title: Characterization of NF-κB Reporter U937 Cells and Their Application for the Detection of Inflammatory Immune-Complexes.
abstract: Our study tested the hypothesis that immunoglobulins differ in their ability to activate the nuclear factor-κB pathway mediated cellular responses. These responses are modulated by several properties of the immune complex, including the ratio of antibody isotypes binding to antigen. Immunoassays allow the measurement of antigen specific antibodies belonging to distinct immunoglobulin classes and subclasses but not the net biological effect of the combination of these antibodies. We set out to develop a biosensor that is suitable for the detection and characterization of antigen specific serum antibodies. We genetically modified the monocytoid U937 cell line carrying Fc receptors with a plasmid encoding NF-κB promoter-driven GFP. This clone, U937-NF-κB, was characterized with respect to FcR expression and response to solid-phase immunoglobulins. Human IgG3, IgG4 and IgG1 induced GFP production in a time- and dose-dependent manner, in this order of efficacy, while IgG2 triggered no activation at the concentrations tested. IgA elicited no response alone but showed significant synergism with IgG3 and IgG4. We confirmed the importance of activation via FcγRI by direct stimulation with monoclonal antibody and by competition assays. We used citrullinated peptides and serum from rheumatoid arthritis patients to generate immune complexes and to study the activation of U937-NF-κB, observing again a synergistic effect between IgG and IgA. Our results show that immunoglobulins have distinct pro-inflammatory potential, and that U937-NF-κB is suitable for the estimation of biological effects of immune-complexes, offering insight into monocyte activation and pathogenesis of antibody mediated diseases.
authors.affiliation: Department of Immunology, Eötvös Loránd University, H-1117, Pázmány Péter s. 1/C, Budapest, Hungary.
authors.forename: Csilla
authors.initials: C
authors.lastname: Kecse-Nagy
authors.affiliation: MTA-ELTE Immunology Research Group, H-1117, Pázmány Péter s. 1/C, Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Szittner
authors.affiliation: MTA-ELTE Immunology Research Group, H-1117, Pázmány Péter s. 1/C, Budapest, Hungary.
authors.forename: Krisztián
authors.initials: K
authors.lastname: Papp
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Magyar tudósok körútja 2, Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Hegyi
authors.affiliation: Department of NeuroFarBa, Section of Pharmaceutical Sciences and Nutraceutics, Laboratory of Peptide and Protein Chemistry and Biology, University of Florence, Via Ugo Schiff 6, 50019, Sesto Fiorentino, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rovero
authors.affiliation: Clinical Immunology Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Migliorini
authors.affiliation: Department of Rheumatology and Immunology, Clinic Center, PTE, Pécs, Hungary.
authors.forename: Veronika
authors.initials: V
authors.lastname: Lóránd
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Magyar tudósok körútja 2, Budapest, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Homolya
authors.affiliation: MTA-ELTE Immunology Research Group, H-1117, Pázmány Péter s. 1/C, Budapest, Hungary.|Research and Development Laboratory, Diagnosticum Inc., H-1047, Budapest, Attila út 146, Hungary.
authors.forename: József
authors.initials: J
authors.lastname: Prechl
mesh_terms: D000936:Antigen-Antibody Complex / Q000276:immunology / Q000378:metabolism*; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007249:Inflammation / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D017452:Receptors, IgG / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D020298:U937 Cells
chemical_list: D000936:Antigen-Antibody Complex; C489017:FCGR1A protein, human; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D008070:Lipopolysaccharides; D016328:NF-kappa B; D017452:Receptors, IgG; D049452:Green Fluorescent Proteins