Pemphigus antibody action on skin explants: kinetics of acantholytic changes and stability of antigens in tissue cultures of normal monkey skin explants.

The pathogenic effect of the intercellular antibodies of pemphigus was studied by using organ culture of monkey skin. Skin explants that were grown on sera with intercellular antibody titers of 320 or greater fixed these antibodies within one day as demonstrated by direct immunofluorescence for IgG. None of the control sera gave such staining patterns. Following the binding of intercellular antibodies, characteristic histologic changes appeared, notably separation of individual epidermal cells and acantholysis. These histologic changes became more marked in two to five days. During this period, the bound antibodies and the intercellular antigens decreased and disappeared. These temporal relationships of immunofluorescence and histologic findings suggest that pemphigus antibodies play a role in the induction of acantholysis.     

Immunopathologic and Histologic Studies on Skin from Pemphigus Patients in Tissue Culture

Explants of skin from patients with pemphigus vulgaris and pemphigus foliaceus taken in the active stages of the disease had in vivo bound IgG in the intercellular area. After 24-48 h incubation of these explants in normal sera acantholytic bullae developed above the stratum basale, thus indicating that the bound IgG is probably in vivo bound pemphigus antibody. In both cases, skin from the inactive stage of the disease contained no in vivo bound pemphigus antibodies. Explants of these skin specimens failed to develop acantholysis in culture.     

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.     

The use of two substrates to improve the sensitivity of indirect immunofluorescence in the diagnosis of pemphigus

The aim of this study was to re-evaluate indirect immunofluorescence (IIF) comparing two substrates, normal human skin (HS) and monkey oesophagus (MO) using serum from 29 pemphigus patients classified according to the presence of serum autoantibodies to either desmoglein (Dsg) 1 or Dsg3 detected by enzyme-linked immunosorbent assay (ELISA). Overall, the sensitivity of IIF was 83% on HS and 90% on MO. When data from both substrates were combined, the sensitivity increased to 100%. When sera from pemphigus foliaceus (PF) patients were studied, which contained Dsg1 antibodies only, the sensitivity of IIF was greatest on HS and titres were on average 4.8 doubling dilutions higher than on MO. In contrast, when sera containing autoantibodies only to Dsg3 from pemphigus vulgaris (PV) patients was studied, the sensitivity was greatest on MO and titres were on average 4.4 doubling dilutions higher than on HS. There was a significant correlation between Dsg1 antibody levels and IIF titres on HS and between Dsg3 antibody levels and IIF titres on MO. The investigation of immunobullous disorders in the future is likely to move towards antigen-specific techniques such as the Dsg ELISAs used in this study. However, in laboratories which currently rely on IIF for detecting pemphigus autoantibodies, the data presented in this study strongly suggest that two substrates should be used for IIF screening: one rich in Dsg1, such as HS, and the other rich in Dsg3, such as MO. This combination of substrates should not only increase the sensitivity of detecting pemphigus antibodies, but will aid in the differentiation of PV from PF. It is also possible that the data might be more useful for disease monitoring.     

An organ culture model for the study of pemphigus acantholysis

An in vitro model for the study of pemphigus acantholysis has been developed. The histological changes of pemphigus vulgaris were reproduced in vitro in organ culture by growing normal human skin in the presence of pemphigus vulgaris or pemphigus foliaceus sera. At 24 h a suprabasilar split was noted and at 72 h extensive suprabasilar acantholysis developed. Direct immunofluorescent tests demonstrated that pemphigus antibody became bound to the epidermal intercellular space antigen(s) during the first 6-12 h. As acantholysis increased the presence of tissue-fixed antibody decreased. The fixation of the pemphigus antibody to the skin prior to the development of acantholysis provides strong evidence for the pathogenetic role of this antibody in the production of acantholysis. The data suggest that complement is not required in this model for the production of the acantholytic changes of pemphigus since heating the serum for 30 min at 56 degrees C did not destroy the acantholytic activity and no complement (C3) could be detected by DIF of organ culture explants.     

Comparison of the reactivity of various epithelial substrates for the titration of pemphigus antibodies by indirect immunofluorescence

Indirect immunofluorescence was used to compare the reactivity of thirteen different squamous epithelia for the titration of serum antibody in five patients with active pemphigus vulgaris. The most useful epithelia, as judged by pemphigus antibody titres, were, in order of effectiveness, human skin, human tonsil, monkey oesophagus, monkey lip, guinea-pig oesophagus, rabbit-oesophagus, guinea-pig lip and rabbit lip. The choice of substrate is of importance in the sensitivity of the indirect immunofluorescence test in pemphigus. In view of the availability and high reactivity of human tonsils, we would recommend their use as a substrate for pemphigus antibody estimation.     

Deposition of the membrane attack complex of complement in pemphigus vulgaris and pemphigus foliaceus skin

The present study was performed to determine whether complement activation in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) results in the assembly of the terminal complement sequence or membrane attack complex (MAC) in skin lesions. Biopsy specimens of skin lesions from five patients with PV and three patients with PF contained C5, C7, C9, and the MAC related neoantigen (C5b-9 neoantigen) in intercellular substance areas (ICS), as well as IgG and the early complement components Clq, C4, and C3. The presence of these late complement components and the C5b-9 neoantigens in ICS sites of the skin lesions is indicative of complement activation by the pemphigus antibody, with subsequent assembly of the MAC. The binding of IgG and early complement components to ICS was observed in both non-lesional (normal appearing) skin and in skin lesions. However, no MAC could be detected in the normal appearing skin of our pemphigus patients. It was also noted that the MAC could be generated in vitro on cryostat sectioned normal human skin by pemphigus antibody in the presence of complement. Results of these studies suggest that complement activation may be related to membrane damage of epidermal cells in both PV and PF.     

Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris-acetate-buffered saline (TAS), TAS with 5 mM CaCl+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.     

Experimental acantholysis by complement-fixing intercellular antibodies

Summary Complement-fixing intercellular antibodies were detected in 10 of 17 sera from untreated pemphigus patients. The role of complement in the organ culture system was investigated using these sera. Ten sera possessing complement-fixing intercellular antibodies showed IgG binding to the intercellular substance in the organ-cultured skin and acantholysis-like changes were observed in eight cases. C3 deposition was not seen in any case. However, after treatment of the sections of cultured skin with fresh normal human serum, complement fixation of the intercellular substance by bound IgG was revealed in all the ten cases. No significant differences in the grade of acantholysis-like changes between the complement-depleted system and the complement-supplied system were observed. Complement does not appear to be necessary in the acantholytic process in the in vitro organ culture system, even though we considered the presence of complement-fixing intercellular antibodies.This work was supported by research grants from the Ministry of Health and Welfare of Japan and from Professor Kato Memorial Fund for Physiology and Medicine     

Distinct types of IgG and IgA anti-intercellular autoantibodies from patients with pemphigus and vesiculopustular dermatosis

The pattern of binding of two different types of IgA class pemphigus-like antibodies was compared with that of the true IgG class pemphigus antibody. The IgG antibodies from pemphigus vulgaris (PV) and pemphigus foliaceus (PF) sera bound to the intercellular spaces in normal human epidermis, whereas only the PV antibody reacted with monolayers of keratinocytes derived from human foreskin. Both IgG and IgA antibodies from a patient with PF were found in the intercellular spaces of the epidermis and only the IgA antibody reacted with the cultured keratinocytes. The IgA antibody from a patient with a vesiculopustular eruption and whose serum contained IgA intercellular space antibody alone, bound to the upper epidermis but not to the monolayers of keratinocytes. These findings indicate that PV but not PF antigens can be expressed by monolayers of cultured human keratinocytes and that there are at least two distinct types of IgA intercellular antibodies.     

Study of desmoglein 1 and 3 antibody levels in relation to disease severity in Indian patients with pemphigus

OBJECTIVES: To conduct a cross-sectional study to compare Dsg1 and Dsg3 antibody levels independently with severity of disease activity in pemphigus vulgaris (PV) and pemphigus foliaceus (PF). METHODS: Blood samples from 44 patients with pemphigus (PV-38, PF-6) were analyzed using ELISA. The severity of skin and mucosal disease was graded using a score from 0 to 3. RESULTS: A statistically significant correlation between increase in Dsg 3 antibody titres with severity of oral involvement and Dsg 1 titres with severity of skin involvement was found in both PV and PF patients (p < 0.01). However, we were unable to demonstrate a relationship between increased titres of Dsg1 and Dsg 3 antibodies with oral and skin involvement respectively. CONCLUSION: This study suggests that the severity of skin and oral disease in pemphigus is determined by the quantities of Dsg1 and Dsg3 antibodies respectively.     

Elution of antibodies from the lymphocyte membrane in certain dermatoses

The lymphocyte eluates of three patients with discoid lupus erythematosus and two patients with pemphigoid possessed basement membrane antibody of the IgG class either for normal human skin or guinea-pig lip. The lymphocyte eluates from one patient with pemphigus did contain antibody (IgG) reactive with the intercellular substances of monkey oesophagus, and that of a patient with a drug reaction showed antibody reactive with basal cells of stratified epithehum. These results indicate either antibody synthesis by the lymphocytes themselves, or that these cells may provide antigenic receptors reactive with specific antibody.     

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.     

Molecular mechanisms of pathogenesis

The first clue to the etiology of pemphigus was established in 1964, when Beutner and Jordon¹ 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 mice⁵ or in grafts of human oral mucosa on nude mice⁶ 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.     

Pemphigus foliaceus, myasthenia gravis, thymoma and red cell aplasia

A 68-year-old man developed pemphigus foliaceus, myasthenia gravis with a spindle cell thymoma, and later died with red cell aplasia. At autopsy, pemphigus affected the oesophageal mucosa, and this finding was confirmed by direct immunofluorescence. In order to clarify the relationship between pemphigus and myasthenia gravis or thymoma, sera from 38 patients with myasthenia gravis were examined. Intercellular epithelial antibodies (IC-AB) at titres of 10-40 were found in 8, when monkey oesophagus was employed as the substrate. IC-AB in sera of patients with pemphigus and or myasthenia gravis did not react to any part of human thymoma, human hyperplastic thymus or monkey normal thymus. Deposits of immunoglobulins or complement were not demonstrated in the human thymoma.     

The pathogenic mechanisms of blister formation in bullous pemphigoid

Normal human skin was cultured with sera, IgG fractions, and blister fluids (BF) from patients with bullous pemphigoid. Antibody binding (IgG) was observed by immunofluorescence techniques at the dermal-epidermal junction of all skin explants culture with sera, IgG fractions, and BF. Dermal-epidermal separation was observed only in the skin explants cultured with BF. Dermal-epidermal separation was observed in 19 out of 20 explants cultured with BF obtained from fresh bullae. In addition, dermal-epidermal separation can be produced in vivo 6 hr after the injection of BF into the dorsal skin of Hartley guinea pigs. Dermal-epidermal separation was not observed in skin explants cultured with heat-inactivated (56 degrees, C 30 min) BF, although antibody binding was observed. In addition, dermal-epidermal separation did not occur when the BF were preincubated with rabbit antihuman C1, C3, C4, and C5 antibodies. These observations suggested that both antibody and complement were essential for the production of dermal-epidermal separation. Since patient sera failed to produce dermal-epidermal separation, other factor(s) present in BF but absent from serum might be necessary for the production of dermal-epidermal separation. The addition of the proteinase inhibitors, pepstatin, EDTA, and soy bean trypsin inhibitor, did not inhibit the formation of dermal-epidermal separation. In contrast, the presence of alpha 2-macroglobulin inhibited dermal-epidermal separation.     

Epidermal intercellular binding of concanavalin a and pemphigus antibody.

The binding sites of Concanavalin A (Con A) and pemphigus antibody in the intercellular spaces of the human epidermis have been investigated. Extraction of human skin with phosphate-buffered saline (PBS) and 70% ethanol resulted in complete loss of the binding sites of the pemphigus antibody, whereas the extracted skin still retained its reactivity to Con A. Preincubation with more than 0.25 mg/ml of Con A with normal skin caused a reduction in the binding of pemphigus sera. However, preincubation of pemphigus sera with human skin caused no inhibition of the subsequent binding of fluorescein-labelled Con A. These results indicate that Con A binding sites might be related to, but not identical with those of pemphigus antibody.     

Pemphigus controlled by dapsone

Three uncomplicated cases of pemphigus were clinically controlled by dapsone. Improvement was associated with decreasing titres of circulating intercellular antibodies. Interestingly, the sera from one case of pemphigus foliaceus contained intercellular antibodies found in the Malpighian and basal cell layers using the fluorescent technique and in the granular layer using the peroxidase technique. These findings suggest that the intercellular antibodies in pemphigus vulgaris and pemphigus foliaceus are similar but bind at different anatomical site     

Increased antibody levels to desmogleins 1 and 3 after administration of carbamazepine

Desmoglein (Dsg) 1 and Dsg3 are recognized as the autoantigens in pemphigus foliaceus and pemphigus vulgaris. Pemphigus-like syndromes have been reported to occur in individuals after exposure to a variety of drugs, but pemphigus caused by carbamazepine is not common. We found that anti-Dsg1 and anti-Dsg3 antibody titres were increased in three individuals administered carbamazepine. Antibody titres against Dsgs 1 and 3 were measured by enzyme-linked immunosorbent assay (ELISA). Of 42 serum samples (25 patients administered carbamazepine, eight patients administered valproic acid and nine healthy volunteers) tested by ELISA, three patients administered carbamazepine showed positive reactivity against both Dsg1 and Dsg3. The patient with the highest titre against Dsg1 and Dsg3 (the index values of anti-Dsg1 and anti-Dsg3 were 79.3 and 86.4, respectively) was a 23-year-old woman (Case 1). The other two patients with positive reactivity were a 5-year-old boy (Case 2) and a 65-year-old man (Case 3). In addition, indirect immunofluorescence study showed intercellular antibodies to the cell surface of the whole epidermis with a titre of 1 : 64 in Case 1 and 1 : 2 in Cases 2 and 3. However, no skin or mucosal involvement was found in any of these cases. There was no difference in the serum concentrations of carbamazepine between the three positive cases and the 22 negative cases of carbamazepine administration. From these facts, the lack of skin diseases may be explained by relatively low values of anti-Dsg 1 and 3 antibodies in Cases 2 and 3. However, it cannot be excluded that undefined exogenous and/or endogenous factors are involved in an outbreak of pemphigus. Furthermore, these findings might be helpful for preventing susceptible individuals from exposure to the suspect drugs.     

Is Pemphigus Herpetiformis an Entity?

Clinical, histologic, and immunofluorescence studies were performed in 15 patients with pemphigus herpetiformis. The initial diagnosis was dermatitis herpetiformis, IgA linear bullous dermatosis or bullous pemphigoid. The histology varied depending on the character of skin lesions, and showed eosinophilic spongiosis or slight acantholysis and/or polymorphonuclear papillary microabscesses. Direct immunofluorescence showed invariably intercellular IgG staining, and indirect immunofluorescence on monkey esophagus substrate was positive, at some periods, in five cases. About half of the patients responded to therapy with sulfones and prednisone, and only one patient responded to sulfones alone. Half of the patients required combined therapy with prednisone and cyclophosphamide or with higher doses of prednisone. In consecutive relapses, nine patients retained the pattern of pemphigus herpetiformis: in the others, lesions were mostly of pemphigus seborrheicus-foliaceus type.