Vogelgesang Antje, Lange Claudia, Blümke Lara, Laage Georg, Rümpel Sarah, Langner Sönke, Bröker Barbara M, Dressel Alexander, Ruhnau Johanna
Department of Neurology, University Medicine Greifswald, Fleischmannstraße 41, FC3, 17475, Greifswald, Germany.
Department of Diagnostic Radiology and Neuroradiology, University Medicine Greifswald, Greifswald, Germany.
J Neuroinflammation. 2017 Jul 21;14(1):140. doi: 10.1186/s12974-017-0914-6.
Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here.
Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy.
MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA.
Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.
中风会引发免疫改变，如氧化爆发受损和中性粒细胞胞外陷阱（NETs）释放减少。我们推测这些防御机制的关键酶在缺血性中风中可能会发生改变。因此，我们分析了患者血清以及粒细胞和单核细胞中髓过氧化物酶（MPO）和中性粒细胞弹性蛋白酶（NE）的细胞内和细胞外含量。由于自主神经系统被认为介导中风诱导的免疫改变，我们还研究了应激激素和乙酰胆碱对MPO和NE的影响。除血栓切除术外，重组组织型纤溶酶原激活剂（r - tPA）快速再通是缺血性中风唯一可用的治疗方法，本文探讨了其对粒细胞和单核细胞抗菌防御机制的影响。
体外实验：在中风后第0、1、3和5天，通过流式细胞术或酶联免疫吸附测定（ELISA）测量细胞内和血清中的MPO和NE，并与对照组进行比较。体内实验：将健康供体的血液与儿茶酚胺、地塞米松和乙酰胆碱一起孵育，通过免疫组织化学法定量产生NET的细胞百分比和NET覆盖的面积。通过流式细胞术或ELISA对细胞内和细胞外的MPO和NE进行定量。将健康供体的血样与r - tPA一起孵育，通过流式细胞术、ELISA和显微镜检查对氧化爆发、吞噬作用、NETosis、细胞因子释放、MPO和NE进行定量。
与对照组相比，中风患者粒细胞中的MPO减少，但血清中MPO增加。NE在细胞内未发生改变，但在患者血清中升高。应激激素降低了产生NET的中性粒细胞百分比，而乙酰胆碱则使其增加。激素处理未改变细胞内的MPO和NE；然而，肾上腺素和乙酰胆碱诱导NE释放。r - tPA导致体外粒细胞和单核细胞的吞噬作用和氧化爆发降低。NETosis、MPO释放和细胞因子未发生改变，而r - tPA增强了NE释放。
细胞内MPO的减少可能是中风患者NETosis降低的原因。中风患者血清中MPO和NE水平升高的影响应在未来的研究中加以探讨。r - tPA在体外损害了抗菌防御功能。因此，再通治疗失败的患者可能感染风险更高，这应在未来的研究中进行分析。中风患者中r - tPA作用引起的免疫改变也应进行研究。
