Characterization of NF-κB reporter U937 cells and their application for the detection of inflammatory immune-complexes

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

Expression of Ig receptors on the U937-NF-κB reporter cell line.

<p>U937 cells express FcγRI, FcγRII, FcαRI of Fc receptors (A). Transfection with NF-κB-GFP did not alter the Fc receptor expression patterns in the U937 cell line (B). U937 and U937-NF-κB cells were labeled with PE-conjugated anti-FcγRI, FcγRII, FcγRIII and FcαRI, antibodies. The histograms show the unlabeled cells (filled histogram), the corresponding isotype control labeled cells (dashed line) and the specific antibody stained cells (solid line).</p

Activation of U937-NF-κB cells occurs mainly through FcγRI.

<p>The U937-NF-κB cells were activated on anti-FcγR-coated surfaces (A). FcγRI cross-linking induces strong, while FcγRII results in weaker activation. The cell activation was measured on IgG4-coated surfaces (B, C, D) in the presence of soluble IgG1(B), IgG3 (C) and IgG4 (D) in three doses (10, 20, 30 μg/ml). The untreated cells on IgG4 coat served as control (black bars).Results are mean % ± SEM % (n = 9) *p<0.05 versus control (untreated cells).</p

Detection of NF-κB activation by U937-NF-κB cells.

<p>Cells were treated with 10 μg/ml LPS (A), and the activation was detected for 24 h. Cells subjected to the medium only served as a negative control. The activation reached the maximum at 12 h. Cells were also activated on 10 μg/ml human immunoglobulin G (hIgG) coated surface (B). For negative controls, cells subjected to the medium only or to 10 μg/ml soluble human immunoglobulin (shIgG) on uncoated surface were used. The activation was tested on human immunoglobulin coat in the presence of 10 μg/ml goat-anti human immunoglobulin (F(ab’)₂).</p

Time- and dose-dependent activation of U937-NF-κB cells.

<p>The activation of U937-NF-κB cells was screened on plates coated with BSA, human IgG1, IgG2, IgG3, IgG4, IgA, or IgM in two-fold serial dilution from 20 μg/ml to 1.25 μg/ml concentration. Following a one hour adhesion period, the cells were imaged for 12 hours by HCS (A-F). The results were normalized to the fluorescence intensities of cells measured on BSA coat. Soluble form of LPS served as positive control (G).</p