Department of Vascular and Endovascular Surgery, Heinrich Heine University, Düsseldorf, Germany.
Department of Immunology, Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany; and.
Blood Adv. 2022 Jan 11;6(1):129-137. doi: 10.1182/bloodadvances.2021005721.
Neutrophil extracellular traps (NETs) are networks of extracellular fibers primarily composed of DNA and histone proteins, which bind pathogens. We investigated NET formation in 12 patients with myelodysplastic syndrome (MDS) and 15 age-adjusted normal controls after stimulation with phorbol-12-myristate-13-acetate (PMA). Histones and neutrophil elastase were visualized by immunostaining. Since NET formation is triggered by reactive oxygen species (ROS), mainly produced by reduced NADP-oxidase and myeloperoxidase (MPO), ROS were analyzed by flow cytometry using hydroethidine, 3'-(p-aminophenyl) fluorescein, and 3'-(hydroxyphenyl) fluorescein. On fluorescence microscopy, PMA-stimulated MDS neutrophils generated fewer NETs than controls (stimulated increase from 17% to 67% vs 17% to 85%) (P = .02) and showed less cellular swelling (P = .04). The decrease in mean fluorescence intensity (MFI) of 4',6-diamidino-2-phenylindole, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (ΔMFI 3467 vs ΔMFI 4687, P = .03). In addition, the decrease in MFI for fluorescein isothiocyanate, indicating release of neutrophil elastase from cytoplasmic granules, was diminished in patients with MDS (P = .00002). On flow cytometry, less cell swelling after PMA (P = .02) and a smaller decrease in granularity after H2O2 stimulation (P = .002) were confirmed. PMA-stimulated ROS production and oxidative burst activity did not reveal significant differences between MDS and controls. However, inhibition of MPO activity was more easily achieved in patients with MDS (P = .01), corroborating the notion of a partial MPO defect. We conclude that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of hypochloride, impaired ROS production may not be the only cause of deficient NETosis in MDS.
中性粒细胞胞外诱捕网（NETs）是由 DNA 和组蛋白蛋白组成的细胞外纤维网络，可与病原体结合。我们在 12 名骨髓增生异常综合征（MDS）患者和 15 名年龄匹配的正常对照者中，在佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激后，研究了 NET 的形成。通过免疫染色观察组蛋白和中性粒细胞弹性蛋白酶。由于 NET 的形成是由活性氧（ROS）触发的，ROS 主要由还原型 NADP-氧化酶和髓过氧化物酶（MPO）产生，因此我们使用羟乙基噻吩嗪、3'-(对氨基苯)荧光素和 3'-(羟苯基)荧光素通过流式细胞术分析 ROS。在荧光显微镜下，与对照组相比，PMA 刺激的 MDS 中性粒细胞生成的 NET 较少（刺激后从 17%增加到 67%与 17%增加到 85%）（P =.02），且细胞肿胀程度较轻（P =.04）。MDS 中性粒细胞的 4'，6-二脒基-2-苯基吲哚（4'，6-diamidino-2-phenylindole，DAPI）的平均荧光强度（MFI）下降（表示染色质解凝）明显低于对照组（ΔMFI 3467 与ΔMFI 4687，P =.03）。此外，从细胞质颗粒中释放的中性粒细胞弹性蛋白酶的异硫氰酸荧光素 MFI 下降在 MDS 患者中减弱（P =.00002）。流式细胞术证实，PMA 后细胞肿胀减少（P =.02），H2O2 刺激后颗粒度下降减少（P =.002）。MDS 和对照组之间 ROS 产生和氧化爆发活性没有明显差异。然而，在 MDS 患者中更容易实现 MPO 活性的抑制（P =.01），这证实了 MPO 部分缺陷的概念。我们得出结论，MDS 中性粒细胞的 NET 形成明显受损。尽管我们发现 MPO 依赖性次氯酸盐生成异常，但 ROS 产生受损可能不是 MDS 中 NET 形成不足的唯一原因。
