FcγRIIA and FcγRIIIB Are Required for Autoantibody-Induced Tissue Damage in Experimental Human Models of Bullous Pemphigoid  Xinhua Yu, Karoline Holdorf, Brigitte Kasper, Detlef Zillikens, Ralf J. Ludwig, Frank Petersen  Journal of Investigative Dermatology  Volume 130, Issue 12, Pages 2841-2844 (December 2010) DOI: 10.1038/jid.2010.230 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The role of Fcγ receptors (FcγRs) in autoantibody-mediated dermal separation ex vivo. Human skin cryosections were incubated with sera derived from bullous pemphigoid patients for 1hour, as previously described (Sitaru et al., 2002b). Subsequently, freshly isolated human peripheral leukocytes from healthy donors, preincubated with the anti-FcγR antibodies indicated or a corresponding isotype control, were added. Sections of a representative experiment are shown (a). Arrows indicate the dermis–epidermis separations. Bar=200μm. Furthermore, skin separation was quantified as percentage of epidermis detached from dermis by an observer blinded to the applied sera/antibodies (b). Data are presented as mean±SEM of three independent experiments. *Indicates statistically significant differences (P<0.01). Journal of Investigative Dermatology 2010 130, 2841-2844DOI: (10.1038/jid.2010.230) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Effect of blocking antibodies to Fcγ receptors (FcγRs) on neutrophil activation by immobilized immune complexes (ICs) in vitro. (a) Expression of FcγRs on human neutrophils. Neutrophils were stained against FcγRI, FcγRII, FcγRIII, or a corresponding isotype control, respectively, before (upper panel) or after (lower panel) exposure to IC. (b) Reactive oxygen species (ROS) production. Neutrophils were pretreated for 30minutes with blocking antibodies against FcγRs (10μgml−1) or a corresponding isotype control and exposed to IC. Generation of ROS was determined by measurement of chemiluminescence. One representative result out of three is given. (c) Neutrophil exocytosis. Degranulation was determined by the amount of lactoferrin released (Kasper et al., 2004). Release rates of IC-treated cells (8.1–18.5% of total lactoferrin) were set as 100%, and release rates of antibody-treated cells as well as unstimulated samples (negative control) are given as percentage of these control cells. Data are presented as mean±SEM, n=3, *P<0.05 and **P<0.001. (d) Morphology of neutrophils exposed to uncoated (negative control) or IC-coated surfaces in the presence of different antibodies. Photographs of one representative experiment out of three are given. Bar=25μm; RLU, relative light unit. Journal of Investigative Dermatology 2010 130, 2841-2844DOI: (10.1038/jid.2010.230) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions