Repetitive Immunization Breaks Tolerance to Type XVII Collagen and Leads to Bullous Pemphigoid in Mice (1)
Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. Auto- antibodies in BP are directed against two hemides- mosomal proteins, BP230 and BP180 (type XVII collagen). Autoantibody reactivity against BP180 has been explored in various experimental animal models mimicking human disease, mostly by transfer of anti-BP180 autoantibodies in neonatal rodents. However, no model has been available in which tolerance to BP180 has been lost and in which clinical disease was present in immunocompetent animals over a prolonged time period.
12 female SJL/J mice were immunized at the hind footpad with 60 mg murine BP180 (mCOL17A fused with GST) emulsified in the nonionic block copolymer adjuvant TiterMax. Immunization was performed at week 0. Mice were either immunized once or received additional boosts 3 and 9 wk after initial immunization.
All immunized SJL/J female mice developed Ag-specific IgG and peaked at 4 wk after the initial immunization, though no correlation with disease severity was observed. Circulating IgG from mice immunized against murine BP180 bound to the epidermal side of the split. In contrast, serum from mice immunized with murine type VII collagen bound to the dermal side. Immunogold EM using murine BP180-immunized mice sera showed binding of autoantibodies mainly to the plasma membrane of the hemidesmosomal complex of nonimmunized control mice.
Six out of 11 female SJL/J mice immunized with murine BP180 developed skin lesions, including erythema, erosions, and crust between 6 and 14 wk after the initial immunization.Histopathologically, in all diseased female SJL/J mice, lesional skin biopsies showed subepidermal blisters accompanied by a dense inflammatory infiltrate in the underlying dermis. Mouse IgG deposits at the DEJ peaked at week 6, and declined until week 14, whereas C3 deposits at the DEJ were weaker at week 6 compared with week 14.
At week 6, IgG binding was observed in 10 out of 11 female SJL/J mice immunized against murine BP180, whereas complement C3 deposits to the DEJ were found in 7 out of 11 female mice and 2 out of 6 male mice at week 14. All female SJL/J mice with clinical skin lesions showed IgG deposits at week 6 and 14, and C3 deposits at the DEJ at week 14. In none of other strains, even in B6.SJL-H2s, which have the same H2s haplotype as SJL/J mice, development of skin lesions was observed even though most mice developed circulating autoantibodies.
IgG1, IgG2b, and IgG2c demonstrated linear deposits of these Ab subclasses at the DEJ by direct IF microscopy. Stainings for IgG3 were negative in both groups. By ELISA, IgG1, IgG2b, and IgG2c Abs to mCOL17A were detected in sera from all immunized SJL/J mice, whereas levels were not significantly different between diseased and nondiseased immunized mice.
IgG purified from six diseased SJL/ J or six nondiseased BALB/c mice was injected into the ears of healthy C57BL/6 mice. Macroscopically, erythema, erosion, and crust were observed in mice injected with IgG purified from SJL/J, but not with IgG from BALB/c, mice. Deposition of IgG1 at the DEJ was found in mice injected with IgG from both SJL/J and BALB/c mice. However, deposits of IgG2b and IgG2a/c were only observed in mice injected with IgG purified from SJL/J but not from BALB/c mice.
As B6-SJL.H2s mice also carrying H2s did not develop BP, it is likely that genes outside of the MHC locus contribute to disease susceptibility, which has also been noted in humans (e.g., association of BP with low-affinity FcgRIIIa gene in white patients). SJL/J mice are used for induction of experimental autoimmune encephalitis (EAE), supporting the immunological uniqueness of this strain. Incidence of immunization-induced EAE is significantly higher in female mice. This observation is paralleled by elevated levels of circulating CD4+ T cells in female, as compared with male SJL/J mice. In contrast, castration of C57BL/6 males showed no change in disease severity. An androgen treatment reduced EAE disease severity in female SJL/J mice.