Adhesion of flowing neutrophils to cultured endothelial cells after hypoxia and reoxygenation in vitro

Abstract

Using a novel in-line deoxygenating system linked to an in vitro flow-based adhesion assay and video microscopy, we have studied neutrophil recruitment and migration after hypoxia and reoxygenation of cultured human umbilical vein endothelial cells (HUVEC). Unstimulated purified neutrophils were perfused over reoxygenating HUVEC immediately after various periods of endothelial hypoxia. Adhesion to HUVEC was dependent on the duration of hypoxia, with 30, 60, and 100 min of exposure causing graded increments in neutrophil recruitment. The degree of hypoxia also markedly influenced the endothelial response. Severe hypoxia (O2 < 2.5%) induced stationary attachment and then migration of neutrophils, in contrast to rolling adhesion alone under a less intense regime (O2 = 2.5-4.0%). Judged from studies with monoclonal antibodies, P-selectin was essential for adhesion after severe hypoxia, and neutrophil immobilization was attributable to the activation of neutrophil beta 2-integrin. Perfusion of neutrophils with an antibody against interleukin-8 or a platelet-activating factor antagonist reduced levels of adhesion. However, IL-8 appeared to be the dominant agent involved in the immobilization from flow, whereas platelet-activating factor was the more potent agent involved in initiating subendothelial migration. Thus endothelial cells alone can initiate all stages of adhesion and migration of flowing neutrophils after hypoxia and reperfusion.

Polymorphonuclear leucocyte (PMN)-derived inflammatory cytokines—regulation by oxygen tension and extracellular matrix

“…The importance of altered oxygen tensions in regulating PMN function is well established and has been implicated in ischaemia/ reperfusion injury [12,29]. Alterations in oxygen tension have been shown to effect cell adhesion and interactions with extracellular matrix [11], endotoxin tolerance [20], and macrophage cytokine elaboration [17,30]. Therefore, we designed our studies to investigate the dynamics of the modulating effect of the normoxia/ hypoxia/reoxygenation transition on the integrin-controlled sensitivity of PMN cytokine elaboration with external stimulation at the level of cell surface receptor-ligand interactions (fMLP and LPS) as well as at the post-receptor level (direct stimulation of PKC by PMA).…”

Regulation of leukocyte adherence and migration by glycosylphosphatidyl-inositol-anchored proteins

“…Between rolling and the firm adhesion of leukocytes to the endothelium, triggering the arrest of leukocytes is necessary. This process is mediated by chemokines [4] and other chemotactic factors, such as plateletactivating factor (PAF) [5], on the surfaces of ECs. Expression of chemokines on EC surfaces is ascribed either to their production by ECs themselves [6] or to the retention on ECs of chemokines produced by other cells [7].…”

Differential Ability of Exogenous Chemotactic Agents to Disrupt Transendothelial Migration of Flowing Neutrophils

“…This concept gains support from findings that infusion of IL-8 or fMLP into rabbits actually impaired neutrophil transmigration induced by separate localized application of chemotactic agents (13,14), although these effects were linked to the ability of these agents to inhibit adhesion to stimulated endothelium (15,16) rather than to disruption of migration per se. IL-8 is generated by endothelial cells exposed to cytokines or to hypoxia and reoxygenation, and in these cases it has been shown to promote neutrophil activation, adhesion, and migration (17)(18)(19). Thus, it may be hypothesized that for efficient migration, activating signals must have a correct sequence or mode of presentation (e.g., on the endothelial surface rather than in the fluid phase), and that exposure of neutrophils to activating agents in the blood might cause them to fail to cross the endothelial barrier.…”