Evidence that Anti-Type VII Collagen Antibodies Are Pathogenic and Responsible for the Clinical, Histological, and Immunological Features of Epidermolysis Bullosa Acquisita (1)
Epidermolysis bullosa acquisita (EBA) is an incurable autoimmune blistering disease of the skin characterized by skin fragility, blisters in trauma-prone sites, and scarring with milia formation and nail dystrophy. EBA autoantibodies bind to type VII collagen (especially its NC1 domain) within anchoring fibrils and this binding is associated with a diminution of normal anchoring fibrils in the patient’s skin and subsequent epidermal–dermal disadherance. Type VII collagen is composed of three identical α chains. Each α chain consists of a central collagenous domain flanked by a 145 kDa non-collagenous amino-terminal domain (NC1) and a 30 kDa carboxyl-terminal domain.
After the cells were transfected with the pRC/CMV vector containing human cDNA for the NC1 domain of type VII collagen, the cells synthesized and secreted the 145 kDa NC1 domain of type VII collagen. The recombinant NC1 was then used to immunize rabbits to produce polyclonal anti-NC1 antibodies. The anti-NC1 IgG only labeled the 145 kDa NC1 and did not label other matrix proteins including type I collagen, type IV collagen, fibronectin, laminin-1, and laminin-5. The purified IgG fraction from the rabbit anti-NC1 sera strongly labeled the BMZ of both human and mouse skin.
SKH1 mice were given daily intradermal injections of purified IgG fractions prepared from the rabbit anti-NC1 antisera and control rabbit sera at doses from 0.1 to 1 mg per g body weight. animals injected with the rabbit anti-NC1 IgG showed numerous erosions and crusts forming from ruptured blisters as early as 4 d after the initial injection at the injection site. All of the mice developed blisters and then erosions at the injection site, and most, but not all mice, developed bullae and erosions on the ears, hips, and paws. Nail loss was also observed in 80% of injected mice. A greater percentage of the total body surface area was involved with erosions and blisters when the mice were given higher (>1 mg per g body weight) doses of rabbit anti-NCI IgG. Animals given doses less than 0.2 mg per g body weight of immune IgG did not develop any skin lesions.
Histological examination of mouse skin injected intradermally with rabbit anti-NC1 IgG demonstrated a full sub-epidermal blister with a clean separation between the epidermis and dermis.
Direct immunofluorescence (DIF) of perilesional and lesional skin of all mice injected with rabbit anti-NC1 IgG showed strong deposition of tissue-bound, rabbit, anti-NC1 IgG at the DEJ between the epidermis and dermis of the mouse skin. Within the dermal inflammatory infiltrate, neutrophils could be detected in small numbers.
Serum from the diseased mice had rabbit antibodies that bound to the DEJ of normal human skin, and the dermal side of the salt-split human skin, reminiscent of EBA patient sera.
Patients with systematic lupus erythematosus (SLE) develop autoantibodies to the EBA antigen, they develop skin blisters and fall into a subset called “bullous SLE”. EBA patients often develop blisters and erosions in oral mucosa. In these studies, we did not examine formally whether the experimental mice had significant involvement with the oral mucosa. The immune IgG-injected mice, however, did not exhibit significant weight loss even though they had many months of active skin lesions. This suggests that the animals did not have significant oral mucosal involvement that inhibited their ability to eat and drink. Specific domains within the NC1 domain of the type VII collagen α chain have affinity for laminin-5, type IV collagen, and fibronectin. These interactions may be necessary for keeping the DEJ intact. EBA antibodies are more potent activators of complement than are antibodies in the sera of patients with bullous pemphigoid, a prototypic inflammatory autoimmune bullous disease. The presence of complement-activating IgG autoantibodies does not correlate with the inflammatory or non-inflammatory EBA clinical phenotypes.