Abstract
purpose. To examine the effects of oxidized low-density lipoproteins (oxLDL) on
phagocytosis and processing of photoreceptor outer segments (OS) by
retinal pigment epithelial (RPE) cells.
methods. Confluent cultures of RPE-J cells were pretreated with oxLDL or LDL,
and the effects of such treatment on the processing of added OS was
determined. Processing was determined either by the degradation of 125I-labeled OS to trichloroacetic acid-soluble label or by
the cleavage of rhodopsin observed on Western blot analysis of cell
lysates separated by sucrose density gradient fractionation. Binding to
and uptake of OS by RPE-J cells was assessed by determining the
fluorescence of FITC-labeled OS before and after quenching with trypan
blue.
results. OxLDL induced a significant decrease in the degradation of 125I-OS in RPE-J cells but no reductions in either binding
or uptake, when a 24-hour recovery period was inserted between
treatment with oxLDL and challenge with OS. Rhodopsin cleavage
increased in a time-dependent manner after phagocytosis of OS by RPE-J
cells. The small guanosine triphosphatase (GTPase), Rab5, was first
found in phagosome fractions containing rhodopsin and its cleavage
products, and at later times of challenge, in more dense fractions
representing phagolysosomes. These were assessed by the colocalization
of rhodopsin cleavage products in density fractions with cathepsin D, a
marker of lysosomes. OxLDL induced a reduction in rhodopsin cleavage
product formation and in phagosome-lysosome fusion (maturation). It
also reduced the time-dependent shift of rhodopsin to higher density
fractions containing more cathepsin D without any detectable reduction
in the expression of cathepsin D or in acid protease activity.
conclusions. OxLDL induces a reduction in the processing of OS by RPE by perturbing
the fusion of lysosomes with phagosomes.
The retinal pigment epithelial (RPE) cell layer acts as a
support cell for photoreceptors performing such functions as nutrient
and waste transport, as well as phagocytosis and processing of shed
photoreceptor outer segments (OS).
1 2 3 4 5 However, this
processing has been suggested to become perturbed by the prooxidant
environment of the retina
6 7 and to be responsible for the
intralysosomal formation and accumulation of lipofuscin, a
characteristic of RPE cells in vitro
8 9 and in
vivo.
4 10 Although oxidative events in the RPE have been
linked to such disease states as age-related macular degeneration
(AMD),
7 the evidence for a causative relationship remains
circumstantial. We therefore sought to develop a model system to
directly assess the effects of oxidized lipids on a key cell biological
function of RPE cells—the processing of isolated OS by the rat RPE-J
cell line—and to better understand the underlying mechanisms. We used
Cu
2+-oxidized plasma low-density lipoproteins
(oxLDL)
11 as the major source of oxidized lipids. Our goal
was to assess whether ox-lipids affect processing of OS, and if so,
whether the ox-lipid–induced effect is the result of inhibition in
their binding, uptake, and/or degradation by the cells. In preliminary
studies,
12 we found that inhibition was at the level of
degradation, and we therefore sought to determine whether this was due
to a reduction in the total cellular acid protease activity in RPE or
to an inhibition of the fusion of phagosomes containing OS and
lysosomes.
4 5 We report that oxLDL induced a deficiency in
the degradation of OS by RPE-J cells, that the pertubation was
primarily at the level of OS degradation after phagocytosis, and that
it was most likely due to an inhibition of the phagosome–lysosome
fusion event.
4 5