Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes.

The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.

Formation and origin of basal lamina and anchoring fibrils in adult human skin.

The purpose of this investigation was to study the formation and origin of basal lamina and anchoring fibrils in adult human skin. Epidermis and dermis were separated by "cold trypsinization." Viable epidermis and viable, inverted dermis were recombined and grafted to the chorioallantoic membrane of embryonated chicken eggs for varying periods up to 10 days. Basal lamina and anchoring fibrils were absent from the freshly trypsinized epidermis before grafting although hemidesmosomes and tonofilaments of the basal cells remained intact. Basal lamina and anchoring fibrils were absent from freshly cut, inverted surface of the dermis. Beginning 3 days after grafting, basal lamina was noted to form immediately subjacent to hemidesmosomes of epidermal basal cells at the epidermal-dermal interface. From the fifth to the seventh day after grafting, basal lamina became progressively more dense and extended to become continuous in many areas at the epidermal-dermal interface. Anchoring fibrils appeared first in grafts consisting of epidermis and viable dermis at five day cultivation and became progressively more numerous thereafter. In order to determine the epidermal versus dermal origin of basal lamina and anchoring fibrils, dermis was rendered nonviable by repeated freezing and thawing 10 times followed by recombination with viable epidermis. Formation of basal lamina occurred as readily in these recombinants of epidermis with freeze-thawed, nonviable dermis as with viable dermis, indicating that dermal viability was not essential for synthesis of basal lamina. This observation supports the concept of epidermal origin for basal lamina. Anchoring fibrils did not form in recombinants containing freeze-thawed dermis, indicating that dermal viability was required for anchoring fibrils formation. This observation supports the concept of dermal origin of anchoring fibrils.

Calcium-induced assembly of adherens junctions in keratinocytes.

Extracellular calcium concentration has been shown to control the stratification of cultured keratinocytes, presumably by regulation of formation of desmosomes. Previous studies have shown that keratinocytes cultured in medium containing 0.1 mM Ca++ form loose colonies without desmosomes. If the Ca++ is raised to 1 mM, desmosomes are assembled and the distribution of keratin filaments is altered. We have examined the disposition of vinculin and actin in keratinocytes under similar conditions. Using immunofluorescence microscopy we show that raising [Ca++] in the medium dramatically alters the distribution of vinculin and actin and results in the formation of adherens-type junctions within 15 min after switching to high calcium medium. Borders of cells at the edge of colonies, which are not proximal to other cells, are not affected, while cells in the interior of the colony form junctions around their periphery. Attachment plaques in keratinocytes grown in low calcium medium are located at the ventral plane of the cell, but junctions formed after switching to high calcium are not, as demonstrated by interference reflection microscopy. In cells colabeled with antibodies against vinculin and desmoplakin, vinculin-containing adherens junctions were visible before desmosomal junctions when cells were switched to high calcium. Although newly formed vinculin-containing structures in high calcium cells, like desmosomes, colocalize with phase-dense structures, superimposition of video fluorescence images using digitized fluorescence microscopy indicates that adherens junctions and desmosomes are discrete structures. Adherens junctions, like desmosomes, may play an essential role in controlling stratification of keratinocytes.

Uninvolved skin from psoriatic patients develops signs of involved psoriatic skin after being grafted onto nude mice.

Clinically involved psoriatic epidermis maintains its histological appearance, increased labeling index, and increased level of plasminogen activator after being grafted onto athymic nude mice. Uninvolved psoriatic epidermis develops increases in plasminogen activator activity after being grafted onto athymic nude mice; this is accompanied by an increased labeling index. Thus, psoriatic skin can develop markers of psoriasis independent of the host.

Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice.

Patients with a recently identified subepithelial blistering disease have IgG anti-laminin 5 autoantibodies. To determine if such antibodies can be pathogenic in vivo, we developed and characterized rabbit anti-laminin 5 IgG, and passively transferred these antibodies to neonatal mice. Immune rabbit IgG specifically bound human and murine epidermal basement membranes, immunoblotted and immunoprecipitated all laminin 5 subunits from extracts of human and murine keratinocytes, and showed no reactivity to other keratinocyte proteins or epithelial basement membranes that do not contain laminin 5. Mice (n = 29) receiving purified anti-laminin 5 IgG developed, in a dose-related fashion, circulating anti-laminin 5 antibodies, deposits of rabbit IgG and murine C3 in epidermal basement membranes, and subepidermal blisters of skin and mucous membranes. No alterations developed in controls (n = 14) receiving identical amounts of normal rabbit IgG. Passive transfer of anti-laminin 5 (but not control) IgG to neonatal C5- (n = 3) or mast cell-deficient (n = 3) mice produced subepidermal blisters with the same clinical, histologic, and immunopathologic features as those documented in BALB/c mice. These studies establish an animal model of a human blistering disease that can be used to define disease mechanisms and treatment modalities.

Epidermolysis bullosa acquisita antigen is the globular carboxyl terminus of type VII procollagen.

Epidermolysis bullosa acquisita (EBA) is a severe, chronic blistering disease of the skin. EBA patients have circulating and tissue-bound autoantibodies to a large (Mr = 290,000) macromolecule that is localized within the basement membrane zone between the epidermis and dermis of skin, the site of blister formation. The "EBA antigen" is known to be distinct from laminin, heparan sulfate proteoglycan, fibronectin, the bullous pemphigoid antigen, elastin, and collagen types I, II, III, IV, and V. Sera from patients with EBA, two monoclonal antibodies to the EBA antigen, and a monoclonal antibody to the carboxyl terminus of type VII procollagen identically label human amnion and skin by immunofluorescent and immunoelectron microscopy. Western immunoblots of the EBA antigen extracted from skin and of type VII procollagen labeled with the above sera and antibodies are identical. None of the sera or antibodies labels Western blots of pepsinized type VII collagen which is missing the globular amino and carboxyl terminal domains. These data show that the EBA antigen is the carboxyl terminus of type VII procollagen.

Specific affinity between fibronectin and the epidermolysis bullosa acquisita antigen.

Autoantibodies in the skin and sera of patients with epidermolysis bullosa acquisita bind to a large matrix molecule within the lamina densa region of skin basement membrane. At the site of these immune complexes, the epidermis separates from the dermis, which creates a subepidermal blister just below the lamina densa. The target molecule for the autoantibodies is in close apposition to fibronectin, a major extracellular matrix molecule that is abundant in the upper dermis of skin. In this report, we show specific affinity between fibronectin and the 290,000-D chain of the epidermolysis bullosa acquisita antigen, and that this affinity is mediated by the gelatin/collagen-binding domain of fibronectin (Mr = 60,000). Since blistering in epidermolysis bullosa acquisita often occurs in the absence of clinical and histological inflammation, a direct interruption in the fibronectin-epidermolysis bullosa acquisita antigen bond may be involved in the pathogenesis of epidermal-dermal disadherence that occurs in this bullous disease.

Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease.

Epiligrin, the major component of human keratinocyte extracellular matrix, serves as the preferred integrin ligand for alpha 3 beta 1 in plasma membranes and focal adhesions, and colocalizes with alpha 6 beta 4 in hemidesmosomes. In human skin, epiligrin is found in the lamina lucida subregion of epidermal basement membrane, where it is thought to be associated with anchoring filaments. We have identified three patients with an acquired mucosal predominant subepidermal blistering disease who have IgG anti-basement membrane autoantibodies that bind the lamina lucida/lamina densa interface of epidermal basement membrane, stain cultured human keratinocyte extracellular matrix, and immunoprecipitate disulfide linked polypeptides of 170, 145, 125, and 95 kD in human keratinocyte culture media in a pattern identical to that of P1E1, a murine monoclonal antiepiligrin antibody. Comparative immunoprecipitation studies of patient sera, P1E1, and GB3 monoclonal antibody show that epiligrin is identical to the antigen (i.e., BM600 or GB3 antigen) previously reported to be absent from the skin of patients with lethal junctional epidermolysis bullosa, an inherited subepidermal blistering disease. Moreover, skin from a fetus with this disease shows no evidence of reactivity to patient antiepiligrin autoantibodies or P1E1. These studies show that antiepiligrin autoantibodies are a specific marker for a novel autoimmune blistering disease and that the epidermal basement membrane antigen absent in patients with lethal junctional epidermolysis bullosa is epiligrin.

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.

Epidermolysis bullosa dystrophica-recessive: a possible role of anchoring fibrils in the pathogenesis.

The purpose of this study was to define the ultrastructural defects and pathogenesis of epidermolysis bullosa dystrophica-recessive (EBD-R). The only consistent ultrastructural alteration found in EBD-R was an absence of anchoring fibrils. In many specimens of nonblistered, nontraumatized EBD-R skin, absence of anchoring fibrils was the only ultrastructural abnormality observed. The possibility that lack of anchoring fibrils was a secondary change resulting from previous blistering and scarring was eliminated by our observation that anchoring fibrils were consistently absent in the never previously blistered skin of two newborns with EBD-R. In experimentally traumatized skin, the epidermis and dermis separated in the region of the epidermal-dermal junction normally occupied by anchoring fibrils. Basal lamina and dermal microfibril bundles appeared to be normal. Using recombinant grafts, we demonstrated that anchoring fibrils were not formed by EBD-R dermis when combined with EBD-R epidermis or normal epidermis. Anchoring fibrils were formed when normal dermis was combined with normal and EBD-R epidermis. These studies indicate that the defect in EBD-R resides in the dermis and that the defect may be associated with impaired formation of anchoring fibrils.

Absence of specific histologic changes in guinea pig skin treated with bullous pemphigoid antibodies.

Previous studies have reported that intradermal injections of bullous pemphigoid antibodies into guinea pigs can reproduce the histologic and immunohistologic features of bullous pemphigoid lesions. In this study we examined this model to determine its reproducibility and suitability for testing other types of anti-BMZ antibodies. Twenty guinea pigs were injected intradermally with 0.1, 0.3, or 0.5 ml of either bullous pemphigoid serum or IgG fraction containing high-titer complement-binding anti-BMZ antibodies or an equivalent volume of normal human serum or IgG fraction as control. Sites were biopsied at intervals after injection and were examined by routine histology and direct immunofluorescence. The results showed (a) no difference in the incidence of dermal epidermal separation or type of inflammation in experimental and control sites; (b) no evidence of an eosinophil-rich inflammatory reaction typical of bullous pemphigoid; (c) an absence of linear BMZ deposits of IgG and complement in the majority of sites injected with bullous pemphigoid antibodies; and (d) no correlation between dermal-epidermal separation and deposition of immune reactants at the BMZ. These results suggest the histologic changes seen in guinea pigs that are administered intradermal injections of bullous pemphigoid antibodies are nonspecific and that the model is not suitable for testing the pathogenicity of anti-BMZ antibodies in sera or IgG fractions.

Bullous SLE: a phenotypically distinctive but immunologically heterogeneous bullous disorder.

Bullous systemic lupus erythematosus (SLE) is a rare blistering disease with a distinctive combination of clinical, histologic and immunopathologic features that together constitute a unique bullous disease phenotype. There appear to be at least two immunologically distinct subtypes of bullous SLE characterized by the presence or absence of circulating and/or tissue-bound basement membrane zone autoantibodies that recognize type VII collagen. The two subtypes are not clearly distinguishable except by indirect immunofluorescence and/or direct immunoelectron microscopy. In patients without circulating antibodies, immunoelectron microscopy is required to distinguish between the two subtypes. Patients with autoantibodies to type VII collagen are similar but not identical to patients with epidermolysis bullosa acquisita--another bullous disease associated with autoantibodies to type VII collagen. Autoantibodies to type VII collagen in patients with bullous SLE is only one of several lines of evidence that indicate autoimmunity to that protein and susceptibility to SLE are associated phenomena. In addition, there is emerging evidence for an association between epidermolysis bullous acquisita and SLE. There is also evidence that autoantibodies to type VII collagen are pathogenic in bullous SLE (and epidermolysis bullosa acquisita) and that their production is regulated by the class II major histocompatibility complex DR beta 1 allele, 1501 and possibly other DR beta 1 alleles that share a similar sequence of amino acids in the second hyper-variable region.

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.

Lamellar ichthyosis: long-term graft studies on congenitally athymic nude mice.

Whole-skin grafts from a patient with lamellar ichthyosis were maintained on athymic nude mice for periods of 6 weeks to 4 months with excellent preservation of all gross and histologic features of the disease. In recombinant grafts composed of lamellar ichthyosis epidermis and normal dermis grown for periods up to 87 days on nude mice, the abnormal epidermis retained all the features of lamellar ichthyosis. Similar results were found in recombinants of lamellar ichthyosis epidermis and dermis. In recombinant grafts of normal epidermis and lamellar ichthyosis dermis, the epidermis remained normal and did not become ichthyotic. These observations support the hypothesis that the defective gene in lamellar ichthyosis acts directly on the epidermis as opposed to acting indirectly through a systemic abnormality or through an effect on neighboring dermis.

The epidermal-dermal junction.

Ultrastructurally, the epidermal-dermal junction is composed of four component areas:(1) the basal cell plasma membrane with its specialized attachment devices or hemidesmosomes, (2) an electron-lucent area, the lamina lucida, (3) the basal lamina, and (4) the sub-basal lamina fibrous components, including anchoring fibrils, dermal microfibril bundles, and collagen fibers. The light microscopic "basement membrane" comprises only the sub-basal lamina fibrous zone. Other cell types, including melanocytes and Merkel cells, are also found at the epidermal-dermal junction. Structures at the junction derive their origin from the epidermis and dermis: the basal lamina is primarily of epidermal origin, the anchoring fibrils of dermal origin. The junction serves the following functions: (1) epidermal-dermal adherence, (2) mechanical support for the epidermis, and (3) a barrier to the exchange of cells and of some large molecules across the junction.

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.

Epidermolysis bullosa acquisita antigen: relationship between the collagenase-sensitive and -insensitive domains.

To clarify the relationship of the 290 and 145 kDa chains of the epidermolysis bullosa acquisita (EBA) antigen, we subjected urea extracts of skin basement membrane zone (BMZ) proteins and isolated 290 and 145 kDa chains of the EBA antigen cut out of sodium dodecyl sulfate polyacrylamide gels to treatment with clostridial collagenase. When the reaction products were electrophoresed, transblotted, and reacted with EBA patient sera or two monoclonal antibodies to the EBA antigen, the 290 kDa chain was degraded into the 145 kDa band that was resistant to cleavage with collagenase. The 145 kDa domain, isolated after collagenase treatment of the whole BMZ extract, was resistant to degradation by hyaluronidase, chondroitinase ABC, heparinase, and heparitinase but was readily degraded by V-8 protease. These data suggest that the EBA antigen consists of collagen and noncollagen domains of identical size (Mr 145,000), and that the 145 kDa noncollagen domain is generated via degradation of the native 290 kDa species by collagenase.

Epidermolysis bullosa acquisita of the immunopathological type (dermolytic pemphigoid).

Epidermolysis bullosa acquisita (EBA) of the immunopathological type is a distinct disease entity which we propose to name dermolytic pemphigoid. Clinical features of this disease are heterogeneous. An inflammatory phase may mimic bullous pemphigoid or, less commonly, mucosal pemphigoid or dermatitis herpetiformis. The noninflammatory mechanobullous phase equates with classic EBA and features marked skin fragility, bullae and/or erosions at sites of trauma, which result in scarring and milia. The inflammatory or the noninflammatory phase may occur separately or in combination. Transition from inflammatory to noninflammatory phases has been seen. Linear basement membrane zone (BMZ) immune deposits of immunoglobulin G (IgG) are present in the lesional and uninvolved skin of affected patients by immunofluorescence and are essential for the diagnosis. Many patients also have circulating antibasement membrane zone IgG antibodies. Immunoelectron microscopy localizes the immune deposits in the lamina densa and sublamina densa zone and serves to distinguish this disease from diseases with lamina lucida antibodies, such as bullous pemphigoid and mucosal pemphigoid. The EBA antigen has recently been identified and partially characterized from human skin using circulating antibasement membrane zone antibodies from patients with EBA. The EBA antigen consists of 2 components of Mr 290,000 and 145,000, and were shown to be distinct from other known basement membrane components. Mouse monoclonal antibody, H3a, recognizes the same 290 kilodaltons (kd) and 145 kd proteins and localizes to the lamina densa and sublamina densa zone of human skin. The EBA antigen is a newly recognized basement membrane component that is restricted in its distribution to the BMZ of stratified squamous epithelium of both keratinizing and nonkeratinizing types.

Transplantation of psoriatic skin onto nude mice.

Involved psoriatic epidermis maintains its histologic appearance, increased labeling index, and increased level of plasminogen activator after grafting onto athymic nude mice. Epidermis from clinically uninvolved psoriatic skin develops an increase in plasminogen activator activity after grafting for 6 weeks on nude mice and demonstrates an increased labeling index. Normal control skin maintains its low level of plasminogen activator and labeling index after grafting. These results indicate that psoriatic skin can maintain and develop markers of psoriatic skin independent of the host.

Transplantation of Psoriatic Skin onto Nude Mice.

Involved psoriatic epidermis maintains its histologic appearance, increased labeling index, and increased level of plasminogen activator after grafting onto athymic nude mice. Epidermis from clinically uninvolved psoriatic skin develops an increase in plasminogen activator activity after grafting for 6 weeks on nude mice and demonstrates an increased labeling index. Normal control skin maintains its low level of plasminogen activator and labeling index after grafting. These results indicate that psoriatic skin can maintain and develop markers of psoriatic skin independent of the host.