Characterization of NF-κB Reporter U937 Cells and Their Application for the Detection of Inflammatory Immune-Complexes

• Published in: PloS one 2016-05, Vol.11 (5), p.e0156328-e0156328
• Main Authors: Kecse-Nagy, Csilla, Szittner, Zoltán, Papp, Krisztián, Hegyi, Zoltán, Rovero, Paolo, Migliorini, Paola, Lóránd, Veronika, Homolya, László, Prechl, József
• Format: Article
• Language: English
• Subjects: Antigen-Antibody Complex - immunology; Antigen-Antibody Complex - metabolism; Antigen-antibody complexes; Antigens; Arthritis; Biological effects; Biology and Life Sciences; Biosensors; Cell activation; Citrulline; Cloning; Cytokines; Fc receptors; Female; Genetic modification; Green Fluorescent Proteins - genetics; Humans; Immunoassays; Immunoglobulin A; Immunoglobulin A - immunology; Immunoglobulin G; Immunoglobulin G - immunology; Immunoglobulins; Immunology; Inflammation; Inflammation - immunology; Isotypes; Laboratories; Lipopolysaccharides - pharmacology; Male; Medicine and Health Sciences; Monoclonal antibodies; Monocytes; NF-kappa B - genetics; NF-kappa B - metabolism; NF-κB protein; Pathogenesis; Peptides; Protein Transport - drug effects; R&D; Receptors; Receptors, IgG - metabolism; Research & development; Research and Analysis Methods; Response Elements - genetics; Rheumatoid arthritis; Rheumatology; Synergism; Synergistic effect; Tumor necrosis factor-TNF; U937 Cells
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0156328

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.