Abstract

Neutrophil (PMN) activation and recruitment are coordinated by ligand-operated surface receptors. These responses are involved in the tissue injury that follows hypoxia/reoxygenation. Here, we report that inflammatory mediators each evoke distinct and characteristic extracellular acidification rates (EAR) in both PMN and endothelial cells (EC) as measured by a Cytosensor microphysiometer. Leukotriene B₄ (LTB₄) and the peptide N-formylmethionyl-leucyl-phenylalanine were the most potent activators of EAR, whereas other potent stimuli including interleukin-8 and platelet-activating factor only weakly stimulated EAR in PMN. In contrast, other lipid-derived PMN mediators such as prostaglandin E₂ and lipoxin A₄ (LXA₄) did not evoke EAR. Ligand-operated EAR exhibited desensitization as well as ligand specificity and sensitivity to pertussis toxin. Human endothelial cell agonists including histamine, prostacyclin stable analog and LXA₄ each gave sharply different EAR responses, with only histamine evoking an EAR in these cells. Hypoxia/reoxygenation did not alter ligand-operated EAR from PMN, and similarly LTB₄-stimulated PMN transendothelial migration, a functional response, was not influenced by either PMN or EC exposure to intervals of hypoxia/reoxygenation. LXA₄ stable analogs inhibited PMN transendothelial migration (1 nM–1 μM), and this PMN-EC responsiveness to inhibition by a lipoxin stable analog (e.g., 16-phenoxy-LXA₄) was enhanced ∼2 log orders of magnitude after hypoxia/reoxygenation. Results demonstrate that ligand-receptor interactions evoke characteristic profiles of EAR and that some well-characterized ligand-receptor pairs (including interleukin-8, platelet-activating factor, prostaglandin E₂ or LXA₄) on these cell types either weakly activate the EAR pathway or are silent. Furthermore, hypoxia/reoxygenation did not alter LTB₄ PMN responses but did heighten responsiveness to 16-phenoxy-LXA₄, which suggests a potential protective role in leukocyte-mediated injury.