Retinal microglial activation and chemotaxis by docosahexaenoic acid hydroperoxide.
OBJECTIVE
Peroxynitrite generated during the early phase of experimental autoimmune uveoretinitis (EAU) causes peroxidation of docosahexaenoic acid (22:6), a principal unsaturated fatty acid of the photoreceptor membrane, to its hydroperoxide (22:6HP). During this phase, microglia migrate to the site of photoreceptors. The effect of 22:6HP on the migration of isolated retinal microglia was investigated.


METHODS
Retinal microglia were isolated and cultured from newborn Lewis rats and identified immunohistochemically by OX42 antibody staining. Chemotactic activity of the microglia toward 22:6HP was assayed and compared with 22:6 and other control cultures. The effect of 22:6HP on the organization of actin fibers and on the expression of Rac and Iba1 in the microglia was studied by confocal microscopy. The gene and protein expression of Rac and Iba1 in these microglia was analyzed by real-time PCR and Western blot.


RESULTS
Ninety-five percent of isolated microglia stained for OX42 and a chemotactic assay showed that 22:6HP was a potent chemoattractant for these cells. Exposure to hydroperoxide resulted in reorganization of F-actin with intense staining within the lamellipodia. Iba1 and Rac were upregulated in these activated cells and localized in the cell periphery and the lamellipodia.


CONCLUSIONS
22:6HP can activate retinal microglia and is a potent chemoattractant for microglial migration in response to the activation of Rac and reorganization of actin cytoskeleton. In EAU, microglial migration toward the photoreceptors may be mediated by 22:6HP formed in the photoreceptors, and these migrating cells could help modulate the inflammation.
Saraswathy
Sindhu
S
Doheny Eye Institute and the Department of Ophthalmology, The Keck School of Medicine, University of Southern California, Los Angeles, 90033, USA.
Wu
Gueyshuang
G
Rao
Narsing A
NA
D000199:Actins / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects*; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D008054:Lipid Peroxides / Q000494:pharmacology*; D008840:Microfilament Proteins; D017628:Microglia / Q000502:physiology*; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D012160:Retina / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D008054:Lipid Peroxides; D008840:Microfilament Proteins; D012333:RNA, Messenger; D004281:Docosahexaenoic Acids; C037169:docosahexaenoic acid hydroperoxide; D020744:rac GTP-Binding Proteins