Department for Pulmonary Medicine, Allergology, Sleep Medicine and Intensive Care, Hospital Bethanien, Universitaet Witten/Herdecke, Solingen, Germany.
Respir Res. 2011 Feb 27;12(1):24. doi: 10.1186/1465-9921-12-24.
Neutrophil influx into the airways is an important mechanism in the pathophysiology of the inflammatory process in the airways of patients with chronic obstructive pulmonary disease (COPD). Previously it was shown that anticholinergic drugs reduce the release of non-neuronal paracrine mediators, which modulate inflammation in the airways. On this basis, we investigated the ability of the long-acting anticholinergic tiotropium bromide to inhibit a) alveolar macrophage (AM)-mediated chemotaxis of neutrophils, and b) cellular release of reactive oxygen species (ROS).
AM and neutrophils were collected from 71 COPD patients. Nanomolar concentrations of tiotropium bromide were tested in AM cultured up to 20 h with LPS (1 μg/ml). AM supernatant was tested for TNFα, IL8, IL6, LTB4, GM-CSF, MIPα/β and ROS. It was further used in a 96-well chemotaxis chamber to stimulate the migration of fluorescence labelled neutrophils. Control stimulants consisted of acetylcholine (ACh), carbachol, muscarine or oxotremorine and in part PMA (phorbol myristate acetate, 0.1 μg/ml). Potential contribution of M1-3-receptors was ascertained by a) analysis of mRNA transcription by RT-PCR, and b) co-incubation with selective M-receptor inhibitors.
Supernatant from AM stimulated with LPS induced neutrophilic migration which could be reduced by tiotropium in a dose dependent manner: 22.1 ± 10.2 (3 nM), 26.5 ± 18,4 (30 nM), and 37.8 ± 24.0 (300 nM, p < 0.001 compared to non-LPS activated AM). Concomitantly TNFα release of stimulated AM dropped by 19.2 ± 7.2% of control (p = 0.001). Tiotropium bromide did not affect cellular IL8, IL6, LTB4, GM-CSF and MIPα/β release in this setting. Tiotropium (30 nM) reduced ROS release of LPS stimulated AM by 36.1 ± 15.2% (p = 0.002) and in carbachol stimulated AM by 46.2 ± 30.2 (p < 0.001). M3R gene expression dominated over M2R and M1R. Chemotaxis inhibitory effect of tiotropium bromide was mainly driven by M3R inhibition.
Our data confirm that inhibiting muscarinic cholinergic receptors with tiotropium bromide reduces TNFα mediated chemotactic properties and ROS release of human AM, and thus may contribute to lessen cellular inflammation.
嗜中性粒细胞流入气道是慢性阻塞性肺疾病（COPD）患者气道炎症过程病理生理学的重要机制。先前的研究表明，抗胆碱能药物可减少非神经旁分泌介质的释放，从而调节气道炎症。在此基础上，我们研究了长效抗胆碱能药物噻托溴铵抑制 a）肺泡巨噬细胞（AM）介导的嗜中性粒细胞趋化性，和 b）细胞内活性氧物质（ROS）释放的能力。
从 71 例 COPD 患者中收集 AM 和嗜中性粒细胞。将 LPS（1μg/ml）培养 20 小时的 AM 中测试纳摩尔浓度的噻托溴铵。测试 AM 上清液中的 TNFα、IL8、IL6、LTB4、GM-CSF、MIPα/β 和 ROS。进一步将其用于 96 孔趋化性室中以刺激荧光标记的嗜中性粒细胞的迁移。对照刺激物包括乙酰胆碱（ACh）、卡巴胆碱、毒蕈碱或 Oxotremorine，部分为佛波醇肉豆蔻酸乙酯（PMA，0.1μg/ml）。通过 a）通过 RT-PCR 分析 mRNA 转录和 b）与选择性 M 受体抑制剂共孵育，确定 M1-3 受体的潜在贡献。
用 LPS 刺激的 AM 上清液诱导嗜中性粒细胞迁移，噻托溴铵可呈剂量依赖性降低迁移：22.1±10.2（3 nM），26.5±18.4（30 nM），和 37.8±24.0（300 nM，与非 LPS 激活的 AM 相比，p<0.001）。同时，刺激的 AM 中 TNFα 的释放减少了对照的 19.2±7.2%（p=0.001）。在这种情况下，噻托溴铵溴化物不影响细胞 IL8、IL6、LTB4、GM-CSF 和 MIPα/β 的释放。噻托溴铵（30 nM）可使 LPS 刺激的 AM 中 ROS 释放减少 36.1±15.2%（p=0.002），并使 carbachol 刺激的 AM 中 ROS 释放减少 46.2±30.2%（p<0.001）。M3R 基因表达超过 M2R 和 M1R。噻托溴铵溴化物的趋化抑制作用主要由 M3R 抑制驱动。
我们的数据证实，用噻托溴铵抑制毒蕈碱能乙酰胆碱能受体可降低 TNFα 介导的人 AM 的趋化性和 ROS 释放，从而可能有助于减轻细胞炎症。
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