Chemokine Fractalkine Mediates Leukocyte Recruitment to Inflammatory Endothelial Cells in Flowing Whole Blood

Abstract

Background-The membrane-bound chemokine fractalkine (CX 3 CL1) is expressed on various cell types such as activated endothelial cells and has been implicated in the inflammatory process of atherosclerosis. The aim of the present study was to dissect the role of fractalkine in leukocyte recruitment to inflamed endothelium under arterial shear forces. Methods and Results-With the use of immunofluorescence and laminar flow assays, the present study shows that human umbilical vein endothelial cells stimulated with tumor necrosis factor-␣ and interferon-␥ abundantly express CX 3 CL1 and promote substantial leukocyte accumulation under arterial flow conditions. In the presence of high shear, firm adhesion of leukocytes to inflamed endothelial cells is reduced by Ϸ40% by a function-blocking anti-fractalkine antibody or by an antibody directed against the fractalkine receptor (CX 3 CR1). With the use of intravital video-fluorescence microscopy we demonstrate that inhibition of fractalkine signaling attenuates leukocyte adhesion to the atherosclerotic carotid artery of apolipoprotein E-deficient mice, which suggests that the CX 3 CL1-CX 3 CR1 axis is critically involved in leukocyte adhesion to inflamed endothelial cells under high shear forces both in vitro and in vivo. Surprisingly, platelets were strictly required for fractalkine-induced leukocyte adhesion at high shear rates. Correspondingly, specific inhibition of platelet adhesion to inflamed endothelial cells also significantly reduced leukocyte accumulation. We show that both soluble and membrane-bound fractalkine induces platelet degranulation and subsequent surface expression of P-selectin, which thereby promotes direct platelet-leukocyte interaction.

Measuring and interpreting platelet-leukocyte aggregates

“…Microfluidic assays represent another microscopy-based approach to study platelet-leukocyte interactions in real time (11,69–74). Typically, isolated cells or whole blood are perfused through a chamber coated with immobilised proteins or cells, allowing precise control of soluble and cellular input.…”

Novel Methods for Assessment of Platelet and Leukocyte Function Under Flow – Application of Epifluorescence and Two-Photon Microscopy in a Small Volume Flow Chamber Model

“…Platelets were labelled with the fluorescent dye 5-carboxyfluorescein diacetate succinimidyl ester (DCF; Invitrogen). Thereafter, platelets were washed and resuspended in Tyrodes-HEPES buffer [11] obtaining a platelet count of 1 10 7 cells/ml. To provide a near-physiological distribution of platelets in the perfused fluid isolated erythrocytes (final hematocrit 35%) were added.…”

“…/ Promochem) [16] were cultured directly in chamber slides and grown until confluence. Cells were either left in their resting state or treated with medium that contained 50 ng/mL tumor necrosis factor (TNF)-and 20 ng/mL interferon (IFN)-for 20 hours prior to the experiments [11]. Platelets and leukocytes were perfused together over bEnd.3 cells at an arterial shear rate of 500/s [15].…”

“…1). By alternately switching the air-valves in defined time intervals Fluorescent-tagged cells were visualized under flow using either a Zeiss Axiotech microscope (20x water immersion objective, W 20x/0.5, Zeiss) with a 100W HBO mercury lamp for epi-illumination as reported previously [5,11]. For two-photon microscopy we used a high-speed 2-photon microscope (TriM Scope, LaVision BioTec, Bielefeld, Germany).…”

“…We [11] and others [25] have shown previously, that endothelial cells can be cultured on chamber slides, activated by cytokine-stimulation to obtain an inflammatory phenotype and then perfused with isolated human leukocytes. Due to the small amount of volume necessary for cell perfusion, we were able to establish a murine vascular inflammation model.…”