Association of superoxide anions with retinal pigment epithelial cell apoptosis induced by mononuclear phagocytes.
OBJECTIVE
Oxidative stress of the retinal pigment epithelium by reactive oxygen species and monocytic infiltration have been implicated in age-related macular degeneration. The purpose of this study was to determine the role of superoxide anions (O(2)(-)) in mononuclear phagocyte-induced RPE apoptosis.


METHODS
Mouse RPE cell cultures were established from wild-type and heterozygous superoxide dismutase 2-knockout (Sod2(+/-)) mice. The intracellular reactive oxygen species, O(2)(-) and hydrogen peroxide, were measured by using dihydroethidium assay and 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate, acetyl ester assay, respectively. RPE apoptosis was evaluated by Hoechst staining and terminal deoxynucleotidyltransferase dUTP nick-end labeling assay. Changes in mitochondrial membrane potential were detected by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide dye. Activated caspases and caspase-3 were detected in situ by FITC-VAD-fmk staining and caspase-3 substrate, respectively.


RESULTS
Mononuclear phagocytes and interferon-gamma-activated mononuclear phagocytes induced the production of intracellular RPE O(2)(-), a decrease in RPE mitochondrial membrane potential, caspase activation, and apoptosis of mouse RPE cells. All theses changes were significantly enhanced in the Sod2(+/-) RPE cells. Activated mononuclear phagocytes induced more of these oxidative and apoptotic changes in RPE cells than did unstimulated mononuclear phagocytes.


CONCLUSIONS
The authors have shown that the decreased expression of SOD2 and increased superoxide production correlate with RPE apoptosis induced by unstimulated and activated mononuclear phagocytes. The authors suggest that elevated O(2)(-) levels due to genetic abnormalities of SOD2 or immunologic activation of mononuclear phagocytes lead to greater levels of RPE apoptosis. The present study could serve as a useful model to characterize RPE/phagocyte interaction in AMD and other retinal diseases.
Yang
Dongli
D
Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, Michigan 48105-0714, USA.
Elner
Susan G
SG
Lin
Li-Ren
LR
Reddy
Venkat N
VN
Petty
Howard R
HR
Elner
Victor M
VM
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D006861:Hydrogen Peroxide / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007371:Interferon-gamma / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012157:Mononuclear Phagocyte System / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism*
D017382:Reactive Oxygen Species; D013481:Superoxides; D007371:Interferon-gamma; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; C505409:Casp3 protein, mouse; D053148:Caspase 3