AB0043 Effect of Macrophage Polarization in the Response to Uric Acid Crystals

BackgroundMacrophages have been involved in both initiation and resolution of gout flares.1,2 These cells are characterised by their plasticity as the environment modulates their phenotype exerting inflammatory or anti-inflammatory functions depending on their activation or polarisation state.3 Macrophages in the presence of interferon-γ and lipopolysaccharide (LPS), what is known as classical activation, acquire an inflammatory phenotype and are also termed M1 macrophages. On the other hand, M2 or alternatively activated macrophages with IL-4 exhibit anti-inflammatory and homeostatic functions. Equivalently, in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) macrophages become M1 or M2, respectively. As M-CSF is present in the blood stream at steady state, some authors propose that M2 macrophages polarised with M-CSF could represent the population of resident macrophages.ObjectivesTo investigate the response of polarised macrophages to monosodium urate (MSU) crystals in vitro.MethodsMacrophages were derived from peripheral blood monocytes of healthy donors after informed consent. Peripheral blood mononuclear cells were separated from whole blood by centrifugation with a density gradient. Monocytes were then isolated by negative selection with magnetic beads and cultured for 1 week with GM-CSF (1000 I.U./mL) or M-CSF (20 ng/mL) to obtain M1 or M2 macrophages, respectively. Macrophages were then stimulated with MSU crystals (200 μg/ml), LPS (100 ng/ml) or both for 18 hours and quantification of IL-1β and IL-10 in supernatants was performed by ELISA. Activation of caspase-1 was analyzed by flow cytometry with the Caspase-1 FLICA™ Detection Kit (Immunochemistry Technologies). Cytoplasmic pro-caspase-1 and pro-IL-1β were analysed by western blot. Flow cytometry and statistics analysis were performed with the FACSDiva and GraphPad Prism 5.ResultsAs expected, M1 macrophages produced inflammatory cytokines in response to LPS, whereas M2 macrophages produced IL-10. Both M1 and M2 failed to produce IL-1β after MSU stimulation. However, when challenged with MSU and LPS, M2 macrophages produced IL-1β and reduced IL-10 production. Resting M2 macrophages exhibited lower levels of active caspase-1 and pro-caspase-1 compared with M1. MSU stimulation increased active caspase-1 levels in both M1 and M2 macrophages and the presence of MSU and LPS had a synergic effect in pro-IL-1β.ConclusionsM1 and M