Autoimmunity of the dermal-epidermal junction

ARTICLE

The human dermal-epidermal junction (DEJ) is at the same time the target of a group of autoimmune blistering diseases collectively called subepidermal blistering diseases and of a group of heritable blistering disorders named inherited epidermolysis bullosa (Fig. 1 and Table 1) [1-5]. The clinical findings shared by these diseases demonstrate that the main function of the DEJ is to insure adherence of the epidermis to the underlying dermis. Within the framework of this common function, many distinct, yet interconnected protein components of the DEJ have been identified in the past few years. Interestingly, some of the DEJ constituents were discovered because they represented the autoantigens of autoimmune diseases. The isolation of these autoantigens has broadened our knowledge of the molecular organization of the DEJ. The term inherited epidermolysis bullosa (EB) encompasses a group of disorders which are clinically characterized by blister formation as the result of varying degrees of trauma and due to genetic mutations resulting in the defective production of various DEJ proteins [4, 5]. The identification of the specific DEJ proteins altered in the different clinical forms of EB has facilitated our understanding of the mechanisms underlying blister formation in these diseases and of the functional role of the same proteins.

Well-characterized autoimmune subepidermal blistering diseases include bullous pemphigoid (BP), herpes gestationis (also named pemphigoid gestationis, PG), cicatricial pemphigoid (CP), linear IgA bullous dermatosis (LABD), epidermolysis bullosa acquisita (EBA), and bullous eruption of systemic lupus erythematosus (BSLE) [1-3]. Autoantibodies targeting different skin and mucosal basement membrane zone components result in slightly different clinical pictures and in the histopathological findings common to all subepidermal blistering diseases, that is a blister which occurs just below the epidermis/epithelium, usually with a mixed inflammatory infiltrate. The hallmark of this disease group is the linear deposition of Ig, mainly IgG, and complement components along the cutaneous/mucosal basement membrane zone, as detected by direct immunofluorescence microscopy of perilesional skin. Differential diagnosis of various autoimmune subepidermal blistering diseases can be obtained using a simple and practical method of direct or indirect immunofluorescence testing on frozen skin sections separated within the lamina lucida by treatment with 1 mol/l NaCl (salt-split skin), in combination with direct and indirect immunoelectron microscopy and target antigen determination using immunoblot or immunoprecipitation studies [6].

Dermal-epidermal junction structure and function

The DEJ can be divided in four ultrastructurally distinct areas: the hemidesmosome/upper lamina lucida, the lower lamina lucida, the lamina densa, and the anchoring fibril-containing sub-lamina densa. The hemidesmosomes (HD) and the anchoring fibrils (AF) are the two highly specialized attachment structures characteristic of the basement membrane zone of the skin and other stratified epithelia [7]. HD appear as small, electron dense domains of the plasma membrane on the basal surface of basal keratinocytes [5, 7, 8]. Their most conspicuous component is a bipartite cytoplasmic plaque, to which the bundles of keratin intermediate filaments are attached. HD are associated with a sub-basal dense plate in the upper lamina lucida and are connected via thread-like anchoring filaments to the lamina densa. In its turn, the latter seems to be anchored to the underlying papillary dermis by the cross-banded anchoring fibrils. These various morphological structures, i.e. intermediate filaments, HD, anchoring filaments and AF, constitute a functional unit that provides stable adherence of keratinocytes to the underlying mesenchyme.

The molecular organization of HD is based on three classes of proteins: the cytoplasmic plaque proteins acting as linkers for elements of the cytoskeleton at the cytoplasmic side of the plasma membrane, the transmembrane proteins serving as cell receptors connecting the cell interior to the extracellular matrix, and, finally, the basement membrane-associated proteins of the extracellular matrix.

The HD cytoplasmic plaque components include the 230 kDa bullous pemphigoid antigen (BP230, also named bullous pemphigoid antigen 1, BPAG1), plectin and other less well characterized proteins [8]. BPAG1 and plectin belong to the plakin family of proteins implicated in the organization of the cytoskeletal architecture, in particular by linking the keratin intermediate filaments to the cell surface and specifically to desmosomes and hemidesmosomes [9]. Other members of this family include the desmosomal proteins desmoplakin I and II, periplakin and envoplakin [9, 10].

BPAG1 was first recognized as a target antigen in the autoimmune blistering disorder of the skin called bullous pemphigoid (see below). BPAG1 is involved in the anchorage of keratin intermediate filaments to the plasma membrane: HD of BPAG1 null-mutant mice lack the inner cytoplasmic plaque and the connection of intermediate filaments with HD is severely impaired [11]. In addition, in vitro studies have provided evidence that BPAG1 interacts with the cytoplasmic domain of the 180-kDa bullous pemphigoid antigen [12] and probably also of the ß4 integrin subunit [13], the two transmembrane constituents of HD.

Plectin is a large protein expressed in various cell types which acts as a multifunctional cytoskeletal linker [14]. In epithelial cells, plectin is involved both in the attachment of keratin IF to the plasma membrane and in interactions with other hemidesmosomal components, in particular the ß4 integrin subunit [15]. Mutations in plectin gene in humans result in a variant of EB, EB simplex with muscular dystrophy, characterized by reduced keratin filament attachment to HD which almost completely lack the inner cytoplasmic plaque [4, 5]. In addition, knock-out of plectin gene in mice leads to a phenotype similar to the human disease [16].

The transmembrane constituents of HD include the alpha6ß4 integrin and the 180-kDa bullous pemphigoid antigen (BP180, also termed bullous pemphigoid antigen 2 ­ BPAG2 ­ or type XVII collagen). The long cytoplasmic tail of the ß4 integrin subunit contains binding sites for plectin [15] and BPAG2 [13]. The extracellular domain of alpha6ß4 is crucial for cell adhesion. Antibodies directed against the alpha6ß4 integrin prevent the assembly of HD and induce dermal-epidermal separation [8]. In addition, mutations in humans in the alpha6 and ß4 integrin genes [17, 18] or targeted disruption of the same genes in mice [19-21] result in severe phenotypes with HD impairment and extensive blistering of the skin and mucous membranes of the digestive, respiratory and genito-urinary tracts. The human disease is a variant of junctional EB (JEB), named JEB with pyloric atresia [17, 18]. The alpha6ß4 integrin is a receptor for various laminin variants, but it binds with high affinity to laminin 5, a laminin isoform selectively expressed in epithelia [8]. In addition, a growing body of evidence indicates that alpha6ß4 is implicated in transducing signals from the ECM to the cell interior that do not only control the assembly of HD and cytoskeleton organization but are also involved in the regulation of cell proliferation and differentiation [22, 23].

BPAG2 is a collagenous molecule with a type II membrane orientation [24]. The intracellular NH2-terminal domain of BPAG2 interacts with the ß4 integrin [13] and the incorporation of BPAG2 into HD is probably stabilized by additional interactions with BPAG1 and plectin. The extracellular COOH-terminal portion of BPAG2 contains 15 collagenous triple helical domains (C1 through C15) interrupted by non-collagenous sequences (NC1, located at the C-terminus, through NC16A, immediately adjacent to the membrane-spanning domain) that form collagen triple helices [7, 24]. The structure of BPAG2, i.e. its large extracellular collagenous domain, implies a role of this protein in epithelial-stromal adhesion. This idea is supported by the observation that acquired (see below) or congenital defects of BPAG2 expression are associated with impairment of dermal-epidermal cohesion. In particular, mutations in the gene, COL17A1, encoding BPAG2, result in a variant of JEB, named generalized atrophic benign epidermolysis bullosa, characterized by blisters predominantly localized to the skin and by abnormal HD [25]. Finally, evidence has been recently provided that the extracellular domain of BPAG2 undergoes proteolytic processing resulting in the formation of a 120 kDa fragment that is incorporated into the basement membrane [26, 27]. The biological significance of this processing remains unclear.

The lower lamina lucida and the lamina densa regions of the basement membrane zone are formed by a scaffolding of two network polymers consisting of laminin isoforms and type IV collagen, in which diverse glycoproteins such as nidogen, perlecan and fibulins, act as stabilizing bridges [28]. Laminin 5 represents a conspicuous component of this network in the basement membrane zone of epithelial tissues. This laminin isoform consists of three chains, alpha3, ß3 and gamma2, two of which (alpha3 and gamma2) undergo extracellular processing [28]. Laminin 5 supports epithelial cell binding and spreading and is the major ligand for the alpha6ß4 integrin. This interaction is crucial for the maintenance of stable epithelial adhesion, as inferred from clinical observations and cell biological studies. Keratinocytes with a defective expression of laminin 5 have reduced adhesive properties and the assembly of hemidesmosomes is severely impaired, as shown in the Herlitz (lethal) variant of JEB [4, 5, 29]. However, upon re-expression of laminin 5, the keratinocyte normal phenotype and ability to form hemidesmosomes is restored [30]. Laminin 5 interacts with the NC-1 domain of collagen VII, the major constituent of anchoring fibrils [31]. Hence, laminin 5 serves as a bridge between the alpha6ß4 integrin and components of the dermal matrix.

Anchoring fibrils (AF) are cross-banded fibrillar structures that extend from the lamina densa into the papillary dermis. The main structural component of AF is type VII collagen [28]. This molecule is synthesized as a procollagen form comprising a large N-terminal globular domain
(NC-1), a central triple alpha-helical collagenous domain, and a small C-terminal globular propeptide (NC-2). After secretion, procollagen VII monomers form antiparallel dimers with overlapping NC-2 domains which are then proteolytically processed. Antiparallel dimers aggregate laterally to form anchoring fibrils in which the central collagenous domain contributes to the cross-banded region. The NC-1 domain mediates attachment to the basement membrane and interacts with laminin 5 [31, 32]. The role of type VII collagen in maintaining epidermal-dermal adhesion is illustrated by epidermolysis bullosa acquisita (EBA), an acquired autoimmune bullous skin disease characterized by circulating and tissue-bound autoantibodies against type VII collagen (see below), and by inherited dystrophic EB (DEB) which results from mutations in the type VII collagen gene [4, 5].

Bullous pemphigoid (BP)

BP is the most frequent immune mediated blistering disease of the skin and predominantly affects the elderly population [1-3, 33]. BP is clinically characterized by generalized, tense blisters that occur on healthy skin or on an erythematous base and, in a few cases, by blisters or erosions of the mucous membranes. The histological features of BP include a subepidermal blister with an inflammatory infiltrate that often is rich in eosinophils but may also contain lymphocytes, histiocytes, or neutrophils; the typical immunopathological finding is a linear deposition of IgG and C3 at the DEJ. A hallmark of BP is the presence of circulating autoantibodies directed against BPAG1 and BPAG2 molecules [34]. The BPAG1 was first identified in 1981 by Stanley and colleagues as the major antigenic target of BP autoantibodies [35] and subsequent studies confirmed that 50-90% of BP sera contain autoantibodies able to immunoprecipitate or immunoblot the BPAG1. The BPAG2 antigen is also recognized by a high percentage of BP sera. The autoantibody production in BP is polyclonal and most antibodies are of the IgG4 subclass even though IgG1 are also present [36, 37].

In the past ten years, cloning of the BPAG1 and BPAG2 cDNAs has enabled epitope mapping studies in BP. The major antigenic epitopes of BPAG1 appear to map within the COOH-terminal end of the protein [38]. However, the pathological role of anti-BPAG1 antibodies still remains unknown. As to BPAG2, epitope-mapping studies have at first identified a 14 amino acid stretch (designated MCW-1) within the NC16A region as an immunodominant epitope recognized by the majority of BP sera, as well as by sera of patients affected with the closely related, pregnancy-associated disease herpes gestationis (also named pemphigoid gestationis, PG) [39, 40]. In addition to the MCW-1 epitope, a set of epitopes tightly clustered in the N-terminal 45 amino acid stretch of NC16A has been subsequently identified [41]. On the basis of these findings, it has been suggested that the reactivity to BPAG2 in BP and PG patients is largely restricted to this region.

However, recent studies have shown that some BP sera react with the COOH-terminal region of BPAG2, explaining the previous observation that the preabsorption of BP sera with the NC16A region does not always completely abolish their reactivity against the extracellular domain of BPAG2 [42, 43]. Finally, BPAG2-reactive sera containing IgG autoantibodies that bind to the intracellular domain of BPAG2 can also be found [43]. The identification of different regions of autoantibody reactivity on BPAG2 is not surprising since it is well-known that during the course of an autoimmune disease B and T cell responses are not restricted to a unique "immunodominant" epitope, but additional "secondary" epitopes within the same protein are recognized [44, 45]. This phenomenon termed "epitope spreading" appears to have importance for the perpetuation and progression of the disease. The observation that in BP two distinct molecules, BPAG1 and BPAG2, are targeted, on which multiple epitopes are recognized, suggests that epitope spreading also occurs in this disorder.

As to the pathogenetic role of BP autoantibodies, the repeated failure to induce disease by passive transfer of BP IgG into neonatal mice has been subsequently explained by sequence divergence in the region of the MCW-1 epitope between human and murine BPAG2 molecules which is responsible for the lack of cross-reactivity between BPAG2 antibodies recognizing human MCW-1 and the murine protein [46]. To circumvent this problem, rabbit antibodies were raised against the murine BPAG2 region homologous to the human MCW-1 epitope; when these antibodies were tested by passive transfer experiments in neonatal mice they induced a blistering disorder closely mimicking BP [46]. A difference between this animal model and the human disease is in the inflammatory infiltrate which in mice is predominantly composed of neutrophils and almost completely lacking in eosinophils, typically present in large numbers in human BP lesions. Nevertheless, these results supported the pathogenetic role of autoantibodies against the NC16A region of the extracellular domain of BPAG2 [47]. Subsequent studies aimed at further characterizing the immunopathogenic mechanism operating in this animal model of BP have demonstrated that the triggering of subepidermal blisters by anti-BPAG2 antibodies is dependent on complement activation and subsequent neutrophil recruitment to the skin via a C5-dependent pathway [48, 49]. Finally, it has been shown that gelatinase B-deficient mice injected with rabbit anti-BPAG2 antibodies do not develop subepidermal blistering although they show antibody deposition at the DEJ and neutrophil infiltration thus implicating neutrophil-derived gelatinase-B in the pathogenesis of experimental BP [50]. It can be concluded that the binding of anti-BPAG2 antibodies to their target site in the DEJ initiates a cascade of inflammatory events that leads to subepidermal blister formation through complement activation, neutrophil recruitment, and proteolytic enzyme release. In order to apply this pathogenetic model to human BP several issues still need to be clarified, in particular those concerning the role of eosinophils, which appear to be recruited and undergo degranulation prior to blister formation in human lesional BP skin [51]. These data have also led to the hypothesis that, while autoantibodies directed against the extracellular NC16A domain of BPAG2 have an initiatory role in the development of BP, antibodies to cytoplasmic antigenic determinants arise as a secondary event. However, experimental findings supporting this hypothesis are missing.

Autoreactive T cells may provide help to B cells to produce autoantibodies that are critical in the pathogenesis of several organ-specific autoimmune diseases, such as myastenia gravis, autoimmune thyroiditis and pemphigus vulgaris [52-54]. According to a recent study [55], most BP patients have autoreactive T cells to the extracellular domain of BPAG2 and the majority of these BPAG2-reactive BP patients carry the DQB1*0301 allele which has been reported to be prevalent in BP [56]. However, autoreactive T cell responses to BPAG2 can also be detected in healthy individuals carrying the DQB1*0301 allele [55], in keeping with previous findings that autoreactive T cell clones specific for the autoantigen of other autoimmune diseases, such as pemphigus vulgaris [53] or myasthenia gravis [57], are present in healthy individuals carrying the disease-associated HLA class II allele. Autoreactive T cell lines from BP patients have been found to secrete Th1 and Th2 cytokines suggesting that both autoreactive Th1 and Th2 cells may be involved in the regulation of the production of pathogenic autoantibodies by B cells in BP [55]. The predominance of IgG4 antibodies [36, 37] and the presence of IgE antibodies [58] in the sera of BP patients might be related to autoreactive IL-4 and IL-13 producing Th2 cells, while the presence of complement-binding IgG1 in BP sera may be associated to IFN-gamma producing Th1 cells. Finally, autoreactive Th2 cells may be critically involved in the initiation of the production of pathogenetic autoantibodies in BP since BPAG2-reactive healthy donors exhibited only Th1 responses to BPAG2 [55].

Cicatricial pemphigoid (CP)

Among autoimmune subepidermal blistering diseases, CP is clinically characterized by mucous membrane involvement that often leads to scar formation [2, 3]. CP typically affects the oral or ocular mucosa, but lesions of the nasal, pharyngeal, laryngeal, esophageal, or anogenital regions can occur. Skin involvement, present in 10-25% of patients, is usually a minor component of the disease. Despite the common immunopathological finding of linear deposition of IgG and C3 at the DEJ, CP is now recognized to be an immunologically heterogeneous group of diseases with similar clinical phenotypes. Several subsets of patients with characteristic antibody systems have been identified. The majority of CP patient sera recognizes the BPAG2 antigen, and recent data indicate, in addition to the NC16A domain, a region nearer to the COOH-terminus as more frequently targeted by circulating IgG autoantibodies [59, 60]. Fewer patients have antibodies to the BPAG1 [60]. A small, about 5%, but well-characterized subset of patients have IgG antibodies directed against laminin 5, as shown by immunoprecipitation and immunoblot studies [61, 62]. These antibodies bind to the dermal side of salt split skin. In particular, the large majority of anti-laminin 5 CP sera recognize the alpha3 chain alone [62]. In vitro data showing that monoclonal anti-laminin 5 antibodies block keratinocyte adhesion and produce epithelial detachment in organotypic skin cultures have suggested a possible pathogenetic role of anti-laminin 5 antibodies in CP-affected patients [63]. An animal model has then been developed in which passive transfer of purified rabbit anti-laminin 5 IgG (human anti-laminin 5 antibodies do not bind murine DEJ) to neonatal mice induces non-inflammatory subepithelial blisters of skin and mucous membranes in a concentration-related fashion [64]. Moreover, the same lesions developed in C5- or mast cell-deficient mice, indicating that anti-laminin 5 antibodies elicit blister formation in this experimental model directly and independently of an inflammatory cascade based on complement activation or mast cell degranulation [64].

Among the remaining antigenic molecules possibly recognized by CP sera, the cytoplasmic domain of the ß4 integrin subunit has been reported to represent the target antigen in patients affected with ocular CP, a clinical subset of CP with prominent ocular involvement [65]. A further putative CP mucosal antigen of 168 kDa (M168) has been identified using oral mucosa protein extracts in a subset of CP patients whose sera did not react with epidermal or dermal extracts [66].

Taken altogether, these data clearly show the existence of different immunological subsets of CP that are associated with distinct target molecules involved in interactions between epithelial cells and extracellular matrix proteins of the basement membrane zone. In addition, the finding of CP antibodies not only directed against different epitopes of a single antigen but also recognizing more than one target molecule at the same time in the same patient [67] illustrates the complexity of the pathogenetic process in CP. The relevance of intermolecular epitope spreading of closely associated molecules in the production of these antibodies as well as the respective pathogenetic role of the different antibodies remain to be established.

Epidermolysis bullosa acquisita (EBA)

Common clinical features of another subepidermal autoimmune blistering disease, EBA, include the occurrence of trauma-induced blisters, which are predominantly localized at the extensor surfaces of legs, arms and hands, and heal with scarring and/or milia formation [68]. These findings correspond to the classical EBA phenotype and closely mimic those of an inherited bullous skin disease, dominant DEB. However, EBA can also present with a pemphigoid-like phenotype or a cicatricial pemphigoid-like phenotype. Direct immunofluorescence of peri-lesional EBA skin shows a wide IgG band along the DEJ. Both direct and indirect immunofluorescence on salt-split skin provide evidence that the IgG bind to the dermal side of the separated skin. Direct and indirect immunoelectron microscopy show that IgG bind to anchoring fibrils in the sub-lamina densa of the DEJ. A percentage of EBA patients have detectable circulating IgG autoantibodies that recognize a 290 kDa antigen corresponding to type VII collagen [68, 69].

Four major immunodominant epitopes localized within the NH2-terminal, non-collagenous NC-1 domain of type VII collagen have been identified in EBA patients and also in patients affected with bullous systemic lupus erythematosus (BSLE), a small subset of LES patients who develop blisters in their skin [70]. In addition, sera from three children affected with an inflammatory form of EBA have recently been reported to recognize the central triple-helical domain and not the NC-1 domain of type VII collagen, suggesting that different clinical forms of EBA might correspond to different antibody specificity [71].

The role of the circulating IgG autoantibodies in the pathogenesis of EBA remains to be elucidated. Several attempts to passively transfer the disease by injecting human antibodies into the SCID mice with a human skin graft have been unsuccessful and a single report exists of passive transfer in neonatal mice of high doses of purified IgG from a patient with severe EBA that resulted in alterations mimicking some aspects of this disease (i.e. deposits of immunoreactants in DEJ, granulocyte-rich infiltrate in dermal papillae, and pronounced dermal edema), yet failed to produce clinical or microscopic subepidermal blisters in vivo [72].

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