Characterization of CD16+ Monocyte Inflammatory Profile

The isolated CD16⁻ and CD16⁺ monocytes were double-stained with anti-CD16-PE (BD Biosciences, USA) and anti-TLR4-APC (BD Biosciences, USA) antibodies. In a dose-finding preliminary experiment, the most potent stimulation of TLR4 expression on cells analyzed by flow cytometry (FACScan, BD Biosciences) was presented at 100ng/mL of Escherichia Coli LPS (Sigma-Aldrich, St, Louis, MO). Therefore, all the following acute in vitro experiments were divided into un-stimulated and stimulated [LPS, 100ng/mL] groups. The HLA-DR expression on CD16^- (classical) and CD16⁺ (non-classical) monocytes were measured by staining with anti-HLA-DR-APC, anti-CD14-FITC (BD Biosciences, USA) and anti-CD16-PE antibodies.
Our preliminary experiments revealed that the TLR4 expression was not different between CD16^- (classical) monocytes between groups. It is well-established that classical monocyte are phagocytic with no inflammatory attributes [15 (link)–17 (link)], so the LPS-stimulated extra- and intra-cellular inflammatory cytokines production and corresponding signals were only evaluated in cultured CD16⁺ (non-classical) monocytes.