Background/Purpose: Macrophages have been involved in both initiation and resolution of gout flares. Accordingly, these cells are characterized by their plasticity as the environment modulates their phenotype exerting inflammatory or anti-inflammatory functions depending on their activation or polarization state. Macrophages in the presence of interferon-ɤ 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 have 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 polarized with M-CSF could represent the population of resident macrophages. In this work we investigated M2 macrophages response to monosodium urate (MSU) crystals in vitro.

Methods: Macrophages 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 (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 in M1 and M2 macrophages was analyzed by flow cytometry with the Caspase-1 FLICA™ Detection Kit (Immunochemistry Technologies). Cytoplasmic pro-caspase-1 and pro-IL-1β were analyzed by western blot. Flow cytometry and statistics analysis were performed with the FACSDiva and GraphPad Prism 5 respectively.

Results: As expected, M1 macrophages produced inflammatory cytokines in response to LPS, whereas M2 macrophages were unable. Both M1 and M2 failed to produce IL-1β after MSU stimulation. However, when challenged with MSU and LPS, M2 macrophages produced IL-1β (mean+/-SEM, LPS 2.59+/-1.37 pg/ml, MSU+LPS 111.4+/-30.44 pg/ml, p= 0.0078) and reduced IL-10 production (mean+/-SEM, LPS 3738+/-230 pg/ml, MSU+LPS 1587+/-386.4 pg/ml, p= 0.0039). Resting M2 macrophages exhibited lower levels of active caspase-1 and pro-caspase-1. 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.

Conclusion: M1 and M2 macrophages failed to produce inflammatory cytokines after MSU challenging, according with the fact that MSU crystals can be found in asymptomatic joints. However, after MSU phagocytosis, M2 macrophages were able to produce IL-1β after LPS stimulation, explaining the requirement of a trigger, such as a copious meal or alcohol intake, for the initiation of an acute flare in gout. M2 macrophages also had lower levels of caspase-1 and pro-caspase-1, than M1 macrophages.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/macrophages-mediated-response-to-uric-acid-crystals-is-modulated-by-their-functional-polarization/