Studies on the pathogenesis of bullous pemphigoid in vivo in a xenogeneic SCID-hu transplantation model and in vitro in cryosections of human skin
Project management at the University of Würzburg:
Pemphigus and bullous pemphigoid (BP) are blistering skin diseases associated with IgG autoantibodies to desmosomal and hemidesmosomal components. When autoantibodies to desmoglein 1 and 3 from patients with pemphigus foliaceus (PF) and pemphigus vulgaris (PV) or rabbit antibodies against the murine hemidesmosomal component BP180 are passively transferred into neonatal mice, they induce blisters in the skin of the mice. To develop an animal model that would duplicate the findings in the skin of the patients more closely, we grafted full-thickness human skin from healthy volunteers onto SCID mice. Injection of the purified IgG fraction from the serum of PF and PV patients led to subcorneal and suprabasal splits in the human grafts and human IgG was deposited intercellularly in the upper and lower layers of the epidermis, respectively. Interestingly, anti-BP180 autoantibodies purified from serum of BP patients and from a rabbit immunized with recombinant human BP180 strongly bound to the basement membrane zone of the grafts (n=32), fixed murine complement, led to the recruitment of neutrophils to the upper dermis of the graft but did not induce subepidermal blisters. We report a novel experimental model for PF and PV which should greatly facilitate further studies dissecting the immunopathological mechanisms in these diseases. Specifically, this model can be used to identify pathogenically relevant epitopes on human desmoglein 1 and 3 and to develop novel strategies for the treatment of pemphigus.
Subsequently, to analyze the pathogenic effect of antibodies from patients with BP, we used a different strategy. Most sera from BP patients recognize epitopes within the N-terminal NC16A portion of the BP180 ectodomain. Previously, using cryosections of human skin, patients? sera had been shown to generate dermal-epidermal separation when co-incubated with leukocytes and complement from healthy volunteers. However, the specificity of pathogenic autoantibodies in BP patients had not yet been elucidated. By the use of a modified version of the cryosection model, we showed that sera from all of 13 BP patients and from 2 rabbits, immunized against BP180 NC16A, induced dermal-epidermal separation. This finding was confirmed with the use of IgG purified from patients? sera, while sera and purified IgG from healthy controls were not pathogenic. The induction of subepidermal splits in this experimental model was shown to be dependent on the presence of neutrophils, but not complement. Interestingly, patients? autoantibodies affinity-purified against a recombinant form of BP180 NC16A retained their blister-inducing capacity, whereas patients? IgG depleted of reactivity to NC16A lost this ability. F(ab?)2 fragments of antibodies specific to NC16A, lacking the Fc portion, did not induce splits. In addition, patients? autoantibodies purified against a recombinant fragment of the C-terminus of BP180 as well as rabbit antibodies to the intracellular portion of BP180 and to BP230 did not cause dermal-epidermal separation. Our in vitro results support the idea that autoantibodies to BP180 from patients with BP are of pathogenic relevance.
Projekt period: from 01.2000 to 12.2002
DFG ,Granting date: 01.09.1999