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Home , Acantholysis, Bullous pemphigoid, Dermatitis herpetiformis, IgA pemphigus, Paraneoplastic pemphigus, Pemphigus erythematosus

Reviews 13 (2014) 482–489

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Autoimmunity Reviews

journal homepage: www.elsevier.com/locate/autrev

Review Diagnosis and classification of autoimmune blistering diseases

Sharon Baum ⁎,1, Nicole Sakka 1,Ofir Artsi, Henri Trau, Aviv Barzilai

Department of , Chaim Sheba Medical Center, Tel-Aviv University, Sackler School of Medicine, Tel Hashomer, Israel article info abstract

Article history: Blistering skin diseases are a group of autoimmune disorders that are characterized by against Received 7 October 2013 structural proteins of the or the dermal–epidermal junction and clinically by and erosions Accepted 13 November 2013 on skin and/or mucous membranes. Since clinical criteria and histopathological characteristics are not sufficient Available online 13 January 2014 for diagnosis, direct immunofluorescence microscopy of a specimen or serological tests are needed for exact diagnosis. The differentiation between the various disorders became more important since prognosis as Keywords: well as different treatment options are nowadays available for the various diseases. Moreover, some bullous Blistering skin diseases diseases may indicate the presence of an underlying malignancy. The detection of serum autoantibodies have been shown to correlate with disease activity and thus may be helpful in deciding treatment options for these Autoantibodies patients. Direct immunofluorescence © 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction...... 482 2. Clinical classification...... 483 3. Pemphigus group — diseasesofintraepidermallossofadhesion...... 483 3.1. Pemphigusvulgaris(PV)...... 483 3.2. Pemphigusfoliaceus(PF)...... 484 3.3. Pemphiguserythematosus(PE)...... 484 3.4. IgApemphigus...... 484 3.5. Paraneoplasticpemphigus(PNP)...... 484 4. Pemphigoid diseases — diseasesofsubepidermallossofadhesion...... 485 4.1. Bullouspemphigoid(BP)...... 485 4.2. Mucousmembraneorcicatricialpemphigoid(MMP)...... 485 4.3. LinearIgAbullousdermatosis(LAD)...... 485 4.4. Epidermolysisbullosaacquisita(EBA)...... 485 4.5. Dermatitisherpetiformis(DH)...... 485 5. Pemphigoidgestationis(PG)...... 486 6. Histology...... 486 7. Pemphigus group — diseasesofintraepidermallossofadhesion...... 486 8. Pemphigoid Diseases — diseasesofsubepidermallossofadhesion...... 487 9. Direct immunofluorescencemicroscopy...... 487 10. Indirect immunofluorescencemicroscopy...... 487 11. Target -specific serological confirmatorytestsbyELISAandWesternblotting...... 488 References...... 489

1. Introduction

⁎ Corresponding author at: Department of Dermatology, Chaim Sheba Medical Centre, Autoimmune blistering dermatoses comprise a heterogeneous Tel Hashomer, Tel Aviv, Israel, 52621. Tel.: +972 52 666 6182; fax: +972 3 530 3515. E-mail address: [email protected] (S. Baum). group of diseases that are characterized by autoantibodies directed 1 Contributed equally. against adhesion molecules of the skin and adjacent mucous membranes.

1568-9972/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autrev.2014.01.047 S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 483

According to the skin level at which the occurs and by the struc- erosions of the skin and/or mucous membranes, intraepidermal blistering tural proteins that the autoantibodies target, autoimmune blistering and the production of IgG autoantibodies against the adhe- diseases can be categorized into different groups i.e. in pemphigus sion molecules. The major subtypes are; and pem- group, autoantibodies target desmosomal proteins that results in phigus foliaceus, and aless frequent form, IgA pemphigus. Pemphigoid loss of cell adherence between keratinocytes, in pemphigoid diseases, diseases are characterized by autoantibodies directed against structural hemidesmosomal proteins of the dermo-epidermal junction are tar- proteins of the dermal–epidermal junction that clinically can manifest geted, and in herpetiformis, autoantibodies bind to epider- with urticarial , tense blisters and erosions which may involve mal and tissue (Diagram 1). the mucous membranes. Diseases in this group include bullous pemphi- The diagnosis of autoimmune blistering dermatoses is based on a goid, , pemphigois, linear Ig constellation of clinical and laboratory findings and due to their rarity A bullous dermatosis, herpes gestationis and and heterogeneous clinical features, they often pose a major diagnostic acquisita (Table 1, Diagram 1). , although clas- challenge. Diagnosis cannot be based solely on clinical signs and the sified as a type of pemphigus based on its clinical presentation, shows histopathological findings and requires the detection of tissue bound against both intraepidermal and subepidermal components. and circulating autoantibodies which still remains the diagnostic gold standard in the detection of autoantibodies. Furthermore, sensitive 3. Pemphigus group — diseases of intraepidermal loss of adhesion and specific serological assays have been developed to allow the detec- tion of serum antibodies which are used as diagnostic tools as well as for 3.1. Pemphigus vulgaris (PV) disease activity monitoring. Pemphigus vulgaris (“pemphigus” stems from the Greek word 2. Clinical classification pemphix for “blister”) is the most frequent representative of the group of pemphigus diseases with an incidence of 0.1–0.5/100 000 pop- Autoimmune bullous diseases have a broad spectrum of clinical ulation [1–3]. The mean onset of PV is usually around middle age with manifestations and a variety of morphological lesions. Based on the an age peak in the fourth and fifth decade of life and a higher level of the skin blister formation — intraepidermal or subepidermal, in patients of Jewish or Mediterranean ancestry [1–4]. PV is a chronic they can be classified into pemphigus group and pemphigoid group of autoimmune intraepithelial blistering disease and potentially life- diseases respectively. Pemphigus is characterized by flaccid vesicles and threatening condition. Despite advances in management, the mortality

Table 1 Diagnostic algorithm of clinical, histological, immunofluorescence and autoantibodies against structural for autoimmune bullous disorders. Abbreviations: DEJ: dermo-epidermal junction zone; Dsg: ; Ig: immunoglobulins; DC: desmocollin; TTG: tissue transglutaminase, LAD-1: soluble ectodomain of BP180; PF: ; PV: pemphigus vulgaris; PNP: paraneoplastic pemphigus; BP: ; MMP: mucous membrane pemphigoid; EBA: epidermolysis bullosa acquisita; and DH: dermatitis herpetiformis. 484 S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489

Fig. 1. (a) Widespread flaccid blisters and erosions on the trunk of a patient with pemphigus vulgaris. (b) Mucosal involvement; erosions on the buccal mucosa and hard palate. rate is currently estimated at 5 to 10% [5]. In the majority of the cases accumulation of [7]. Lesions have a distinct tendency to co- (70–90%) PV presents itself with painful and long-persisting erosions alesce and there is an associated pruritus. Frequently affected sites for of the mucous membranes, especially of the oral(buccal) mucosa [1,4]. IgA pemphigus are distal trunk and proximal limbs as well as Other sites such as the throat, , conjunctivae, nasal mucosa, intertriginous sites [7]. Mucosal involvement is rare. vagina, vulva, penis and anus can also be involved [3]. In general, cuta- neous involvement follows oral or genital lesions by three and more 3.5. Paraneoplastic pemphigus (PNP) months [2,6]. On the skin, PV is characterized by widespread fragile fl accid blisters that arise on normal appearing skin and are easily rup- Paraneoplastic pemphigus is a rare autoimmune blistering disease fi tured to evolve into super cial erosions that become crusted (Fig. 1). that typically manifests with hemorrhagic stomatitis and extensive mu- Favored sites include the head (mainly the scalp), upper trunk and cous membrane erosions, associated with obvious or occult neoplasia. intertriginous areas [3]. PV can involve only the mucosa (mucosal The oral mucosa is most severely affected but other mucous membranes dominant type) or can cause extensive cutaneous and mucosal lesions such as the nose, pharynx, larynx, esophagus, conjunctivae, and genitals (mucocutaneous type). In the active phase of PV, both direct Nikolsky may also be affected [8]. Oral lesions in PNP characteristically involve sign (slight rubbing of the perilesional skin results in exfoliation of the the vermilion border of the lips [3]. Cutaneous manifestations present – outermost layer) and indirect Nikolsky sign (also known as Asboe after the appearance of the mucous membrane lesions [8–10],and Hansen sign or bulla spread sign; inwhichanintactblistercanbe have heterogeneity in their presentation including; polymorphic lesions shifted laterally and enlarged by digital pressure) can be elicited. such as blisters, lichen-planus like skin changes [8] as well as lichenoid palmoplantar exanthema and multiforme-like lesions resem- 3.2. Pemphigus foliaceus (PF) bling toxic epidermal necrolysis [1]. Less commonly, psoriasiform and

Pemphigus foliaceus (from the Latin word folium meaning “leaf”)is considered a superficial form of pemphigus. Lesions usually begin with multiple, pruritic, crusted, circumscribed patches in seborrheic areas such as the scalp as well as the face and trunk. In PF, blisters are more superficial than PV and erosions are more prominent than blisters. Untreated lesions do not heal and slowly develop into hyperkeratotic scales. Lesions can become confluent and can resemble exfoliative erythroderma. PF, in contrast to PV, does not affect the oral mucosa and has a more favorable prognosis than PV [5]. There are two clinical variants of superficial pemphigus: pemphigus foliaceus (including the endemic form known as fogo selvagum) and . Fogo selvagem, an endemic variant of PF, predominantly affects young women in endemic regions in Brazil and in North Africa. Fogo selvagem shares the same clinical, histological and immunological profile of classic PF [3].

3.3. Pemphigus erythematosus (PE)

Pemphigus erythematosus (PE) is a localized form of PF, affecting more commonly the elderly, is characterized by superficial erosions, erythema and crusts of the malar area of the face and seborrheic regions. Clinical features may resemble cutaneous erythematosus and in about 80% of cases antinuclear antibodies (ANA) can be detected, with- out the presence of anti-ds-DNA antibodies.

3.4. IgA pemphigus

IgA pemphigus is an intraepidermal blistering disease, a rare entity among the pemphigus diseases. Clinically it manifests with fragile Fig. 2. Tense blisters and erosion arising from urticarial plaque, on the chest and breast of a clear fluid-filled blisters that transform into pustules, owing to the patient with bullous pemphigoid. S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 485 pustular variants as well as palmoplantar have been re- membrane presents with , followed by recurrent ported [11,12]. The most frequent associated with PNP in blistering that heals with scarring. Ocular involvement usually starts two-thirds of the cases are non-Hodgkin's and chronic unilaterally with conjunctivitis, entropium and trichiasis. The conse- lymphocytic leukemia, followed by Castleman's disease, , quences of the disease to the mucous membranes can be severe and dev- Waldenström macroglobulinemia, sarcoma and carcinomas of the pan- astating; potentially MMP can lead to blindness, airway constriction, creas, colon and prostate [8,12–15]. PNP may precede the manifestation dysphagia and urinary and sexual dysfunction [1,4]. Cutaneous findings of the malignancy hence, when paraneoplastic pemphigus is suspected occurs in 20–30% of cases with lesions clinically resembling bullous clinically, a comprehensive work-up is mandatory. If there is remission pemphigoid with generalized blistering [1,4].MMPcanalsobelocalized of , improvement of the lesions is possible. presenting itself on the scalp and upper trunk which can lead to scarring alopecia and atrophic scars respectively. This is known as the Brunsting- 4. Pemphigoid diseases — diseases of subepidermal loss of adhesion Perry variant of localized BP.

4.1. Bullous pemphigoid (BP) 4.3. Linear IgA bullous dermatosis (LAD)

Bullous pemphigoid is the most common acquired bullous autoim- Linear Ig A dermatosis is one of the rarer subepidermal blistering dis- mune dermatosis with an estimated annual incidence of 6 to 14 new eases, with an estimated incidence of only 0.5 per million in western cases per 1 million population and a peak manifestation at the age of Europe [25]. LAD has two peaks of onset, in childhood and in adulthood. 70 years [1,2,16,17,29]. It is primarily a disease of the elderly with an It represents the most common autoimmune bullous disorder of child- equal incidence in men and women. It is the only hood with age peak of 4– 5years[1,2]. In adulthood it usually manifests in which incidence increases with age [2,18]. between the 40th and 60th year of life [1]. Typical lesions present as The clinical presentation includes tense blisters with serous or rarely pearl necklace-like grouped, centrifugally arranged tense blisters at hemorrhagic content which may arise on an erythematous base or on the edge of inflammatory erythema (Fig. 3). Extensive pruritus usually normal skin. The degree of itchiness varies but severe pruritus is often exists. Annular or multiforme-like lesions can also occur [2]. In adults, present. Blisters are often preceded over a period of weeks to months, LAD lesions comprise of urticarial plaques and with vesicles by an intractable pruritic eczema-like or urticarial eruption, so-called and blisters; the string-of- pearls grouping of blisters is less common nonbullous phase (Fig. 2). Lesions may be localized or generalized [25]. Predilection sites of LAD are the trunk and limbs. Iliosacral location with a predilection for the abdomen and flexural aspects of the limbs. or large body folds can also be affected [1,25]. Mucosal involvement is Scarring is usually not observed. The indirect Nikolsky sign is always pos- common (up to 50%) especially the oral mucosa, with erosions and itive whereas the direct Nikolsky sign can be elicited only on perilesional ulcers[1,25]. If there is involvement of the conjunctiva healing can result skin [16].In10–30% of the cases there is an involvement of the oral with scarring formation. Hoarseness may indicate pharyngeal involve- mucous membranes, usually in the form of erosions, more rarely as blis- ment. In most cases LAD starts spontaneously but drug-induced cases ters [2,16,18]. Atypical and localized forms of BP have been described are recognized, with vancomycin frequently being the culprit drug with a variety of different denominations, such as palmoplantar pemphi- [11]. In individual cases an association with lymphoproliferative dis- goid, prurigo nodularis-like, prurigo-like, erythroderma-like, ecthyma orders was found even after remission of the skin disease [1,26–28].The gangrenosum-like, intertrigo-like, and toxic epidermal necrolysis-like therapeutic response to diaminodiphenylsulfone () is good, in lesions [19–21]. contrast to systemic which is poor.

4.2. Mucous membrane or cicatricial pemphigoid (MMP) 4.4. Epidermolysis bullosa acquisita (EBA)

MMP is a chronic inflammatory subepithelial blistering disease with EBA is a rare autoimmune subepidermal bullous disease that involves a predilection for the elderly population and an estimated incidence of the skin and mucous membranes. EBA can manifest at any age, with a 1.6 per million population [1,22–24]. MMP is characterized by recurrent peak incidence between 40 and 60 years of age and an estimated inci- blistering of the mucous membrane but also of the skin. Lesions tend to dence of 0.2 and 0.5 new cases per million inhabitants per year [1,17]. heal with deforming scars. MMP primarily involves the oral and ocular The disease presents with four clinical variants: classical, inflammatory, mucosa, but the nasopharynx, esophagus, larynx, and anogenital muco- cicatricial pemphigoid-like, and Brunsting-Perry pemphigoid-like forms sa may also be involved [4]. Initially, involvement of the oral mucous [30]. The classical variant is the mechanobullous, noninflammatory form, in which slight trauma elicits blistering and erosions of the skin. Sites of predilection are mechanically irritated acral locations such as the hands and feet, but also the elbows and knees. In contrast to BP, healing of the lesions results with , milia, scars and pigmentation but distinction can be made with immunofluorescence studies [1,30]. Severe cases are characterized by fibrosis of the hands and feet, scarring alopecia and dystrophy even leading to acquired anonychia. The gen- eralized inflammatory form of EBA, clinically resembling BP, is character- ized by tense blisters usually on an erythematous, urticarial base. The cicatricial (localized) variant usually presents itself on the mucous membranes and clinically resembles MMP. The fourth variant presents with head and neck involvement, scarring and minimal mucosal disease resembling the Brunsting-Perry variant of cicatricial pemphigoid.

4.5. Dermatitis herpetiformis (DH)

Dermatitis herpetiformis (Duhring's disease) is a chronic, polymor- phic, and very pruritic skin disease that can manifest at any age, but Fig. 3. Linear IgA bullous dermatoses: centrifugally arranged tense blisters, ‘necklace-like’ often the onset is between the age of 25 and 55 years [1,31,32]. grouped lesions. Men are affected almost double as frequently as women. The annual 486 S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 incidence was 0.4 to 2.6 cases per 100,000 [32–34].Typically,DHlesions are grouped, 1- to 3-mm large papules, vesicles, (hence the name “herpetiform”), erosions and excoriations. Only rarely blisters are seen. Lesions often heal with lichenification, hypopigmentation or . DH is a polymorphic skin disease and usually pre- sents with symmetrical skin involvement at sites of predilection such as the extensor surfaces of the extremities with accentuation of the elbows and knees as well as the gluteal region and back. The oral muco- sa is rarely involved. DH is closely related to -sensitive enteropa- thy and clinically manifest in 5–10% of patients with celiac disease [1,2,31]. Although most of the patients are asymptomatic in 90% endo- scopic examination can reveal minimal duodenal involvement and submucosal endoscopic can reveal infiltration and jejunum atrophy [1,2]. The rapid improvement of pruritus and inflammatory skin lesions after administration of gluten free diet and dapsone are characteristic and dapsone is considered the first line treatment for DH. Unlike, celiac disease that does not respond to dapsone [31].

5. Pemphigoid gestationis (PG)

PG is a rare autoimmune subepidermal bullous dermatosis of pregnancy. This disease shares some similarities with BP on clinical, histological and pathophysiological aspects with most of the patients de- veloping antibodies against hemidesmosomal proteins, BP180 and less frequently BP230 [1,2]. The trigger for the development of autoantibodies remains unknown. The placenta is known to be the main source of pater- nal antibodies and can thus present an immunologic target during gesta- tion. Incidence is estimated to be 0.5 cases per million people per year. PG typically manifests during late pregnancy, with an abrupt onset of ex- tremely pruritic urticarial papules and blisters on the abdomen and trunk. No increase in fetal or maternal mortality has been demonstrated but a greater prevalence of premature and small-for-gestational-age (SGA) babies was reported with PG. 5–10% of infants may present with transient cutaneous involvement that resolves quickly.

6. Histology

Histopathologically examination is used to interpret the level of adhesion loss i.e. intraepidermal or subepidermal, but also for charac- terization of the cutaneous inflammatory infiltrate. Tissue biopsy should be obtained from the edge of an active and include some adjacent healthy skin so that the level of the blister can be accurately identified (Table 1, Diagram 1).

7. Pemphigus group — diseases of intraepidermal loss of adhesion

In PV, as in all forms of pemphigus, the basic abnormality is the loss of adhesion, known as . In PV there is intraepidermal, suprabasal acantholysis. This process leads to the formation of a cleft within the epidermis, which then enlarges into a bulla [3] (Fig. 4a). The presence of residual basal keratinocytes on the dermo-epidermal junction zone, at the site of the blister's floor is “ ” known as tombstone effect . In older blisters, neutrophilic and eosino- Fig. 4. (a). Pemphigus vulgaris: Histopathology specimen of the skin demonstrates philic granulocytes are seen with scant perivascular infiltrate. In early intraepidermal blister. The basal cell layer is separated from the spinous layer by a lesions eosinophilic may be present, even as the sole histo- suprabasal cleft. Acantholytic cells are seen within the blister and the basal cells stand fl logical manifestation. like a row of tombstones on the oor of the blister. (b). Bullous pemphigoid: Histopathol- ogy specimen of the skin demonstrates subepidermal blister with a superficial Light microscopy of lesional biopsies in pemphigus foliaceus reveals perivascular and interstitial inflammatory infiltrate containing . Eosinophils subcorneal acantholysis in the upper or granulosum are seen also within the blister. (c) Dermatitis herpetiformis: Histopathology specimen with slight inflammatory infiltration in the upper . Acantholytic of the skin demonstrates neutrophils and neutrophilic nuclear dust within edematous der- cells can also be seen in other bullous conditions that need to be differ- mal papillae. Eosinophils are also present. Papillary microabscesses form and progress to entiated, especially chronic that clinical and histological can re- subepidermal and vesicle formation. semble PF [3]. In impetigo, lesions are usually fewer, asymmetrically distributed and generally, improve rapidly with therapy; and interface dermatitis with vacuolar degeneration of basal circulating or tissue-fixed intercellular antibodies are absent. Histolog- keratinocytes and keratinocyte (dyskeratotic cells) [3,18]. ically in paraneoplastic pemphigus there is suprabasal acantholysis Often there is a dense lichenoid lymphocytic inflammatory infiltrate S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 487

Diagram 1. Schematic representation of autoantigens localization in autoimmune bullous disorders with the pemphigus group of diseases. The production of autoantibodies is against structural antigens of the desmosome and the pemphigoid group with characterized by the production of autoantibodies against hemidesmosomal proteins. in the vicinity of the zone. IgA pemphigus of the dermoepidermal basement membrane zone. Tissue-bound auto- is characterized histopathologically by slight acantholysis and an antibodies are detected by direct immunofluorescence microscopy intraepidermal or subcorneal infiltration of neutrophils. Acantholysis (DIF) (Table 1, Diagram 1). in IgA pemphigus is much milder and may be absent. If present, clefts In both PV and PF DIF of perilesional skin show intercellular suste- can be localized in the subcorneal region, in subcorneal pustular derma- nance deposition in a typical net-like pattern (honey comb like) of IgG tosis-type IgA pemphigus, whereas in intraepidermal neutrophilic and/or C3 [36]. In PV an accentuation is seen in the suprabasal epidermis dermatosis-type IgA pemphigus they can be present in the entire or while in PF the accentuation is subcorneal [32]. In about 90% of patients midepidermis. Neutrophilic spongiosis, i.e. epidermal spongiosis with with pemphigus tissue fixed intercellular antibodies are present and are neutrophils in the epidermis can be the only histological finding in IgA considered uncommon finding in individuals without pemphigus, pemphigus. therefore DIF can be used as an important tool in the diagnosis [3,37]. PE has also granular deposits along BM. In PNP, in addition to the above 8. Pemphigoid Diseases — diseases of subepidermal loss of adhesion findings, an abnormal band-like deposits of immunoglobulin and/or complement are detected in the dermal–epidermal junction [3,32]. Histopathologically, hematoxylin and eosin staining of an early bulla In IgA pemphigus depositions of IgA predominate in the cell–cell in bullous pemphigoid reveals subepidermal blistering with dense contact region [(17,32]. DIF in pemphigoid diseases with subepidermal inflammatory infiltrate consisting predominantly of eosinophils, but loss of adhesion, such as BP, MMP, PG and EBA, in most cases is found as also and neutrophils. Eosinophils are found within the linear staining of IgG and/or C3 seen at the dermo-epidermal junction blister as well as in the edematous papillary dermis [32]. In the early zone [32,36]. To further distinguish between these subepidermal bul- nonbullous phase, subepidermal clefts and eosinophilic spongiosis (epi- lous diseases, the salt split DIF technique is employed [38]. In general, dermal spongiosis with eosinophils within the epidermis) can be in this technique, the punch biopsy sample is incubated in a solution found [32] (Fig. 4b). A cell poor variant also exists. Mucous membrane of NaCl (1 mol/L) at 4 °C for 24 h, followed by a gentle manual separa- pemphigoid is characterized by noninflammatory subepidermal blister- tion of the epidermis from the dermis. On this specimen the DIF is ing in early lesion. Later, the inflammatory infiltration of the upper performed. The level of the IgG/C3 deposit at the blister formed serves dermis similar to that in BP can appear [32]. Linear IgA dermatosis has to distinguish between the diseases: In BP deposits are seen at the blis- less characteristic histologic features. There is subepidermal blistering ter roof, in EBA at its floor and in MMP in either roof or floor depending with a variable infiltrate of lymphocytes and neutrophils and even on the location of the targeting antigen [39]. In patients with DH, DIF sometimes intrapapillary microabscesses are present [25,32]. Skin shows granular IgA and C3 deposits along the dermoepidermal junction, biopsy in epidermolysis bullosa acquisita demonstrates subepidermal and especially in the tips of the papillary dermis (pathognomonic for DH) blisters with dermal inflammatory infiltration of the papillary dermis as well as along dermal blood vessels [34,38,39]. In the perilesional skin consisting primarily of neutrophilis and mononuclear cells. Bullous of LAD, DIF shows linear deposition of IgA along the basement mem- pemphigoid-like features can often be found [30]. Dermatitis herpeti- brane zone and, in some instances IgG, Ig M, or C3 are also seen [25]. formis in the blistering stage is characterized by pathognomonic subepidermal cleft and papillary consisting predominantly 10. Indirect immunofluorescence microscopy of neutrophils at the tips of dermal papillae accompanied by a peri- vascular mixed inflammatory infiltrate [32,35] (Fig. 4c). Indirect immunofluorescence microscopy (IIF) serves to detect and quantitate circulating autoantibodies in patients' serum and in most 9. Direct immunofluorescence microscopy cases the patterns observed in IIF resemble those in DIF but with lower sensitivity (Table 1). The diagnostic gold standard of autoimmune bullous diseases is the All forms of pemphigus are associated with the presence of circulating detection of autoantibodies in patients' epidermis or mucosal epitheli- autoantibodies against keratinocyte cell-surface antigens (Diagram 1). A um. Autoantibodies production in bullous diseases results as a patho- variety of substrates are used according to the subtypes. In most cases genic immune response against structural proteins of keratinocytes or guinea pig esophagus or monkey esophagus are employed as substrate 488 S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 as they are the most sensitive in detecting circulating intraepidermal au- vulgaris both desmoglein 3 and desmoglein 1 autoantibodies can be toantibodies of PV and PF. Rat bladder epithelium is the most appropriate found [37]. titre usually correlates with disease activity substrate for screening for antibodies to PNP due to its high expression of [2,3,16,33] and even though they are not widely available in routine plakins [18,32]. In pemphigus diseases IIF reveals circulating IgG autoan- clinical practice, are appropriate for follow-up monitoring. It has been tibodies in the serum of the patients in a lace-like pattern of fluorescence shown that autoantibodies of the IgG4 as well as IgG1 class are indica- within the epidermis [13,38]. Circulating intraepidermal antibodies are tive of active rather than remittent disease [2]. Western blotting analy- present in about 80% of patients with active pemphigus disease, and ses are not useful in the diagnosis of pemphigus based on the fact that their titre usually correlates with disease activity. [3,38]. Low titres can most of the on desmoglein 1 and 3, are conformational. Fur- also be found in patients with antibodies to ABO blood group antigens thermore, acetylcholine receptors have been reported as autoantigens or with burns, fungal infections, or allergic drug reactions. In IgA pemphi- in pemphigus vulgaris but their pathogenetic relevance is unclear. gus cultured skin is used as a substrate and circulating intercellular IgA PF with its two subsets of endemic (fogoselvagem) and non- autoantibodies can be detected in only about 50% of cases [32]. endemic forms is characterized by exclusive involvement of desmoglein In autoimmune bullous dermatoses with subepidermal loss of adhe- 1 autoantibodies, but not against desmoglein 3 or against plakins sion, up to 70% of patients present with circulating IgG–anti-BMZ (Diagram 1). In PE antinuclear antibodies are present [36].Inmost autoantibodies (Diagram 1). Similar to DIF, IIF on salt-split human cases of PNP IgG autoantibodies against desmoglein 3 and 1 are found split-thickness skin (SSST)–induced by incubation in one molar sodium but PNP is characterized by the detection of autoantibodies against chloride solution–has been identified as the optimal substrate and is desmosomal proteins of the plakin family. Envoplakin and periplakin used to differentiate between serum autoantibodies that bind to the are the most specific target antigens followed by 1 and 2, roof and those that stain the floor of the artificial split, reflecting the dif- plectin, and a 170 kDa [17,32](Diagram 1). IgA pemphigus is character- ferent autoantibody specificities [36]. This split at the level of the lamina ized by the detection of IgA autoantibodies against desmoglein 1 and lucida of the dermo-epidermal junction zone allows the differentia- desmoglein 3 in the IEN type and against desmocollin 1 in the SPD tion between different pemphigoid diseases i.e. in BP there is linear type [17,32] (Diagram 1). Bullous pemphigoid is characterized by auto- fluorescence on the epidermal side of the split, while in EBA linear fluo- antibodies that target 2 hemidesmosomal structure proteins, BP180 and rescence is found on the dermal side. In around 80% to 85% of patients BP230. BP180 is a transmembrane glycoprotein of which the extracellu- with BP, IIF microscopy reveals circulating IgG autoantibodies [16]. lar portion–16th non-collageneous (NC16A)–was identified as the In MMP and LAD mixed fluorescence both on the epidermal as well immunodominant region in patients with BP (Diagram 1). Serum levels as on the dermal side has been reported [32]. Low titres of IIF are found of anti-BP180 NC16A antibodies can be detected using ELISA and it has in LAD and are more frequently encountered in children (74%) rather been shown that antibodies levels correlate with disease activity in BP than in adults (30%) [25]. Unlike pemphigus diseases, no correlation patients [40,41]. In several reports, ELISA has been demonstrated to be exists between the autoantibody titer and disease activity [38].Now- highly sensitive and specific. It should be taken into consideration that adays further research includes visualization on electron microscopic a considerable proportion of older patients with polymorphic pruritic examination. This procedure is termed direct and indirect immuno- skin diseases also show reactivity of IgG autoantibodies against BP230 electron microscopy which ultrastructurally localizes in vivo bound IgG and BP180. Hence, the diagnosis of BP is not based solely on the detec- autoantibodies (direct immunoEM) or the binding site of circulating tion of IgG against BP230 and/or BP180 but also on the identification IgG autoantibodies (indirect immunoEM) at the basement membrane. of tissue-bound autoantibodies in DIF. ELISA test systems for the detec- For DH, ELISA-based assays are the preferred tests, but IIF may also tion of IgG against BP230 are available commercially. based on be used to demonstrate IgA reactivity directed against the endomysium recombinant proteins encoded by BP230 and BP180 have been devel- and are visualized on substrates containing smooth muscle, such as oped but are not commercially available and offer an investigational monkey esophagus [32,36,38,39]. The titer of circulating autoantibodies tools. In MMP, IgG autoantibodies are found against BP180; as well as, reflects the disease activity [38,39] but the method is expensive, time autoantibodies against 332 (=laminin 5), α6andβ4- integrin consuming and not routinely available in all laboratories. and occasionally against BP230 (Diagram 1). Autoantibodies can be detected using Western blotting, and ELISA. It 11. Target antigen-specific serological confirmatory tests by ELISA has been reported that autoantibodies against laminin 332 can be asso- and Western blotting ciated with malignancy whereas autoantibodies against α6-intergrin with oral involvement and against β4-integrin with ocular involvement Enzyme-Linked Immuno Sorbent Assay (ELISA), immunoblotting or [41]. The autoantigen of linear IgA dermatosis is a 120 kDa large extra- immunoprecipitation are recently developed, highly sensitive and cellular product ectodomain of the BP180, named LAD-1, while in only specific detection systems of autoantibodies utilizing recombinant 20% of patients sera recognize BP180 NC16A [42,43] (Diagram 1). autoantigens or keratinocyte extracts from healthy . Unlike EBA is characterized by autoantibodies directed against type VII col- immunoblotting, immunoprecipitation is performed with native, rather lagen (Diagram 1). VII is the main component of the anchoring than denatured, protein and is more sensitive. The latter assays, in addi- fibrils of the dermoepidermal junction zone. Autoantibodies can be tion to serving as serological confirmatory tests (in many cases used as a detected by immunoblotting with extract of human dermis and in definitive diagnostic step for autoimmune bullous dermatoses) are used almost all patients IgG reactivity against the NC1 domain of type VII col- as a valuable tool for immunoserological follow-up. These above named lagen is detected [44]. Besides EBA collagen type VII autoantibodies can tests are able to detect IgG or IgA autoantibodies against desmoglein 1, be detected in bullous systemic . desmoglein 3, BP180, BP230, tissue transglutaminase as well as against Epidermal transglutaminase has been identified as the autoantigen envoplakin and are available commercially. Immunopercipitation is of DH (Diagram 1). In gluten-sensitive persons after oral gluten exposi- more difficult to perform and is generally less available than immuno- tion, IgA autoantibodies develop in the epithelium of the small intestine blotting (Diagram 1). Due to the increasing availability of the assays, against a complex consisting of gluten and tissue transglutaminase. The the diagnostic spectrum of autoimmune bullous skin diseases has autoantibodies cross-react with epidermal transglutaminase, which been expanded considerably in recent years and has gained both in sen- participates as an isoenzyme in keratinocyte differentiation. Immune sitivity as well as specificity (Table 1). complexes of IgA and epidermal transglutaminase are found as diagnos- Diseases of the pemphigus group are characterized by the presence tic markers in the papillary dermis. ELISA systems are available for of autoantibodies against desmosomal structure proteins (Diagram 1). detecting IgA reactivity against tissue transglutaminase in DH [45]. Fur- Desmoglein 3 autoantibodies can be detected in PV with exclusive mu- thermore, ELISA employing -analogous multimeric fusion pro- cosal involvement whereas in the mucocutaneous variant of pemphigus teins are available. S. Baum et al. / Autoimmunity Reviews 13 (2014) 482–489 489

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