Abstract
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, in vitro 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.
Highlights
The physiological response of the endothelium to inflammatory signals is a graded process of rolling, tight binding, and extravasation of leukocytes into inflamed tissue sites that comprise the initial step of the inflammatory cascade [1,2,3]
In the work presented here, we aim to establish a device which allows for mimicking the blood flow and physiological shear stress to investigate the influence of ionizing radiation on adhesion of peripheral blood lymphocytes (PBL) to endothelial cells (EC) in vitro
In order to be able to analyze a larger number of pictures and to avoid a possible bias of manual counting, we developed a semiautomated, software-based analysis method to evaluate the data from PBL adhesion assays
Summary
The physiological response of the endothelium to inflammatory signals is a graded process of rolling, tight binding, and extravasation of leukocytes into inflamed tissue sites that comprise the initial step of the inflammatory cascade [1,2,3]. The endothelium is exposed to laminar shear stress, which is exerted by the blood flow. These hemodynamic forces determine the functional properties of the endothelium and contribute to the integrity of the blood vessel wall [7]. For the assessment of an inflammatory response, the adhesion of leukocytes, i.e., peripheral blood lymphocytes (PBL), to endothelial cells (EC) is used as a read-out in vitro and in vivo [8, 9]. We hypothe size that this accounts even more, when primary cells are used instead of established cell lines in order to be more close to the p hysiological situation
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