Cell Analysis Centre, Scientific Affairs, Sysmex Corporation, Kobe, Japan.
Blood Transfus. 2013 Jan;11(1):53-60. doi: 10.2450/2012.0141-11. Epub 2012 Jun 28.
Transfusion-related acute lung injury (TRALI) is associated with vascular endothelial cell injury following neutrophil activation. Recently, it has been suggested that haem-related molecules induce activation of neutrophils and that erythrocyte-derived substances contained in blood preparations are involved in TRALI. We observed the morphological effects and reactive oxygen species (ROS) production of haem-related molecules and investigated the effects of signal transduction inhibitors on haem-induced neutrophil activation.
The polymorphonuclear cell fraction was isolated and stimulated using a control stimulant, PMA or fMLP, or by haem-related molecules, haemin, ferric citrate, or protoporphyrin IX. After stimulation, neutrophil was analysed using electron microscopy, a flowcytometer (FCM) and confocal laser scanning microscope to determine the fluorescent intensity of aminophenyl fluorescein (to detect ROS).
In FCM analysis, haemin and protoporphyrin IX, both of which have a porphyrin ring, induced ROS production in neutrophils. Ferric citrate, which has no porphyrin ring, did not induce neutrophil activation. Haemin alone induced ROS production at relatively high concentrations, whereas low-level fMLP acted as an agonist in the presence of low concentrations of haemin. Haem-related molecules induced ROS production in neutrophil granules through signal transduction in a way similar to PMA. However, in electron microscopy studies, haemin stimulated neutrophils showed minute process at their surface and did not show the vacuolation observable following stimulation with PMA or fMLP.
We suggest that low concentrations of haem-related molecules with porphyrin rings in the presence of other stimulating agent are important for ROS production and possibly the onset of TRALI. The ROS production induced by these molecules is dependent on a signal transduction pathway in a way similar to PMA.
输血相关的急性肺损伤（TRALI）与中性粒细胞激活后血管内皮细胞损伤有关。最近，有人提出与血红素相关的分子可诱导中性粒细胞激活，并且血液制剂中含有的红细胞衍生物质可能与 TRALI 有关。我们观察了血红素相关分子的形态效应和活性氧（ROS）产生，并研究了信号转导抑制剂对血红素诱导的中性粒细胞激活的影响。
分离并刺激多形核细胞部分，使用对照刺激物 PMA 或 fMLP 或血红素相关分子血红素、柠檬酸铁或原卟啉 IX 进行刺激。刺激后，使用电子显微镜、流式细胞仪（FCM）和共聚焦激光扫描显微镜分析中性粒细胞，以检测氨基苯荧光素（检测 ROS）的荧光强度。
在 FCM 分析中，血红素和原卟啉 IX 都具有卟啉环，可诱导中性粒细胞产生 ROS。柠檬酸铁没有卟啉环，不会诱导中性粒细胞激活。血红素本身在相对较高的浓度下诱导 ROS 产生，而低浓度的 fMLP 在低浓度血红素存在下充当激动剂。血红素相关分子通过与 PMA 相似的信号转导途径诱导中性粒细胞颗粒中 ROS 的产生。然而，在电子显微镜研究中，血红素刺激的中性粒细胞表面出现微小突起，而不像 PMA 或 fMLP 刺激后那样出现空泡化。
我们认为，在存在其他刺激物的情况下，具有卟啉环的低浓度血红素相关分子对于 ROS 产生和可能的 TRALI 发病机制很重要。这些分子诱导的 ROS 产生依赖于与 PMA 相似的信号转导途径。
