Since human and murine BP180 antigens are not immune cross-reactive, a humanized mouse strain expressing the BP immunodominant epitopes, BP180NC16A domain, is needed to study the humoral immune response in BP disease immunopathology. In this work, we created such a mouse strain (NC16A+/+) and demonstrated that anti-BP180NC16A autoantibodies in BP are indeed pathogenic. These findings are in agreement with a recent report by Nishie et al [38
], in which anti-BP180 autoantibodies induce BP in mice humanized with whole BP180 instead of only the NC16A domain.
The NC16A humanized mouse model also sheds light on the structure/function of BP180. This transmembrane collagen is a key component of the junctional adhesion complex, or hemidesmosome, and is thought to function in cell-matrix adhesion [3
] The homologous human BP180NC16A and murine BP180NC14A domains are not cross-reactive immunologically and exhibit only 59% identity and 71% similarity, compared with the relatively high overall homology exhibited by the human and murine BP180 proteins (81% identity, 86% similarity) [3
] Nevertheless, the humanized NC16A mice are phenotypically normal and develop normal hemidesmosomes at both the microscopic and ultrastuctural levels. Our findings suggest that domain-specific replacement of mBP180NC14A with hBP180NC16A does not impair the expression and function of this protein in vivo
, indicating that this model system might well prove useful in future investigations into the biological roles of BP180. In addition, domain-specific humanization could be a useful approach for a human protein that may lead to a dominant negative phenotype when introduced into mice as a whole molecule.
We previously showed that an intact complement cascade, degranulation of resident mast cells, and activation of infiltrating neutrophils are required in the dermal-epidermal separation induced by experimentally generated rabbit anti-mBP180 antibodies [42
] While the rabbit anti-mBP180 IgG passive transfer model has provided invaluable insight to the key steps in BP disease development, it has been debated whether the rabbit anti-mBP180 IgG faithfully mirrors effector functions of pathogenic anti-BP180 autoantibodies since rabbit IgG is a single isotype while the BP autoantibodies are a mixture of IgG1, IgG3 and IgG4. Our present study offers us direct evidence that the key innate immune system players, complement and inflammatory cells, also play a critical role in BP autoantibody-mediated subepidermal blistering. Involvement of these same components in the immuno-pathogenic response to BP autoantibodies has also been supported by the results of previous clinical and in vitro
studies. Intact and degranulating mast cells, eosinophils, and neutrophils are seen in the dermis, suggesting these cells have been activated locally [24
] In vitro
dermal-epidermal junction separation of human skin sections induced by human BP autoantibodies depends on complement and leukocytes (predominantly neutrophils) [50
] Human BP blister fluids contain high levels of both neutrophil elastase and gelatinase B, but BP180 degradation by blister fluid depends on neutrophil elastase activity [52
] Thus, the present findings provide important information concerning potential therapeutic targets for BP.
While human BP and humanized murine BP closely mimic each other at the clinical, histological, and immunological levels, the IgG passive transfer models do not reflect the large number of eosinophils typically found in the inflammatory infiltrate of human BP lesional skin. Therefore, the question of the role of eosinophils in BP cannot be addressed in our humanized BP180NC16A mouse model.
In conclusion, this study provides direct evidence that anti-BP180NC16A autoantibodies are pathogenic and their tissue injury activity depends on innate immune responses. This novel experimental mouse model will be useful for dissecting the immunopathology of BP, and will aid in the future investigation of some important questions which could not be addressed before. These include the role of different isotypes and IgG subclasses of anti-BP180 autoantibodies since BP180-specific IgA, IgE, IgG1, IgG3, and IgG4 are present in BP [39
] This humanized mouse strain may also provide an in vivo
system to elucidate the pathogenic mechanisms of other subepidermal blistering diseases with an autoimmune response to BP180, such as herpes gestationis, mucous membrane pemphigoid, linear IgA bullous dermatosis, and lichen planus pemphigoides [18