Pemphigus Acantholysis Research Articles

Autoantibodies against desmoglein 2 are not pathogenic in pemphigus

BackgroundAnti-desmoglein 1 and 3 autoantibodies justify acantholysis in pemphigus; however, the pathogenesis of anti-desmoglein 2 is hypothetical. ObjectiveTo compare the participation of desmogleins 1, 2 and 3 through the production of serum autoantibodies, and protein and gene expression in the skin/mucosa of patients with pemphigus foliaceus and pemphigus vulgaris. MethodsThe autoantibodies were titrated by ELISA in 202 samples of pemphigus foliaceus, 131 pemphigus vulgaris, 50 and 57 relatives of patients with pemphigus foliaceus and pemphigus vulgaris, respectively, and 114 controls. Protein and gene expressions were determined by immunohistochemistry and qPCR in the skin/mucosa of 3 patients with pemphigus foliaceus and 3 patients with pemphigus vulgaris. ResultsHigher titers of anti-desmoglein 2 (optical density) resulted in pemphigus foliaceus and pemphigus vulgaris, when compared to controls (0.166; 0.180; 0.102; respectively; p < 0.0001). There was a correlation between anti-desmoglein 2 and anti-desmoglein 1 titers in pemphigus foliaceus (r = 0.1680; p = 0.0206). There was no cross-reaction of anti-desmoglein 2 with desmoglein 1 and 3. Protein overexpression of desmoglein 2 was observed in intact and lesional skin of patients with pemphigus compared to the skin of controls. Internalization granules of desmoglein 1 and 3, but not of desmoglein 2, were observed in lesions of pemphigus foliaceus and pemphigus vulgaris, respectively. Gene overexpression of desmoglein 2 was observed in the mucosa. Study limitationsSmall sample size for the statistical analysis of protein and gene expression. ConclusionAutoantibodies against desmoglein 2 are not pathogenic in pemphigus; protein and gene overexpression of desmoglein 2 in the skin and mucosa may be involved in acantholysis repair.

Identification of novel therapeutic targets for blocking acantholysis in pemphigus.

Background and PurposePemphigus is caused by autoantibodies against desmoglein (Dsg) 1, Dsg3, and/or non‐Dsg antigens. Pemphigus vulgaris (PV) is the most common manifestation of pemphigus, with painful erosions on mucous membranes. In most cases, blistering also occurs on the skin, leading to areas of extensive denudation. Despite improvements in pemphigus treatment, time to achieve remission is long, severe adverse events are frequent and 20% of patients do not respond adequately. Current clinical developments focus exclusively on modulating B cell function or autoantibody half‐life. However, topical modulation of PV autoantibody‐induced blistering is an attractive target because it could promptly relieve symptoms.Experimental ApproachTo address this issue, we performed an unbiased screening in a complex biological system using 141 low MW inhibitors from a chemical library. Specifically, we evaluated PV IgG‐induced Dsg3 internalization in HaCaT keratinocytes. Validation of the 20 identified compounds was performed using keratinocyte fragmentation assays, as well as a human skin organ culture (HSOC) model.key ResultsOverall, this approach led to the identification of four molecules involved in PV IgG‐induced skin pathology: MEK1, TrkA, PI3Kα, and VEGFR2.Conclusion and ImplicationsThis unbiased screening revealed novel mechanisms by which PV autoantibodies induce blistering in keratinocytes and identified new treatment targets for this severe and potentially life‐threatening skin disease.

Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway Ca2+/Mn2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.

Locations of acantholysis in 71 cases of pemphigus

Objective To analyze locations of acantholysis in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) , so as to explain why acantholysis in pemphigus occurs in different locations of the epidermis. Methods Clinical data, histopathological and immunopathological findings, and pemphigus antibody level values were collected from 43 patients with PV and 28 with PF, and retrospectively analyzed. Results Of the 43 patients with PV, 35 showed acantholysis in the upper basal layer, 8 in the middle-to-upper epidermis. Of the 28 patients with PF, 25 showed acantholysis in the granular layer and the upper prickle cell layer, 3 in the middle-to-lower epidermis. Patients with PF showed significantly higher levels of anti-Desmoglein 1 (Dsg1) antibody compared with patients with PV (P= 0.047) . However, there were no significant differences in the levels of anti-Dsg1 and anti-Dsg3 antibodies between PV patients who had acantholysis in the middle-to-upper epidermis and those in the upper basal layer. Conclusion Histopatho-logical examinations of PV and PF lesions show that acantholysis can occur in the middle-to-upper epidermis, as well as in the middle-to-lower epidermis, and locations of acantholysis may be associated with levels of anti-Dsg1 and anti-Dsg3 antibodies. Key words: Pemphigus; Acantholysis; Desmoglein 1; Desmoglein 3; Antibodies; Enzyme-linked immunosorbent assay

Mechanisms underlying the reversal of acantholysis in pemphigus by a cholinergic receptor agonist

Objective To evaluate the reversal effect of a cholinergic receptor agonist on acantholysis in pemphigus, and to investigate its mechanism. Methods Human HaCaT keratinocytes were co-cultured with pemphigus vulgaris immunoglobulin G(PV-IgG) to establish a cell model of pemphigus, then classified into two groups to be incubated with the cholinergic receptor agonist carbachol for 12 hours (test group) or remain untreated (control group). Cell dissociation assay was performed to quantitatively estimate the reversal effect of carbachol on acantholysis, and immunofluorescence assay to qualitatively assess the changes of desmosomal proteins. Radio-immunoprecipitation assay (RIPA) lysis buffer and Triton X-100 were used to lyse HaCaT cells to obtain total proteins and cytoplasmic proteins, and Western blot was conducted to determine the expression levels of adhesion-related proteins desmoglein 3(Dsg3) and plakoglobin (PG) on the surface of HaCaT cells, as well as the phosphorylation levels of p38 mitogen activated protein kinase (p38 MAPK) and epidermal growth factor receptor (EGFR) at different time points. Quantitative polymerase chain reaction (qPCR) was performed to detect the mRNA expressions of the above surface proteins, and co-immunoprecipitation assay to qualitatively evaluate the interaction between Dsg3 and PG. Results The number of cell debris was significantly lower in the test group than in the control group (18.67±2.52 vs. 46.67±2.03,t=11.22, P<0.01). Immunofluorescence assay showed that carbachol could reverse the internalization of desmosomal molecules induced by PV-IgG. In the pemphigus cell model, the levels of total Dsg3 and PG as well as non-desmosomal Dsg3 were decreased, while the level of non-desmosomal PG increased, and the interaction between Dsg3 and PG was attenuated. When the pemphigus cell model was co-cultured with carbachol, these above changes were reversed. Carbachol also increased the mRNA levels (expressed as 2-ΔΔCt) of Dsg3 and PG from 1.428±0.215 and 1.563±0.247 in the control group to 4.974±0.948 (t=3.65,P=0.01) and 13.420±1.715 (t=6.85, P<0.01) in the test group respectively. In phosphorylation assay, carbachol inhibited the phosphorylation of EGFR, but had no significant effect on that of p38 MAPK. Conclusions The cholinergic receptor agonist carbachol can reverse acantholysis in pemphigus, likely by inhibiting the internalization of Dsg3 and PG, enhancing their expressions and interaction, and suppressing the phosphorylation of the key signaling molecule for acantholysis, EGFR. Key words: Pemphigus; Cholinergic agonists; Acantholysis; Desmoglein 3; gamma Catenin; HaCaT cells

Alteration of Cholinergic System in Keratinocytes Cells Produces Acantholysis: A Possible Use of Cholinergic Drugs in Pemphigus Vulgaris

Human epidermis shows a non-neuronal cholinergic system including keratinocyte (kc) acetylcholine (Ach) axis which is composed by enzymes and two families of Ach receptors (muscarinic and nicotinic receptors). The activity of these two receptors can regulate the interkeratinocytes and kcs-extracellular matrix adhesion modifying the regulation of intercellular adhesion molecules like cadherins and integrins. Some authors demonstrate that acantholysis in pemphigus depends not only on anti desmogleins antibodies (abs) (mostly IgG) but even on other abs directed against kc membrane antigens (e.g. anti Ach receptors Abs). In the early phase of pemphigus pathogenesis, anti Ach receptors Abs block Ach signaling essential for cell shape and intercellular adhesion and increase the phosphorylation of adhesion molecules. Combined with the action of abs antidesmogleins, anti Ach receptors Abs cause the acantholytic phenomenon. In vitro experiments show that high doses of Ach in acantholytic kcs can rapidly reverse this pathologic event. In vivo experiments using neonatal mice model of Pemphigus have demonstrated that cholinergic agonists reduce these lesions. Therapy with pyridostigmine bromide and Nicotinamide per os or pilocarpine used topically, drugs that present cholinomimetic effects, has lead to encouraging results in patients affected by Pemphigus disease. Cholinergic agents could have a strategic role in the therapy of pemphigus since they could be responsible for the early stage of acantholytic diseases.

Jean-Claude Bystryn 1938-2010. An obituary

Jean-Claude Bystryn, M.D., passed away on 19 August, 2010. Dr. Bystryn's research interests encompassed a large group of dermatologic conditions. He has earned a worldwide recognition for his innovative works on autoimmune blistering diseases, melanoma and alopecia areata. The most significant impact Dr. Bystryn's research has made is on our understanding of the mechanisms of epidermal cell detachment (acantholysis) in pemphigus and development of adequate treatment. During the last decade, he chaired the Medical Advisory Board of the International Pemphigus and Pemphigoid Foundation. Dr. Bystryn was an innovative physician-scientist whose scientific contributions will be long recalled and admired both by patients and colleagues.

The cause of acantholysis in pemphigus: further support for the ‘basal cell shrinkage’ hypothesis

Conflicts of interest: none declared. Sir, The article ‘The ultrastructure of acantholysis in pemphigus vulgaris’ by Drs Diercks, Pas and Jonkman1 confirms prior electron microscopic observations made by multiple investigators in patients with pemphigus2 3–4 and in in vitro and in vivo models of pemphigus acantholysis.5 6–7 These and the article by Diercks et al.1 show that the initial event in pemphigus acantholysis is the separation of keratinocytes from each other at sites where there are no desmosomes. Desmosomes do not separate from each other until very late in acantholysis, if at all. Often, desmosomes continue to stick so tightly to each other that they are ripped off from acantholytic keratinocytes without coming apart. We have previously summarized these and other observations and their implications.8, 9 They lead to two important conclusions: (i) acantholysis in pemphigus is not caused by loss of desmosomal adhesion due to autoantibodies blocking the adhesive properties of desmogleins; and (ii) rather, they suggest acantholysis occurs because the cytoskeleton of keratinocytes is disrupted causing these cells to collapse and to shrink more than desmosomes can hold them together. We call this the ‘basal cell shrinkage hypothesis’.8 Recently published experimental results have further substantiated this hypothesis.10 We are pleased the observations of Diercks et al.1 provide further support for it.

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.

Peptides Targeting the Desmoglein 3 Adhesive Interface Prevent Autoantibody-induced Acantholysis in Pemphigus

Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the self-designed cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment.

Sulfasalazine and Pentoxifylline: Potential for Adjunctive Treatment of Pemphigus Vulgaris

Evidence shows that tumor necrosis factor α (TNF-α) contributes to acantholysis in pemphigus, and several reports document the efficacy of the TNF inhibitors infliximab and etanercept in autoimmune blistering disease. Sulfasalazine and pentoxifylline both inhibit TNF-α, although by different mechanisms: Pentoxifylline reduces its production, whereas sulfasalazine inhibits TNF-α mRNA levels and binds to circulating TNF-α. In this study, researchers examined sulfasalazine plus …

Differential Coupling of M1 Muscarinic and α7 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.

At Least Three Phosphorylation Events Induced by Pemphigus Vulgaris Sera are Pathogenically Involved in Keratinocyte Acantholysis

Intercellular adhesion among keratinocytes is guaranteed by desmosomes. Disruption of desmosomal integrity leads to cell-cell detachment or acantholysis, as it classically occurs in pemphigus vulgaris (PV), an autoimmune blistering disease of skin and mucous membranes. While purified PV IgG seems to trigger intracellular signaling that crucially involves p38 MAPK, keratinocyte acantholysis induced by whole PV serum may recruit a number of additional signals. In this study, the Pro-Q Diamond Phosphoprotein Assay was used to investigate the overall changes in protein phosphorylation levels in an in vitro model of PV. We showed that keratinocytes exposed to whole PV sera underwent at least three early and transient phosphorylation events. Two bands with apparent molecular masses of 35 and 45 kDa were found to be phosphorylated within 1 min after incubation with PV sera. A third band of about 80 kDa reached the peak of phosphorylation level after 3 hours. Morphologic evidence of cell shrinkage and acantholysis were late events and did not correlate temporally with kinase activation, suggesting that cytoskeleton reorganization is a downstream phenomenon. Interestingly, pharmacological abrogation of PV-specific protein phosphorylation was able to inhibit the cell-cell detachment, rounding up, and redistribution of Dsg3 in keratinocytes. Thus, at least three phosphorylation events are pathogenically involved in pemphigus acantholysis.

Changes in desmoglein 1 expression and subcellular localization in cultured keratinocytes subjected to anti‐desmoglein 1 pemphigus autoimmunity

The complexity of pemphigus acantholysis together with the weak expression of desmoglein 1 (Dsg1) in cultured keratinocytes have made the study on the pathogenic action of anti-Dsg1 antibodies quite difficult. The pathophysiology of the acantholytic phenomenon could depend on the reduction of Dsg1 adhesion function occurring after its massive internalization or decrease of its synthesis. Here, we have investigated this hypothesis by using sera of patients having antibodies against Dsg1 or monoclonal anti-Dsg1 antibodies to simulate pemphigus autoimmunity in Dsg1-rich keratinocytes. Similar to pemphigus foliaceus (PF) and vulgaris (PV) sera, monoclonal anti-Dsg1 antibodies induced transient internalization of Dsg1 and reduced the adhesion strength among keratinocytes. However, binding of IgG to Dsg1 did not determine its early depletion from the adhesion complexes but reduced the amount of Dsg1 found in the Triton X-100 soluble pool of proteins. Taken together, our results represent the first demonstration that anti-Dsg1 antibodies induce similar alterations on the subcellular distribution of Dsg1 irrespective of the disease where they come from. Furthermore, the present study provides insight into the mechanisms underlying epithelial blistering observed in the skin type of pemphigus.

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Corticosteroids, aurothioglucose and soybean trypsin inhibitor do not prevent pemphigus antibody-induced acantholysis in vitro

Hydrocortisone, triamcinolone acetonide, aurothioglucose and soybean trypsin inhibitor were added to normal human skin explants cultured with IgG from pemphigus serum to determine if acantholysis could be prevented. At the therapeutic concentrations used none of these compounds prevented binding of the autoantibody to the epidermal target cells, and none prevented acantholysis. These experiments support the concepts that the pemphigus antibody alone is responsible for producing the acantholytic lesions of pemphigus, that the therapeutic effectiveness of steroids and gold salts is probably due to their ability to reduce serum autoantibody titres and that pemphigus acantholysis is probably not caused by a serine proteinase.

How does acantholysis occur in pemphigus vulgaris: a critical review

Pemphigus vulgaris is a life-threatening autoimmune blistering disease targeting skin and mucous membranes, characterized by disruption of keratinocytes' adhesion termed acantholysis. Today multiple classes of targets are considered to play a role in the genesis of the acantholysis; of these, the classical pemphigus antigens, desmosomal cadherins (desmoglein 1 and 3) are the best characterized and considered as the most important. Additional antigens include the novel epithelial acetylcholine receptors (alpha9 and pemphaxin). Thus, acantholysis in pemphigus seems to result from a cooperative action of antibodies to different keratinocyte self-antigens, but the mechanisms by which epithelial cleft occurs are not yet clearly understood. In fact, the binding of the autoantibodies to these targets generates a plethora of biological effects due, on one hand, to their direct interference with adhesive function and, on the other, to more complex events involving intracellular pathways that modify proteases activity or calcium metabolism, leading to loss of cell-cell adhesion.

Mechanisms of Acantholysis in Pemphigus: Mechanical or Inflammatory?

Pemphigus is a rare autoimmune disease, involving the skin and mucous epithelia, characterized by flaccid blisters and erosions. Histologically, the basic abnormality in all forms of pemphigus is the separation of keratinocytes from one another, a process known as acantholysis. There is direct evidence that autoantibodies against desmoglein, a transmembrane desmosomal component, are critical in its pathogenesis, but the exact mechanism that induces acantholysis is yet unknown. Actually, different studies suggest three possible mechanisms: sterical impedance, intracellular signalling and apoptosis. Understanding these processes should show new therapeutic perspective.

Pemphigus in the XXI Century: New life to an old story

In this new century of pemphigus research, the search for novel treatments is switching from a monospecific approach, focused on immunosuppression, to a polyspecific approach that includes drugs acting on novel pathophysiologic pathways. Current research argues that acantholysis in pemphigus occurs as an active process resulting from intracellular signaling triggered as a result of IgG binding to the keratinocyte membrane antigens in a receptor-ligand fashion. Recent progress regarding the pathophysiology of pemphigus acantholysis led to, or was accompanied by, breakthrough discoveries of safer treatments. Both the identification of cell-surface receptors to acetylcholine among the nondesmoglein (Dsg) targets for pemphigus antibodies, and the elucidation of the cholinergic control of keratinocyte cell adhesion provide an explanation for the therapeutic efficacy of cholinomimetics in patients with pemphigus. In patients' skin, Fas-L, TNFα, and, probably, IL-1α act as autocrine/paracrine co-factors for anti-keratinocyte IgG. Thus, it appears that an array of interconnected signaling cascades is responsible for acantholysis and cell death in pemphigus. Future studies should define the signaling pathways mediating acantholysis that occur in individual pemphigus patients and identify the membrane proteins (receptors) triggering signaling along a specific pathway upon their ligation by autoantibodies. It will be important to determine which pathway 1) leads directly to a loss of cell-cell adhesion (primary pathway), 2) which is being activated due to cell shrinkage/detachment (secondary pathway), 3) which contributes to utilization of altered proteins and organelles (scavenging pathway), and 4) which represents the cell defense (protective pathway). To dissect out the signaling pathways originating from binding of pemphigus IgG to non-Dsg targets on the keratinocyte plasma membrane experiments should be performed in cultures of murine keratinocytes grown from the Dsg3-/- mice or human keratinocytes with the knocked-down expression of the Dsg1 and/or Dsg3 gene by the RNA interference.