mechanism of skin blistering in pemphigus vulgaris linking the apoptotic pathways to basal cell shrinkage and suprabasal acantholysis

Abstract:  Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a 'tombstone' appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering.     

Induction of senescence pathways in Kindler syndrome primary keratinocytes

Background Individuals with Kindler syndrome (KS) have loss‐of‐function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin‐1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated. Objectives To investigate how kindlin‐1 deficiency affects the proliferative potential of primary human keratinocytes. Methods  We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony‐forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin‐1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology. Results We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi‐1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle‐arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin‐1 proved that its deficiency was directly responsible for p63, Bmi‐1 and pRb downregulation and p16 induction. Conclusions Our data directly implicate kindlin‐1 in preventing premature senescence of keratinocytes.