CHARACTERIZATION OF HUMAN NEUTROPHIL AND ENDOTHELIAL-CELL LIGAND-OPERATED EXTRACELLULAR ACIDIFICATION RATE BY MICROPHYSIOMETRY - IMPACT OF REOXYGENATION

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 infla mmatory mediators each evoke distinct and characteristic extracellular acidification rates (EAR) in both PMN and endothelial cells (EC) as m easured by a Cyto-sensor microphysiometer. Leukotriene B-4 (LTB4) and the peptide N-formylmethionyl-leucyl-phenylalanine were the most poten t activators of EAR, whereas other potent stimuli including interleuki n-8 and platelet-activating factor only weakly stimulated EAR in PMN. In contrast, other lipid-derived PMN mediators such as prostaglandin E -2 and lipoxin A(4) (LXA(4)) did not evoke EAR. Ligand-operated EAR ex hibited desensitization as well as ligand specificity and sensitivity to pertussis toxin. Human endothelial cell agonists including histamin e, prostacyclin stable analog and LXA(4) each gave sharply different E AR responses, with only histamine evoking an EAR in these cells. Hypox ia/reoxygenation did not alter ligand-operated EAR from PMN, and simil arly LTB4-stimulated PMN transendothelial migration, a functional resp onse, was not influenced by either PMN or EC exposure to intervals of hypoxia/reoxygenation. LXA(4) stable analogs inhibited PMN transendoth elial migration (1 nM-1 mu M), and this PMN-EC responsiveness to inhib ition by a lipoxin stable analog (e.g., 16-phenoxy-LXA(4)) was enhance d similar to 2 log orders of magnitude after hypoxia/reoxygenation. Re sults demonstrate that ligand-receptor interactions evoke characterist ic profiles of EAR and that some well-characterized ligand-receptor pa irs (including interleukin-8, platelet-activating factor, prostaglandi n E-2 or LXA(4)) on these cell types either weakly activate the EAR pa thway or are silent. Furthermore, hypoxia/reoxygenation did not alter LTB4 PMN responses but did heighten responsiveness to 16-phenoxy-LXA(4 ), which suggests a potential protective role in leukocyte-mediated in jury.