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Nitrogen-containing Bisphosphonates and Lipopolysaccharide Mutually Augment Inflammation via ATP- and IL-1β-mediated Production of NETs

Kanan Bando 1Toshinobu Kuroishi 2Hiroyuki Tada 2Takefumi Oizumi 3 4Yukinori Tanaka 5Tetsu Takahashi 3Itaru Mizoguchi 1Shunji Sugawara 2Yasuo Endo 3

J Bone Miner Res doi: 10.1002/jbmr.4384. 


Among the bisphosphonates (BPs), nitrogen-containing BPs (N-BPs) have much stronger anti-bone-resorptive actions than non-N-BPs. However, N-BPs have various side-effects such as acute influenza-like reactions after their initial administration and osteonecrosis of the jawbones after repeated administration. The mechanisms underlying such effects remain unclear. To overcome these problems, it is important to profile the inflammatory nature of N-BPs. Here, we analyzed the inflammatory reactions induced in mouse ear-pinnas by the N-BPs alendronate (Ale) and zoledronate (Zol). We found the following. (i) Ale and Zol each induced two phases of inflammation (early weak and late strong ear-swelling). (ii) Both phases were augmented by lipopolysaccharides [LPS; cell-surface constituent of gram-negative bacteria (including oral bacteria)], but prevented by inhibitors of the phosphate transporters SLC20/34. (iii) Macrophages and neutrophils were involved in both phases of Ale+LPS-induced ear-swelling. (iv) Ale increased or tended to increase various cytokines, and LPS augmented these effects, especially that on IL-1β. (v) ATP was involved in both phases, and Ale alone or Ale+LPS increased ATP in ear-pinnas. (vi) The augmented late-phase swelling induced by Ale+LPS depended on both IL-1 and neutrophil extracellular traps (NETs, neutrophil-derived net-like complexes). (vii) Neutrophils, together with macrophages and dendritic cells, also functioned as IL-1β-producing cells, and upon stimulation with IL-1β, neutrophils produced NETs. (viii) Stimulation of P2X7 receptors by ATP induced IL-1β in ear-pinnas. (ix) NET formation by Ale+LPS was confirmed in gingiva, too. These results suggest that (1) N-BPs induce both early- and late-phase inflammation via ATP-production and P2X7-receptor stimulation, (2) N-BPs and LPS induce mutually augmenting responses both early and late phases via ATP-mediated IL-1β production by neutrophils, macrophages, and/or dendritic cells, and (3) NET production by IL-1β-stimulated neutrophils may mediate the late phase, leading to prolonged inflammation. These results are discussed in relation to the side effects seen in patients treated with N-BPs.