Aggregation of the High-Affinity IgE Receptor FcεRI on Human Monocytes and Dendritic Cells Induces NF-κB Activation
2002; Elsevier BV; Volume: 118; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.2002.01757.x
ISSN1523-1747
AutoresStefan Kraft, Natalija Novak, Thomas Bieber, Norito Katoh, Rudolf A. Rupec,
Tópico(s)Immune Cell Function and Interaction
ResumoIn contrast to mast cells and basophils, the high-affinity IgE receptor (FcεRI) on monocytes and dendritic cells (DC), including epidermal Langerhans cells, is not constitutively expressed and lacks the β-chain. FcεRI is upregulated on Langerhans cells of atopic individuals, particularly in atopic dermatitis skin. Although FcεRI provides IgE-mediated antigen focusing on monocytes and DC/Langerhans cells, its relevance for cell activation remains elusive, and the transcription factors regulating FcεRI-induced genes are unknown. We show that NF-κB, known to regulate genes essential for inflammatory responses and DC differentiation and function, is activated upon FcεRI ligation in primary human monocytes and DC. In Langerhans cells isolated from epidermis, NF-κB activation is restricted to donors expressing high FcεRI amounts. FcεRI-induced NF-κB complexes in monocytes and DC contain p50 and p65, but no other NF-κB subunits despite increased RelB expression during differentiation. NF-κB activation is preceded by serine phosphorylation and degradation of its inhibitory protein IκB-α without involving other IκB proteins. Finally, we show that FcεRI ligation on monocytes and DC leads to synthesis and release of tumor necrosis factor-α and monocyte chemoattractant protein-1, which is decreased by two mechanistically distinct inhibitors of NF-κB activation. Thus NF-κB activation represents a novel mechanism by which FcεRI on monocytes and DC potentially controls inflammatory reactions. In contrast to mast cells and basophils, the high-affinity IgE receptor (FcεRI) on monocytes and dendritic cells (DC), including epidermal Langerhans cells, is not constitutively expressed and lacks the β-chain. FcεRI is upregulated on Langerhans cells of atopic individuals, particularly in atopic dermatitis skin. Although FcεRI provides IgE-mediated antigen focusing on monocytes and DC/Langerhans cells, its relevance for cell activation remains elusive, and the transcription factors regulating FcεRI-induced genes are unknown. We show that NF-κB, known to regulate genes essential for inflammatory responses and DC differentiation and function, is activated upon FcεRI ligation in primary human monocytes and DC. In Langerhans cells isolated from epidermis, NF-κB activation is restricted to donors expressing high FcεRI amounts. FcεRI-induced NF-κB complexes in monocytes and DC contain p50 and p65, but no other NF-κB subunits despite increased RelB expression during differentiation. NF-κB activation is preceded by serine phosphorylation and degradation of its inhibitory protein IκB-α without involving other IκB proteins. Finally, we show that FcεRI ligation on monocytes and DC leads to synthesis and release of tumor necrosis factor-α and monocyte chemoattractant protein-1, which is decreased by two mechanistically distinct inhibitors of NF-κB activation. Thus NF-κB activation represents a novel mechanism by which FcεRI on monocytes and DC potentially controls inflammatory reactions. atopic dermatitis dendritic cell(s) epidermal cell(s) high-affinity IgE receptor low-affinity IgE receptor IκB kinase monocyte chemoattractant protein-1 monocyte-derived DC n-acetylcystein nuclear factor-κB tumor necrosis factor-α n-tosyl-L-phenylalanin-chloromethyl-ketone Among professional antigen-presenting cells (APC), dendritic cells (DC) show the unique capacity to initiate primary immune responses (Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (11832) Google Scholar). Immature DC form the outposts of the immune system in peripheral tissues and are characterized by their high antigen uptaking capacity. Upon various stimuli, they differentiate into highly stimulatory mature DC. The prototype of immature DC are Langerhans cells, which as epidermal sentinels take part in host defense or in pathologic conditions like allergic contact dermatitis. The study of DC functions has been facilitated since the in vitro generation of DC from peripheral monocytes by culture with GM-CSF and IL-4 has been reported (Sallusto and Lanzavecchia, 1994Sallusto F. Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4396) Google Scholar). Langerhans cells, blood DC, and monocytes variably express a trimeric form of the high-affinity receptor for IgE (FcεRI) containing one α- and two γ-chains, but lack the β-chain characteristic for tetrameric FcεRI on mast cells and basophils (Bieber et al., 1992Bieber T. de la Salle H. Wollenberg A. et al.Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FcεRI).J Exp Med. 1992; 175: 1285-1290Crossref PubMed Scopus (417) Google Scholar;Wang et al., 1992Wang B. Rieger A. Kilgus O. et al.Epidermal Langerhans cells from normal human skin bind monomeric IgE via FcεRI.J Exp Med. 1992; 175: 1353-1365Crossref PubMed Scopus (326) Google Scholar;Maurer et al., 1994Maurer D. Fiebiger E. Reininger B. et al.Expression of functional high affinity immunoglobulin E receptors (FcεRI) on monocytes of atopic individuals.J Exp Med. 1994; 179: 745-750Crossref PubMed Scopus (317) Google Scholar,Maurer et al., 1996Maurer D. Fiebiger E. Ebner C. et al.Peripheral blood dendritic cells express FcεRI as a complex composed of FcεRIα- and FcεRIγ-chains and can use this receptor for IgE-mediated allergen presentation.J Immunol. 1996; 157: 607-616PubMed Google Scholar;Kinet, 1999Kinet J.P. The high-affinity IgE receptor (FcεRI): from physiology to pathology.Annu Rev Immunol. 1999; 17: 931-972Crossref PubMed Scopus (822) Google Scholar). In contrast to the latter, there is ample evidence that FcεRI+ APC are involved in IgE-mediated delayed-type hypersensitivity reactions in atopic diseases: First, FcεRI surface expression on Langerhans cells is enhanced in patients with atopic dermatitis (AD) (Bieber, 1997Bieber T. FcεRI-expressing antigen-presenting cells: new players in the atopic game.Immunol Today. 1997; 18: 311-313Abstract Full Text PDF PubMed Scopus (86) Google Scholar). Second, Langerhans cells/DC and monocytes can take up, process, and present antigens to T cells more efficiently using FcεRI (Mudde et al., 1990Mudde G.C. Van Reijsen F.C. Boland G.J. de Gast G.C. Bruijnzeel P.L. Bruijnzeel-Koomen C.A. Allergen presentation by epidermal Langerhans' cells from patients with atopic dermatitis is mediated by IgE.Immunology. 1990; 69: 335-341PubMed Google Scholar;Maurer et al., 1995Maurer D. Ebner C. Reininger B. Fiebiger E. Kraft D. Kinet J.P. Stingl G. The high affinity IgE receptor (FcεRI) mediates IgE-dependent allergen presentation.J Immunol. 1995; 154: 6285-6290PubMed Google Scholar,Maurer et al., 1996Maurer D. Fiebiger E. Ebner C. et al.Peripheral blood dendritic cells express FcεRI as a complex composed of FcεRIα- and FcεRIγ-chains and can use this receptor for IgE-mediated allergen presentation.J Immunol. 1996; 157: 607-616PubMed Google Scholar). Nuclear factor-κB (NF-κB) family transcription factors are DNA-binding dimers consisting of p50, p52, p65, RelB, and/or c-Rel subunits and play a pivotal role in the regulation of gene expression (Baeuerle and Henkel, 1994Baeuerle P.A. Henkel T. Function and activation of NF-kappa B in the immune system.Annu Rev Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4509) Google Scholar;Ghosh et al., 1998Ghosh S. May M.J. Kopp E.B. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses.Annu Rev Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4456) Google Scholar). A wide range of stimuli lead to NF-κB-mediated activation of target genes involved in acute-phase responses, inflammation, or cell growth and differentiation, e.g., MHC molecules, adhesion molecules, cytokines, and chemokines. Inactive NF-κB is retained by inhibitory IκB molecules in the cytoplasm and, upon stimulation, undergoes serine phosphorylation by the IκB kinase (IKK) complex followed by its proteasomal degradation (Karin and Ben-Neriah, 2000Karin M. Ben-Neriah Y. Phosphorylation meets ubiquitination. the control of NF-κB activity.Annu Rev Immunol. 2000; 18: 621-663Crossref PubMed Scopus (3911) Google Scholar). Mature DC constitutively express high levels of NF-κB subunits with RelB required for differentiation of myeloid DC and possibly relevant for antigen presentation (Burkly et al., 1995Burkly L. Hession C. Ogata L. et al.Expression of relB is required for the development of thymic medulla and dendritic cells.Nature. 1995; 373: 531-536Crossref PubMed Scopus (646) Google Scholar;Pettit et al., 1997Pettit A.R. Quinn C. MacDonald K.P. et al.Nuclear localization of RelB is associated with effective antigen-presenting cell function.J Immunol. 1997; 159: 3681-3691PubMed Google Scholar;Banchereau and Steinman, 1998Banchereau J. Steinman R.M. Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (11832) Google Scholar;Wu et al., 1998Wu L. D'Amico A. Winkel K.D. Suter M. Lo D. Shortman K. RelB is essential for the development of myeloid-related CD8α- dendritic cells but not of lymphoid-related CD8α+ dendritic cells.Immunity. 1998; 9: 839-847Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). FcεRI signal transduction pathways have been explored in mast cells and basophils with a major FcεRI-induced regulator being NF-AT (Hutchinson and McCloskey, 1995Hutchinson L.E. McCloskey M.A. FcεRI-mediated induction of nuclear factor of activated T-cells.J Biol Chem. 1995; 270: 16333-16338Crossref PubMed Scopus (71) Google Scholar;Prieschl et al., 1995Prieschl E.E. Gouilleux-Gruart V. Walker C. Harrer N.E. Baumruker T. A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation.J Immunol. 1995; 154: 6112-6119PubMed Google Scholar;Turner et al., 1995Turner H. Reif K. Rivera J. Cantrell D.A. Regulation of the adapter molecule Grb2 by the FcεRI in the mast cell line RBL2H3.J Biol Chem. 1995; 270: 9500-9506Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), while the induction of a complex binding to NF-κB sites, but containing unknown proteins different from members of the NF-κB family, has been described (Pelletier et al., 1998Pelletier C. Varin-Blank N. Rivera J. et al.FcεRI-mediated induction of TNF-α gene expression in the RBL-2H3 mast cell line: regulation by a novel NF-κB-like nuclear binding complex.J Immunol. 1998; 161: 4768-4776PubMed Google Scholar;Marquardt and Walker, 2000Marquardt D.L. Walker L.L. Dependence of mast cell IgE-mediated cytokine production on nuclear factor-kappaB activity.J Allergy Clin Immunol. 2000; 105: 500-505Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). As a result of FcεRI ligation on these cells, the release of histamine, production of eicosanoids, proinflammatory cytokines and Th2 cytokines are induced (Gordon et al., 1990Gordon J.R. Burd P.R. Galli S.J. Mast cells as a source of multifunctional cytokines.Immunol Today. 1990; 11: 458-464Abstract Full Text PDF PubMed Scopus (162) Google Scholar;Razin et al., 1983Razin E. Mencia-Huerta J.M. Stevens R.L. Lewis R.A. Liu F.T. Corey E. Austen K.F. IgE-mediated release of leukotriene C4, chondroitin sulfate E proteoglycan, beta-hexosaminidase, and histamine from cultured bone marrow-derived mouse mast cells.J Exp Med. 1983; 157: 189-201Crossref PubMed Scopus (165) Google Scholar;Lorentz et al., 2000Lorentz A. Schwengberg S. Sellge G. Manns M.P. Bischoff S.C. Human intestinal mast cells are capable of producing different cytokine profiles. role of IgE receptor cross-linking and IL-4.J Immunol. 2000; 164: 43-48Crossref PubMed Scopus (148) Google Scholar). FcεRI on APC, however, lacks the β-subunit, which plays a crucial role in signal transduction in mast cells and basophils. In addition, FcεRI-bearing APC are proposed to exert a different functional role, i.e., in antigen focusing, and may activate different genes upon FcεRI ligation. Thus, it is not clear whether FcεRI in these cells involves similar pathways as in effector cells of anaphylaxis. We have previously reported that cross-linking of FcεRI on Langerhans cells leads to rapid tyrosine phosphorylation of several proteins and only Langerhans cells from patients with AD show calcium mobilization (Jürgens et al., 1995Jürgens M. Wollenberg A. Hanau D. de la Salle H. Bieber T. Activation of human epidermal Langerhans cells by engagement of the high affinity receptor for IgE, FcεRI.J Immunol. 1995; 155: 5184-5189PubMed Google Scholar). Later events in FcεRI signaling, like transcription factor activation, are unknown in APC. In this study we asked whether FcεRI ligation on primary human monocytes, monocyte-derived DC (MoDC), and epidermal Langerhans cells leads to the activation of NF-κB and how NF-κB complexes are composed. In addition, we investigated the pathways leading to NF-κB activation and analyzed the expression of genes mediated by FcεRI and NF-κB. PE-labeled T6/RD1 (Coultertronics, Krefeld, Germany), BL6 (Immunotech, Marseille, France) mouse monoclonal antibody (MoAb) recognizes CD1a. Rabbit anti-human IgE was from DAKO (Glostrup, Denmark). Human myeloma IgE was obtained from Calbiochem (San Diego, CA). The MoAb 22E7 (a kind gift from Dr. R. Chizzonite, Roche, Nutley, NJ) and CRA1 (kindly provided by Dr. C. Ra, Juntendo University School of Medicine, Tokyo, Japan) recognize the FcεRI α-subunit. MoAb specific for FcεRII/CD23 were clones MHM6 (DAKO) and 9P.25 (Jackson, West Grove, PA). FITC-labeled F(ab′)2 fragments of goat anti-mouse were also purchased from Jackson. FITC- or PE-labeled anti-CD14, PE-labeled anti-CD-117, and mouse IgG1 were from BD (Mountain View, CA) and PE-labeled anti-203c was from Immunotech. Sheep anti-mouse coated magnetic beads (M-280) were from Dynal (Oslo, Norway). VLE RPMI 1640 medium was from Biochrom (Berlin, FRG). Antibodies against IκB and NF-κB subunits were from Santa Cruz (Santa Cruz, CA). Phospho-IκB-α antibody was purchased from New England Biolabs (Beverly, MA). PE-labeled MoAb against tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), isotype controls, and GolgiPlug (Brefeldin A) were from Pharmingen (San Diego, CA). All other chemicals were from Sigma unless otherwise indicated. Skin specimens were obtained from our surgery department. Written informed consent was obtained from all donors. Epidermal cell (EC) suspensions were prepared as described inBieber et al., 1992Bieber T. de la Salle H. Wollenberg A. et al.Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FcεRI).J Exp Med. 1992; 175: 1285-1290Crossref PubMed Scopus (417) Google Scholar. Then EC were purified by positive selection with anti-CD1a bound magnetic beads according to the manufacturer's protocol. The purity of the Langerhans cell preparations was controlled by microscopy, and the procedure was stopped when unbound cells were completely removed (> 98% Langerhans cells). Monocytes were isolated from peripheral blood with a modified density gradient protocol using Nycoprep (Nycomed, Oslo, Norway) according to the manufacturer's instructions. Briefly, erythrocytes were sedimented from whole EDTA blood with 1/10 (wt/vol) 6% Dextran 500 in 0.9% NaCl. Plasma was gently layered over Nycoprep and centrifuged for 20 min at 600 × g. After separation, the interphase and Nycoprep were collected and washed four times with 0.9% NaCl + 0.13% EDTA + 1% BSA. The purity of the isolated monocytes in a typical experiment was 94% CD14+ cells, 2.4% CD56+ cells, 0.25% CD3+ CD4+ cells, 0.27% CD3+ CD8+ cells, 1.72% CD83+ cells, 0.03% CD19+ cells. Then, monocytes were used for experiments or cultured for 4–8 d in RPMI 1640 + 10% fetal calf serum + 1% L-glutamine + 1% antibiotics/antimycotics + 500 U GM-CSF per ml (Genzyme, Cambridge, MA) + 500 U IL-4 per ml (Life Technologies) to yield immature MoDC. Final maturation was achieved by incubation with TNF-α (100 IU per ml) for 2 d. Labeling for FcεRI was performed as previously reported inBieber et al., 1992Bieber T. de la Salle H. Wollenberg A. et al.Human epidermal Langerhans cells express the high affinity receptor for immunoglobulin E (FcεRI).J Exp Med. 1992; 175: 1285-1290Crossref PubMed Scopus (417) Google Scholar. Briefly, EC were washed with PBS + 0.5% fetal calf serum + 0.1% NaN3 followed by incubation with anti-FcεRIα MoAb 22E7, anti-FcεRII/CD23 MoAb 9P.25, or isotype control. After washing, EC were stained with FITC-conjugated secondary antibody, washed again, and blocked with 5% mouse serum. Finally, staining against CD1a (Langerhans cells or MoDC) or CD14 (monocytes) was performed using PE-labeled antibodies. Acquisition and analyzes were performed on a FACScalibur flow cytometer (BD). CD1a-negative and dead cells (determined by 7-amino-actinomycin D uptake) were gated out manually. Flow cytometric staining of intracellular RelB in saponin-permeabilized cells using a rabbit polyclonal antibody followed by staining with a FITC-conjugated donkey anti-rabbit (Jackson) and PE-conjugated anti-CD14 or anti-CD1a antibodies was performed as described inWollenberg et al., 1996Wollenberg A. Kraft S. Hanau D. Bieber T. Immunomorphological and ultrastructural characterization of Langerhans cells and a novel, inflammatory dendritic epidermal cell (IDEC) population in lesional skin of atopic eczema.J Invest Dermatol. 1996; 106: 446-453Crossref PubMed Scopus (328) Google Scholar. Cross-linking of FcεRI was done with IgE/anti-IgE as previously described (Jürgens et al., 1995Jürgens M. Wollenberg A. Hanau D. de la Salle H. Bieber T. Activation of human epidermal Langerhans cells by engagement of the high affinity receptor for IgE, FcεRI.J Immunol. 1995; 155: 5184-5189PubMed Google Scholar). Monocytes, MoDC, and Langerhans cells were tested for their FcεRI, FcεRII/CD23, and CD14/CD1a expression. Then, cells were incubated for 60 min with 5 µg monomeric IgE per ml at 37°C (monocytes were allowed to adhere for 3 h before stimulation). After washing with culture medium, 20 µg rabbit anti-human IgE per ml was added and cells were incubated for an adequate period of time for subsequent ELISA, flow cytometric staining, or lysis. For flow cytometric determination of cytokine synthesis, GolgiPlug (brefeldin A) was added to the culture. When indicated, NAC (n-acetylcystein) (30 mM) or TPCK (n-tosyl-L-phenylalanin-chloro methyl-ketone) (5 µM) were added to inhibit NF-κB activation. For EMSA and IκB degradation experiments, cells were incubated with 0.5 U neuraminidase per ml followed by 0.02 M lactose to enhance IgE binding to FcεRI before stimulation (Wollenberg et al., 1993Wollenberg A. de la Salle H. Hanau D. Liu F.T. Bieber T. Human keratinocytes release the endogenous β-galactoside-binding soluble lectin immunoglobulin E (IgE-binding protein) which binds to Langerhans cells where it modulates their binding capacity for IgE glycoforms.J Exp Med. 1993; 178: 777-785Crossref PubMed Scopus (73) Google Scholar;Reischl et al., 1996Reischl I.G. Corvaia N. Effenberger F. WolffWiniski B. Kromer E. Mudde G.C. Function and regulation of FcεRI expression on monocytes from non-atopic donors.Clin Experiment Allergy. 1996; 26: 630-641Crossref PubMed Scopus (40) Google Scholar). Alternatively, specific stimulation of IgE receptors was achieved with the anti-FcεRI MoAb 22E7 or CRA1, anti-FcεRII/CD23 MoAb MHM6 or isotype control (10 µg per ml) or TNF-α as positive control (100 U per ml). Production of TNF-α or MCP-1 by monocytes was assessed by intracellular staining and ELISA. Intracellular staining was performed with the Cytofix/Cytoperm kit (Pharmingen) according to the manufacturer's instructions. Then, the cells were analyzed on a FACScalibur flow cytometer (BD). RFI (relative fluorescence index) values were calculated using MFI (mean fluorescence index) values as follows:RFI=MFI(specific)−MFI(isotype control)MFI(isotype control). For determination of released cytokines, ELISA from Bender Medical Systems (Vienna, Austria) for TNF-α and R&D Systems (Minneapolis, MN) for MCP-1 were used according to the manufacturer's instructions. After lysis with 10 mM Tris + 10 mM MgCl2 + 1% Triton X-100 in the presence of protease inhibitors, the samples were centrifuged at 12 000 × g. Supernatants were collected and boiled after adding 25% 5 × Laemmli buffer. For determination of IκB-α phosphorylation, cells were lysed directly in Laemmli buffer. Samples were fractionated by SDS-PAGE and electrotransferred to nitrocellulose membranes. After blocking, proteins were identified using NF-κB and IκB antibodies. The bands were visualized with peroxidase-conjugated secondary antibodies followed by an enhanced chemiluminescence detection protocol (Amersham, IL). Cells were lysed with 10 mM Tris/HCl pH 7.5, 50 mM NaCl, 50 mM NaF, 5 µM ZnCl2, 1% Triton X-100, and 0.5 mM phenymethylsulfonyl fluoride. After centrifugation at 14 000 × g, the supernatants were subjected to binding reactions (total volume 20 µl) in 10 mM Hepes pH 7.2, 40 mM KCl, 0.05 mM EDTA, 0.025% NP-40, 4% Ficoll 400, 2% glycerol, 1 µg BSA per µl, 25 ng poly(dL-dC) per µl, 1 mM dithiothreitol, 0.05 mM phenymethylsulfonyl fluoride, and 20 000 cpm of a 32P-labeled double-stranded NF-κB consensus oligonucleotide (Promega, Madison, WI) for 25 min at room temperature. Labeling of the oligonucleotide was performed with a T4 polynucleotide kinase kit (New England Biolabs). For supershifts, lysates were incubated with 200 µg per ml of NF-κB-specific antibodies (Santa Cruz) for 1 h on ice, followed by 25 min of incubation with the oligonucleotide. DNA-protein complexes were separated from unbound DNA probe on 4.5% polyacrylamide gels. NF-κB oligonucleotide sequence: 5′-AGTTGAGGGGACTTTCCCAGGC-3′ 3′-TCAACTCCCCTGAAAGGGTCCG-5′ Statistical analysis using Wilcoxons' test was performed with SPSS 9.0 for Windows (SPSS, Chicago, IL). Calculated values shown were mean ± standard error of mean (SEM). NF-κB transcription factors are essential for DC differentiation and APC functions (Burkly et al., 1995Burkly L. Hession C. Ogata L. et al.Expression of relB is required for the development of thymic medulla and dendritic cells.Nature. 1995; 373: 531-536Crossref PubMed Scopus (646) Google Scholar;Pettit et al., 1997Pettit A.R. Quinn C. MacDonald K.P. et al.Nuclear localization of RelB is associated with effective antigen-presenting cell function.J Immunol. 1997; 159: 3681-3691PubMed Google Scholar;Rescigno et al., 1998Rescigno M. Martino M. Sutherland C.L. Gold M.R. Ricciardi-Castagnoli P. Dendritic cell survival and maturation are regulated by different signaling pathways.J Exp Med. 1998; 188: 2175-2180Crossref PubMed Scopus (587) Google Scholar;Wu et al., 1998Wu L. D'Amico A. Winkel K.D. Suter M. Lo D. Shortman K. RelB is essential for the development of myeloid-related CD8α- dendritic cells but not of lymphoid-related CD8α+ dendritic cells.Immunity. 1998; 9: 839-847Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). To determine if NF-κB is expressed in our system, we analyzed p50, p52, p65, RelB, and c-Rel expression using monocytes, immature, and mature MoDC. Peripheral monocytes were isolated at high purity (>85%) using a modified density gradient protocol avoiding adhesion of cells prior to isolation. Immature MoDC were obtained after culture with GM-CSF and IL-4. In addition, we used TNF-α to induce further maturation of DC. Contamination of the monocyte and DC preparations with effector cells of anaphylaxis such as mast cells and basophils was excluded by staining with anti-CD117 (c-kit) and anti-CD203c (ectonucleotide pyrophosphatase/phosphodiesterase-3) antibodies. We found all five subunits to be expressed during the differentiation stages observed (Figure 1a). In addition, we detected higher c-Rel levels at both MoDC stages compared with monocytes and a continuous upregulation of RelB during differentiation, which was confirmed by flow cytometric analysis of permeabilized cells (Figure 1b). This is in accordance with its formerly described essential role in DC development and antigen presentation and recently published data using DC generated from adherent monocytes (Neumann et al., 2000Neumann M. Fries H. Scheicher C. et al.Differential expression of Rel/NF-κB and octamer factors is a hallmark of the generation and maturation of dendritic cells.Blood. 2000; 95: 277-285Crossref PubMed Google Scholar). Because of the prominent expression of NF-κB subunits in primary human APC and NF-κB's role in the regulation of genes relevant for inflammatory processes (Baeuerle and Henkel, 1994Baeuerle P.A. Henkel T. Function and activation of NF-kappa B in the immune system.Annu Rev Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4509) Google Scholar;Ghosh et al., 1998Ghosh S. May M.J. Kopp E.B. NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses.Annu Rev Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4456) Google Scholar), we looked for its activation following cross-linking of FcεRI-bound IgE by anti-IgE anti bodies. Monocytes from our donor group (n = 5) expressed significant amounts of FcεRI (24.90% ± 2.99%; mean ± SEM) and no or very low low-affinity IgE receptor FcεRII/CD23 (4.20% ± 3.69%), whereas MoDC were consistently positive for both FcεRI (21.05% ± 10.34%) and FcεRII/CD23 (84.54% ± 4.19%). Cross-linking of FcεRI via IgE/anti-IgE induced a strong activation of NF-κB DNA-binding activity (Figure 2a). Whereas in monocytes the activation peaked after 60 min, the maximum was reached after 10 min in MoDC. TNF-α, a known inducer of NF-κB used as a positive control, reached approximately the same level of activation after 30 min of stimulation. To compare our results with effector cells of anaphylaxis bearing tetrameric FcεRI, we performed control experiments with RBL-48 cells transfected with human FcεRIα (Gilfillan et al., 1992Gilfillan A.M. Kado-Fong H. Wiggan G.A. Hakimi J. Kent U. Kochan J.P. Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.J Immunol. 1992; 149: 2445-2451PubMed Google Scholar). Stimulation with IgE/anti-IgE similarly resulted in a DNA-binding NF-κB complex (data not shown). Because the low-affinity IgG receptor CD32 is present both on monocytes and on MoDC (Pickl et al., 1996Pickl W.F. Majdic O. Kohl P. et al.Molecular and functional characteristics of dendritic cells generated from highly purified CD14+ peripheral blood monocytes.J Immunol. 1996; 157: 3850-3859PubMed Google Scholar), stimulation via IgE/anti-IgE might theoretically also activate pathways mediated by this receptor. To confirm that the NF-κB induction observed is indeed mediated by FcεRI, we used two different MoAb specific for FcεRI for stimulation, compared with an irrelevant IgG1 isotype control. Figure 2(b) shows that FcεRI is able to induce NF-κB activation in the cell types investigated. From these experiments we conclude that FcεRI cross-linking activates NF-κB in APC. Having determined that NF-κB is a mediator of FcεRI-derived signals in in vitro generated immature DC, we then analyzed epidermal FcεRI+ Langerhans cells, which are assumed to play a key role in AD. Therefore, Langerhans cells were freshly isolated from normal looking human skin, purified, and stimulated with IgE/anti-IgE followed by determination of NF-κB DNA binding. They did not express the low-affinity receptor FcεRII/CD23 but displayed various levels of FcεRI, as determined by flow cytometry (n = 6). Langerhans cells expressing low but significant amounts of FcεRI (18.5% ± 2.8%; n = 3) did not show any activation of NF-κB in EMSA after activation with anti-IgE-antibody (Figure 3, lower panel). In contrast, in Langerhans cells with high amounts of FcεRI (56.7% ± 16.0%; n = 3), a strong induction of NF-κB DNA binding could be observed after activation under the same conditions (Figure 3, upper panel). From these experiments we conclude that FcεRI induces NF-κB DNA binding in freshly isolated human epidermal Langerhans cells, i.e., immature DC isolated ex vivo, provided that they substantially express the high-affinity IgE receptor. Because NF-κB complexes can be composed of various homo- and heterodimers potentially targeting different genes, we analyzed the NF-κB subunits induced by IgE/anti-IgE stimulation in monocytes and DC by supershift assays using antibodies specific for NF-κB subunits. In monocytes and MoDC p50-specific antibodies produced a retarded band and p65-specific inhibitory antibodies directed against the DNA-binding domain abrogated the NF-κB complex (Figures 4a, b); however, despite significant expression of c-rel and even upregulated levels of RelB (Figure 1) in MoDC, none of these factors could be detected in the FcεRI-induced complexes in MoDC. p52 was also not detected in any NF-κB complex activated by FcεRI (data not shown). Taken together, these data imply that FcεRI on APC activates complexes containing the most abundant p50 and p65 subunits. Two different activation pathways for NF-κB have been described so far. In the best-characterized pathway IκB-α is phosphorylated at Ser32/36 and becomes subsequently degraded via the ubiquitin-proteasome pathway (Karin and Ben-Neriah, 2000Karin M. Ben-Neriah Y. Phosphorylation meets ubiquitination. the control of NF-κB activity.Annu Rev Immunol. 2000; 18: 621-663Crossref PubMed Scopus (3911) Google Scholar). Degradation of other IκB family members like IκB-β has also been shown to lead to NF-κB activation (Ghosh et al., 1998Ghosh S. May M.J. Kopp E.B. NF-kappa B and Rel proteins: evolutionarily conserved mediators of i
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