Pemphigus autoimmunity: Hypotheses and realities

The goal of contemporary research in pemphigus vulgaris and pemphigus foliaceus is to achieve and maintain clinical remission without corticosteroids. Recent advances of knowledge on pemphigus autoimmunity scrutinize old dogmas, resolve controversies, and open novel perspectives for treatment. Elucidation of intimate mechanisms of keratinocyte detachment and death in pemphigus has challenged the monopathogenic explanation of disease immunopathology. Over 50 organ-specific and non-organ-specific antigens can be targeted by pemphigus autoimmunity, including desmosomal cadherins and other adhesion molecules, PERP cholinergic and other cell membrane (CM) receptors, and mitochondrial proteins. The initial insult is sustained by the autoantibodies to the cell membrane receptor antigens triggering the intracellular signaling by Src, epidermal growth factor receptor kinase, protein kinases A and C, phospholipase C, mTOR, p38 MAPK, JNK, other tyrosine kinases, and calmodulin that cause basal cell shrinkage and ripping desmosomes off the CM. Autoantibodies synergize with effectors of apoptotic and oncotic pathways, serine proteases, and inflammatory cytokines to overcome the natural resistance and activate the cell death program in keratinocytes. The process of keratinocyte shrinkage/detachment and death via apoptosis/oncosis has been termed apoptolysis to emphasize that it is triggered by the same signal effectors and mediated by the same cell death enzymes. The natural course of pemphigus has improved due to a substantial progress in developing of the steroid-sparing therapies combining the immunosuppressive and direct anti-acantholytic effects. Further elucidation of the molecular mechanisms mediating immune dysregulation and apoptolysis in pemphigus should improve our understanding of disease pathogenesis and facilitate development of steroid-free treatment of patients.


Introduction
Autoimmune pemphigus is al ife-threatening mucocutaneousb listeringd isease associated with IgG antibodiest argeting several types of keratinocyte antigens and eliciting epidermal clefting (acantholysis) via intracellular signalingactivating apoptotic enzymes (apoptolysis) [1]. Systemic administration of glucocorticosteroid hormones is essential to establish controlofdiseaseduring the acute stage [2]. Although glucocorticosteroid treatment is life-saving,i tm ay causes everes idee ffects, including death [ 3,4]. Therefore,p emphigus patients need drugs thatc an replace glucocorticosteroids. Thed evelopment of non-steroidalt reatment has been hampered by al ack of clear understandingo ft he mechanisms leading to keratinocyte detachment and death in pemphigus. This overview of recent advances in the knowledge of pemphigusa utoimmunity challengest he existing dogmas, helps resolve old controversies, andidentifies newp erspectives fort reatment.I te ncompasses knowledge on pemphigusv ulgaris (PV)a nd pemphigus foliaceus (PF), but specifically excludes reports on paraneoplastic pemphigus, or PNP, originally described by Anhalte ta l. [5], because this disease is not related to PV and PF.T he notion that PNP represents avariant of classical pemphigus stems from the facts that patients with PV or PF sometime have concomitant neoplasms [6][7][8]and that some patients with PNP develop anti-desmoglein (Dsg) 1a nd/or 3 antibodies-the hallmark of classical pemphigus [9]. In fact, PNP represents only one manifestation of the heterogeneousautoimmune syndrome-termed paraneoplastic autoimmune multiorgan syndrome (PAMS)-targeting both tegumental epithelium and internalo rgans [10]. In marked contrast to classical pemphigus, PAMS has an overall mortality more than 90% despite therapy,w ith progressive respiratory failure with clinical features of bronchiolitis obliterans being them ostf requentc ause of death [ 11]. Sloughing of bronchial epithelial cells contributes to the occlusion of the small airways that provides a potential mechanismf or the respiratoryf ailure [ 10]. Patients with PAMS develop mucocutaneous lesions that resemble pemphigoid,e rythemam ultiforme, lichen planus, andg raft vs. host disease, as well as the pemphigus-like variant that was termed PNP in the index patient with PAMS.Oral mucosal lesionsof painful, progressive stomatitis are the hallmark of the diseasea nd usually are the initial manifestation of PAMS [12]. The proposedp athogenesis of PAMS continues to evolve. It is clear that the immunopathologicm echanisms differ appreciably from those responsible for the lesionso fc lassical pemphigus. The spectrumofP AMSincludes patients with disease predominantly or exclusively mediated by the cellmediated autoimmunity effectorsand those with both autoantibodies and cellular cytotoxicity [13].

Pemphigusa utoantigens
Following the discoveryo fI gG autoantibodies in patients with PV [14]a nd PF [15], numerous attempts havebeen madetoidentify targetedantigens. The patient's serum and isolated IgG fraction were utilized in the immunoprecipitation and immunoblotting experiments using the epidermal or keratinocyte culturep roteins as well as saliva andu rine as substrates. Although the low-sensitivity approaches, such as fluorography with metabolically labeled keratinocyte proteins preabsorbedw ith human serum [16], demonstrated single protein bands,a more sensitiveb ut less specific immunoblotting technique revealed more thanadozen of targeted keratinocyte proteins [17]. An enhanced sensitivity immunoprecipitation assay demonstrated that different PV or PF patients produceantibodies recognizing both commonand unique antigens [18].
Identification of the nature of proteins targeted by pemphigus autoimmunity is as ubject of intense research. Originally,i tw as assumed that the proteins with the MW of approximately 60 kD or less are "contaminating"k eratinst hatd on ot represent meaningful targets.H owever, recent studies demonstrated that only 2% of pemphigus and normal sera contain anti-keratin antibodies [39]. Furthermore, a 66 kD antigen recognized by PV IgG-am embrane glycoproteinc omposed of two apparently identical subunits of 33 kD-was used to raise rabbit antibody that induced PV-likep henotype in neonatal mouse [27]. Nevertheless, the candidates for the pathophysiologically relevant PV and PF antigens were selected among af ew bands migratingw ith ah igherM W, wherein the 130 and 160 polypeptides were most commonly seen [16,29]. The antigens with these MWs were identified as Dsg 3 [ 17] and Dsg 1 [ 40], respectively. Thereafter, exploration of the nature of pemphigusa ntigensh as been hampered by a simplistic (or" monopathogenic" [41]) explanation of pemphigus pathophysiology through the "Dsg compensation" hypothesis placing Dsg 1/3 in the center of the pathophysiologic loop [42].
The Dsgc ompensation hypothesis maintains that anti-Dsg 1a nd 3a ntibody profiles in pemphigus sera and the normal epidermal distributions of Dsg 1and 3 determine the sites of blisterf ormation and that eitherD sg 1o rD sg 3a lone is sufficientt om aintain keratinocyte adhesion [42]. The threep ostulates of this hypothesis are as follows: (1) in the superficial epidermis of PF patients, where Dsg 1without Dsg 3 is expressed, anti-Dsg 1a ntibody alone can cause blisters;(2) Dsg 3antibodyalone is sufficienttocause Figure 1. Characterization of anti-keratinocyte antibody profiles of PV and PF sera by immunoprecipitation with proteins from cultures of human epidermal keratinocytes resolvedb y7 .5% SDS-PAGE. Modified from Ref. [18].
suprabasal splitinthe oral mucosa of PV patients that lacks Dsg 1; and (3) skin lesionsi nP Vp atients develop when both Dsg 1a nd Dsg 3a ntibodies are present. The major flaw of this hypothesis is an assumptiont hatt he integrity of thes tratified squamous epithelium enveloping skin ando ral mucosa relies entirely on Dsg 1a nd 3m olecules. If that would be the case,t he epidermis would have disintegrated to as ingle cell suspension in the PV patientswho develop both anti-Dsg1and 3antibodies ( Figure 2).
The monopathogenic explanation of localization of intraepidermal clefts in PV and PF through Dsg compensation hypothesis ignorest he complexity of homo-andh eterophilic interactionso fs even known desmosomal cadherins, i.e. Dsg 1-4 and desmocollin (Dsc)1 -3.I nr eality,D sg 3a lone cannot sustain epidermal cohesion. This is evidentfrom the facts that Dsg 3c annot compensate for al oss of Dsc 3i nt he conditional Dsc 3 null mutant mouset hate xhibits suprabasal acantholysis ando verts kin blistering [43]. Furthermore, the in vitro experiments demonstrated that extracellular domain of Dsg 3m ediates only aw eak homophilica dhesion [44]. Lack of skin blistersi np atientsw iths triate palmoplantar keratoderma featuring adeletion mutation in the extracellular domain of Dsg 1a nd in micew ith engineered or spontaneous mutations of Dsg3(reviewed in [45,46]) clearly indicate that the integrity of epidermis does not depend solely on Dsg 1a nd 3. The electron microscopic studies demonstrated that keratinocytes deprived of endogenous production of Dsg1or3due to gene silencing via RNA interferencec ontinue to formd esmosomes [47]. Apparently,t he redundancy of desmosomal cadherins rendersthe external integumentsufficient integrity and durability.
The first evidence that keratinocyte antigens other than Dsg 1a nd 3a re pathophysiologically relevant in pemphigus was provided by experiments showing the ability to induce suprabasal acantholysis andgross skin blistersin Dsg3 2 / 2 neonates by passivetransfer of PV patients' antibodies [ 48]. In this model, murine epidermis lacked Dsg 3a nd the passively transferred PV IgGs lacked anti-Dsg1antibody.H ence, the injected PV antibodiesthat caused blisterscouldtarget only the non-Dsg 1a nd 3a ntigenst hat mediated and/or regulated keratinocyte adhesion. This observation promptedfurther investigations into the nature of pemphigusa ntigens( reviewed in [41]). By now, over 50 humanp roteinsh ave been reported to specifically react with pemphigus IgG (Table I). In addition to the known desmosomal cadherins and several othertypes of adhesion molecules,the hitherto identified pemphigus antigens include cell membrane (CM)r eceptors, immunologic/hematologic antigens, neuronal/oncologic antigens, and thyrogastric cluster antigens.
Of particulari nterest is ar ecently discovered autoimmunity against an ovelm embero ft he peripheralm yelin protein( PMP)-22/gas3f amily termed PERPa sw ell as the structurallyr elated PMP-22 [49,50]. Knockoutm icel acking PERP display ap henocopy of PV [51], which gaver ise to a notion that the biologic function of PERPislimited to desmosomal stabilization [52]. However, PERP is expressed in various types of cells that do not form desmosomes [53], which argues against its exclusive biologic function in desmosomal adhesion. Recent findings and its putative tetraspan transmembrane topology implicate ar ole for PERPi nt he extrinsic apoptotic pathway that involves direct interaction between adaptor proteins andthe receptor complexes activating caspase 8, i.e. PERPi sanovel cell death receptor [54]. Hence, dissolution of desmosomes and PV-like intraepidermal split in Perp 2 / 2 mice apparently result from aberranti nside-out signalinga long the altered cell death pathways.
The types of otherA ChRs targetedb yp emphigus autoimmunity have been investigated using various experimental approaches. Reactivity of PV IgG with the mixedm uscarinic and nicotinic a 9A ChR was observed in the immunofluorescence blockinge xperiments, wherein stainingo fm onkey esophagus by rabbit anti-a 9a ntibodyw as preventedd ue to preincubation of the substrate with PV antibodies [36]. Recently,t he proteomics approach has demonstrated that PV antibodiesreact with a 10 subunit that can be ap arto ft he pentameric a 9 a 10 nAChR (unpublished).T herefore,t he binding sitef or a 9 antibody in the heteromeric a 9 a 10 nAChR also can be hindered by anti-a 10 antibody present in PV sera. Whena dded to keratinocytem onolayer,a nti-a 9 antibody produced acantholysis [36],i ndicating that the alteration of either a 9o r a 10 subunit inactivates functioning of the a 9 a 10 channel coupled to the regulation of keratinocyte adhesion.
Al arge spectrumo fo rgan-non-specific antigens that can be targeted by pemphigusa utoimmunity Nicotinamide/nicotinic acid mononucleotide adenylyltransferase2 [49] Unclassified neuronal antigen [348] Peripheral myelin protein 22 [49] Thyrogastric cluster antigens Gastric parietal cell antigen [348] Glutamic acid decarboxylase (GAD65) [348] Proline dehydrogenase 1 [ 50] Microsomal antigen [348] Thyroperoxidase [349] ( Table I) has been recently reviewed [39,65,66]. Although contribution of organ-non-specific antigens to keratinocyte detachment and death in pemphigus remainst ob ee lucidated, it is plausible to speculate that some of them, e.g. tumorn ecrosis factor (TNF) receptor superfamily member 5, are involved in the activation of the extrinsic apoptotic pathway and others,e.g. NADHdehydrogenase-like protein, in the activation of the intrinsic pathway [37]. Of particular interest is av eryh igh intensity of reactivity of PV IgG with Fc-IgG 2 [49]. The fact that it has . 95% homology with Fc-IgG 1 maye xplain ah itherto mysterious ability of the chimeric baculoproteins containing the constant region of human IgG 1 and Dsg 1orDsg 3, in contrast to the extracellular portion of Dsg 3a lone, to absorb out all disease-causing antibodies [67 -69].T hus,i tc an be concluded that pemphigus autoimmunity is directedagainst multiple organ-specific and non-organ-specific proteins, some of whicha re also targeted in othert ypes of autoimmune diseases.

Pemphigusa utoantibodies
Explorationo fn ovel self-antigensr eactingw ith pemphigusa ntibodiesr emains oneo ft he top priorities in pemphigus research, because binding of patients' IgGs to these antigens triggersk eratinocyte detachment and death in PV and PF.A lthough them echanism of blistering in patients' skin andm ucosai nvolvesv arious factors, including cell-mediatedc ytotoxicity,p roteolytic enzymes, and pro-inflammatorya nd pro-apoptotic cytokines, the principal role of anti-keratinocyte antibodies in the pathophysiology of autoimmune pemphigus has been well documented. The major lines of evidence are as follows: (1) occurrence of transient pemphigus-like skin lesionsi nn eonates born by mothersw ith active pemphigus; (2) induction of pemphigus-like phenotype upon passivet ransfero fp atients' IgGs to neonatal mice; and (3) elimination of diseasec ausing activity of patients' IgG fraction due to absorption with antigenic constructs. Despite enormouse fforts to single out an autoantibody responsible for eitherPV or PF,n one of hitherto reported results provides compelling evidence in favor of the monopathogenic hypothesis of pemphigus immunopathology. Afi rsts uccessful attempt to reproduce disease phenotype in animal model was reported by Peterson and Wuepper [27]w ho observed small vesicles and limiteda reaso fs uprabasal acantholysis in the skino f neonatal mouse 36 ha fter intraperitoneal injection of averyhigh (40 mg) dose of rabbit IgG antibody raised against the 66 kD pemphigus antigen.U nfortunately, that antibody was not affinity purified on the antigenic peptiden or wasi ts reactivity with keratinocyte proteins characterized. Some microscopic blisters also could be induced by pemphigusI gG eluted from ar ecombinant amino-terminus of Dsg 3 [ 67]. That IgG fraction uniquely recognized a1 30 kD protein of SDS-PAGE-resolved keratinocyte proteins. Although the anti-Dsg 3antibody did not cause gross skin blisters,t he obtainedp henotype was deemed significant and the antibody termed "pathogenic". Paradoxically, grossb listersi nt hats tudy were induced,a lbeitb yt he pemphigus IgGf raction depleted of Dsg 3a ntibody,i ndicatingt hat non-Dsg 3a ntibodies were actually pathogenic. This finding, however, was interpreted as evidence that diseasecausing antibodies are directedt oo ther" conformational" epitopesofD sg 3 [ 67].
Next, it was hypothesized that the extracellular domain of Dsg 3s hould be combined with the Fc-portiono fh uman IgG 1 to create ap roper conformation of the disease-specificD sg 3a ntigen [68]. Indeed, the obtained chimeric protein absorbed out alld isease-causing antibodiesa nd injection of preabsorbed PV IgG fraction to neonatalm ice did not producee xtensive skin blisters. Furthermore, the modified chimerat hatc ontainedH is residues attached to the Dsg3-Ig construct eliminatedd isease-causing antibodies from serao fp atientsw ith PAMS,a nd the eluted antibodies caused gross skin blisters in neonatalmice [70]. Thatsame strategy was applied to create a"proper" conformational epitope of the Dsg1antigen capable of eliminating the diseasecausing antibodies from PF sera [69]. Surprisingly, the antibodies eluted from neither chimeric construct were examined for their reactivities against keratinocyte proteins to confirm their unique specificityfor the 130 kD Dsg3and 160 kD Dsg 1a ntigens.
Successive studies demonstrated that the results obtained in experiments with recombinant( r)Dsg1-Ig-His and rDsg3-Ig-His chimeras that led to a conclusion thata nti-Dsg1a nd anti-Dsg 3a ntibodies are pathogenic in PF and PV,r espectively,w ere complicated by the presence of non-Dsg antibodies [18]. Figure 3s hows that antibodies eluted from the chimeric constructs react with am ixture of keratinocyte proteins, includinganon-Dsg 31 30 kD protein produced in the Dsg3 2 / 2 keratinocytes used as a sourceo fa ntigens. Noteworthy,t he fact that the rDsg3-His construct was recognized by PV antibodies indicated that it actually had aproper confirmation. In contrast, the Fc-containing Dsgc onstructs evidently did nothavep roper confirmation.
These constructs were ill designed,b ecause the ability of Fc fragment to mediatet he Fc-Fc interactions known as Fc-mediated immune precipitation [71,72] was ignored. It has been recently demonstrated that the CH 2 and CH 3 domain regions of the Fc fragment used by Amagai et al. [68 -70] in their pathogenic antibodye limination experiments provide an interface for the antigen-unspecific binding with another IgG moleculei ni mmune complexes [73]. Thus,a na ntigen-unspecific,F c-mediated immune precipitation cane xplaina dsorptiono f multiplea ntibodies on the chimeric Dsg baculoproteins. However, one of the antibodies evidently was absorbed specifically.Aprotein arrayhas revealed that PV patients produce anti-Fc-IgG antibody [49].
Anti-Dsg 3a ntibody produces ad esmosomal split without keratin retraction, apparently due to steric hindrance of Dsg 3f rom opposingc ells [ 84,85]. In marked contrast, numerous classical [86 -89] and contemporary [55,90] electron microscopic studies of PV patients' skin vividly demonstrated that desmosomes remain intact till the late stages of acantholysis when they are cleaved behind the desmosomal plaque, due to shearing forces produced by collapsing cells, andfl oat freei nt he intercellular space. Thus, althought he alterations of keratinocyte adhesion in recipient Rag2 2 / 2 mice are different from those taking place in patient's skin, these mice are still useful for studying the regulation of adaptive immuner esponse to Dsg 3.
Although anti-Dsg 1a nd 3a ntibodies, alone or in combination,a re note xclusively responsiblef or triggering intraepidermal blistering in patient'ss kin, they have ad iagnostic utility.T he Dsg 1a nd Dsg 3 ELISAsp rovide as implea nd highlys ensitive approach to confirm the initial diagnosis of autoimmunep emphigus and differentiate it from other blistering diseases. The true value of ELISA results for patient management andprognosis, however, remains uncertain. The Dsg 1a nd Dsg 3I gG antibody titers do nota lways correlatew ithp emphigusd isease activity [91,92] nor do they predict exacerbation and relapse of the disease [ 93]. Dsg3a ntibody can be absent in PV patientsw ith active diseasea nd present during remission [ 94 -96].F urthermore, anti-Dsg 1 or 3antibodieshavebeen detected in healthy subjects, relatives of pemphigus patients, andp atientsw ith irrelevant medical conditions [97 -109].
It was originally thought that the clinical phenotype of pemphigus is defined by the anti-Dsgautoantibody profilea sf ollows: anti-Dsg 1a ntibodya lone is associated with PF,a nti-Dsg 3a ntibody alone-with mucosal variant of PV,a nd both antibodies-with mucocutaneous varianto fP V [ 115]. Although Dsg 1 antibody indeed appearst ob eareliable serologic marker of PF and Dsg3antibody-that of PV,u pt o 58% of PF patients and 12% of patients with endemic PF (FogoS elvagem) were reported to develop antibodiesa gainst both Dsg1andD sg 3 [108,116,117]. Furthermore, it has been conclusively demonstrated that Dsg 1a nd Dsg3t esting cannot differentiate between various morphologic subtypes of PV [96,118,119]. In one study,f or instance, 46% of PV patients did not havet he PV phenotype (mucosal or mucocutaneous) predicted by their Dsg antibody profile [118]. In another study,t he Dsg 1 þ /Dsg 3 þ patternw as observedi n1 5% of PV patientsw ith exclusive mucousmembrane involvement [96].
Ar ecento bservation that an increase in Dsg 1 antibody titer in aP Vp atient haso ccurreda lready after the patient had startedt reatment and went into clinical remission [120] supports the notion that anti-Dsg antibodies "witness" rathert han trigger PV,i .e. that production of these autoantibodies is the result rathert han the causeo fe pidermal blistering in pemphigus [121]. Reactivity of pemphigus autoantibodiesw ith both extracellulara nd intracellular domains of Dsg1a nd Dsg 3 [ 122] suggests that these antibodies are produced alreadyafter the whole Dsg molecules have been released from the CM of damaged keratinocytes into the intercellular space and became available to antigen-presenting cells. The presence of the N-terminal portion of Dsg 3inhuman sera [123] lends additionals upportf or the Dsg sloughing hypothesis.
Perhaps other desmosomal cadherins are also shed from the CM of damaged keratinocytes. The presence of anti-Dsc1 -3 antibodiesi nP Vp atientsh as been discovered relatively long ago [124,125], but the interest to these antibodies was diminishedb yt he reports that none of 45 [126] or 74 [127] PV patients tested by ELISAh ad anya nti-Dsca ntibodies. However, ar ecent study showing that the Dsc3loss of function model exhibitsaphenocopy of PV [43] suggested that anti-Dsc3antibody contributes to PV. As expected, the monoclonal antibodyr aised against the extracellular domain of Dsc 3c aused intraepidermal blistering in an in vitro model of human skin and al oss of cell -cell adhesion in the keratinocyte culture [128].
In the most recent study,6out of 38 PV and1out of 85 normal serum samples immunoprecipitatedDsc 3 [129]. Furthermore, while incubation of patient'sIgG with human keratinocytes caused the loss of intercellular adhesion, adsorption with rDsc 3p revented this effect [ 129]. Thus, while antibodiest ot he desmosomal cadherins mayb ep laying as cavenging role by eliminating CM debris from the intercellular spaces of damaged epidermis theym ay obstruct homophilic andh eterophilicb inding between the neighboringk eratinocytes, thus contributing to acantholysis.
The initial insult that triggersk eratinocyte damage in pemphigusi sa pparently sustained by autoantibodiest ot he cell membrane receptorsw hose ligation causes cell shrinkage-the earliest sign of keratinocyte detachment in pemphigus lesions [55,86 -90] that leads to ripping desmosomes off the CM. Indeed,i th as been demonstrated that rabbit anti-a 9A ChR antibody causes keratinocyte shrinkagea nd roundingu p [ 36].N oteworthy, pemphigusI gG produces similar morphologic changes in keratinocyte monolayers [ 130].
Although the hypothesis of primaryi nvolvement of autoantibodies against canonical AChR subtypes in pemphigus pathophysiology is awaiting its in vivo confirmation, the alreadyc ompleted studies have demonstrated essential roleo fa ntibody against the non-canonical ligand of AChRs termed pemphaxin [62]. Preabsorption of PV sera with recombinant pemphaxin eliminated acantholytic activity and eluted antibody immunoprecipitatedn ativep emphaxin. Although anti-pemphaxina ntibodya lone did not causes kinb listers in vivo, itsa ddition to the preabsorbed PV IgG fraction restoredthe acantholytic activity of passively transferred antibodies [62].
These observations indicate that pemphaxin is an essential parto ft he pool of keratinocyte cell surface antigens that shouldb es imultaneously targeted by autoantibodies to induce acantholysis. The antimitochondrial antibodies from different PV patients that recognized distinct combinationso fa ntigens with apparent MWs of 25, 30, 35, 57, 60, and 100 kD evidently area lsop athogenic, because their absorption abolished the ability of PV IgG to causek eratinocyte detachment both in vitro and in vivo [37].
Involvement of multiple autoantibody specificities in pemphigus pathogenesis is explained through the "multiple hit" hypothesis [134] as follows: anti-AChR antibodies trigger acantholysis by weakening cohesion of neighboring keratinocytes due to inhibition of the physiologic control of their polygonal shape and intercellular attachment. The affected keratinocytes shrink, causing desmosomes to be sloughed in the intercellular space. The adhesion molecules floating free in the intercellular spacebring aboutareciprocal production of scavenger antibodies that, in turn, saturate epidermis, thus preventing nascent desmosome formation by steric hindrance. Thus,a ccording to the multiplehit hypothesis, pemphigus results from as ynergistic and cumulative effects of autoantibodies targetingk eratinocyteC Ma ntigens of different kinds including (i) molecules that regulate cell shape and adhesion (e.g. AChRs); and (ii)m olecules that mediate cell-cell adhesion (e.g. desmosomal cadherins). Severity of the disease and exactc linical picture dependo nt he ratio of different kinds of autoantibodiesine achparticular patient.
In conclusion, different patients develop distinct constellationso fa utoantibodiesw hich,t ogether with thei ndividual's re-epithelialization abilities, determine clinical severityo fd isease, its natural course, and response to treatment. The Dsg 1a nd 3 antibodies are the sensitive markersofpemphigus, but their primaryr ole in the pathogenesis of PF and PV, respectively,i so verestimated. Therefore,n ot surprisingly,c linical trial of the Dsg3peptides (PI-0824 vaccine) has not shown the anticipated clinical or immunologic activity (reviewed in [135]). Apparently, an attack by ac onstellationo fa utoantibodies simultaneously targeting several keratinocyteproteins is requiredt od isrupt the integrity of epidermis. The multipleh it hypothesis reconciles findings of anti-AChR autoimmunity with the fact that pemphigus patients also develop autoantibodiest oa dhesion molecules as well as various other proteins. The hypothetical sequence of immunopathologic and biochemical events leading to acantholysis in pemphigus is shown in Figure 4. Future studies shoulddefine the autoantibodies that sustain an initial insult triggering keratinocyte detachment andt hose produced as ar esult of primaryc ell damage to clean up the proteins released in the intercellular spaces by damaged cells.

Regulationofp emphigus autoimmunity
Although autoimmunity is an ormal event, autoimmuned iseases result from an aberration of the normal phenomenon [136]. The etiology of this switch in pemphigus is apparently multifactorial, with the final common pathway being aloss of normalselftolerancei nt he stratifieds quamouse pithelium. Analysis of genetic factorsc ontributingt oP Va nd PF has shown thatt he sameg enetic regions can contribute to both forms of the disease( reviewed in [137]).T he HLAg enes arep robably them ost significantg enetic predisposition factors, because they play an important role in the antigen presentation process, whereas other loci may participate in an additive or epistastic manner.
Population studies have consistently shown al ink between certain class II HLA alleles andd istinct ethnic groups of pemphigus patients. Ar ecent study involving al arge cohorto fW hite European and Indo-Asian patients with PV confirmed associations with the alleles HLA DRb 1 * 0402 and 1404, and DQb 1 * 0302 and 0503 [138]. The DRb 1 * 1404 was the strongestr isk factor in the Indo-Asian groupa nd DRb 1 * 0402-in the White European group.InWhite Europeans, as ignificant association was also shown fort he novel allele DRb 1 * 1454 [138]. Also,i t has been documentedthat HLA-DRb 1 * 0402 is associated with PV in Jewish andH LA-DQb 1 * 0503 in non-Jewish populations [139 -141]. Although HLA studies have shown that susceptibility to PF also correlates with the presence of DR4, DR14,a nd DR1 alleles, in contrast to PV,n os ingle DR4 or DR14 allele was found to be overrepresented in PF patients [137]. The HLA-DR/DQ distributions does not differ among PV patients according to the presence or absence of anti-Dsg 1c oexisting with anti-Dsg3 [ 142].
Although the basis for autoimmunityinpemphigus remainsunrecognized, the regulation of anti-keratinocyte autoimmunity in PV and PF has been studied basedo nt he assumption that Dsg 1/3 antibodies solely represent pemphigus autoimmunity.I tw as reportedthat the Th1 and Th2 cell recognition of Dsg 3p eptides is restricted by HLA-DR b 1 * 0402 and/or HLA-DQ b 1 * 0503, and that the proliferative response of autoreactive Th cellsc an be blocked by anti-DR and anti-DQ antibodies, respectively [143 -146].
Aloss of self-toleranceagainst Dsg 3inboth Tand Bl ymphocytes was found to be required for efficient production of anti-Dsg3IgG antibodies [147 -149]. The anti-Dsg3antibodyp roduction in micew as inhibited by the anti-CD154 monoclonal antibody that blocksC D40L-CD40 interaction [150].T he CD8 þ Tcellsspecific for Dsg 3were also detected in PV patients [151], which is in keeping with earlier observation of the autoreactive cytotoxic Tl ymphocytes are sensitized to putative keratinocyte antigens in PV patients [152].
Autoimmunity to certain epitopes of Dsg 3may be a normal event, because Dsg 3-reactive Bcellsaswellas Th1 and Th2 cells are present in normali ndividuals [97,100,101,146,151,153,154].T he presence of autoreactive Bc ellsi se videnced by production of anti-Dsg3antibodies by healthy relatives of PV patients [ 97,100,101,154]. The presence of Dsg3reactive Th1 cells has been demonstrated in healthy carriersofPV-associated HLA class II alleles, andthe Dsg3 -reactiveT h1 clones derivedf rom these individuals were restricted by HLA-DRb 1 * 0402 and DQb 1 * 0503 [146,151,153].
There is ap redominance of autoreactive Dsg 3reactive Th1 cells in healthy individuals and Dsg 3reactive Th2 cellsinPVpatients [146]. However, Dsg 3-reactive Th2 cells are detected at similar frequencies in acuteonset, chronic active, and remittent PV,while the numbero fa utoreactive Th1 cells exceeds that of Th2 cells in chronic active PV [146]. Likewise, Dsg 1-responsiveTh1 and Th2 cells were also found both in patientswithP Fand in healthy individuals [155].
Defectsi nT regs haveb een reported in aw ide varietyofhumanorgan-specific autoimmune diseases [156]. Whilethe inductionofTreg suppressive activity is specific and requiresa ntigenic stimulation through T-cell receptor, the suppression exerted by Tregs is antigen nonspecific [157]. Various Tregs can employ distinct mechanismst oc ollaboratively regulate the duration and magnitude of an immuner esponse [158]. It has been postulated that active immune suppression operates in healthy individuals possessing Dsg 1-and Dsg 3-reactive Tc ells, and that an imbalance of the putative relationship between autoreactive Th and Tregs (Tr1) cellsi sc ritical to the development of pemphigus [159]. In support, the CD4 þ CD25 þ hi Tregs are decreased in peripheral bloodo fP Vp atients [160]. Furthermore, as ubset of Dsg 3-reactive, interleukin (IL)-10-secreting Tr1cells was found in the majority of healthy carrierso f PV-associated HLA class II alleles and only in , 20% of PV patients [161]. However, ar ecent study demonstrated thatP Vs kinl esions contain both Foxp3-expressing cellsa nd IL-17 producing CD4 þ cells [162].
Thus, deficiencyofTregs in PV patient's bloodisnot accompaniedbyadecrease in Tregs in PV lesions, and adecrease in Tregs in peripheral blood mayresult from accumulation of Tregs in skin lesionsa nd draining lymphn odes [162]. Alternatively,o ra dditionally, pemphigus autoimmunity may be triggeredv ia the pathway involving activation of toll-liker eceptors (TLRs). This mechanism has been suggested by a recent demonstrationo fr eversible relapseo fP F triggeredb yt he TLR7a gonist imiquimod [163]. In this scenario, B-cell tolerance is broken due to ligation of B-cell receptor andT LR by self-antigen/TLR ligand, leading to breakage of T-cell tolerance and activation of autoreactive Band Tcells [164].
In conclusion, regulation of Dsg 1a nd 3a ntibody production agrees with the basic postulates of fundamentali mmunology on Tc ell-Bc ellc ooperation. Th1 andT h2 cellsf ound in patientsw ith PV and healthy carriersofPV-associated HLA class II allelesr ecognize identical epitopeso ft he Dsg 3 ectodomain presented by antigen-presenting cells. Ad ecrease in Tregs in peripheral bloodo fP V patients does not validatet he postulated deficiency of the immunosuppressive activity,b ecause Tregs are present in PV lesions. Future studies of the immunoregulatorymechanisms of PV should characterize ther eputed interplay betweenT regs and Th17 cells (Figure 5), andi dentify the role for TLRs that can regulate function of Th1, Th2, Th17 cells, and Tregs.

Molecular mechanisms of keratinocyte detachment in pemphigus
Several hypotheses haveb een put forward to explain them echanism of pemphigus acantholysis.T he classical explanation through the hypothesis of steric hindrance of Dsg 1-and Dsg 3-mediated adhesion by respective antibodies [165,166] has been challengedb yn umerous reports showing activation of specific signalingpathways in keratinocytes exposed to pemphigus IgGs. The steric hindrance hypothesis is basedo nt he erroneous assumptions that binding of PV IgG in the epidermis is limited to desmosomes [167]a nd that the phenocopies of PF andP Vi n mouse skinc an be induced by passive transfer of PV IgGs recognizing uniquely the desmosomal cadherins Dsg 1a nd 3, respectively [68,69].
In fact,a lthough Dsgm olecules arei ndeed predominantly localized to desmosomes [168], binding of PV IgG extends well beyond the desmosomes decorating the entire surface of keratinocytes [169]. As alreadym entioned, the Dsg-Fc-IgG constructs were found to be not specific for Dsg 1or3antibodies [18,36]. Furthermore, PF IgG has recentlyb een shown to caused issociation of keratinocytes without blocking Dsg1homophilic transinteraction [170]. Surprisingly, this cumulative evidence that non-Dsg molecules are targeted by pathogenic PV IgG in the interdesmosomal areasi sb eing interpreted as targeting of Dsg3located outside of the desmosomes [171].
Thea lternativeD sg 3a lterationh ypotheses proposed by different authors [ 171 -173] haveo ne commont heme. They are basedo nt he assumption that all outside-in signals elicited due to binding of PV IgG to keratinocytes emanateexclusively from Dsg3. To account foraverybroad spectrumofsignals, it was inferred that Dsg 3can act as both adhesion receptor and signal transmitter [171 -173].T he following hypothetical chain of events was envisioned: (1) PV IgG signaling is initiated by ligationo fn onfunctional pool of Dsg3o utside of the desmosome; (2) when ligated by an autoantibody,t hese putative nonfunctional Dsg3molecules, eitherm embrane associated or internalized or both, send signals thatimpair Dsg 3 trafficking intoa nd out of desmosome; and (3)t he supposedly impaired Dsgt raffickings pecifically depletes Dsg 3f rom desmosomes without changes in otherf unctionalproteins.
The notion aboutprincipal roleofDsg 3inPVIgG signaling stems from experiments in whicht he whole PV IgG fraction, ratherthan affinity purified patient's anti-Dsg 3a ntibody, was used to elicit biologic responses [174][175][176][177][178][179][180][181][182][183].W hen interpreting results, it was assumed that the whole plethora of PV IgG effects, includingDsg 3e ndocytosis andactivation of various signaling cascades, resulted exclusivelyf rom Dsg 3ligation by an autoantibody.However, Jennings et al. [184] have recentlyd emonstrated an explicit biologic effect of PV IgGo nt he keratinocytes expressing the Dsg 3m olecules whose "pathogenic" epitopes were hidden due to cell preincubation at 4 8 C with the anti-Dsg3monoclonal antibody AK23 that reportedly reproduces PV phenotype [81,185] and induces PV IgG-like signaling [186]. Therefore, depletiono fD sg 3i sa pparently a secondaryevent resulting from an inside-out signaling caused by keratinocyte response to the pathogenic autoantibodies that deliver an initiali nsult. The primaryr ole of anti-PEPR antibodyi nt he depletion of Dsg 3was suggested by an observation that binding of PV antibodiest riggers internalization of PERP, whiche nhancesd epletion of desmosomal Dsg 3a nd intercellular adhesion defects [187]. Thus, endocytosis of the immunec omplexes containing non-junctional Dsg 3a nd depletion of Dsg 3f rom desmosomes apparently represent two independent events, with the former being an atural outcome of formation of antigen -antibody complex on the CM and the latter resulting from the inside-out signalinga ltering function andtrafficking of desmosomal cadherins.
The outside-in signaling elicited due to binding of PV IgGst ok eratinocytes proceeds viad ifferent pathways, consistent with simultaneous ligation of several types of cell surface receptorsb yd istinct antibodies produced by pemphigus patients. Different researchg roups reported engagement of Src, epidermal growth factor receptor (EGFR) kinase (EGFRK), cAMP,protein kinases Aand C(PKC), phospholipase C, mTOR, p38 MAPK, JNK, other tyrosine kinases, and calmodulin [37,47,175,188 -191].The preferential signaling pathway downstream of targeted selfantigensi sa pparently determined by au nique composition of the pool of anti-keratinocyte antibodiesp roduced by each PV patient,b ecause IgG fractions from different PV patientsexhibit distinctive time patterns of kinase activation [37].
Timing of kinase activation is critical for understanding the hierarchy of signaling events leading to acantholysis. The time course studies demonstrated that the activities of Src and EGFRK peak at 30 -60 min after exposure to PV IgG [37,47], suggesting that engagement of Src/EGFRK is ak ey step that relays signals emanating from ligated antigens to the intracellular effectorsa ffecting keratinocyte adhesion and viability.
Activation of EGFRK due to binding of PV IgG to keratinocytes is followed by phosphorylation of its downstream substrates, the MAPkinase ERK and the transcriptionfactor c-Jun [189]. Activation of PKC is also one of the earliest events in PV IgG-induced acantholysis [192]. The elevation of p38M APK activity caused by antibodies from some PV patients can be observed alreadyat15min, while the majority of PV IgGs activate p38M APK after ap rolonged incubation [37]. Thus,i ti sb ecoming evidentt hat an array of interconnected signaling cascadese manating from different cell surface antigens simultaneously targeted by ac onstellation of patients' anti-keratinocyte antibodies triggera cantholysis andk eratinocyte death in pemphigus.
Twod ifferent approaches have been used to elucidate involvemento fD sg 3i nt he PV IgG-mediated signaling and define relevant pathways. Through one approach, the Dsg1 or Dsg3 genes in cultured human keratinocytes were silencedusing the RNA interference technology [47]. Transfection with small interfering RNAst hat inhibited expression of eitherD sg 1o rD sg 3o rb oth in allc ases blocked approximately 50% of p38M APK activity,b ut only slightlyaltered the PV IgG-dependent raise in Src and EGFRK activities. To avoid any possible contribution to PV IgG signaling by residualD sg 3p rotein, a separate series of experiments employed keratinocytes grown from the epidermis of neonatalDsg 3knockout mice [ 37]. It was documented that lack of Dsg 3d id not affect the ability of PV IgG to activate Src and EGFRK.
However, in the absence of Dsg 3, the PV IgGdependent activation of both p38 MAPK andJ NK was significantly reduced. Because both p38 MAPK and JNK can be activated secondaryt ok eratinocyte shrinkage and detachment [193][194][195],a nd since keratinocyte damage in PV is associated with activation of the cell deathp rogram (reviewed in [196]), it was important to determine whether p38 MAPK andJ NK activationp recedeso rf ollows launching of the apoptotic cascade. Inhibitorso ft he executioner caspases abolished activation of JNK and the late p38 MAPK peak [37], indicating that these activities were indeed triggeredbythe cell injuryrather than by the PV IgG binding to keratinocyte antigens. This supposition has been recently corroborated by a reportthat p38 MAPK activation occursdownstream at the loss of intercellular adhesion in PV [197].
Thus,a lthough thep ool of anti-keratinocyte antibodies produced by PV patientsc ontains anti-Dsg 1a nd/or 3a ntibodies, published studies indicate that non-Dsg antibodies are the major contributors to early signaling events.E arly activation of the Src/EGFRK and PKC-dependent pathwaysi sa pparently pathogenic because it leads to acantholysis [47,189], late activation of p38MAPK is secondaryto cell detachment [197], whereas activation of the cAMP/proteink inase As tepa ppears to have a protective function [191].
The principal signaling event leading to acantholysis is triggered due to antibody interference with the physiologic control of keratinocyte survival, shape, and adhesion. Fori nstance, blocking of keratinocyte AChRs interferesw itha uto/paracrinec ontrol of assembly/disassembly of intercellular junctions, cell shape,motility,p roliferation, apoptosis, and differentiation (reviewed in [198]). Both muscarinic and nicotinic antagonists haveb een shown to wident he intercellular spaces in epidermis and cause overt acantholysis in vitro through the mechanism that may involve alterations of both production and phosphorylationo fk eratinocyte adhesion molecules (reviewed in [55]). It is well known that phosphorylation of adhesion molecules plays an important role in assembly/disassembly of intercellularj unctions [199 -203], and that phosphorylation of cadherin [204 -206], g -catenin [ 207], desmoplakin [ 208,209], andDsg [210 -212]isassociatedwithaloss of adhesion.
Some intercellular junction proteins are phosphorylated on serine, someo nt yrosine, and someo nb oth residues.T he seminalw orks by Aoyama et al. [212,213] demonstrated that binding of PV IgG to cell surface antigens induces phosphorylation of Dsg3 ,i ts dissociationf romp lakoglobin,a nd formation of Dsg 3-depleted desmosomes. These findings were corroborated by the results showing that in additiont oD sg 3, PV IgG also increases the level of phosphorylation of keratinocyte E-cadherin as well as b -, g -, and p120 catenins [214,215]. These experimentsa lsod emonstratedt hatk eratinocyte dyshesion correlates closely with the degree of tyrosine phosphorylation of p120-catenin by Src and serine phosphorylationo f b -catenin by classic PKC isoforms [215].
The Src-dependentc ascade is also responsible for keratinocyte shrinkage (cell volume reduction) and keratin aggregation [47]. The cytoskeletal collapse has been reporteda sa ne arly eventi np emphigus acantholysis that precedes visibles eparationo f keratinocytes [47,179,216 -218]. Thus, it appears that PV IgG-induced phosphorylation of adhesion molecules and structuralp roteins leadst ow eakening of intercellular junctions andc ollapse of the cytoskeleton, respectively.The chronological scheme of early signaling and pathobiologic events in keratinocytes exposed to PV IgG are shown in Figure 6.
Numerous classical andm odernc linical and experimental studiesi np emphigusd emonstrated thatd esmosomes separatew hent he intercellular spaces are alreadyw idened [ 87,90,[219][220][221][222].D esmosomes do nots plit and disappear until late in acantholysis when keratinocytes are almost completely separatedf rome acho ther.A te arly stages of acantholysis,t he half-desmosomes remain invisible because they are firmlyadhering to each other. In late acantholysis,s omeh alf-desmosomesa dhere to each others os trongly that they can be rippedo ff one cell while remaining adherent to their counterparto nt he opposite cells.
Hence,d isruptiono fi ntercellularb ridges results fromr ipping intact desmosomes offt he plasma membrane of collapsing keratinocytes by shearing forces.T he intactd esmosomes ripped offf rom neighboring cellsc an be seen floatingf ree in the intercellular space, which is in keeping with the sloughing of desmosomal cadherins thatg ivesr ise to scavenging autoantibodies. The "basal cell shrinkage" hypothesis reconciles the time course of acantholysis in PV with the characteristic appearance of acantholytic epidermis, knowna s" tombstoning" [223]. According to this hypothesis: (1)k eratinocytes separate because they shrink more than can be held together by desmosomes; (2) the suprabasal clefting occursbecause basal cells shrink more than suprabasal keratinocytes; and (3) pharmacologic inhibition of the principal signalingp athways leading to cytoskeletal disorganization shouldp revent pemphigus acantholysis. Figure 6. Hypothetical scheme of early signaling steps during first 6h after PV IgG binding to keratinocytes and their correlation with the major intracellular pathobiologice vents. Abbreviations: EGFR, epidermal growth factor receptor; Dsg,d esmoglein; NDAgs, non-Dsg antigens.From Ref. [47].
One of the most important recent advances in pemphigus research was elucidation of the molecular mechanisms that selectively target basal cells in PV,as predicted by the basalcell shrinkage hypothesis. Pretel et al. [190] demonstrated that pretreatment with the mTOR inhibitors irolimus prevented suprabasal acantholysis in the epidermis of neonatalmice injected with PV IgG. In this model, PV antibodiesc aused unopposed upregulation of mTOR selectively in basal keratinocytes, which was associated with the appearance of signs of apoptosis that were also abolished by sirolimus [190].
Downstream of mTOR, the induction of keratinocyte apoptosis by PV antibodies apparently proceeds through the c-Myc-dependent pathway [218]. c-Mycinduced apoptosis involves caspase 9 [ 224], which is activated in keratinocytes treated with PV IgG [225]. In agreement with the fact that cyclin-dependent kinase 2( Cdk2) is required by c-Myc to induce apoptosis [226], PV sera induces accumulation of Cdk2 that contributes to acantholysis in the mouse model of PV [227]. Indeed, sirolimus has beenshown to inhibit expression of both c-Myc [228]a nd Cdk2 [229]. Ta kentogether, these observations help explain why epidermal clefting in PV always occursjust above basal cells( tombstoning), despite depositiono fI gG antibodies throughout the entire epidermis.
Both extrinsic andi ntrinsic pathwayso fc elld eath triggered in keratinocytes by PV IgGs can lead to the structural damagem anifested by basal cell shrinkage. It has been documented by different research groups that in the skin of PV patients, keratinocytes exhibit signs of apoptosis that precede their detachment and blister formation, andt hat PV IgG ands era induce biomolecular markerso fa poptosis and oncosis in keratinocytem onolayersa nd skino rgan cultures [225,230 -235]. Twog roups of PV patients, each producing autoantibodies activating predominantly either apoptotic or oncotic cell deathp athway,h ave been identified [225].
The anti-PERP PV antibody may launch the cell deathp athwaysi nk eratinocytes, because PERP expression leads to activation of an extrinsic receptor-mediateda poptotic pathwayw ith ap ossible subsequent engagement of the intrinsic apoptotic pathway [54]. Anti-mitochondrial PV antibodies also can trigger intrinsic apoptotic cascade in keratinocytes [37].O thert ypes of autoantibodiesa nd soluble mediatorso fi nflammation can activate cell death pathways in keratinocytes.D rP incelli'sg roup demonstrated that Fasl igand( FasL) in pemphigus sera induces keratinocyteapoptosis through activation of caspase8 [231], andt hatF asL neutralizing antibody prevents PV IgG-induced apoptosis both in vitro and in vivo [236].
Furthermore, it hasbeen shown that TNFa mRNA is abundantly expressed in PV skin lesions [237,238]; serum TNF a levelsc orrelate closely with disease activity and autoantibody titers [ 239,240]; and anti-TNF a antibodyi nhibits acantholysis induced by PV autoantibodies in vitro [237]. The synergistic acantholytic effects of PV IgG, FasL, and TNFa were documentedi ne xperiments with keratinocyte monolayers andf ull thickness equivalents of human epidermis [241]. These mediatorso fa poptosis as well as the increased amounts of activated kallikreins [242] and several types of inflammatoryc ytokines [237,239,243,244] found to be elevated in pemphigus sera maya ccount for am inor acantholytic activity of the PV serum depletedo fI gG, as reported by Cirillo et al. [245].
It is well known that blisterfluid and/or perilesional skin of PV patientsc ontains high levels of proteases and various inflammatorycytokines that may contribute to acantholysis [242,248 -250].T hus,asimultaneous autoimmune attack by the threec lasses of autoantibodies against desmosomal, mitochondrial, and otherk eratinocyte autoantigens, such as AChRs and PERP, mayb er equired to induce pathologic changesi np atient's skin.T he anti-keratinocyte antibodies synergize with the effectorso fa poptotic pathway FasL and TNFa as well as proinflammatory/ cytotoxic serum and tissue factors, such as serine proteases and cytokines, that altogether overcome the natural resistancea nd activate cell death pathways in keratinocytes (Figure 7). Acantholysis andc ell death (apoptosis/oncosis) are inseparable in PV because both processes are triggered by the same signal effectorsa ctivated due to PV IgG binding to keratinocytes and mediatedbythe sameset of cell deathenzymes. This is evident from the reports that inhibitorso fS rc, EGFRK, p38 MAPK, and mTOR blockb otha cantholysis anda poptosis [1,47,176,177,189,190,215,251] and caspase inhibitorsp revent acantholysis both in vitro and in vivo [190,225,246]. Moreover, it has been demonstrated that apoptotic enzymes cleaveD sg 1, 2, and 3 [236,252,253].
Fori nstance, when PV IgG wasa ddedt o keratinocytes in the presence of anti-FasL neutralizing antibody,t he cleavage of the intracellular portion of Dsg 3a nd its degradation decreased [1]. The fact that structural damagea nd deatho fk eratinocytes in PV are mediated by the sames et of enzymes has justified introduction of the new term "apoptolysis" to distinguish the unique mechanism of autoantibodyinduced keratinocyte damage in PV from other known forms of cell death [1]. The apoptolysis hypothesis links the basal cell shrinkage to suprabasal acantholysis and cell death, and emphasizes that apoptotic enzymes contributet oa cantholysis in terms of both molecular events andc hronologic sequence. It postulates that cell detachment and death in PV develop through the following five major steps (Figure 8): . Step1 : bindingo fp athogenica ntibodiest o keratinocytes via ar eceptor -ligandt ype of interaction sends an arrayofthe agonist-and antagonistlike signals; .
Step 3: suprabasal acantholysis starts when basal cells shrink due to reorganization of corticala ctin filaments,c ollapse and retraction of the tonofilaments (TFs) cleaved by executioner caspases, as well as dissociation and internalization of intercellular adhesion complexes caused by phosphorylation of adhesion molecules and their cleavage by caspases; .
Step 4: acantholysis advances due to continued degradationa nd massive collapse of structural proteins by the same cell deathenzymes, leading to separationand rending of preexisting desmosomes from the CM by shear forces, thus separating the collapsing cells ands timulating productiono f secondary(scavenging) antibodies; .
Step 5: rounding up and death of acantholytic cells in the lower epidermal compartment follows irreversible damage of mitochondrial and nuclear proteins.
In conclusion, the mechanism of apoptolysis in PV encompasses several tierso fe vents triggeredt hrough distinct antigen -antibody systems. Because apoptolysisd evelops in as tepwise fashion, late morphologic features of apoptosis, sucha sr ounding up, nuclear fragmentation, and plasma membrane blebbing,d o not become apparent until the keratinocyte death in Step 5. Signaling mechanisms may vary from patient to patient because aunique composition of the pool of autoantibodies determines the principal pathway.The Dsg 3-dependent late peak of p38 MAPK activity and activation of JNK represent the pathways mediating processing and utilization of internalized Dsg 3, rather than ap rimary downstreams ignalinge manating from the CM.T he apoptolysis hypothesis has several important implications:(1) it links togetheranumber of apparently unrelated, and previously heldc ontradictory, observations on thee ventss urrounding acantholysis;(2) it opens new avenues of investigation into the pathomechanism of pemphigus; and (3)i t creates new approaches to the treatment of pemphigus basedo ni nterfering with or blocking the signaling pathways ande nzymatic processes that lead to blistering.

Treatment of pemphigus
The high-dose, long-terms ystemicg lucocorticoid therapy remainsthe mainstay of currenttherapy of PV and PF patients. Corticosteroidh ormones (usually prednisone tablets)are essential to establish control of PV during the acute stage [2,254,255]. Optimal dosingp roved to be variable and couldn ot be predicted at the outset in any given patient [4]. Some patientsr espond rapidly and completely to treatment with moderatedoses of oral prednisone (1 mg/kg/day), others are rather refractorya nd require muchh igher doses. If there is no response after 5-7days, the dose is increased by 50 -100% [256,257]. Once the control of the diseasei sa chieved (lack of new lesions, epithelialization of existing erosions and negative Nikolskiy sign [258,259]),t he prednisone dosage is decreased in a"logarithmic fashion",i.e. by 10 -20% every7 -15d ays.C hronic PV patientsw ith disease exacerbation are treated exactlyt he same way as new patients, i.e. prednisone dose is increased until disease controlisa chieveda nd than tapered [256,257].
The first reportofadministration of glucocorticoids to ap emphigus patient is datedt o1 940 [260], i.e. some 25 yearsearlier than pemphigus antibodies were discovered. Adrenocortical extractw as triedf or treatment, because it hadb een noticed that pemphigus is associated with changes in patients' blood chemistryc haracteristic of abnormal (deficient) function of the adrenal gland producing cortisone. Thes ynthetic cortisonew as introduced to the treatment of pemphigus approximately 10 yearsl ater [261]. Prior to the introduction of therapy with oral corticosteroids in the 1950s, the diseaseh ad ad ismal natural course with a50% mortality rate at 2yearsand 100% mortality rate by 5y earsa fter the onseto ft he disease. Although there has been asignificant decrease in mortality nowadays [262], it remains at arelatively high level of approximately 12% [263], with death being almost invariably related to complications of therapy.
The early adverseeffects of systemicglucocorticosteroidst hata re essentially unavoidablei nclude enhanced appetite, fluid, and salt retention leading to weight gain and neuropsychiatric disorderssuch as Step 4: massivecleavageofcellular proteins by activated cell death enzymes leads to collapse of the cytoskeleton and tearing off desmosomes from the CM with subsequent productionofscavenging (i.e. secondary) autoantibodies mainly to sloughed adhesion molecules.
Unfortunately,t hese therapiesd on ot allow reliable control of acutep emphigus without systemic glucocorticosteroids, indicatingt hati na ddition to immunosuppression the therapeutic actiono fc orticosteroids in pemphigusi ncludes otherm echanisms, such as directa nti-acantholytic effecto nk eratinocytes. Moreover, althought here is ab ulk of evidence that PV is predominantly aT h2-type autoimmune disease, at least with regard to the anti-Dsg3antibody production [ 113,144,159,287], thed atao nt he mechanisms of immunomodulatorya ction of glucocorticosteroidsc ounterintuitively demonstrate that these drugs foster Th2 polarization of CD4 þ Tc ells [288 -290].
Direct anti-acantholytic effects of the corticosteroids methylprednisolone andh ydrocortisone on keratinocytes were discovered in in vitro experiments, in which high doses of these drugs blocked PV IgGinduced acantholysis [291,292]. Because antibodyproducing cells were notp resent in cultures, these drugs couldn ot exhibit their anti-acantholytic effects by way of acting upon lymphocytes. Subsequent in vivo experimentsd emonstratedt hata dministration of methylprednisolone significantly decreased the extent of acantholysis in the epidermis of 3-5-day-old nude miceinjected with PV IgG [214]. This was in keeping with thec linical observations that blistering in pemphigus patients stops within 24 -48h rs after initiation of ah ighd ose, "pulse" therapyw ith methylprednisolone or dexamethasone [293][294][295][296], while the major decline in autoantibody titerso ccurs 3-4 weeks after initiation of glucocorticoid therapy [297]. It is well known thatp emphigus therapy improves disease earlier than decreasing the antibody titers [298].
Also, local administration of a0 .05% clobetasol propionate cream cani nitially control cutaneous lesionsi nm ild cases of PV [299]. The directe ffects of corticosteroids that can protect keratinocytes from PV IgG mayi nclude alterations in gene expression, as revealed by aD NA microarraya ssay [214]. PV IgG downregulated and methylprednisoloneu pregulated expression of the genes encoding the keratinocyte adhesionm oleculesD sg 3a nd periplakin, regulatorso fc ell cycle progression and apoptosis, differentiation markers, protein kinases and phosphatases, serine proteases and their inhibitors, ands ome otherg enes.F urthermore,m ethylprednisolone blocked phosphorylation of Dsg 3, E-cadherin, and b -a nd g -catenins induced by PV IgG [214].
These pharmacologic effects of methylprednisolone help explain the dose-dependent therapeutic actionof corticosteroids in pemphiguspatients. It is well known that extremely high corticosteroiddoses are sometime required to attain control of acantholysis in the acute stage of disease. Thus,i na ddition to their immunosuppressive and anti-inflammatorya ctions in pemphigus, glucocorticoids can also regulate adhesion and viability of keratinocytes through ac ombination of their genomica nd non-genomic effects.
Historically,the earliest attempt to treat pemphigus patientsb yp rotecting the target cells( keratinocytes) from the PV IgG-induced damagew as made using the proteinase inhibitors aprotinin (Contrykal) and 1 -aminocaproica cid [242]. Af ew yearslater, Dobrev et al. [300] administered p -aminomethylbenzoic acid. In both studies, the addition of protease inhibitors allowed to achieve therapeutic effects at lower doses of systemic corticosteroids, thus decreasing the risk for adverser eactions. More recently, TNFa inhibitors haveb een tried.
The importance of nicotinergic stimulation for the treatment of pemphigus was first suggested by the reporto faPV patient whose disease worsened when he stopped smoking and improved shortly after he resumeds moking [314]. The epidemiologic studies confirmedt he beneficiale ffect of smokingo n pemphigus. Brenner et al. [315] reported that 25.9% of 126 patientsw ere smokers vs. 48.5% of controls. According to Sullivane ta l. [316], only 15.3% of 59 patientswere current or former smokers,comparedto 47.4% in the general population. These findings have recently been validated in as tudy involving 199 patientswith PV,11with PF,and 205 control subjects [317]. Also, it has been reportedthat smokerswith PV achieve partial remission more frequently than nonsmokers at the end of the 1st year of treatment, and that the number of patients in remission at the end of the 2nd year of therapy is significantly higherf or smokers than for non-smokers [ 318]. The cigarette smoke contains the nicotinergic agent nicotine that not only upregulates epithelialization in vitro [131] but also facilitates healing of skine rosions [319]. In additiont os timulation of epithelialization, nicotine may exhibit its therapeutic effect in pemphigus by affecting the immune system.
The perspective for the development of steroidsparing therapy employing cholinergic drugs is very promising,b ecause cholinergica gonistso fb oth nicotinic and muscarinic classesh avea lreadys hown their therapeutic activities in pemphigus patients. The nAChRs in keratinocytes can be directly activated by pyridostigmine bromide (Mestinon) [325]. Besides its underappreciated nicotinergic action, pyridostigmine bromide is reversible acetylcholinesterase inhibitor [326] that can elevate tissuel evelso fa uto/paracrine ACh, thus augmenting signalingt hroughb oth muscarinica nd nicotinic pathways in thec ells secreting ACh,l ike humank eratinocytes [ 327]. Pyridostigmine bromide has been shown to antagonize the effects of PV antibodies in both in vitro [328] and in vivo experiments [329]. Most importantly,a clinical trial of Mestinon in the treatment of eight pemphigusp atients brought encouragingr esults [330]. Three patientss howed av eryg ood response, andfi ve patients did nots howa ny significant improvement. One patient was able to discontinue glucocorticosteroids and immunosuppressive medications, andcontrol the disease using Mestinon only.
Pilocarpine preferentially binds to and activate the M 1 molecular subtype of keratinocyte mAChRs [333] that has been recently found to be specifically targeted by PV antibodies [49]. Pilocarpine blocks PV IgGinduced phosphorylationo fp 120-and b -catenins in keratinocytes, because it elevates both serine/threonine andt yrosine phosphatase activities [ 215]. Furthermore, the anti-acantholytic activity of pilocarpine synergizes with that of the a 7n AChR agonist AR-R17779 thatb oth activates tyrosine phosphatase and inhibitsSrc [215]. Ta ken together, these findings identify novel paradigm of regulation of signaling kinases associated with cholinergic receptorsa nd provide mechanistice xplanationo ft herapeutic activity of cholinomimeticsinP Vp atients. Al arge variety of steroid-sparing effects reported thus fari nt he literature suggests that it should be possible to replace corticosteroids by combining the steroid-sparing drugs and/or treatment modalities that can provide for simultaneous inhibition of antibody production andp rotection of keratinocytesf rom autoantibody action. Unfortunately,s uch putative combination has not been devised yet, though a combination of rituximaba nd IVIg allows to treat certain PV patients without corticosteroids [334]. Most recently,i th as been reported that the mTOR inhibitor sirolimus( Rapamune,R apamycin w )c ombinedw ithI VIga llowed rapida nd complete withdrawal of systemicg lucocorticosteroids in aP Vp atient whod eveloped diseasee xacerbation that could notb ec ontrolledw ith4 0mg/day of prednisone [120]. Sirolimus is an aturally occurring lipophilic microcyclic lactone isolated from Streptomyces hygroscopicus discovered at Rapa Nui (Easter Island). It bindst oi mmunophilin and FK binding protein-12.
As already mentioned, the mTOR pathway is activated in keratinocytes exposed to PV IgG and mTOR inhibitionprevents acantholysis in the murine passive transfer model of PV IgG [190]. Additionally, sirolimus mayp revent damage of keratinocytes and enforce their adhesive function by inhibiting reorganization of the actin cytoskeleton andp hosphorylation of focala dhesion proteins, andu pregulating E-cadherine xpression [337,338]. The clinical trial of sirolimus in pemphigusp atientsi sc urrently underway at University of California Irvine (http:// clinicaltrials.gov/ct2/show/NCT01313923).
Ta ble II. Hypotheses and realities in the knowledge of pemphigus.

Hypotheses Realities
The epidermal integrity is mediated exclusively by the Dsg 1and 3adhesion molecules Neither Dsg 1n or Dsg 3c an solely sustain keratinocyte adhesion in epidermis.P atients with striate palmoplantar keratoderma featuring N-terminal deletion in Dsg 1donot develop skin blisters [350]. In turn, the conditionalDsc3 null mutant mouse developsP Vp henotype despite the presence of intact Dsg 3 [43]. If the integrity of epidermis would rely exclusively on Dsg 1a nd 3, the epidermis should disintegrate to asingle cell suspension in the PV patients who develop both anti-Dsg 1a nd 3antibodies ( Figure 2) Acantholysis is PV is caused by steric hindrance of Dsg 3b yautoantibodies Electron microscopic studies of limited acantholysis produced by anti-Dsg 3a ntibody in murine epidermis revealed that steric hindrance of Dsg 3leads to adesmosomal split without keratin retraction [84,85]. The ultrastructural changes in the skin of PV patients are quite different. Desmosomes remain intact till the late stages of acantholysis when they are cleaved behind the desmosomal plaque, due to shearing forces produced by collapsing cells, and float free in the intercellular space [55,[86][87][88][89][90]. Acantholysis is PV results from PV antibody-dependent signaling events collectively described by the termapoptolysis [1] Clinical and histological features of PF and PV can be reproduceds olely by Dsg 1a nd 3antibodies, respectively The experiments using the Dsg1-Ig and Dsg3-Ig chimeras that absorbed out all disease causing pemphigus antibodies,thus giving arise to anotion that anti-Dsg 1/3 antibodies are the sole cause of pemphigus,werefl awed by the presence of non-Dsg antibodies ( Figure 3) An interplay between Dsg 1and 3 antibodies determines the mucocutaneous phenotype in patients with autoimmunep emphigus PF patients can developantibodies against both Dsg 1and Dsg 3 [ 108,116,117], and the Dsg 1/3 antibody patternd oes not match the predicted morphologic phenotype of PV [96,118,119] The titers of anti-Dsg 1or3antibodies correlate closely with the severely of the disease The Dsg 1/3 antibody titers do not correlate with disease activity [91][92][93]. While Dsg 3 antibody can be absent in PV patients in active stage of disease, it can be present in PV patients during remission [94][95][96] as well as in healthysubjects and patients with irrelevant medical conditions [97][98][99][100][101][102][103][104][105][106][107][108][109] The sera of patients with autoimmune pemphigus contain autoantibodies only to the Dsg 1/3 targets More than 50 organ-specific and non-organ-specific proteins haveb een reported to date as specific targets for autoantibodies produced by PV and/or PF patients (Table I) Systemic corticosteroids treat pemphigus patients exclusively by inhibiting autoantibody production The therapeutic effect of "pulse" therapy with methylprednisolone commenceswithin a few days, whereas autoantibody titers decline within 3-4w eeks [294][295][296]. The rapid therapeutic effect is apparently mediated by direct anti-acantholytic action of glucocorticosteroids that protects keratinocytes from an autoantibody-induced damage [214] Paraneoplastic pemphigus (PNP) is a variant of classical pemphigus PNP is not related to PV and PF,but represents aclinical variant of the paraneoplastic autoimmune multiorgan syndrome (PAMS) in which patients, in addition to small airway occlusion, may display aspectrum of at least five clinical variants, i.e. pemphigus like (a.k.a. PNP), pemphigoid like, erythema multiforme like, graft vs. host disease like, and lichen planus like [10,12,13] In conclusion, corticosteroids remain an essential componento fp emphigust reatment. Earlya nd aggressiveu se of corticosteroidsi sr equired to decrease the duration of treatment and avoid relapses. Adjuvant drugs allow ad ecrease in the total dose of corticosteroids. The natural course of pemphigus has improved with new therapies. Cholinomimetics can achieve asteroid-sparing effect in pemphigus patients by both stimulating epithelialization and inhibiting autoimmune aggression. The dualistic pharmacologic actiono fs irolimus that affects both effectorso f autoimmunityand targetcellsapparently mediatesits therapeutic effect in pemphigus. Further elucidation of the molecular mechanisms mediating aberrant signaling along the mTOR pathway in PV should improveo ur understandingo ft he pathogenesis and lead to novel therapeutic approachesf or the developmentofs teroid-free treatment of pemphigus.

Summary
(1) Recent advances of knowledge on pemphigus autoimmunity scrutinize old dogmas,r esolve controversies, and open novel perspectives for treatment (Table II). (2) The initial insult is sustained by the autoantibodiest ot he cell-membraner eceptor antigens triggering thei ntracellulars ignalingb yS rc, EGFRK, PKC, phospholipase C, mTOR, p38 MAPK,o ther tyrosine kinases, and calmodulin that cause basalc ells hrinkage andr ipping desmosomes off the CM. (3) Autoantibodies synergize with the effectorso f apoptotic and oncoticpathways, serine proteases and inflammatoryc ytokines to overcome the natural resistance and activate the cell death program in keratinocytes. (4) The process of keratinocyte shrinkage/detachmenta nd death via apoptosis/oncosish as been termed apoptolysis to emphasizet hati ti s triggeredb yt he same signal effectorsa nd mediated by the same cell deathe nzymes. (5) Although ahigh-dose, long-termsystemicglucocorticoid therapy remainsthe mainstay of current treatment of patientsw ith PV or PF,c ausing severe adverse effects, as ubstantial progress has been made towardd evelopment of steroidsparing therapies combining the immunosuppressive anddirectanti-acantholytic effects. (6) Theo nset of acantholysis in drug-induced pemphigusi np atients takinga ngiotensinconverting enzyme inhibitors, such as captopril, apparently involves adrop in the concentration of auto/paracrine ACht hat sustainsk eratinocyte shape and cohesiveness, due to as trongu pregulation of the AChd egrading enzyme acetylcholinesterase [ 351].
Declaration of Interest:T he author reports no conflicts of interest.T he author alone is responsible for the contentand writing of the paper.