Optimization of IgG1 immune complexes to stimulate cytokine production in innate cells

Abstract

Fcγ receptors are key immunoreceptors, that when bound by IgG immune complexes, trigger activation of downstream signaling pathways. However, there are limited in vitro assays that stimulate innate cells via Fcγ receptors that allow for evaluation of drugs or chemicals on antibody-triggered signaling. Our study investigated the activation of Fcγ receptors in innate cells using immune complexes. Our findings revealed that immobilized IgG antibodies did not elicit a significant immune response, so we designed two IgG1 immune complexes: trinitrophenyl-bovine serum albumin (TNP-BSA)/TNP-IgG1 and streptavidin-biotinylated IgG1 (Strept-Biotin IgG1). Strept-Biotin IgG1 immune complex was particularly effective, significantly enhancing IL-6, TNFα, and C3a levels, whereas TNP-BSA/TNP-IgG1 immune complex showed a modest IL-6 increase. Both TNP-BSA/TNP-IgG1 and Strept-Biotin IgG1 stimulated CD86 marker expression on F4/80+ macrophages. We also confirmed the binding of Strept-Biotin IgG1 to innate cells with fluorochrome-conjugated streptavidin. To further understand the Fcγ receptor-mediated activation of innate cells, we blocked the downstream phosphatidylinositol 3-kinase (PI3K) pathway. We found out that the PI3K inhibitor successfully suppressed IL-6 cytokine release and C3a production. However, specific Fcγ receptor-blocking antibodies failed to block IL-6 cytokine production and only modestly suppressed TNFα cytokine release, suggesting either that the antibodies were not effective blockers or that these immune complexes use other receptors. Regardless, the use of the Strept-Biotin IgG1 immune complex to stimulate cytokine production and other immune signaling was consistent with Fcγ receptor activation on innate cells which might be useful in assessing the effects of drugs or chemicals in innate cells.