Pemphigus Acantholysis Research Articles

A Possible Mechanism for Phenol‐Induced Pemphigus

A possible mechanism for phenol-induced pemphigus lesions in genetically predisposed individuals is proposed that accounts for in vitro observations and cases of biochemical acantholysis, as well as the in vivo acantholysis in pemphigus induced by phenols. The mechanism involves the induction of interleukin-1a and tumor necrosis factor-a release by keratinocytes. These cytokines in turn have been shown to be involved in the regulation and synthesis of complement and proteases like plasminogen activator, which have been implicated in the pathogenesis of acantholysis in pemphigus vulgaris.

Synergistic actions of pemphigus vulgaris IgG, Fas-ligand and tumor necrosis factor-α during induction of basal cell shrinkage and acantholysis

This study tested a recently proposed “Basal Cell Shrinkage” hypothesis of pemphigus acantholysis through a quantitative analysis of individual and cooperative effects of pemphigus vulgaris (PV) IgG, Fas-ligand (Fas-L) and tumor necrosis factor-α (TNFα) on keratinocyte (KC) volume (i.e. cell size) and adhesive properties. Exposure of KC monolayers and MatTek EpiDermFT™ tissues cultures to the physiologic concentrations of Fas-L, TNFα or IgGs from two PV patients resulted in various degrees of reversible changes, which were not observed in control cultures either exposed to normal IgG or left intact. Within 12–24 h of exposure, basal cells in experimental cultures lost their ability to form stress fibers, retracted cytoplasmic aprons and formed keratin aggregates, indicating that their cytoskeleton collapsed. The cell volume decreased significantly (p < 0.05) as the polygonal cell shape changed to a round one. The shrunk cells detached from their neighbors and the substrate, resulting in a reciprocal increase of both the areas of acantholysis and the number of detached KCs, respectively. Since in the skin of PV patients, KCs are targeted by autoantibodies concomitantly with being exposed to autocrine and paracrine pro-apoptotic and pro-inflammatory cytokines, we combined PV IgG with Fas-L and/or TNFα in the cell culture experiments. This amplified several fold an ability of PV IgG to cause basal cell shrinkage and detachment. The obtained results demonstrated for the first time that PV IgG works together with Fas-L and TNFα to induce acantholysis via basal cell shrinkage, which provides a novel mechanism explaining successful treatment of PV patients with TNFα inhibitors.

Phosphatidylinositol-specific-phospholipase C cleaves urokinase plasminogen activator receptor from the cell surface and leads to inhibition of pemphigus-IgG-induced acantholysis in DJM-1 cells, a squamous cell carcinoma line.

We showed previously that pemphigus IgG enhanced both the activity of urokinase plasminogen activator (uPA) in cultured cells and the expression of its receptor (uPAR) on uPA-binding keratinocytes. In the present study, to clarify whether uPAR and uPA-activated plasmin are actually involved in the blistering process after pemphigus IgG binding to the cell surface, we examined the effects of the following on uPAR expression and on cell-cell detachment in DJM-1 cells, a squamous cell carcinoma line: (i) phosphatidylinositol-specific phospholipase C (PI-PLC) - which releases uPAR from the membrane surface into the culture medium by cleaving the glycosylphosphatidylinositol anchor thus inhibiting uPAR activity, and (ii) uPA inhibitors (tranexamic acid, aprotinin, p-aminobenzonic acid and dexamethasone). Preincubation with PI-PLC decreased dramatically the pemphigus IgG-induced uPAR expression in a dose-dependent manner, and inhibited pemphigus IgG-induced cell-cell detachment at 10 microg/mL. On the other hand, tranexamic acid (15 mM) inhibited pemphigus IgG-induced cell-cell detachment without reduction of uPAR expression, although aprotinin, p-aminobenzonic acid and dexamethasone failed to alter either of these parameters. Although uPAR expression on the pemphigus IgG-bound cell surface and uPA activation may contribute significantly to the pathogenesis of acantholysis in pemphigus, the mechanisms are complicated and should be defined further.

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.

Incidental acantholysis.

The incidental finding of microscopic foci of acantholytic dyskeratosis, reproducing the histological pattern of Darier's disease, has been the subject of a number of papers in the last few years. In contrast, the incidental finding of other types of acantholysis has not been mentioned in the literature. In the last 5 years, we have noticed such a microscopic finding in 14 biopsy specimens (0.15% of our cutaneous specimens). they were from 13 patients (7 men and 6 women); their ages ranged from 37 to 79 years (mean, 63). Three lesions were located on the head, 7 on the trunk, and 4 on the limbs. The histological diagnoses were basal cell carcinoma (6 cases), keratoacanthoma (2 cases), psoriasis (2 cases), elastolytic granuloma, acral arteriovenous angioma, tinea corporis, and leukocytoclastic vasculitis (1 case each). The histological patterns of the acantholytic foci simulated pemphigus vulgaris (9 cases), superficial pemphigus (1 case), Hailey-Hailey disease (3 cases), and unclassifiable acantholysis (1 case). None of the patients had familial or personal history of acantholytic disorders. We propose a classification of the primary acantholytic disorders based on both their acantholytic pattern and their clinical extent.

An immunohistological study of desmosomal components in pemphigus

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune diseases in which there is loss of cohesion between keratinocytes (acantholysis) and blistering within the epidermis. PV is characterized by acantholysis predominantly between the epidermal basal cells and suprabasal layers, whereas in PF intraepidermal cleavage is higher in the epidermis. Adhesion between keratinocytes is dependent on the function of transmembrane glycoproteins of the cadherin family present in specialized adhesion junctions, the desmosomes. The pathogenesis of acantholysis in pemphigus is uncertain, but the pemphigus autoantibodies bind to epithelial cadherins. We have used monoclonal antibodies to desmosomal components to investigate their distribution in different forms of pemphigus. Our results show that the localization of desmosomal components is abnormal in intact perilesional epidermis, intact epidermis above the blisters in PV and intact epidermis below the blisters in PF. We suggest that autoantibody binding may have a direct effect on the function of specific epithelial cadherins, but will only cause cell separation where the antigen is the principal adhesion molecule.

Involvement of Membrane Attack Complex of Complement in UV-B-Induced Acantholysis in Pemphigus

The initial acantholytic process induced by irradiation of UV-B on uninvolved skin of patients with pemphigus was immunohistologically studied using monoclonal antibodies that bind to antigenic determinants present in the C5b9 complex. Membrane attack complex of complement-related antigens in suprabasal intercellular sites could be detected after 5 hours of irradiation, at which time there was no acantholysis. At 24 hours, the staining intensity markedly increased and suprabasal clefts with acantholytic epidermal cells could be seen. In contrast, acantholysis did not develop in the absence of membrane attack complex formation at intercellular sites even 24 hours following UV-B irradiation. These findings suggest that UV-B can induce membrane attack complex assembly in the epidermis, which may itself be involved in the process of acantholysis and provide additional evidence for involvement of the complement system in pemphigus.

Involvement of membrane attack complex of complement in UV-B-induced acantholysis in pemphigus

Involvement of membrane attack complex of complement in UV-B-induced acantholysis in pemphigus

In Vitro Pemphigus Vulgaris Model Using Organotypic Cultures of Human Epidermal Keratinocytes

Using the Combi-ring-dish (CRD), a new culture device, organotypic cultures of human epidermal keratinocytes were grown on bovine eye lens capsules. In these highly differentiated cultures, typical suprabasal acantholysis was induced by pemphigus vulgaris antibodies. This in vitro pemphigus vulgaris model may be used to analyse keratinocyte-derived factors causing acantholysis in experimental pemphigus.

Proteinase Inhibitors Block Formation of Pemphigus Acantholysis in Experimental Models of Neonatal Mice and Skin Explants: Effects of Synthetic and Plasma Proteinase Inhibitors on Pemphigus Acantholysis

In organ culture experiments, the induction of pemphigus acantholysis is known to be blocked by the addition of serine proteinase inhibitors. Recently, nontoxic synthesized low molecular weight proteinase inhibitors have been clinically available for the treatment of disseminated intravascular coagulation and pancreatitis. To determine if these drugs are useful aids to treat patients with pemphigus, we examined the effect of omega-guanidino ester analogues, i.e., 1) gabexate mesilate, 2) camostat mesilate, and 3) nafamostat mesilate, on experimental pemphigus acantholysis in both organ culture and neonatal BALB/c mice. Furthermore, the effect of plasma natural proteinase inhibitors (alpha-1-proteinase inhibitor) isolated from human plasma was similarly examined. Results revealed that synthesized low molecular weight inhibitors (drugs) were able to inhibit the induction of acantholysis in organ culture system, but had little or no effect on lesion formation in the neonatal mouse system. By contrast, alpha-1-proteinase inhibitor could completely inhibit acantholysis formation in mice. These findings implied a possible new therapeutic approach using proteinase inhibitors for patients with pemphigus.

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

Summary 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.

Plasmin induces acantholysis in skin organ cultures.

Addition of human plasminogen to three different pemphigus plasma samples showed a synergistic effect on acantholysis in the skin organ culture model. Human plasmin itself, without addition of pemphigus plasma, induced typical acantholytic changes in the skin explants, causing different types of acantholysis in a dose- and time-dependent manner: in the presence of 3 CU plasmin per ml culture medium, focal suprabasilar acantholysis of pemphigus vulgaris type could be detected after 72 h incubation, whereas 15 CU/ml caused extended acantholysis of pemphigus foliaceus type in the upper epidermal layers after 24 h, and extended acantholysis of benign chronic pemphigus (Hailey-Hailey disease) type comprising all layers of the epidermis after 48 h incubation. Plasminogen activator levels (Mr 55,000 urokinase type) in tissue extracts of skin explants and in culture media were reduced after 24 and 48 h incubation with pemphigus IgG as compared to control experiments with normal human IgG; this probably resulted from urokinase inactivation by reaction with inhibitors. These results lend support to the hypothesis proposed by Hashimoto et al. in 1983 that the plasminogen activator-plasmin system could play an essential role in the protease mechanisms of pemphigus acantholysis.

Concurrent features of cutaneous lupus erythematosus and pemphigus erythematosus following myasthenia gravis and thymoma

A case that exhibits the pathologic features of cutaneous lupus erythematosus and the immunopathologic characteristics of pemphigus erythematosus following myasthenia gravis and thymoma is presented, and the literature pertaining to these disease associations is reviewed. The findings in this case were analyzed with respect to the accepted diagnostic criteria for lupus erythematosus, pemphigus, and pemphigus erythematosus. The pathogenic mechanisms that lead to acantholysis in pemphigus are examined in an attempt to explain why acantholysis did not occur in this case.

Molecular mechanisms of pathogenesis

The first clue to the etiology of pemphigus was established in 1964, when Beutner and Jordon 1 demonstrated that serum from pemphigus patients contained autoantibodies that bound to an intercellular substance (ICS) of skin and mucosa. Subsequently, skin biopsies revealed in vivo deposition of auto-antibodies in the epidermis of pemphigus patients. 2 While these results were intriguing, they did not prove that antibody played a role in the acantholytic process. The following three lines of evidence do support a role for antibody in the induction of acantholysis in pemphigus: correlation of antibody titers with disease activity 3,4; induction of acantholytic lesions in the skin of neonatal mice 5 or in grafts of human oral mucosa on nude mice 6 following passive transfer of patient IgG; and induction of acantholytic lesions in expiants of normal human skin following incubation with patient IgG. 7,8 The first two lines of evidence have been discussed in other chapters of this volume. It is our intention to describe experimental results from several laboratories confirming the pathologic events induced in normal human skin by serum or IgG preparations from pemphigus patients and to discuss the molecular events leading to the pathophysiology of pemphigus.

Pathophysiology of Pemphigus

Three lines of evidence support a role for antibody in the induction of acantholysis in pemphigus: correlation of antibody titers with disease activity, induction of acantholytic lesions in neonatal mice by passive transfer of patient IgG, and induction of acantholytic lesions in explants of normal human skin following incubation with patient IgG in the presence and absence of plasminogen. The authors discuss all three lines of evidence and describe the model they have developed in vivo to explain induction of acantholytic lesions by antibody.

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.

Pemphigus antibody interaction with human epidermal cells in culture.

The mechanism of pemphigus acantholysis has been studied with an in vitro system. Freshly prepared human skin epidermal cells were incubated in F-10 medium which contained the immunoglobulin G fraction from either pemphigus serum or normal human serum. During 18-h incubation periods, the pemphigus antibody became bound to the surface of the epidermal cells, caused the destruction of 75% of the viable cells as compared to only 14% in the normal immunoglobulin G controls (trypan blue exclusion), prevented the accumulation of newly synthesized proteins by nearly 60% as determined by radioactive tracer studies, and caused a dramatic shift in distribution of the newly synthesized proteins from an insoluble cell-associated fraction to an extracellular soluble fraction. These effects on the accumulation and partitioning of newly synthesized proteins were antibody concentration-dependent. Kinetic studies showed that at a fixed pemphigus antibody concentration the inhibition of protein accumulation preceded solubilization by about 1 h, at which time rapid solubilization of up to 70% of the insoluble cellular material occurred. Several lines of evidence suggested that this phenomenon was caused by enzymatic activity. Epidermal extracts solubilized a prepared substrate of radioactivity labeled insoluble epidermal cell material. This activity was heat labile and pH dependent, with pH optima ranging from 4.5 to 6.5. Enzymes with pH optima between 6 and 6.5 were recovered in the culture medium after a 2-day incubation of pure, intact epidermis with the pemphigus antibody. We proposed the following hypothesis to account for pemphigus acantholysis. The pemphigus antibody reacts with the epidermal cell surface and produces such a severe disturbance that the integrity of the cell surface is lost. As a result of these primary perturbations, the cell is killed and during the process, responds by release or activiation of soluble hydrolytic enzymes. This autolytic process results in the characteristic acantholysis of pemphigus.

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.

Pemphigus acantholysis in tissue culture: studies on photo-induction

It has previously been shown tha UV irradiation can give rise to fresh skin lesions in the uninvolved skin of patients with pemphigus. In this study no increase of acantholytic activity was found in an organ culture model system for pemphigus in vitro, after in vitro and in vivo irradiation under various experimental conditions. By immunofluorescence no increase in IgG binding in irradiated specimens was observed. From these in vitro findings, which contrast the effect of UV light in pemphigus in vivo, it may be concluded that the circulatory system of the skin plays a major role in the photo induction of acantholysis in pemphigus.

Capability of complement fixation by in vivo bound antibodies in pemphigus skin lesions

Summary Skin biopsy was performed in five cases of pemphigus; direct immunofluorescence (IF) studies demonstrated intercellularly bound IgG and C3 in each case. Sections of lesional skin, containing in vivo bound IgG were, in two cases, capable of further binding C3in vitro from normal human serum, an effect most marked in relation to areas of acantholysis. This work provides additional evidence that the complement system is involved in the process of pemphigus acantholysis.