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.
Footnotes
-
Send reprint requests to: Dr. Charles N. Serhan, Director, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.
-
↵1 This work was supported in part by grants from the National Institutes of Health GM38765, P01-DK50305 (to C.N.S.) and DK50189 (to S.P.C.), and a discovery research grant from Ono Pharmaceutical Co., Japan (to C.N.S.).
-
↵2 Recipient of a postdoctoral fellowship from the National Arthritis Foundation.
- Abbreviations:
- DPBS
- Dulbecco’s phosphate-buffered saline
- EAR
- extracellular acidification rate
- EC
- endothelial cells
- fMLP
- N-formylmethionyl-leucyl-phenylalanine
- GM-CSF
- granulocyte-macrophage colony-stimulating factor
- HUVEC
- human umbilical vein endothelial cells
- IL-8
- interleukin 8
- LTB4
- leukotriene B4
- LXA4
- 5(S),6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid
- 15 (R/S)-methyl-LXA4
- 5(S),6(R), 15(R/S)-trihydroxy-15-methyl-7,9,13-trans-11-cis-eicosatetraenoic acid
- 16-phenoxy-LXA4
- 16-phenoxy-17,18,19,20-tetranor-LXA4
- MD
- modified
- ME
- carboxy methyl ester
- MPO
- myeloperoxidase
- O2−
- superoxide anion
- PAF
- platelet-activating factor
- PGE2
- prostaglandin E2
- pHi
- intracellular pH
- PMN
- polymorphonuclear leukocytes
- PTX
- pertussis toxin
- Received June 5, 1997.
- Accepted December 24, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|