Pathogenicity of Autoantibodies in Anti-p200 Pemphigoid
Figure 3
Human IgG specific to laminin γ1 is not pathogenic ex vivo.
Using recombinant forms of the C-terminus of laminin γ1 (hLAMC1-cterm) and full length laminin γ1 (LAMC1-FL) IgG specific for hLAMC1-cterm (a, c; lane 3) and LAMC1-FL (b, lane 3; c lane 5 ) was generated from anti-p200 pemphigoid serum (a, b, c; lane 2), as well as serum depleted from anti-hLAMC1-cterm (a, c, lane 4) and LAMC1-FL reactivity (b, lane 4; c lane 6) reactivity, respectively, as shown by immunoblotting with recombinant hLAMC1-cterm (a), LAMC1-FL (b), and extract of human dermis-(c). Interestingly, serum depleted from anti-hLAMC1-cterm and LAMC1-FL reactivity, respectively, (a, b; lane 4) still labeled the p200 protein in dermal extract (c, lane 4, lane 6). Monoclonal antibody against LAMC1 (a, b, c; lane 1) and serum from a healthy volunteer (a, b; lane 5; c lane 7) were used as controls. Arrows indicate the positions of the proteins and bars the molecular weight markers (a, 34 kD and 26 kD; b, 200 kD; c, 200 kD). hLAMC1-cterm-specific (h, i) and LAMC1-FL-specific patients IgG (l, m) and the monoclonal anti-LAMC1 antibody (d, e) labeled the dermal-epidermal junction (DEJ) by indirect immunofluorescence (IF) microscopy but did not induce dermal-epidermal separation (DES). In contrast, serum depleted from reactivity against hLAMC1-cterm (j, k), and LAMC1-FL (n, o), respectively, as well as patient serum (f, g) resulted in DES (black triangles mark base of the split). While untouched patient serum (f) as well as serum depleted from anti-hLAMC1-cterm (j) and LAMC1-FL reactivity (n), respectively, stained the DEJ of human skin in a linear pattern, the monoclonal anti-hLAMC1-antibody (d), hLAMC1-cterm-specific patient-IgG (h), and hLAMC1-FL-specific patient IgG showed an additional staining of basal keratinocytes. Serum from a healthy volunteer was used as control (p, q). Magnification: x200.