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FcγRIIA and FcγRIIIB Are Required for Autoantibody-Induced Tissue Damage in Experimental Human Models of Bullous Pemphigoid

      Abbreviations

      BP
      bullous pemphigoid
      IC
      immune complex
      ROS
      reactive oxygen species
      TO THE EDITOR
      Bullous pemphigoid (BP) is an autoimmune bullous disease of the elderly, characterized by subepidermal blister formation. Patients with BP have circulating and tissue-bound antibodies directed against antigens of the dermal–epidermal junction, including BP230 (BPAG1) and BP180 (BPAG2) (
      • Kasperkiewicz M.
      • Zillikens D.
      The pathophysiology of bullous pemphigoid.
      ). Binding of autoantibodies initiates an inflammatory cascade resulting in dermal–epidermal separation and blister. An essential role of neutrophils in the pathogenesis of BP has been demonstrated in mouse studies in vivo (
      • Liu Z.
      • Giudice G.J.
      • Zhou X.
      • et al.
      A major role for neutrophils in experimental bullous pemphigoid.
      ). In BP patients, eosinophils and, to a lesser extent, neutrophils are found in lesional skin (
      • Borrego L.
      • Maynard B.
      • Peterson E.A.
      • et al.
      Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins.
      ). There is increasing evidence that eosinophils may contribute to the pathogenesis of the human disease (
      • Zone J.J.
      • Taylor T.
      • Hull C.
      • et al.
      IgE basement membrane zone antibodies induce eosinophil infiltration and histological blisters in engrafted human skin on SCID mice.
      ), whereas neutrophils have been reported to be important for dermal–epidermal separation in an ex vivo model using BP patient's serum and cryosections of human skin (
      • Gammon W.R.
      • Merritt C.C.
      • Lewis D.M.
      • et al.
      An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement.
      ;
      • Sitaru C.
      • Kromminga A.
      • Hashimoto T.
      • et al.
      Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin.
      ,
      • Sitaru C.
      • Schmidt E.
      • Petermann S.
      • et al.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ).
      Neutrophil activation in BP is supposed to be a consequence of Fcγ receptor (FcγR) binding to immune complexes (ICs) (
      • Sitaru C.
      • Kromminga A.
      • Hashimoto T.
      • et al.
      Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin.
      ,
      • Sitaru C.
      • Schmidt E.
      • Petermann S.
      • et al.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ;
      • Zhao M.
      • Trimbeger M.E.
      • Li N.
      • et al.
      Role of FcRs in animal model of autoimmune bullous pemphigoid.
      ). Using neonatal mice deficient in different genes for FcγRs,
      • Zhao M.
      • Trimbeger M.E.
      • Li N.
      • et al.
      Role of FcRs in animal model of autoimmune bullous pemphigoid.
      ) showed that FcγRIII is required for neutrophil activation and subsequent disease development in a passive transfer mouse model of BP. However, the relevance of these findings and their impact on the disease in humans has not been evaluated. In addition, mice and man differ significantly in their FcγRs with regard to cell-specific expression and cellular functions. Quiescent human neutrophils express FcγRIIA and FcγRIIIB (
      • Li M.
      • Wirthmueller U.
      • Ravetch J.V.
      Reconstitution of human Fc gamma RIII cell type specificity in transgenic mice.
      ;
      • Veri M.C.
      • Gorlatov S.
      • Li H.
      • et al.
      Monoclonal antibodies capable of discriminating the human inhibitory Fcgamma-receptor IIB (CD32B) from the activating Fcgamma-receptor IIA (CD32A): biochemical, biological and functional characterization.
      ), whereas quiescent murine neutrophils express FcγRIII and FcγRIV and the inhibitory IgG receptor FcγRIIB (
      • Nimmerjahn F.
      • Ravetch J.V.
      Fcgamma receptors as regulators of immune responses.
      ). FcγRIII represents the murine orthologue of human FcγRIIA, a corresponding counterpart of the human FcγRIIIB is absent in mice (Supplementary Table S1 online). Murine FcγRIV, however, is represented in humans by its ortholoque FcγRIIIA. Therefore, concerning the role of FcγR, a direct transfer of findings derived from mouse models of BP to the human disease is not possible. In this study, we aimed at defining the role of human FcγRs in the activation of neutrophils in BP.
      In a first set of experiments, we attempted to determine the contribution of individual FcγR to the pathogenesis of BP using the ex vivo cryosection model of the disease (
      • Sitaru C.
      • Schmidt E.
      • Petermann S.
      • et al.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ). The role of individual FcγRs was analyzed by preincubation of leukocytes with blocking antibodies directed against FcγRI, FcγRII, or FcγRIII, respectively, or an appropriate isotype control. Cells were subjected to skin cryosections treated with patient sera, and dermal–epidermal separation was determined by light microscopy. Although a strong separation was seen in sections where leukocytes received no or isotype control antibodies (78 and 60% separation of the entire length of the dermal–epidermal junction, respectively), no splits were detected in sections incubated with normal human serum (Figure 1). Treatment of leukocytes with antibodies directed against FcγRII or FcγRIII resulted in a dramatic decrease in dermal–epidermal separation (86 and 88% reduction as compared with the positive control, respectively). However, no such inhibition was observed when leukocytes received antibodies to FcγRI (Figure 1). These results indicate that both FcγRII and FcγRIII, but not FcγRI, mediate autoantibody-induced dermal–epidermal separation. Although the results derived from the ex vivo model of BP clearly show that FcγRII and FcγRIII are involved in tissue damage, the individual cellular functions contributing to activation of the different receptors remained unclear. To address this question, we established an in vitro assay allowing to analyze neutrophil functions, including exocytosis and production of reactive oxygen species (ROS), which have been shown to be essential for blistering in autoimmune bullous diseases (
      • Chiriac M.T.
      • Roesler J.
      • Sindrilaru A.
      • et al.
      NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.
      ). To mimic tissue-bound ICs, plastic surfaces were coated with recombinant murine type XVII collagen (mCOL17) and subsequently incubated with rabbit anti-mCOL17 IgG to form immobilized ICs (Supplementary Materials and Methods online). Freshly isolated neutrophils were exposed to these IC and neutrophil exocytosis, production of ROS as well as neutrophil morphology were monitored (Supplementary Materials and Methods online). Exposure of neutrophils to immobilized IC resulted in an immediate production of ROS, neutrophil exocytosis, and neutrophil spreading, whereas untreated control cells or cells, which had received mCOL17 in the presence of normal rabbit IgG, remained quiescent (Supplementary Figure S1 online).
      Figure thumbnail gr1
      Figure 1The 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 C.
      • Schmidt E.
      • Petermann S.
      • et al.
      Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin.
      ). 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).
      Next, we analyzed the individual contribution of different FcγRs to IC-mediated neutrophil activation. Flow cytometric analysis revealed that neutrophils expressed high levels of FcγRIII and moderate levels of FcγRII on their surface, whereas FcγRI could not be detected under our experimental conditions (Figure 2a), confirming that human neutrophils express the low-affinity IgG receptors FcγRIIA and FcγRIIIB. Stimulation with IC did not induce the expression of FcγRI or modulate FcγRIIIB, whereas a slight reduction of FcγRIIA was observed. Furthermore, ROS production induced by immobilized IC was completely inhibited in the presence of either anti-FcγRII or anti-FcγRIII antibodies, whereas preincubation of cells with anti-FcγRI blocking antibody or isotype control IgG did not affect the formation of ROS (Figure 2b). In addition, blocking antibodies directed against FcγRII and FcγRIII also significantly reduced neutrophil exocytosis in response to IC by 95 and 73%, respectively, as compared with the control cells (Figure 2c). A combination of both antibodies did not further increase this effect (data not shown). Irrelevant control IgG or anti-FcγRI antibody did not affect the exocytosis response. Finally, we determined the effect of FcγR blocking antibodies on neutrophil morphology. Although IC-activated neutrophils, in the absence of antibodies or in the presence of either anti-FcγRI-antibodies or an isotype control, spread on the surface, preincubation of cells with antibodies directed against FcγRII or FcγRIII drastically inhibited neutrophil spreading. Interestingly, eosinophils express FcγRIIA on their surface and produce ROS in response to IC (Supplementary Figure S2 online). Although this may indicate that eosinophils can principally contribute to the disease, because of their limited numbers (1–3% of total cells) in our neutrophil preparations, their role in our study can be neglected.
      Figure thumbnail gr2
      Figure 2Effect 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 B.
      • Brandt E.
      • Bulfone-Paus S.
      • et al.
      Platelet factor 4 (PF-4)-induced neutrophil adhesion is controlled by src-kinases, whereas PF-4-mediated exocytosis requires the additional activation of p38 MAP kinase and phosphatidylinositol 3-kinase.
      ). 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.
      In summary, our results show that FcγRIIA and FcγRIIIB are required for neutrophil-mediated dermal–epidermal separation in the ex vivo model of human BP. Our findings extend previous studies in murine models of the disease, where FcγRIII has been identified as key regulator of tissue damage. The observed differences between human and murine cells may be highly relevant for the development of future therapeutic strategies to BP and other antibody-mediated autoimmune diseases.

      ACKNOWLEDGMENTS

      We thank Dr Norito Ishii and Martin Hänsel for their help with the cyrosection assays. This work was supported by Deutsche Forschungsgemeinschaft, Cluster of Excellence “Inflammation at Interfaces” (EXC 306/1).

      SUPPLEMENTARY MATERIAL

      Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

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