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.
Highlights
Understanding immunoglobulin-mediated activation of myeloid cells is of great interest in the study of autoimmune conditions
In our work we focused on the nuclear factor-κB (NF-κB) transcription factor [3] mediated activation in monocytic cell line U937 [4] in response to immunoglobulin isotypes
The Fc receptor expression patterns remained unchanged in U937 cells after transfection (Fig 1)
Summary
Understanding immunoglobulin-mediated activation of myeloid cells is of great interest in the study of autoimmune conditions. Different cell types in this group may mediate various effector functions through their Fc receptors (FcRs) after engagement with multimeric immunoglobulins (Ig). These include phagocytosis, expression of cytokines [1] and chemokines, as well as release of inflammatory mediators and reactive oxygen species [2]. The system we applied here is based on the work of the Rosales group They were the first to show, that among many other ligands, multimeric IgG (or mimicking it through cross-linking Fc receptors) initiates the signaling of the NF-κB pathway through FcRs in monocytes [5], and identified the steps leading to activation of genes under the control of NF-κB responsive elements [6]
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