Apoptosis is not required for acantholysis in pemphigus vulgaris

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

p38MAPK Signaling and Desmoglein-3 Internalization Are Linked Events in Pemphigus Acantholysis

Pemphigus vulgaris (PV) is an autoimmune blistering disease in which antibodies against the desmosomal cadherin, DSG3 (desmoglein-3), cause acantholysis. It has become increasingly clear that loss of cell-cell adhesion in PV is a complex and active process involving multiple signaling events such as activation of p38MAPK. It has also been demonstrated that incubating keratinocytes with PV IgG causes a redistribution of DSG3 from the cell surface to endosomes, which target these proteins for degradation. This study was undertaken to determine the relationship between p38MAPK and DSG3 endocytosis in pemphigus. In this work, we confirm that PV IgG causes internalization of cell-surface DSG3 into endosomes (as early as 4 h), which are then depleted from both detergent-soluble and detergent-insoluble pools. Cell-surface DSG3 internalization and depletion from both the detergent-soluble and detergent-insoluble fractions were blocked by the p38MAPK inhibitor SB202190. These data suggest that p38MAPK is capable of regulating PV IgG-mediated DSG3 internalization and that previously isolated mechanistic observations may be linked to a common pathway by which pemphigus autoantibodies lead to acantholysis.     

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.Pemphigus is a group of related autoimmune diseases characterized by blistering in the skin. The histologic hallmark of these disorders is termed acantholysis, which describes the loss of adhesion between adjacent epithelial cells. The two major variants are pemphigus foliaceus (PF)² and pemphigus vulgaris (PV). In PF, acantholysis is observed beneath the stratum corneum and within the granular layer of epidermal epithelia, whereas in PV, blister formation occurs above the basal layer of epidermal epithelia and mucosal epithelium. Passive transfer of IgG purified from both PV and PF patient sera reproduces the clinical, histological, and immunologic features of the human diseases, demonstrating that these autoantibodies are pathogenic (1, 2). In PF, autoantibodies target the desmosomal cadherin desmoglein (dsg) 1, whereas in PV, autoantibodies initially target dsg3 (3, 4) in mucosal PV and then subsequently target both dsg1 and dsg3 in mucocutaneous PV (5-7).The mechanism by which pemphigus autoantibodies induce blistering has been under investigation. Work from a number of laboratories has suggested that activation of intracellular events is induced by binding of PF or PV IgG to dsg1 and dsg3, respectively (8-14). Previously, we have reported that PV IgG activate p38MAPK and heat shock protein (HSP) 27 in human keratinocyte tissue cultures (15). Significantly, p38MAPK inhibitors blocked PV IgG-induced keratin filament retraction and actin reorganization in human keratinocyte tissue cultures. Furthermore, we have demonstrated that both PV and PF IgG induce phosphorylation of p38MAPK and HSP25, the murine HSP27 homologue, in mouse models and that inhibitors of p38MAPK block blistering in both the PV (16) and the PF (17) passive transfer mouse models. Additionally, in human skin biopsies from both PV and PF patients, phosphorylation of p38MAPK and HSP27 has been observed (18). Collectively, these observations suggest that activation of p38MAPK within the target keratinocyte contributes directly to loss of cell-cell adhesion induced by pemphigus autoantibodies.Both p38MAPK and HSP27 have been implicated in the regulation of the intermediate filament and actin cytoskeletons (19-25); the ability of p38MAPK inhibitors to block both pemphigus IgG-activated cytoskeletal reorganization and pemphigus IgG-activated blistering suggests that p38MAPK may be acting upstream of the cytoskeleton in the mechanism of acantholysis; however, p38MAPK signaling has been implicated in other cellular responses (reviewed in Ref. 26). For example, there is abundant evidence for p38MAPK involvement in apoptosis (27-29); however, the role of p38MAPK in apoptosis seems to be cell type- and stimulus-dependent. Although p38MAPK signaling promotes cell death in some cell lines, it also functions to enhance survival, growth, and differentiation in other cell lines (30). Several reports describe increased apoptosis of keratinocytes in pemphigus (31-35); however, the relationship between PV IgG-mediated p38MAPK signaling, the induction of apoptosis, and the relationship of apoptosis to blistering has not been defined. This study was undertaken to investigate the relationship between p38MAPK activation, apoptosis, and acantholysis.     

Serum from pemphigus vulgaris reduces desmoglein 3 half-life and perturbs its de novo assembly to desmosomal sites in cultured keratinocytes

Defects of cell-cell adhesion underlie disruption of epithelial integrity observed in patients with pemphigus vulgaris (PV), an autoimmune disease characterized by severe mucosal erosions and skin blisters. Pathogenic PV autoantibodies found in patients' sera target desmoglein 3 (Dsg3), a major component of the desmosome, but how does this phenomenon affect Dsg-dependent adhesion and lead to acantholysis still remains controversial. Here, we show that PV serum determines a reduction of Dsg3 half-life in HaCaT keratinocytes, although the total amount of Dsg3 remains unchanged. Immunofluorescence studies suggest that PV IgG exert their effect prevalently by binding non-desmosomal Dsg3 without causing its massive internalization. Furthermore, PV IgG targeting desmosome-assembled Dsg3 do not induce depletion of Dsg3 from the adhesion sites. Conversely, incorporation of PV IgG-Dsg3 complexes into new forming desmosomes appears perturbed. With our study, the basic biochemical changes of Dsg3 in an in vitro model of PV have been defined.     

Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell-cell detachment and processing of adhesion molecules via mechanisms that involve PKC

In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder.     

Differential coupling of M1 muscarinic and alpha7 nicotinic receptors to inhibition of pemphigus acantholysis

The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120- and beta-catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of beta-catenin and Src-tyrosine phosphorylation of p120-catenin. The M(1) muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M(1) antagonist MT7. The alpha7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The alpha7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of beta-catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgG-induced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M(1) abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas alpha7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.     

Defining the role of complement in experimental pemphigus vulgaris in mice

Parenteral passive transfer of human pemphigus vulgaris IgG (PV IgG) into neonatal mice reproduces the cutaneous disease. We used this model to study the role of complement in the development of acantholysis in three steps. Peptic F(ab')2 fragments were prepared from PV IgG and were injected into seven newborn mice, and all animals developed acantholytic skin blisters without local complement activation, as shown by direct immunofluorescence. These fragments were reduced and alkylated to produce Fab' fragments with equivalent in vitro binding activity. The monovalent fragments were given in an identical fashion to five littermates but failed to produce disease even though they were bound in the epidermis in vivo. Intact PV IgG was injected in 20 genetically C5-deficient neonates (B10-D2-OSN strain) and 20 control neonates (B10-D2-NSN, normal complementemic). Extensive blistering, with a positive Nikolsky sign, was produced in all 40 animals. PV IgG was given to 34 BALB/c neonates that were complement depleted by pretreatment with cobra venom factor (CoF) for 24 hr, and to 38 untreated neonates from the same litters. There was no difference in the disease produced after CoF treatment in animals that received high doses of PV IgG (5 to 15 mg/g/day). In animals receiving 2.5 mg PV IgG/g/day, blister formation was delayed and the final extent of the cutaneous lesions was less in CoF-treated mice (n = 12) than in normal complementemic controls (n = 12, p less than 0.02). These results show that complement activation is not an essential mechanism in PV IgG-induced acantholysis in vivo, but it does have an amplifying effect on the development of cutaneous lesions under certain conditions, and lesions can be induced in vivo by bivalent F(ab')2 fragments of PV IgG, but not by the monovalent Fab' fragments, suggesting that cross-linking of the cell surface antigen is an initiating signal in acantholysis.     

Preclinical Studies Identify Non-Apoptotic Low-Level Caspase-3 as Therapeutic Target in Pemphigus Vulgaris

The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients’ biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including cancer.     

Targeted Disruption of the Pemphigus Vulgaris Antigen (Desmoglein 3) Gene in Mice Causes Loss of Keratinocyte Cell Adhesion with a Phenotype Similar to Pemphigus Vulgaris

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell–cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 −/− mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8–10 d weighed less than DSG3 +/− or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and “tombstoning” of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 −/− mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.     

Protease inhibitors prevent plasminogen-mediated,but not pemphigus vulgaris-induced,acantholysis in human epidermis

Pemphigus is an autoimmune blistering disease of the skin and mucous membranes. It is caused by autoantibodies directed against desmosomes, which are the principal adhesion structures between epidermal keratinocytes. Binding of autoantibodies leads to the destruction of desmosomes resulting in the loss of cell-cell adhesion (acantholysis) and epidermal blisters. The plasminogen activator system has been implicated as a proteolytic effector in pemphigus. We have tested inhibitors of the plasminogen activator system with regard to their potential to prevent pemphigus-induced cutaneous pathology. In a human split skin culture system, IgG preparations of sera from pemphigus vulgaris patients caused histopathologic changes (acantholysis) similar to those observed in the original pemphigus disease. All inhibitors that were tested (active site inhibitors directed against uPA, tPA, and/or plasmin; antibodies neutralizing the enzymatic activity of uPA or tPA; substances interfering with the binding of uPA to its specific cell surface receptor uPAR) failed to prevent pemphigus vulgaris IgG-mediated acantholysis. Plasminogen-mediated acantholysis, however, was effectively antagonized by the synthetic active site serine protease inhibitor WX-UK1 or by p-aminomethylbenzoic acid. Our data argue against applying anti-plasminogen activator/anti-plasmin strategies in the management of pemphigus.     

Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a life-long, potentially fatal IgG autoantibody-mediated blistering disease targeting mucocutaneous keratinocytes (KCs). PV patients develop pathogenic anti-desmoglein (Dsg) 3 ± 1 and antimitochondrial antibodies (AMA), but it remained unknown whether and how AMA enter KCs and why other cell types are not affected in PV. Therefore, we sought to elucidate mechanisms of cell entry, trafficking, and pathogenic action of AMA in PV. We found that PVIgGs associated with neonatal Fc receptor (FcRn) on the cell membrane, and the PVIgG-FcRn complexes entered KCs and reached mitochondria where they dissociated. The liberated AMA altered mitochondrial membrane potential, respiration, and ATP production and induced cytochrome c release, although the lack or inactivation of FcRn abolished the ability of PVIgG to reach and damage mitochondria and to cause detachment of KCs. The assays of mitochondrial functions and keratinocyte adhesion demonstrated that although the pathobiological effects of AMA on KCs are reversible, they become irreversible, leading to epidermal blistering (acantholysis), when AMA synergize with anti-Dsg antibodies. Thus, it appears that AMA enter a keratinocyte in a complex with FcRn, become liberated from the endosome in the cytosol, and are trafficked to the mitochondria, wherein they trigger pro-apoptotic events leading to shrinkage of basal KCs uniquely expressing FcRn in epidermis. During recovery, KCs extend their cytoplasmic aprons toward neighboring cells, but anti-Dsg antibodies prevent assembly of nascent desmosomes due to steric hindrance, thus rendering acantholysis irreversible. In conclusion, FcRn is a common acceptor protein for internalization of AMA and, perhaps, for PV autoantibodies to other intracellular antigens, and PV is a novel disease paradigm for investigating and elucidating the role of FcRn in this autoimmune disease and possibly other autoimmune diseases.     

Immune modulation in pemphigus vulgaris: role of CD28 and IL-10

Pemphigus vulgaris (PV) is an autoimmune bullous skin disease characterized by Abs to the desmosomal cadherin desmoglein-3. Although the autoantibodies have been shown to be pathogenic, the role of the cellular immune system in the pathology of pemphigus-induced acantholysis is unclear. To further delineate the potential role of T cell-signaling pathways in the pathogenesis of PV, we performed passive transfer experiments with PV IgG in gene-targeted mutant mice. Our results demonstrated that CD28-deficient mice (lacking a costimulatory signal for T cell activation) are 5-fold more sensitive to the development of PV than wild-type mice. To evaluate whether the higher incidence of disease was due to an impairment in intercellular adhesion of keratinocytes, we performed an in vitro acantholysis, using CD28-/- mice keratinocytes. No alteration in in vitro adhesion was detected in CD28-/--type keratinocytes. Because the CD28 molecule plays a pivotal role in the induction of Th2 cytokines, we examined the levels of a prototypic Th2 cytokine (IL-10) in CD28-/- mice. Lower levels of IL-10 mRNA were found in lesions from CD28-/- mice. To determine whether pemphigus susceptibility in CD28-/- was related to IL-10 deficiency, we performed passive transfer experiments in IL-10-/- mice that demonstrated increased blisters compared with controls. To confirm that IL-10 is involved in the pathogenesis, rIL-10 was given with PV IgG. IL-10 significantly suppressed the disease activity. These data suggest a potential role of IL-10 in PV.     

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.     

Cytokine profiles in pemphigus vulgaris patients treated with intravenous immunoglobulins as compared to conventional immunosuppressive therapy

Pemphigus vulgaris (PV) is a potentially fatal blistering disease of the skin and mucous membranes, characterized by the presence of autoantibodies against adhesion molecules (desmoglein, Dsg3) present on the surface of keratinocytes, which lead to the loss of cellular adhesion or acantholysis. The mainstay of treatment is conventional immunosuppressive therapy (CIST), i.e. high dose, long-term systemic corticosteroids with or without immunosuppressive drugs. Intravenous immunoglobulin (IVIg) has been used in patients refractory to CIST, and its use has resulted in long-term clinical remission. Since cytokines play an important role in the immunopathogenesis of PV, it would be useful to compare how both IVIg and CIST therapies affect cytokine levels in the serum of PV patients. Thus, the goal of this study was to conduct a comparative analysis of levels of various cytokines, during an 18 month consecutive period, after the initiation of CIST or IVIg treatment in PV patients, with similar extent and severity of disease in the two study groups, with 11 patients in each group. The cytokines measured were IL-1β, IL-6, IL-8, IFN-γ, IL-4 and IL-10. The levels of most of these cytokines were higher in the sera of untreated patients in both groups, compared to normal controls. The cumulative data collected over an 18 month period of treatment demonstrates that there is a gradual reduction in the levels of these cytokines, until they are at levels observed in normal individuals. The conclusions from this limited number of patients, prospectively studied, would suggest that both CIST and IVIg therapies are similar in their ability to influence a panel of cytokines in patients with pemphigus vulgaris.     

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.     

K14、K19在寻常型银屑病发病中的作用机制研究

目的:研究细胞角蛋白K14、K19在寻常型银屑病(PV)发病中的作用机制。方法:选择从2012年1月到2016年12月在西安交通大学第二附属医院皮肤科就诊的PV患者60例纳入本次研究。将PV患者在皮损区及未受累皮肤区分别取1份皮肤,制成存档蜡块,分别记为PV皮损组和PV未受累皮肤组各60例。另选同期在我院接受外伤手术治疗的健康皮肤60例作为对照组,分析K14、K19在PV中的阳性表达率以及表达水平,分析K14及K19在PV皮损区阳性表达的相关性。结果:各组的K14及K19阳性表达率以及表达水平比较差异有统计学意义(P0.05)。PV皮损组和PV未受累皮肤组的K14阳性表达率及表达水平明显高于对照组,K19阳性表达率及表达水平明显低于对照组,差异有统计学意义(P0.05)。PV皮损组的K14阳性表达率及表达水平明显高于PV未受累皮肤组,K19阳性表达率及表达水平明显低于PV未受累皮肤组,差异有统计学意义(P0.05)。根据Spearman法分析相关性发现,K14及K19在PV皮损区阳性表达中呈负相关(r=-0.871,P=0.000)。结论:K14及K19均在PV发病过程中出现异常表达,其中K14阳性表达较强,而K19阳性表达较弱,二者在PV皮损区阳性表达中呈负相关。临床上可考虑监测K14及K19的阳性表达水平,从而更好地辅助临床诊断及病情的预估,值得临床医师关注与重视。     

Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies

The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease.     

Pemphigus vulgaris antibody identifies pemphaxin. A novel keratinocyte annexin-like molecule binding acetylcholine

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte lambdagt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting approximately 40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced in Escherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [(3)H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.