Pemphigus Vulgaris and Its Active Disease Mouse Model (1)
Pemphigus is a group of chronic blistering skin diseases in which autoanti- bodies are directed against the cell surface of keratinocytes, resulting in the loss of cell-cell adhesion of keratinocytes through a process called acantholysis. Pemphigus can be divided into three major forms: pemphigus vulgaris (PV), pemphigus foliaceus (PF) and paraneoplastic pemphigus. The hallmark of pemphigus is the finding of IgG autoantibodies against the cell surface of keratinocytes. The PV and PF antigens are 130-kDa and 160-kDa transmembrane glycoproteins, termed desmoglein 1 (Dsg1) and Dsg3, respectively. Anti-Dsg3 and anti-Dsg1 IgG autoantibodies were necessary for efficient PV blister formation in the skin of neonatal mice and were generally correlated with disease activity. In both PF and PV patients, major epitopes were mapped to the respective N-terminal 161 residues of Dsg1 and Dsg3. Although there was no single major epitope on Dsg1 and Dsg3, dominant epitopes were further mapped to residues 26–87 of Dsg1 and 25–88 of Dsg3, both of which are in the N-terminal EC1 domain.
The conventional approach to develop an autoimmune mouse model is forced immunization of autoantigens in various strains of mice with various kinds of adjuvants. If it were possible to remove the antigen during the development of the immune system, or if the antigen were not present from the start, toler- ance against the removed or absent molecule would not be acquired. The transferred lymphocytes from the autoantigen knockout mice should be persistently stimulated by the endogenous autoantigen in the recipient mice and should therefore produce antibodies against the autoantigen with resultant phenotypes of the human disease.
The PV model mice were generated from a panel of anti-Dsg3 IgG monoclonal antibodies (AK mAbs) . AK19 mAb and AK23mAb induced blister formation after passive transfer, while the remaining mAbs failed to display pathogenic activities. In the ascites formation assay, only AK23 mAb induced blisters, and the phenotypes of the mice that received AK23 hybridoma cells were virtually identical to those of PV model mice and Dsg3-/- mice. AK23 and AK19 are capa- ble of inducing both the loss of cell-cell adhesion of keratinocytes and blister formation with different potencies, while the other AK mAbs apparently lack pathogenic activities.
The epitopes of AK19 and AK23, which possessed pathogenic activities, were calcium dependent and were located in residues 89–161 and 1–63, respectively. Subsequent extensive studies with point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognizes a conformational epitope that consists of the V3, K7, P8 and D59 residues of Dsg3.
Residues V3, K7, P8 and D59 of the epitope of AK23, which is the most potent pathogenic mAb tested, were located precisely on the Dsg3-specific residues of the adhesive interface. In contrast, the AK mAbs which lacked appar- ent pathogenic activities recognize the middle to C-terminal portion of the extracellular domain, a region in which no direct molecular interaction is predicted.
Ten anti-Dsg3 IgG mAbs were isolated from PV model mice that received naive Dsg3/ splenocytes (NAK series). Although eight of ten NAK mAbs showed pathogenic activity in the passive transfer assay, none induced the PV phenotype in the ascites formation assay, indicating that none were as potent as AK23 mAb, which could induce the PV phenotype in adult mice upon hybridoma inoculation. The minimal combination tested that was sufficient to induce the phenotype in all mice tested was NAK1, 2, 7 and 11 mAbs or NAK2, 3, 5 and 11 mAbs. Thus, NAK mAbs recognizing the middle to C-terminal extracellular domain of Dsg3 showed synergistic effects with mAbs reacting with the N-terminal domain.