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Epidermolysis bullosa acquisita and bullous systemic lupus erythematosus are blistering skin diseases characterized by IgG autoantibodies that predominantly target the noncollagenous domain 1 of type VII collagen, a skin basement membrane component. The basic immunologic events leading to the blistering processes in these diseases remains unclear. We defined the subclass and light chain compositions of the IgG autoantibodies in 15 patients, in order to gain insight into the blistering mechanism. Immunofluorescence correlated the patients' in vivo-bound and circulating antibasement membrane autoantibodies. Four eukaryotic recombinant proteins, including one full-length and three truncated noncollagenous domain 1 proteins generated by sequential deletion of C-terminal amino acids, were used to perform enzyme-linked immunosorbent assay to detect the patients' anti-type VII collagen autoantibodies. The majority of patients' autoantibodies contained both complement-activating and non-complement-activating IgG subclasses. The presence or absence of complement-activating IgG autoantibody subclasses did not correlate with the inflammatory or noninflammatory clinical phenotype. The majority of tested sera contained both kappa and lambda light chain autoantibodies. All sera that reacted to the full-length noncollagenous domain 1 also reacted to the smallest truncated protein containing the cartilage matrix protein and the first three fibronectinlike repeats. The patients' anti-type VII collagen autoantibodies, likely to be polyclonal in nature, may contribute to the pathogenesis of the blistering process by both complement-dependent inflammatory injury and complement-independent mechanical disruption of the anchoring function of type VII collagen. The N-terminal region of the noncollagenous domain 1 may contain an important antigenic epitope targeted by the IgG autoantibodies.

Background: Atrophic scars of the forehead can result from various pathologic processes including morphea en coup de sabre as well as trauma. A variety of surgical techniques can be used to correct these atrophic scars., Objective: Soft tissue augmentation for correction of atrophic scars of the forehead using en bloc autologous dermal fat graft., Methods: Use of en bloc autologous dermal fat graft harvested from the hip and inserted into a pocket created under the atrophic scar in two patients with depressed scars of the forehead., Results: Overcorrection of the scars with en bloc autologous dermal fat grafts resulted in the treated areas becoming level with the adjacent skin within 3 months. Follow-up for a period of 12 months showed a perfectly level and stable graft with no further resorption., Conclusion: En bloc autologous dermal fat grafting appears to be a safe technique that provides excellent cosmetic results for the correction of small to medium depressed scars of the forehead.

Background: Hailey-Hailey disease is an inherited acantholytic disorder affecting the intertriginous areas of the body which is exacerbated by sweat, moisture, and friction. The disease is frequently resistant to conventional nonsurgical treatment., Objective: To evaluate whether chemodenervation of sweat glands would improve the course of the disease in a patient with Hailey-Hailey., Methods: We used low-dose treatment of the left axilla with botulinum toxin type A, the right axilla being used as a control, followed by treatment of both axillae with the optimal dose routinely used for the treatment of axillary hyperhidrosis., Results: After one treatment with a low dose of botulinum toxin type A, we observed partial improvement of the treated axilla. With subsequent treatment of both axillae with the recommended dose for axillary hyperhidrosis, we observed a sustained complete remission of the disease in the treated axillae., Conclusion: Botulinum toxin type A may be an effective and safe nonsurgical alternative for the treatment of benign familial pemphigus in intertriginous areas such as the axillae.

Background: Bullous systemic lupus erythematosus is a generalized subepidermal blistering skin eruption in patients suffering from systemic lupus erythematosus. Type VII collagen was initially identified as the target antigen., Observation: We studied an unusual patient who had bullous systemic lupus crythematosus. The patient fulfilled the criteria of systemic lupus with an antinuclear antibody titer of 1:5120. Immunopathological testing revealed in vivo deposition of all IgG subclasses, secretory IgA1, and both light chains at the patient's skin basement membrane. The in vivo-bound IgG and IgA were localized at the hemidesmosomes and lamina densa. The patient's IgG and IgA circulating autoantibodies labeled both the epidermal roof and the dermal floor of salt-split skin and recognized the hemidesmosomal protein BP230 as well as the full-length native form and the recombinant noncollagenous domain 1 of type VII collagen (anchoring fibril). In addition, the patient's IgG autoantibodies recognized the anchoring filament proteins laminin-5 and laminin-6 (alpha3 chain and gamma2 chain)., Conclusions: We conclude that patients with bullous systemic lupus erythematosus may have autoantibodies to multiple basement membrane components critical for epidermal-dermal junctional adhesion. Possible pathogenic mechanisms in this patient's clinical diseases include provocation of organ-specific disease (bullous disease) by systemic autoimmunity (lupus) and the "epitope spreading" immune phenomenon.

A 105-kD lower lamina lucida antigen (p105) has been detected by autoantibodies (anti-p105) from patients with a novel immunobullous disease. To distinguish p105 from other known lamina lucida components, we performed comparative immunoblotting on purified human amniotic laminin-5 (kalinin), 804G matrix (enriched in laminin-5), and keratinocyte and fibroblast proteins using anti-804G matrix antibody (J-18) and anti-p105. J-18 labeled the truncated laminin-5 gamma 2 chain in amniotic laminin-5, 804G matrix, and keratinocyte conditioned medium, but did not label fibroblast cytosol. Conversely, anti-p105 did not label amniotic laminin-5 or 804G matrix, but did label p105 in both keratinocyte conditioned medium and fibroblast cytosol. J-18 labeled the 105-kD laminin-5 gamma 2 chain in reduced keratinocyte proteins and a 400-kD laminin-5 complex under non-reducing conditions. In contrast, anti-p105 labeled p105 under both reducing and non-reducing conditions but did not label a 400-kD protein complex. Similarly, comparative immunoblotting on keratinocyte proteins using anti-p105 and anti-laminin-1 revealed no commonly labeled protein bands. Electrophoretic fractionations by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of these fractions revealed that the peak fractions of keratinocyte proteins reactive with anti-p105 are different from those reactive with J-18. Furthermore, keratinocyte proteins fractionated by Mono Q anion-exchange chromatography revealed fractions immunoreactive with anti-p105, whereas J-18 showed no reactivity with these fractions. Two-dimensional gel electrophoresis and immunoblotting with anti-p105 revealed p105 to be an acidic protein with isoelectric points between 5.7 and 6.3, distinct from the isoelectric points of laminin-5 gamma 2 chain. We conclude that p105 is an acidic protein located in the lamina lucida and distinct from the truncated laminin-5 gamma 2 chain and the laminin-1 family.

A 9-year-old girl newly diagnosed with systemic lupus erythematosus (SLE) developed a localized linear papulovesicular eruption over the right dorsal hand and ulnar forearm. The skin findings were clinically suggestive of herpes zoster, lichen striatus, or lichen planus-lupus erythematosus overlap. However, histologic, immunofluorescent, immunoelectron microscopic, and immunoblot studies revealed findings compatible with bullous SLE. Our patient is noteworthy because she is the first one reported with bullous SLE presenting in a localized linear pattern. She is also the second-youngest reported patient with bullous SLE.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulate keratinocyte migration on collagen by up-regulating the alpha 2 subunit of the collagen integrin, alpha 2 beta 1. Interleukin-1 (IL-1) is an autocrine factor, produced by keratinocytes themselves, that is modulated by ultraviolet light and increases the proliferative potential of keratinocytes in culture. The autocrine nature of keratinocyte-derived IL-1 alpha is emphasized by the fact that it induces the keratinocyte to synthesize IL-1 alpha and TGF-alpha, a cytokine known to induce keratinocyte motility. Further, topical application of IL-1 alpha has been shown to promote wound healing in animals. In this study, we used a well-defined keratinocyte migration assay to assess the effect of IL-1 alpha on keratinocyte motility and to examine whether the IL-1 alpha/TGF alpha pathway is involved. The addition of recombinant human IL-1 alpha to keratinocytes produced a statistically significant and concentration-dependent increase in migration on matrices of collagen types I and IV, but not on laminin. Maximal levels of keratinocyte migration obtained on these matrices with IL-1 alpha were comparable to those obtained with stimulation by EGF and TGF-alpha. The effects of TGF-alpha and IL-1 alpha on keratinocyte migration are additive; however, the maximal level of migration achieved by using IL-1 alpha and TGF-alpha in combination never exceeds the maximal level of migration found by using either cytokine alone. The time course of keratinocyte migration induced by IL-1 alpha is delayed (onset of migration 9-12 h after addition) as compared with that induced by TGF-alpha (onset of migration 6-9 h after addition) even if the cells are preincubated in IL-1 alpha. Flow cytometry analysis demonstrated no change in surface expression of integrin subunits, specifically that of integrin subunit alpha 2, previously shown to be up-regulated by EGF/TGF-alpha. These results suggest that IL-1 alpha stimulates keratinocyte migration on collagen via a mechanism distinct from that of EGF/TGF-alpha.

Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (M(r) 33,000, 31,000, and 27,000). These inhibitors protected the degradation of gelatin by trypsin and elastase, and of casein by plasmin. In contrast, the gelatinolytic activities of thermolysin and papain were not inhibited. Compared to untreated cells, cells treated with phorbol 12-myristate 13-acetate showed a two- to 10-fold increase in the expression of these inhibitors. Cycloheximide and actinomycin D decreased the cellular expression of these inhibitors, suggesting the involvement of de novo protein and mRNA synthesis. Antitrypsin activity of these inhibitors was resistant to heat and sodium dodecylsulfate, but was lost after reduction of disulfide bonds. The inhibitors bound specifically to trypsin and could be eluted from a trypsin column in active form. Collectively, these data suggest that the extracellular matrix deposited by keratinocytes and dermal fibroblasts contains active serine protease inhibitors.

The term bullous drug eruption connotes several heterogeneous diseases in which blisters occur as a complication of the administration of drugs. Blisters may occur in bullous erythema multiforme, fixed drug eruption, or severe dermatitis medicamentosa with blisters. The common denominator is thought to be a hypersensitivity reaction to a systemic medication. Nevertheless, little has been written about the blisters in these disorders, and neither common nor distinct pathogenic mechanisms have been proposed. We describe a patient who had a rapidly progressive bullous eruption that occurred within hours of receiving intravenous trimethoprim-sulfamethoxazole. Routine histologic study of lesional skin demonstrated subepidermal blisters. Transmission electron microscopy and immunomapping of various basement components revealed that the cleavage plane of the blister was well below the lamina densa. After healing of the blistering process, no scarring or milia formation was observed.

Type VII collagen is the major component of anchoring fibrils, structures within basement membranes beneath stratified squamous epithelium thought to mediate the adherence of the epidermis to the dermis of human skin. Type VII collagen has affinity for fibronectin. The interaction between type VII collagen and fibronectin is mediated through the collagen-binding domain on the amino terminus of fibronectin. Heretofore, the domain on the type VII collagen molecule that binds to fibronectin was not known. In this study, we mapped the binding site of fibronectin to a specific subdomain of the triple helical collagenous region of type VII collagen, immediately adjacent to the small carboxyl terminal non-collagenous domain. This fibronectin-binding site within the type VII collagen molecule lies between nucleotide residues 615 and 1161.

Type VII collagen is the major component of anchoring fibrils, structures in human skin that mediate the adherence of the epidermis to the underlying dermis. Dystrophic forms of epidermolysis bullosa, a group of inherited mechanobullous disorders of the skin, are linked to the type VII collagen gene. Several mutations in the recessive form of this inherited disorder have been delineated. In this study, we mapped the epitopes of two commercially available monoclonal antibodies (clone I, 185 and LH 7.2) within the amino-terminal, non-collagenous domain of type VII (anchoring fibril) collagen. The precise localizations of the epitopes for these two monoclonal antibodies which are widely used to diagnose dystrophic epidermolysis bullosa, will be useful for the confirmation of gene mutations at the protein level.

Several cases have been reported of patients with immunemediated subepidermal blistering disorders whose autoantibodies react to antigens present on both the dermal and epidermal side of 1 M NaCl-split skin. In this report, we identify, localize, and characterize the basement membrane zone antigen corresponding to the dermal staining in a patient whose serum stains both the dermal and epidermal side of 1 M NaCl-split skin. This patient's serum contains autoantibodies directed against a 105-kilodalton(kDa) dermal antigen and the 230-kDa epidermal (bullous pemphigoid) antigen. This novel 105-kDa protein was previously identified as the sole antigen in another patient with a unique bullous disease whose autoantibodies were directed against only the dermal side of 1 M NaCl-split skin. This 105-kDa antigen was identical by one- and two-dimensional immunoblot analysis in these two patients. By immunoblot analysis, autoantibodies from our patient labeled a 105-kDa protein within various extracts of human skin basement membrane. Immunoblot analyses using epitope-selected autoantibodies directed against the 105-kDa protein demonstrated that this antigen is independent and distinct from other known basement membrane antigens. The 105-kDa antigen is an extracellular matrix component of the basement membrane, which is synthesized and secreted by both keratinocytes and fibroblasts. Identical electrophoretic migration of cellular and secreted forms of the protein suggested there is no major post-translational modification of the protein. Immunomapping of normal human skin fractured through the dermal-epidermal junction by incubation in 1 M NaCl or by suction blistering demonstrated that the location of the 105-kDa antigen within the basement membrane zone is between the bullous pemphigoid antigens and two other lamina lucida components, laminin and nicein. These data demonstrate clearly that a subepidermal autoimmune bullous disease may have autoantibodies directed against two distinct components of the dermal-epidermal junction.

Recessive dystrophic epidermolysis bullosa is a blistering skin disease characterized by diminished anchoring fibrils within the cutaneous basement membrane zone. Because anchoring fibrils are composed of type VII collagen, we compared the synthesis of type VII collagen by keratinocytes from patients and normal subjects. By Western blot analysis and immunoprecipitation of cell extracts, we found that both cell types express type VII collagen alpha chains of equivalent size (Mr = 290,000). Transforming growth factor-beta enhanced type VII collagen in both cell types. In contrast, the expression of type VII collagen within the culture medium was markedly diminished or absent in cultures of keratinocytes from patients. The patients' keratinocytes are capable of synthesizing type VII collagen of a normal molecular size, but they cannot assemble or secrete type VII collagen properly into the extracellular space, or they secrete a product that is abnormally sensitive to proteolytic digestion.