The vesiculobullous reaction pattern

Adhesion molecules

As indicated previously, there are four families of adhesion molecules. They are sometimes divided into two groups—those that mediate cell–cell adhesion, such as the cadherins and immunoglobulin superfamily, and those that mediate cellular adhesion to matrix molecules, such as the selectins. The integrins are capable of mediating both types of adhesion. The cadherins are the most important group in keratinocyte cohesion, although the integrins have a role in basal cells, particularly in the hemidesmosomes. Cadherins and integrins are important for the maintenance of tissue integrity and in signal transduction during skin development. Only brief mention is made of the other families in the discussion that follows.

Cytoplasmic plaques

Cytoplasmic plaques are dense, submembranous regions of the intercellular junctions, measuring 14 to 20 nm in thickness. They are composed of filament-binding proteins that connect with the cytoplasmic domain of the cadherins and the filaments of the cytoskeleton. In desmosomes, the plaques connect with keratin intermediate filaments, whereas in the adherens junctions they connect with actin filaments.

The composition of the cytoplasmic plaques is different in the two junctions; only plakoglobin is common to both. In the adherens junctions, the cytoplasmic plaques contain α– and β- catenins . In desmosomes, the cytoplasmic plaques consist primarily of desmoplakins 1 and 2 , antibodies to which are found in paraneoplastic pemphigus. The desmoplakins play a pivotal role in anchoring the network of intermediate filaments to desmosomes. Other proteins (e.g., IFAP-300) may enhance this association. The first mutation to be described in desmoplakin was in a family with autosomal dominant striate palmoplantar keratoderma (OMIM 125647 ) (see p. 320 ). An autosomal recessive mutation in desmoplakin gives rise to Carvajal syndrome (OMIM 605676 ), which comprises dilated cardiomyopathy, woolly hair, and keratoderma (see p. 319 ). The desmoplakins, along with the bullous pemphigoid antigen 1, plectin, and the cell envelope proteins envoplakin and periplakin, belong to the so-called “intermediate filament-associated proteins,” now known as the plakin family. An absence of plectin from the hemidesmosomes has been found in a variant of epidermolysis bullosa simplex with associated muscular dystrophy. Autoantibodies to plectin (450 kDa) have been reported in a patient with a bullous pemphigoid–like eruption. One study suggests that inflammation in inflammatory bowel disease may expose plectin, prompting a secondary immune response that can cross-react with skin, resulting in bullous pemphigoid as a form of “epitope spreading” in patients who have undergone colostomy or ileostomy. Other constituents of the desmosomal cytoplasmic plaque include the armadillo proteins plakoglobin and plakophilins 1, 2, 3, and 4. Plakophilins are essential for the formation and function of desmosomes because they are involved in the recruitment and normal association of the other desmosomal proteins. Plakophilin 1 is expressed primarily in the suprabasal layers of stratifying epithelia. It is absent in patients with ectodermal dysplasia–skin fragility syndrome (OMIM 604536 ). Mutations in the plakoglobin gene result in the palmoplantar keratoderma known as Naxos disease (see p. 319 ). Absence of plakophilin 1 has been associated with skin fragility and hypohidrotic ectodermal dysplasia.

Tight junctions

Tight junctions play an important role in the formation of the epidermal barrier. Their role in cell cohesion is minimal. They are a gatekeeper of the paracellular pathway, forming a circumferential, belt-like structure involving cells of the granular layer, in normal epidermis. Tight junctions contain the proteins occludin; ZO-1; cingulin; claudin-1, -4, -5, and possibly others; and JAM-1, a member of the immunoglobulin superfamily. It appears that ZO-1 acts as a link between JAM-1 and the claudins. Tight junction proteins are destroyed in various inflammatory diseases of the skin, including psoriasis and atopic dermatitis.

In summary, epidermal cohesion is a complex process involving the adhesion molecule families—cadherins and integrins—concentrated in areas known as desmosomes and adherens junctions. They have extracellular domains (homophilic in the case of cadherins) and intracellular ones that connect with filament-binding proteins (plakoglobin and catenins or desmoplakins) in cytoplasmic plaques. These proteins connect with filaments of the cytoskeleton, such as actin and keratin intermediate filaments.

Histopathology

In impetigo, there are subcorneal collections of neutrophils. A few acantholytic cells are sometimes present, particularly in bullous impetigo, as a result of the action of enzymes released from neutrophils. Acantholysis is never as prominent in impetigo as it is in pemphigus foliaceus. Gram-positive cocci can usually be demonstrated in impetigo, another distinguishing feature of this condition.

Subcorneal pustules, sometimes resembling impetigo, can be seen in some cases of listeriosis.

Histopathology

It is usually difficult to obtain an intact blister in SSSS because the stratum corneum may be cast off during the biopsy procedure or the subsequent processing of the specimen. A few acantholytic cells and neutrophils are usually present in intact blisters or on the surface of the epidermis if its roof has been shed ( Fig. 7.1 ). Organisms are not usually present in the affected skin, in contrast to bullous impetigo. There is usually only a sparse inflammatory cell infiltrate in the upper dermis, in contrast to bullous impetigo and pemphigus foliaceus in which the infiltrate is usually heavier.

Pathogenesis

Pemphigus foliaceus results from the formation of autoantibodies, mainly of the IgG4 subclass, that react with several different antigenic epitopes on the amino-terminal region of dsg1, a 160-kDa transmembrane glycoprotein, which is present in desmosomes. The amino-terminus of dsg1 appears to be a key region for the intercellular adhesion of cadherins. In certain cases, the antibodies appear to react with desmocollins, the other subtype of desmosomal cadherins, or other antigens. The expression of dsg1 is highest in specimens from the upper torso, excluding the scalp. It is expressed primarily in the upper layers of the epidermis. Dsg1 is expressed at a much lower level than dsg3 in oral mucosa. This suggests that dsg3 may be sufficient for cell–cell adhesion in oral mucosa, with consequently no oral involvement in patients with pemphigus foliaceus. Neonates are protected from the effect of the passive transfer of maternal autoantibodies across the placenta by possessing dsg3 in the upper levels of the epidermis. The antibodies found in patients with drug-induced pemphigus, and in some patients with pemphigus vulgaris, also react with dsg1. The antibodies themselves are pathogenic, but complement, including the terminal complement sequence (membrane attack complex), is also an important mediator in the detachment of the epidermal cells. The use of plasminogen activator knockout mice suggests that there is no requirement for proteases in blister formation. Antibody levels fluctuate during the course of the disease and have some correlation with disease activity. However, a study has shown that the predominant binding of anti-dsg1 antibodies to the amino-terminus of dsg1 persists despite the activity stage of the disease, including periods of remission. The availability of recombinant dsg1 has facilitated the development of antigen-specific plasmapheresis as a therapeutic strategy. A subset of pemphigus foliaceus patients exhibits pathogenic autoantibodies against both dsg1 and dsg3. The figure is less than 7%.

Cytokines also have a pathogenic role in this disease. Tumor necrosis factor-α (TNF-α) levels are found in the serum and blister fluid. Increased cytokine production is the likely mechanism of the localized pemphigus foliaceus produced by topical imiquimod. Epidermal keratinocytes can be induced to produce proinflammatory cytokines and other mediators such as bradykinin. The kinin system might have a potential role in the pathogenesis of acantholysis in pemphigus foliaceus, along with TNF-α.

Treatment of pemphigus foliaceus

Corticosteroids are the treatment of choice, but because this is a chronic disease that often requires long-term therapy, other adjuvants have been added for their steroid-sparing effect. Sometimes these other agents are used as monotherapy. They include azathioprine, cyclophosphamide, gold, methotrexate, chlorambucil, cyclosporine (ciclosporin), mycophenolate mofetil, dapsone, hydroxychloroquine, plasmapheresis, extracorporeal photopheresis, intravenous immunoglobulin, and topical epidermal growth factor. Isolated cases, refractory to other therapies, have been successfully treated with etanercept and rituximab. There is experimental evidence that ectopic superficial expression of desmoglein 2 can limit epidermal blister formation produced by pemphigus foliaceus antibodies and exfoliative toxin-A. The plant lectin jacalin specifically binds to O -linked Thomsen–Friedenreich carbohydrate structures on dsg1 and inhibits its interaction with pemphigus foliaceus IgG, thereby interfering with the latter's pathogenicity; this may have potential therapeutic value.

Histopathology

Established lesions of pemphigus foliaceus of both endemic and nonendemic forms show a superficial bulla with the split high in the granular layer or directly beneath the stratum corneum. The bulla contains fibrin, some neutrophils, and scattered acantholytic keratinocytes. No bacteria are present, unlike in bullous impetigo. A localized clinical variety with a peau d'orange clinical appearance was characterized by acantholysis exclusively involving follicular infundibula. The earliest change appears to be the formation of vacuoles in the intercellular spaces in the upper layers of the epidermis. These expand, leading to cleft formation. Uncommonly, eosinophilic spongiosis is seen as a precursor lesion, and transitions between this picture and that of pemphigus foliaceus may be seen. Eosinophilic spongiosis appears to be more common in pemphigus foliaceus in some African races. Neutrophilic spongiosis is a rare occurrence in pemphigus foliaceus ; its occurrence is usually related to the deposition of IgA or the herpetiform type (herpetiform pemphigus). Neutrophilic pustules are another uncommon manifestation of pemphigus foliaceus.

In late lesions of pemphigus foliaceus, the epidermis may be hyperplastic, with overlying focal parakeratosis and some orthokeratosis. Dyskeratotic cells with hyperchromatic nuclei and somewhat resembling the “grains” found in Darier's disease are a distinctive feature of the granular layer ( Fig. 7.2 ). In the rare mixed forms of the disease, both suprabasal acantholysis and subcorneal clefting with acantholysis are present.

The superficial dermis is edematous with a mixed inflammatory cell infiltrate that usually includes both eosinophils and neutrophils. In drug-induced lesions, eosinophils may predominate in the dermal infiltrate.

With direct IF, there is intercellular staining for IgG and C3 in both affected and normal skin. Sometimes the staining is localized to the upper levels of the epidermis ( Fig. 7.3 ). Rarely, these immunoreactants may be present along the basement membrane, even in cases that clinically resemble pemphigus foliaceus rather than pemphigus erythematosus (discussed later). In a few instances, IgA rather than IgG has been present in the intercellular regions. The term IgA pemphigus has been used for these cases. Indirect IF demonstrates circulating antibodies in nearly 90% of cases of nonendemic pemphigus foliaceus. A subset of cases of pemphigus foliaceus also has positive indirect IF using a rat bladder epithelial substrate—a feature more typical of paraneoplastic pemphigus. A new biochip IF test for the serological diagnosis of pemphigus foliaceus (as well as one for pemphigus vulgaris) has been developed that tests simultaneously for multiple diagnostically relevant antibodies (e.g., desmogleins 1 and 3 and bullous pemphigoid antigens), serving as a fast and cost-effective screening tool.

Trichoscopy can be of value in the differential diagnosis of scalp lesions of pemphigus foliaceus; the most common findings include extravasations and yellow hemorrhagic crusts, yellow dots with whitish halos, white polygonal structures, and, less commonly, vascular anomalies such as linear serpentine vessels. Reflectance confocal microscopy can be used to suggest an initial diagnosis of pemphigus, including pemphigus foliaceus, although it is not considered a replacement for histopathological and IF studies. The diagnosis is suggested when two of three criteria are met: acantholytic clefts in a lesion, acantholytic clefts in normal-appearing adjacent skin, and multiple dilated blood vessels in lesional skin.

Histopathology

The appearances are identical to those of pemphigus foliaceus, with a subcorneal blister containing occasional acantholytic cells (discussed previously). Eroded and crusted lesions may develop.

Direct IF usually demonstrates IgG and/or complement, both in the intercellular spaces and at the dermoepidermal junction. The intercellular staining may be more pronounced in the upper layers of the epidermis. The lupus band test is sometimes positive in uninvolved skin. A possible explanation for the basement membrane zone deposits has been provided in a study of three cases of pemphigus foliaceus that were subjected to UV-A phototherapy. Oktarina et al. found evidence for release of desmoglein 1 fragments, which appear to form immune complexes with circulating anti–desmoglein 1 IgG antibodies and deposit along the basement membrane zone. A similar mechanism may occur in pemphigus erythematosus and would also explain the localization of these lesions to sun-exposed skin.

Histopathology

The characteristic features of herpetiform pemphigus are eosinophilic spongiosis with the formation of intraepidermal vesicles. Variable numbers of neutrophils may be present, leading to the formation of neutrophilic spongiosis or subcorneal pustules with both eosinophils and neutrophils. Acantholysis is minimal or absent in some cases, but it was present in half of the cases reported in a study at the University of Toronto.

Direct IF shows the deposition of IgG, with or without C3, on the cell surfaces of keratinocytes, as seen in pemphigus. There is usually superficial accentuation, but in cases with antibodies to desmoglein 3, the deposition may be more prominent on the lower layers of the epidermis.

Histopathology

The subcorneal pustule is filled with neutrophils, with an occasional eosinophil. Neutrophils also migrate through the epidermis, but they do not form spongiform pustules. The pustule appears to “sit” on the epidermis ( Fig. 7.4 ), and usually it causes no depression of the latter. An occasional acantholytic cell may be present in older lesions, a result of the activity of the proteolytic enzymes released from neutrophils. Mitotic figures are usually absent within the epidermis, unlike pustular psoriasis.

A mixed superficial perivascular inflammatory cell infiltrate is present in the underlying dermis. In early lesions, the infiltrate includes quite a few neutrophils.

Direct IF is usually negative, although immunoreactants have been described in the epidermis in rare cases. Several cases have been reported of a condition resembling subcorneal pustular dermatosis clinically but in which a biopsy of the lesions showed intercellular IgA. Such cases are now classified as IgA pemphigus, although a case could be made for the retention of some of them as variants of subcorneal pustular dermatosis. This subject is discussed further in the following section.

Histopathology

In the SPD type, there are subcorneal pustules with variable but usually mild acantholysis, whereas in the IEN type there are intraepidermal pustules. There is usually a mixed inflammatory cell infiltrate in the underlying dermis. A case of IgA pemphigus (foliaceus) has been reported that resembled the more usual cases of pemphigus foliaceus without any neutrophilic infiltration. Neutrophilic spongiosis, suggestive of IgA pemphigus, has been reported in a case of pemphigus foliaceus devoid of IgA. A rare variant of the IEN type, which occurred in a child on immunosuppressive drugs, had features of pemphigus vegetans with epidermal hyperplasia and intraepidermal pustules.

Direct IF shows intercellular deposition of IgA in the epidermis. There is usually increased intensity of staining in the upper layers of the epidermis in the SPD type. In the IEN type, the IgA staining is usually throughout the entire epidermis.

IgG/IgA pemphigus usually shows a lesser degree of pustule formation and more acantholysis than seen in cases of IgA pemphigus. However, there have also been cases with subcorneal pustules and acantholytic mid-epidermal blisters with neutrophilic and eosinophilic spongiosis, and prominent dermal neutrophils with lining up of these cells along the junctional zone. Direct IF shows intercellular staining for IgG and IgA, but another difference from IgA pemphigus is that there are also intercellular deposits of C3.

Differential diagnosis of intracorneal and subcorneal forms of pemphigus

Because of the level of splitting—subcorneal, often through the granular cell layer— pemphigus foliaceus can be confused with bullous impetigo and subcorneal pustular dermatosis . Acantholysis is usually, but not always, more evident in pemphigus foliaceus, and occasionally, suprabasilar acantholysis can also be observed. Pemphigus foliaceus tends to be less pustular than those two diseases, with occasional exceptions, such as some cases of herpetiform pemphigus. Direct IF can permit a definitive diagnosis because among these diseases, positive intercellular IgG and C3 staining is seen only in pemphigus foliaceus. The same superficial epidermal acantholysis is also characteristic of pemphigus erythematosus, and this finding permits separation from lupus erythematosus, which it can resemble both clinically and, partially, on direct IF because basement membrane zone staining resembling a “lupus band” may also be observed. Paraneoplastic pemphigus shows changes resembling erythema multiforme, but most often there are at least foci of suprabasilar acantholysis, and intercellular staining on direct IF is confirmatory of the diagnosis. The morphological findings of the subcorneal pustular dermatosis –like variant of IgA pemphigus can be virtually identical to those of the disease it resembles. However, classical subcorneal pustular dermatosis clearly lacks positive intercellular IgA deposition on direct IF study or circulating antibodies to epidermal antigens. It appears that to recognize this variant of IgA pemphigus, IF studies are essential.

Histopathology

In early lesions, there is an intraepidermal pustule containing neutrophils and sometimes varying numbers of eosinophils. This progresses to form a subcorneal pustule. There is a sparse perivascular mixed inflammatory cell infiltrate in the upper dermis.

In congenital erosive and vesicular dermatosis with reticulated supple scarring , the histology has been variable with no consistent pattern. There may be well-defined vesicles in the papillary dermis. The epidermis may be intact, eroded, or ulcerated. Other cases have shown a mixed dermal infiltrate without vesicles or edema with a predominantly neutrophilic infiltrate. Scarring with loss of appendages, an overlying atrophic epidermis, and/or a polymorphous dermal infiltrate is seen in older lesions. In another case, there were many “Civatte's bodies in the dermis suggesting previous keratinocytes’ necrosis.” There was subepidermal cleavage.

Histopathology

There are subcorneal or intraepidermal pustules, filled with eosinophils and related to the orifices of the pilosebaceous follicles. An inflammatory infiltrate composed predominantly of eosinophils is present in the upper dermis in the vicinity of the follicles, and there is some exocytosis of these cells into the epithelium of the involved follicles.

The histological appearances resemble those of eosinophilic pustular folliculitis, but the clinical features of the two conditions differ.

Histopathology

The vesiculopustules are intracorneal or subcorneal collections of neutrophils, admixed with fibrin and a few eosinophils. There may be a mild infiltrate of inflammatory cells around vessels in the upper dermis.

The pigmented macules show increased melanin in the basal and suprabasal keratinocytes but, surprisingly, there is no melanin in the dermis.

Histopathology

The usual picture is a subcorneal or superficial intraepidermal pustule with mild spongiform pustulation at the margins ( Fig. 7.5 ). This latter change is never as prominent as the spongiform pustules seen in pustular psoriasis. The pustules often contain eosinophils in addition to neutrophils. There is usually some exocytosis of neutrophils adjacent to the pustules. Scattered apoptotic keratinocytes are often present.

The papillary dermis is usually edematous, and there is a heavy mixed inflammatory cell infiltrate in the upper dermis. Eosinophils are often present, a distinguishing feature from pustular psoriasis. In a small number of cases, a leukocytoclastic vasculitis is present; this was found in only one case out of a large series of 102 hospitalized patients. Less commonly, there is subepidermal pustulation that may be in continuity with the intraepidermal pustules. Similar cases have been reported in the past as acute generalized pustulosis or included with the pustular vasculitides. A case clinically presenting as AGEP, associated with clindamycin and levofloxacin therapy, showed subcorneal pustules but also an atypical mononucleated dermal infiltrate composed of CD3 ⁺ and CD30 ⁺ cells, consistent with a lymphomatoid drug eruption. The eruption resolved within 1 week of discontinuing both drugs.

Differential diagnosis

Acute generalized pustulosis, or pustulosis acuta generalisata, an eruption caused by upper respiratory streptococcal infection, can also present with acute onset of widespread lesions and may have eosinophils in the dermal infiltrate. However, the intraepidermal pustules tend to be larger, and there are more pronounced dermal infiltrates and more obvious leukocytoclastic vasculitis than would be expected in AGEP. The pustules of pustular psoriasis are usually larger and show more prominent spongiform pustulation, whereas the degrees of acanthosis in AGEP are generally less than would be expected in fully developed plaque-type psoriasis. An increase in Ki-67-positive keratinocytes is seen in both pustular psoriasis and AGEP, with no significant difference in numbers or distribution of these cells. In comparing these two dermatoses, AGEP often has eosinophils in the pustules or in the dermis, necrotic keratinocytes, a neutrophil-rich but mixed interstitial and mid-dermal infiltrate, and a lack of tortuous dilated blood vessels. Despite an occasional clinical resemblance to erythema multiforme , pustules are generally not observed in the latter disease, whereas apoptosis is usually more extensive than in lesions of AGEP. As noted previously, a resemblance has been noted between AGEP and toxic epidermal necrolysis. This has been mostly based on clinical features. Although one study reported a potential histopathological resemblance as well, the photomicrographs of that case show an intraepidermal pustule with necrosis of the overlying epidermis and adjacent spongiosis—features more in keeping with AGEP—whereas other potentially differentiating features, such as the presence or absence of eosinophils, were not described. Infection with Candida or a dermatophyte could show overlapping features, but periodic acid–Schiff (PAS) or silver methenamine stains would be positive for microorganisms in those disorders.

Histopathology

The vesicle forms within or directly beneath the stratum corneum. It is centered on the acrosyringium.

Histopathology

The earliest lesion is a spongiotic vesicle in the lower malpighian layer that contains mononuclear cells and some neutrophils. This progresses to a unilocular, well-delimited pustule within the epidermis and extending upwards to the undersurface of the stratum corneum ( Fig. 7.6 ). There may be overlying, focal parakeratosis. In the dermis, a mixed perivascular and diffuse infiltrate of inflammatory cells is present.

Differential diagnosis

Controversy exists as to whether it is possible to differentiate pustular psoriasis and palmoplantar pustulosis on histopathological grounds. Spongiform pustulation is often present at the upper margins of the pustule in palmoplantar pustulosis, but the focus is usually much smaller than that seen in pustular psoriasis. Sometimes there is spongiosis without associated pustulation; this feature is not present in pustular psoriasis. Eosinophils may be present in palmoplantar pustulosis, a finding not usually seen in pustular psoriasis.

Immunoreactants are present in the stratum corneum in some cases of palmoplantar pustulosis. Regarding the histopathological differentiation from pompholyx, Yoon et al. found that loss of the granular cell layer, thinning of suprapapillary plates, eosinophils in pustules or vesicles, tortuosity of capillaries, capillaries touching the undersurface of the epidermis, and extravasated erythrocytes were statistically significant features of palmoplantar pustulosis, and psoriasiform epidermal features also tended to favor palmoplantar pustulosis. On the other hand, pompholyx tended to have multiple foci of parakeratosis, irregular acanthosis, and thinning of rete ridges. These two disorders also show a different pattern of epithelial membrane antigen staining within the epidermis; both show staining along the edge of intraepidermal vesicles, but there is little staining of the surrounding epidermis in palmoplantar pustulosis, whereas there is strong and diffuse staining of neighboring keratinocytes in pompholyx.

Histopathology

There are high intraepidermal spongiform pustules in an acanthotic epidermis. There is usually overlying parakeratosis containing neutrophil debris. Exocytosis of neutrophils into the epidermis is present. A mixed perivascular infiltrate of neutrophils and lymphocytes, with nuclear dust, is present in the upper dermis. There is marked overexpression of Bcl-2 in lesional skin.

Histopathology

Intact lesions show spongiotic pustules at a high level in the epidermis. They eventually become subcorneal or intracorneal in location. There is usually some acanthosis and a mixed dermal infiltrate of neutrophils, lymphocytes, and a few plasma cells. Erosions are common; they have a heavy mixed inflammatory cell infiltrate in the base.

Histopathology

The blister usually forms just beneath the stratum granulosum. The keratinocytes in the base of the blister show variable edema and pallor and even degenerative changes.

Etiology and pathogenesis

The antibodies in pemphigus vulgaris are directed against dsg3, a 130-kDa polypeptide that, like the pemphigus foliaceus antigen (dsg1), is a member of the desmoglein subfamily of the cadherin supergene family. Genetic variations in DSG3 , the gene controlling dsg3 on chromosome 18q12, appear to be an additive risk factor predisposing to pemphigus vulgaris. Dsg3 is localized to the desmosomes of keratinocytes, particularly the extracellular domain. Epitope mapping suggests that a segment containing amino-terminal residues 1–161 of dsg3 contains the critical antigens recognized by the autoantibodies in pemphigus vulgaris sera. This segment is considered to include structures essential for cell–cell adhesion. The cytoplasmic “tail” of the pemphigus vulgaris antigen, like other desmogleins, binds to the plakoglobin inside keratinocytes. Plakoglobin delocalization from the nucleus may play a role in the production of acantholysis (discussed later). More than 50% of sera from patients with pemphigus vulgaris also contain autoantibodies against dsg1. These antibodies also appear to be pathogenic. There is a shift over time in the profile of antidesmoglein antibodies in pemphigus vulgaris. The antibodies against both dsg1 and dsg3 have predominant IgG4 subclass specificity. Antibodies in the IgA and IgE class have also been detected. The majority of patients with mucosa-dominant disease have antibodies only against dsg3, but a significant number also have antibodies to dsg1. In fact, there is evidence that anti-Dsg1 autoantibodies may be better predictors of disease progression, and therefore motoring of anti-Dsg1 levels may be valuable for this purpose. The development of antibodies to dsg1 in some patients with dsg3-related mucosal disease is sometimes accompanied by changes in the clinical expression of the disease, such as the development of generalized cutaneous lesions. Conversely, lesions limited to the skin may occur in the presence of predominant dsg1 autoantibodies and weaker levels of dsg3 autoantibodies, but antibody levels are not the complete explanation for this phenomenon. Antidesmoplakin antibodies, which are usually found in paraneoplastic pemphigus, have also been reported in patients with mucosal dominant pemphigus vulgaris, who also had dsg3 autoantibodies. Although it has been thought that typical pemphigus does not have autoantibodies to the desmocollins, both desmocollins 2 and 3 have been detected in mucosal-dominant pemphigus vulgaris with severe symptoms, and studies by Mao et al. have shown that at least a subset of patients with identical clinical presentations have desmocollin 3 as the pathogenic autoantigen. Among other findings, IgG from these patients incubated with human keratinocytes causes loss of intercellular adhesion, and prior adsorption with recombinant desmocollin 3 prevents this effect. In pemphigus vulgaris, the antibodies produced by circulating B cells tend to bind preferentially to desmosomes in the lower epidermis, reflecting differences in the relative expression of the different cadherins in different levels of the epidermis. This antibody binding may be followed by internalization of some desmosomes. Antibodies will only cause cell separation (acantholysis) where the antigen is the principal adhesion molecule and others are unable to compensate. Cholinergic receptors on keratinocytes also play a role in cell adhesion. Antibodies to the acetylcholine receptors α-9 acetylcholine receptor and pemphaxin are present in humans with pemphigus vulgaris. Autoantibodies to these receptors can induce clinical features resembling pemphigus in dsg3-deficient knockout mice. Cholinergic agonists ameliorate acantholysis in mice. Antibody levels to the acetylcholine receptor are somewhat elevated in pemphigus vulgaris patients and appear to correlate with disease activity. There is also upregulation of P-cadherin expression in lesional skin. E-cadherin autoantibodies have also been found in the sera of patients. Another protein involved in cellular adhesion is focal adhesion kinase (p125 ^fak ). Its upregulation in lesional skin may be a reactive or reparative response to acantholysis. Using a passive transfer mouse model of pemphigus vulgaris, Espana et al. found that neural nitric oxide synthase (nNOS) may be involved in epidermal growth factor (EGF) receptor–mediated acantholysis via upregulation of Rous sarcoma kinase (Src), mammalian target of rapamycin (mTOR), and focal adhesion kinase, with eventual upregulation of caspase-9 and caspase-3. The desmosomal damage and consequent acantholysis may follow the activation of complement or local stimulation of the plasminogen–plasmin system, independent of complement (see later). However, studies using plasminogen activator knockout mice suggest that this system is not necessary for blister formation. Experimental evidence suggests that antibodies may eventually generate desmosomes lacking dsg3, resulting in acantholysis. Other mechanisms involved in acantholysis include the release of keratinocyte-derived cytokines such as IL-1 and TNF-α. In addition, the role of flotillins in this disease has recently been explored, and there is evidence that loss of flotillin expression is associated with weakened desmosomal adhesion and reduced expression of Dsg3.

These traditional views that acantholysis results from a defect in desmosomes caused by antibody/complement deposition have been challenged by several groups. The binding of antibodies leads to intracellular events that precede the acantholysis. Bystryn and Grando believe that keratinocytes separate because cytoskeletal collapse results in the inability of desmosomes to hold cells together any longer, not because of a defect in the desmosomes. The shrinkage is limited to basal cells because they are less rigid and shrink easily. It has been suggested subsequently that this cytoskeletal collapse is due to keratin intermediate filament retraction linked to plakoglobin-dependent signaling or nuclear delocalization. The entire process of antibody binding to keratinocytes, keratin retraction, cell shrinkage, and eventual acantholysis occurs very rapidly. There may be signaling events other than plakoglobin, such as heat shock protein, that trigger this cytoskeletal collapse. The importance of intracellular signaling cascades in producing blisters in pemphigus vulgaris has been confirmed in an animal model. Other studies indicate that reorganization of the actin cytoskeleton is an important factor in pemphigus vulgaris IgG–related dissociation among keratinocytes.

Activated mononuclear cells are present in lesional skin and may contribute to the pathogenesis. Autoreactive T lymphocytes that respond to epitopes located on dsg3 have been identified, confirming a role for cell-mediated as well as humoral responses in pemphigus vulgaris. This Th2 response directs autoantibody production. T cells that react against dsg1 are sometimes present. Various inflammatory mediators have been isolated from blister fluid, and these presumably have a role in eliciting the accompanying inflammatory response. Osteopontin may be one of the cytokines that drives this immune response. What stimulates the formation of antibodies is unknown, although minor trauma, surgery, ionizing radiation, thermal burns, PUVA therapy, viral infection, and exposure to chemicals have occasionally been documented before the onset of the disease. Cutaneous lesions limited to a radiation portal have been reported. Antidesmoglein autoantibodies are sometimes found in patients with no bullous disease. For example, they have been found in some patients with silicosis and in relatives of patients with pemphigus vulgaris. The term contact pemphigus has been used for cases that have followed contact with chemicals, usually pesticides. Pemphigus foliaceus has been found in dogs that were treated with the topical flea preventative metaflumizone–amitraz. Contact pemphigus has also followed the application of imiquimod. Smoking seems to have a protective function. Drugs producing pemphigus vulgaris are listed in Table 7.3 . Drugs with thiol groups have a particular capacity to precipitate or exacerbate pemphigus. Despite the long list of drugs, a recent study concluded that drugs are a rare precipitating factor. Incidental acantholysis has been found in two biopsies from a patient on enalapril who had no clinical features of pemphigus vulgaris. Carbamazepine can result in the production of antibodies to dsg1 and dsg3 without producing lesions.

The role of various foods in the precipitation of pemphigus has been studied. Possible candidates include thiol-containing foods (garlic, onion, leek, and shallots), thiocyanates (mustards), phenols (artificial sweeteners, preservatives, and colorings), and tannins (certain fruits).

The finding of HHV-8 DNA sequences in lesional skin may indicate trophism for pemphigus lesions rather than a causal role.

The desmoglein ELISA was positive in all 29 cases of pemphigus vulgaris in one series. It was superior to indirect IF as a diagnostic tool. Other studies have confirmed the reliability of the ELISA test. It may be used to monitor disease activity because the titer of circulating antibodies tends to parallel this activity. Nevertheless, circulating anti-Dsg autoantibodies are still found in some pemphigus patients in remission, particularly if their titers during the active phases of the disease were high.

Treatment

Now that a consensus statement has been produced defining aspects of the disease, including severity and end points, comparative treatment studies can be undertaken. More trials are needed to assess some of the newer therapies ; several recent reviews have been published. Therapies that have been used include systemic corticosteroids, usually in a high dose; a pulse protocol using intravenous betamethasone and oral prednisone ; cyclophosphamide ; azathioprine; prednisolone alone or combined with azathioprine, cyclophosphamide, or mycophenolate mofetil ; dapsone ; high-dose intravenous immunoglobulin, alone or as a steroid-sparing agent ; plasmapheresis; protein A immunoadsorption combined with other therapies ; gold ; rituximab ; etanercept ; thalidomide ; topical tacrolimus combined with cyclophosphamide and prednisolone ; and EGF in 0.1% silver sulfadiazine cream.

Histopathology

Established lesions of pemphigus vulgaris are suprabasal bullae with acantholysis ( Fig. 7.7 ). The clefting may extend down adnexal structures. The basal cells lose their intercellular bridges, but they remain attached to the dermis, giving a “tombstone appearance.” The blister cavity usually contains a few acantholytic cells that often show degenerative changes. Occasionally, a few eosinophils or neutrophils are present in the cavity.

The earliest changes in pemphigus vulgaris consist of edema and disappearance of the intercellular bridges of keratinocytes in the lower epidermis ( Fig. 7.8 ). This leads to acantholysis and subsequent suprabasal blisters. These changes may also occur at an early stage in hair follicles. Eosinophilic spongiosis and subepidermal splitting are two rare presentations of pemphigus vulgaris ( see Fig. 7.7C ). Vegetative lesions show epidermal hyperplasia but a paucity of eosinophils, in contrast to pemphigus vegetans. Acanthomas resembling seborrheic keratosis may form at the sites of previous blisters.

Dermal changes are of little significance. There is usually a mild, superficial, mixed inflammatory cell infiltrate that usually includes scattered eosinophils. There are no histological features that differentiate drug-associated pemphigus vulgaris from idiopathic cases.

Immunostaining with the monoclonal antibody 32-2B, which detects desmogleins 1 and 3, can be used in many cases to distinguish drug-induced pemphigus from idiopathic pemphigus. Whereas a patchy pattern of staining was observed in idiopathic pemphigus, the staining was more often normal in drug-induced cases, although patchy staining was found in 30% of cases (70% sensitivity).

Direct IF usually demonstrates IgG in the intercellular regions of the epidermis in and around the affected parts of the skin or mucous membrane ( Fig. 7.9 ). IgG1 and IgG4 are the subclasses of IgG found most commonly in patients with active lesions ; C3, IgM, and IgA are present less often. Direct IF can also be performed on plucked anagen hair. Intercellular staining is seen between cells of the outer root sheath. Patients in clinical remission, who have positive direct IF on a skin biopsy, are more likely to relapse than those with negative IF findings. Circulating intercellular antibodies are present in 80% to 90% of patients with pemphigus vulgaris, although they may be absent in early cases. Their demonstration depends to some extent on the substrate used; monkey esophagus gives a higher yield of positive results than guinea pig esophagus or human skin. Pemphigus-like antibodies have also been reported in a wide range of inflammatory dermatoses, as well as after burns.

Cytology preparations obtained from smears of bullae show acantholytic cells with ameboid cytoplasmic projections, as well as eosinophils, basophils, and lymphocytes, some of which have abnormal nuclear contours. Older bullae possess dysplastic epithelial cells with high nuclear-to-cytoplasmic ratios and prominent nucleoli. Acantholytic cells can also be appreciated with reflectance confocal microscopy.

Using a technique known as structured illumination microscopy , a form of super-resolution fluorescence microscopy , Stahley et al. found altered desmosomal protein organization, co-localization of patient IgG with markers for lipid rafts and endosomes, and reduction in size of desmosomes (in cell culture models, the pemphigus vulgaris IgG-Dsg complex is internalized from the plasma membrane through “lipid raft”–mediated endocytosis, with resultant degradation of Dsg3).

A newly developed biochip mosaic-based indirect IF technique, using recombinant antigenic substrates and transfected cells, can detect autoantibodies against Dsg3 in the serum of about 98% of patients with pemphigus vulgaris. This method has high sensitivity and specificity comparable to ELISA methods and could serve as an initial diagnostic screening test. Saliva can be used as an alternative to serum in selected circumstances, but its lack of reliable correlation with serum may make it a less than ideal substrate.

Differential diagnosis

Pemphigus vulgaris can be potentially confused with other acantholytic disorders, particularly Hailey–Hailey disease (familial benign chronic pemphigus), an inherited acantholytic disorder, and a similar but localized disorder that appears to be acquired, acantholytic dermatosis of the genitocrural region . However, those conditions usually show much more extensive suprabasilar acantholysis, with separation of keratinocytes in the spinous layer producing the appearance of a “dilapidated brick wall.” Some dyskeratosis can be seen in Hailey–Hailey disease—a feature not often seen in pemphigus. Importantly, the acantholysis in Hailey–Hailey disease “respects” the hair follicles; that is, follicular epithelia in acantholytic lesions tend to be spared. This is not the case in pemphigus. Dermal infiltrates containing eosinophils are common in pemphigus, and eosinophilic spongiosis may occur in early lesions. This cell type is less common in Hailey–Hailey disease; although eosinophils can occasionally be seen, Hailey–Hailey disease is not associated with eosinophilic spongiosis. Examples of focal acantholytic dyskeratosis, particularly transient acantholytic dermatosis (Grover's disease), can have a pemphigus vulgaris–like configuration, but often some dyskeratosis can be identified. Actinic keratoses with marked acantholysis might occasionally mimic a lesion of pemphigus vulgaris, but some degree of basilar keratinocyte atypia is usually present, and the clinical presentation would be quite different from that of pemphigus. Immunofluorescent studies would be definitive because among these conditions, only pemphigus shows positive intercellular fluorescence. Although a characteristic feature of paraneoplastic pemphigus is positive indirect IF using murine bladder epithelium (see later), some cases of nonneoplastic pemphigus vulgaris and pemphigus foliaceus are also positive using this substrate.

Histopathology

In the early pustular lesions of the Hallopeau type, the appearances resemble those of eosinophilic spongiosis with transmigration of eosinophils into the epidermis and the formation of spongiotic microvesicles and eosinophilic microabscesses. Charcot–Leyden crystals have been seen within these microabscesses. Sporadic acantholytic cells may be present, although they are not usually seen in Tzanck smears prepared from the pustules.

In early lesions of the Neumann type, there are intraepidermal vesicles with suprabasal acantholysis but no eosinophilic microabscesses.

The vegetative lesions of both types of pemphigus vegetans are similar, with hyperkeratosis, some papillomatosis, and prominent acanthosis with downward proliferation of the rete ridges ( Fig. 7.10 ). There are suprabasal lacunae containing some eosinophils. A few acantholytic cells may be present, particularly in the Neumann type.

In both types of pemphigus vegetans, the upper dermis contains a heavy infiltrate of lymphocytes and eosinophils, together with some neutrophils. There may be edema of the papillary dermis.

The direct IF findings in pemphigus vegetans are similar to those in pemphigus vulgaris with the intercellular deposition of IgG and C3. Circulating antibodies to the intercellular region are usually detectable by indirect IF.

Differential diagnosis

The histological features of PPV show some similarities to pemphigus vegetans. There are intraepithelial and subepithelial microabscesses with an admixture of neutrophils and eosinophils ( Fig. 7.11 ). There is a moderately heavy, mixed inflammatory cell infiltrate in the underlying dermis. The lesions are usually more inflammatory than the lesions of pemphigus vegetans. Epithelial hyperplasia is variable and usually more marked in oral lesions. Acantholysis is focal and never severe. Direct IF is negative, if misdiagnosed cases of pemphigus vegetans are excluded. However, there is a report of two cases presenting clinically as PPV, associated with inflammatory bowel disease, in which direct IF showed intercellular IgA deposition. It remains to be determined whether other cases presenting as PPV might also show IgA deposits.