Aggregation of the High-Affinity IgE Receptor FcεRI on Human Monocytes and Dendritic Cells Induces NF-κB Activation  Stefan Kraft, Natalija Novak, Thomas Bieber  Journal of Investigative Dermatology  Volume 118, Issue 5, Pages 830-837 (May 2002) DOI: 10.1046/j.1523-1747.2002.01757.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of NF-κB subunits during human DC differentiation. Monocytes, MoDC, and mature DC were generated as described in Materials and Methods. (A) Immuno blotting was performed using antibodies specific for NF-κB subunits. (B) Flow cytometric data were obtained after permeabilization with saponin and double staining with anti-RelB followed by a FITC-labeled secondary antibody and a PE-labeled anti-CD14 or anti-CD1a antibody for monocytes or DC, respectively. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 FcεRI ligation induces NF-κB DNA binding in monocytes and MoDC.(A) Cells were loaded with IgE and stimulated as described in Materials and Methods. TNF-α was used as a positive control. For EMSA assays, cell lysis and incubation with a 32P-labeled oligonucleotide representing a conserved NF-κB binding sequence were performed. Electrophoretic separation of the DNA/protein-complexes was followed by autoradiography. The arrows indicate the retarded complexes representing NF-κB proteins bound to the labeled oligonucleotide. The data shown are representative for six experiments. (B) EMSA assays showing NF-κB induction using MoAb stimulation against FcεRI (10 µg per ml) for 60 min. The data shown are representative for three experiments. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 NF-κB induction in human Langerhans cells is restricted to FcεRIhigh Langerhans cells. FcεRI expression was monitored by flow cytometry as described in Materials and Methods (see histograms on the left side). Purified Langerhans cells were stimulated with IgE/anti-IgE and EMSA assays were performed as described in Figure 2. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The FcεRI-induced NF-κB complex in monocytes and MoDC contains p50 and p65 subunits. Monocytes (A) and MoDC (B) were loaded with IgE, washed, and then stimulated with anti-IgE antibody for 30 min. For supershift analyzes, lysates were incubated with antibodies specific for NF-κB subunits. This was followed by incubation with a 32P-labeled oligonucleotide specific for NF-κB. DNA/protein-complexes were separated by polyacrylamide gel electrophoresis and detected by autoradiography. NF-κB subunits were identified by further retardation of bands (p50) or competition of NF-κB DNA binding (p65, RelB, c-Rel) caused by the addition of specific antibodies. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 FcεRI ligation on monocytes and MoDC induces serine phosphorylation of IκB-α followed by its degradation, but not IκB-β degradation. Cells were loaded with IgE, washed, and then stimulated with anti-IgE antibody for various time periods. After cell lysis, immunoblotting was performed using anti-IκB-α-phosphoserine (A), anti-IκB-α(B), and (shown for monocytes) IκB-β(C) antibodies. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 FcεRI ligation on monocytes and MoDC induces the release of TNF-α and MCP-1, which is sensitive to NAC and TPCK. (A) TNF-α; (B) MCP-1. Experimental conditions are identical to that of Table I. After culture for 6 h and 18 h, the supernatants were analyzed using an ELISA kit for TNF-α and MCP-1, respectively, according to the manu facturer's instructions. Journal of Investigative Dermatology 2002 118, 830-837DOI: (10.1046/j.1523-1747.2002.01757.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions