Activation of NADPH oxidase by docosahexaenoic acid hydroperoxide and its inhibition by a novel retinal pigment epithelial protein.
OBJECTIVE
In an earlier study, a novel retinal pigment epithelial protective protein (RPP) was described, which suppresses the superoxide generation of activated polymorphonuclear leukocytes (PMNs). In experimental autoimmune uveitis, docosahexaenoic acid hydroperoxide (22:6OOH) has been shown to be the major lipid peroxidation product in photoreceptors. This hydroperoxide was also found to be chemotactic to PMNs. This study was undertaken to evaluate the activation capability of 22:6OOH in resting PMNs and the possible inhibition of this activation by RPP.


METHODS
The 22:6OOH was obtained from pure 22:6 and was purified by thin-layer and high-performance liquid chromatography. Intact rabbit peritoneal PMNs (7-8 X 10(5)) were coincubated with 0.5 microM formyl-methionyl-leucyl-phenylalanine (fMLP), 1.3 microM 22:6OOH, or 5.0 microM 22:6. These systems were coincubated with and without 0.25 microg/ml RPP. From PMN cell-free preparations, the reconstitutes each containing 21 microg plasma membranes and 276 microg cytosolic factors were coincubated with arachidonate, 22:6OOH, or 22:6, each at 100 microM. The inhibition of superoxide production was estimated by adding 0.20 microg/ml RPP. Superoxide generation was measured by superoxide dismutase-inhibitable cytochrome C reduction.


RESULTS
In 30 minutes, 22:6OOH-activated PMNs produced 11.10 +/- 0.68 nanomoles superoxide, and production was suppressed 72% by RPP. Under the same conditions, fMLP induced production of 34.6 +/- 2.77 nanomoles superoxide, and RPP inhibited 60% of production. In the PMN cell-free systems, 100 microM 22:6OOH induced 74.7 nanomoles superoxide per milligram plasma membrane proteins per 5 minutes, and RPP suppressed 50% of production. These results were comparable with those generated by arachidonate, a known stimulator for this system. RPP was effective only when it was added before assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.


CONCLUSIONS
The inflammation-mediated retinal peroxidation product 22:6OOH significantly activates resting PMNs, either in intact cells or in cell-free preparations, to increase further the release of superoxide from PMNs, thus accelerating inflammation-mediated tissue damage. This profound amplification process seems to be effectively downregulated by an RPE-generated protein RPP.
Wu
G S
GS
Doheny Eye Institute and the Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles 90033-1088, USA.
Rao
N A
NA
D000818:Animals; D002474:Cell-Free System; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004789:Enzyme Activation / Q000187:drug effects; D005136:Eye Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000494:pharmacology*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018375:Neutrophil Activation / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects* / Q000201:enzymology; D010857:Pigment Epithelium of Eye / Q000737:chemistry*; D011817:Rabbits; D013481:Superoxides / Q000378:metabolism
D004338:Drug Combinations; D005136:Eye Proteins; D008054:Lipid Peroxides; D013481:Superoxides; D004281:Docosahexaenoic Acids; D009240:N-Formylmethionine Leucyl-Phenylalanine; C037169:docosahexaenoic acid hydroperoxide; D019255:NADPH Oxidases