Definition of bullous pemphigoid antibody binding to intracellular and extracellular antigen associated with hemidesmosomes

Bullous pemphigoid (BP) antibodies are deposited predominantly in the lamina lucida in vivo; however, circulating BP antibodies bind in vitro to the cytoplasmic plaque of basal cell hemidesmosomes. We examined the ability of IgG in nine BP sera to bind to intracellular or extracellular antigen. On skin cryosections, indirect IF showed IgG bound to basement membrane zone (BMZ) and indirect ImmunoEM confirmed intracellular binding on the cytoplasmic plaque of hemidesmosomes. In contrast, when normal skin was exposed to BP serum in organ culture, direct IF showed fainter linear deposition of IgG along the BMZ, and direct ImmunoEM demonstrated extracellular IgG binding in the lamina lucida, predominantly beneath hemidesmosomes. Four of nine sera showed complement fixation on indirect IF samples (IgG bound to intracellular antigen) and three showed complement fixation on direct IF specimens (IgG bound to extracelluar antigen). Three of the nine sera con tained complement fixing antibodies detectable only in antibody populations specific for intracellular or extracellular antigen. Western immunoblots showed that five of nine sera recognized a 240-kD protein and four of nine recognized a 180-kD protein. There was no correlation between the presence (or absence) of either band and the detection of complement fixing antibodies specific for intracellular or extracellular antigen. BP autoantibodies bind both intracellular and extracellular antigen, and IgG binding exclusively to extracellular antigen that mimics the in vivo situation can be detected by using organ culture. Complement fixation may be restricted to antibodies specific for intracellular or extracellular antigen. These findings underscore the complexity of the autoantibody-antigen system in BP and have implications regarding the proposed pathogenicity of the autoantibodies.