diagnosis and classification of autoimmune blistering...
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Autoimmunity Reviews 13 (2014) 482–489
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Review
Diagnosis and classification of autoimmune blistering diseases
Sharon Baum ⁎,1, Nicole Sakka 1, Ofir Artsi, Henri Trau, Aviv BarzilaiDepartment of Dermatology, Chaim Sheba Medical Center, Tel-Aviv University, Sackler School of Medicine, Tel Hashomer, Israel
⁎ Corresponding author at: Department of DermatologTel Hashomer, Tel Aviv, Israel, 52621. Tel.: +972 52 666 6
E-mail address: [email protected] (S. B1 Contributed equally.
1568-9972/$ – see front matter © 2014 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.autrev.2014.01.047
a b s t r a c t
a r t i c l e i n f oArticle history:Received 7 October 2013Accepted 13 November 2013Available online 13 January 2014
Keywords:Blistering skin diseasesPemphigusPemphigoidAutoantibodiesDirect immunofluorescence
Blistering skin diseases are a group of autoimmune disorders that are characterized by autoantibodies againststructural proteins of the epidermis or the dermal–epidermal junction and clinically by blisters and erosionson skin and/or mucous membranes. Since clinical criteria and histopathological characteristics are not sufficientfor diagnosis, direct immunofluorescence microscopy of a biopsy specimen or serological tests are needed forexact diagnosis. The differentiation between the various disorders became more important since prognosis aswell as different treatment options are nowadays available for the various diseases. Moreover, some bullousdiseases may indicate the presence of an underlying malignancy. The detection of serum autoantibodies havebeen shown to correlate with disease activity and thus may be helpful in deciding treatment options for thesepatients.
© 2014 Elsevier B.V. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4822. Clinical classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4833. Pemphigus group — diseases of intraepidermal loss of adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
3.1. Pemphigus vulgaris (PV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4833.2. Pemphigus foliaceus (PF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4843.3. Pemphigus erythematosus (PE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4843.4. IgA pemphigus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4843.5. Paraneoplastic pemphigus (PNP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
4. Pemphigoid diseases — diseases of subepidermal loss of adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4854.1. Bullous pemphigoid (BP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4854.2. Mucous membrane or cicatricial pemphigoid (MMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4854.3. Linear IgA bullous dermatosis (LAD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4854.4. Epidermolysis bullosa acquisita (EBA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4854.5. Dermatitis herpetiformis (DH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
5. Pemphigoid gestationis (PG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4866. Histology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4867. Pemphigus group — diseases of intraepidermal loss of adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4868. Pemphigoid Diseases — diseases of subepidermal loss of adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4879. Direct immunofluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
10. Indirect immunofluorescence microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48711. Target antigen-specific serological confirmatory tests by ELISA and Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
y, Chaim Sheba Medical Centre,182; fax: +972 3 530 3515.aum).
ghts reserved.
1. Introduction
Autoimmune blistering dermatoses comprise a heterogeneousgroup of diseases that are characterized by autoantibodies directedagainst adhesionmolecules of the skin and adjacentmucousmembranes.
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According to the skin level at which the blister occurs and by the struc-tural proteins that the autoantibodies target, autoimmune blisteringdiseases can be categorized into different groups i.e. in pemphigusgroup, autoantibodies target desmosomal proteins that results inloss of cell adherence between keratinocytes, in pemphigoid diseases,hemidesmosomal proteins of the dermo-epidermal junction are tar-geted, and in dermatitis herpetiformis, autoantibodies bind to epider-mal and tissue transglutaminase (Diagram 1).
The diagnosis of autoimmune blistering dermatoses is based on aconstellation of clinical and laboratory findings and due to their rarityand heterogeneous clinical features, they often pose a major diagnosticchallenge. Diagnosis cannot be based solely on clinical signs and thehistopathological findings and requires the detection of tissue boundand circulating autoantibodies which still remains the diagnostic goldstandard in the detection of autoantibodies. Furthermore, sensitiveand specific serological assays have been developed to allow the detec-tion of serum antibodieswhich are used as diagnostic tools aswell as fordisease activity monitoring.
2. Clinical classification
Autoimmune bullous diseases have a broad spectrum of clinicalmanifestations and a variety of morphological lesions. Based on thelevel of the skin blister formation — intraepidermal or subepidermal,they can be classified into pemphigus group and pemphigoid group ofdiseases respectively. Pemphigus is characterized by flaccid vesicles and
Table 1Diagnostic algorithmof clinical, histological, immunofluorescence and autoantibodies against stjunction zone; Dsg: desmoglein; Ig: immunoglobulins; DC: desmocollin; TTG: tissue transglutvulgaris; PNP: paraneoplastic pemphigus; BP: bullous pemphigoid; MMP: mucous membrane
erosions of the skin and/ormucousmembranes, intraepidermal blisteringand the production of IgG autoantibodies against the keratinocytes adhe-sion molecules. The major subtypes are; pemphigus vulgaris and pem-phigus foliaceus, and aless frequent form, IgA pemphigus. Pemphigoiddiseases are characterized by autoantibodies directed against structuralproteins of the dermal–epidermal junction that clinically can manifestwith urticarial lesions, tense blisters and erosions which may involvethe mucous membranes. Diseases in this group include bullous pemphi-goid, dermatitis herpetiformis, mucous membrane pemphigois, linear IgA bullous dermatosis, herpes gestationis and epidermolysis bullosaacquisita (Table 1, Diagram 1). Paraneoplastic pemphigus, although clas-sified as a type of pemphigus based on its clinical presentation, showsantibodies against both intraepidermal and subepidermal components.
3. Pemphigus group— diseases of intraepidermal loss of adhesion
3.1. Pemphigus vulgaris (PV)
Pemphigus vulgaris (“pemphigus” stems from the Greek wordpemphix for “blister”) is the most frequent representative of thegroup of pemphigus diseaseswith an incidence of 0.1–0.5/100 000 pop-ulation [1–3]. The mean onset of PV is usually around middle age withan age peak in the fourth and fifth decade of life and a higher prevalencein patients of Jewish or Mediterranean ancestry [1–4]. PV is a chronicautoimmune intraepithelial blistering disease and potentially life-threatening condition. Despite advances in management, the mortality
ructural antigens for autoimmune bullous disorders. Abbreviations: DEJ: dermo-epidermalaminase, LAD-1: soluble ectodomain of BP180; PF: pemphigus foliaceus; PV: pemphiguspemphigoid; EBA: epidermolysis bullosa acquisita; and DH: dermatitis herpetiformis.
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.
Fig. 2. Tense blisters and erosion arising from urticarial plaque, on the chest and breast of apatient with bullous pemphigoid.
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rate is currently estimated at 5 to 10% [5]. In the majority of the cases(70–90%) PV presents itself with painful and long-persisting erosionsof the mucous membranes, especially of the oral(buccal) mucosa [1,4].Other sites such as the throat, esophagus, conjunctivae, nasal mucosa,vagina, vulva, penis and anus can also be involved [3]. In general, cuta-neous involvement follows oral or genital lesions by three and moremonths [2,6]. On the skin, PV is characterized by widespread fragileflaccid blisters that arise on normal appearing skin and are easily rup-tured to evolve into superficial erosions that become crusted (Fig. 1).Favored sites include the head (mainly the scalp), upper trunk andintertriginous areas [3]. PV can involve only the mucosa (mucosaldominant type) or can cause extensive cutaneous and mucosal lesions(mucocutaneous type). In the active phase of PV, both direct Nikolskysign (slight rubbing of the perilesional skin results in exfoliation of theoutermost layer) and indirect Nikolsky sign (also known as Asboe–Hansen sign or bulla spread sign; in which an intact blister can beshifted laterally and enlarged by digital pressure) can be elicited.
3.2. Pemphigus foliaceus (PF)
Pemphigus foliaceus (from the Latin word folium meaning “leaf”) isconsidered a superficial form of pemphigus. Lesions usually begin withmultiple, pruritic, crusted, circumscribed patches in seborrheic areassuch as the scalp as well as the face and trunk. In PF, blisters are moresuperficial than PV and erosions are more prominent than blisters.Untreated lesions do not heal and slowly develop into hyperkeratoticscales. Lesions can become confluent and can resemble exfoliativeerythroderma. PF, in contrast to PV, does not affect the oral mucosa andhas amore favorable prognosis than PV [5]. There are two clinical variantsof superficial pemphigus: pemphigus foliaceus (including the endemicform known as fogo selvagum) and pemphigus erythematosus. Fogoselvagem, an endemic variant of PF, predominantly affects young womenin endemic regions in Brazil and in North Africa. Fogo selvagem sharesthe same clinical, histological and immunological profile of classic PF [3].
3.3. Pemphigus erythematosus (PE)
Pemphigus erythematosus (PE) is a localized form of PF, affectingmore commonly the elderly, is characterized by superficial erosions,erythemaand crusts of themalar area of the face and seborrheic regions.Clinical features may resemble cutaneous lupus erythematosus and inabout 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 entityamong the pemphigus diseases. Clinically it manifests with fragileclear fluid-filled blisters that transform into pustules, owing to the
accumulation of neutrophils [7]. Lesions have a distinct tendency to co-alesce and there is an associated pruritus. Frequently affected sites forIgA pemphigus are distal trunk and proximal limbs as well asintertriginous sites [7]. Mucosal involvement is rare.
3.5. Paraneoplastic pemphigus (PNP)
Paraneoplastic pemphigus is a rare autoimmune blistering diseasethat typically manifests with hemorrhagic stomatitis and extensivemu-cous membrane erosions, associated with obvious or occult neoplasia.The oralmucosa ismost severely affected but othermucousmembranessuch as the nose, pharynx, larynx, esophagus, conjunctivae, and genitalsmay also be affected [8]. Oral lesions in PNP characteristically involvethe vermilion border of the lips [3]. Cutaneous manifestations presentafter the appearance of the mucous membrane lesions [8–10], andhave heterogeneity in their presentation including; polymorphic lesionssuch as blisters, lichen-planus like skin changes [8] as well as lichenoidpalmoplantar exanthema and erythemamultiforme-like lesions resem-bling toxic epidermal necrolysis [1]. Less commonly, psoriasiform and
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pustular variants as well as palmoplantar hyperkeratosis have been re-ported [11,12]. The most frequent neoplasms associated with PNP intwo-thirds of the cases are non-Hodgkin's lymphoma and chroniclymphocytic leukemia, followed by Castleman's disease, thymoma,Waldenströmmacroglobulinemia, sarcoma and carcinomas of the pan-creas, colon and prostate [8,12–15]. PNPmay precede themanifestationof the malignancy hence, when paraneoplastic pemphigus is suspectedclinically, a comprehensive work-up is mandatory. If there is remissionof neoplasm, improvement of the lesions is possible.
4. Pemphigoid diseases — diseases of subepidermal loss of adhesion
4.1. Bullous pemphigoid (BP)
Bullous pemphigoid is the most common acquired bullous autoim-mune dermatosis with an estimated annual incidence of 6 to 14 newcases per 1 million population and a peak manifestation at the age of70 years [1,2,16,17,29]. It is primarily a disease of the elderly with anequal incidence in men and women. It is the only autoimmune diseasein which incidence increases with age [2,18].
The clinical presentation includes tense blisters with serous or rarelyhemorrhagic content which may arise on an erythematous base or onnormal skin. The degree of itchiness varies but severe pruritus is oftenpresent. Blisters are often preceded over a period of weeks to months,by an intractable pruritic eczema-like or urticarial eruption, so-callednonbullous phase (Fig. 2). Lesions may be localized or generalizedwith a predilection for the abdomen and flexural aspects of the limbs.Scarring is usually not observed. The indirect Nikolsky sign is always pos-itive whereas the direct Nikolsky sign can be elicited only on perilesionalskin [16]. In 10–30% of the cases there is an involvement of the oralmucous membranes, usually in the form of erosions, more rarely as blis-ters [2,16,18]. Atypical and localized forms of BP have been describedwith a variety of different denominations, such as palmoplantar pemphi-goid, prurigo nodularis-like, prurigo-like, erythroderma-like, ecthymagangrenosum-like, intertrigo-like, and toxic epidermal necrolysis-likelesions [19–21].
4.2. Mucous membrane or cicatricial pemphigoid (MMP)
MMP is a chronic inflammatory subepithelial blistering disease witha predilection for the elderly population and an estimated incidence of1.6 permillion population [1,22–24]. MMP is characterized by recurrentblistering of themucous membrane but also of the skin. Lesions tend toheal with deforming scars. MMP primarily involves the oral and ocularmucosa, but the nasopharynx, esophagus, larynx, and anogenital muco-sa may also be involved [4]. Initially, involvement of the oral mucous
Fig. 3. Linear IgA bullous dermatoses: centrifugally arranged tense blisters, ‘necklace-like’grouped lesions.
membrane presentswith desquamative gingivitis, followed by recurrentblistering that heals with scarring. Ocular involvement usually startsunilaterally with conjunctivitis, entropium and trichiasis. The conse-quences of the disease to themucousmembranes can be severe and dev-astating; potentially MMP can lead to blindness, airway constriction,dysphagia and urinary and sexual dysfunction [1,4]. Cutaneous findingsoccurs in 20–30% of cases with lesions clinically resembling bullouspemphigoid with generalized blistering [1,4]. MMP can also be localizedpresenting itself on the scalp and upper trunkwhich can lead to scarringalopecia and atrophic scars respectively. This is known as the Brunsting-Perry variant of localized BP.
4.3. Linear IgA bullous dermatosis (LAD)
Linear Ig A dermatosis is oneof the rarer subepidermal blisteringdis-eases, with an estimated incidence of only 0.5 per million in westernEurope [25]. LAD has two peaks of onset, in childhood and in adulthood.It represents the most common autoimmune bullous disorder of child-hood with age peak of 4–5 years [1,2]. In adulthood it usually manifestsbetween the 40th and 60th year of life [1]. Typical lesions present aspearl necklace-like grouped, centrifugally arranged tense blisters atthe edge of inflammatory erythema (Fig. 3). Extensive pruritus usuallyexists. Annular or multiforme-like lesions can also occur [2]. In adults,LAD lesions comprise of urticarial plaques and papules with vesiclesand blisters; the string-of- pearls grouping of blisters is less common[25]. Predilection sites of LAD are the trunk and limbs. Iliosacral locationor large body folds can also be affected [1,25]. Mucosal involvement iscommon (up to 50%) especially the oral mucosa, with erosions andulcers[1,25]. If there is involvement of the conjunctiva healing can resultwith scarring formation. Hoarseness may indicate pharyngeal involve-ment. In most cases LAD starts spontaneously but drug-induced casesare recognized, with vancomycin frequently being the culprit drug[11]. In individual cases an association with lymphoproliferative dis-orderswas found even after remission of the skin disease [1,26–28]. Thetherapeutic response to diaminodiphenylsulfone (dapsone) is good, incontrast to systemic corticosteroids which is poor.
4.4. Epidermolysis bullosa acquisita (EBA)
EBA is a rare autoimmune subepidermal bullous disease that involvesthe skin and mucous membranes. EBA can manifest at any age, with apeak incidence between 40 and 60 years of age and an estimated inci-dence of 0.2 and 0.5 new cases per million inhabitants per year [1,17].The disease presents with four clinical variants: classical, inflammatory,cicatricial pemphigoid-like, and Brunsting-Perry pemphigoid-like forms[30]. The classical variant is the mechanobullous, noninflammatoryform, in which slight trauma elicits blistering and erosions of the skin.Sites of predilection are mechanically irritated acral locations such asthe hands and feet, but also the elbows and knees. In contrast to BP,healing of the lesions results with atrophy, milia, scars and pigmentationbut distinction can be made with immunofluorescence studies [1,30].Severe cases are characterized by fibrosis of the hands and feet, scarringalopecia andnail 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. Thecicatricial (localized) variant usually presents itself on the mucousmembranes and clinically resembles MMP. The fourth variant presentswith head and neck involvement, scarring and minimal mucosal diseaseresembling 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, butoften the onset is between the age of 25 and 55 years [1,31,32].Men are affected almost double as frequently as women. The annual
Fig. 4. (a). Pemphigus vulgaris: Histopathology specimen of the skin demonstratesintraepidermal blister. The basal cell layer is separated from the spinous layer by asuprabasal cleft. Acantholytic cells are seen within the blister and the basal cells standlike a row of tombstones on the floor of the blister. (b). Bullous pemphigoid: Histopathol-ogy specimen of the skin demonstrates subepidermal blister with a superficialperivascular and interstitial inflammatory infiltrate containing eosinophils. Eosinophilsare seen also within the blister. (c) Dermatitis herpetiformis: Histopathology specimenof the skin demonstrates neutrophils andneutrophilic nuclear dustwithin edematousder-mal papillae. Eosinophils are also present. Papillary microabscesses form and progress tosubepidermal vacuolization and vesicle formation.
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incidencewas 0.4 to 2.6 cases per 100,000 [32–34]. Typically, DH lesionsare grouped, 1- to 3-mm large papules, vesicles, (hence the name“herpetiform”), erosions and excoriations. Only rarely blisters areseen. Lesions often heal with lichenification, hypopigmentation orhyperpigmentation. DH is a polymorphic skin disease and usually pre-sents with symmetrical skin involvement at sites of predilection suchas the extensor surfaces of the extremities with accentuation of theelbows and knees aswell as the gluteal region and back. The oralmuco-sa is rarely involved. DH is closely related to gluten-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 andsubmucosal endoscopic biopsies can reveal lymphocyte infiltrationand jejunum atrophy [1,2]. The rapid improvement of pruritus andinflammatory skin lesions after administration of gluten free diet anddapsone are characteristic and dapsone is considered the first linetreatment for DH. Unlike, celiac disease that does not respond todapsone [31].
5. Pemphigoid gestationis (PG)
PG is a rare autoimmune subepidermal bullous dermatosis ofpregnancy. 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 lessfrequently BP230 [1,2]. The trigger for the development of autoantibodiesremains unknown. The placenta is known to be themain source of pater-nal antibodies and can thus present an immunologic target during gesta-tion. Incidence is estimated to be 0.5 cases permillion people per year. PGtypically manifests during late pregnancy, with an abrupt onset of ex-tremely pruritic urticarial papules and blisters on the abdomen andtrunk. No increase in fetal or maternal mortality has been demonstratedbut a greater prevalence of premature and small-for-gestational-age(SGA) babies was reported with PG. 5–10% of infants may presentwith transient cutaneous involvement that resolves quickly.
6. Histology
Histopathologically examination is used to interpret the level ofadhesion loss i.e. intraepidermal or subepidermal, but also for charac-terization of the cutaneous inflammatory infiltrate. Tissue biopsy shouldbe obtained from the edge of an active lesion and include some adjacenthealthy 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 theloss of keratinocyte adhesion, known as acantholysis. In PV there isintraepidermal, suprabasal acantholysis. This process leads to theformation of a cleft within the epidermis, which then enlarges into abulla [3] (Fig. 4a). The presence of residual basal keratinocytes on thedermo-epidermal junction zone, at the site of the blister's floor isknown as “tombstone effect”. In older blisters, neutrophilic and eosino-philic granulocytes are seen with scant perivascular infiltrate. In earlylesions eosinophilic spongiosis may be present, even as the sole histo-logical manifestation.
Light microscopy of lesional biopsies in pemphigus foliaceus revealssubcorneal acantholysis in the upper stratum spinosum or granulosumwith slight inflammatory infiltration in the upper dermis. Acantholyticcells can also be seen in other bullous conditions that need to be differ-entiated, especially chronic impetigo that clinical and histological can re-semble PF [3]. In impetigo, lesions are usually fewer, asymmetricallydistributed and generally, improve rapidly with antibiotic therapy;circulating or tissue-fixed intercellular antibodies are absent. Histolog-ically in paraneoplastic pemphigus there is suprabasal acantholysis
and interface dermatitis with vacuolar degeneration of basalkeratinocytes and keratinocyte necrosis (dyskeratotic cells) [3,18].Often there is a dense lichenoid lymphocytic inflammatory infiltrate
Diagram 1. Schematic representation of autoantigens localization in autoimmune bullous disorders with the pemphigus group of diseases. The production of autoantibodies is againststructural antigens of the desmosome and the pemphigoid group with characterized by the production of autoantibodies against hemidesmosomal proteins.
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in the vicinity of the dermoepidermal junction zone. IgA pemphigusis characterized histopathologically by slight acantholysis and anintraepidermal or subcorneal infiltration of neutrophils. Acantholysisin IgA pemphigus is much milder and may be absent. If present, cleftscan be localized in the subcorneal region, in subcorneal pustular derma-tosis-type IgA pemphigus, whereas in intraepidermal neutrophilicdermatosis-type IgA pemphigus they can be present in the entire ormidepidermis. Neutrophilic spongiosis, i.e. epidermal spongiosis withneutrophils in the epidermis can be the only histological finding in IgApemphigus.
8. Pemphigoid Diseases— diseases of subepidermal loss of adhesion
Histopathologically, hematoxylin and eosin staining of an early bullain bullous pemphigoid reveals subepidermal blistering with denseinflammatory infiltrate consisting predominantly of eosinophils, butalso lymphocytes and neutrophils. Eosinophils are found within theblister as well as in the edematous papillary dermis [32]. In the earlynonbullous phase, subepidermal clefts and eosinophilic spongiosis (epi-dermal spongiosis with eosinophils within the epidermis) can befound [32] (Fig. 4b). A cell poor variant also exists. Mucous membranepemphigoid is characterized by noninflammatory subepidermal blister-ing in early lesion. Later, the inflammatory infiltration of the upperdermis similar to that in BP can appear [32]. Linear IgA dermatosis hasless characteristic histologic features. There is subepidermal blisteringwith a variable infiltrate of lymphocytes and neutrophils and evensometimes intrapapillary microabscesses are present [25,32]. Skinbiopsy in epidermolysis bullosa acquisita demonstrates subepidermalblisters with dermal inflammatory infiltration of the papillary dermisconsisting primarily of neutrophilis and mononuclear cells. Bullouspemphigoid-like features can often be found [30]. Dermatitis herpeti-formis in the blistering stage is characterized by pathognomonicsubepidermal cleft and papillary abscesses consisting predominantlyof neutrophils at the tips of dermal papillae accompanied by a peri-vascular mixed inflammatory infiltrate [32,35] (Fig. 4c).
9. Direct immunofluorescence microscopy
The diagnostic gold standard of autoimmune bullous diseases is thedetection of autoantibodies in patients' epidermis or mucosal epitheli-um. Autoantibodies production in bullous diseases results as a patho-genic immune response against structural proteins of keratinocytes or
of the dermoepidermal basement membrane zone. Tissue-bound auto-antibodies are detected by direct immunofluorescence microscopy(DIF) (Table 1, Diagram 1).
In both PV and PF DIF of perilesional skin show intercellular suste-nance deposition in a typical net-like pattern (honey comb like) of IgGand/or C3 [36]. In PV anaccentuation is seen in the suprabasal epidermiswhile in PF the accentuation is subcorneal [32]. In about 90% of patientswith pemphigus tissue fixed intercellular antibodies are present and areconsidered uncommon finding in individuals without 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 abovefindings, an abnormal band-like deposits of immunoglobulin and/orcomplement are detected in the dermal–epidermal junction [3,32].
In IgA pemphigus depositions of IgA predominate in the cell–cellcontact region [(17,32]. DIF in pemphigoid diseases with subepidermalloss of adhesion, such as BP, MMP, PG and EBA, inmost cases is found aslinear staining of IgG and/or C3 seen at the dermo-epidermal junctionzone [32,36]. To further distinguish between these subepidermal bul-lous diseases, the salt split DIF technique is employed [38]. In general,in this technique, the punch biopsy sample is incubated in a solutionof NaCl (1 mol/L) at 4 °C for 24 h, followed by a gentle manual separa-tion of the epidermis from the dermis. On this specimen the DIF isperformed. The level of the IgG/C3 deposit at the blister formed servesto distinguish between the diseases: In BP deposits are seen at the blis-ter roof, in EBA at its floor and in MMP in either roof or floor dependingon the location of the targeting antigen [39]. In patients with DH, DIFshows granular IgA and C3 deposits along the dermoepidermal junction,and especially in the tips of the papillary dermis (pathognomonic forDH)as well as along dermal blood vessels [34,38,39]. In the perilesional skinof LAD, DIF shows linear deposition of IgA along the basement mem-brane zone and, in some instances IgG, Ig M, or C3 are also seen [25].
10. Indirect immunofluorescence microscopy
Indirect immunofluorescence microscopy (IIF) serves to detect andquantitate circulating autoantibodies in patients' serum and in mostcases the patterns observed in IIF resemble those in DIF but withlower sensitivity (Table 1).
All forms of pemphigus are associatedwith the presence of circulatingautoantibodies against keratinocyte cell-surface antigens (Diagram 1). Avariety of substrates are used according to the subtypes. In most casesguinea pig esophagus or monkey esophagus are employed as substrate
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as they are the most sensitive in detecting circulating intraepidermal au-toantibodies of PV and PF. Rat bladder epithelium is themost appropriatesubstrate for screening for antibodies to PNP due to its high expression ofplakins [18,32]. In pemphigus diseases IIF reveals circulating IgG autoan-tibodies in the serum of the patients in a lace-like pattern of fluorescencewithin the epidermis [13,38]. Circulating intraepidermal antibodies arepresent in about 80% of patients with active pemphigus disease, andtheir titre usually correlates with disease activity. [3,38]. Low titres canalso be found in patients with antibodies to ABO blood group antigensorwith burns, fungal infections, or allergic drug reactions. In IgA pemphi-gus cultured skin is used as a substrate and circulating intercellular IgAautoantibodies can be detected in only about 50% of cases [32].
In autoimmune bullous dermatoses with subepidermal loss of adhe-sion, up to 70% of patients present with circulating IgG–anti-BMZautoantibodies (Diagram 1). Similar to DIF, IIF on salt-split humansplit-thickness skin (SSST)–induced by incubation in onemolar sodiumchloride solution–has been identified as the optimal substrate and isused to differentiate between serum autoantibodies that bind to theroof and those that stain the floor of the artificial split, reflecting the dif-ferent autoantibody specificities [36]. This split at the level of the laminalucida of the dermo-epidermal junction zone allows the differentia-tion between different pemphigoid diseases i.e. in BP there is linearfluorescence on the epidermal side of the split, while in EBA linear fluo-rescence is found on the dermal side. In around 80% to 85% of patientswith BP, IIF microscopy reveals circulating IgG autoantibodies [16].
In MMP and LAD mixed fluorescence both on the epidermal as wellas on the dermal side has been reported [32]. Low titres of IIF are foundin LAD and are more frequently encountered in children (74%) ratherthan in adults (30%) [25]. Unlike pemphigus diseases, no correlationexists between the autoantibody titer and disease activity [38]. Now-adays further research includes visualization on electron microscopicexamination. This procedure is termed direct and indirect immuno-electron microscopy which ultrastructurally localizes in vivo bound IgGautoantibodies (direct immunoEM) or the binding site of circulatingIgG autoantibodies (indirect immunoEM) at the basement membrane.
For DH, ELISA-based assays are the preferred tests, but IIF may alsobe used to demonstrate IgA reactivity directed against the endomysiumand are visualized on substrates containing smooth muscle, such asmonkey esophagus [32,36,38,39]. The titer of circulating autoantibodiesreflects the disease activity [38,39] but the method is expensive, timeconsuming and not routinely available in all laboratories.
11. Target antigen-specific serological confirmatory tests by ELISAandWestern blotting
Enzyme-Linked Immuno Sorbent Assay (ELISA), immunoblotting orimmunoprecipitation are recently developed, highly sensitive andspecific detection systems of autoantibodies utilizing recombinantautoantigens or keratinocyte extracts from healthy human skin. Unlikeimmunoblotting, immunoprecipitation is performedwith native, ratherthan denatured, protein and is more sensitive. The latter assays, in addi-tion to serving as serological confirmatory tests (inmany cases used as adefinitive diagnostic step for autoimmune bullous dermatoses) are usedas a valuable tool for immunoserological follow-up. These above namedtests are able to detect IgG or IgA autoantibodies against desmoglein 1,desmoglein 3, BP180, BP230, tissue transglutaminase as well as againstenvoplakin and are available commercially. Immunopercipitation ismore difficult to perform and is generally less available than immuno-blotting (Diagram 1). Due to the increasing availability of the assays,the diagnostic spectrum of autoimmune bullous skin diseases hasbeen expanded considerably in recent years and has gained both in sen-sitivity as well as specificity (Table 1).
Diseases of the pemphigus group are characterized by the presenceof autoantibodies against desmosomal structure proteins (Diagram 1).Desmoglein 3 autoantibodies can be detected in PV with exclusive mu-cosal involvementwhereas in themucocutaneous variant of pemphigus
vulgaris both desmoglein 3 and desmoglein 1 autoantibodies can befound [37]. Autoantibody titre usually correlates with disease activity[2,3,16,33] and even though they are not widely available in routineclinical practice, are appropriate for follow-up monitoring. It has beenshown that autoantibodies of the IgG4 as well as IgG1 class are indica-tive of active rather than remittent disease [2]. Western blotting analy-ses are not useful in the diagnosis of pemphigus based on the fact thatmost of the epitopes on desmoglein 1 and 3, are conformational. Fur-thermore, acetylcholine receptors have been reported as autoantigensin pemphigus vulgaris but their pathogenetic relevance is unclear.
PF with its two subsets of endemic (fogoselvagem) and non-endemic forms is characterized by exclusive involvement of desmoglein1 autoantibodies, but not against desmoglein 3 or against plakins(Diagram 1). In PE antinuclear antibodies are present [36]. In mostcases of PNP IgG autoantibodies against desmoglein 3 and 1 are foundbut PNP is characterized by the detection of autoantibodies againstdesmosomal proteins of the plakin family. Envoplakin and periplakinare the most specific target antigens followed by desmoplakin 1 and 2,plectin, and a 170 kDa [17,32](Diagram 1). IgA pemphigus is character-ized by the detection of IgA autoantibodies against desmoglein 1 anddesmoglein 3 in the IEN type and against desmocollin 1 in the SPDtype [17,32] (Diagram 1). Bullous pemphigoid is characterized by auto-antibodies that target 2 hemidesmosomal structure proteins, BP180 andBP230. BP180 is a transmembrane glycoprotein of which the extracellu-lar portion–16th non-collageneous (NC16A)–was identified as theimmunodominant region in patients with BP (Diagram 1). Serum levelsof anti-BP180 NC16A antibodies can be detected using ELISA and it hasbeen shown that antibodies levels correlate with disease activity in BPpatients [40,41]. In several reports, ELISA has been demonstrated to behighly sensitive and specific. It should be taken into consideration thata considerable proportion of older patients with polymorphic pruriticskin diseases also show reactivity of IgG autoantibodies against BP230and BP180. Hence, the diagnosis of BP is not based solely on the detec-tion of IgG against BP230 and/or BP180 but also on the identificationof tissue-bound autoantibodies in DIF. ELISA test systems for the detec-tion of IgG against BP230 are available commercially. ELISAs based onrecombinant proteins encoded by BP230 and BP180 have been devel-oped but are not commercially available and offer an investigationaltools. In MMP, IgG autoantibodies are found against BP180; as well as,autoantibodies against laminin 332 (=laminin 5), α6 and β4- integrinand occasionally against BP230 (Diagram 1). Autoantibodies can bedetected using Western blotting, immunoprecipitation and ELISA. Ithas been reported that autoantibodies against laminin 332 can be asso-ciated with malignancy whereas autoantibodies against α6-intergrinwith oral involvement and against β4-integrin with ocular involvement[41]. The autoantigen of linear IgA dermatosis is a 120 kDa large extra-cellular product ectodomain of the BP180, named LAD-1, while in only20% of patients sera recognize BP180 NC16A [42,43] (Diagram 1).
EBA is characterized by autoantibodies directed against type VII col-lagen (Diagram1). Collagen VII is themain component of the anchoringfibrils of the dermoepidermal junction zone. Autoantibodies can bedetected by immunoblotting with extract of human dermis and inalmost all patients IgG reactivity against the NC1 domain of type VII col-lagen is detected [44]. Besides EBA collagen type VII autoantibodies canbe detected in bullous systemic lupus erythematosus.
Epidermal transglutaminase has been identified as the autoantigenof DH (Diagram 1). In gluten-sensitive persons after oral gluten exposi-tion, IgA autoantibodies develop in the epithelium of the small intestineagainst a complex consisting of gluten and tissue transglutaminase. Theautoantibodies cross-react with epidermal transglutaminase, whichparticipates as an isoenzyme in keratinocyte differentiation. Immunecomplexes of IgA and epidermal transglutaminase are found as diagnos-tic markers in the papillary dermis. ELISA systems are available fordetecting IgA reactivity against tissue transglutaminase in DH [45]. Fur-thermore, ELISA employing gliadin-analogous multimeric fusion pro-teins are available.
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