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

A study on the involvement of zoledronate in innate immunity via TLR4 signaling

要旨 緒言 材料と方法 結果 考察 謝辞 参考文献Made available in DSpace on 2013-05-01T02:58:30Z (GMT). No. of bitstreams: 1 dent591.pdf: 3996228 bytes, checksum: bcfac261c9660d064ae227505816395d (MD5) Previous issue date: 2013-03-26歯学府_歯学専攻ビスフォスフォネート(bisphosphonate: BP)製剤は骨粗鬆症など骨の脆弱化を特徴とする疾患に対する有効な治療薬として用いられるが、重篤な副作用としてビスフォスフォネート関連顎骨壊死(bisphosphonate-related osteonecrosis of the jaw: BRONJ)を生じる事例が報告されている。BRONJ発症メカニズムの詳細は未だ不明であるが、その発症は顎骨に限定されており、さらに口腔内は細菌が豊富であるため、細菌感染に対する免疫系の関与も示唆され始めている。一般に細菌に対する免疫反応が惹起されると、その初期段階で自然免疫が重要な働きをする。この自然免疫における代表的な受容体として Toll 様受容体(toll-like receptors: TLRs)が知られている。本研究では、口腔内細菌に多いグラム陰性菌の菌体外多糖(lipopolysaccharide: LPS)と、その受容体であるTLR4を介した炎症反応へのBPの影響を明らかにするために、自然免疫の代表的細胞であるマクロファージを含む各種細胞に対する、窒素含有BPであるゾレドロネート(zoledronate: ZOL)の影響を検討した

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

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