The role of neutrophil density in neutrophils-mediated inflammatory response induced by monosodium urate crystals

Abstract

Objective: To study the role of neutrophil density and molecular mechanism in neutrophils-mediated inflammatory response induced by monosodium urate (MSU) crystals. Methods: Polymorphonuclear neutrophils (PMNs) isolated from healthy human peripheral blood were treated with MSU crystals at different density (5×10(6)/ml, 20×10(6)/ml, 100×10(6)/ml) in vitro. The mean fluorescence intensity (MFI) of PMNs and production of reactive oxygen species (ROS) were detected by flow cytometry. The distribution of MSU crystals was observed by polarized light microscopy. The neutrophil extracellular traps (NETs) formation was detected by immune fluorescence. The cytokines in cell supernatant were measured by beads assay including interleukin 1β (IL-1β) , tumor necrosis factor α (TNFα) , interleukin 8 (IL-8) , interferon inducible protein 10 (IP-10) , macrophage inflammatory protein 1 (MIP-1) , monokine induced by interferon-γ (MIG) , macrophage inflammatory protein 1α (MIP-1α) , macrophage inflammatory protein 1β (MIP-1β) . Results: (1) After MSU crystal intervention, the side scatters (SSC) of neutrophils with medium-cell density (20×10(6)/ml) and high-cell density (100×10(6)/ml) were 128±13 and 93±9 respectively, both significantly lower than 170±19 in low-cell density (5×10(6)/ml) group.(2) Similarly, compared with low-cell density group, the MFI (lucifer yellow) of PMNs with high-cell density was 1.8±0.2, also significantly decreased (P<0.05). When co-treated with oxygenated adenosine triphosphate (oxATP), MFI of PMNs were all enhanced consistently. (3) In MSU crystals stimulated PMNs, after adding 2',7'-dichlorodihydrofluorescein diacetate, the MFI values were 0.85±0.32, 2.49±0.78, 4.54±1.02 in low cell density groups, medium cell density groups, and high cell density groups respectively, indicating that the generation of ROS was positively correlated with the increase of PMN density (P<0.05). After the intervention of oxATP, the ROS production was significantly reduced. (4) MSU crystal induced NETs formation, especially at high cell density. NETs formation promotes MSU crystal aggregation, which could be partially overcome by oxATP pretreatment. (5) The expression of cytokines were all significantly decreased in the supernatant of PMNs at high cell density exposed to MSU crystals compared with PMNs at medium cell density (P<0.05) . Conclusion: The PMN-mediated inflammation induced by MSU crystals is cell density dependent, and ATP may play a role in partially overcoming the process.