Autoantibodies to BPAG1e trigger experimental bullous pemphigoid in mice (1)
Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering disease characterized by autoantibodies in BP patients against the dermal-epidermal junction (DEJ). IgG antibodies target two hemidesmosomal proteins, BP180 and BP230/bullous pemphigoid antigen 1e (BPAG1e). BP230, also known as BPAG1e or dystonin (DST)-e, is a cytoplasmic protein of the hemidesmosomal plaques that belong to a family of intermediate filament-binding plakin proteins. BPAG1e is composed of the plakin domain, a central coiled-coil rod domain, and an intermediate filament-binding domain (IFBD). BPAG1e C-terminal portion is the most frequent target region for autoantibodies in BP patients with anti-BP230 antibodies. The antibody titer after immunization was 25,600, which was significantly higher compared with before immunization and wild-type mice.
Approximately 4 weeks after splenocyte transplantation, some mice expressed scaling and erosion on their ears, dorsum, and tail, and the symptoms gradually worsened. Histological examination by hematoxylin and eosin (H&E) staining showed subepidermal blisters with hyperkeratosis and acanthosis. The DIF analyses showed only IgG1 antibody deposition at the DEJ. Furthermore, CD4+ cells significantly infiltrated in the dermis compared with CD8+ cells.
Immunoblots using sera and whole protein from cultured murine keratinocytes showed a weak band over the 250-kDa marker, which was consistent with a band by BPAG1-specific antibodies. Furthermore, reaction with their sera and recombinant BPAG1e-C showed a strong band; however, the control K5-Cre:Bpag1-/- mice did not have antibodies for either BPAG1e or the recombinant BPAG1e-C protein. The recipient Rag2-/- mice, which expressed BP-like erosions, yielded more significant bands than the donor mice. The binding activities for recombinant BPAG1e-C were also potentiated in the recipient mice. Rag2-/- mice possessed no antibodies against BPAG1e-C. Forty-eight hours after injection, the mice clinically exhibited no skin fragility. However, most of the sera-injected mice (4/5) had microscopic subepidermal blisters in the dorsal skin and IgG deposition at the DEJ. In contrast, the control mice with murine IgG injection induced neither blister formation nor IgG deposition at the DEJ.
Compared with the nonwounded control group, the wounded group showed a significantly increased incidence of clinical symptoms. Furthermore, the period from injection to the development of BP-like symptoms was clearly shorter in the wounded than in the nonwounded group. The clinical score was measured every week and revealed that compared with the nonwounded recipient mice, the wounded recipient mice exhibited severe symptoms on their feet and tails, and a high risk of death. For the observation period of 8 weeks after adoptive transfer, of the 15 mice that developed a BP-like phenotype, 6 mice died of a severe BP-like phenotype and 9 mice were alive with erosion and crusting. Early development of the BP-like phenotype was not relevant to disease severity. The recipient Rag2-/- mice with the BP-like symptoms showed a significantly higher ELISA index compared with the asymptomatic mice.
The pathogenic importance of BPAG1e remains controversial because BPAG1e is an intracellular protein. It has been questioned whether antibodies for BPAG1e result from epitope spreading after a reaction to the BP180 antigen in BP patients. Regarding BP, adoptive transfer of splenocytes, which were immunized with human BP180, into Rag2-/- mice expressing human BP180 generated antibodies against various sites of the human BP180 protein, including the NC16A domain, as well as the plakin domain of BPAG1e. Autoantibodies directed to BPAG1e directly contribute to skin blistering and tissue damage, either independently and/or in synergy with anti-BP180 antibodies. In general, BP patients with antibodies against only BPAG1e showed significantly lower severity than those with antibodies against BP180. Exposure of the antigen, including due to trauma, might induce serious conditions of BP, even if it was generated solely by the anti-BPAG1e antibodies.