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.

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.

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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.

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 .