The two major diseases in the group of subepidermal/epithelial AIBD with cleavage in the LL are bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP), formerly called cicatricial pemphigoid (CP) [12]. In spite of their similarity, BP and CP exhibit several differences when a fine analysis by TEM is performed. The HDs at the roof of the cleavage are absent, whereas the ultrastructure of the floor is normal in BP (Fig. 19.3a–c). In contrast, the HDs along the cytoplasmic membrane of basal keratinocytes are normal, but a thickening or focal interruptions of the LD in the floor of the cleavage are observed in the CP (Fig. 19.3d) [13–15].

The studies using direct IEM allow differentiating the BP of CP in most of the cases. The first case reports with studies by direct IEM were published between 1975 and 1982 and large prospective series in 1987 and 1998 [4, 16–18]. In the skin of patients with BP, the immune deposits along unseparated zone of DEJ appear very thin. They occupy exclusively the LL and the LD is easily identified beneath them. They are very close to the plasma membrane of basal keratinocytes and stop in front of melanocytes (Fig. 19.4). Sometimes, they overflowed into the HD attachment plaque of necrotic basal keratinocytes. An infraclinical cleavage of DEJ is rarely observed in the skin of BP patients processed for IEM. If present, it is then seen in LL, and immune deposits decorate its roof (Fig. 19.5). In the skin or mucous membranes of patients with classical mucocutaneous CP, the immune deposits along unseparated zone of DEJ or CEJ appear thick, approximately two to three times the width of the deposits seen in BP. They are irregular in thickness, mostly overlying the LD and occasionally the LL (Fig. 19.6) [19]. A clear space is frequently seen between immune deposits and cytoplasmic membrane of keratinocytes. The labeling of DEJ/CEJ stops where the LD is destroyed. A subclinical split of DEJ or CEJ is frequently observed in the skin or mucous membranes of CP patients processed for IEM. It is then seen in LL, and immune deposits decorate both its roof and floor. Concordant results were obtained for testing of skin and different mucous membranes in the same patient and for ultrastructural localization of Ig and C3 in the same sample. Very few studies by direct IEM of anti-laminin-332 (LN-332) MMP (formerly called anti-epiligrin and anti-laminin 5 CP) have been published [20–22]. In this form of CP, immune deposits localized to the lower LL at its interface with the LD, exclusively to the floor of cleavage in split zone. In patients with a pure ocular CP, the localization of immune deposits in their conjunctiva may be similar to that observed in the skin of patients with a BP. Very thin deposits are observed at the upper LL region immediately subjacent to the HD (Fig. 19.7) [23]. The fineness of deposits can explain the negativity of direct IF in some patients [24]. This pattern is consistent with studies showing that the target antigen of Ab in these pure ocular CP is the chain β[beta]4 of integrin α[alpha]6β[beta]4, a transmembrane molecule of the HD [25].

The studies using indirect IEM show also differences between the sera of patients with BP and CP (Fig. 19.8). In BP, target antigens of circulating auto-Ab are clearly associated with HD and distributed in two pools. On the one hand, there is a large intracellular pool, closely related to the attachment plaque of HD; on the other hand a small extracellular pool, in the upper lamina lucida close to HD [5, 26, 27]. More recent studies demonstrated that the first one corresponds mainly to the BP230 antigen and the second to the NC16a epitope of the BP180 antigen on which auto-Ab bind in vivo in the patient’s skin [7, 28]. In the skin of patients with BP, the intracellular BP230 antigen is not recognized by auto-Ab because large molecules such as Ig cannot penetrate the cytoplasmic membrane of keratinocytes. In the CP, the target antigens of circulating Ab are exclusively extracellular in most cases, located at the lower part of the LL and on the LD, on the anchoring filament beneath the HD, averaging 40–48 nm from the cytoplasmic membrane of keratinocytes [5, 7]. In double labeling, they colocalize with the carboxy-terminal region of BP180 antigen and LN-332. In anti-LN-332 MMP, studies by indirect IEM showed immune deposits localized to the lower LL at its interface with the LD, as in direct IEM [20]. The differences in localization patterns exhibited by BP and CP autoantibodies might well help to explain the observed clinical differences in these diseases, e.g., scarring in the CP versus non-scarring in the BP.

Pemphigoid gestationis (PG) is another, much rarer subepidermal AIBD with cleavage in the LL. It occurs during the last trimester of pregnancy and is usually regressive within 3 months after delivery. There is now evidence that it is secondary to IgG1 directed against the epitope NC16a of the BP180 antigen. Therefore, the localization of immune deposits in direct and indirect IEM is completely superimposable to that observed in the BP, namely, deposits in the upper part of the LL [29, 30].

The main disease in the group of subepidermal/epithelial AIBD with cleavage below the LD is epidermolysis bullosa acquisita (EBA). As early as 1979 the TEM studies in EBA showed a cleavage under the LD clearly different from that in the LL observed in the BP and CP. A careful analysis in unseparated zone also showed destruction of AF or the presence of a broad weakly electron-dense amorphous deposit (corresponding to immune deposits sufficiently intense to be seen by TEM) (Fig. 19.9) [31–33].

The first case reports of EBA studied by direct IEM with peroxidase labeling were published in the 1980s [33–35] and large prospective series of 231 patients including 30 EBA and of 19 EBA in black patients of African descent later, respectively, in 1996 and 2011 [18, 36]. In the skin of patients with EBA, the immune deposits along unseparated zone of DEJ appear extremely thick. They are most often located in the AF zone, away from the LD in which they are separated by a clear space. Duplication of the LD and AF zone underlying can be observed (Fig. 19.10). Sometimes, particularly in the inflammatory form of the disease are deposits both in the AF zone and overlying the LD (Fig. 19.11). An infraclinical cleavage of DEJ is very frequently seen in the skin of EBA patients processed for IEM. It sits generally under deposits which therefore are located on the roof of cleavage (Figs. 19.10 and 19.11).

The studies by indirect IEM with peroxidase labeling of the target antigens of circulating auto-Ab in patients with EBA give a slightly different pattern. Deposits are seen below the LD, but they only delicately decorated the AF, usually at their ends (Fig. 19.12).

Direct and indirect IEM with gold particle studies confirm, in most patients, the labeling of AF ends that corresponds to auto-Ab directed against the type VII collagen N-terminal noncollagenous (NC) 1 domain [37–41]. However, there are discrepancies between these different studies on the precise location of AF ends. For Karpati et al. and Ishiko et al., they are localized both at the lower part of the LD and on the dermal anchoring plaques (Fig. 19.12), while for Mc Millan et al. and Ishiii et al., they are exclusively on the LD. A labeling of the central banded portion of the AF has also been reported by Tanaka in three patients whose sera reacted with the central triple-helical domain of type VII collagen [8]. Lastly, an exclusive dermal labeling has been reported by Ishii and Hashimoto [41, 42]. Ishii et al. clearly demonstrated that these patients’ sera, showing an immunoreactivity in the dermis 300–360 nm below the LD, reacted with the type VII collagen N-terminal NC2 domain.

Bullous systemic lupus erythematosus is another, much rarer subepidermal AIBD with cleavage below the LD. Patients fulfill the American College of Rheumatology diagnostic criteria for systemic lupus erythematosus and have concomitantly a vesiculobullous eruption, in relation to auto-Ab against type VII collagen [43]. Therefore, direct and indirect IEM analyses show both granular immune deposits in the superficial dermis which correspond to the large “lupus band” observed by indirect IF and linear immune deposits below the LD, as in EBA (Fig. 19.13) [44]. Sometimes immune deposits are also seen in the capillary walls.

Linear IgA bullous diseases (LABD) are subepidermal/epithelial AIBD, characterized by linear deposition of IgA auto-Ab at the DEJ. They are a quite heterogeneous group under the EM. Indeed, LABD may not be a single entity but may possess target antigens shared with other subepidermal AIBD, namely, the BP, CP, and EBA [45].

Direct IEM shows many different patterns of Ab deposition in LABD. All studies have been reported before 1999 and referred by Egan et al. [45, 46]. IgA auto-Ab were localized to the LL, either on the basal surface of keratinocytes or adjacent to the HD, the LD, and the AF zone or on each side of the LD in a “mirror image” pattern, i.e., both within the LL and the sub-LD region, in respectively 26, 4, 49, and 20 % of a total of 67 patients (Fig. 19.14).

All studies by indirect IEM in LABD have been reported between 1994 and 1998, apart from that of Bhogal in 1987 [45, 47]. They cover a total of 38 patients who were selected on the presence of a high titer of Ab in their serum. The vast majority (82 %) of these patients had sera labeling the roof of salt-split skin (SSS) by indirect IF and LL by indirect IEM [9, 10, 48, 49]. A minority (18 %) had sera labeling the floor of SSS by indirect IF and the AF zone by indirect IEM [9, 42, 47]. In one of these last cases, the serum labeled the AF dermal end but did not label the LD [42]. To our knowledge, no study by indirect IEM with LABD sera labeling both sides of SSS by indirect IF [50] has been reported.

Correlation of immunoblotting and immunogold EM studies shows that all the auto-Ab recognizing proteins in epidermal extracts of 180 kDa (or its breakdown products of 97 or 120 kDa), 200 kDa, or 230 kDa bound to the LL [9, 10, 48, 51]. The LABD97 antigen is a conformation-sensitive epitope of BP180 which needs a study by immunogold EM with cryoultramicrotomy to be demonstrated instead of cryosubstitution [10]. In contrast, auto-Ab binding to the AF zone and blotting type VII collagen has been reported in only four case reports [42, 52–54]. Indeed, dermal target antigens of auto-Ab in LABD are still only partially characterized. On the one hand, Dmochowski et al. and Egan et al. reported, respectively, 4 and 17 LABD whose sera had dermal binding of IgA on IIF and did not react with type VII collagen on immunoblot [45, 55]. Unfortunately, these sera have not been tested by indirect IEM. On the other hand, most of the patients with dermal binding of IgA on direct IEM had no circulating auto-Ab allowing indirect IEM, immunoblotting, and ELISA studies [46, 47]. These data may explain the apparent discrepancy in the LL labeling versus that of the AF zone, between direct and indirect IEM studies reported in the literature.

In most forms of EBS, the split occurs through the lower part of basal keratinocytes beneath their nuclei (Fig. 19.16). The distance, usually very small between the cleavage and BMZ itself, explains that in most cases the cleavage seems to be subepidermal/epithelial in light microscopy. Other ultrastructural changes can be observed. They are particularly useful when no cleavage is seen by TEM. In both subtypes of dominant classical EBS (localized and generalized EBS formerly called EBS Weber–Cockayne and Koebner), at first, the cytoplasm with the cell organelles appears to become diluted, and then holes develop in the cytoplasm, merge, and finally form cytolytic blisters. Although due to mutations in the genes encoding K5 or K14 keratins, the tonofilaments as well as all other constituents of the basal keratinocytes look perfectly normal [56, 57]. In contrast, in other EBS subtypes, ultrastructural abnormalities of tonofilaments can clearly provide clues to the underlying molecular defects of keratins. In generalized EBS Dowling–Meara, they can form clumps within basal keratinocytes that are due to abnormalities in the N- or C-terminal segment of the rod domain of K5 or K14 keratins, instead of the central part in classical EBS [58, 59]. In generalized autosomal recessive EBS caused by null mutations in the K14 gene, the cytoplasm in the basal keratinocytes is clarified, devoid of tonofilaments. A thin meshwork of filaments made of thin protofilaments is seen in places [60–62]. In EBS with muscular dystrophy (EBS-MD) or pyloric atresia (EBS-PA), the cleavage is located just above the HDs whose attachment plaques are small or absent. This pattern is explained by the plectin or integrin α[alpha]6β[beta]4 defects underlying this subtype of EB [63–66]. However, this pattern is not pathognomonic and may be misleading because a similar pattern has been reported in EBS without muscular dystrophy or pyloric atresia related to defect of cytoplasmic domain of integrin β[beta]4 [67] or BP180/collagen XVII [68, 69] or BPAG1-e/BP230 antigen [70].

All the JEB are characterized by separation through the LL of the BMZ (Fig. 19.17). Nevertheless, they display a remarkable clinical, genetic, and ultrastructural heterogeneity [71]. They include two subtypes: the very severe lethal JEB Herlitz (JEB-H) and JEB other (JEB-O) including the JEB non-Herlitz. In the first subtype, the majority of the mutations have been identified in the three genes encoding LN-332, while in the second subtype, they are usually in LN-332 or collagen XVII genes. Extracellular portion of collagen XVII and LN-332 are both components of anchoring filaments beneath HDs whose defect explains very well the cleavage in the LL. JEB-O include also some EB with pyloric atresia, named JEB-PA, that results from mutations in integrin α[alpha]6 and β[beta]4 genes [66]. A number of studies had shown qualitative and quantitative ultrastructural changes of HDs which had been initially considered as the primary defects and of diagnostic value [72–76]. In view of the current genetic data, they now appear to be secondary rather than primary. Usually HDs are sparse and small with poorly formed sub-basal plate in JEB-H, while they are normal in number and appearance in JEB-O. However, normal HDs have been reported in the JEB-H subtype and vice versa hypoplastic HDs in the JEB-O one [77–80]. Finally, an abnormal insertion of tonofilaments on the HD attachment plaque has been described in some patients [76, 81]. It may explain the extension of LL cleavage at the basal pole of keratinocytes. Therefore, even though the HD abnormalities are more frequent in the JEB-H, they do not correlate sufficiently well with the clinical outcome to be useful as a prognostic indicator.

DEB is characterized by cleavage immediately below the LD (Fig. 19.18). Several studies using EM and/or IEM and trying to correlate clinical and ultrastructural phenotypes and genotype were published before the new nomenclature of EB [1, 82–86]. All authors agree that most patients with HS-RDEB/RDEB-sev-gen have no AF in EM and no immunostaining of DEJ by IF and IEM using a monoclonal Ab (LH 7:2) that binds to the NC-1 globular domain of type VII collagen. This is explained by the absence or drastic reduction of type VII collagen synthesis, which itself is the result of mutations in the type VII collagen gene (COL7A1) leading to premature stop codons. Nevertheless, some patients with RDEB-sev-gen and normal or near-normal collagen VII immunostaining have been reported [86]. They had fewer AF which were shorter and thinner than normal, without characteristic, centrosymmetric banding pattern in EM. IEM showed no type VII collagen immunostaining or a normal labeling of both the LD and the dermal anchoring plates or a labeling restricted to the LD (Fig. 19.19). This last aspect suggests a defect in the alignment of two type VII collagen molecules into antiparallel dimers. A contrast between a quite normal type VII collagen immunostaining in IF and absent AF in EM has also been reported in RDEB-I [87, 88]. In DDEB and RDEB-O, the variability in the clinical phenotype is accompanied by heterogeneous type VII collagen immunostaining by IF and anchoring fibril morphology by EM: labeling by LH7:2 may be normal or decreased and present AF more or less rare, short, and thin (Fig. 19.18d). A detailed quantitative analysis failed to find significant differences in AF between these two subtypes [82]. In conclusion, although RDEB-sev-gen are usually recognized by the combination of negative type VII collagen immunostaining in IF and absence of AF in EM, the DDEB and RDEB-O subtypes cannot be differentiated on the basis of IF and TEM studies.