1. Autoantibodies to BP180 Associated with Bullous Pemphigoid Release Interleukin-6 and Interleukin-8 from Cultured Human Keratinocytes 
Enno Schmidt, Stanislaus Reimer, Silke Jainta, Eva-Bettina Bröcker, Detlef Zillikens 
Journal of Investigative Dermatology 
Volume 115, Issue 5, Pages (November 2000)
DOI: /j x
Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Schematic diagram of recombinant forms of the human BP180 protein. The transmembrane region of BP180 is labeled TM, the cytoplasmatic domain is shown to the left and the C-terminal ectodomain, consisting of 15 interrupted collagen domains (shaded boxes), is shown to the right. Amino acid residue numbers are shown next to the boxes. The NC16A domain has been subdivided into five regions, each of approximately 15 amino acids in length. Each region represents one epitope designated MCW-0 to MCW-5. The bright box located to the lower right indicates the position of the homologous mouse epitope recognized by pathogenic antibodies identified in the passive transfer mouse model (Liu et al. 1995). The bright box at the lower left represents the position of the pathogenic epitopes on human BP180 as defined in this report.
Journal of Investigative Dermatology  , DOI: ( /j x)
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3. Figure 2
BP sera used in this study react with different epitopes within the N-terminal 45 amino acids of BP180 NC16A. Three BP sera (panels a–c) and one NHS (d) were tested for immunoblot reactivity with the BP180 NC16A fusion proteins NC16A1–5 (lane 1), NC16A1–3 (lane 2), NC16A3 (lane 3), NC16A2.5 (lane 4), NC16A2 (lane 5), and NC16A1 (lane 6). Migration positions of molecular weight markers of 31 and 21 kDa are indicated to the left and right.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
BP sera react with cultured NHEK but not with GABEB cells by indirect IF microscopy. NHEK (a–d) and keratinocytes from a patient with GABEB (e–h) were incubated with serum R594 (generated against human BP180 NC16A2–4; a, e), serum from a patient with BP (b, f), normal human serum (c, g), and with antibody GB-3 to the laminin 5 γ2 chain (d, h) for 30 min. The staining of the BP serum is representative for the three BP sera used in this study. In two BP sera, an additional punctate staining pattern was observed. Scale bar: 10 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
NHEK secrete IL-6 and IL-8 in a concentration- and time-dependent manner after incubation with BP IgG. NHEK were treated with IgG from patient BP1 (black columns) and from a healthy control subject (white columns). Striped columns represent the culture medium without any IgG. Levels of IL-6 (A, B) and IL-8 (C, D) were assayed using 4 mg IgG per ml after incubation periods of 1.5, 3, 6, 9, 12, and 24 h, respectively (A, C). In (B) and (D), data are given for a 12 h incubation period with IgG concentrations from 0.4 to 8.0 mg IgG per ml. Cytokine levels in the supernatant were analyzed in quadruplicate by ELISA. Bars indicate mean ± SD (pg per ml). Asterisks indicate statistical significance between BP IgG-treated and normal IgG-treated cells (***p < 0.001; **p < 0.01; *p < 0.05). This pattern is representative of the patterns seen in three separate experiments with keratinocytes from different donors.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
GABEB keratinocytes are not induced to secrete IL-8 by BP IgG. BP180-deficient GABEB cells were grown in 0.15 mM Ca2+ (dark columns) and 1.5 mM Ca2+ culture medium (bright columns), respectively. Cells were incubated for 12 h with 4 mg IgG per ml from three BP patients (BP IgG), one pemphigus vulgaris (PV IgG) and one pemphigus foliaceus patient (PF IgG), two healthy volunteers (Normal IgG), and with medium alone (Medium). Culture supernatants were assayed in quadruplicate by ELISA. Bars indicate mean ± SD (pg per ml). Asterisks indicate statistical significance between patient IgG-and normal IgG-treated cells (**p < 0.01; *p < 0.05). This pattern is representative of the patterns seen in two separate sets of experiments with NHEK from different donors.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
Incubation of NHEK with rabbit IgG to BP180 NC16A (R594) results in increased IL-6 and IL-8 protein and mRNA levels. Cultured NHEK were stimulated for 3 h, 6 h, and 9 h, respectively, with 4 mg IgG per ml from rabbit R594 (immunized against human BP180 NC16A; black columns), with preimmune rabbit IgG (white columns), and with medium alone (striped columns). Culture supernatant was assayed for IL-8 (a) and IL-6 (b) reactivity in quadruplicate by ELISA. Bars indicate mean ± SD (pg per ml). IL-6 mRNA (b) was detected by reverse transcription–PCR and IL-8 mRNA (a) by semiquantitative reverse transcription–PCR (Taq-Man procedure). The number of mRNA molecules (IL-6) and Ct values (IL-8) per 106 β-actin mRNA molecules are shown as means of triplicate determinations and SD. A difference in Ct of 3.5 cycles corresponds to a 10-fold difference in mRNA concentration. Asterisks indicate statistical significance between R594 IgG-treated and preimmune IgG-treated cells (***p < 0.001; **p < 0.01; *p < 0.05).
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
Immunoadsorption with recombinant fragments of NC16A reduces the secretion of IL-8 from NHEK after stimulation with BP IgG. NHEK were stimulated for 12 h with 4 mg IgG per ml from three BP patients (bright columns) after immunoadsorption with recombinant GST, a cocktail containing recombinant forms of NC16A1, NC16A1–3, and NC16A2–5 (cocktail) or NC16A fragments NC16A1, NC16A2, NC16A2.5, respectively. Negative controls included 4 mg IgG per ml from three healthy volunteers (normal IgG, dark columns) that was immunoadsorbed against equimolar amounts of recombinant GST. Culture supernatants were assayed in quadruplicate by ELISA. Bars indicate mean ± SD (pg per ml). Asterisks indicate statistical significance of samples preadsorbed with GST or various recombinant NC16A fragments (NC16A1, NC1A2, NC16A2.5) compared with BP IgG preadsorbed with the cocktail of NC16A fusion proteins (*p < 0.001).
Journal of Investigative Dermatology  , DOI: ( /j x)
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