Mv. Miceli et al., EVALUATION OF OXIDATIVE PROCESSES IN HUMAN PIGMENT EPITHELIAL-CELLS ASSOCIATED WITH RETINAL OUTER SEGMENT PHAGOCYTOSIS, Experimental cell research, 214(1), 1994, pp. 242-249
To investigate the nature of the oxidative event that occurs during ph
agocytosis of retinal outer segments (ROS) by cultured human retinal p
igment epithelial (RPE) cells, cells were incubated with isolated bovi
ne ROS labeled with either the fluorescent probe carboxy-SNAFL-2 or th
e nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H
2DCF). The increase in fluorescence following phagocytosis was measure
d by a flow cytometer. Other measurements included: oxygen consumption
using a Clark-type oxygen electrode, extracellular superoxide release
by superoxide dismutase inhibitable lucigenin chemiluminescence, intr
acellular hydrogen peroxide (H2O2) production, and the effect of catal
ase inhibition on cellular thiobarbituric acid-reactive substances (TB
ARS) caused by phagocytosis. The activities of the enzymes NADPH oxida
se and palmitoyl-CoA oxidase were also measured. H2DCF attached to bov
ine ROS was oxidized during phagocytosis with a time course suggesting
oxidation subsequent to ROS uptake. Measurements of oxygen consumptio
n showed a time-dependent increase of 10%, 4 h after ROS feeding, attr
ibutable to a doubling of the cyanide-resistant oxygen consumption. In
tracellular H2O2 production also doubled 4 h after ROS phagocytosis. R
OS uptake by RPE cells produced no significant extracellular superoxid
e, while extracellular superoxide production was readily demonstrated
in a control macrophage cell line. Enzyme activity measurements showed
that incubation of RPE cells with ROS doubled catalase activity witho
ut affecting superoxide dismutase or glutathione peroxidase activities
. Inhibition of catalase during ROS uptake increased TBARS by 66%. Oth
er enzyme activity measurements showed that human RPE cells possess bo
th NADPH oxidase and palmitoyl-Coh oxidase activities. We conclude tha
t ROS phagocytosis subjects RPE cells to an oxidative event on the sam
e order of magnitude as measured in a macrophage. The event is not an
extracellular macrophage-type respiratory burst and may be due to intr
acellular H2O2 resulting from an NADPH oxidase in the phagosome or fro
m beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, t
he enzyme catalase appears to be essential in protecting the RPE cell
against reactive oxygen species produced during phagocytosis. (C) 1994
Academic Press, Inc.