Passive transfer of collagen XVII-specific antibodies induces sustained blistering disease in adult mice (1)
Bullous pemphigoid (BP) is characterized by the presence of tissue-bound and circulating autoantibodies directed against the dermal-epidermal junction (DEJ). Antibodies in BP patients mainly recognize BP180 (bullous pemphigoid antigen of 180kDa) also known as collagen (C)XVII, a transmembrane protein with a type II orientation that spans the lamina lucida, and to a lesser extent BP230 (bullous pemphigoid antigen of 230kDa), an intracellular hemidesmosomal protein. Antibodies against mouse and human BP180/ CXVII induce subepidermal blisters when passively transferred into wild-type or CXVII-humanized mice, respectively; in addition, antibodies to human type XVII collagen cross the placenta of immunized mice and induce disease in type XVII collagen-humanized neonates. Autoantibodies in the majority of BP patients recognize the non-collagenous 16th (NC16)A domain of BP180/ CXVII and pre-adsorption of pathogenic sera with NC16A abolished their pathogenic potential both in vivo and ex vivo. Four fragments of murine BP180/ CXVII cloned in a prokaryotic expression vector were expressed in E. coli. The proteins, purified by glutathione-affinity chromatography, migrated consistently with their calculated masses of 37, 32, 39 and 57 kDa when separated by SDS-PAGE.
Antibodies from both rabbit and sheep showed a linear staining of the basal membrane by IF microscopy using murine skin as a substrate. In contrast, antibodies obtained before the first immunization did not bind to the DEJ. When incubated with 1M NaCl–split mouse skin, antibodies from immunized rabbits and sheep stained the epidermal side of the substrate. By immunoblot analysis, antibodies from immune sera, in contrast to control sera, recognized recombinant forms of BP180/ CXVII.
In mice receiving 10 mg/injection of BP180/ CXVII-specific rabbit IgG lesions first appeared 9–10 days after the primary injection. In these mice, initial blisters evolved into erosions partly covered by crusts on an erythematous background. With 10 mg/injection of sheep BP180/ CXVII-specific antibodies every second day, initial lesions, including blisters and erosions appeared after 5–6 days in mice injected with these sheep antibodies. Lesions developed at different sites, including the ears, the snout, hind limbs, abdomen, and the back. In general, more extensive disease developed around the injection sites, but few lesions also developed at distant sites. deposits of complement C3 were detected at the DEJ of mice injected with rabbit and sheep BP180/ CXVII-specific IgG.
By Spearman’s rank correlation test, titres of serum antibodies strongly correlated with the extent of skin disease both in mice receiving rabbit (r = 0.96) and sheep antibodies respectively (r=0.90). Sheep immune serum showed higher BP180/ CXVII-specific reactivity compared with rabbit immune sera.
Mice receiving BP180/ CXVII-specific sheep IgG showed an earlier onset of disease phenotype and higher scores at any time point when compared to mice injected with the same amount of BP180/ CXVII-specific rabbit IgG whereas mice receiving pre-immune antibodies showed no clinical phenotype.
Since only about 85% of the BP sera react with the immunodominant NC16A domain of BP180/ CXVII by ELISA and most BP sera also recognize other epitopes. This may be due to the higher titre of injected sheep antibodies and/or to a better in vivo activation of murine innate immune players, including complement and inflammatory cells. A further major advantage of the newly developed mouse model of BP is the fact that it allows for induction of sustained disease and longer observation times of weeks, which can be likely extended to months.