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.     

Plasmin induces acantholysis in skin organ cultures

Summary 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.This work was supported by a grant from the Swiss National Science Foundation 3.075-1.84. Presented in part at the 67th Congress of the Swiss Society of Dermatology and Venereology, October 19, 1985, Zürich, Switzerland     

Pemphigus,a pathomechanism of acantholysis

Autoantibodies to the desmosomal proteins desmoglein 1 and 3 cause pemphigus foliaceus and pemphigus vulgaris, which are characterised by keratinocyte dissociation (acantholysis) and intraepidermal blister formation. The passive transfer of pathogenic anti‐desmoglein antibodies induces blisters in mice in vivo and the loss of keratinocyte adhesion in vitro. The pathogenetic mechanisms of acantholysis due to anti‐desmoglein autoantibodies are not fully understood. However, recent studies have revealed that signalling‐dependent and signalling‐independent pathways are operative in the loss of cell adhesion. In this review, we focus on the pathomechanism of acantholysis due to autoantibodies to desmogleins and recent therapeutic approaches.     

Bilateral herpes simplex virus keratitis in a patient with pemphigus vulgaris

Pemphigus is a group of chronic blistering diseases in which acantholysis and blister formation occur within the epidermis. Immunoglobulins and complement are found in the circulation and are bound to the cell surfaces of keratinocytes. Pemphigus is classified into several types hut may he divided into two major variants, pemphigus vulgaris and pemphigus foliaceus. The primary skin lesion of pemphigus vulgaris which was often fatal before the introduction of systemic glucocorticoid therapy, is a flaccid, fragile blister which can occur anywhere. The most common skin lesions arc erosions, which are often painful; suprabasal elefting within the epidermis is also present. In the majority of these patients, painful mucous membrane erosions will be the first symptom, while sometimes the conjunctiva is affected but corneal involvement is very rare.     

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.     

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.     

Diagnosis and clinical features of pemphigus vulgaris

Autoimmune bullous diseases are associated with autoimmunity against structural components that maintain cell-cell and cell-matrix adhesion in the skin and mucous membranes. They include those where the skin blisters at the basement membrane zone and those where the skin blisters within the epidermis (pemphigus vulgaris, pemphigus foliaceus, and other subtypes of pemphigus). The variants of pemphigus are determined according to the level of intraepidermal split formation. There are 5 main variants of pemphigus: pemphigus vulgaris, pemphigus foliaceus, pemphigus erythematosus, drug-induced pemphigus, and paraneoplastic pemphigus. This review focuses only on pemphigus vulgaris.     

A clinico-pathological study of 70 cases of pemphigus

A clinicopalhological study of 70 cases of pemphigus observed over a span of four and a half years from January 1992 to June 1996 at the Sir J.J. Group of Hospitals and Grant Medical College, Mumbai is reported. Pemphigus vulgaris constituted the single largest group of 43 cases, followed by pemphigus foliaceus (25 cases) and pemphigus vegetans (2 cases). Majority of the cases were seen in the age group of 21-60 years, with a slight male predominance. The youngest patient was 14 years while the eldest was aged 75 years. Mucosal involvement was seen in 31 cases of pemphigus vulgaris, as opposed to only 5 cases of pemphigus foliaceus. Flaccid bullae were present in 100% cases. Pruritus was complained of in 14 cases, though it was more common in pemphigus vegetans and vulgaris. Salient histopathological features of pemphigus vulgaris observed were (I) intraepidermal suprabasal blisters (35 cases), (2) presence of acantholytic cells (40 cases), (3) "Row of tombstone appearance" (I8 cases) and (4) acantholysis involving follicular sheath (20 cases). Main histopathological features of pemphigus foliaceus were (1) subcorneal blister (15 case), (2) acantholysis (24 cases) and (3) bulla cavity containing inflammatory infiltrate (12 cases). Both cases of pemphigus vegetans showed hyperkeratosis, papillomatosis and irregular acanthosis with intra-epidermal eosinophilic abscesses besides suprabasal lacunae.     

Pemphigus--paradigm of autoantibody-mediated autoimmunity

Pemphigus encompasses a group of life-threatening autoimmune blistering diseases due to a loss of adhesion between keratinocytes, called acantholysis, which is caused by autoantibodies (AAb) against intercellular adhesion structures of epidermal keratinocytes. In pemphigus vulgaris (PV), the blisters are located in the suprabasal layer whereas in pemphigus foliaceus (PF), a clinically less severe disease, the blisters occur within the upper layers of the epidermis. In PV and PF, the AAb target the extracellular portions of desmoglein 3 (Dsg3) and Dsg1, respectively. AAb production in PV and PF is polyclonal and most AAb are of the IgG4 subclass in acute onset or active disease while patients in remission have mainly AAb of the IgG1 subtype. Evidence for the pathogenicity of these circulating AAb is provided by the observations that (1) the activity of pemphigus correlates with AAb titers, (2) newborns of mothers with active pemphigus temporarily exhibit blisters due to the transplacentar transfer of maternal AAb and (3) pemphigus-like lesions are induced in neonatal mice by transfer of IgG from PV patients. Clinically, pemphigus is characterized by extensive cutaneous blisters and erosions of the mucous membranes (PV). Patients with untreated pemphigus are prone to infections, loss of body fluids and proteins and to weight loss due to painful oral and esophageal erosions. The major therapeutic strategy in pemphigus is chronic immunosuppressive therapy with glucocorticosteroids in combination with immunosuppressive adjuvants.     

Plasminogen activation in lesional skin of Pemphigus vulgaris type Neumann

Zymographic and immunological studies revealed that primarily tissue-type plasminogen activator and to a lesser extent urokinase-type plasminogen activator were present in fluids of pemphigus vulgaris (type Neumann) skin blisters. Furthermore, plasmin activity was detected in pemphigus blister fluids using chromogenic peptide substrate assays. In pemphigus, but not in control, suction blister fluids plasmin/₂-antiplasmin and plasmin/ ₂-macroglobulin complexes were found by immunoprecipitation or by testing in immunoassays after fractionation by molecular-sieve chromatography. Plasmin activity, detected by a low molecular weight chromogenic peptide assay, was ascribed to plasmin/₂-macroglobulin complexes. Since formation of plasmin/inhibitor complexes requires active plasmin, the finding indicates previous activation of plasminogen in pemphigus lesions.     

The anti-desmoglein 1 autoantibodies in pemphigus vulgaris sera are pathogenic

Pemphigus vulgaris and pemphigus foliaceus are two closely related, but clinically and histologically distinct, autoimmune skin diseases. The autoantigens for pemphigus vulgaris and pemphigus foliaceus are desmoglein 3 and desmoglein 1, respectively. The anti-desmoglein 1 antibodies in pemphigus foliaceus and anti-desmoglein 3 antibodies in pemphigus vulgaris are pathogenic as determined by immunoglobulin G passive transfer animal models. More than 50% of pemphigus vulgaris sera also contain anti-desmoglein 1 autoantibodies; however, the pathogenicity of the anti-desmoglein 1 autoantibodies in pemphigus vulgaris remains unknown. In this study, we used soluble recombinant extracellular domains of desmoglein 1 and desmoglein 3 to obtain affinity-purified anti-desmoglein 1 and anti-desmoglein 3 autoantibodies from pemphigus vulgaris sera and examined the pathogenicity of each fraction separately using the passive transfer mouse model. By immunoprecipitation, the purified anti-desmoglein 1 and anti-desmoglein 3 showed no cross-reactivity. The anti-desmoglein 1 autoantibodies in pemphigus vulgaris induced typical pemphigus foliaceus lesions in neonatal mice, whereas the anti-desmoglein 3 fraction induced pemphigus vulgaris-like lesions. In addition, the pathogenic anti-desmoglein 1 and anti-desmoglein 3 autoantibodies in pemphigus vulgaris had predominant IgG4 subclass specificity. These findings suggest that the anti-desmoglein 1 antibodies in pemphigus vulgaris are pathogenic.     

Internalization of constitutive desmogleins with the subsequent induction of desmoglein 2 in pemphigus lesions

Acantholytic blisters in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are caused by a dissociation of desmosomes mediated by autoantibodies against desmoglein (Dsg) 3 and Dsg 1, respectively. The blistering occurs at the suprabasilar level in PV and at the subcorneal level in PF, which corresponds to the distribution of target antigens in the epidermis: there is a more prominent expression of Dsg 1 in the upper layer, whereas Dsg 3 is more prominent in the lower layer. To elucidate the histogenesis of acantholysis, we studied the alterations of the desmosomal components and the expression pattern of Dsg isoforms in the lesional and perilesional epidermis of pemphigus patients. The results demonstrated an internalization of the desmosomes in the lower epidermis of PV, PF and pemphigus vegetans. A similar phenomenon was induced in monolayers of keratinocytes cultured with PV sera. However, little change was observed in E-cadherin expression until acantholysis became manifest. This internalization occurred prior to overt acantholysis, and was frequently associated with the induction of Dsg 2 expression in the basilar or lower layers of the epidermis. These findings indicate an alteration of Dsg isoform expression in subclinical pemphigus lesions, which might be related to the characteristic acantholytic patterns: the suprabasilar layer in PV and the upper epidermis in PF.     

Curvicircular intracytoplasmic membranous structures in keratinocytes of pemphigus foliaceus

We noticed intracytoplasmic membranous, annular, or circular structures in the lesion of pemphigus foliaceus and studied these by regular transmission electron microscopy and immuno-electron microscopy. These curvicircular bodies were observed in the preacantholytic keratinocytes of the blister wall as well as in acantholytic cells in 6 out of 6 patients with pemphigus foliaceus. They were absent in samples from 3 patients with pemphigus vulgaris. These structures were about 60–70 nm wide and consisted of 4 electron-dense layers. They were continuous with intact desmosomal structures and gap junctions in the periphery of the keratinocytes. These curvicircular membranous bodies were well labeled with immunogold particles for desmoglein, plakoglobin, connexin 43, and IgG. In contrast to pemphigus vulgaris, splitting of desmosomes through dissolution of intercellular desmoglea was seldom observed in all 6 specimens of pemphigus foliaceus. These findings suggest that in pemphigus foliaceus 1) curvicircular bodies are derived from internalized desmosomes and gap junctions, and 2) cell-to-cell adhesions are weakened by this internalization and acantholysis is initiated, while in pemphigus vulgaris the dissolution of clesmoglea is the initial event. It is suggested that in pemphigus foliaceus the binding of autoantibody induces internalization of many intact desmosomes and gap junctions rather than splitting them.     

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.     

71例天疱疮的棘层松解位置分析

目的 探讨天疱疮的棘层松解位置,为天疱疮出现不同位置棘层松解的表现提供解释.方法 收集43例寻常型天疱疮和28例落叶型天疱疮患者的临床资料、组织病理、免疫病理、天疱疮抗体指标值进行分析.结果 寻常型天疱疮中有35例棘层松解的位置发生在基底层上方,8例发生在表皮中上部,落叶型天疱疮中有25例棘层松解的位置发生在颗粒层、棘层上方,3例发生在表皮中下部,落叶型天疱疮患者抗Dsg1抗体指标较寻常型天疱疮患者显著升高(P=0.047),寻常型天疱疮棘层松解的位置发生在表皮中上部的患者抗Dsg1、3抗体指标值与棘层松解发生在基底层上方的患者相比有差异,但无统计学意义.结论 寻常型天疱疮及落叶型天疱疮患者组织病理中,棘层松解的位置可发生于表皮中上部、表皮中下部.棘层松解的位置可能与抗Dsg1抗体和抗Dsg3抗体指标值等相关.     

Pemphigus foliaceus induced by exposure to sunlight. Report of a case and analysis of photochallenge-induced lesions

BACKGROUND: Although there are many reports on photo-induced pemphigus, careful analysis in the development of acantholytic blister have rarely been performed in the lesions induced by photochallenge. OBJECTIVE: The study was intended to elucidate the mechanisms by which ultraviolet light (UV) radiation causes the skin lesions of pemphigus foliaceus. METHODS: Photochallenge-induced lesions were examined histologically and immunohistochemically over the time course with three biopsy specimens. Neutrophil adhesion assay was performed using the specimens prepared from the photochallenged lesions as substrates. RESULTS: A small number of neutrophils were seen at 5 h after photochallenge in the dermis and epidermis, and slight acantholysis was detected at 72 h. In neutrophil adhesion assay, greater numbers of neutrophils adhered to the upper epidermis of the skin obtained at 5 h after challenge. CONCLUSIONS: Our results suggest that both enhanced binding of autoantibodies to the epidermis after UV radiation and preferential adhesion of neutrophils to the UV-irradiated epidermis contribute to the development of acantholysis in photo- induced pemphigus foliaceus. Intercellular adhesion molecule-1 (ICAM-1) expression on keratinocytes is not primarily responsible for the epidermal migration of neutrophils.     

胫前大疱性表皮松解症并发落叶型天疱疮1例

报告胫前大疱性表皮松解症并发落叶型天疱疮1例。患者男,64岁。全身红斑、瘙痒1年,出现松弛性水疱、糜烂、结痂5个月。患者自10岁始四肢出现瘙痒性疱疹,渐形成红色瘢痕,并持续至今。皮损组织病理检查：大疱处示棘层上部大疱,疱中有嗜酸性纤维蛋白网、大量中性粒细胞及棘层松解细胞;胫前处皮损示表皮下裂隙性水疱,较多成纤维细胞和结缔组织。间接免疫荧光检查抗表皮细胞间基质抗体（＋）,滴度为1：40。     

An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid

Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-fesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n=4) and in suction blisters (n=2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.     

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.     

用红斑型天疱疮患者血清IgG诱导新生小鼠天疱疮的实验研究

本文系用红斑型天疱疮血清IgG,被动转移给BaIb/c新生小鼠,诱导新生小鼠天疱疮的实验研究.2例患者血清天疱疮抗体的滴度均为1:128.患者血清IgG按每日每克体重5 mg经腹腔接射给生后4及8小时的Balb/c小鼠,注射后24小时及48小时5只小鼠有3只皮肤见到小水疱,直接免疫荧光检查表皮细胞间有IgG沉着,常规组织病理检查见到表皮内水疱及棘细胞松解.本实验证明:天疱疮自身抗体在人类红斑型天疱疮发病中的作用,这种执体可被动转给新生小鼠.