Biomedical subjects
Publications and source records attributed to W R Gammon.
The epidermolysis bullosa acquisita antigen is a newly discovered major extracellular matrix component within basement membranes beneath stratified squamous epithelia. Human keratinocytes cultured without mesenchymal cells synthesize the major 290 kd chain of 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.
Circulating and tissue-deposited IgG antibodies to the cutaneous basement membrane zone (BMZ) were detected in 3 patients with the clinical, pathologic, and immunologic features of bullous eruption of systemic lupus erythematosus (SLE). The antibodies were present in sera and IgG fractions in all cases and in eluates of cutaneous immune deposits from one of the cases. The antibodies were easily detected in sera by indirect immunofluorescence on adult human thigh skin separated through the lamina lucida by incubation in 1.0 M NaCl but were less easily detected on intact neonatal foreskin. The antibodies had features of epidermolysis bullosa acquisita (EBA) anti-BMZ antibodies including binding to the dermal side of the BMZ in separated skin, binding to the cutaneous but not vascular or glomerular basement membranes, binding to and just below the lamina densa, and binding to 290 or 290 and 145 kD dermal proteins previously identified as components of the EBA autoantigen. The antibodies were relatively specific for SLE patients with features of bullous eruption of SLE since they were detected in 3 of 4 of those cases and in only 1 of 20 SLE patients without blisters. These results show anti-BMZ antibodies with features of EBA antibodies are present in patients with bullous eruption of SLE and suggest there may be a close relationship between that disease and EBA. The results also suggest that EBA antibodies may be part of the autoantibody spectrum of SLE and that separated skin is more sensitive than intact skin for their detection.
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.
The vasculitides are a heterogeneous group of disorders characterized by inflammation and necrosis of blood vessels. They have been classified into several major groups on the basis of the size and type of vessels affected and the type of inflammation. Urticarial vasculitis is a recently recognized but relatively common presentation of cutaneous necrotizing vasculitis. This article discusses its clinical, pathologic, and immunologic features, its association with other disease states, and its differentiation from nonvasculitic urticaria.
Epidermolysis bullosa acquisita is an acquired chronic blistering disease of the skin, in which separation of the skin occurs in the basement-membrane zone between the epidermis and the dermis. There is evidence that blistering is initiated by an immune process. Using serum samples from nine patients as a source of antibodies, we have identified a major protein of the basement membrane of human skin that serves as the antigen (or target) for autoantibodies in this disorder. This previously unrecognized protein, which consists of two components of 290,000 and 145,000 daltons, is distinct from other known components of the basement membrane. These studies provide evidence that epidermolysis bullosa acquisita is a specific disease that is different from other primary bullous diseases, such as bullous pemphigoid and pemphigus vulgaris, and suggest that the basement-membrane component that has been identified may have a role in normal epidermal-dermal adherence.
Epidermolysis bullosa acquisita (EBA) is an immunologically distinctive disease with characteristic clinical features. These include extreme skin fragility, trauma-induced blisters and erosions, and healing with scars and milia. These clinical features are currently the major means of routinely distinguishing EBA from bullous pemphigoid (BP) and cicatricial pemphigoid (CP). In this study we describe five patients with EBA who presented with or developed clinical as well as histologic and immunohistologic features indistinguishable from those characteristic of BP or CP. At different times during the course of their disease, it closely resembled classic BP or CP and cases of BP or CP with atypical features or evolving features of EBA. To determine how often EBA might present and be misdiagnosed as BP, we reviewed our experience with bullous diseases and screened eighty-five "BP" sera for EBA antibodies. The results of this study show EBA can mimic the clinical, histologic, and immunohistologic features of BP and CP, suggest that a BP-like clinical presentation occurs in as many as 50% of EBA patients, and suggest that 10% of patients referred to medical centers and diagnosed as BP have EBA.
Sixty-one bullous disease sera containing IgG anti-BMZ antibodies were examined by indirect immunofluorescence on intact skin and skin separated through the lamina lucida by incubation in 1.0 M NaCl. All sera produced an indistinguishable pattern of linear immunofluorescence on intact skin at dilutions of 1:10 or higher. On separated skin, antibodies bound to either the epidermal (epidermal pattern), dermal (dermal pattern), or epidermal and dermal (combined pattern) sides of the separation. The binding patterns were consistent on separated skin from several donors and titers of anti-basement membrane zone antibodies on separated skin were comparable to those on intact skin. Sera from 3 patients with herpes gestationis (HG), 36 patients with bullous pemphigoid (BP), and 1 patient with clinical and histologic features of epidermolysis bullosa acquisita (EBA) showed an epidermal pattern. Sera from 9 patients with BP showed a combined pattern and sera from 6 patients with EBA and 6 patients with clinical and histologic features of BP showed a dermal pattern. Indirect immunoelectron microscopy of selected sera showed antibodies producing the epidermal and combined patterns were anti-lamina lucida antibodies and those producing the dermal pattern were anti-sublamina densa antibodies. These results show indirect immunofluorescence on separated skin is a dependable method for differentiating bullous disease anti-lamina lucida and anti-sublamina densa antibodies and that differentiating between the antibodies is essential for accurate diagnosis in some patients. The results also suggest BP anti-lamina lucida antibodies may have more than one antigenic specificity.
Bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) are chronic blistering diseases associated with circulating complement (C)-binding anti-basement membrane zone (BMZ) antibodies and tissue-deposited immune complexes at the BMZ. Experimental evidence supporting a role for C-activating immune complexes in the pathogenesis of dermal inflammation and blisters has been reported in BP but not in EBA. In this study tissue-deposited immune complexes composed of EBA or BP antibodies were tested for generation of C-dependent chemotactic activity and the capacity to cause dermal leukocyte infiltration and dermal-epidermal separation (DES). Chemotactic activity was measured by the leukocyte attachment (LA) method. The capacity of complexes to mediate leukocyte infiltration and DES was examined in vitro using a newly described organ culture method. The results of LA showed immune complexes formed in vivo in EBA skin or in vitro by treating normal human skin with EBA antibodies were significantly more active in mediating C-dependent chemotaxis than complexes in BP skin or those formed with BP antibodies of equivalent or higher C-binding titers. Furthermore EBA antibodies and C caused leukocyte infiltration and DES in organ culture while BP antibodies did not. These results support a role for C-binding anti-BMZ antibodies in the pathogenesis of EBA lesions and demonstrate differences in the capacity of BP and EBA immune complexes to generate C-dependent chemotactic activity. These results suggest factors in addition to C-binding titers are important in the activation of C by BP and EBA immune complexes and suggest chemotactic factors other than those derived from C activation may be important in the recruitment of leukocytes in BP.
In vivo suppression of neutrophil chemotaxis in humans by orally and topically administered tetracycline (TCN) was examined using both modified Boyden chamber and skin chamber assays. Chemoattractants were derived from serum complement and bacterial culture supernates. The results showed that oral TCN caused significant suppression of neutrophil chemotaxis when measured by both assays and both sources of chemoattractant. Furthermore, application of a commercially available topical preparation containing TCN caused local suppression of chemotaxis as measured by the skin chamber assay. These results show that TCN does suppress neutrophil migration in vivo, and they provide support for an anti-inflammatory effect of TCN mediated in part by suppression of neutrophil chemotaxis.
Evidence supporting an immune complex pathogenesis of bullous lesions in systemic lupus erythematosus includes immune deposits, acute inflammation, and blister formation at the cutaneous basement membrane zone. Since cutaneous immune deposits are a general feature of lupus, an attempt has been made to determine whether deposits in lupus patients with blisters are functionally different from those in patients without blisters. Skin was obtained from 4 consecutive patients with blisters and 14 controls. The groups were matched for clinical and serologic features, duration and activity of disease, and treatment. Skin was examined by direct immunofluorescence for immune deposits and by the leukocyte attachment assay for quantification of complement-activating immune complexes. Clinically normal, viable skin from 1 patient with blisters and 1 patient without blisters was incubated in organ culture with normal human leukocytes and serum complement. All patients in both groups had immune deposits at the basement membrane zone with an equivalent incidence of the major Ig classes. Deposits in patients with blisters were slightly more intense and a linear pattern of fluorescence seen in 75% of these patients was not seen in controls. The leukocyte attachment assay showed significantly greater (p less than .02) cell attachment in patients with blisters (mean = 167) than in patients without blisters (mean = 64) and greater cell attachment in peribullous than normal skin from the same patient. Organ culture showed complement-dependent migration of leukocytes and histologic features similar to those in spontaneous lesions in skin from the patient with blisters but not in skin from the patient without blisters. These results provide evidence supporting immune complex and complement-dependent inflammation in the pathogenesis of bullous lesions in systemic lupus erythematosus.
Immune deposits at the cutaneous basement membrane zone are a characteristic feature of systemic lupus erythematosus. Previous studies using immunofluorescent methods to detect complement components have provided evidence that some deposits contain immune complexes capable of activating complement. However, this important biologic property of complexes has not been detected or measured using functional assays, and it has not been determined whether immune deposits can activate complement at the basement membrane zone. In this study immune deposits in biopsies of lupus skin have been examined using direct immunofluorescence for the third component of complement (C3) to detect complement deposited in vivo. In addition, the deposits have been studied using the leukocyte attachment assay and indirect C3 binding immunofluorescence to detect and measure complement activation at the basement membrane zone in vitro. The results show that complement activation occurs at the basement membrane in some but not all lupus skin containing immunoglobulin deposits, that deposits differ quantitatively in their ability to activate complement, and that direct C3 immunofluorescence is a relatively insensitive method for detecting complement-activating complexes. The results provide functional evidence suggesting that immune deposits in some lupus skin are complement-activating complexes and potentially capable of activating complement at the basement membrane in vivo. Furthermore, the results suggest functional assays for evaluating complement-activating complexes may be valuable supplements to immunofluorescence in exploring the relationship between immune deposits and systemic and cutaneous disease.
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Epidermolysis bullosa acquisita is a subepidermal bullous disease of the skin with distinctive clinical, histologic, immunologic, and ultrastructural features. A case of epidermolysis bullosa acquisita is presented in which the onset of the disease was heralded by inflammatory blisters similar to herpes simplex or herpes zoster. The immunofluorescent findings were indistinguishable from bullous pemphigoid. Although some inflammatory blisters persisted throughout the evolution of the disease, the patient eventually developed noninflammatory blisters that healed with milia formation and scarring, lesions typical of classic epidermolysis bullosa acquisita. The diagnosis of epidermolysis bullosa acquisita was definitely made by electron and immunoelectron microscopy which showed a sub-lamina-densa blister cleavage plane and immune deposits beneath the lamina densa. This case illustrates that some cases of epidermolysis bullosa acquisita may have an inflammatory stage and immunofluorescent findings that make it difficult to distinguish from inflammatory bullous diseases. The value of electron microscopy and immunoelectron microscopy in making a firm diagnosis of epidermolysis bullosa acquisita is emphasized.
Current evidence strongly supports the theory that the lesions of pemphigus are due to binding of pemphigus antibody to an antigen in or near the epidermal cell membrane, which causes a release of at least one enzyme which results in dissolution of the intercellular attachments and acantholysis. Similarly, strong evidence supports the hypothesis that pemphigoid blisters are due to binding of antibody at the basement membrane, followed by activation of complement and release of anaphylatoxins which activate tissue mast cells to release eosinophil chemotactic factor. These eosinophils then release tissue-destructive enzymes and reactive oxygen intermediates directly onto the basement membrane zone, with loss of dermoepidermal adherence and formation of blisters.
Two patients with established systemic lupus erythematosus developed bullous dermatoses clinically suggestive of bullous pemphigoid (BP). Direct immunofluorescence demonstrated linear staining at the dermoepidermal junction for IgG and C3 in a pattern typical for BP. However, immunoelectron microscopy demonstrated deposits of IgG largely below the basal lamina, with little if any deposit present within the lamina lucida. These findings are consistent with systemic lupus erythematosus (SLE) and effectively rule out BP. In addition, the leukocyte attachment assay, an in vitro assay of the functional activity of tissue-deposited immune complexes, demonstrated strong attachment of leukocytes to the dermoepidermal junction of patients' skin, suggesting that the immune reactants were functional immune complexes of possible importance to the pathogenesis of the bullous lesions. To our knowledge, this is the first time that immunoelectron microscopy has been performed on skin from patients suspected of manifesting concurrent SLE and BP. Our findings cast doubt on previous such reports by demonstrating the existence of a bullous form of SLE resembling BP both clinically and by direct immunofluorescence.
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Previous immunofluorescent studies showing deposits of immunoglobulin and complement at the cutaneous basement membrane zone have provided evidence supporting a role for immune complexes in the pathogenesis of bullous pemphigoid. In this study the functional activity of the deposits has been examined using leukocyte attachment, a method for detecting and quantitating the biological activity of complement-activating immune complexes in tissues. When peripheral blood leukocytes suspended in serum complement were incubated with cryostat sections of lesional and adjacent normal-appearing skin from 9 patients with pemphigoid, skin from 11 normal controls and lesional skin from 14 nonpemphigoid disease controls there was significantly greater attachment of leukocytes to the basement membrane zone of lesional bullous pemphigoid skin compared to normal-appearing pemphigoid skin and skin of both control groups. A significant reduction in attachment in the absence of serum complement suggested the reaction was dependent on activation of complement by tissue-deposited complexes. Although leukocyte attachment was greater in lesional than normal-appearing pemphigoid skin, a comparison of the incidence and intensity of cutaneous IgG and complement immunofluorescence between the 2 groups showed no significant differences. Furthermore, no correlation between leukocyte attachment and serum titers of immunoglobulin G or complement-binding anti-basement membrane zone antibodies was observed. These results suggest that immune reactants in lesional pemphigoid skin are functional complement-activating immune complexes, that differences exist between the activity of complexes in lesional and normal-appearing pemphigoid skin and may explain why lesions develop at some sites and not others.