Noninvasive molecular imaging reveals role of PAF in leukocyte-endothelial interaction in LPS-induced ocular vascular injury.
ABSTRACT Uveitis is a systemic immune disease and a common cause of blindness. The eye is an ideal organ for light-based imaging of molecular events underlying vascular and immune diseases. The phospholipid platelet-activating factor (PAF) is an important mediator of inflammation, the action of which in endothelial and immune cells in vivo is not well understood. The purpose of this study was to investigate the role of PAF in endothelial injury in uveitis. Here, we use our recently introduced in vivo molecular imaging approach in combination with the PAF inhibitors WEB 2086 (WEB) and ginkgolide B (GB). The differential inhibitory effects of WEB and GB in reducing LPS-induced endothelial injury in the choroid indicate an important role for PAF-like lipids, which might not require the PAF receptor for their signaling. P-selectin glycoprotein ligand-1-mediated rolling of mouse leukocytes on immobilized P-selectin in our autoperfused microflow chamber assay revealed a significant reduction in rolling velocity on the cells' contact with PAF. Rolling cells that came in contact with PAF rapidly assumed morphological signs of cell activation, indicating that activation during rolling does not require integrins. Our results show a key role for PAF in mediating endothelial and leukocyte activation in acute ocular inflammation. Our in vivo molecular imaging provides a detailed view of cellular and molecular events in the complex physiological setting.
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ABSTRACT: Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows that PAF receptor antagonists block the action of both PAF and these PAF-like lipids.Journal of Biological Chemistry 07/1991; 266(17):11104-10. · 4.65 Impact Factor
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ABSTRACT: The interaction between leukocytes and vascular endothelial cells plays an important role in various inflammatory disorders. This study evaluated leukocyte behavior in the retina during endotoxin-induced uveitis (EIU) in vivo. EIU was induced in female Lewis rats by footpad injection of lipopolysaccharide (LPS). The time-course changes of retinal leukocyte behavior were followed at 1.5, 3, 4.5, 6, 12, 24, 48, 72, and 120 hours after LPS treatment using acridine orange digital fluorography, consisting of high-resolution images from a scanning laser ophthalmoscope and a fluorescent nuclear dye of acridine orange. Major retinal vessels were significantly dilated (P < 0.05) at 4.5 hours after LPS injection. The vasodilation, marked in veins, became maximum at 24 hours and subsided at 72 hours. Leukocytes were observed rolling along the walls of major veins at 4.5 hours. The number of rolling leukocytes gradually increased and reached a peak level of 33.8 +/- 3.4 cells/minute per major vein at 12 hours. Leukocyte rolling was still observed at 72 hours. No rolling of leukocytes was observed along the arterial walls throughout any experiments. The velocities of rolling leukocytes were determined at 6, 12, 24, and 48 hours. The leukocyte rolling velocity at 6 hours was significantly slower (33.3 +/- 2.8 microns/second, P < 0.05) than at the other three times (average, 46.6 microns/second). Cellular infiltration into the vitreous cavity was detected at 24 hours and reached its maximum at 48 hours. This study demonstrates that it is possible to evaluate EIU by investigating retinal leukocyte behavior and that vasodilation of major retinal vessels and leukocyte-endothelial interactions precede inflammatory cell emigration into the vitreous. This method may be useful to quantify the severity of inflammation in EIU.Investigative Ophthalmology & Visual Science 12/1996; 37(13):2708-15. · 3.44 Impact Factor
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ABSTRACT: The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle.Journal of Biological Chemistry 07/2008; 283(25):17099-106. · 4.65 Impact Factor