Monoclonal Antibodies to Anchoring Fibrils for the Diagnosis of Epidermolysis Bullosa

Murine monoclonal antibodies to human anchoring fibrils reacted with human and monkey cervix, tongue, esophagus, and vagina. Rat, mouse, and guinea pig tissues were negative. In 11 patients with dystrophic recessive epidermolysis bullosa there was no reaction by immunofluorescence and immunoelectron microscopy. Other forms of epidermolysis bullosa had normal reactivity

Epidermal-dermal Interactions in Adult Skin

Epidermal-dermal interactions are important determinants of embryonic development in skin. This review examines the role of such epidermal-dermal interactions in the conservation of epithelial specificity in adult skin. The basic epidermal keratinization program as defined as a proliferative basal cell population, orderly stratified cytodifferentiation of the cells and production of stratum corneum can be expressed by adult epidermis without the continued presence of a specific dermis. This is evidenced by the ability of epidermis to differentiate fully in association with nondermal connective tissues. Several other epithelia including cornea and esophagus express their specific differentiative characteristics without the continued presence of specific connective tissue. On the other hand, certain regional epithelial specificities in adult skin may be determined by the dermis. These regional epidermal specificities involve alterations in epidermal thickness, size of epidermal compartments including the stratum corneum, and the folding pattern at the epidermal-dermal interface. Possible mechanisms by which dermis could control these regional characteristics are discussed. Epidermal-dermal recombination techniques have been used to define the site of gene action in a variety of inherited skin abnormalities in animals. Similar studies are reported utilizing adult human skin in long-term cultivation on nude athymic mice. The abnormal gene in lamellar ichthyosis exerts its effect directly on the epidermis. Generalized exfoliative psoriasis was also studied by recombination techniques. Both epidermis and dermis were required for the maintenance of psoriatic morphology, suggesting a complex polygenic mechanism or one involving both genetic and environmental factors

Functional Heterogeneity of Immune Complexes in Epidermolysis Bullosa Acquisita

Epidermolysis bullosa acquisita is an inflammatory subepidermal bullous disease characterized by circulating and tissue-bound complement-binding anti-basement membrane zone autoantibodies to type VII procollagen. Lesions are characterized by neutrophil-predominant inflammation in some patients, but not in others. These features suggest complement activation and generation of complement-derived chemotactic factors for leukocytes by basement membrane zone immune complexes may contribute to inflammation, but that complexes may be heterogeneous in the ability to express that function. In this study, we measured the ability of basement membrane zone complexes from patients with (n = 4) and without (n = 6) neutrophil predominant inflammation to activate complement and generate complement-derived chemotactic activity using a complement-dependent neutrophil attachment assay. The results showed considerable heterogeneity in neutrophil attachment among EBA patients and that both the incidence (4/4 vs 2/6) and magnitude (81 +/- 34 vs 12 +/- 10 neutrophils/mm basement membrane zone) of attachment were greater in patients with neutrophil-predominant inflammation. Functional heterogeneity appeared to be due to differences in the amounts of complement-activating complexes formed at the basement membrane zone, which in turn appeared to be due to differences in the availability of circulating complement-binding anti-basement membrane zone antibodies. This was suggested by a positive correlation (r = 0.72, p less than 0.01) between neutrophil attachment and complement-binding anti-basement membrane zone antibody titers and the observation that high levels of neutrophil attachment could be generated in skin from patients with epidermolysis bullosa acquisita who did not have neutrophil-predominant inflammation by treating their skin in vitro with complement-binding anti-basement membrane zone antibodies. These results suggest tissue complexes in epidermolysis bullosa acquisita are heterogeneous in the ability to activate complement and generate complement-derived chemotactins (C5a, C5a des arg), and that functional heterogeneity contributes to histologic heterogeneity. The functional immunologic-pathologic correlations observed in this study suggest epidermolysis bullosa acquisita is an autoimmune "collagen" disease

Adherens Junctions: Demonstration in Human Epidermis

Adherens junctions are intercellular and cell-matrix junctions that, like desmosomes and hemidesmosomes, mediate adhesion of cells to each other or to matrix structures. These junctions have been detected recently in cultured human keratinocytes, indicating that they may be of importance in epidermis. To investigate the localization of adherens junctions in normal epidermis, we examined human epidermis, human oral mucosa, and monkey esophagus for the presence of vinculin, a major protein of the intracellular plaques of adherens junctions that is thought to be present in all adherens junctions. 'Western blot analysis demonstrated vinculin in extracts of epidermis. Immunohistochemistry of vinculin in these tissues displayed two distinct locations for adherens junctions: i) at the dermal-epidermal junction, and ii) in the region of cell-cell contacts in all layers of the epidermis. The location of vinculin in the region of the epidermal-dermal junction is reminiscent of the distribution of vinculin-containing focal contacts in cultured keratinocytes, and the intercellular staining of vinculin in epidermis is consistent with the presence of vinculin in adherens junctions in cultured keratinocytes at sites of cell-cell contact. These results demonstrate that adherens junctions are present in human epidermis, oral mucosa, and monkey esophagus. Vinculin-containing junctions in epidermis may be important in the pathogenesis, of skin diseases involving alterations in intercellular integrity

A Mouse Monoclonal Antibody Against a Newly Discovered Basement Membrane Component, the Epidermolysis Bullosa Acquisita Antigen

A mouse monoclonal antibody, H3a, directed against the newly described epidermolysis bullosa acquisita (EBA) antigen was obtained using hybridoma techniques. The distribution of the monoclonal antibody is identical to that of the polyclonal serum antibody of patients with EBA. By immunofluorescence, a linear band is seen at the dermal-epidermal junction and, by immunoelectron microscopy, immune reaction products are present in the lamina densa and sublamina densa regions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot analysis shows that the monoclonal antibody recognizes 290 and 145 kilodalton proteins present in the immunizing junctional extract, identical with the newly discovered EBA antigen. This monoclonal antibody should be useful in the further isolation and characterization of the EBA antigen

Neonatal Foreskin Substrate Has Limitations for the Immunofluorescent Screening of Monoclonal Antibodies

Two monoclonal antibodies to type IV collagen showed a marked decrease in the labeling of the dermal-epidermal junction of neonatal foreskin while the basement membrane around dermal blood vessels was brightly stained. In contrast, these antibodies labeled the junction and dermal blood vessels with approximately equal intensity when adult skin of nonforeskin site was used as substrate. Other antibodies to matrix molecules (bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and laminin) showed excellent staining of both the dermal-epidermal junction and dermal blood vessels in both neonatal foreskin and adult skin. Further, the ultrastructural appearance of the substrates appeared identical. The implication is that neonatal foreskin is not a good substrate to use for the routine screening of monoclonal antibodies to matrix components by indirect immunofluorescence since a "false negative" evaluation may occur

Epidermolysis Bullosa Acquisita Antigen, a Major Cutaneous Basement Membrane Component, Is Synthesized by Human Dermal Fibroblasts and Other Cutaneous Tissues

The epidermolysis bullosa acquisita (EBA) antigen is identified as 2 chains: a 290,000-dalton protein and a less prominent 145,000-dalton protein. The 290,000-dalton chain is synthesized by human keratinocytes in culture. In this study, we show that the 290,000-dalton chain is synthesized by human skin fibroblasts and cutaneous human tumors. In contrast, HT1080 cells, a human sarcoma cell line known to produce matrix molecules (such as laminin and type IV collagen), does not synthesize the EBA antigen. Further, the EBA antigen is absent from serum and blood components, placenta, amnion, lung, and the EHS tumor, a murine sarcoma that produces large amounts of laminin, type IV collagen, nidogen, entactin, and basement membrane proteoglycan but is present in cutaneous tumors of adnexal and epithelial origin. These data suggest that while the EBA antigen is synthesized by both human skin keratinocytes and fibroblasts and is therefore not specific for a primordial germ layer, it does appear to be specific for tissue containing a stratified squamous epithelium

Autoantibodies to Type VII Collagen Recognize Epitopes in a Fibronectin-Like Region of the Noncollagenous (NC1) Domain

Autoantibodies to type VII collagen are characteristic of the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus (SLE). Blisters in those diseases are due to defective adhesion of the lamina densa subregion of the epithelial basement membrane to the underlying dermis. Previous studies indicating that type VII collagen contributes to lamina densa-dermal adhesion by cross-linking lamina densa and dermal matrix proteins suggests that autoantibodies may contribute to blisters by interfering with type VII collagen function. That hypothesis is supported by previous studies showing autoantibodies from a small number of epidermolysis bullosa acquisita patients recognize proteolytic fragments containing the 145-kD noncollagenous domain of type VII collagen. In this study, we examined reactivity of autoantibodies from a large number of epidermolysis bullosa acquisita and bullous SLE patients with fusion proteins representing most of the noncollagenous domain of type VII collagen and that those regions are homologous to type III repeats of fibronectin. These results suggest autoantibodies binding to fibronectin homology regions within the 145-kD noncollagenous domain may interfere with the adhesion function of type VII collagen and contribute to lamina densa-dermal dysadhesion in epidermolysis bullous acquisita and bullous SLE