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.

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.

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.

Heavy and light chain isotypes of immunoglobulin in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a chronic acquired blistering disease with characteristic clinical, pathologic, and immunopathologic features. The disease is characterized immunopathologically by circulating and tissue-bound IgG class autoantibodies (EBA antibodies) to the basement membrane zone of stratified squamous epithelium. Previous studies have shown that circulating and tissue-bound EBA antibodies are heterogenous in their ability to activate complement and have raised the possibility that functional heterogeneity might be related to IgG subclass restriction. In this study, we have characterized the IgG subclasses of the circulating and tissue-bound EBA antibodies by immunofluorescence and have examined the relationship between IgG subclass and complement binding. The results show that EBA antibodies belonging to all IgG subclasses are present in the skin of EBA patients. The results also show that EBA antibodies belonging to all IgG subclasses are present in the sera of most patients, including sera with and without complement binding EBA antibodies.

The presence of intra-lamina lucida blister formation in epidermolysis bullosa acquisita: possible role of leukocytes.

In evaluating patients we have noted disparity between the locations of bound immunoreactants and the level of blistering in epidermolysis bullosa acquisita (EBA). We examined 10 consecutive EBA patients by routine histology, direct (DIF) and indirect (IIF; intact and NaCl-split skin) immunofluorescence, immunofluorescence mapping (IM), and/or direct immunoelectron microscopy (DIEM). DIF was positive in each. IIF was positive in 3/8 and 6/7 patients when intact and split skin were used as substrates. DIEM revealed immunoreactants within the lamina densa (LD) in 6/10, sub-LD in 1/10, and both LD and sub-LD in 3/10 patients. In contrast, by DIEM and IM, blister formation was noted within the lamina lucida (LL) in 7/9 and 8/10, sub-LD in 1/9 and 1/10, and within both LL and sub-LD in 1/9 and 1/10, respectively. In the presence of neutrophils within the upper dermis (n = 6), cleavage occurred within the LL in 5 specimens; in one additional specimen containing predominantly neutrophils, cleavage occurred within both LL and sub-LD. In the presence of mononuclear cells (n = 2), intra-LL cleavage occurred. In the presence of eosinophils, cleavage occurred within both LL and sub-LD. In the one specimen lacking any infiltrate, the cleavage plane was exclusively sub-LD. Intra-LL cleavage planes are more common than sub-LD ones in at least early cases of EBA. These findings likely represent the intra-LL-separating effect of leukocyte-derived proteolytic enzymes, when such cells are chemoattracted to the dermoepidermal junction by bound immuno-reactants.

Demonstration of the complement regulating protein, beta 1H, in skin biopsies from patients with bullous pemphigoid.

beta 1H-globulin is a recently characterized plasma protein which regulates the biologic activities of the major fragment of the third complement component, C3b. The major function of this protein is to act as a co-factor for C3b Inactivator (C3bINA) in the cleavage of C3b to an intermediate molecule, C3b', consisting of an intact beta-chain covalently bound by disulfide bridges to 2 alpha-chain fragments of 40,000 and 67,000 daltons. Final cleavage of C3b' to the C3c and C3d fragments requires an additional protease such as plasmin or elastase. Additionally, beta 1H interferes with the activity of the alternative pathway convertases, C3bBb and C3bBbP, by displacing or competing with the binding of factor B. In this study, perilesional skin biopsies from 10 patients with active bullous pemphigoid were examined for the presence of beta 1H at the dermal-epidermal junction by immunofluorescent methods. The protein was found in 8 of 9 biopsies in which C3 also was deposited. In a single case where C3 was not found, beta 1H was not seen. These findings suggest that beta 1H plays a role in the in vivo control of C3b and provides additional evidence for the participation of the complement system in the pathogenesis of bullous pemphigoid.

Differences in complement-dependent chemotactic activity generated by bullous pemphigoid and epidermolysis bullosa acquisita immune complexes: demonstration by leukocytic attachment and organ culture methods.

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.

Double immunofluorescence microscopy: a method for localizing immune deposits in skin diseases associated with linear basement membrane zone immunofluorescence.

Direct immunofluorescence microscopy has shown that a linear pattern of immunoglobulin and/or complement deposition at the cutaneous basement membrane zone is a characteristic feature in a number of acquired bullous diseases and is occasionally observed in systemic lupus erythematosus. Immunoelectron microscopy has shown the linear pattern of immunofluorescence may be produced by immune deposits located either above the basal lamina (in the lamina lucida) or below the basal lamina (in the upper dermis). Distinguishing between these sites of immune reactant deposition may be of value in differential diagnosis. In this study we report a double immunofluorescent method by which skin biopsies with linear IgG immunofluorescence due to deposits above the basal lamina (bullous pemphigoid) could be distinguished from biopsies with deposits beneath the basal lamina (bullous systemic lupus erythematosus and epidermolysis bullosa acquisita). When skin sections were treated sequentially with rhodamine-labeled anti-human IgG followed by fluorescein-labeled antilamina lucida (pemphigoid) antibody and examined by fluorescence microscopy, the following results were obtained. In biopsies with IgG deposits in the lamina lucida, a single green fluorescent band was observed. In tissues with subbasal lamina deposits, either parallel and contiguous bands of green and yellow-orange fluorescence or a single band of yellow-orange fluorescence was observed. The method is simpler, quicker, and less expensive than immunoelectron microscopy and should be a useful technique for evaluating skin diseases with linear immunofluorescence at the basement membrane zone.

Recombinant human cytokines stimulate neutrophil adherence to IgG autoantibody-treated epithelial basement membranes.

We investigated the ability of the purified recombinant human cytokines: tumor necrosis factor-alpha (rTNF), granulocyte-macrophage colony-stimulating factor (rGM-CSF), interleukin-1 beta (rIL-1), interleukin-3, and tumor necrosis factor-beta (rTNF-beta) to stimulate neutrophil adherence (NA) to basement membranes (BMs) of stratified squamous epithelia pretreated with autoantibodies (ABM) specific for the BM matrix protein, type-VII collagen. rTNF, rGM-CSF, rIL-1, and rTNF-beta, but not IL-3, stimulated NA and stimulation was ABM- and cytokine-concentration-dependent. Stimulation was cytokine-specific and not due to endotoxin since it was significantly inhibited by cytokine-specific antibodies but not by polymyxin B (PB). rTNF and rGM-CSF were the most potent stimulators, were effective at concentrations less than 0.067 ng/ml, and stimulated NA greater than 600%. Relative potency was: rTNF = rGM-CSF greater than rTNF-beta greater than rIL-1. Stimulation by rTNF was due to a rapid, time-dependent effect on the neutrophil, and NA appeared to be dependent, in part, on the low-affinity neutrophil receptor for IgG, Fc(gamma)RIII, because it could be specifically inhibited by monoclonal antibody (3G8) to Fc(gamma)RIII. These results suggest that rTNF, rGM-CSF, rIL-1, and rTNF-beta may contribute individually or in combination to immune-mediated inflammation and tissue injury by stimulating immune adherence of neutrophils to tissue-bound autoantibodies and immune complexes.

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.

Inhibition of neutrophil adherence to antibody by dapsone: a possible therapeutic mechanism of dapsone in the treatment of IgA dermatoses.

Dapsone is frequently effective in cutaneous diseases characterized by antibody deposition and accumulation of neutrophils. We hypothesized that this mechanism of action of dapsone may involve the inhibition of neutrophil adherence to antibody. The neutrophil adherence assay, which measures the binding of neutrophils to basement membrane zone-bound antibody on skin sections, was used to evaluate the effect of dapsone on neutrophil adherence to immunoglobulin A and immunoglobulin G. We evaluated the effect of dapsone on adherence of normal neutrophils to immunoglobulin A and immunoglobulin G from sera of linear immunoglobulin A bullous dermatosis and bullous pemphigoid patients, respectively. Linear immunoglobulin A bullous dermatosis or bullous pemphigoid antibody were bound to the basement membrane zone of normal skin sections as a substrate for the neutrophil adherence assay. Dapsone was added directly to the neutrophils or to the antibody source in concentrations of 0-50 micrograms/ml (pharmacologic range). Addition of dapsone to neutrophils produced an incremental inhibition of neutrophil adherence up to 75% at 50 micrograms/ml. Dapsone produced similar inhibition when added directly to the antibody itself, despite washing prior to usage in the neutrophil-adherence assay. Control specimens including irrelevant fractions of patient sera failed to demonstrate binding. Serum from a patient on dapsone therapy also showed inhibition of neutrophil adherence compared to the same patient on no therapy. We conclude that dapsone inhibits the adherence of neutrophils to basement membrane zone antibody in a dose-dependent manner. This may be related to an effect directly on antibody. This inhibition may contribute to the clinical efficacy of dapsone in antibody-mediated diseases.

Normal molecular weight of type VII collagen produced by recessive dystrophic epidermolysis bullosa keratinocytes.

Studies of the recessive dystrophic form of epidermolysis bullosa (RDEB) have suggested that an abnormality in type VII collagen may be involved in the pathogenesis of this disorder. Indirect immunofluorescence studies have shown that the staining for type VII collagen along the dermal-epidermal junction is markedly reduced or absent in all but rare cases of severe, generalized RDEB. These findings imply that the genetic defect may involve type VII collagen but do not exclude the possibility that the alterations demonstrated are secondary, for example, to nonspecific proteolysis of type VII collagen. To evaluate the ability of cells of affected patients to produce type VII collagen, we cultured keratinocytes from a severely affected patient and immunoprecipitated type VII collagen from the cells. Keratinocytes were metabolically labelled with 35S-methionine, and solubilized cell extracts were reacted with antibody to type VII collagen. The results indicate that the patient's keratinocytes synthesize type VII collagen and that the M(r) of the protein synthesized does not differ from that of an unaffected control. Because cultured cells from a patient severely affected with recessive dystrophic epidermolysis bullosa produce type VII collagen, the genetic defect, at least in this patient, is unlikely to reside in a major truncation of the type VII collagen molecule.

Immunodominant autoepitopes of type VII collagen are short, paired peptide sequences within the fibronectin type III homology region of the noncollagenous (NC1) domain.

Autoantibodies to type VII collagen are associated with the blistering diseases epidermolysis bullosa acquisita and bullous systemic lupus erythematosus. We showed previously that these autoantibodies recognize epitopes within the noncollagenous (NC1) region of type VII collagen. That region is composed of fibronectin type III homology units that may contribute to intermolecular cross-linking and basement membrane adhesion functions of type VII collagen. In this study, we defined the specific amino acid sequences recognized by these autoantibodies. By fusion protein analysis, sera from patients with epidermolysis bullosa acquisita and bullous lupus were found to react with two regions within the fourth (E-1) and eighth (E-2) fibronectin homology repeats, each consisting of approximately 100 amino acids. Affinity purification studies showed E-1 and E-2 to be independent and non-cross-reactive epitope regions. These regions were probed further by enzyme-linked immunosorbent assay analysis of overlapping octapeptide sets derived from the amino acid sequences of E-1 and E-2. The results showed two reactive, closely associated octapeptide sequences within each region, both lying in amphipathic portions of fibronectin type III homology repeats. These studies identify short peptide sequences within the NC1 domain of type VII collagen that are targeted independently by autoantibodies. These sequences may play a direct role in determining the properties of type VII collagen that influence adhesion between this molecule and other basement membrane proteins, and their alteration by antibody binding may be the immunopathogenic event underlying epidermolysis bullosa acquisita and bullous lupus.

Production of fibronectin by epithelium in a skin equivalent.

Although human keratinocytes in vitro have been shown to produce fibronectin, whether keratinocytes can contribute fibronectin to the dermal-epidermal junction or wound matrix is unknown. In order to approach this problem experimentally, we used the "skin equivalent" model composed of a native collagen gel populated with cultured fibroblasts and covered by cultured keratinocytes. By using bovine fibroblasts to populate the gel, fetal bovine serum in the culture medium, and human keratinocytes to form the epithelium, we were able to be certain that any human fibronectin produced in the culture was synthesized by the keratinocytes. A monoclonal antibody to fibronectin was found to recognize human but not bovine fibronectin. When the skin equivalent was stained by indirect immunofluorescence with antifibronectin, fibronectin was visible as an intensely staining band at the dermal-epidermal junction. In sections in which the dermis and epidermis had separated, the staining was usually limited to the dermal aspect of the skin equivalent. The results indicate that epithelium can contribute fibronectin to the dermal-epidermal junction and suggest that dermal staining in skin sections may originate from the epidermis. Since the developing skin equivalent has a rapidly growing epithelium and simulates a healing wound, contribution of fibronectin by the epithelium, in addition to that possibly contributed by serum and fibroblasts, may be of importance in wound healing.

Evidence supporting a role for immune complex-mediated inflammation in the pathogenesis of bullous lesions of systemic lupus erythematosus.

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.

Complement-activating immune deposits in systemic lupus erythematosus skin.

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.

Pemphigoid antibody mediated attachment of peripheral blood leukocytes at the dermal-epidermal junction of human skin.

It has been proposed that cutaneous inflammation and blister formation in bullous pemphigoid is caused by antibodies to the cutaneous basement membrane zone which active complement, thereby, attracting leukocytes to the dermal-epidermal junction. There is, however, no functional evidence which supports a role for pemphigoid antibodies in complement activation or leukocyte activity in skin. This study describes the in vitro attachment of human peripheral blood leukocytes to the dermal-epidermal junction of cryostat skin sections treated with 9/13 pemphigoid sera containing antibodies to the cutaneous basement membrane zone. A requirement for complement in the reaction was supported by the findings that only complement-fixing pemphigoid sera mediated the leukocyte response, a strong correlation existed between complement-fixation titers and leukocyte attachment titers and only leukocytes suspended in fresh serum but not buffer or heat inactivated serum attached at the junction. A requirement for antibody was supported by the observation that IgG fractions of 4 pemphigoid sera were as effective as whole sera in mediating leukocyte attachment. The leukocyte response was shown to be specific for complement-fixing pemphigoid sera since it was not observed with noncomplement-fixing sera or sera from 15 normal human and 22 nonpemphigoid disease controls. This study offers functional evidence for an interaction between pemphigoid antibody, complement and leukocytes in the immunopathogenesis of bullous pemphigoid and demonstrates that complement-fixing antibasement membrane zone antibodies may be important in initiating the cellular inflammatory events observed near the dermal-epidermal junction in vivo.

Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin.

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.