Introduction to Autoimmune Bullous Diseases

1. Introduction

Bullae are formed as a result of the damage of skin integrity due to various reasons, including bacterial or viral infections, trauma, genetic disorders and autoantibodies and fluid accumulation in the different layers of the skin; subcorneal, suprabasilar, dermal-epidermal junction and upper dermis [1]. Autoimmune bullous diseases (ABD) are a heterogeneous group of rare but fatal or debilitating skin diseases characterized by varying degrees of mucosal and cutaneous blister formation due to autoantibodies directed against the structural proteins of epidermis or the dermal-epidermal junction [2, 3]. ABD are classified according to the location of the bullae in the skin and the antigens targeted by the antibodies. They are simply examined in four main groups: pemphigus, pemphigoid, acquired epidermolysis bullosa and dermatitis herpetiformis [1].

It is important to know the structure of the skin and antigens targeted by autoantibodies in order to better understand the ABD. The epidermal stratified squamous epithelium is a complex structure which includes several layers of keratinocytes. Cohesion among these cells is needed to preserve the epidermal architecture and function [4]. Epidermal integrity is provided by three types of junctional structures: (1) anchoring junctions (desmosomes and adherens junctions), major adhesive cell–cell junctions of epithelial cells that function with each other to hold epithelial sheets together. Both are connected with the cytoskeleton and represent sites of mechanical coupling between cells. (2) Tight junctions (zonula occludens) that constitute a diffusion barrier. (3) Gap junctions, where intercellular channels allowing for the direct exchange of small molecules between cells [4, 5]. While suprabasal, differentiating keratinocytes adhere to each other, undifferentiated basal keratinocytes are anchored to the dermis and interact with extracellular matrix. Basal cell surfaces not in contact with basement membrane have desmosomes which attach adjacent keratinocytes [1].

2. Desmosomes

Desmosomes are disc-like strong cell–cell adhesion complexes that act as anchors linking the intermediate filament (IF) cytoskeletons of neighboring cells in tissues that undergo large amounts of mechanical strain such as the heart and skin [6, 7]. In addition to their adhesive role, desmosomes are dynamic structures that regulate normal physiological processes such as proliferation and differentiation during development, tissue morphogenesis and wound healing [3, 6, 8–10].

Desmosomes are described as small dense nodules at the contact points between neighboring cells. “Desmos” means “bond” and “soma” means “body.” Electron microscopic investigations and newly developed procedures have supplied detailed knowledge about their structures and major protein components [3].

Desmosomes are 0.2–0.5 μm in diameter in human epidermis and consist of dense plaques located symmetrically on the plasma membranes of adjoining cells. Extracellular domain, a dense midline separates the membranes [8, 11].

Desmosomes, calcium-dependent junctions, have five major component proteins such as the desmosomal cadherins (DCs) [desmoglein (dsg) and desmocollin (dsc)], the plakin family [desmoplakins, (DP)], and the armadillo proteins [plakoglobin (PK) and plakophilin (PP)] [6, 8].

3. Desmosomes in diseases

The role of desmosomes in maintaining tissue integrity is defined by the large number of diseases in which one or more of its constituents are impaired [4]. The impairment of adhesive functions of desmosomal cadherins results from either development of autoantibodies against desmosomal cadherins or by gene mutations. Pemphigus is a family of blistering skin disorders caused by autoantibodies against desmosomal cadherins [5].

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are two most common forms of pemphigus family and potentially fatal disorders characterized by blister formation in skin and mucous membranes (in PV) due to the acantholysis, loss of keratinocytes cell–cell adhesion. Immunochemical studies showed that in PV, autoantibodies are immunoglobulin (Ig) G type and are directed against dsg3, 130 kD glycoprotein, or both dsg3 and dsg1, 160 kD antigen [5, 13], while in PF, they directed to only dsg1 [1, 12–14]. IgG1 and 4 type autoantibodies are indicators for active disorder while IgG2 is found in remission [1, 3]. Dsg3 and dsg1 show different expression patterns throughout epidermis. Dsg1 is expressed throughout epidermis and oral mucosa but it is more predominant in superficial epidermis than in deep epidermis. In contrast, dsg3 is expressed throughout the oral mucosa but it is only present in basal and lower epidermal cells. In PF, anti-dsg IgG antibodies cause blistering in the superficial epidermis, but not in the mucosa or deep epidermis because dsg3 expression compensates loss of function due to the anti-dsg1 antibodies. In PV, anti-dsg3 antibodies cause blister development in the deepest layer of mucosa, where dsg1 expression is minimal. Mucocutaneous type PV results from both anti-dsg1 and anti-dsg3 antibodies [14–16]. But, in this type, diffuse intercellular blisters throughout epidermis do not occur. A cause of it may be that cell–cell adhesion might be weaker at the basal and intermediate suprabasal layers, where there are fewer desmosomes. Another reason may be that the lower layer of epidermis might have better access for autoantibodies which penetrate from the dermis. The main postulate of this monopathogenic theory (compensation theory) is that anti-dsg3 and 1 antibodies-dependent disabling of cell–cell adhesion is adequate to cause detachment of keratinocytes and form the blisters [3, 17]. However, data demonstrated that inactivation of dsg3 gene or depletion of dsg3 from keratinocytes could not induce gross blistering in the skin. In striate palmoplantar keratoderma which is due to N-terminal deletion of dsg1 acantholysis or skin blisters are not seen. Thus, compensation theory is still controversial [3, 15, 18]. Multipathogenic theory works to explain blister formation by multiple hit hypothesis. According to this hypothesis, a simultaneous and synchronized inactivation of physiological mechanisms of cell–cell adhesion causes disruption of epidermal detachment. Non-dsg antibodies may be pathogenic because they cause cell shrinkage, loss of adhesion at keratinocytes and/or proapoptotic signaling [17]. Additionally, IgA and IgE classes of Anti-dsg3 antibodies have been found in the sera of PV patients [3].

T-helper cells have critical role in the formation of pemphigus autoantibodies. Activation of autoreactive T cells (losing self-tolerance to dsg) responsive to pemphigus antigens lead to induction of IgG antibodies from B cells [3, 19]. Autoimmunity to certain epitopes of dsg3, dsg-reactive T and B cells may be seen in normal individuals particularly, the relatives of PV patients. There are dsg3 reactive Th1 cells in healthy relatives but there are Th2 cells in PV patients. Th2 reactive cells are detected at similar frequencies in the acute, chronic active and remittent phases of the disease but Th1 cells are increased in chronic active PV. It was demonstrated that Treg cells were decreased in the serum of PV patients [3, 18]. However, there is no decrease in Treg cells in PV skin lesions because Treg cells accumulate in the skin lesions and the draining lymph nodes. It has been thought that pemphigus autoimmunity can be triggered by Toll-like receptors (TLR) because of the activation of PF by TLR7 agonist, imiquimod [3].

4. Mechanism of acantholysis

It was shown that the number and size of desmogleins are reduced in PV and PF [19]. Data demonstrated that pemphigus autoantibodies bind to conformational epitopes formed by the N-terminal 161 amino acids and stabilized by calcium on desmogleins, and that these binding areas are responsible for the pathogenicity but C-terminal extracellular domain is not the pathogenic domain [14]. Previous data showed that PV IgG most likely directly cause the loss of adhesion via the disruption of desmogleins by steric hindrance (cis or trans interaction) [12, 13, 18]. Interestingly, the detachment of keratinocytes from each other first occurs in the interdesmosomal area, and desmosomal detachment is seen in late acantholysis. Recent studies have demonstrated that the loss of desmosomal function is not only related to the steric hindrance, it may be related with other mechanisms [5, 13]. PV IgG bound to unassembled desmosomal cadherins does not prevent desmosomal generation rather, it causes internalization and degradation of IgG-antigen complex [15].

It has been shown that polyclonal PV IgG causes the retraction of keratin IF and intercellular detachment in vitro in keratinocytes obtained from wild type mice. But PG has critical importance for keratin retraction and detachment of cells [14]. PV IgG binding results in the depletion of dsg3 from keratinocytes and is followed by its internalization and degradation and depriving the not yet assembled desmosome of dsg3 [12, 14]. This suggestion was supported by the demonstration of a reduction in dsg3 levels in cell lysates. In contrast, some studies showed that dsg3 levels increase in cell lysates due to the reduction of anchorage of dsg3 to the cytoskeleton caused PV IgG antibodies [15]. Recent studies in mice have not shown the loss of dsg3 in split desmosomes or keratin retraction in acantholytic areas and that dsg3 is not depleted from desmosome before acantholysis [14].

Early studies showed that non-lysosomal proteases like plasminogen activator released by antibody binding caused the development of blisters but later, investigations in mice did not support this hypothesis and demonstrated that plasmin and plasminogen activators were not necessary for IgG-mediated acantholysis in mice in PV. Recent studies in vivo and in vitro have shown that selective proteases such as MMPs disrupt the adhesion of keratinocytes leading to proteolysis of adhesion molecules. While dsg3 is digested by MMP-9, a member of MMPs family, cell adhesion molecules like dsg1 and E-cadherin are digested by members of ADAM family of MMP during apoptosis [15]. In cultured keratinocytes, it has been shown that PV IgG induces apoptosis resulting in acantholysis. Apoptotic keratinocytes, reduced antiapoptotic factors and increased proapoptotic factors were detected in the epidermis in PF. Thus, induction of apoptosis may be a primary factor responsible for acantholysis and loss of intercellular adhesion. Caspases, apoptosis enzymes that have a role in acantholysis, are the other proteases. It was shown that activated caspase 3 was found in the epidermis before the blister formation, and it could cleave desmosomal proteins such as dsg1, dgs2 and dsg3. Caspases also cause the disruption of plaque proteins such as PP and DP1 and DP2, plektin and periplakin [8, 15]. Moreover, caspase inhibitors may block blister formation [8]. Shortly, these data suggested that caspases have fundamental role in apoptolysis [15]. FasL and CytC activate both extrinsic and intrinsic apoptotic signaling pathways in keratinocytes treated with PV IgGs [17]. Tumor necrosis factor alpha receptor superfamily member 5 and NADAH dehydrogenase-like protein are involved in the extrinsic and intrinsic apoptotic pathways, respectively [3].

Acantholysis is an active and complex process. Interaction of cell and PV IgG causes activation of phosphatidylcholine-specific phospholipase C, an increase in inositol 1,4,5 triphosphate (IP3) and diacylglycerol production and protein kinase C (PKC) activity. It also causes an increase in intracellular calcium concentration [15]. It has been shown that PV IgG causes serine-phosphorylation of dsg3, and the phosphorylation leads to the loss of PG binding. Data suggested that PG, a cytoplasmic plaque constituent, plays a critical role in keratin retraction because PG binding is essential for targeting dsg3 to desmosomes [8, 14]. Recently, a lot of protein kinase and signaling molecules, including p38 MAPK, PKC, c-myc, Src, Rho A, PERK, FAK, Akt/mTOR, and cdk2 have been demonstrated [11, 15]. For example, it was shown that p38 phosphorylation facilitates the retraction of IF and detachment of the cells [13]. Desmocollin genes encoded N-glycosylated type 1 transmembrane proteins belong to Ca-dependent cell adhesion molecules of cadherin family. Similar to dsg3, dsc3 is expressed in the basal and suprabasal layers of the epidermis. It was demonstrated that anti-dsc3 antibodies might induce the loss of adhesion of epidermal cells and contribute to blister development in pemphigus. In addition to dsg and dsc3 antibodies, reactivity to dsc1, several muscarinic and nicotinic acetylcholine receptor subtypes, HLA molecules, a number of mitochondrial proteins, thyroid peroxidase and hSPCA1 encoded ATP2C1 gene were shown. Moreover, anti-non-dsg antibodies may show the synergistic effects with anti-dsg antibodies, in other words, they may potentially amplify the activity of anti-dsg antibodies [17].

Anti-mitochondrial antibodies (AMA) target the mitochondrial nicotinic acetylcholine receptors that prevent apoptolysis in keratinocytes. AMA with anti-dsg antibodies can induce acantholysis, AMA/anti-dsg1 induces subcorneal splitting and AMA/anti-dsg3 induces suprabasal acantholysis. Recent studies showed that FcRn receptors exist on the keratinocytes and are a single target for PV IgG. PVIgG/FcRn complexes become internalized and are transmitted to mitochondria. Mitochondria are damaged via AMA and apoptotic signals are triggered for cell shrinkage. This shrinkage resulting in cytoskeleton collapse is an outcome of energy failure due to the damaged mitochondria [17].

According to a recent hypothesis, anti-dsg antibodies are not the reason but the result because reactivity to dsg1/3 develops in both extracellular and intracellular domains, and this gives rise to the thought that dsg molecules are released to intercellular space from damaged keratinocytes and become available to antigen presenting cells [3]. Consequently, pemphigus autoimmunity is directed to multiple organ-specific and non-organ-specific proteins.

Paraneoplastic pemphigus (PNP) is a rare and serious form of pemphigus. It is different from other OBD because it can affect multiple organs as well as skin [11, 20]. It has unusual clinical features, including severe mucosal involvement, bronchiolitis and a wide range of skin rash (pemphigus-like, bullous pemphigoid-like, erythema multiforme-like, graft versus host disease-like and lichen planus-like) [21]. It also shows unusual histopathological and immunological findings. PNP lesions are extremely painful and may be localized on the palm and soles, conjunctiva and simple squamous epithelia. The lesions are resistant to therapy. PNP is usually associated with malignancies such as lymphoma and leukemia. The mortality rate of PNP is high (90%) [11]. It may also be associated with myasthenia gravis and thymomas [22]. Because of cutaneous and noncutaneous pathologies associated with neoplasia it is called as paraneoplastic autoimmune multiorgan syndrome [21].

In PNP, targets of autoantibodies are more than one: dsgs, dscs, DP1 and 2, bullous pemphigoid antigen (BPAg)1, PF, PP, envoplakin, plectin, epiplakin and alpha-2-macroglobulin-like-1 (A2ML-1) that is a broad range protease inhibitor expressed stratified epithelia and other damaged tissues in PNP [11, 20, 22]. Characteristic autoantibodies in PNP target the plakin family proteins that are molecules localized in the intracellular plaque of desmosomes and hemidesmosomes [20]. Also, anti-acetylcholine receptor autoantibodies and acetylcholinesterase autoantibodies were detected in 35 and 28% of PNP patients, respectively. High levels of these autoantibodies correlated with dyspnea in PNP patients. These antibodies target not only epidermal proteins but also other antigens in neural and bronchial tissues [22].

It is currently unclear why there are multiple autoantibodies in PNP. In patients associated with thymoma, it has been thought that defective thymocyte maturation might lead to the production of autoreactive T cells that can induce B-cell proliferation and autoantibody production. In hematologic malign tumors, aberrant immunological conditions caused by tumors might cause the production of many autoantibodies [22]. Another theory is that autoantibodies against the neoplastic antigens cross-react to epithelial antigens [21]. In PNP, responsible immunity is not solely humoral immunity, also cellular immunity plays a role in the pathogenesis. Therefore, histopathology shows individual keratinocyte necrosis with lymphocyte exocytosis in addition to deposits of autoantibodies in direct immunofluorescence (DIF) examination [20].

Lymphoid tumors may produce antibodies to desmosome and hemidesmosome components. But this solely cannot be explained with the pathogenesis of PNP. It is thought that tumors may express proteins that cross-react with epithelial proteins such as plakins. Another mechanism is dysregulated cytokine production by the tumor cells. The levels of interleukin (IL)-6 which promotes B-cell differentiation and Ig production is increased in PNP. Epitope spreading may explain antibodies against multiple proteins found in PNP [20].

In PNP, accumulation of activated CD8+ T cells and increased interferon gamma and tumor necrosis factor alpha levels were shown in the epidermis locally. Also, natural killer cells were detected in the affected tissues. Consequently, both humoral and cellular immunity play a role in the development of PNP [20].

Another subtype of pemphigus is IgA pemphigus characterized by IgA antibodies to desmosomal and non-desmosomal keratinocyte cell surface constituents. It has two subtypes: subcorneal pustular dermatosis type in which there are antibodies to dsc1 and very rarely to dsc 2 and 3, and intraepidermal neutrophilic type in which target antigen is still unknown but in rare cases, anti-dsg1 and 3 antibodies are the target antigens [11, 21, 23]. The mechanism of the development of skin lesions is not clear. It is thought that IgA antibodies might bind to the Fc receptor CD89 on monocytes and granulocytes resulting neutrophil chemotaxis and subsequent proteolytic cleavage of keratinocyte cell–cell junction [21]. Recently, IgG/IgA pemphigus which is an overlapping variant of classic IgG pemphigus and IgA pemphigus has been defined. Histopathological findings are acantholysis, blister formation localized on subcorneal or entire layer of epidermis and neutrophilic infiltration [11].

Pemphigus herpetiformis (PH) is a pemphigus form clinically resembling dermatitis herpetiformis and histopathologically pemphigus. In PH, autoantibodies against dsg1, dsg3, both dsg1 and dsg3 and more recently, dsc1, dsc3 and an unknown 178-kDa protein were recognized. PH autoantibodies may recognize functionally less important epitopes of dsg1 or 3; therefore, it does not lead acantholysis directly. It is thought that autoantibodies cause the attraction of the inflammatory cells to tissue inducing by signaling pathway of cytokine production by keratinocytes [21] (Table 1).

5. Basement membrane zone

Basement membranes are highly specialized forms of extracellular matrix composed of a distinct set of glycoproteins and proteoglycans [24]. They underlie all epithelia and endothelia, enveloping nerves, muscle fibers, distinct cell compartments and whole organs [24]. Basement membranes of various tissues differ ultrastructurally, biochemically and functionally. They act as substrates for attachment of cells, templates for tissue repair, matrices for cell migration, substratum to influence differentiation, morphogenesis and apoptosis of epithelial cell layers and permeability barriers for cells and macromolecules [25]. Basement membranes consist of lamina densa, a central electron-dense region, adjacent to a less dense area which is lamina lucida or lamina rara [24].

Human skin is the body’s largest organ, which provides mechanical and immunological barrier against the external environment [26]. The interface between the lower part of the epidermis and the top layer of dermis is the dermoepidermal basement zone (BMZ) which maintains the structure and integrity of the skin by anchoring the overlying epidermis to the dermal matrix below [27]. The importance of the correct assembly of the components of BMZ for skin integrity is apparent from the multiple skin blistering disorders caused by mutations in genes coding for proteins associated with the epidermal BMZ and from autoimmune disorders where autoantibodies target these molecules. These proteins are also important in tissue homeostasis, repair and regeneration [28].

The epidermal BMZ can be divided into four zones. The first zone contains the cytoskeleton, hemidesmosomes and plasma membranes of basal keratinocytes. The second zone is lamina lucida which contains filaments connecting hemidesmosomes in basal keratinocytes to the lamina densa. The third zone is lamina densa and the fourth zone is sublamina densa region which contains anchoring fibrils, anchoring plaques and fibrillin containing microfibrils [25, 29]. The biochemical components of BMZ are synthesized by basal keratinocytes and dermal fibroblasts [30]. Molecular components of epidermal BMZ are shown in Table 2.

The basal keratinocytes are anchored to the basal lamina via the keratin intermediate filaments and hemidesmosomes. The molecules within the basal lamina connect the basal keratinocyte to the basal lamina which anchors the BMZ to the underlying collagenous matrix of the superficial dermis [31]. Hemidesmosomes are small, regularly spaced electron dense structures on the inner surface of the basal pole of the keratinocytes [32]. They extend from the intracellular compartment of basal keratinocytes to the lamina lucida in the upper portion of the dermal epidermal basement membrane. The intracellular domains within the basal keratinocytes attach to the keratin intermediate filament network, and within the lamina lucida, they are contiguous with anchoring filaments [30]. The anchoring filaments transverse the lamina lucida and insert it into the lamina densa. Beneath the lamina densa, the anchoring fibrils extend beneath the basement membrane within the papillary dermis. The hemidesmosomes, anchoring fibrils and anchoring filaments form the hemidesmosome-anchoring filament complex [25, 32]. The hemidesmosome-anchoring filament complex forms a continuous link between the basal keratinocyte intermediate keratin filaments and the underlying BMZ and dermal components [32, 33].