S79 Localised hypoxia enhances neutrophil extravasation and activation in interstitial lung disease
S79 Localised hypoxia enhances neutrophil extravasation and activation in interstitial lung disease
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S79 间质性肺疾病中局部缺氧增强中性粒细胞外渗和活化
DOI：
10.1136/thoraxjnl-2017-210983.85
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Khawaja A
中科院分区：
文献类型：
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作者：
Khawaja A
背景中性粒细胞炎症在多种疾病中普遍存在，并可能参与间质性肺疾病（interstitial lung disease，ILD）的病理生理过程，但其机制尚不完全清楚。局部组织缺氧往往伴随着炎症，这可能会改变细胞反应，以驱动免疫病理学。假设我们提出，缺氧调节中性粒细胞功能，包括整合素激活，中性粒细胞外渗和中性粒细胞胞外陷阱（NET）释放可能导致ILD患者的肺损伤。方法采用氟咪唑（FMISO）-ILD患者的PET扫描和免疫组化HIF-1 α评估肺缺氧。ILD和对照肺切片中的1a染色。为了检查缺氧的影响，在实验之前在缺氧（1%氧）或常氧（21%氧）下培养分离的嗜中性粒细胞。使用流式细胞术评估神经元整合素表达。使用基于荧光的测定来测量神经元粘附和跨内皮迁移。使用酶测定法测量活性氧物质（ROS）的产生，以评估过氧化氢（H2 O2）的产生。使用新的捕获ELISA测量NETosis。支气管肺泡灌洗（BAL）从ILD或对照组患者和评估NETs. ResultsFMISO-PET扫描表明，在ILD患者的纤维化区域局部缺氧。还在肺切片中测定了缺氧，其在ILD中而非对照肺切片中显示HIF-1 a阳性。与对照组相比，ILD-BAL的NETS水平显著更高（p=0.0039）。缺氧不影响b1整合素的表达，但增加了aM和aX亚基的表面表达（p分别为0.0001和0.0179）。未受刺激，PMA和LPS刺激的中性粒细胞粘附到两者？静息和激活的内皮细胞单层增强下？缺氧缺氧时中性粒细胞跨静息和活化内皮细胞的跨内皮迁移也更大（p<0.05）。有趣的是，虽然活性氧生成不受？缺氧，自发性和PMA诱导的NETosis在缺氧条件下均增加（p分别<0.05和0.001）。此外，在ILD-BAL中发现NET升高，表明异常中性粒细胞活化在ILD病理学中的作用。缺氧可增加中性粒细胞整合素的表达、与内皮细胞单层的粘附、跨内皮细胞迁移和NETosis。目前正在进一步研究ILD中性粒细胞活化的信号通路。
BackgroundNeutrophilic inflammation is common in various diseases and may contribute to the pathophysiology of interstitial lung disease (ILD), however the underlying mechanism are not fully understood. Localised tissue hypoxia is often accompanied with inflammation, which may alter cellular responses to drive immunopathology.HypothesisWe propose that hypoxia modulates neutrophil functions including integrin activation, neutrophil extravasation and neutrophil extracellular trap (NET) release that may contribute to pulmonary damage in ILDMethodsPulmonary hypoxia was assessed using both fluoromisonidazole (FMISO)-PET scanning of ILD patients and immunohistochemical HIF-1a staining in ILD and control lung sections. To examine the effects of hypoxia, isolated neutrophils were cultured under hypoxia (1% oxygen) or normoxia (21% oxygen) prior to experimentation. Neutrophil integrin expression was evaluated using flow cytometry. Neutrophil adhesion and trans-endothelial migration were measured using fluorescence-based assays. Reactive oxygen species (ROS) production was measured using an enzymatic assay to assess hydrogen peroxide (H2O2) generation. NETosis was measured using a novel capture ELISA. Bronchoalveolar lavage (BAL) were obtained from ILD or control patients and assessed for NETs.ResultsFMISO-PET scans indicated localised hypoxia in fibrotic regions in ILD patients. Hypoxia was also determined in lung sections, which demonstrated positive HIF-1a in ILD but not control lung sections. ILD-BAL had significantly greater levels of NETS compared to control (p=0.0039). Hypoxia did not affect b1integrin expression, however increased surface expression of the aMand aX subunits (p=0.0001 and 0.0179 respectively). Unstimulated, PMA- and LPS-stimulated neutrophil adhesion to both ?resting and activated endothelial monolayers were enhanced under?hypoxia. Neutrophil trans-endothelial migration, across both resting and activated endothelial cells, was also greater in hypoxia (p<0.05). Interestingly, whilst ROS generation was not affected by?hypoxia, both spontaneous and PMA-induced NETosis were increased under hypoxic conditions (p<0.05 and 0.001 respectively).ConclusionsWe have demonstrated that localised hypoxia is a feature of the ILD lung. Moreover, elevated NETs were found in ILD-BAL, suggesting a role for aberrant neutrophil activation in ILD pathology. Hypoxia increased neutrophil b2integrin expression, adhesion to endothelial monolayers, trans-endothelial migration and NETosis. Further work is underway to investigate the signalling pathways underlying neutrophil activation in ILD.