In vitro acantholysis by captopril and thiopronine

The possible acantholytic property of captopril and thiopronine has been investigated using in vitro tissue cultures. Normal human breast skin explants have been cultured in Hanks' balanced salt solution containing 40% normal inactivated human serum with the addition of L-cysteine, or reduced glutathione (GSH), or captopril, or thiopronine, at four different concentrations (1, 5, 10, 15 mM). Patterns of diffuse, mainly suprabasal acantholysis, with formation of bullae, were observed in the skin explants cultured with captopril or thiopronine at a 15-mM concentration after 5 days of culture; intraepidermal splits were present also at a 10-mM concentration. Focal acantholysis was seen in specimens cultured with L-cysteine or GSH at a 15-mM concentration. No lesions occurred in the samples treated with lower concentrations of the above substances, nor in controls. The results show a biochemical acantholytic potential of both captopril and thiopronine, resembling that of penicillamine in similar experimental conditions, and consonant with clinical observations of pemphigus induced by drugs containing thiol groups in their molecule (SH drugs).     

In vitro acantholysis induced by D-penicillamine, captopril, and piroxicam on dead de-epidermized dermis

Drug-induced pemphigus has been recognized for 20 years, but the mechanisms leading to acantholysis are still unclear. It has recently been demonstrated that penicillamine, captopril, and thiopronin may produce acantholytic lesions, either by direct toxic or biochemical effect, in human skin explants. Our work confirms that penicillamine and captopril may induce acantholysis on the model of keratinocyte culture on dead, de-epidermized dermis. Moreover, it demonstrates that piroxicam, a new non-steroidal anti-inflammatory drug, of which one side effect is a pemphigus vulgaris-like eruption, is also able to produce in vitro acantholysis.     

Appearance of "pemphigus acantholysis factor" in human skin cultured with pemphigus antibody

These studies deal with the mechanism of pemphigus IgG-induced epidermal acantholysis. When normal human skin was culted with defined medium containing IgG from pemphigus serum, extensive epidermal acantholysis developed and heat-labile proteolytic enzyme(s) were recovered in the culture medium. The enzyme(s) displayed maximal activity at pH 6.5 when a 3H-amino acid-labeled, insoluble epidermal cell material was used as substrate. The enzyme activity increased during the first 3 days of culture and the appearance of maximal activity coincided with the time of onset of acantholysis. Acantholysis did not occur in control cultures incubated with normal IgG and the enzyme did not appear in the medium or in aqueous extracts of cultured tissues. The enzyme(s) is probably not of lysosomal origin because low pH-active proteases characteristic of these organelles remained within the cells. The effects of puromycin on appearance of enzyme activity, acantholysis and cell viability was studied. At cytotoxic concentrations, the appearance of the enzyme(s) and acantholysis were prevented, whereas at less toxic concentrations enzyme activity and acantholysis were not prevented. Because inhibition of protein synthetic rates by puromycin could not be dissociated from the cytotoxic effects, it is uncertain whether enzyme appearance and acantholysis were dependent upon living tissue or on specific protein synthesis. After pemphigus IgG was removed from the conditioned medium by DEAE cellulose and affinity column chromatography, the remaining material contained enzyme activity and caused acantholysis in fresh skin explants. Similar activities were not present in normal IgG-containing conditioned medium or unfractionated epidermal extracts from normal skin. These data indicate that when the pemphigus IgG autoantibody interacts with epidermal cell surface antigens, the cell responds by synthesis or activation of a non-IgG "pemphigus acantholysis factor" (PAF) which may be a nonlysosomal proteolytic enzyme. It is suggested that PAF causes loss of adhesion between keratinocytes and ultimately produces the characteristic acantholytic cells of pemphigus.     

Drug-induced pemphigus: autoantibodies directed against the pemphigus antigen complexes are present in penicillamine and captopril-induced pemphigus

Pemphigus is an autoimmune blistering disease characterized by circulating autoantibodies directed against the keratinocyte cell surface. The two variants, pemphigus foliaceus and pemphigus vulgaris, can be distinguished at the molecular level by immunochemical studies. The large majority of patients with pemphigus develop the disease spontaneously; however, there is a small group of patients who develop pemphigus after treatment with certain medications, of which penicillamine and captopril are the best documented. Most patients with drug-induced pemphigus have circulating and/or tissue bound epidermal cell surface autoantibodies; however, the molecular specificity of these autoantibodies has not been studied. We performed immunoprecipitation studies utilizing extracts of 125I-labeled suction blister epidermis and the sera of three patients with drug-induced pemphigus foliaceus (two due to penicillamine and one due to captopril) and one patient with captopril-induced pemphigus vulgaris. We found that the three patients with drug-induced pemphigus foliaceus had circulating autoantibodies that are directed against the pemphigus foliaceus antigen complex and that the one patient with drug-induced pemphigus vulgaris had circulating autoantibodies that are directed against the pemphigus vulgaris antigen complex. This study demonstrates that autoantibodies from drug-induced pemphigus patients have the same antigenic specificity, on a molecular level, as do autoantibodies from other pemphigus patients.     

Activation of keratinocyte muscarinic acetylcholine receptors reverses pemphigus acantholysis

We studied neuroendocrine mediator regulation of adhesion and motility of human epidermal keratinocytes (EK) and found that cholinergic compounds control EK cell-matrix and cell-cell attachment. In this study, we tested the anti-acantholytic activity of muscarinic agonists in pemphigus and non-pemphigus acantholysis, and investigated the effects of pemphigus antibody (Pab) on the the keratinocyte muscarinic acetylcholine receptors (mAChR). Acantholysis produced by Pab from two patients with pemphigus vulgaris was compared with acantholysis induced by the serine protease trypsin. the calcium chelator EOT A or the muscarinic antagonist atropine. Trypsinized EK first lost contact with microplate surface and then retracted their intercellular filaments. EDTA-treated cells first detached from each other and then from the dish. EK cultures treated with Pab or atropine rounded up and retracted their intercellular filaments simultaneously, although it took hours to obtain acantholysis with Pab treatment compared to several seconds with atropine treatment. Addition of acetylcholine or other muscarinic agonists (bethanechol. carbachol or methacholine) to acantholytic cultures reversed both pemphigus and non-pemphigus acantholysis. Acantholysis induced by atropine reversed spontaneously. Short-term preexposure of EK to Pab significantly increased, and long-term preexposure significantly decreased, the [³H]atropine binding to keratinocyte mAChR. We conclude that muscarinic agonists reverse various types of acantholysis. including acantholysis induced by Pab, and that binding of Pab to EK may affect the ability of keratinocyte mAChR to bind its ligands.     

ENALAPRIL: A POWERFUL IN VITRO NON-THIOL ACANTHOLYTIC AGENT

Drugs containing sulfhydryl groups (thiol drugs) (e.g., penicillamine, captopril, thiopronine) can induce pemphigus in vivo and provoke acantholysis in vitro. Enalapril, like captopril, is an angiotensin-converting enzyme (ACE) inhibitor largely used as an antihypertensive drug; it has recently been reported to induce pemphigus, though it is not a thiol drug. In this study we investigated the possible in vitro acantholytic effect of enalapril on normal human skin from donors. The drug induced severe acantholytic changes of keratinocytes and complete suprabasal splitting at one tenth the concentration required by thiol drugs in similar experiments, even after a shorter period of culture. All skin samples from different donors was highly susceptible to the acantholytic effect of enalapril. In our experience, enalapril is the most powerful acantholytic drug in vitro.     

Functional involvement of urokinase-type plasminogen activator receptor in pemphigus acantholysis

Urokinase-type plasminogen activator (uPA) has been well documented in the development of pemphigus acantholysis. The function of its receptor (uPA-R) in pemphigus acantholysis has only recently attracted attention. Increased expression of uPA-R has been demonstrated in pemphigus vulgaris. In this study, we have further explored the functional involvement of uPA-R in pemphigus acantholysis. Our results show that uPA-R expression is significantly increased in acantholytic foci of pemphigus vulgaris and pemphigus foliaceus but not in bullous pemphigoid or normal skin specimens; the expression of uPA-R in cultured human keratinocytes is subjected to regulation by pemphigus vulgaris IgG but not by pemphigoid IgG or normal human IgG; furthermore, anti-uPA-R monoclonal antibody effectively inhibits pemphigus vulgaris IgG induced acantholysis in skin organ cultures. These data suggest that uPA-R may play an important role in the pathogenesis of pemphigus acantholysis.     

Pemphigus and pemphigoid-like effects of nifedipine on in vitro cultured normal human skin explants

BACKGROUND: A variety of drugs have been implicated in the onset and exacerbation of pemphigus and bullous pemphigoid. The demonstration of biochemical acantholysis in skin explants to various drugs in the absence of autoantibodies, in which the tested drugs evoke a biochemical reaction that leads to desmosomal function loss, may be a valuable adjunct to patient management by confirming the suspicion of drug-related pemphigus or bullous pemphigoid. OBJECTIVE: To determine whether a skin explant model might serve as a possible in vitro correlate of drug-induced pemphigus and pemphigoid-like effects related to the calcium channel blocker nifedipine. METHODS: Normal human breast skin obtained from nonpemphigus and nonpemphigoid patients undergoing mastectomy was cultured with nifedipine at final concentrations of 2, 4, and 8 mM. The drug effect on skin explants evidenced by morphologic changes was evaluated by microscopy by three observers. RESULTS: Five out of seven explants cultured with nifedipine at concentrations ranging from 2 to 8 mM exhibited obvious morphologic changes of two types: intraepithelial (or pemphigus-type) splittings and subepithelial (or pemphigoid-type) splittings. Two explants showed no acantholysis and no subepithelial splittings. Control cultures without polyethylene glycol 200 (PEG) showed no changes. Skin control samples cultured in medium supplemented with 10% PEG displayed vacuolar degeneration throughout the entire epidermis, but no sign of cell-cell dyshesion or dermo-epidermal detachment. CONCLUSIONS: A type of skin susceptibility to nifedipine may be genetically determined, with some nifedipine-treated patients developing an acantholytic reaction and others a subepidermal bullous eruption.     

High doses of antigen-nonspecific IgG do not inhibit pemphigus acantholysis in skin organ cultures

Summary A patient suffering from severe pemphigus vulgaris was treated using large-volume plasma exchange in combination with an immunosuppressive regimen. As some recent reports have shown evidence that polyclonal, polyspecific human IgG in high doses through the i.v. route (IGIV) protect target platelets in idiopathic thrombocytopenic purpura from attack by antiplatelet autoantibodies and/or immune complexes, we also administered IGIV to this pemphigus-vulgaris patient. In order to test the hypothesis that IGIV might protect in vitro-cultured human skin from acantholysis induced by pemphigus antibodies, studies with skin organ cultures were carried out using plasma from another pemphigus-vulgaris patient who had undergone plasma exchange. The preincubation of either the skin explants or the pemphigus plasma with various concentrations of IGIV (ranging from 0.15 to 15 mg/ml in the culture medium) did not prevent acantholysis induced by the pemphigus plasma nor did it inhibit the binding of the specific antibodies visualized by direct immuno-fluorescence. Thus, the assumption that IGIV may coat the pemphigus antigens on epidermal cells making them inaccessible to pathogenic autoantibodies was not substantiated by our tests in vitro; likewise, the hypothesis of functionally blocking autoantibody activity by means of anti-idiotype effects of IGIV cannot be supported.Abbreviations IGIV Human immunoglobulin G i.v. - DIF direct immunofluorescence - IIF indirect immunofluorescence Presented in part at the International Symposium on Models to Study Function and Disease of Skin, March 17, 1984, Cardiff, United Kingdom     

Signalling pathways in pemphigus vulgaris

Acantholysis in pemphigus vulgaris is induced by binding of autoantibodies to desmoglein 3 (Dsg3). The roles of signalling pathways on development of acantholysis have recently been extensively studied. In the study by Sayar et al., recently published in Exp Dermatol, epidermal growth factor receptor (EGFR) signalling was activated in both in vivo and in vitro pemphigus vulgaris experimental models. However, while EGFR inhibitors suppressed activity of p38 mitogen‐activated protein kinase (p38MAPK) linearly, they suppressed activity of c‐Myc and acantholysis in a non‐linear, V‐shaped relationship. These findings indicated complicated interactions among EGFR, p38MAPK and c‐Myc in pemphigus vulgaris pathology.     

Pityriasis rubra pilaris: the clinical context of acantholysis and other histologic features

Background Acantholysis has been described in biopsies of pityriasis rubra pilaris (PRP), but this has not been emphasized in the dermatology literature. It is helpful for dermatologists to associate acantholysis with PRP in the correct clinical setting. Objective This study aims to further elucidate the clinical context and associated histologic features of acantholysis in PRP. Methods Eight cases of PRP with acantholysis, 16 other cases of PRP, 26 cases of psoriasis, and 17 cases of erythroderma of different causes were studied in an academic setting. Results The presence of acantholysis initially confounded the diagnosis in two cases of PRP. Acantholysis was focal or extensive and resembled Darier’s disease, Hailey–Hailey disease, or pemphigus vulgaris. Acantholysis was seen in biopsies from early, isolated papulosquamous lesions from the trunk as well as from erythroderma. By comparison, 26 cases of psoriasis and 17 cases of erythroderma showed only focal acantholysis in two cases of erythroderma. Conclusions Acantholysis is a histologic feature of PRP and can serve as a histologic clue to the diagnosis of PRP before the onset of erythroderma. Eosinophils and/or a lichenoid infiltrate may also be evident.     

Paraneoplastic pemphigus triggered by radiotherapy

Paraneoplastic pemphigus is a recently described autoimmune disease characterized by painful mucosal ulceration and polymorphous skin lesions in association with an underlying neoplasm. Distinct autoantibodies bind desmoplakin I, desmoplakin II, bullous pemphigoid antigen and an uncharacterized 190kDa antigen. A case is presented of paraneoplastic pemphigus that developed after radiotherapy for non- Hodgkin's lymphoma in a 53 year old man. Multiple skin biopsies showed a lichenoid reaction without acantholysis. Immunofluorescence and mucosal biopsies were required to establish the correct diagnosis. Corneal opacities resembling lichenoid graft-versus-host disease and retinal haemorrhages, which developed in the patient, have not been previously documented. Despite high doses of immunosuppressive agents and plasmaphoresis, the patient eventually died from respiratory failure.     

Pemphigus

The term pemphigus refers to a group of autoimmune intraepidermal blistering diseases of the skin and mucous membranes. Several clinical variants of pemphigus are recognized. The major histologic feature of all variants is acantholysis, the disruption of normal cell-to-cell adhesion, which leads to intraepidermal blister formation. Most patients with pemphigus demonstrate IgG autoantibodies directed against an antigen located on the surface of keratinocytes. Although the stimulus for autoantibody production is unknown, several mechanisms have been proposed to explain the pathogenesis of acantholysis. One popular model proposes that pemphigus antibodies induce acantholysis through local stimulation of the plasminogen-plasmin system. Another model proposes that pemphigus antibodies fix complement and thereby alter cell membrane integrity to produce acantholysis. Prior to the availability of corticosteroids, pemphigus vulgaris was commonly fatal. Treatment with glucocorticosteroids has drastically improved the prognosis. Immunosuppressive agents and plasmapheresis have been used successfully in some patients with severe disease.     

Different patterns of in vitro acantholysis in normal human skin samples explanted from different sites of the body

Background The factors that contribute to a preferential anatomic localization of pemphigus lesions are not well known. In particular, the question arises as to whether certain skin areas may be more acantholysis-prone than others.
Objective To verify whether, in pemphigus patients, a different susceptibility to acantholysis exists among different cutaneous regions, the technique of tissue cultures was used.
Methods Normal human skin explants from two distinct anatomic regions (back and buttocks) of two former pemphigus patients were cultured in vitro in the presence of enalapril (6 mM) or cystamine (10 mM), two substances with a proven biochemical acantholytic effect. After 4 days of culture, the tissues were processed for standard histology.
Results Diffuse acantholysis, with large intraepidermal splits, was observed in the explants taken from the backs of both subjects and cultured with either enalapril or cystamine. Mild to moderate acantholytic changes were detected in the explants taken from the buttocks of both subjects and cultured with either enalapril or cystamine. No structural changes were seen in the control cultures.
Conclusions Pemphigus patients present different thresholds of acantholysis in different areas of their bodies. This might explain, at least in part, certain preferential anatomic localizations of pemphigus lesions.     

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.     

Involvement of urokinase-type plasminogen activator in acantholysis induced by pemphigus IgG

Pemphigus IgG induces acantholysis in skin organ culture without the involvement of complement. Urokinase-type plasminogen activator, a proteolytic enzyme, has been implicated in the development of acantholysis. To test this hypothesis, we prepared a rabbit anti-urokinase antibody, which inhibited the plasminogen activator activity in normal human epidermis and in cultured keratinocytes. When added to skin organ cultures along with pemphigus IgG, anti-urokinase IgG completely prevented the development of acantholysis. Normal or preimmune rabbit IgG had no effect on pemphigus IgG-induced acantholysis. Plasminogen activator converts the zymogen plasminogen to its active form plasmin, a broad specificity serine proteinase. When high concentrations of plasminogen alone were added to skin organ culture, acantholysis of the pemphigus foliaceous type was induced. Anti-urokinase antibody also inhibited plasminogen-induced acantholysis. These results strongly support a pivotal role for plasminogen activator in the development of acantholysis.     

天疱疮发病机制的研究进展

天疱疮是自身免疫引起表皮棘层细胞松解导致的慢性复发性表皮内大疱性皮肤病,典型表现为红斑基础上的疱壁松弛性水疱、糜烂、尼氏征阳性.依据天疱疮的临床表现,分为寻常型、增殖型、落叶型和红斑型天疱疮.天疱疮主要的发病机制是患者存在针对角质形成细胞桥粒芯(糖)蛋白的自身抗体,但棘层松解的详细机制尚不清.近年来,随着对蛋白组学、免疫学和分子生物学技术的发展以及天疱疮发病机制研究的不断深入,发现天疱疮的发病机制中除传统的桥粒芯(糖)蛋白自身抗体外,非桥粒芯(糖)蛋白抗体因素也参与了棘层松解的形成,为天疱疮提供了新的潜在治疗靶位.     

Aprotinin inhibition of experimental pemphigus in Balb-c mice following passive transfer of pemphigus foliaceus serum

The effect of aprotinin, a plasmin inhibitor, is evaluated in experimental pemphigus. Serum with indirect immunofluorescence IgG litres of 1:1280 was obtained from a pemphigus foliaceus patient. Twenty neonatal Balb-c mice less than 1-day-old were injected intraperitoneally every 24 h with 0·1 ml of pemphigus foliaceus serum. Ten were also inoculated subcutaneously with 0·1 ml physiological saline solution (Group 1), and another ten animals received aprotinin 0·1 ml subcutaneously (Group 2). Five hours later serum and skin samples from the mice were obtained for histological study and indirect and direct immunofluorescence. Clinically extensive disease and histologically subcorneal pemphigus with extensive acantholysis were observed in Group 1. In Group 2 no clinical manifestations were seen apart from minimal acantholysis in four mice. The direct immunofluorescence for intercellular epidermal IgG was positive in both groups. Similar titres of IgG were seen in mouse serum in both groups (1:160-1:320). We conclude that aprotinin is able to inhibit experimental pemphigus in neonatal Balb-c mice by passive transfer of pemphigus foliaceus serum. It also suggests that plasmin plays an important role in the pathophysiology of the experimental disease. Pemphigus is a potentially fatal autoimmune blistering disease. It is characterized by a single autoantibody, pemphigus IgG which binds to a specific antigen belonging to the cell surface of differentiated cells in stratified epithelium of birds and mammals. ^1–2 The experimental pemphigus model in Balb-c mice, was successfully developed by Anhalt et al. in 1982. ³ They showed that purified IgG from patients with pemphigus vulgaris, injected intraperitoneally in mice, may reproduce pemphigus clinically, histologically, immunologically and ultrastructurally. Since then, other papers have appeared describing the use of the murine experimental model. ^4–6 Other in vitro studies (epidermal cell cultures) indicate that the IgG of pemphigus vulgaris and pemphigus foliaceus produce a significant increase in plasminogen activator. Hashimoto et al.⁷ reported that an increase in the activity of plasminogen activator could be an important step in the development of acantholysis after the reaction of pemphigus IgG with antigen. Plasminogen activator would act on the plasminogen of the epidermis generating plasmin, which would degrade the adhesive components of the cell surface. The addition of corticosteroids to epidermal cell cultures, incubated with pemphigus autoantibodies inhibited the increase of plasminogen activator significantly but failed to block the acantholytic process. ⁸ In other studies inhibition of acantholysis was obtained. ⁹ If plasmin inhibitors such as lima bean trypsin inhibitor and aprotinin, which do not inhibit plasminogen activator, are added to cell cultures treated with pemphigus immunoglobulins, acantholysis is not produced. These findings, are consistent with the theory that plasmin is the enzyme that produces acantholysis in pemphigus. ¹⁰ The present study attempts to evaluate the in vivo effect of a plasmin inhibitor such as aprotinin. To our knowledge this problem has not been investigated in the murine model of the disease.     

Complement-fixing pemphigus antibodies.

The serum of a patient with a typical case of pemphigus vulgaria contained complement-fixing intercellular autoantibodies (pemphigus antibodies). Complement-fixing pemphigus antibodies were demonstrated only during the untreated active stage of the disease and titrated lower than corresponding IgG autoantibodies, which paralleled well with the disease activity. Pemphigus lesional skin, which contained in-vivo-bound IgG, showed the capability of further binding C3 in vitro from normal human serum. It was suggested from these findings that the complement system may play an active role in pemphigus acantholysis through complement-fixing pemphigus antibodies.     

Fosinopril as a possible pemphigus-inducing drug

Fosinopril has recently been added to the angiotensin-converting enzyme inhibitors inducing pemphigus. The observation of a patient in whom pemphigus vulgaris (PV) worsened after taking fosinopril prompted us to study an experimental way to assess its responsibility. Slices of normal human skin (NHS) were simultaneously incubated for 2, 6, 12 and 24 h at 4 degrees C with progressively diluted fosinopril and captopril solutions and used as indirect immunofluorescence (IIF) substrates for 2 sera containing anti-desmoglein-3 (anti-Dsg3) antibodies at a dilution of 1/160. With captopril, IIF was negative, irrespective of dilution and time of incubation. Only at 1/40,000 dilution was IIF positive. With fosinopril, IIF was negative for the 2- and 6-hour-long incubations but turned positive after 12 h and so remained with all other solutions and incubation times. IIF negativity with captopril suggests that anti-Dsg3 antibodies contained in the PV sera were unable to find molecules in NHS to bind to. Captopril would therefore induce acantholysis by blocking the adhesion molecules. With fosinopril, instead, a partial block of the adhesion molecules was seen only with the very concentrated solution, unlikely to occur in vivo. Fosinopril, therefore, is probably unable to block the adhesion molecules in vivo. Our method might be used to verify the acantholytic properties of a drug.