Objective: To study rolling of mouse neutrophils on E-selectin and ICAM-1 in an ex vivo flow chamber system.
Methods:The authors developed a small autoperfused flow chamber (20 × 200-µm cross section) that allows direct visualization of cells with and without fluorescent labeling and does not require recirculation of blood.Results: Neutrophils rolled on E-selectin alone, but were unable to interact with immobilized ICAM-1. When ICAM-1 was co-immobilized with E-selectin, the number of cells that rolled was doubled, but no significant firm adhesion was observed. This phenomenon was specific for E-selectin, and no enhancement of rolling was observed when P-selectin was immobilized with ICAM-1. The increased neutrophil rolling seen on E-selectin and ICAM-1 substrates required β 2 integrins. Treating mice with antibodies to the β 2 integrins LFA-1 and Mac-1 showed that LFA-1 was primarily responsible for mediating rolling on ICAM-1 in this model. Increased rolling on E-selectin and ICAM-1 was significantly reduced following administration of a specific p38 mitogen-activated protein kinase (MAPK) inhibitor.
Conclusion:The data show that neutrophil rolling on E-selectin leads to partial activation of LFA-1, enabling LFA-1-dependent rolling on ICAM-1. This mechanism is likely to amplify and accelerate neutrophil recruitment in inflammation. To access inflamed tissue, circulating leukocytes must tether to the endothelium, roll, become firmly adherent, and transmigrate through the vessel wall. Rolling is mediated primarily by selectins (4,22,31) to slow rolling in combination with selectins and can support rolling upon partial activation (9,10,24,41). As leukocytes roll, chemokines and other signaling pathways activate integrins, leading to firm adhesion (5,8,16,46). It is not known whether the partial integrin activation necessary for rolling on ICAM-1 is induced by incomplete chemokine-dependent activation or by selectin engagement.Rolling on selectins allows leukocytes to come in contact with other factors, such as chemokines, that can induce activation and lead to firm adhesion (24,33). There is also evidence that selectin ligation can directly mediate integrin activation. Cross-linking L-selectin on the surface of human neutrophils enhanced the ability of chemotactic stimuli to activate β 2 integrins (16,50). Furthermore, when isolated human neutrophils were perfused over a monolayer of E-selectin and ICAM-1 expressing L-cells, 60% of the interacting cells became firmly adherent, and adhesion was inhibited by blocking the p38