Zoledronic Acid Enhances Lipopolysaccharide-Stimulated Proinflammatory Reactions through Controlled Expression of SOCS1 in Macrophages

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a serious side effect of nitrogen-containing bisphosphonate (NBP) use. Many studies have shown that BRONJ is limited to the jawbone and does not occur in the other bones. We hypothesized that BRONJ is related to local bacterial iections and involves the innate immune system. To examine the relationship between BRONJ and innate immunity, we examined the effects of NBPs on macrophages, one of the important cell types in innate immunity. The expression of toll-like receptor-4 (TLR4) in cells after pretreatment with zoledronic acid (ZOL) did not considerably differ from that in untreated control cells. However, cytokine levels and nitric oxide (NO) production increased after pretreatment with ZOL. Furthermore, ZOL induced NF-κB activation by enhancing IκB-α degradation. Lipopolysaccharide (LPS)-induced apoptosis also increased after pretreatment with ZOL. This effect was mediated by a reduction of suppressor of cytokine signaling-1 (SOCS1), which is a negative regulator of myeloid differentiation primary response gene 88 (MyD 88)-dependent signaling. These results suggest that ZOL induced excessive innate immune response and proinflammatory cytokine production and that these processes may be involved in the bone destruction observed in BRONJ

Effects of ZOL on LPS-induced cell apoptosis.

<p>RAW264.7 cells were incubated for the indicated times (0, 24, 48, or 72 h) with control medium, LPS (100 ng/mL), ZOL (10 µM), or ZOL+LPS. annexin V and PI were added to the cultures prior to flow cytometry. See the methods for a detailed explanation of the contour plots. (B) Plot of apoptosis in (▴) controls, (•) LPS-treated, (□) ZOL-treated, and (▪) ZOL+LPS-treated cells. After stimulation with LPS, apoptosis increased compared to that in unstimulated controls. A similar amount of LPS-stimulated apoptosis was observed after ZOL pretreatment, and a significant increase in apoptosis was observed after further LPS stimulation. Significant differences from the cikb ontrols that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.01).</p

Effects of ZOL on NO release from LPS-stimulated macrophages.

<p>RAW264.7 cells were cultured with or without ZOL for 24 h and then with LPS for an additional 0, 1, 2, 4, or 6 h. After treatment, iNOS expression was examined by real-time PCR. LPS-stimulated production of iNOS from RAW264.7 cells increased significantly after ZOL pretreatment. Significant differences from the negative controls that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.05). (B) RAW264.7 cells were cultured with or without ZOL (10 µM) and LPS (100 ng/mL) for 24 h. NO release was measured by the Griess method. NO production was significantly higher in ZOL-pretreated cells than in LPS-treated positive controls. ZOL had no effect on the release of NO. Significant differences from the negative controls that were not treated with ZOL are indicated by an asterisk (*<i>P</i><0.05).</p

Effects of ZOL on LPS-induced TLR4-mediated activation of NF-κB cytokines.

<p>(A) RAW264.7 cells were cultured with or without ZOL (10 µM) for 24 h and then with LPS (100 ng/mL) for the indicated times (0, 15, 30, or 60 min). The levels of phospho-IκB-α, IκB-α, and ERK2 were analyzed by western blotting. These data indicated that ZOL treatment increased the levels of phosphorylated IκB-α and enhanced the degradation of IκB-α. (B) RAW264.7 cells were cultured with ZOL for the indicated times (0, 1, 4, 8, or 12 h). STAT1 protein levels decreased over time after ZOL treatment. The levels of Phospho-STAT1 increased 1 h after ZOL treatment and then decreased in a time-dependent manner. The levels of SOCS1, which suppresses TLR4 signaling, increased 1 h after ZOL treatment and then decreased in a time-dependent manner. The levels of MyD88 protein increased with time after ZOL treatment.</p