Novel leukocyte agonists are released by endothelial cells exposed to peroxide.
Reactive oxygen species do not activate isolated neutrophils, yet in vivo, such oxidants promote their adhesion to, and subsequent migration through, the vascular wall. We show human endothelial cells exposed to t-butylhydroperoxide shed large, sealed membrane vesicles that contained potent neutrophil agonists. This activity migrated on TLC like platelet-activating factor (PAF). Since neutrophils have a receptor for this phospholipid, which recognizes its unique characteristics including the short sn-2 acetyl residue, we examined the effect of PAF receptor antagonists and PAF acetylhydrolase on this activity. Structurally unrelated PAF receptor antagonists blocked neutrophil stimulation by vesicular phospholipids, and digestion with PAF acetylhydrolase, which is specific for short sn-2 residues, destroyed this activity. However, metabolic labeling, inhibition of synthesis, phospholipase A1 digestion, and high performance liquid chromatographic studies demonstrated that the vesicles did not contain PAF. Instead, the bioactivity migrated on high performance liquid chromatography like the phospholipids generated by oxidative fragmentation of synthetic arachidonoyl phosphatidylcholine that we have shown previously (Smiley, P. L., Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11104-11110) to stimulate neutrophils through their receptor for PAF. Thus, peroxide treatment of endothelial cells fragments cellular phosphatidylcholines, forming novel PAF-like phospholipids, and induces the shedding of membrane vesicles that contain these bioactive phospholipids.
Patel
K D
KD
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84112.
Zimmerman
G A
GA
Prescott
S M
SM
McIntyre
T M
TM
D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005663:Furans / Q000494:pharmacology; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D010545:Peroxides / Q000494:pharmacology*; D010741:Phospholipases A; D054466:Phospholipases A1; D010743:Phospholipids / Q000378:metabolism; D010972:Platelet Activating Factor / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled; D010738:Type C Phospholipases; D015854:Up-Regulation; D020122:tert-Butylhydroperoxide
D005663:Furans; D010545:Peroxides; D010743:Phospholipids; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; C054893:L 659989; D007531:Isoflurophate; D020122:tert-Butylhydroperoxide; D010741:Phospholipases A; D054466:Phospholipases A1; D010738:Type C Phospholipases