Figure 6

Effect of PF-IgGs on binding of Dsg1-coated beads to HaCaT cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of EGTA (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion (n = 6 for each condition).