Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen (1)
Epidermolysis bullosa acquisita (EBA), a severe chronic subepidermal blistering disease of skin and mucous membranes, is characterized by tissue-bound and circulating IgG antibodies specific to the dermal-epidermal junction (DEJ). Patients’ serum autoantibodies bind to the 290-kDa type VII collagen, the major component of anchoring fibrils. Epitopes recognized by the majority of EBA sera were mapped to the noncollagenous 1 (NC1) domain of type VII collagen.
Three fragments of murine type VII collagen NC1, purified by glutathione-affinity chromatography, migrated consistently with their calculated masses of 38.8, 38.2, and 49.5 kDa when separated by SDS-PAGE.
Circulating IgG from immune rabbit sera, but not preimmune and normal rabbit sera, bound to the DEJ, as shown by IF microscopy using murine skin as substrate at a titer of 20,480. In addition, immune sera stained intact human skin. By immunoblot analysis, IgG antibodies from immune sera targeted both cell-derived and recombinant forms of type VII collagen.
Adult nude mice were injected with different doses of IgG purified from rabbits immunized with type VII collagen or from normal rabbits. Within 24–48 hours after the first injection, mice that received 15 mg and 7.5 mg of IgG specific to type VII collagen per injection developed single blisters on their tails and ears, often accompanied by mild erythema. In mice that received 3.75 mg of IgG specific to type VII collagen per injection, initial skin lesions were observed 48–72 hours after the first injection. 15 mg of IgG specific to type VII collagen per injection to BALB/c (n = 2) and C57BL/6 mice. Single blisters developed initially 6 days after the first injection on tail base, snout, and ears and then spread to limbs and trunk. Widespread lesions, including blisters, erosions, and crusts, were seen after 9 days in all mice injected with pathogenic IgG. Adult mice with skin disease showed significant weight loss (mean of 15% of initial weight, P < 0.001).
IF microscopy of perilesional mouse skin revealed linear deposits of rabbit IgG and murine complement C3 at the DEJ in adult mice that received IgG specific to type VII collagen.
In all adult mice injected with IgG specific to type VII collagen, light microscopic analysis of skin biopsies revealed extensive dermal-epidermal separation accompanied by different degrees of inflammatory infiltrates that were dominated by neutrophils.
Significantly more extensive disease was induced in mice injected with 7.5 mg or 15 mg immune IgG compared with mice treated with 3.75 mg IgG (Figure 7). Mice injected with 7.5 mg or 15 mg immune IgG showed no significant difference with regard to the extent of the disease.
Mice treated with F(ab′)2 preparations, used at the same molar concentration as unfragmented IgG, failed to induce dermal-epidermal separation. By IF microscopy, IgG deposits were found in perilesional skin of mice injected with intact pathogenic IgG using both antibodies to IgG-Fab and IgG-Fc. In contrast, in mice receiving F(ab′)2 fragments, positive staining for Fab but not for Fc.
Like C57BL/6 and BALB/c mice, C5-sufficient mice developed initial blisters 6 days after the first injection of 15 mg of rabbit IgG specific to type VII collagen, and widespread disease was observed 9 days after the first injection.
When injected into adult nude, BALB/c, and C57BL/6 mice, rabbit IgG specific to type VII collagen bound to the DEJ of mouse skin, activated complement, and induced subepidermal blisters. Lesions first developed in friction-exposed regions distant from the injection site, including tail and ears, and then on legs and trunk. BALB/c and C57BL/6 strains are most commonly used for genetic manipulations. The induction of an EBA phenotype in these strains will allow the use of various knockout and transgenic mice to study the pathogenesis of blister formation in experimental EBA. Since C5-deficient mice are resistant to the induction of blisters by antibodies specific to type VII collagen, activation of terminal complement components is required for blister formation in experimental EBA.