Abstract
Recently, the C-terminus of laminin γ1 has been identified as target antigen in anti-p200 pemphigoid and the disease was renamed as anti-laminin γ1 pemphigoid. However, the pathogenic relevance of these autoantibodies has not yet been demonstrated. Therefore, we employed an ex vivo model of autoantibody-mediated leukocyte-dependent neutrophil activation and dermal-epidermal separation (DES) using cryosections of human skin. We showed that anti-p200 pemphigoid sera (n = 7) induced DES in a time-dependent manner, in contrast to sera from healthy controls. Furthermore, laminin γ1-specific IgG and serum depleted from anti-laminin γ1 reactivity were generated using the recombinant C-terminus of laminin γ1 (LAMC1-term; amino acids 1364 to 1609). Interestingly, both fractions labeled the dermal-epidermal-junction (DEJ) by indirect immunofluorescence microscopy on human foreskin and recognized a 200 kDa protein by immunoblotting with dermal extract. Human and rabbit IgG against LAMC1-cterm failed to attract neutrophils at the DEJ and to induce DES. In contrast, patient serum depleted from LAMC1-cterm reactivity led to the same extent of DES as non-depleted IgG. Repeated injection of rabbit anti-murine LAMC1-cterm IgG into both neonatal and adult C57BL/6mice as well as repetitive immunization of various mouse strains with murine LAMC1-cterm failed to induce macro- and microscopic lesions. In all mice, circulating anti-LAMC1-cterm antibodies were present, but only in some mice, IgG deposits were seen at the DEJ. We conclude that autoantibodies in anti-p200 pemphigoid sera are pathogenic while pathogenicity is not mediated by autoantibodies against laminin γ1. Further studies are needed to identify the pathogenically relevant autoantigen in anti-p200 pemphigoid.
Highlights
Anti-p200 pemphigoid is an autoimmune subepidermal blistering disease which was first described in 1996 [1,2]
We previously developed an ex vivo model in which incubation of IgG from patients with bullous pemphigoid (BP) and epidermolysis bullosa acquisita (EBA) or rabbit antibodies raised against the target antigens BP180 and type VII collagen, respectively, induced leukocyte-dependent dermal-epidermal separation in cryosections of human skin [10,16]
Serum samples from anti-p200 pemphigoid patients (n = 7) as well as sera from a patient with BP and from healthy volunteers were incubated with cryosections of human skin in the presence of leukocytes purified from healthy donors
Summary
Anti-p200 pemphigoid is an autoimmune subepidermal blistering disease which was first described in 1996 [1,2]. The disease is characterized by tense blisters and resembles bullous pemphigoid, the most frequent autoimmune blistering disease patients with anti-p200 pemphigoid tend to be younger [3]. Autoantibodies in patients’ skin localize along the dermalepidermal junction (DEJ) by direct immunofluorescence (IF) microscopy. Serum IgG autoantibodies label the dermal side of 1 M NaCl-split human skin by indirect IF microscopy and recognize a 200 kDa protein by immunoblotting of human dermal extract [1,2]. Dainichi et al showed reactivity with anti-laminin c1 in about 90% of patients’ sera and coined the term anti-laminin c1 pemphigoid [6,7]. The Cterminus of laminin c1 was identified as the immunodominant region of this protein, a finding that we recently confirmed by developing an ELISA using a recombinant monomeric C-terminal fragment of laminin c1 [8]
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