Search

In this study, an in vitro model of immune complex-mediated basement membrane zone separation caused by periphigoid antibodies, serum complement, and peripheral blood leukocytes is described. When cryostat sections of fresh-frozen normal human skin were treated with either of 4 bullous pemiphigoid sera containing complement-activating anti-basement membrane zone antibodies and subsequently incubated at 37 degrees C with normal human peripheral blood leukocytes and fresh human serum, leukocytes attached to 96% of the basement membrane zone in 100% of sections. Sixty-seven percent of the sections developed focal areas of basement membrane zone separation resembling dermal-epidermal separation described in early pemphigoid lesions. In control sections in which either leukocytes, pemphigoid antibody or fresh human serum were omitted, significantly less leukocyte attachment and basement membrane zone separation occurred. Evidence that leukocytes caused separation was supported by an absolute requirement for viable leukocytes during incubation, a high correlation between leukocyte attachment and separation and experiments showing that leukocytes attached to the basement membrane zone were activated. This study provides the first in vitro evidence directly supporting a functional role for immune-complex mediated inflammation in the pathogenesis of basement membrane zone separation and blisters in bullous pemphigoid.

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.

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.

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.

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

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.

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

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

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.

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