Erbeldinger Nadine, Rapp Felicitas, Ktitareva Svetlana, Wendel Philipp, Bothe Anna S, Dettmering Till, Durante Marco, Friedrich Thomas, Bertulat Bianca, Meyer Stephanie, Cardoso M C, Hehlgans Stephanie, Rödel Franz, Fournier Claudia
Department of Biophysics, GSI Helmholtz Center for Heavy Ion Research, Darmstadt, Germany.
Department of Biology, Technical University Darmstadt, Darmstadt, Germany.
Front Immunol. 2017 Jun 1;8:627. doi: 10.3389/fimmu.2017.00627. eCollection 2017.
The vascular endothelium interacts with all types of blood cells and is a key modulator of local and systemic inflammatory processes, for example, in the adhesion of blood leukocytes to endothelial cells (EC) and the following extravasation into the injured tissue. The endothelium is constantly exposed to mechanical forces caused by blood flow, and the resulting shear stress is essential for the maintenance of endothelial function. Changes in local hemodynamics are sensed by EC, leading to acute or persistent changes. Therefore, assessment of EC functionality should include shear stress as an essential parameter. Parallel-plate flow chambers with adjustable shear stress can be used to study EC properties. However, commercially available systems are not suitable for radiation experiments, especially with charged particles, which are increasingly used in radiotherapy of tumors. Therefore, research on charged-particle-induced vascular side effects is needed. In addition, α-particle emitters (e.g., radon) are used to treat inflammatory diseases at low doses. In the present study, we established a flow chamber system, applicable for the investigation of radiation induced changes in the adhesion of lymphocytes to EC as readout for the onset of an inflammatory reaction or the modification of a pre-existing inflammatory state. In this system, primary human EC are cultured under physiological laminar shear stress, subjected to a proinflammatory treatment and/or irradiation with X-rays or charged particles, followed by a coincubation with primary human lymphocytes (peripheral blood lymphocytes (PBL)). Analysis is performed by semiautomated quantification of fluorescent staining in microscopic pictures. First results obtained after irradiation with X-rays or helium ions indicate decreased adhesion of PBL to EC under laminar conditions for both radiation qualities, whereas adhesion of PBL under static conditions is not clearly affected by irradiation. Under static conditions, no radiation-induced changes in surface expression of adhesion molecules and activation of nuclear factor kappa B (NF-κB) signaling were observed after single cell-based high-throughput analysis. In subsequent studies, these investigations will be extended to laminar conditions.
血管内皮与所有类型的血细胞相互作用，是局部和全身炎症过程的关键调节因子，例如，在血液白细胞与内皮细胞（EC）的黏附以及随后向受损组织的外渗过程中。内皮细胞不断受到血流产生的机械力作用，由此产生的剪切应力对于维持内皮功能至关重要。局部血流动力学的变化由内皮细胞感知，导致急性或持续性变化。因此，评估内皮细胞功能应将剪切应力作为一个重要参数。具有可调剪切应力的平行板流动腔可用于研究内皮细胞特性。然而，市售系统不适用于辐射实验，尤其是对于带电粒子，而带电粒子在肿瘤放射治疗中越来越常用。因此，需要研究带电粒子诱导的血管副作用。此外，α粒子发射体（如氡）被用于低剂量治疗炎症性疾病。在本研究中，我们建立了一个流动腔系统，适用于研究辐射诱导的淋巴细胞与内皮细胞黏附变化，以此作为炎症反应起始或对已存在炎症状态的改变的读出指标。在这个系统中，原代人内皮细胞在生理层流剪切应力下培养，进行促炎处理和/或用X射线或带电粒子照射，随后与原代人淋巴细胞（外周血淋巴细胞（PBL））共同孵育。通过对显微镜图像中荧光染色的半自动定量分析来进行分析。用X射线或氦离子照射后获得的初步结果表明，在层流条件下，两种辐射质量下PBL与EC的黏附均降低，而在静态条件下PBL的黏附不受照射的明显影响。在静态条件下，基于单细胞的高通量分析未观察到辐射诱导的黏附分子表面表达变化和核因子κB（NF-κB）信号通路激活。在后续研究中，这些研究将扩展到层流条件。
