The carboxyl-terminal domain of type VII collagen is present at the basement membrane in recessive dystrophic epidermolysis bullosa

Recent studies showing that type VII collagen is a component of anchoring fibrils suggests that the absence of anchoring fibrils in recessive dystrophic epidermolysis bullosa may be due to a defect in the synthesis, secretion, and deposition of type VII collagen. That hypothesis is further supported by recent studies suggesting that monoclonal antibodies to type VII collagen do not react with the basement membrane in most patients. To investigate further, we examined skin from 12 patients by electron microscopy and by immunohistology and immunoelectron microscopy using a concentrated and purified monoclonal antibody to the carboxy-terminal domain of Type VII collagen. Although anchoring fibrils were not detected by electron microscopy, the results of immunohistology showed definite, but reduced, binding of the monoclonal antibody to type VII collagen at the basement membrane in a linear pattern in 11 of 12 patients. By immunoelectron microscopy, reduced deposition of anti-type VII collagen antibody was detected beneath the lamina densa. The results of this study show that the carboxyl-terminal domain of type VII collagen is synthesized, secreted, and deposited at the basement membrane zone in 11 of 12 patients with recessive dystrophic epidermolysis bullosa and suggest that the absence of anchoring fibrils may be due either to deposition of abnormal type VII collagen, reduced levels of normal type VII collagen, defective assembly of type VII collagen into anchoring fibrils, or destruction of the collagenous domain of type VII collagen.     

Type VII collagen is expressed but anchoring fibrils are defective in dystrophic epidermolysis bullosa inversa

A patient with dystrophic epidermolysis bullosa inversa was studied using electron microscopy and indirect immunofluorescence using antibodies to matrix macromolecules of the dermoepidermal junction zone. There was splitting below the lamina densa with an apparently normal basement membrane, but a lack of intact anchoring fibrils and with a disarranged papillary connective tissue. Indirect immunofluorescence examination with antibodies to type VII collagen, the major structural protein of anchoring fibrils, showed a normal linear staining pattern. Synthesis of type VII collagen which is unable to form stable, resistant anchoring fibrils may be a distinct feature of this subtype of recessive dystrophic epidermolysis bullosa.     

Anchoring fibrils, collagen VII, and neutral metalloproteases in recessive dystrophic epidermolysis bullosa inversa.

Structure of the anchoring fibrils, expression of collagen VII, and gelatinolytic activity in skin fibroblasts were assessed in six patients with epidermolysis bullosa dystrophica (EBD) inversa and in control groups consisting of probands with other EBD subtypes and healthy individuals. All six patients with EBD inversa, as well as the patients with generalized non-mutilating and localized EBD, showed positive staining with antibodies to collagen VII, the major anchoring fibril protein. Four patients with severe generalized mutilating EBD exhibited negative staining. Ultrastructurally, normal anchoring fibrils were demonstrable in uninvolved skin of patients with localized, inversa, and generalized non-mutilating subtypes. At the same time, a high degree of variability was observed in the amount and quality of anchoring fibrils in the various stages of lesional skin, including co-existence of normal and partially degraded anchoring fibrils. Of all 12 patients only one localized and two inversa cases showed an increased gelatinolytic activity in vitro. However, the high activity was associated with neither the severity of the disease nor the inversa subtype. In addition, intact collagen VII could be extracted from the dermis of one inversa patient. The present data show no correlation between increased in vitro gelatinolytic activity and abnormalities of the anchoring fibrils or collagen VII in skin of patients with recessive EBD, and therefore suggest molecular heterogeneity of the causative pathogenetic mechanisms.     

Molecular basis for the dystrophic forms of epidermolysis bullosa: mutations in the type VII collagen gene

Significant progress has recently been made in understanding the molecular basis of heritable skin diseases, such as epidermolysis bullosa, a group of mechano-bullous genodermatoses. In particular, the dystrophic forms of epidermolysis bullosa have been shown to result from distinct mutations in the gene encoding type VII collagen, the major, if not the exclusive, component of the anchoring fibrils. These mutations result in deficient synthesis and/or altered assembly of the anchoring fibrils, thus compromising the integrity of the cutaneous basement membrane zone. The mutations in the type VII collagen gene have implications for understanding the structure-function relationships of the type VII collagen molecule, and also provide the basis for prenatal DNA-based diagnosis in families at risk for recurrence of the disease. Finally, understanding the genetic basis of dystrophic forms of EB sets the stage for gene therapy approaches for the treatment of these devastating skin diseases.     

Molecular biology and pathology of type VII collagen.

Type VII collagen is a genetically distinct member of the collagen family of proteins. Type VII collagen has been shown to be the major component of anchoring fibrils, attachment complexes which secure the cutaneous basement membrane of the skin to the underlying dermis. Understanding of the structure of type VII collagen has been advanced by recent cloning of the corresponding gene. Chromosomal mapping of the gene to the short arm of chromosome 3 and identification of intragenic polymorphic markers have allowed demonstration of strong genetic linkage between the type VII collagen locus and the dystrophic forms of EB (epidermolysis bullosa). This overview summarizes the progress made in the molecular genetics of type VII collagen.     

Keratinocytes and fibroblasts from a patient with mutilating dystrophic epidermolysis bullosa synthesize drastically reduced amounts of collagen VII: lack of effect of transforming growth factor-beta.

Keratinocytes and fibroblasts derived from skin of a patient with recessive dystrophic mutilating epidermolysis bullosa (EB) did not synthesize collagen VII as assessed by indirect immunofluorescence staining or immunoblotting, but expressed another basement membrane protein, laminin, in a normal manner. In contrast to control cells, no stimulation of collagen VII production was achieved in co-cultures of EB keratinocytes and fibroblasts. Further, treatment of normal keratinocytes or co-cultures with TGF-beta 2 significantly increased their expression of collagen VII, whereas the cytokine failed to induce its synthesis in the EB cells. Mixed co-cultures were constructed with normal fibroblasts and EB keratinocytes and vice versa. Both combinations showed strong expression of collagen VII in the normal cells but no synthesis in the EB counterparts. These results suggest that in this patient with severe mutilating dystrophic EB, inability of cutaneous cells to synthesize sufficient amounts of collagen VII underlies the lack of anchoring fibrils and skin fragility.     

Lack of type VII collagen in unaffected skin of patients with severe recessive dystrophic epidermolysis bullosa

Type VII collagen, the major structural component of the anchoring fibrils, was assayed in normal unaffected skin of patients with different forms of hereditary epidermolysis bullosa. Immunofluorescence staining with affinity-purified polyclonal antibodies to type VII collagen revealed a complete absence of staining in the skin of patients with severe dystrophic recessive epidermolysis bullosa. In all other forms, localized recessive dystrophic, dominant dystrophic, junctional and simplex forms there was an intense continuous linear staining of type VII collagen at the dermoepidermal junction. Also, obligate heterozygote carriers of the gene for severe dystrophic recessive form showed a normal pattern of staining. As internal controls and to define the clinical diagnosis, staining with antibodies to type IV collagen, laminin and bullous pemphigoid antigen was also performed. All these antibodies showed a normal staining pattern indicating an intact general morphology of the dermoepidermal junction zone. These results suggest that there is a defect of type VII collagen in patients with severe recessive dystrophic epidermolysis bullosa. The data also suggest that the group of recessive dystrophic epidermolysis bullosa may be heterogeneous not only clinically, but also at the molecular level.     

Evidence for a structural abnormality of collagen VII in a patient with dystrophic epidermolysis bullosa inversa.

Recent studies indicate that in skin of patients with dystrophic epidermolysis bullosa (EB) inversa, anchoring fibrils have an abnormal ultrastructure, but the major protein of these fibrils, collagen VII, is expressed and detectable with antibodies at the dermo-epidermal junction. For molecular characterization of this rare EB phenotype, skin biopsies from a patient with dystrophic EB inversa were investigated with indirect immunofluorescence, immunoelectron microscopy, and immunoblotting. Ultrastructural analysis of clinically uninvolved skin showed sublamina densa splitting. In unblistered areas, focal groups of anchoring fibrils that appeared loosely polymerized and without a distinct crossbanding pattern were observed. Indirect immunofluorescence staining with antibodies to collagen VII exhibited a linear fluorescence at the dermo-epidermal junction and at the roof of a spontaneous blister. Immunoelectron microscopy demonstrated staining of the poorly assembled anchoring fibrils, but no significant reaction in areas where no fibrillar structures could be discerned. In contrast to normal control skin, immunoblotting showed immunoreactive collagen VII in both epidermal and dermal extracts. Moreover, the dermis-associated collagen VII appeared as a distinct doubleband that contained the tissue form of collagen VII (250-300 kD) and an additional band with a slightly smaller molecular weight. In epidermal extracts one band, of the size of the tissue form, was detected. The studies on the present patient suggest that a structural abnormality of collagen VII that prevents its aggregation to stable dimers or polymerization to distinct anchoring fibrils may contribute to the etiopathogenesis of dystrophic EB inversa.     

Immunomapping of EBA sera to multiple epitopes on collagen VII: further evidence that anchoring fibrils originate and terminate in the lamina densa

Epidermolysis bullosa acquisita (EBA) is an autoimmune blistering disease with circulating antibodies to type VII collagen, a major component of anchoring fibrils located at the dermal-epidermal junction. The purpose of this study was to further confirm the ultrastructural organisation of anchoring fibrils and to assess the relationship between the clinical phenotype of EBA and target site of their autoantibodies on anchoring fibrils. We studied the ultrastructural binding site of circulating autoantibodies from two patients with atypical clinical features who predominantly presented with oral lesions, and compared this with two patients with clinically typical forms of EBA. Immunoblotting of whole dermal extracts showed labelling of 290-kDa bands consistent with that of type VII collagen as well as the non-collagenous (NC-1) domain fusion protein in three out of four patients' sera. Postembedding immunoelectron microscopy (IEM) using Lowicryl K11M embedded normal human skin and patients' sera demonstrated the majority of labelling within the lamina densa, not below the lamina densa. We conclude that EBA autoantibodies in these patient's sera bind to the NC-1 domain of collagen VII situated in the lamina densa of the epidermal basement membrane, regardless of the EBA clinical phenotype. This confirms the previous notion that anchoring fibrils originate and terminate in the lamina densa.     

Fibrillin immunoreactivity is associated with normal or fragmented elastic microfibrils at the dermal-epidermal junction in recessive dystrophic epidermolysis bullosa

Elastic microfibrils containing fibrillin are a part of the fibroreticular network of normal epidermal basement membrane. In dystrophic epidermolysis bullosa (DEB) at least one other fibroreticular component, anchoring fibrils, which contain type VII collagen, is known to be abnormal. We therefore questioned whether elastic microfibrils and fibrillin expression might also be abnormal in DEB. By indirect immunofluorescence, and pre-embedding immunogold electron microscopy using a monoclonal antifibrillin antibody, we found no difference from control samples in either the quantity of the labelling or in the ultrastructural appearances of the immunolabelled fibrils in intact DEB skin. In areas of dermal-epidermal separation, however, we observed a number of thin, fragmented sublamina densa wisp-like structures, which still labelled for fibrillin despite lacking the typical ultrastructural features of normal elastic microfibril bundles. As a consequence of blistering in DEB, elastic microfibril bundles are disrupted, and fragmented microfibrils may still remain attached to the blister roofs. Many of these elastic microfibrils cannot be distinguished from rudimentary or altered anchoring fibrils on morphology alone, and might therefore account for misinterpretation of ultrastructural disorders of the dermal-epidermal junction. We postulate that, in intact skin, elastic microfibrils might contribute to dermal-epidermal adherence, in the absence of normalfunctioning anchoring fibrils.     

Animal model for dermolytic mechanobullous disease: sheep with recessive dystrophic epidermolysis bullosa lack collagen VII

A severe congenital mechanobullous disease with dermolytic blistering and recessive inheritance is described in sheep. The affected animals of wild and inbred flocks of the breed Weisses Alpenschaf (WAS) have blisters of skin, oral mucosa, tongue, and esophagus at birth or within the first week of life. Exungulation occurs early, and severe erosions in the mouth lead to difficulty in feeding. Electron microscopic examination revealed sub-lamina densa splitting in natural or fresh friction blisters and absence of identifiable anchoring fibrils in clinically uninvolved skin. Antigen mapping localized laminin and collagen IV to the blister roof. Indirect immunofluorescence staining with antibodies to collagen VII, the major structural component of the anchoring fibrils, demonstrated a complete absence of reaction in clinically uninvolved tissues of the affected sheep, whereas in normal sheep a strong linear fluorescence was seen at the epithelial-mesenchymal basement membrane zone. Dermal extracts of normal sheep contained intact collagen VII, but epidermal and dermal extracts from the affected sheep lacked this collagen or its fragments in immunoblotting experiments. Based on genetic, clinical, ultrastructural, and immunochemical findings, the sheep disorder corresponds to the severe mutilating subtype of recessive dystrophic epidermolysis bullosa in humans and can be used as an animal model to investigate the human disorder.     

Immunofluorescent analysis of the basement membrane zone in lichen planus suggests destruction of the lamina lucida in bullous lesions

Lichen planus is an inflammatory dermatosis which is characterized histologically by an intense lymphocytic infiltrate at the dermal epidermal junction. This frequently results in disruption of the basement membrane zone, occasionally causing clinical blisters. In order to better understand the specific portion of the basement membrane zone which is disrupted by the lymphocytic infiltrate, we examined 7 cases of lichen planus with antibodies directed against anchoring filaments (GB3), the bullous pemphigoid antigen, anchoring fibrils (type VII collagen) and type IV collagen. In lesions without separation at the BMZ, all antibodies were strongly expressed, as in normal skin. In lesions with early separation, there was a focal decrease in GB3 staining, but types VII and IV collagen labelled normally. In lesions resulting in blisters, GB3 staining was essentially absent, and anti-types IV and VII collagen remained, but stained in a disrupted, less discrete pattern. The bullous pemphigoid antigen showed only slight deviation from the normal staining pattern. These findings suggest that the basement membrane zone in lichen planus is disrupted in the lamina lucida region. The lamina densa and sub-lamina clensa zones remain intact even in bullous lesions of lichen planus.     

IgA-epidermolysis bullosa acquisita in a child resulting in blindness

Epidermolysis bullosa acquisita (EBA) is an acquired subepidermal bullous disease characterized by IgG autoantibodies directed against type VII collagen, the major component of anchoring fibrils. The classical phenotype of EBA is a non-inflammatory, mechanobullous disease resembling the dystrophic forms of inherited epidermolysis bullosa. Mucous membrane involvement is frequent but usually mild. We report a 1-year-old girl suffering from IgA-EBA, who presented with an initial eruption of disseminated urticarial lesions and tense blisters of the skin but subsequently developed severe oral and ocular lesions reminiscent of cicatricial pemphigoid. Direct immunofluorescence of the skin and buccal mucosa revealed linear IgA and C3 at the basement membrane zone (BMZ). IgA anti-BMZ autoantibodies stained the dermal side of salt-split skin by indirect immunofluorescence and recognized a dermal protein of 290 kDa co-migrating with type VII collagen by immunoblotting. Direct and indirect immunoelectron microscopy revealed IgA deposits overlying the anchoring fibrils. The ocular involvement led to total blindness in spite of intense treatment. This case of childhood IgA-EBA is particularly striking because of the cicatricial pemphigoid phenotype with severe ocular involvement which resulted in blindness. It reinforces the necessity to use modern immunological methods to classify autoimmune bullous diseases in order to allow early and appropriate treatment.     

A novel homozygous splice site mutation in COL7A1 in a Chinese patient with severe recessive dystrophic epidermolysis bullosa and squamous cell carcinoma

Background Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in COL7A1 gene encoding type VII collagen, the major component of anchoring fibrils in the dermo–epidermal junction. The development of cutaneous squamous cell carcinoma (SCC) is one of the most serious complications of this disease. We report herein a Chinese patient with the severe generalized subtype of RDEB (RDEB‐sev gen) complicated by SCC. Methods Skin biopsies were examined for histology, basement membrane ultrastructure, and type VII collagen expression. Genomic DNA was extracted from the peripheral blood samples and subjected to polymerase chain reaction amplification and direct automated DNA sequencing. Results Histopathological examination of the patient’s skin revealed an undetectable expression of type VII collagen polypeptides in the basement membrane zone. Mutation analysis identified a novel splice site mutation in intron 64 (IVS64+5g‐>a) of COL7A1 gene, which resulted in an in‐frame deletion of exon 64 in both alleles. Conclusions This report contributes to the expanding database of COL7A1 mutations and emphasizes the need to elucidate the underlying genetic mechanisms associated with the increased incidence of SCC in RDEB patients.     

A Case of Pretibial Dystrophic Epidermolysis Bullosa: Decreased Expression of the Non-helical Domain of Type VII Collagen Molecule

A 27-year-old man with an ataxic gait due to infantile cerebral paralysis exhibited recurrent blistering caused by mechanical stimuli on the pretibial areas of both legs from the age of 20. His parents were not consanguineous, and he had no relatives who suffered from blistering. The histology showed a subepidermal bulla due to dermolytic epidermal-dermal separation. The anchoring fibrils were sparse and rudimentary in the predilection area. An LH 7:2 monoclonal antibody against the non-helical domain of the type VII collagen molecule stained the basement membrane zone of the patient's skin at a weaker intensity than the staining of normal human skin, but at a distinctively stronger intensity than the staining of skin from a patient with recessive dystrophic epidermolysis bullosa. Immunoelectron microscopy revealed that LH 7:2-immuno-reactants were distributed irregularly within the lamina densa and sparsely in the sublamina densa region. The patient was diagnosed with pretibial dystrophic epidermolysis bullosa.     

Epidermolysis bullosa acquisita antigen and the carboxy terminus of type VII collagen have a common immunolocalization to anchoring fibrils and lamina densa of basement membrane

In this study a variety of immunoelectron microscopic methods were used to define the precise ultrastructural binding site of epidermolysis bullosa acquisita antibodies (EBA-Ab). We used two EBA sera which immunoblotted with the same skin-extracted protein as that labelled by a monoclonal antibody (LH7.2) which is known to react with the carboxy terminus of type VII collagen. Gold-conjugated antibodies were used in two different immunoelectron microscopic procedures to compare the labelling characteristics of EBA-Ab and LH7.2 in normal human skin. Antibody incubations were performed using ultra-thin cryosections of unfixed skin and thin slices of fresh skin (en bloc technique) before conventional fixation and embedding in Epon. Both methods showed similar labelling features for both EBA-Ab and LH7.2. With ultra-thin cryosections there was labelling of the lamina densa and an undefined component of the sublamina densa region. With the en bloc technique, labelling of dermal ends of anchoring fibrils and of amorphous material recently defined as 'anchoring plaques' was evident. There was no labelling of the central banded portions of anchoring fibrils. We conclude that EBA-Ag is localized to the dermal ends of anchoring fibrils in addition to the lamina densa and possibly anchoring plaques, and thus has the same distribution as the carboxy terminus of type VII collagen.     

Intra-epidermal retention of type VII collagen in a patient with recessive dystrophic epidermolysis bullosa

An infant born with severe blisters on the limbs, face, trunk, and oral mucosa was diagnosed by light and electron microscopy to have recessive dystrophic epidermolysis bullosa. Transmission electron microscopy showed that the basal lamina remained with the epidermis and that the floor of the blister was exposed collagen of the papillary dermis. No banded anchoring fibrils were observed along either the roof or the floor of the blister; however, small filamentous structures, possibly immature anchoring fibrils, extended down from the lamina densa along the blister roof. Some basal and suprabasal keratinocytes contained large vesicles filled with filamentous matrix of variable electron density. Immunofluorescent staining of skin for type VII collagen showed sparse and irregular staining of type VII collagen along the blister roof, and intense intracellular labeling for type VII collagen in clusters of epidermal cells in basal and suprabasal layers. Type VII collagen appeared to be synthesized by keratinocytes but not secreted.     

Pathogenesis of mechanobullous disorders

Recent advances in the molecular biology of the dermo-epidermal basement membrane zone have contributed greatly to our understanding of the etiopathogenetic pathways underlying mechanobullous disorders. Genetic linkage was established between the keratin gene clusters and epidermolysis bullosa simplex, and keratin mutations were identified in several patients. Anchoring filaments and the alpha 6 beta 4 integrin are likely to be affected in junctional EB. Genetic linkage was established between the collagen VII gene and both dominant and recessive subtypes of dystrophic epidermolysis bullosa, and different molecular abnormalities of collagen VII leading to formation of non-functional, rudimentary anchoring fibrils were observed in several families. These discoveries that led to definition of mutations underlying EB also help us to understand the normal physiology and function of the affected structures. They may also point the way to new therapeutic strategies for common acquired blistering diseases and disturbances of epithelialization in general.     

Ontogenesis of the basement membrane zone after grafting cultured human epithelium: a morphologic and immunoelectron microscopic study.

Sheets of cultured epithelial cells have been successfully used as autografts for the permanent coverage of patients with full-thickness burns and as allografts to stimulate the healing of chronic skin ulcers. The basement membrane zone (BMZ), composed of lamina lucida, lamina densa, and anchoring fibrils, plays a pivotal role in the firm adherence of the epidermis to the dermis. The present study describes the ultrastructural development during various stages of wound healing after resurfacing different wound areas by cultured epithelial grafts. For this purpose, biopsies were obtained from five patients 5 d to 4.5 years after resurfacing full-thickness burns with cultured autografts, and from five patients at various stages after treatment of excised tattoos and chronic skin ulcers with cultured allografts. One biopsy was taken from a spontaneously healed burn wound 30 years post-injury. Ultrathin sections were prepared for transmission and immunoelectron microscopy, using monoclonal antibodies against type IV and VII collagen. Findings were compared to controls of age- and site-matched normal skin. Eleven days after grafting, the first BMZ features had regenerated, including lamina lucida, a discontinuous lamina densa, hemidesmosomes, and sparse anchoring fibrils. The process of de novo synthesis of BMZ components had begun, and within 4 to 5 weeks complete reformation of BMZ was observed, including normal distribution of anchoring fibrils. Immunolabeling of type VII collagen was first observed upon the lower part of lamina densa at day 11 and steadily increased, reaching normal values 5 weeks after grafting. In contrast, gold deposition of type IV collagen upon lamina densa was strongly increased at day 19 compared to normal. This high expression reduced a little at 5 weeks, but remained high up to 30 years after injury. Long-term burn scars exhibited pseudopodia-like extensions of all basal cells, abundant anchoring fibrils, and an increased amount of arching anchoring fibrils. These features might compensate for the lack of proper rete ridges.     

Palmar Erythema Ab Igne without Detectable Type IV Collagen at the Basement Membrane Zone

A 42-year-old baker had hyperkeratotic lesions on reticular erythema of his palms and volar digits. Attachment between the epidermis and the dermis was loose at the hyperkeratotic sites, but no blisters had formed. After he stopped work for 2 months, the skin lesions disappeared, leaving a slight scar. Immunofluorescence studies showed a normal distribution of type VII collagen and laminin, but there was almost no fluorescence for type IV collagen in the basement membrane zone. Disappearance of cytoplasmic organelles and aggregated tonofibrils was evident under electron microscopy in the basal cells which appeared shrunken under light microscopy. The structures of lamina lucida, basal lamina and anchoring fibrils were disarranged. The loose dermo-epidermal attachment is probably due in part to this disorganized basement membrane structure lacking type IV collagen, which may have been digested by heat-activated matrix metalloproteinases. In addition, his work-related chronic heat exposure might have caused hyperproliferation of keratinocytes.