Update on the pathogenesis of bullous pemphigoid: An autoantibody-mediated blistering disease targeting collagen XVII

Journal of Dermatological Science 73 (2014) 179–186

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Invited review article

Update on the pathogenesis of bullous pemphigoid: An autoantibodymediated blistering disease targeting collagen XVII Wataru Nishie * Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 November 2013 Accepted 4 December 2013

Bullous pemphigoid (BP) is a common autoimmune blistering skin disorder that tends to affect the elderly. Autoantibodies (autoAbs) from BP patients react with two hemidesmosomal components: transmembrane collagen XVII (BP180 or BPAG2) and plakin family protein BP230 (BPAG1). Of these, collagen XVII (COL17) is thought to be a major autoantigen. The binding of autoAbs to COL17 following the activation of complements and inflammatory pathways eventually leads to the degradation of COL17, and this has been regarded as the main pathogenesis of BP. However, recent investigations have suggested other pathways, including a complement-independent pathway and a pathway involving IgEautoAbs. BP-autoAbs can directly deplete COL17, leading to fragility of the dermal–epidermal junction. In addition, IgE-autoAbs to COL17 may be involved in the formation of itchy urticarial erythema associated with eosinophilic infiltration. This article summarizes the update on pathogenesis of BP, with a special focus on blister formation by autoAbs to COL17. ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Complement activation Autoantibody IgE Model mice Protease

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical, histological and immunohistological features of BP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of BP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Clinical features of BP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Histopathological and immunological features of BP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. BP-autoAbs target two hemidesmosomal proteins: COL17 and BP230 . . . . . . . . . . . . . . . . . . . . . . . . . . COL17 is a major autoantigen for BP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juxtamembranous extracellular NC16A domain of COL17 contains major pathogenic epitopes. 4.1. Other epitopes on COL17, and epitope spreading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Complement-dependent inflammatory pathways in BP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Complement-independent pathways in BP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct roles of autoAbs to COL17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Roles of IgE autoAbs to COL17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Pathological cleavage and degradation of COL17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

* Correspondence to: Department of Dermatology, Hokkaido University Graduate School of Medicine, N15W7, Kita-Ku, Sapporo 060-8638, Japan. Tel.: +81 11 706 7387; fax: +81 11 706 7820. E-mail address: [email protected]

Bullous pemphigoid (BP) is the most common autoimmune blistering skin disorder, and it commonly develops in the elderly [1,2]. The autoantibodies (autoAbs) in BP patients target two hemidesmosomal components: transmembrane collagen XVII

0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.12.001

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(BP180 or BPAG2) and plakin family protein BP230 (BPAG1). Of these, collagen XVII (COL17) is thought to be a major autoantigen (autoAg) [2]. The BP pathomechanism is complicated, but binding of autoAbs to COL17 is essential for blister formation. In addition to inflammatory reactions that are known to be initiated by complement activation, recent investigations have suggested other pathways, including a complement-independent pathway and a pathway involving IgE-autoAbs. This article focuses on the pathogenesis of BP, with a special focus on blister formation by autoAbs to COL17 from clinical, histopathological, immunological and molecular observations.

2. Clinical, histological and immunohistological features of BP

BP is known to be unrelated to specific ethnic background, but a significant association with a major histocompatibility complex (MHC) class II loci (HLA-DRB1, DQB1) has been reported in Caucasian patients [5,6]. Although, it is uncertain how a single human leukocyte antigen (HLA) allele may be associated with BP onset. 2.2. Clinical features of BP In typical cases of BP, urticarial erythema with tense blisters on the trunk and extremities associated with moderate to severe pruritus is commonly observed (Fig. 1A). Mucosal involvement is not so common, but 10–20% of patients have oral lesions [2,7]. In some patients, eczema-like erythema may proceed for months or even for many years as a prodromal phase before BP develops.

2.1. Epidemiology of BP 2.3. Histopathological and immunological features of BP BP tends to develop in the elderly of both sexes around the seventh or eighth decade of life. Younger than age 50 rarely develop BP, but it has been reported in small numbers of infants, children and adolescents [2]. Retrospective studies in European countries have found BP to have a prevalence of around 6.6:1,000,000 per year, but recent analyses have shown an increased prevalence of 21.7–66:1,000,000 [2]. This increase is probably due to demographic aging and the development of diagnostic tools such as ELISA assays that can easily detect circulating autoAbs to COL17. Interestingly, patients with a history of neurological diseases, such as cerebrovascular diseases, have a significantly increased prevalence of BP [3]. The precise pathomechanism of patients with neurological diseases having an elevated risk for developing BP remains uncertain, but it should be noted that two autoantigens for BP, COL17 and BP230, are both expressed in the central nervous system [4].

Histologically, the formation of subepidermal blisters with the accumulation of eosinophils is characteristic of BP [2,8] (Fig. 1B). In perilesional skin of blisters, interface dermatitis with eosinophils, and sometimes histological subepidermal blisters, is commonly observed. Therefore, even without distinct blister formation, interface dermatitis with moderate to severe infiltration of eosinophils suggests BP as a histopathological differential diagnosis. Immunologically, the IgG autoAbs from BP patients and C3 bind the dermal–epidermal junction (DEJ) of the skin, which can be detected by direct immunofluorescence (DIF) of cryoskin sections obtained from perilesional skin (Fig. 1C). DIF is the most sensitive investigation, and more than 90% of tested skin shows linear deposition of IgG at the DEJ [9]. To identify circulating autoAbs to the DEJ, indirect immunofluorescence (IIF) with normal human skin as the substrate is usually examined. Since BP230 and COL17

Fig. 1. A typical case of BP with urticarial erythema and tense blisters on the trunk and extremities (A). Histopathology shows subepidermal blister with eosinophilic infiltration (B). DIF on perilesional skin shows deposits of IgG (arrows) and C3 at the DEJ (C). IIF can detect circulating autoAbs, which usually react with the epidermal side (arrowheads) of 1 M NaCl split skin (D). Star: blister.

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are on the epidermal side of the artificial blisters that can be induced by incubating the skin specimen with 1 M NaCl solution, autoAbs from BP patients are known to react with the epidermal side of the blisters (Fig. 1D). In contrast, autoAbs from other autoimmune blistering diseases, including epidermolysis bullosa acquisita and anti-laminin g1 pemphigoid, react with the dermal side of the artificial blisters [2]. Thus, salt-split skin IIF is useful for differentiating between BP and other autoimmune blistering disorders with similar clinical features. 3. BP-autoAbs target two hemidesmosomal proteins: COL17 and BP230 In skin, the epidermis and the dermis are tightly attached by complex structures called hemidesmosomes (HDs) [10]. The HDs are composed of many molecules, including BP230, plectin, a6 and b4 integrins, COL17 and tetraspanin protein CD151. The hemidesmosomal complex interacts with keratin 5/14 in the cytoplasm, and with laminin 332, collagen IV and collagen VII in the extracellular matrix (ECM) (Fig. 2). Of these, COL17 and BP230 are major autoantigens for BP-autoAbs [1,11,12]. The vital biological functions of these molecules have been indirectly demonstrated by the congenital blistering skin disease epidermolysis bullosa, which develops as a consequence of mutations in the genes encoding BP230 [13] or COL17 [14]. In BP, autoAbs preferentially target the two hemidesmosomal molecules COL17 and BP230 [2,15]. In addition, autoreactive T and B cells have been demonstrated to recognize COL17 and BP230 [16,17], suggesting that autoimmunity to these molecules depends on autoreactive T cells that promote the activation of autoreactive B cells. However, BP230 is an intracytoplasmic plakin family protein, in contrast to the transmembrane protein COL17, whose extracellular domain is in the ECM. It is still uncertain whether IgG autoAbs from BP patients can access intracytoplasmic molecules and play pathologic roles for blister formation in BP, although one study has shown the pathogenicity of rabbit IgG Abs targeting BP230 by passive transfer experiments in mice [18]. 4. COL17 is a major autoantigen for BP 4.1. Juxtamembranous extracellular NC16A domain of COL17 contains major pathogenic epitopes COL17 is a 1497-amino acid transmembrane collagen with a type-II orientation. Its amino terminus (N-terminus) and carboxyl

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terminus (C-terminus) are located in the cytoplasm and in the ECM, respectively [14,19]. As described in Fig. 2, the extracellular domain of COL17 interacts with laminin 332 [20,21] and collagen IV [21] in the ECM, and the C-terminal end of COL17 has a flexible tail in vivo [22]. Similar to other transmembrane collagen super families, the extracellular domain of COL17 is constitutively cleaved (shed) from the cell surface by ADAM 9/10/17 [23,24], a process that occurs within the juxtamembranous extracellular non-collagenous NC16A domain [25,26] (Fig. 3). Interestingly, the majority of IgG autoAbs from BP patients react with this NC16 domain, which consists of 77 amino acids, and epitopes cluster tightly in the 45-amino-acid N-terminal stretch of this region [27]. Not only does IgG mainly react with this region, but so do IgE autoAbs [28]. These findings indicate that autoAbs to the NC16A domain are pathogenic for blister formation in BP; however, autoAbs passively transferred into wild-type mice fail to bind with the dermal–epidermal junctions of mice skin and to induce blistering disease [29]. This was thought to be due to significant differences in the amino acid sequences of the NC16A domain (NC14A in the mouse COL17) (Fig. 3D). To address the pathogenicity of BP-autoAbs to the NC16A domain, transgenic (Tg) mice were established, in which the COL17 has been completely [30] or partially [31] humanized. By injecting IgG-autoAbs from BP patients into these mice that express human COL17 autoantigen (COL17-humanized mice), the direct pathogenicity of the autoAbs from BP patients was proven [30,31] (Fig. 4). The reason epitopes are evoked within the NC16A domain remains uncertain, but it is evident that autoimmunity to the NC16A domain is an initial and crucial event. When wild-type mice were exposed to native human COL17 by skin grafting with syngeneic human-COL17-expressing Tg mice skin, they started to produce IgG Abs to human COL17 [32]. Initially, epitopes of the IgG Abs were directed against the extracellular domain of COL17, including the NC16A domain, and later, Abs to other parts including intracytoplasmic regions appeared [32,33]. Also in BP patients, autoAbs to the NC16A domain are usually observed from the early stage of the disease, whose titer correlates closely with disease severity and activity [34,35]. These findings suggest that breaking the immune tolerance to the NC16A domain of COL17 may be the vital and initiating event in the development of BP. In addition, cleavage of the extracellular domain of COL17 may relate to the production of autoAbs. We previously reported that the antigenicities of the NC16A domain change significantly before and after the cleavages within NC16A, which generates new

Fig. 2. Schematic of the molecular composition of HDs.

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Fig. 3. The COL17 ectodomain is cleaved within the NC16A domain by ADAMs (A). Cleaved 120 kDa ectodomain is found in the culture medium, detected by Abs to the NC16A domain (B). Amino acid sequences of the NC16A domain and physiological cleavage sites (arrowheads) (C). The immunological N-terminal 45-amino-acid sequence of the NC16A domain of human and mouse (D).

epitopes (neoepitopes) on the cleaved polypeptides [26]. This finding may answer the mystery of why IgA autoAbs from patients with linear IgA bullous dermatosis (LAD), another autoimmune blistering skin disease related with IgA-class autoAbs targeting COL17, preferentially react with the shed ectodomain but not with full-length COL17. Previous studies have shown that IgA autoAbs from certain LAD patients react with the NC16A domain [36]; thus, neoepitopes on the NC16A domain that appeared after the cleavage of COL17 may be involved in the pathogenesis of LAD. However, epitopes of the IgA autoAbs from some LAD patients were mapped on the 15th collagenous domain, which is immediately downstream of the NC16A region [37]. Therefore, it is likely that significant epitope changes may occur not only within the NC16A domain but also in other regions after the cleavage of COL17. 4.2. Other epitopes on COL17, and epitope spreading In addition to the NC16A domain, BP-autoAbs react with other regions on COL17. It has been reported that more than 90% of BP patients have IgG autoAbs that react with epitopes on the extracellular domain of COL17 other than the NC16A domain [35]. In addition, IgG autoAbs to the intracytoplasmic domain have been reported to be present in around 40–60% of BP patients [35,38]. This phenomenon can be explained by ‘‘intramolecular epitope spreading’’; however, it has been unclear whether this phenomenon is associated with disease progression or prognosis. In 2011, Di Zenzo et al. showed that intramolecular epitope spreading on COL17 is likely to occur from the early stage of the disease, within 3 months after the diagnosis, and that the epitope spreading is found not only within the extracellular domain but also in the intracytoplasmic regions [17]. Importantly, epitope spreading was found to significantly relate to the clinical activity and severity of the disease [17]. In addition to ‘‘intramolecular epitope spreading’’, ‘‘intermolecular epitope spreading’’ can be observed in some BP patients. Usually, IgG autoAbs to COL17 are first found, and then autoAbs to

BP230 start to appear [17]. Of note, it has been reported that IgG reactivity to BP230 appears to be related to the severity of BP [17], although the idea of pathogenic roles of Abs to BP230 is still controversial, as previously mentioned. 5. Complement-dependent inflammatory pathways in BP Activated complements are commonly found in the perilesional skin of BP, which can be observed by DIF (Fig. 1). Interestingly, extensive in vivo deposition of complement at the DEJ is known to be a characteristic of herpes gestationis, a BP-related autoimmune blistering disorder commonly observed in pregnant patients. IgG autoAbs in herpes gestationis patients are known to target the NC16A domain of COL17 [39]. Thus, the activation of complements seems to play a central role in this disorder. In BP, the pathologic relevance of complement activation and the following inflammatory pathways has been extensively studied by using a mouse model that was produced by injecting rabbit Abs directed against the NC14A domain of mouse COL17 (corresponding to the NC16A domain of human COL17) [29]. When rabbit Abs directed against mouse COL17 were passively transferred into wild-type neonatal mice, it was reported that the rabbit Abs bound to the DEJ of the mice skin, following after activation of complements, mast cell degradation, recruitment of neutrophils, and finally epidermal detachment by mild friction was induced. This phenomenon was not reproducible in complement (C) 5-null mice, suggesting that complement activation is essential in this model [40]. In addition, the vital role of neutrophils in lesional skin was proved by the prevention of blistering disease by intraperitoneal injection of IL-8, which sequesters neutrophils in the peritoneal cavity [41]. The infiltration of neutrophils is dependent on the degradation of mast cells, which is induced by the activated complement, and blistering disease is not reproduced in mast cell-deficient mice [42]. Regarding the different pathways of activating complements, both the classical pathways and alternative pathways have been

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Fig. 4. COL17-humanized mice were produced by introducing human COL17A1 cDNA transgene into Col17-null mice with a blistering phenotype and hair abnormalities (A). The COL17-humanized mice were phenotypically normal, and the skin expressed only human COL17 and not mouse COL17 (B). By injecting IgG autoAbs from BP patients into the neonatal COL17-humanized mice, epidermal detachment by gentle friction and histological blister formation associated with the deposition of human IgG (arrows) and activated mouse complements (arrowheads) were induced (C).

thought to be involved, since blistering disease was totally or partially mitigated in mice targeted with C4-null and alternative pathway component factor B, respectively [43]. These findings suggest that the blistering disease in this experimental BP model mice is induced by anti-mouse COL17 IgG which activates complements following mast cell degradation and neutrophil infiltration leading to degradation of COL17 (Fig. 5A). Consistent with these observations, Wang et al. have shown that recombinant Fab fragments of human IgG autoAbs which react with the NC16A domain of COL17 are not pathogenic on humanCOL17-expressing Tg (COL17-humanized) mice. The Fab-IgG lacks the Fc portion, which is essential for complement activation [44], indicating important roles for the Fc portion in blister formation. In addition, Li et al. showed that recombinant IgG1, but not IgG4 human monoclonal Abs to the NC16A domain of COL17, could

induce blistering disease in COL17-humanized Tg mice [45], arguing for a role in complement activation in BP model mice. 6. Complement-independent pathways in BP 6.1. Direct roles of autoAbs to COL17 Histologically, BP lesional skin can show scant infiltrates of inflammatory cells with blister formation, which is known as the ‘‘cell-poor’’ type. Immunologically, IgG4-class autoAbs are commonly observed in BP patients, which cannot activate complements. It has been shown that IgG4 autoAbs from BP patients can induce dermal–epidermal separation in in vitro cryosection assays [46]. In addition, a BP patient with C4 deficiency has been reported [47], although C4-deficient mice were resistant to experimentally

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Fig. 5. Complement-dependent (A) and independent (B) pathways for blister formation in BP. Neonatal BP model mice show that binding of rabbit IgG with COL17 activates complements following mast cell degradation, and neutrophilic and eosinophilic infiltration, which leads to the degradation of COL17. Uncontrolled neutrophil elastase cleaves COL17, which was induced by reduced a1-proteinase. The reduced a1-proteinase was induced by activated-MMP-9 which had been catalyzed by plasmin (A). In a complement-independent manner, COL17 may be depleted by internalization after binding with IgG autoAbs from BP patients (B).

induced BP [43]. These findings suggest that a complementindependent pathomechanism may exist in blister formation in BP patients which is different from those of previous murine models. Supporting this notion, IgG autoAbs from BP patients are known to induce the secretion of IL-8 in cultured normal human epidermal keratinocytes (NHEKs) in vitro, which is a chemotactic cytokine that recruits neutrophils [48]. These observations indicate that IgG autoAb-dependent neutrophil-mediated tissue injury, probably evoked through the Fc portion of the autoAbs, may play an important role in BP blister formation [2]. In addition to identifying the roles of complement-independent neutrophils, recent investigations have made new insights into the pathomechanism of BP. In 2009, Iwata et al. showed for the first time that IgG autoAbs from BP patients reduce the expression of COL17 in cultured NHEKs and that the amount of COL17 in BP perilesional skin is actually reduced [49]. Using an in vivo mouse model, Natsuga et al. revealed in 2012 that mechanical blistering can be induced in the COL17-humanized mice in a complement-independent

manner [50]. The detailed pathomechanism for the depletion of COL17 by Abs to COL17 is still undisclosed, but internalization of immune complex into the cytoplasm is the initial event. Dynamic internalization of IgG and COL17 from the cell surface into the cytoplasm has recently been revealed. Soon after IgG autoAbs from BP patients were added to a culture medium of NHEKs, labeled-IgG autoAbs from BP patients were internalized through a macropinocytic pathway [51]. Further studies will unveil the detailed pathogenesis of complement-independent blister formation in BP (Fig. 5B). 6.2. Roles of IgE autoAbs to COL17 Itchy urticarial erythema, eosinophilic infiltration in the papillary dermis and eosinophilia are commonly observed in the majority of patients with BP. However, these characteristic features are usually lacking in BP model mice that have been administered with IgG (auto) Abs to COL17 [29,30]. In BP patients,

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serum IgE is sometimes elevated, and IgE-class autoAbs reacting with the NC16A domain of COL17 have been demonstrated [28]. In addition, the deposition of IgE autoAbs along the DEJ was observed in certain numbers (41%) of BP patients [52]. These observations indicate that IgE autoAbs may be involved in the pathogenesis of BP. Fairley et al. showed in 2007 that IgE autoAbs from BP patients can induce urticarial erythema and microscopic separation at the DEJ associated with neutrophilic and eosinophilic infiltration in human skin grafted onto nude mice [53]. Furthermore, recent studies have revealed that IgE autoAbs from BP patients can be internalized into cultured NHEKs, where they stimulate IL-8 production and lead to the depletion of hemidesmosomes as observed by BP IgG autoAbs induced by Fc receptors in an independent manner [54]. These studies suggest that IgE is likely to be involved in the pathogenesis of BP and that IgE-class autoAbs will be a focus of future studies. 7. Pathological cleavage and degradation of COL17 In BP lesional skin and blister fluid, several proteolytic enzymes are known to exist, including plasmin, neutrophil elastase and MMP-9. Passive transfer of rabbit Abs reacting with mouse COL17 failed to induce skin fragility in neutrophil elastase-null [55] and MMP-9-deficient mice [56,57]. In addition, only less severe disease was observed in plasminogen-deficient mice [57]. These results suggest that these proteases play vital roles in blister formation in this BP model. Of note, in the plasminogen-deficient mice, the blistering disease was reconstituted by the administration of activated MMP-9 or plasminogen, but not by the proenzyme form of MMP-9. In contrast, in the MMP-9-deficient mice, the blistering disease was reconstituted by the proenzyme form of MMP9 but not by plasminogen. Thus, the plasminogen/plasmin system is thought to be epistatic to the activation of MMP-9 [57], which catalyzes a major suppressor of neutrophil elastase secretion, a1-proteinase inhibitor [58]. These findings suggest that, in the rabbit-IgGinduced BP model mice, released neutrophil elastase is primarily involved, and the induction results from reduced a1-proteinase inhibitor [55,57] catalyzed by plasmin activated-MMP-9 (Fig. 5). However, a direct role of plasmin in the COL17 cleavage of lesional skin has not been completely ruled out. In vitro experiments have revealed that neutrophil elastase [59], MMP-9 [60] and plasmin [61] are able to cleave COL17; however, the molecular consequence of COL17 in the in vivo lesional skin of BP patients is unclear. Recently, the 120 kDa ectodomain of COL17 was found to be present in blister fluid of BP patients, and this ectodomain can be immunoprecipitated by Abs to the NC16A domain of COL17 [26,61]. This finding is notable because the presence of the cleaved ectodomain of COL17 in blister fluid suggests that the protease or proteases in BP lesional skin do not target the collagenous domains of COL17 in vivo, as previously suggested by MMP9 in vitro [60]. In line with this, previous studies reported that blister fluids of BP patients mainly contain the proenzyme form of MMP-9, and not its activated form [62]. Furthermore, we have disclosed that COL17 may be cleaved at different sites within the NC16A domain from those of physiological cleavage by blister fluid of BP [26], suggesting that nonphysiological proteases are involved in the pathological cleavage of COL17 in BP lesional skin. Future studies which dissect the pathomechanism of COL17 cleavage and the responsible proteases in lesional skin may lead to novel therapeutics that target specific proteases in BP. 8. Conclusions BP has been regarded as a well-characterized, organ-specific, autoAb-mediated blistering skin disorder in which complement

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activation is essential. However, recent investigations have revealed BP to be a much more complicated disorder. In addition to the complement-dependent inflammatory pathway, other unproved pathomechanisms must be involved in the pathogenesis of BP.

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