Immunogold localization of the 97-kD antigen of linear IgA bullous dermatosis (LABD) detected with patients'' sera

The classification of linear IgA bullous dermatosis in the group of subepidermal blistering diseases is still a matter of controversy. This situation is due partly to the considerable clinical heterogeneity of the disease but also results from the difficulties in characterization and localization of the specific basement membrane zone antigen(s) recognized by immunoglobulin (Ig)A antibodies. In the present study, we have combined the Western blot detection of circulating autoantibodies with an ultrastructural immunogold labeling of human skin antigens using the same patients' sera. Our results, obtained with a short series of sera showing exclusive IgA class reactivity with the epidermal portion of salt-split skin, indicate that the antibodies recognizing the 97-kD antigen on immunoblot bind to the hemidesmosomal plaques of basal keratinocytes and the adjacent lamina lucida. These homogeneous laboratory results remain in striking contrast to the heterogeneity of clinical pictures in the patients studied, suggesting a participation of complementary, possibly not humoral, phenomena in the pathogenesis of linear IgA bullous dermatosis.     

alpha3beta1 Integrin is required for normal development of the epidermal basement membrane

Integrins alpha3beta1 and alpha6beta4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; alpha6beta4 is present in hemidesmosomes, while alpha3beta1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in alpha3beta1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in alpha3beta1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from alpha3beta1-deficient skin adhered to laminin-5 through alpha6 integrins. However, alpha3beta1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for alpha3beta1 and indicating distinct roles for alpha3beta1 and alpha6beta4. Our findings support a novel role for alpha3beta1 in establishment and/or maintenance of basement membrane integrity, while alpha6beta4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.     

The localization of target antigens and autoantibodies in linear IgA disease is variable: correlation of immunogold electron microscopy and immunoblotting

Linear IgA disease (LAD) of adults and children is a dapsone-responsive, autoimmune subepidermal blistering disease characterized by linear IgA deposits at the basement membrane zone (BMZ) of the skin and mucosa. Circulating IgA antibodies to BMZ components are often present. In this study we investigated the ultrastructural localization of the antigens and autoantibodies in six patients with LAD (five adults and one child). Using a direct postembedding immunogold electron microscopy (EM) technique, three different patterns of IgA antibody deposition were seen in the skin of four patients with LAD. The IgA deposits localized within the uppermost part of the lamina lucida and to the basal surface of the hemidesmosome in two patients, to the lamina lucida in one, and to the lamina densa in the fourth patient. Using an indirect immunogold EM technique and serum or purified blister fluid from two additional LAD patients, we showed that the serum autoantibodies of one patient bound to the hemidesmosome of the BMZ, while the autoantibodies in the blister fluid of the other patient bound to the lamina densa and sublamina densa including the anchoring fibrils in a labelling pattern similar to that of the monoclonal antibody (LH7.2) to collagen VII. All the autoantibodies binding to the hemidesmosome or lamina lucida recognized a protein in epidermal extracts of molecular weight 180 kDa or its breakdown product of 97 kDa, 200 kDa or 230 kDa. The antibodies binding to the lamina densa recognized proteins of 180 and 285 kDa. The antibodies that bound to the lamina densa and anchoring fibrils recognized collagen VII. In this immunogold EM study we have shown four patterns of IgA labelling in six patients with LAD, associated with five different antigens as recognized by immunoblotting. These results, together with our previous immunofluorescence and immunoblotting findings add support to the contention that LAD is a heterogeneous disease as regards both the target antigens and epitopes.     

Linear IgA bullous dermatosis after contact with sodium hypochlorite

The onset of linear IgA bullous dermatosis (LABD) is generally spontaneous, but a number of cases of LABD have been reported either following drug exposure or in association with malignancies. We describe a patient who developed a vesicular eruption shortly after an irritant dermatitis caused by the contact with a detergent containing sodium hypochlorite. Direct immunofluorescence revealed linear deposits of IgA and C3 in the epidermal basement membrane. The patient's serum contained IgA that immunoblotted a 180-kD polypeptide in extracts of human keratinocytes. The patient responded promptly to therapy with dapsone. We suggest a possible pathogenetic relationship between the chemical dermatitis and LABD in this patient.     

Lipoprotein profile, diet and body composition in athletes practicing mixed an anaerobic activities

To investigate the prevalence and possible role of anti-endothelial cell antibodies (AECA) in the pathogenesis of systemic lupus erythematosus (SLE), cell membrane antigen was prepared from cultured human umbilical vein endothelial cells and immunoblotting performed to detect AECA in SLE sera. IgG-AECA could be detected in 41 (86%) of 47 SLE patients. They were highly specific and failed to react with membrane antigens of human peripheral blood mononuclear cells or granulocytes. IgG-AECA reacted with endothelial membrane antigens which ranged from 15 to 200 kDa in molecular size. Further analysis of the antigens reacting with IgG-AECA revealed some interesting correlations between specific species of antibodies with certain clinical manifestations. Thus, patients having lupus nephritis, vasculitis, and hypocomplementemia had IgG-AECA against a 66-kDa membrane antigen; those with thrombocytopenia had IgG-AECA against a 55-kDa antigen; those with pleuritis had IgG-AECA against an 18-kDa antigen. These results indicate that IgG-AECA in the sera of SLE patients consist of heterogenous species.     

Nonsecretory IgA1 autoantibodies targeting desmosomal component desmoglein 3 in intraepidermal neutrophilic IgA dermatosis

Intraepidermal neutrophilic IgA dermatosis, a rare skin disease entity manifested with blisters and pustules clinically and lower epidermal blister, acantholysis, and neutrophilic infiltration pathologically, was first reported in 1985. Although the disease is characterized by IgA autoantibodies targeting the epithelial cell surface component, the target antigen has not been determined. We investigated a patient with this disease by histopathology, direct and indirect immunofluorescence, immunoblotting, and immunoadsorption studies. The pustular lesion was characterized by blister at the lower epidermis, acantholysis, and neutrophilic infiltration. Nonsecretory IgA1 subclass autoantibodies targeting the lower epithelial cell surfaces were detected in the patient's skin and serum. The patient's IgA autoantibodies labeled a recombinant desmosomal protein desmoglein 3 on immunoblotting and the immunolabeling of epithelial cell surfaces was eliminated by preadsorption with desmoglein 3. Thus, desmoglein 3 is identified as a target antigen in intraepidermal neutrophilic IgA dermatosis. The ability of IgA1 autoantibodies to bind neutrophils may be responsible for the prominent neutrophilic infiltration observed histopathologically and for the pustular lesions observed clinically.     

Chronic bullous dermatosis in childhood (linear IgA dermatosis)

A 5-year-old boy presented with disseminated, partly grouped blisters indicative of chronic bullous dermatosis of childhood (CBDC) following a gastrointestinal infection 2 weeks earlier. CBDC has long been differentiated from adult linear IgA disease. Clinical and laboratory studies revealed substantial clinical and immunological overlap between the two blistering disorders, whereas recent investigations suggest heterogeneity of the target antigen involved. Pathohistological and immunofluorescence-microscopical characteristics of a subepidermal blister and linear IgA and granular C3 deposition at the basement membrane together with the typical history and clinical signs were decisive in the differential diagnosis. The disease promptly cleared up after daily administration of 16 mg methylprednisolone-21-acetate tapering and 25 mg dapsone. Immunohistological detection of collagen IV at the base of a blister made it possible to localize the split above the lamina densa. The demonstration of collagen IV stresses the importance of immunodermatopathology in the differential diagnosis of subepidermal blistering diseases.     

Energy balance of rats subjected to continuous and intermittent food restriction

A 64-year-old woman complained of multiple blisters in 1990. She had had a productive cough since 1975. Immunofluorescence study of a specimen obtained from a skin biopsy showed staining in a linear pattern for both IgG and C3 in the epithelial basement membrane zone (BMZ) of the dermal-epidermal junction, and a high titer of anti-BMZ antibody. These findings led to the diagnosis of bullous pemphigoid. Corticosteroid therapy was started, and resulted in some stabilization of the skin lesion. Exertional dyspnea and a productive cough developed gradually, and the patient was referred to our department in 1992. Chest X-ray film and CT findings revealed a diffuse granular shadow and bronchiectatic change, predominantly in the lower lung fields. Laboratory tests showed a high titer of cold hemagglutinin and a high level of serum immunoglobulin A. Pulmonary function tests showed a combined destructive-restrictive defect and hypoxemia (PaO2 = 58.5 Torr). From these findings diffuse panbronchiolitis was diagnosed. A low dose of erythromycin alleviated the patient's pulmonary symptoms and improved the chest radiographic findings.     

A case of nonscarring inflammatory epidermolysis bullosa acquisita: characterization of IgG autoantibodies by immunofluorescence, immunoblotting and immunogold electron microscopy

We report a case of nonscarring inflammatory epidermolysis bullosa acquisita in a 59-year-old Japanese woman. She developed blisters and erosions on her lip, trunk and extremities. Sodium aurothiomalate was effective for the skin lesions. The patient had been free from bullous skin lesions for the last 13 years and had shown no scarring. Indirect immunofluorescence (IF) study on 1 M NaCl-split skin revealed IgG autoantibodies against the dermal side of the split skin. Immunoblotting using normal human dermal extracts disclosed IgG autoantibodies reactive with the 290 and 145 kD antigens. Circulating IgG autoantibodies were deposited on the lamina densa by immunoelectron microscopy. IF mapping using several antibodies for the components of the basement membrane zone revealed blister formation at the lamina densa. These results suggest that the cleavage at the lamina lucida does not necessarily exclude the diagnosis of EBA and that the definite diagnosis of EBA should be confirmed by immunoblotting or immunoelectron microscopic study.     

Erythrodermic bullous pemphigoid is a clinical variant of bullous pemphigoid

Bullous pemphigoid (BP) is an autoimmune blistering disease of the skin. Several variants of BP have been described but until recently the relationship of these variants to generalized BP was unclear. Several studies have shown that pemphigoid nodularis, pemphigoid vegetans, localized BP and vesicular pemphigoid are true variants of BP as the circulating antibodies in these patients recognize the same 230 kDa BP antigen as found in patients with generalized BP. Erythrodermic BP is a very unusual variant characterized by an erythroderma along with blister formation. We describe the third known patient to develop erythrodermic BP and characterize the antigenic specificity of the circulating antibodies in both our newly reported patient with erythrodermic BP and in one of the two other previously reported cases of erythrodermic BP. Both patients with erythrodermic BP had circulating IgG antibodies which bound to the epidermal side of salt-split human skin in a pattern identical to two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to demonstrate detectable circulating IgF autoantibodies. Immunoprecipitation studies revealed that both patients with erythrodermic BP had circulating IgG autoantibodies which recognized, to varying degrees, the same 230 and 180 kDa BP antigens as recognized by sera from two patients with immunopathologically proven generalized BP. Sera from four erythrodermic patients without blisters and from a healthy normal volunteer, as controls, failed to recognize any specific polypeptides. These observations demonstrate that erythrodermic BP is a distinct clinical variant of BP.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 microg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 microm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 microg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along the dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

Inositol 1,4,5-trisphosphate receptor expression in odontoblast cells

Although the exact pathogenesis of mustard gas-induced dermal toxicity remains elusive, morphopathological data gathered in controlled animal and in vitro investigations is providing important clues as to approximate mechanisms. Our laboratory has been studying dermal effects of the chemical warfare agent, sulfur mustard, in a variety of animal models, cultured isolated human cells, and in vitro organotypic skin models. Published anatomical, pathological, and ultrastructural results of these studies have documented consistent cellular and basement membrane zone effects irrespective of the model. Cellular effects include the early targeting of basal cells of the stratum basale to the exclusion of other epidermal cells, with nuclear and cytoplasmic indications of cell injury and cell death. Effects on the basement membrane zone include the formation of characteristic microvesicles in the lamina lucida of those models which possessed structural components of a true basement membrane. We are now investigating effects on proteins of the basement membrane microenvironment and correlate in the present paper the morphopathology of sulfur mustard dermal lesions with immunohistochemical study of bullous pemphigoid antigen, laminin, type IV collagen, and type VII collagen.     

Advantage of the presence of living dermal fibroblasts within in vitro reconstructed skin for grafting in humans

Methods for serial cultivation of human keratinocytes can provide large quantities of epidermal cells, which have the potential of restoring the vital barrier function of the epidermis in extensive skin defects such as burns. To investigate the value of combining an epidermis with a dermal component, fibroblasts originated from the superficial dermis were used to seed a collagen lattice as described by E. Bell (dermal equivalent). Beginning in 1981, we grafted 18 patients (burns and giant nevi) using 35 grafts 10 x 10 cm in size. In the course of this work, the original technique was modified and improved as experience was gained. We began by using small skin biopsy samples as a source of keratinocytes cultured on a dermal equivalent before grafting in a one-step procedure, but this gave poor cosmetic results, because of a nonhomogeneous epidermalization. We then chose to cover the graft bed using a two-step procedure. The first step consisted of grafting a dermal equivalent to provide a dermal fibroblast-seeded substrate for subsequent in vivo epidermalization by cultured epidermal sheets. Whatever the epidermalization technique used, a living dermal equivalent applied to the graft bed was found to reduce pain, to provide good hemostasis, and to improve the mechanical and cosmetic properties of the graft. A normal undulating dermal-epidermal junction reappeared by 3 to 4 months after grafting and elastic fibers were detectable 6 to 9 months after grafting. As a result of the biosynthesis of these products, the suppleness (e.g., elasticity) of the grafts was closer to that of normal skin than the cicatricial skin usually obtained with epidermal sheets grafted without the presence of living dermal cells. This rapid improvement of the mechanical properties of the graft could be attributed to the presence of fibroblasts cultured from the dermis and seeded into the collagen matrix.     

Treatment of carbon monoxide poisoning with hyperbaric oxygen

This report describes differences in humoral immune response of acute and chronic phases of human Chagas disease. The reactivities of IgG, IgM, and IgA anti-Trypanosoma cruzi antibodies in serum samples from both groups of patients were compared by enzyme-linked immunosorbent assay (ELISA) employing either one of four antigenic fractions: mouse laminin (LAM), which reacts through Gal alpha 1-3Gal epitopes expressed on trypomastigote surface: whole intact trypomastigotes (TCT); trypomastigotes excreted/secreted antigens (TESA); and epimastigote alkaline extract (EAE). The selection of T. cruzi antigen preparations was based on their relative content of surface and internal antigens found in trypomastigote forms. The proportion of IgG reactive to carbohydrate epitopes was assessed through the decay of IgG reactivity from acute and chronic sera after m-periodate oxidation of solid-phase bound antigens. Trypomastigote and TESA antigens recognized by IgG from acute and chronic sera were also compared by immunoblotting. ELISA and immunoblotting data showed that: (1) the proportion of IgG directed to trypomastigote surface antigens was higher in acute than in chronic sera, whereas the opposite was found for internal antigens, (2) acute sera contained a higher percentage of IgG reactive to trypomastigote carbohydrate epitopes than chronic sera, and (3) anti-T. cruzi IgA was found exclusively in acute sera and led to 100% positivity when LAM, TCT, and TESA were employed as antigens. IgA ELISA with these antigens and IgG immunoblotting pattern with TESA could be useful as serological markers for the acute phase of human Chagas disease.     

A novel variant of acquired epidermolysis bullosa with autoantibodies against the central triple-helical domain of type VII collagen

Epidermolysis bullosa acquisita and bullous systemic lupus erythematosus are autoimmune bullous disorders, with tissue-bound and circulating autoantibodies reactive with the noncollagenous NC1 domain of type VII collagen (C-VII). Here, we describe a novel acquired bullous dermatosis with autoantibodies against the triple-helical domain of C-VII. Three patients, all Japanese children, presented with widespread inflammatory tense blisters. Histologically, subepidermal tissue separation was noted with inflammatory infiltrate in the superficial dermis. Direct immunofluorescence staining revealed linear IgG/C3 deposits along the dermal-epidermal junction. Circulating IgG anti-basement membrane zone autoantibodies stained the dermal side of normal skin separated with 1 M NaCl. Direct and indirect immunoelectron microscopy using colloidal gold labeling showed that patient sera reacted with anchoring fibrils. The gold particles were localized both near the lamina densa and on the central banded portion of the fibrils. The sera reacted with C-VII in immunoblots. Epitope analyses with natural and recombinant fragments of C-VII disclosed that the sera did not recognize the NC1 domain of C-VII, but the central triple-helical domain of this anchoring fibril protein. Thus, the present probands show a hitherto unrecognized variant of epidermolysis bullosa acquisita, with autoantibodies against epitopes in the collagenous domain of C-VII.     

Hailey-Hailey disease keratinocytes: normal assembly of cell-cell junctions in vitro

The blisters in the inherited disorder, Hailey-Hailey disease, may be caused by defective epidermal junctional complexes. We evaluated these structural complexes in vivo and in vitro. We induced a vesicular lesion in the apparently normal skin of a patient with Hailey-Hailey disease and studied a biopsy of this lesion by transmission electron microscopy. To determine whether acantholysis was related to a defect in the number or assembly of intercellular junctions, we cultured Hailey-Hailey disease keratinocytes in medium containing 0.1 mM Ca2+ and increased the [Ca2+] to 1.1 mM in order to induce assembly of cell-cell junctions. Keratinocytes were examined by double immunofluorescence with antibodies to the desmosome protein, desmoplakin, and the adherens junction protein, vinculin, at intervals after the increase in [Ca2+]. Characteristic Hailey-Hailey disease histopathology was observed by electron microscopy of the patient's skin after trauma, but we found no splitting of desmosomes. Based on the location, intensity, and rate of change of immunofluorescent staining, Hailey-Hailey and normal keratinocytes did not differ in their ability to assemble desmosomes and adherens junctions. Furthermore, we observed no significant morphologic differences between normal and Hailey-Hailey keratinocytes cultured in low and high [Ca2+]-containing media; Hailey-Hailey cells contained abundant normal-appearing desmosomes in 1.1 mM [Ca2+]. Since Hailey-Hailey disease keratinocytes can assemble normal-appearing adherens junctions and desmosomes in vitro, the functional defect may not lie in assembly of cell-cell adhering junctions, or additional perturbation may be required to expose the defect.     

Bullous pemphigoid associated with Shy-Drager syndrome

We report a patient with Shy-Drager syndrome who developed multiple tense blisters mainly on the extremities. Circulating anti-basement membrane zone autoantibodies were detected by the indirect immunofluorescence method. Immunoblot analysis using normal human epidermal extracts demonstrated that this patient's serum reacted only with 230 kD bullous pemphigoid antigen (BPAG1). Concerning the pathoetiology of the association of bullous pemphigoid and Shy-Drager syndrome, we discuss a sequence similarity between BPAG1 and dystonin, a candidate gene for dystonia musculorum.     

Identification of different circulating autoantibodies in patients with bullous pemphigoid and pemphigus vulgaris by means of immunoblotting

Immunoblotting studies with salt-split human epidermis were performed on sera from 15 patients with pemphigus vulgaris and 20 patients with bullous pemphigoid by using peroxidase-labelled antihuman IgG and IgA. Eleven sera of pemphigus vulgaris antigen. Most of the sera gave additional specific bands at 210 and 80 kD, with lower intensity. The sera of 4 patients, 3 of them were in clinical remission, did not yield specific bands. Seventeen sera of the 20 bullous pemphigoid patients yielded a 220-230 kD protein band against the major bullous pemphigoid antigen and 4 of them gave another specific band at 160-180 kD. Five sera produced multiple bands (220, 130, 100 and 75 kD). IgA antibodies against the major bullous pemphigoid antigen were demonstrated in 2 cases with IgA deposits along their basement membrane, as revealed by direct immunofluorescence. The immunoblot patterns correlated only weakly with the clinical findings in bullous pemphigoid. There was a considerable diversity in both clinical findings and immunoblot patterns.     

Characterization of the neutrophil molecules identified by quinine-dependent antibodies from two patients

BACKGROUND: Two patients with episodic pancytopenia and renal failure associated with quinine (Qn) ingestion were previously found to have Qn-dependent antibodies that reacted with red cells, platelets, and neutrophils. The purpose of these studies was to characterize the neutrophil antigens recognized by Qn-dependent antibodies from these two patients. STUDY DESIGN AND METHODS: The neutrophil molecules recognized by the Qn-dependent antibodies in the sera from the two patients were analyzed by immunoprecipitation using 125I-labeled neutrophils. Neutrophils from 13 different donors were tested. RESULTS: The Qn-dependent antibodies from Patient 1 immunoprecipitated a 60-kDa molecule on neutrophils from seven donors and an 85-kDa molecule on neutrophils from three donors. The Qn-dependent antibodies from Patient 2 reacted with a 32-kDa molecule on neutrophils from 5 donors, a 60-kDa molecule on neutrophils from 9 donors, and an 85-kDa molecule on neutrophils from 10 donors. Neutrophil-specific antigen NB1 is also located on a 60-kDa glycoprotein (GP). While the antibody in serum from Patient 1 did not show specificity for NB1, the antibody from Patient 2 detected the 60-kDa molecule on NB1-positive neutrophils from 9 of 11 donors tested and did not detect the 60-kDa molecule on NB1-negative neutrophils from 2 donors. In a monoclonal antibody immobilization of granulocyte antigens assay, the Qn-dependent antibody from both patients reacted with the 60-kDa molecule carrying NB1. The Qn-dependent antibody from a third patient, Patient 3, was previously found to react with an 85-kDa GP and the 60-kDa NB1 GP. To determine if the Qn-dependent antibodies from Patients 2 and 3 recognized the same 85-kDa GP, neutrophils were treated with serum from Patient 3 plus Qn to remove the 85-kDa GP. Then, serum from Patient 2 plus Qn no longer immunoprecipitated the 85-kDa GP. CONCLUSION: The antigens recognized by Qn-dependent neutrophil antibodies were located on molecules of 85, 60, and 32 kDa. Qn-dependent antibodies from two patients reacted with the same 85-kDa GP and those from three patients reacted with the same 60-kDa GP. The 60-kDa molecule recognized by the Qn-dependent antibodies carried the NB1 antigen.