www.elsevier.

com/locate/preteyeres
PII: S1350-9462(99)00003-8

Free Radical Mediated Photoreceptor Damage in Uveitis

Narsing A. Rao* and Guey-Shuang Wu
Doheny Eye Institute and Department of Ophthalmology and Pathology, University of Southern
California, 1450 San Pablo Street, Los Angeles, CA 90033-1088, USA

CONTENTS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2. Uveitis and tissue injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.1. Generation of free radicals in uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.2. Peroxynitrite and uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.3. Photoreceptor lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.4. Ampli®cation of uveitis by lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
2.5. Ampli®cation of uveitis by oxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.6. Antioxidants and their ocular distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.7. Local factors in modulation of uveitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3. Modulation of uveitis by RPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.1. Retinal pigment epithelial protective protein (RPP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3.2. Function of RPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4. Prevention of retinal lipid peroxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

1. INTRODUCTION that participate in chronic uveitis include lympho-

cytes, both T and B cells of various subtypes, and
Uveitis and other intraocular in¯ammations are phagocytic cells, such as macrophages which de-
complex, acute or chronic in¯ammatory processes rive from circulating monocytes. In addition,
that primarily involve the uvea. The process can local tissue cellular components also participate,
extend, however, to involve the retina, intraocular either by enhancing the in¯ammation or by
cavities, optic nerve and other ocular structures. down-regulating the process. In the uvea, these
Chronic intraocular in¯ammation is a major cellular components are vascular endothelia and
cause of blindness. This loss of vision is the result melanocytes. In the retina, the cellular com-
of damage in¯icted by the in¯ammatory cell in®l- ponents that participate in the process include
tration, particularly the release of various cyto- microglia, astrocytes, MuÈller cells, photo-
kines or other mediators, including oxygen receptors, and retinal pigment epithelium (RPE).
metabolites (Rao, 1990). The in¯ammatory cells The latter appears to play an active role in the
modulation of uveitis and other intraocular in-
*Corresponding author. Fax: +1-323-442-6688; E-mail: ¯ammations including intraocular infections
inavarro@hsc.usc.edu. caused by various microbial agents, not only

Progress in Retinal and Eye Research Vol. 19, No. 1, pp. 41 to 68, 2000
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
1350-9462/00/$ - see front matter
42 N. A. Rao and G.-S. Wu
because of its anatomic location but because of
its ability to generate either pro- or anti-in¯am-
matory agents (Elner et al., 1990; Elner et al.,
1992; Planck et al., 1992; Percopo et al., 1990;
Dorey et al., 1989; Forrester et al., 1990;
Campochiaro et al., 1989; Planck et al., 1991).
In recent years, the role of oxygen metabolites
(also known as oxygen free radicals or reactive
oxygen species) in initiating retinal damage in
uveitis has been investigated. Current experimen-
tal studies indicate that phagocyte-generated oxi-
.
dants and nitric oxide ( NO) related species play
an important role in the initiation of peroxidation
of cellular lipid components. These products of
lipid peroxidation also amplify in¯ammatory pro-
cesses locally by various proin¯ammatory mech-
anisms (Rao, 1990; Wu et al., 1997). In this
article, the role of various oxidants in initiation
of retinal damage, their mechanisms in ampli®ca-
tion of in¯ammatory processes in the retina/chor-
oid, the role of RPE in the modulation of
ampli®cation processes, and the approaches taken
to reduce such retina damage will be discussed.
2. UVEITIS AND TISSUE INJURY
In chronic uveitis, tissue injury occurs in the
uveal tract, trabecular meshwork, lens, retina,
optic nerve and other ocular structures (Howes
and Rao, 1996). Retinal damage in the form of
chronic cystoid macular edema and photoreceptor
cell degeneration are the major causes of blind-
ness. Various cytokines, the in¯ammatory me-
diators derived from either plasma proteins or
in¯ammatory phagocytic cells such as polymor-
phonuclear leukocytes (PMNs), macrophages and
possibly microglial cells, can cause this retinal
damage (Okada et al., 1998; Yokoi et al., 1997;
Xu et al., 1997; Ramanathan et al., 1996; Dick et
al., 1996; Dick et al., 1998). Cytokines such as
tumor necrosis factor a (TNF-a), interleukin-1
(lL-1), interleukin-6 (lL-6), interleukin-12 (1L-12)
and others play a role in the ampli®cation of the
in¯ammatory process, primarily by activating and
recruiting various in¯ammatory cells. The in¯am-
matory mediators include arachidonic acid
.
metabolites, proteolytic enzymes, NO, and oxy-
gen metabolites, such as superoxide (Oÿ 2 ), hydro-
.
gen peroxide (H2O2) and hydroxyl radicals ( OH)
(Rao, 1990). Arachidonic acid metabolites are
involved primarily in vascular permeability, che-
motaxis and the ampli®cation of uveitis (Rao et
al., 1987a). Proteolytic enzymes, however, appear
to be only a minor contributing factor in initiat-
ing retinal degeneration (Marak et al., 1985; Rao
et al., 1987b).
In the last few years, attention has been di-
rected to oxygen metabolites, such as Oÿ 2 , H2O2,
.
hypochlorous acid, OH and peroxynitrite
(ONOOÿ), in the initiation of photoreceptor cell
disintegration and the ampli®cation of the in¯am-
matory process in uveitis (Rao et al., 1987b; Rao,
1990; Wu et al., 1992; Wu et al., 1997; Goto et
.
al., 1991). Two of these oxidants, Oÿ 2 and OH,
represent oxygen free radicals. Among these oxi-
.
dants, Oÿ 2 is known to react with NO to form. the
potent oxidant ONOOÿ. The presence of NO
and its product ONOOÿ has been noted in var-
ious forms of experimental uveitis, in particular
those induced by endotoxin and retinal S-antigen
(Zhang et al., 1993; Jacquemin et al., 1996; Hoey
et al., 1997). The latter, a soluble protein also
known as arrestin, is a potent uveitogenic mol-
ecule that readily induces uveitis in various lab-
oratory animals when mixed with Freund's
adjuvant and injected into the animals' footpads
(Rao et al., 1979). Animals, such as Lewis rats,
that are treated in this manner develop uveitis
characterized by the in®ltration of lymphocytes
and phagocytic cells in the uvea and retina
(Marak and Rao, 1982; Nussenblatt, 1991)
(Fig. 1).
2.1. Generation of Free Radicals in Uveitis
In uveitis, the primary in¯ammatory cellular
in®ltration consists of an admixture of phagocytes
and various subtypes of lymphocytes. The former
includes PMNs and macrophages, which on acti-
vation are known to generate superoxide anion
.
radical and NO. A free radical is any molecule,
either organic or inorganic, that carries an
unpaired electron. Superoxide is an example of an
inorganic free radical. It has been documented
that upon phagocytosis or exposure to certain
membrane-active agents, the phagocytes undergo
 Free radical mediated photoreceptor damage in uveitis
Fig. 1. Experimental autoimmune uveitis in Lewis rat. Note the in®ltration of phagocytic cells and destruction of photo-
receptor cells at the site of in®ltration (H & E, 200)
a respiratory burst characterized by increased
consumption of oxygen, increased utilization of
glucose via the hexose monophosphate shunt and
release of Oÿ2 (Weiss and LoBuglio, 1982). The
increase in oxygen consumption may be related to
activation of a membrane-bound nicotinamide
adenine dinucleotide phosphate (NADPH) depen-
dent oxidoreductase enzyme complex, converting
NADH to NADPH, thus shuttling electrons from
the appropriate electron donor to oxygen. The
reduced pyridine nucleotides donate two electrons
via the oxidase to two molecules of oxygen, redu-
cing oxygen molecule to Oÿ2.
NAD P†H ÿ:
2O2 ‡ NAD P †H ÿ4 2O2 ‡ NADP ‡ ‡ H ‡
oxidase
The activation of NADPH oxidase requires
phosphorylation and migration of proteins, such
as p47-phox, p67-phox and Rac1 and 2 to the
plasma membranes (Fig. 2). The membrane active
agents which lead to phosphorylation of p47-
phox and other related proteins including
immune complexes, various peptides and cyto-
kines. It is known that the Oÿ 2 radical itself is
poorly reactive in aqueous solution, and the tissue
43
damaging e€ects are believed to be mediated by
secondary products derived from Oÿ , which
.2
include H2O2, hypochlorous acid and OH (Rao,
1990; Weiss and LoBuglio, 1982) (Fig. 3).
Although human PMNs on activation generate
H2O2, about 80% of this metabolite is derived
from dismutation of Oÿ 2.
Hydrogen peroxide is a strong oxidant, but its
reactions with organic substances are generally
slow (Valentine et al., 1984). The activated phago-
cytes discharge myeloperoxidase along with H2O2
into extracellular milieu. In the presence of H2O2,
myeloperoxidase catalyzes the oxidation of the
halides, including chloride, resulting in generation
of hypochlorous acid. This acid can react with
other oxygen metabolites derived from Oÿ 2 to
.
form OH (Valentine et al., 1984). In addition to
.
this source, OH can form primarily from inter-
ÿ
action of O2 and H2O2 in the presence of iron
salts and their complexes (iron-catalyzed Haber±
.
Weiss reaction) (Halliwell, 1981). The OH and
other oxidants can exert direct cytotoxic e€ects
including peroxidation of cell membrane lipids. In
experimental uveitis, Rao et al. demonstrated
generation of oxygen metabolites by the luminol
44 N. A. Rao and G.-S. Wu
Fig. 2. The signal transduction pathway of respiratory
burst in neutrophils on stimulation by fMLP and 22:6HP.
The diagram depicts a well-documented NADPH-oxidase
activation process induced by fMLP, through phospho-
lipase C ± inositol triphosphate and diacylglycerol ± cal-
cium mobilization ± protein kinase C pathways. An
additional pathway through phospholipase D (phosphati-
dic acid ± protein kinase C is also likely to play a role in
the 22:6HP activation
ampli®ed chemiluminescence assay (Rao, 1990;
Wu et al., 1992). A 20-fold increase in the gener-
ation of oxygen metabolites was demonstrated at
the peak severity of the uveitis. The metabolites
.
detected were Oÿ 2 , H2O2 and OH. The source of
ÿ
O2 and H2O2 was the activated phagocytes in®l-
trating the uvea and retina (Rao, 1990; Wu et al.,
1997).
In recent years several investigators have
.
shown the generation of NO at the site of in¯am-
mation in various experimental models of uveitis
(Zhang et al., 1993; Hoey et al., 1997; Jacquemin
et al., 1996; Wu et al., 1997). Activated phago-
.
cytes generate NO, which is dependent on the ex-
pression of the inducible nitric oxide synthase
(iNOS) gene. Inducible nitric oxide synthase is a
transcriptionally regulated enzyme that catalyzes
. .
synthesis of NO from L-arginine. Nitric oxide
synthesis is up-regulated during the in¯ammatory
Fig. 3. On stimulation, leukocytes release Oÿ2 and H2O2,
.
which give rise to OH in the presence of trace metal. The
.
generation of NO in the presence of Oÿ 2 produces
.
ONOOÿ. Both OH and ONOOÿ can cause peroxidation
of photoreceptor membrane lipid (22:6), resulting in disor-
ganization of photoreceptors and generation of 22:6HP.
These hydroperoxides can function as chemotactic agents
and stimulators of neutrophils and macrophages, leading
to ampli®cation of uveitis
process by H2O2, various cytokines, lipopolysac-
charides and extracellular ground substances.
Cytokines such as interleukin b (IL-1b) are found
to activate transcription factors such as nuclear
transcription factor kappa B (NFkB), which is
activated via selective phosphorylation and degra-
dation of its inhibitor protein I-kB. This process
allows translocation of NFkB into the nucleus
where it up-regulates the transcription of iNOS
(McKee et al., 1997; Jeon et al., 1996; Durante et
al., 1996; Forstermann and Kleinert, 1995; Spink
et al., 1995; Kwon et al., 1995; Griscavage et al.,
1995). Similar mechanisms have been proposed
for in the induction of iNOS by the oxidant
H2O2, as well as by lipopolysaccharide (LPS). All
of these studies, however, have been conducted in
tissue culture systems in vitro. In vivo expression
of iNOS seems to be modulated by local tissue
factors as well as by various cytokines, including
transforming growth factor-b (TGF-b). The lat-
ter, which is produced by RPE and other resident
retinal cells, is known to down-regulate NO pro-
duction (Jun et al., 1995).
 Free radical mediated photoreceptor damage in uveitis 45

In uveitis induced by retinal S-antigen, we

noted the selective expression of iNOS in the acti-
vated phagocytes localized to the outer retina
(Fig. 4A,B). This iNOS expression was minimal
or absent in activated phagocytes in®ltrating the
iris, ciliary body, choroid and limbal tissue. Such
observations have led to the discovery that retinal
soluble proteins such as S-antigen and interpho-
toreceptor retinoid-binding protein (IRBP) can
induce the expression of iNOS in activated
macrophages and possibly in activated retinal
microglia. S-antigen especially was found to be a
potent inducer of iNOS. It has been proposed
that the abundant distribution of these soluble
proteins in the outer retina might contribute to
.
the generation of NO mainly at the site of outer
retinal in¯ammation (Shimizu et al., 1998). In
contrast to these soluble proteins, recoverin phos-
ducin, rhodopsin and myelin basic protein could
not induce expression of iNOS in phagocytes. In
this regard, the stimulation capability of S-anti-
gen peptide(s) also need to be evaluated.
Recently, we noted that activated macrophages
generate Oÿ 2 (Fig. 5) on exposure to retinal sol-
uble proteins such as arrestin (Shimizu et al.,
1998). In the outer retina, Oÿ 2 derived products
.
such as OH can cause peroxidation of photo-
receptor cell membranes, resulting in the for-
mation of hydroperoxides of docosahexaenoic
acid (22:6 HP) (Fig. 6) (Rao, 1990; Wu et al.,
1992). These hydroperoxides can also stimulate
in®ltrating phagocytes, resulting in the pro-
duction of additional Oÿ 2 (Wu and Rao, 1999).
Thus, it is intriguing to note that retinal proteins
and the oxidized lipid products of the outer retina
may enhance the generation of oxidants, superox-
.
ide and NO, primarily at the site of outer retinal
in¯ammation (Fig. 3).

positive stainings are mostly seen in the disrupted areas

Fig. 4. Expression of iNOS in the retina of animals with
S-antigen induced uveo-retinitis. A) Immunohistochemical
localization of iNOS was performed using rabbit anti-
mouse iNOS as the primary antibody and biotinylated
goat anti-rabbit IgG as the secondary antibody. The
of retina. B) Co-localization of ED1 and iNOS cells in
the retina on day 12 after S-antigen injection. The
ED1 + /iNOS+ cells (yellow) appeared mostly in the
outer nuclear layer (ONL) and the inner nuclear layer
(INL). The ED1 + /iNOSÿ cells (green) were noted in
inner limiting membrane (ILM) and nerve ®ber layer
(NFL), 720
46 N. A. Rao and G.-S. Wu
Fig. 5. Superoxide production stimulated by various
agents. Rabbit peritoneal macrophages (1  106 cells) were
incubated with either S-antigen (50 mg/ml), IRBP (50 mg/
ml), or fMLP (5 mM) for 30 min. Superoxide production
was measured by the SOD-inhibitable reduction of cyto-
chrome C. The results are mean 2SD for three determi-
nations
2.2. Peroxynitrite and Uveitis
In in¯ammatory processes such as uveitis, Oÿ 2-
mediated in vivo toxicity was thought to be the
.
result of an interaction with H2O2 to form OH in
2+ .
the presence of tissue Fe . These OH have been
implicated as the ultimate cytotoxic agent because
of their high chemical reactivity. However, cur-
.
rent understanding of this OH theory does not
consistently explain the experimental results gen-
erated. For example, superoxide dismutase (SOD)
was thought to exert its protective e€ect by pre-
venting the reduction of Fe+3 to Fe+2 by Oÿ 2,
thus intercepting the Haber±Weiss reaction
(Weiss and LoBuglio, 1982). However, it has been
shown that this type of reduction can be carried
out equally e€ectively by other biological reduc-
tants, such as cellular ascorbate or glutathione,
which are present at even higher concentrations
in tissues (Babbs and Grin, 1989; Rodenas et
al., 1995; Rubbo and Freeman, 1996; Beckman et
al., 1994).
.
Although the chemical reactivity of NO itself
is low, it has been demonstrated that a bimolecu-
.
lar combination of NO with Oÿ 2 generates
ONOOÿ (Fig. 3), a potent oxidant, capable of
oxidizing and nitrating a variety of cellular
macromolecules. The combination reaction of
.
NO and Oÿ 2 is facilitated by the fact that 1)
under in¯ammatory conditions, phagocytes gener-
.
ate NO and Oÿ 2 simultaneously at a similar rate
(Rodenas et al., 1995); and 2) the reaction pro-
ceeds at a near di€usion-limited rate and is more
than 3 times faster than the enzymatic dismuta-
tion of Oÿ 2 catalyzed by SOD (Rubbo and
Freeman, 1996). Peroxynitrite has a pKa of 6.8
and a half-life of 1 s under physiologic conditions.
When protonated, the resulting peroxynitrous
acid decomposes to form potent and toxic oxi-
.
dants with the reactivity of OH and nitrogen
dioxide. Although the half-life of ONOOÿ is only
1 s, it is known to di€use some distance on a cel-
lular scale (100 mm) and possibly to cross cell
membranes (Beckman et al., 1994). There is evi-
dence that ONOOÿ represents a major pathway
.
for NO cytotoxicity but not other species such as
NO2, N2O3, N2O4 or nitronium cation (NO‡
. 2)
that are formed by the reaction of NO with mol-
ecular oxygen (Rodenas et al., 1995; Rubbo and
Freeman, 1996; Beckman et al., 1994).
The generation of ONOOÿ in tissue depends on
the rate of Oÿ 2 production and the steady state
concentration of Oÿ 2 , which has been shown to
range from 10 pM under basal conditions to
0.01±0.1 mM under pathologic conditions (Zweier
et al., 1989). Therefore, the concentration of
ONOOÿ in tissues may be signi®cant, especially
in in¯ammation.
Moreover, in persistent in¯ammations such as
chronic uveitis, ONOOÿ may remain elevated for
a prolonged period due to a positive feedback
mechanism mediated by cytokines, such as IL-1,
IL-6, TNF-a and interferon-g (IFN-g) and release
of arrestin. These cytokines and the retinal pro-
tein are known to induce iNOS, thus generating
.
NO (Shimizu et al., 1998). Furthermore, ONOOÿ
can inactivate SOD, and such inactivation may
 Free radical mediated photoreceptor damage in uveitis 47

Fig. 6. Selected ion chromatographs from gas chromatography/mass spectometry of derivatized 10±, 11±, 13±, 14±, and
17±22:6HP (m/z 263, 281, 223, 321 and 361, respectively). Hydroperoxides were isolated from retinas of EAU animals.
Ion pro®les of 22:6HP are presented as relative peak areas

result in an increased concentration of Oÿ

2 . In vivo
detection of ONOOÿ in recent years has centered
on the localization of the nitrated tyrosine (Tyr)
product in cellular proteins. Peroxynitrite can
react with Tyr residues at pH 7 to generate
nitroTyr, with the nitration taking place at C3, a
position highly activated by the presence of a phe-
nolic hydroxyl group at C4. This reaction has
been found to be speci®c for ONOOÿ, and no
other in¯ammatory mediator is known to display
this capability.
In experimental uveitis eyes, immunohisto-
chemical studies revealed the presence of nitroTyr
in in¯amed retinas (Wu et al., 1997). The
48 N. A. Rao and G.-S. Wu
Fig. 7. Immunohistochemical localization of peroxynitrite
in EAU retinas. The peroxynitrite in the retinas was
detected as nitrotyrosine by probing with polyclonal rab-
bit antinitrotyrosine antibody as the primary antibody
and goat anti-rabbit IgG conjugated to rhodamine as the
secondary antibody. The peroxynitrite plaques were con-
centrated in the photoreceptors, but they were also visible
in some areas of the inner retina. POS: photoreceptor
outer segments; ONL: outer nuclear layer; INL: inner
nuclear layer; NFL: nerve ®ber layer. Magni®cation, 460
nitroTyr positive staining was localized primarily
in the photoreceptors and, to a lesser extent, in
the inner retina (Fig. 7). Therefore, it appears
that the production of ONOOÿ as well as the con-
sequential Tyr nitration of cellular proteins is
substantial in experimental autoimmune uveitis
(EAU). In other in¯ammatory systems, ONOOÿ
has also been detected in the lungs of patients
with adult respiratory distress syndrome and in
lungs of rats exposed to endotoxin (Haddad et
al., 1994). In addition, ONOOÿ has been found in
pathologic conditions such as acute lung disease,
atherosclerosis, hepatic ischemia reperfusion,
alcoholic liver disease and renal allografts
(Haddad et al., 1994; Skinner et al., 1997;
MacMillan-Crow et al., 1996).
2.3. Photoreceptor Lipid Peroxidation
Hydroxyl radicals derived from Oÿ2 and H2O2,
or from hypochlorous acid generated by myelo-
peroxidase are known to react with various cellu-
lar macromolecules, including lipids, proteins and
.
DNA. The initial step in OH-mediated lipid per-
oxidation is the removal of a hydrogen atom
from one of the methylene groups of the carbon
chain, a process that leaves behind an unpaired
electron on this carbon atom resulting in a lipid
carbon radical. These lipid radicals can rapidly
undergo molecular rearrangement to produce
conjugated dienes. The lipid radicals with diene
conjugation can react rapidly with molecular oxy-
gen, yielding hydroperoxy radicals, which in turn
abstract hydrogen atoms from the neighboring
polyunsaturated fatty acid (PUFA) to form fatty
acid hyroperoxide and another lipid radical. This
newly formed lipid radical then reacts with mol-
ecular oxygen to continue the chain reaction.
Hydroperoxy radical also gives rise to malondial-
dehyde, which reacts with amino groups of pro-
teins, yielding conjugated Schi€ base with
characteristic ¯uorochromes. In experimental
uveitis, analysis of the retina revealed the pre-
sence of all of these lipid peroxidation products,
including conjugated dienes, malondialdehyde,
hydroperoxides of docosahexaenoic acid (22:6)
and ¯uorochrome products (Rao, 1990).
.
The cellular target of ONOOÿ and OH can be
the cellular membrane, the cytoplasm, or the
nucleus. The photoreceptors contain a unique
structural feature: the outer segments have a
stack of membrane discs arranged in an orderly
manner. For example, the rat rod outer segment,
which is approximately 1.5 to 2.0 mm in diameter
and 20 to 40 mm in length, contains approxi-
mately 600 to 1000 bilayer discs (Wiegand et al.,
1984). More than 80% of the membrane protein
is the integral protein rhodopsin and 20% is
made up of peripheral proteins, comprising
mainly enzymes that support the biochemical ma-
chinery of the outer segment. Fifty percent of the
rhodopsin is located at membrane-spanning
helices, and the other 50% is distributed between
the cytoplasmic and intradiscal domains
(Khorana, 1992). Unlike those in any other
organ, the membrane phospholipids of the photo-
receptors contain primarily 22:6, resulting in the
especially ¯uid nature of these membranes. As a
result of these unique features, photoreceptors are
highly susceptible to oxidative damage (Wu et al.,
1992).
 Free radical mediated photoreceptor damage in uveitis
Peroxynitrite-induced membrane lipid peroxi-
dation has been demonstrated in phosphatidyl-
choline liposomes and in linolenic acid emulsions
(Rubbo et al., 1994). The inducing species include
decomposition products from ONOOÿ and perox-
.
ynitrous acid, such as, OH-like species, NO2, and
.
NO2. These species are capable of initiating lipid
peroxidation in the absence of iron and, there-
fore, could constitute readily accessible and more
powerful inducing agents than the agents pro-
duced by Haber±Weiss reaction, the most potent
.
species of these products being OH. The nitro-
gen-containing derivatives of oxidized lipids,
.
including mostly the adducts of NO fragments of
hydroperoxy or alkoxy radicals, have been
detected (Rubbo and Freeman, 1996; Rubbo et
al., 1994). These novel lipid oxidation adducts are
indeed organic ONOOÿ and are expected to
further initiate and propagate oxidative nitration
reactions.
When in¯ammatory processes produce
ONOOÿ around photoreceptors, the easily acces-
sible targets are presumed to be the plasma mem-
branes and disk bilayers. Subsequently, the
oxidatively nitrated 22:6 in a chain reaction pro-
pagates the damage, not only to the neighboring
PUFA molecule, but also to the nearby integral
protein, rhodopsin. It appears that such proteins
located in the cytosol or the cell membranes may
be protected to certain extent by the antioxidant
enzymes present in the cytosol of the photo-
receptors. However, depletion of such enzymes
may lead to mitochondrial oxidation resulting in
ONOOÿ-mediated damage to the stress proteins.
Peroxynitrite has been shown to mediate DNA
strand breaks through the N-nitrosylation of
deoxynucleotides (KroÈncke et al., 1997), and to
cause base modi®cation (Yermilov et al., 1995a).
The DNA strand breaks also lead to the acti-
vation of nuclear enzyme poly (ADP-ribose)
synthetase with concurrent depletion of the cellu-
lar ATP level (Yermilov et al., 1995b; Zhang et
al., 1994; Zingarelli et al., 1996; Szabo et al.,
1996). It appears that such DNA damage could
lead directly to eventual photoreceptor cell death.
However, the oxidative nitration of membrane
lipids and proteins and damage to mitochondria
by ONOOÿ may be equally damaging to cellular
49
structural integrity and could be the initial event
leading to photoreceptor cell loss in uveitis.
Peroxynitrite-induced lipid peroxidation has
been well documented in vitro (Radi et al., 1991).
.
Its decomposition products form OH-like species
in the absence of iron. These products are capable
of initiating lipid peroxidation by hydrogen
abstraction in a manner similar to the reaction
.
that occurs with true OH. Since formation of
.
ONOO as well as OH from the Oÿ
ÿ
2 /H2O2 sys-
tem catalyzed by Fe2+ takes place in uveitis, the
oxidized lipid end products of both mechanisms
may occur in uveitis (Wu et al., 1997).
Considerable peroxidative damage occurs in
the retina in the course of uveitis induced by reti-
nal S-antigen (Rao, 1990; Wu et al., 1992; Wu et
al., 1997). The damaged retina reveals elevated
levels of all of the common lipid peroxidation
parameters, including thiobarbituric acid-reactive
substances, conjugated dienes, ketodienes and ¯u-
orescent Schi€-base compounds (Wu et al., 1991;
Goto et al., 1992). Moreover, the formation of
large amounts of these products correlates with
the severity of uveitis (Rao, 1990; Goto et al.,
1992). However, the major peroxidation product
in severe uveitis was found to be 22:6HP (Rao,
1990; Wu et al., 1992).
In the absence of antioxidant enzymes or other
antioxidants, 22:6, the major PUFA of the photo-
receptors, is especially susceptible to peroxidation
by virtue of its structure and its distribution in
the membrane discs of the outer segments. The
arrangement of these discs favors ready accessibil-
ity of neighboring PUFA molecules in the radical
chain propagation step, where a hydroperoxy rad-
ical abstracts an allylic hydrogen atom from a
neighboring PUFA molecule. Moreover, a large
population of mitochondria in the photoreceptor
inner segment requires a large supply of oxygen,
resulting in a constant ¯ux of oxygen from the
choriocapillaris across the photoreceptor mem-
branes; therefore, once the process is initiated,
these conditions favor lipid peroxidation. In ex-
perimental uveitis studies, several hydroperoxide
isomers from 22:6 were identi®ed (Wu et al.,
1992). These isomers were predominantly those
with the hydroperoxyl group situated at the
middle of the fatty acyl chain, namely 10-, 11-,
50 N. A. Rao and G.-S. Wu

13-, 14- and 17 hydroperoxydocosahexaenoic acid mediated by recruitment of circulating leukocytes

(Fig. 6).
In uveitis, the fatty acid hydroperoxides formed
at the photoreceptors can be degraded to form
carbonyl fragments. These reactive carbonyl com-
pounds bind to protein thiols and amino acids
and do not participate in further reactions (Wu et
al., 1997). The peroxidation products can be loca-
lized with a ¯uorescent derivatization using 3-
hydroxy-2-naphthoic acid hydrazine and Fast
Blue B. In¯amed eyes treated in this manner
revealed localization of carbonyl compounds
exclusively in the photoreceptors (Fig. 8) (Wu et
al., 1997). Such histochemical studies suggest the
high susceptibility of photoreceptors to oxidant-
induced damage.
2.4. Ampli®cation of Uveitis by Lipid Peroxidation
As with any other in¯ammatory diseases, ex-
perimental uveitis characteristically shows three
phases: initiation of the in¯ammatory process,
perpetuation or ampli®cation of the in¯am-
mation, and ®nally chronic phase (Nussenblatt
and Palestine, 1989). Although the initiation pro-
cess remains an enigma for the majority of
patients with uveitis, the ampli®cation process is
to the uvea and surrounding tissues. This recruit-
ment process has been amply studied in the S-
antigen-induced model of uveitis (Nussenblatt,
1991). These animal studies have also revealed the
importance of chemotactic factors, lipid me-
diators and cytokines in the ampli®cation of uvei-
tis (Okada et al., 1998; Yokoi et al., 1997; Xu et
al., 1997; Ramanathan et al., 1996; Dick et al.,
1996; Dick et al., 1998; Rao et al., 1987b;
Nussenblatt, 1991). In addition to these me-
diators, hydroperoxides formed at the site of the
initial phase of in¯ammation in the uvea and ret-
ina may contribute to the recruitment of other
phagocytes leading to ampli®cation (Goto et al.,
1991). The hydroperoxides can be detoxi®ed by
tissue glutathione, which converts hydroperoxides
to the corresponding hydroxy fatty acids.
However, this protective mechanism at the site of
in¯ammation may be destroyed by the initial
in¯ux of reactive oxygen metabolites. The hydro-
peroxides thus formed in the retina are found to
be potent chemotactic agents, attracting phago-
cytes, in particular PMNs. The recruitment of
phagocytes and the subsequent activation of these
cells at the initiation site could lead to further
release of oxygen metabolites such as Oÿ 2 , and
eventually to further recruitment of phagocytes

Fig. 8. Confocal imaging of lipid peroxidation products in in¯amed retina. The cryostat sections of posterior segments
from EAU animals were reacted with a ¯uorescent reagent, 3-hydroxy-2-naphthoic acid hydrazide. A false-color scheme
of blue-green-yellow-red-pale pink was used to represent the increasing ¯uorescence intensity of the positive plaques; blue
being the lowest, pale pink the highest in ¯uorescent intensity. The lipid peroxidation products were localized exclusively
in the photoreceptors. POS: photoreceptor outer segment; ONL: outer nuclear layer; INL:inner nuclear layer.
Magni®cation, 460
 Free radical mediated photoreceptor damage in uveitis 51

with the resultant ampli®cation of the uveitis
(Fig. 3).
In vivo studies of experimental uveitis models
have shown the generation of hydroperoxides
from 22:6; in vitro, however, these hydroperoxides
are found to be potent chemotactic agents for
acute in¯ammatory cells (Goto et al., 1991). In
addition to classic lipid mediators of in¯am-
mation, such as arachidonic acid metabolites,
both cyclo-oxygenase (prostaglandins) and lipox-
ygenase (leukotrienes) products are known to be
generated at the site of uveitis; these metabolites
are also potent chemotactic agents that may play
a role in the ampli®cation of uveitis in concert
with the hydroperoxides of 22:6 (Rao et al.,
1987a).
Of the in¯ammatory mediators stated above,
the in vitro stimulation characteristics of 22:6HP
have recently been evaluated in detail (Wu and
Rao, 1999). Docosahexaenoic acid hydroperox-
ides at a concentration as low as 1.3 mM is
capable of stimulating resting PMNs to release
Oÿ2 . The amount released is less than that pro-
duced by the most potent stimulus, fMLP (0.5
mM), but is several-fold higher than that pro-
duced by the reported fatty acid stimulus, arachi-
donic acid (82 mM) (Fig. 9). The stimulation
induced by 22:6HP was totally inhibited by the
calmodulin antagonist, chlorpromazine (20 mM),
as the inhibition seen in fMLP (Wu and Rao,
1999).
It is generally agreed that in receptor-mediated
activation, the important signal transduction
events for Oÿ 2 generation in PMNs include the
following: After receptor occupancy, phospho-
lipase C (PLC) is activated to release diacylgly-
cerol (DAG) and inositol 1,4,5-tris-
phosphate(IP3). The IP3 then moves into the cyto-
sol to release the calcium ion. The increased level
of calcium, in turn, activates calmodulin and
other calcium-dependent processes, including the
activation of protein kinase C (PKC). The PKC

Fig. 9. Activation of intact rabbit peritoneal PMNs by 22:6HP and its inhibition by retinal pigment epithelial protein
(RPP). PMNs (7.0  105 cells) were activated for 30 min with either fMLP (0.5 mM), 22:6HP (1.3 mM) or 22:6 (5.0 mM),
and the fMLP- and 22:6HP-induced activations were inhibited by 0.25 mg each of RPP The superoxide generation was
measured by SOD-inhibitable reduction of cytochrome C. The data are expressed as mean 2SD of three determinations
52 N. A. Rao and G.-S. Wu
acts to phosphorylate the cytosolic factors
(including p47-phox, p67-phox, Rac1 p21 and
Rac2 p21), and these factors then translocate to
the plasma membrane to integrate with NADPH
oxidase components to form an active Oÿ 2 -gener-
ating complex (Edwards, 1991). Although it has
been known for some time that unoxidized cis-
PUFA can induce respiratory burst in PMNs, the
fatty acid-dependent mechanism of activation has
not been fully elucidated. It was thought that the
structural asymmetry of cis-PUFA was able to
perturb the bilayer order, thus causing activation
of components necessary for the respiratory
burst. Taking into account several new ®ndings in
recent years, it appears that activation of 22:6HP
can best be rationalized by the co-activation of
phospholipase (PLD) and the signal transduction
events that follow (Nakamura and Nishizuka,
1994). The well-documented PLC-pathway alone
is invariably transient and temporary because of
the rapid metabolism of both IP3 and DAG from
the signal transduction cascade. Since the acti-
vation of 22:6HP was sustained for a relatively
long period, the involvement of PLD could sup-
port this notion. The primary product from PLD
activation, phosphatidic acid, has been shown to
activate several protein kinases, thus triggering
the respiratory burst (Limatola et al., 1994)
(Fig. 2).
Docosahexaenoic acid hydroperoxides were
also found to activate PMN cell-free systems. In
the past, arachidonic acid alone was known to
activate the reconstitutes of plasma membrane
and cytosolic factors, both derived from PMN
cell-free preparations (Curnutte, 1985).
Docosahexaenoic acid hydroperoxide (100 mM)
was found to generate an amount of Oÿ 2 /mg
membrane proteins comparable to those gener-
ated by 100 mM arachidonic acid in a 10-min
period. The Oÿ 2 released was much smaller with
100 mM 22:6 at 10 min. In the cell-free system,
the mechanistic pathways for the activation are
not the same as those of intact cells. In this sys-
tem, the phosphorylation of cytosolic factors in
the activation sequence was originally discounted,
and the neutralization of these factors by the
anionic amphiphiles was thought to be enough to
prepare these factors for assembly with cyto-
chrome b558 (Gabig et al., 1987) . However, more
recently in this system, phosphotidic acid was
found to activate protein kinases and induce
phosphorylation of p47-phox (McPhail et al.,
1995). The hydroperoxide-initiated PLD pathway
could also play a role in cell-free activation.
These in vitro studies in both PMN-intact and
cell-free systems therefore, demonstrated that the
226:6HP produced by the reactive oxygen and
nitrogen metabolites on in¯ammatory cells, feeds
back to the in¯ammatory sites to further elicit
PMNs to produce more Oÿ 2 . Therefore, the
22:6HP-derived pathways constitutes the poten-
tial routes leading to the ampli®cation of patholo-
gic retinal degeneration in uveitis.
2.5. Ampli®cation of Uveitis by Oxidants
Recent studies on Oÿ 2 and other oxygen metab-
olites have revealed the importance of H2O2 as an
inducing agent of various cytokines through the
activation of NF-kB (Milligan et al., 1998;
Schmidt et al., 1995; Legrand-Poels et al., 1997;
Bowie and O'Neill, 1997; Schmidt et al., 1996;
Sappey et al., 1995). The cytokines, such as IL-l,
IFN-g, TNF-a and others thus generated, can
amplify the in¯ammatory process. Moreover,
H2O2 has been noted to augment the production
.
of cytokine-induced NO (Milligan et al., 1996).
These in vitro studies indicate the important role
played by oxidants in the initiation, perpetuation
and ampli®cation of uveitis by multiple mechan-
isms, including the induction of pro-in¯ammatory
cytokines and the generation of lipid mediators of
arachidonic acid and hydroperoxides of 22:6.
2.6. Antioxidants and their Ocular Distribution
The eye is endowed with several antioxidants,
both water-soluble and lipid-soluble, as well as
enzymes that scavenge various oxidants (Rose et
al., 1998). In addition, the eye contains metal-
binding proteins with free radicals-scavenging
properties such as transferrin, ceruloplasmin and
albumin. The water-soluble antioxidants include
vitamin C, glutathione, uric acid, cysteine, pyru-
vate and tyrosine (Rose et al., 1998). The lipid
soluble antioxidants consist of tocopherol and
 Free radical mediated photoreceptor damage in uveitis
retinols. The antioxidant/free radical scavenging
enzymes include SOD, catalase, glutathione per-
oxidase, glutathione transferase and others. These
enzymes which are abundantly distributed in var-
ious intraocular structures; as well as other anti-
oxidants are presumed to prevent the damaging
e€ects of oxygen and its metabolites.
Immunohistochemical localization of the anti-
oxidant enzymes, SOD, catalase and glutathione
peroxidase has revealed their presence mainly in
those tissues that are predisposed to oxygen rad-
ical-mediated damage, both in a physiologic state
and in pathologic conditions such as uveitis. For
instance, immunolocalization of these three
enzymes was similar. These enzymes were noted
in corneal epithelium, endothelium, the apical
region of the posterior epithelium of the iris, non-
pigmented inner ciliary epithelium, lens epi-
thelium, the inner segments of photoreceptor cell
layer of the retina and RPE (Rao et al., 1985;
Atalla et al., 1988a; Newsome et al., 1994).
Catalase and glutathine peroxidase were also
found in the choroid (Rao et al., 1985; Atalla et
al., 1988a; Newsome et al., 1994).
The balance between the production and cata-
bolism of oxidants by cells and tissue is critical
for maintaining the biological and structural
integrity of the retina and other intraocular
structures. In the physiologic state, antioxidants
and free radical scavengers may protect the eye
by trapping radicals or by interfering with oxi-
dative chain reactions. However, it is possible
that these protective agents may be overwhelmed
when abundant oxygen metabolites are generated
during uveitis. The overwhelming presence of the
oxidants may lead to structural damage in the
retina, optic nerve, lens and anterior segment
(Guy et al., 1993; Ishimoto et al., 1996). Several
experimental studies have revealed that adminis-
tration of antioxidant enzymes, lipid soluble anti-
.
oxidants or OH scavengers results in the
preservation of ocular structures in uveitis
(Fig. 10) (Rao et al., 1994; de Kozak et al.,
1989; Pararajasegaram et al., 1991; Cid et al.,
1992; Marak et al., 1987; Rao et al., 1988a).
These intervention studies suggest that in patho-
logic conditions like uveitis, native antioxidant
and enzyme levels o€er sub-optimal protection
against free radicals.
53
Moreover, the intracellular distribution of var-
ious antioxidant enzymes may not protect the
extracellular structures of the eye from free rad-
icals liberated at extracellular sites by in¯amma-
tory cells. At these extracellular sites, water-
soluble and lipid soluble antioxidants may play a
role in suppressing the damaging e€ects of the
oxidants. However, the experimental studies on
uveitis disclose that these antioxidants are also no
match for the oxidants/free radicals that are
released at these sites by the in¯ammatory phago-
cytes (Atalla et al., 1988b; Wu et al., 1991; Gritz
et al., 1991).
2.7. Local Factors in Modulation of Uveitis
In in¯ammatory conditions, such as uveitis,
activated phagocytes, particularly macrophages,
generate various cytokines that play a role in the
presentation of antigens, activation of lympho-
cytes and recruitment of other in¯ammatory cells.
The activated lymphocytes also generate various
cytokines and chemokines, which play a role in
the recruitment of other in¯ammatory cells,
including macrophages (Okada et al., 1998,
Yokoi et al., 1997; Xu et al., 1997; Ramanathan
et al., 1996; Dick et al., 1996; Dick et al., 1998).
All these events lead to an up-regulation of the
in¯ammatory process and, if unchecked, can lead
to tissue damage (Rao et al., 1994). Furthermore,
the local environment contributes to either up-
regulation or down-regulation of in¯ammation.
As noted above, the former mechanism is
mediated by soluble retinal proteins in the induc-
tion of iNOS and the generation of Oÿ 2 by the
phagocytes. Moreover, it is possible that resident
retinal cells can participate in the in¯ammation
by producing such speci®c chemokines as
RANTES, lP-10, MIP-1a and MCP-1, resulting
in the recruitment of circulating monocytes and
other in¯ammatory cells.
Various retinal proteins have been found to be
antigenic in experimental studies. These proteins
include arrestin, IRBP, rhodopsin, transducin and
recoverin (Gery et al., 1986). Release of these pro-
teins and exposure to antigen-presenting cells
such as macrophages or retinal microglia could
induce a secondary immune-based intraocular in-
54 N. A. Rao and G.-S. Wu

Fig. 10. Histology of retinas with experimental uveitis. A) Animal with EAU without treatment shows marked in¯amma-
tory cell in®ltration and destruction of photoreceptors. B) Deferoxamine-treated EAU animal shows preservation of retina
and photoreceptors. (H & E, 400)

¯ammation, as noted in humans with toxoplasmic supports such an immune-based perpetuation of

retinochoroiditis. The ®nding of detectable in in¯ammation. Such autoimmune reactions in vivo,
vitro B- and T-cell responses to retinal proteins are believed to perpetuate intraocular in¯am-
 Free radical mediated photoreceptor damage in uveitis
mation/uveitis following a non-relevant triggering
of the initial in¯ammation by various infectious
agents (Rao et al., 1994; Gery et al., 1986;
Nussenblatt et al., 1989).
The RPE is endowed with various uveitogenic
polypeptides that can readily induce intraocular
in¯ammation in sensitized animals similar to that
induced by retinal proteins. These molecules
include RPE-speci®c membrane polypeptide
RPE-65, intrinsic membrane doublet polypeptides
RPE-28/30, and a 43kD peptide (Broekhuyse and
Kuhlmann, 1997; Broekhuyse et al., 1996). All
these polypeptides are able to induce autoimmune
in¯ammation in the uvea primarily directed at
RPE. Animals with such polypeptide-induced in-
¯ammation show an accumulation of macro-
phages along the RPE-Bruch's membrane layer.
According to Broekhuyse and his colleagues, this
in®ltrate is similar to Dalen±Fuchs' nodules,
which are typically seen in severe intraocular in-
¯ammations such as Vogt±Koyanagi±Harada
syndrome, sympathetic ophthalmia and other
granulomatous in¯ammations (Broekhuyse and
Kuhlmann, 1997; Broekhuyse et al., 1996). These
experimental studies suggest that RPE proteins,
like retinal proteins, may also contribute to the
initiation and/or perpetuation of uveitis.
3. MODULATION OF UVEITIS BY RPE
In addition to expressing uveitogenic polypep-
tides (Broekhuyse and Kuhlmann, 1997;
Broekhuyse et al., 1996). RPE cells are believed
to play a pivotal role in the induction and per-
petuation of uveitis. This belief is based on sev-
eral in vitro studies. In the presence of IFN-g and
TNFa, cultured RPE cells express MHC class II
molecules and present antigen to sensitized T-cells
(Percopo et al., 1990). RPE cells produce a potent
leukocyte chemotactic factor, interleukin-8 (IL-8),
and in response to IL-1, they secrete IL-6. The
latter cytokine is a potent mediator of in¯am-
mation. Cultured RPE cells also express adhesion
molecules such as CD54 and intercellular ad-
hesion molecule-1 (ICAM-1), which play a role in
leukocyte tracking. RPE-generated cytokines
such as IL-1, IL-6, IL-8, platelet-derived growth
factor (PDGF-)-like protein, and granulocyte
55
macrophage-colony stimulating factor (GM±
CSF) and the expression of adhesion molecules
indicate pro-in¯ammatory functions attributable
to these cells (Elner et al., 1990; Elner et al., 1992;
Planck et al., 1992; Percopo et al., 1990; Dorey et
al., 1989; Forrester et al., 1990; Campochiaro et
al., 1989; Planck et al., 1991; Ja€e et al., 1995).
In addition to generating various pro-in¯am-
matory cytokines, RPE cells can release Oÿ 2
during phagocytosis. This oxidant may be gener-
ated during the phagocytosis of photoreceptor
outer segments, which are a rich source of S-anti-
gen. Moreover, on exposure to a mixture of cyto-
kines like TNF-a, IL-1b and IFN-g, RPE cultures
.
produce a signi®cant amount of NO (Goureau et
al., 1997; Behar-Cohen et al., 1996; Kutty et al.,
1995). These observations support the general
belief that the RPE contributes to the pathogen-
esis of immune-mediated uveitis and to the per-
petuation of intraocular in¯ammation.
Paradoxically, enucleated eyes of patients with
severe uveitis, including sympathetic ophthalmia
and Vogt±Koyanagi±Harada syndrome, show
preservation of the retina and choriocapillaris at
the site of intact RPE, although the choroid is
heavily in®ltrated by macrophages, lymphocytes
and other in¯ammatory cells (Green, 1985).
These morphologic observations do not support
the in vitro ®ndings suggesting the a proin¯amma-
tory role for RPE mentioned above. It appears
that similar to macrophages, RPE cells can
modulate uveal in¯ammation, either enhancing it
by releasing various cytokines or suppressing the
process by releasing TGF-b and other soluble fac-
tors (Elner et al., 1990; Elner et al., 1990; Planck
et al., 1992; Percopo et al., 1990; Dorey et al.,
1989; Forrester et al., 1990; Campochiaro et al.,
1989; Planck et al., 1991; Kutty et al., 1995).
Although sympathetic ophthalmia and Vogt±
Koyanagi±Harada syndrome are relatively rare
conditions, they o€er well-de®ned clinical and
pathologic entities with an underlying auto-
immune mechanism (Rao and Wong, 1981; Rao
et al., 1983; Moorthy et al., 1995). The antigen
that drives the in¯ammation appears to be a reti-
nal peptide, such as S-antigen, IRBP or other
RPE- or uveal pigment-associated proteins.
Constant features of sympathetic ophthalmia and
related entities include preservation of the chorio-
56
capillaris and retina in spite of extensive uveal
in®ltration by mononuclear cells and other pha-
gocytes (Fig. 11). The presence of such features
can be explained by one or more of the following
mechanisms: 1) RPE cells release a factor(s) that
down-regulates the in¯ammation; 2) endothelial
cells of the choriocapillaris produce cytokines
that down-regulate the in¯ammation; 3) endo-
thelial cells do not express adhesion molecules
required for attachment and migration of leuko-
cytes; and 4) immune complex deposits may not
take place in the choriocapillaris.
A recent study reveals that the RPE releases a
novel soluble protein, which is called ``Retinal
Pigment Epithelial Protective Protein (RPP)'',
that suppresses PMN and macrophage generation
of Oÿ
2 (Wu and Rao, 1996; Wu et al., 1996) As
stated, RPE cells have been demonstrated to pro-
duce a large number of cytokines/growth factors
Fig. 11. Sympathetic Ophthlmia. Note preservation of chorio-capillaris despite the heavy in®ltration of in¯ammatory cells
in the choroid (H & E, 250)
N. A. Rao and G.-S. Wu
and antioxidants, such as SOD, under various
conditions. Therefore, it is possible that the RPP
may be one of these already known antioxidant
agents. However, based on studies by other inves-
tigators as well as our own, the only known pro-
ducts of RPE cells that may have the same
function as RPP, and hence, may be the putative
RPP, are SOD and TGF-b. We conducted exper-
iments to exclude this possibility, by comparing
the e€ects of TGF-b and SOD with that of RPP
in our system, we found that RPP is neither SOD
nor TGF-b (Wu and Rao, 1996; Wu et al., 1996).
In addition, the molecular weight of RPP and its
originally identi®ed function, i.e, suppression of
PMN and macrophage Oÿ 2 production, preclude
it from being a currently known antioxidant or
cytokine/growth factor produced by RPE.
 Free radical mediated photoreceptor damage in uveitis
Fig. 12. Suppression of PMN superoxide generation by
RPE cells, skin ®broblasts and corneal epithelial cells.
PMNs were incubated with A) no other cells; B) RPE
cells (cells) or supernatants (sup); C) skin ®broblasts
(cells) or culture supernatants (sup); D) corneal epithelial
cells (cells) or supernatants (sup). PMNs used were
9.0  106 cells and all other cells were 1.45  106 cells.
Culture supernatants were obtained by incubating equal
number of these cells in HBSS for 30 min. The respiratory
burst was stimulated by 0.5 mM fMLP and superoxide
production was assayed by the SOD-inhibitable cyto-
chrome C reduction
3.1. Retinal Pigment Epithelial Protective Protein
(RPP)
As stated above, RPE secrets a novel protein
(Wu and Rao, 1996; Wu et al., 1996) that acts
directly on neutrophils and macrophages, redu-
cing the release of oxygen metabolites, Oÿ2 during
activation of the phagocytes (Fig. 12), thus limit-
ing the retinal damage in uveitis. This protective
Fig. 13. Partial amino acid sequences of trypsin-generated peptides from puri®ed RPP. Residues within parentheses indi-
cate the most prominent identi®ed amino acid in positions where more than one amino acid was identi®ed. X denotes the
cycle where no amino acid was identi®ed
57
protein (RPP) is not cytotoxic to the in¯amma-
tory phagocytes, and the in vitro inhibitory e€ect
occurs in a dose-dependent manner (Wu and
Rao, 1996). Unlike SOD, there is no evidence of
RPP scavenging SOD that is generated enzymati-
cally by the hypoxanthine-xanthine oxidase sys-
tem, indicating that RPP does not have SOD
activity; rather the protein intervenes in the acti-
vation sequence of neutrophils, particularly in
phosphorylation of neutrophil cytosolic protein,
p47-phox (Rao et al., 1997). It is well recognized
that phosphorylation of p47-phox is a prerequi-
site for assembly of NADPH oxidase and the sub-
sequent generation of Oÿ 2 (Curnutte et al., 1994;
el Benna et al., 1994; Volpp et al., 1989; Ding and
Badwey, 1993; Robinson and Badwey, 1995). Our
preliminary results on the mechanism by which
RPP inhibits Oÿ 2 generation indicates that this
protein is e€ective in blocking p47-phox phos-
phorylation with an eciency comparable to that
of well-documented PKC inhibitor. Such result
suggests that RPP suppresses PMN Oÿ 2 gener-
ation by preventing assembly of NADPH oxidase
(Fig. 2).
Puri®cation studies of the novel RPP have
revealed that it is a doublet with molecular mass
of 69/75 kDa (Wu and Rao, 1996; Wu et al.,
1996). The microsequencing analysis by Edman
degradation revealed that both N-termini were
blocked. Subsequent trypsin digestion followed
by secondary ion mass spectrometry was used to
assess the purity of the tryptic fragments prior to
sequencing analysis. A total of eight polypeptide
58 N. A. Rao and G.-S. Wu

sequences were obtained from the two bands (69
and 75 kDa) (Wu et al., 1996). In three of these
sequences, there was some degree of homology
with the transferrin and transferrin precursor
family of proteins. The remaining ®ve sequences
did not match any sequence in the database.
These chemical and functional studies indicate
that RPP could be a novel protein both in its pri-
mary amino acid sequence (Fig. 13) and in its
function and may belong to the transferrin super-
family.
Retinal pigment protective protein is immuno-
genic and monoclonal antibodies have been gen-
erated against this protein. These antibodies react
speci®cally with the protein, reversing the inhibi-
tory e€ects of RPP on activated phagocytes
(Fig. 14). Immunohistochemical localization stu-
dies on both pigmented and albino rabbits reveal
the restricted distribution of RPP within the cyto-
sol of RPE (Fig. 15A). None of the ocular cells,
including ciliary or iris epithelia, retina, cornea,
conjunctiva or other tissues, express immunohis-
tochemically detectable RPP. It is interesting that
RPP expression abruptly stops anteriorly at the
ora serrata and that neither non-pigmented nor
pigmented epithelia of the pars plana ciliaris
show positive staining (Fig. 15B).
RPP is not restricted to one species since
immune reactive RPP is visualized in the RPE of
rabbits, cattle (Fig. 16), and non-human primates,
as well as that of humans. In all these epithelia
there is constitutive expression of this protein.
However, immunohistochemically the expression
of RPP can be up-regulated by pro-in¯ammatory
agents, including various cytokines. The former
includes LPS and the latter includes IL-1, TNF-a
and IFN-g. These observations in culture systems
were con®rmed by ELISA; RPE exposure to
these cytokines resulted in increased production
of RPP (Matsubara et al., 1997).

Fig. 14. Reversal of RPP activity by antibodies. RPP (0.3 mg) in RPE culture supernatant was co-incubated with either 60
ml of monoclonal antibody, or 2 ml of polyclonal antibody for 45 min. Rabbit peritoneal PMNs, 9.8  105 were used for
the assay. For the controls without antibody, 60 ml of culture medium containing 1% serum and 1 mg of irrelevant mouse
Ig were added to RPP in place of antibody (see PMN + RPP + medium). The respiratory burst was stimulated by 0.5
mM fMLP and the superoxide production was assayed by the SOD-inhibitable cytochrome C reduction
 Free radical mediated photoreceptor damage in uveitis 59

Fig. 15. Immunohistochemical localization of RPP in rabbit eye. A) Section of ciliary body and peripheral retina. Note
positive staining restricted to RPE. B) The ciliary epithelium reveals negative staining (Immunoperoxidase)
60 N. A. Rao and G.-S. Wu
Fig. 16. Immunohistochemical localization of RPP reveals presence of this protein in bovine RPE (Immunoperoxidase)
3.2. Function of RPP
RPP is found to exert its inhibitory functions in
vivo, thus preventing free radical-mediated
damage. Intravitreal administration of this pro-
tein in animals with endotoxin-induced uveitis
results in a suppression of retinal lipid peroxi-
dation, leakage of protein and generation of
superoxide (Numaga et al., 1997). These in vivo
and in vitro studies suggest that RPP could play a
role in protecting the retina in pathologic con-
ditions characterized by in®ltration of macro-
phage or other cellular in®ltrate in the choroid,
sub-RPE region or outer retina. These conditions
include uveitis and various retinal and macular
degenerations.
Although the above observations suggest a pro-
tective role for RPP in intraocular in¯ammations
such as uveitis, this function on phagocytes may
lead to the survival and spread of various infec-
tious agents in the outer retina, RPE-Bruchs'
membrane region in pathologic conditions such
as fungal endogenous endophthalmitis. It is well
recognized that fungi or other infectious agents
are eliminated from the tissue by phagocytic cells,
where the release of oxidants by the phagocytes
leads to destruction of the pathogens. The infec-
tious organisms that localize in the RPE or the
subretinal or sub-RPE space may survive better
due to the release of RPP at these sites. It is inter-
esting that, in the past, Dr. Zimmerman from the
Armed Forces Institute of Pathology pointed out
selective survival of fungal elements in the sub-
retinal and sub-RPE regions in cases with en-
dogenous mycotic endophthalmitis (Fig. 17).
Ocular pathologists are well aware of such obser-
vations which are not limited to fungi, but also
include other infectious agents such as bacteria.
Based on a histologic study of eyes with uveitis or
infectious endogenous endophthalmitis, it is
apparent that the RPE modulates the in¯amma-
tory process around the outer retina and choroid
by releasing anti-in¯ammatory factors such as
RPP and TGF-b and the pro-in¯ammatory cyto-
 Free radical mediated photoreceptor damage in uveitis 61

Fig. 17. Human eye section depicting fungal infection. Note presence of aspergillus hyphae in the sub-RPE along Bruch's
membrane. A) Gomeri methenamine silver staining showing RPE (arrow) and fungi (arrowhead). R: retina and C: chor-
oid. B) Periodic acid-Schi€ stain reveals in¯ammatory in®ltrate in the subretinal space and choroid (C). Note fungi along
Bruch's membrane and preservation of choriocapillaris (arrow). The insert shows higher magni®cation of fungi
62 N. A. Rao and G.-S. Wu
kines mentioned above. A balance between the
generation of these anti-in¯ammatory and pro-in-
¯ammatory factors by local tissue and in®ltrating
in¯ammatory cells, uveitis caused by microbial
infections may result in either a bene®cial or a
damaging e€ect on the neurosensory retina and in
intraocular spread of the infectious agent(s).
4. PREVENTION OF RETINAL LIPID
PEROXIDATION
In experimental uveitis, administration of hy-
droxyl radical scavengers or various antioxidants
can signi®cantly reduce such retinal lipid peroxi-
dation and the severity of intraocular in¯am-
mation (Marak et al., 1985; Rao et al., 1986a;
Rao et al., 1986b; Rao et al., 1986c; Rao et al.,
1986d; Rao et al., 1987b; Rao, 1990). In animals
with uveitis, treatment with deferoxamine, an
iron chelator known to interfere with the gener-
.
ation of OH by Oÿ 2 and H2O2, has been shown
to cause a signi®cant reduction in the formation
of lipid peroxidation products and in the severity
of the ocular in¯ammation (Fig. 10A and
Fig. 10B) (Rao et al., 1986c). These protective
e€ects of deferoxamine are not unique to this
.
agent; other OH hydroxyl radical scavengers,
such as sodium benzoate, 2-3-dihydroxybenzoic
acid and dimethyl thiourea, o€ered similar pro-
tective e€ects. Similarly, animals treated with
SOD, catalase and glutathione peroxidase show a
signi®cant reduction in the severity of uveitis as
well as in the formation of lipid peroxidation pro-
ducts (Rao et al., 1985; Rao et al., 1986a; Rao et
al., 1986b; Rao et al., 1986c; Rao et al., 1986d;
Rao et al., 1987b). Thus these treatment exper-
iments not only substantiate the generation of
.
OH in uveitis; they also provide evidence that
therapeutic agents with free radical scavenging
properties may be bene®cial in the treatment of
uveitis. Such treatment could prevent retinal
damage associated with severe uveitis (Rao et al.,
1990).
.
The protective e€ect of OH scavengers and
antioxidant enzymes was also seen in diverse ocu-
lar and systemic in¯ammatory conditions. In ani-
mals with demyelinating optic neuritis, treatment
with antioxidant enzymes like catalase resulted in
the protection of the optic nerve from demyelina-
tion (Guy et al., 1986; Guy et al., 1989a; Guy et
al., 1989b; Guy et al., 1994a). Such antioxidants
.
and OH radical scavengers have been shown not
only to reduce the severity of optic neuritis and
encephalomyelitis, but to slow the demyelinating
process in animals with myelin basic protein
(MBP)-induced demyelinating encephalomyelitis
(Guy et al., 1994b).
5. FUTURE DIRECTIONS
In experimental uveitis induced by retinal sol-
uble proteins or other retinal proteins, in¯amma-
tory phagocytes in®ltrate the outer retina,
including the photoreceptors. At these sites, the
phagocytes generate and release oxygen metab-
olites, including ONOOÿ. The photoreceptors are
known to be surrounded by uveitogenic extra-
cellular molecules such as IRBP. Hence, the ni-
tration of Tyr residues in IRBP may be the initial
biochemical event in the outer retinal damage in
uveitis. Subsequently, the lipid cell membranes, as
well as cytosolic proteins of the photoreceptors,
may become targets for the oxidants. Since the
radical-mediated damage takes place via a chain
reaction spreading from one molecule to another,
it is conceivable that lipid peroxidation can lead
to the nitration of rhodopsin tyrosine residues,
which in part are located in the photoreceptor
disc membranes. Since rhodopsin is a major com-
ponent of photoreceptors and since it contains 18
Tyr residues, it is apparent that nitration of rho-
dopsin could lead to disrupted retinal functions.
However, further studies are required to docu-
ment the nitration of rhodopsin and IRBP Tyr (s)
and the functional changes that ensue.
Peroxynitrite generation may also mediate modi®-
cation of the bases in the nucleic acid of photo-
receptors, in particular mitochondrial DNA. Such
modi®ed bases can lead to apoptosis or necrosis
of the retinal cells. All these e€ects, including
lipid peroxidation may lead to photoreceptor cell
loss and retinal degeneration in uveitis (Fig. 18).
In contrast to the above photoreceptor cell
damage from peroxidation of cell membrane
lipids that occurs in acute and severe uveitis, in
chronic uveitis the photoreceptors may undergo
 Free radical mediated photoreceptor damage in uveitis
Fig. 18. Macrophages release superoxide and nitric oxide
on activation by S-antigen. Nitric oxide and superoxide
combine to form peroxynitrite, which may cause lipid per-
oxidation of photoreceptor membranes and nitration of
rhodopsin and DNA base modi®cation in photoreceptors.
These insults could lead to photoreceptor necrosis/de-
generation
degenerative changes from mitochondrial damage
caused by the chronic oxidative stress. Such stress
is induced by the depletion of mitochondrial anti-
oxidants such Mn SOD. Unchecked generation of
oxidants in the mitochondria can lead to lipid
peroxidation, protein oxidation and mitochon-
drial DNA mutations. Such damage to lipid cell
membranes of mitochondria can lead to cyto-
chome C release into the cytoplasm and other
alterations that cause apoptotic cascade in the
photoreceptors.
Retinal photoreceptors are well situated to
avoid degeneration from ONOOÿ and this, in
part, is due to RPE generation of antioxidants,
including RPP. Unlike various antioxidant
enzymes, RPP functions as an extracellular pro-
tective protein in and around the RPE sites
including around the photoreceptors. By inhibit-
ing the phosphorylation of phagocyte proteins,
.
RPP interferes with the generation of both NO
ÿ
and O2 by the in®ltrating and activated phago-
cytes around the photoreceptors. It appears that
the balance between generation and catabolism of
.
NO and Oÿ 2 around the photoreceptor is crucial.
Excessive production of these oxidants or lack of
63
adequate generation of RPP may lead to retinal
degeneration. In uveitis, one can suggest that if
RPP generation is enhanced, high levels of RPP
may prevent retinal degeneration even in the pre-
sence of severe uveitis. Either a pharmacologic or
a molecular approach that can lead to upregula-
tion of RPP may be useful, not only in the selec-
tive treatment of uveitis, but also in preventing
any photoreceptor degeneration in¯icted by free
radicals which are known to be generated in var-
ious pathologic conditions a€ecting macula and
other parts of the retina.
6. SUMMARY
Uveitis is a major cause of blindness, with the
visual loss that occurs being due primarily to reti-
nal tissue damage. The tissue damage is mediated
mainly by phagocytic in¯ammatory cells, such as
macrophages, by the release of various proteolytic
enzymes, arachidonic acid metabolites, cytokines
and free radicals. The latter are found to be
potent cytotoxic agents that readily cause tissue
damage by peroxidation of lipid cell membranes.
Recent studies of experimental uveitis indicate
that other potent oxidants are generated in uveitis
by macrophages. One of these is ONOOÿ, which
.
is formed from NO and Oÿ 2 . The macrophages
.
generate NO preferentially in the outer retina fol-
lowing iNOS expression. In these phagocytes,
outer retinal proteins, especially arrestin, are
found to be potent iNOS inducers.
Current studies of RPE show that these cells
protect the retina from ONOOÿ mediated damage
in uveitis by releasing a novel protein called reti-
nal pigment epithelial protective protein. This
.
protein is found to suppress Oÿ 2 and NO gener-
ation by the phagocytes, in both in vitro and in
vivo uveitis models. The protective protein ex-
pression is restricted to RPE, its suppressive e€ect
is a result of the inhibition of the phosphorylation
of cytosolic proteins, p47-phox, required for the
assembly of NADPH and activation of NFkB,
which are required for generation of Oÿ 2 and ex-
pression of iNOS respectively. Either pharmaco-
logically or chemically, up-regulation of RPP
generation could help in preventing retinal de-
generation in uveitis or other degenerative dis-
64 N. A. Rao and G.-S. Wu
orders, which are in¯icted by phagocyte gener-
ation of free radicals.
AcknowledgementsÐThis study is supported in part by grants,
EY10212, EY03040, EY12363, National Institutes of Health
and Research to prevent Blindness, New York.
REFERENCES
Atalla, L.R., Sevanian, A. and Rao, N.A. (1988a)
Immunohistochemical localization of glutathione per-
oxidase in ocular tissue. Curr. Eye Res. 7, 1023±1027.
Atalla, L.R., Sevanian, A. and Rao, N.A. (1988b) Hydrogen
peroxide localization in ocular tissue: an electron
microscopic cytochemical study. Curr. Eye Res. 7, 931±
936.
Babbs, C.F. and Grin, D.W. (1989) Scatchard analysis of
methane sul®nic acid production from dimethyl sulfox-
ide: a method to quantify hydroxyl radical formation
in physiologic systems. Free Radic. Biol. Med. 6, 493±
503.
Beckman, J.S., Chen, J., Ischiropoulos, H. and Crow, J.P.
(1994) Oxidative chemistry of peroxynitrite. Methods
Enzymol. 233, 229±240.
Behar-Cohen, F.F., Heydolph, S., Faure, V., Droy-Lefaix,
M.T., Courtois, Y. and Goureau, O. (1996)
Peroxynitrite cytotoxicity on bovine retinal pigmented
epithelial cells in culture. Biochem. Biophys Res.
Commun. 226, 842±849.
Bowie, A. and O'Neill, L.A. (1997) Studies into the mechan-
ism of NF kappa B activation by IL1, TNF and H2O2
in primary and transformed endothelial cells. Biochem.
Soc. Trans. 25, 125S.
Broekhuyse, R.M., Kuhlmann, E.D., Peters, T.A. and
Kuijpers, W. (1996) Macrophage subpopulations and
RPE elimination in the pathogenesis of experimental
autoimmune pigment epithelial protein-induced uveitis
(EAPU). Exp. Eye Res. 62, 471±480.
Broekhuyse, R.M. and Kuhlmann, E.D. (1997) Uveitogenic
28/30 kD and 43 kD polypeptides in pigment epithelial
membranes of the retina. Ocul. Immunol. In¯amm. 5,
19±26.
Campochiaro, P.A., Sugg, R., Grotendorst, G. and
Hjelmeland, L.M. (1989) Retinal pigment epithelial
cells produce PDGF-like proteins and secrete them into
their media. Exp. Eye Res. 49, 217±227.
Cid, L., Pararajasegaram, G., Sevanian, A., Gauderman, W.,
Romero, J.L., Marak, G.E., Jr. and Rao, N.A. (1992)
Anti-in¯ammatory e€ects of vitamin E on experimental
lens-induced uveitis. Int. Ophthalmol. 16, 27±32.
Curnutte, J.T. (1985) Activation of human neutrophil nicoti-
namide-adenine dinucleotide phosphate, reduced (tri-
phosphopyridine nucleotide, reduced) oxidase by
arachidonic acid in a cell-free system. J. Clin. Invest.
75, 1740±1743.
Curnutte, J.T., Erickson, R.W., Ding, J. and Badwey, J.A.
(1994) Reciprocal interactions between protein kinase
C and components of the NADPH oxidase complex
may regulate superoxide production by neutrophils
stimulated with a phorbol ester. J. Biol. Chem. 269,
10813±10819.
de Kozak, Y., Nordman, J.P., Faure, J.P., Rao, N.A. and
Marak, G.E., Jr. (1989) E€ect of antioxidant enzymes
on experimental uveitis in rats. Ophthalmic Res. 21,
230±234.
Dick, A.D., McMenamin, P.G., Korner, H., Scallon, B.J.,
Ghrayeb, J., Forrester, J.V. and Sedgwick, J.D. (1996)
Inhibition of tumor necrosis factor activity minimizes
target organ damage in experimental autoimmune
uveoretinitis despite quantitatively normal activated T
cell trac to the retina. Eur. J. Immunol. 26, 1018±
1025.
Dick, A.D., Duncan, L., Hale, G., Waldmann, H. and Isaacs,
J. (1998) Neutralizing TNF-alpha activity modulates T-
cell phenotype and function in experimental auto-
immune uveoretinitis. J. Autoimmun. 11, 255±264.
Ding, J. and Badwey, J.A. (1993) Stimulation of neutrophils
with a chemoattractant activates several novel protein
kinases that can catalyze the phosphorylation of pep-
tides derived from the 47-kda protein component of the
phagocyte oxidase and myristoylated alanine-rich C
kinase substrate. J. Biol. Chem. 268, 17326±17333.
Dorey, C.K., Khouri, G.G., Syniuta, L.A., Curran, S.A. and
Weiter, J.J. (1989) Superoxide production by porcine
retinal pigment epithelium in vitro. Invest. Ophthalmol.
Vis. Sci. 30, 1047±1054.
Durante, W., Kroll, M.H., Orlo€, G.J., Cunningham, J.M.,
Scott-Burden, T., Vanhoutte, P.M. and Schafer, A.I.
(1996) Regulation of interleukin-1-beta-stimulated
inducible nitric oxide synthase expression in cultured
vascular smooth muscle cells by hemostatic proteins.
Biochem. Pharmacol. 51, 847±853.
Edwards, S. W. (1991) Regulation of neutrophil oxidant pro-
duction. In: Duncan, C. J. (Ed.), Calcium, Oxygen
Radical and Cellular Damage. Cambridge University
Press, 35±76.
el Benna, J., Ruedi, J.M. and Babior, B.M. (1994) Cytosolic
guanine nucleotide-binding protein Rac 2 operates in
vivo as a component of the neutrophil respiratory burst
oxidase. Transfer of Rac2 and the cytosolic oxidase
components p47-phox and p67-phox to the submem-
branous actin cytoskeleton during oxidase activation. J.
Biol. Chem. 269, 6729±6734.
Elner, V.M., Strieter, R.M., Elner, S.G., Baggiolini, M.,
Lindley, I. and Kunkel, S.L. (1990) Neutrophil chemo-
tactic factor (IL-8) gene expression by cytokine-treated
retinal pigment epithelial cells. Am. J. Pathol. 136, 745±
750.
Elner, S.G., Elner, V.M., Pavilack, M.A., Todd, R.F., III,
Mayo-Bond, L., Franklin, W.A., Strieter, R.M.,
Kunkel, S.L. and Huber, A.R. (1992) Modulation and
function of intercellular adhesion molecule-1 (CD54)
on human retinal pigment epithelial cells. Lab. Invest.
66, 200±211.
Forrester, J.V., Liversidge, J. and Dua, H.S. (1990)
Regulation of the local immune response by retinal
cells. Curr. Eye Res. 9(Suppl), 183±191.
Forstermann, U. and Kleinert, H. (1995) Nitric oxide
synthase: expression and expressional control of the
three isoforms. Naunyn-Schmiedebergs Arch Pharmacol.
352, 351±364.
Gabig, T.G., English, D., Akard, L.P. and Schell, M.J. (1987)
Regulation of neutrphil NADPH oxidase activation in
a cell-free system by guanine nucleotides and ¯uoride.
Evidence for participation of a pertussis and cholera
 Free radical mediated photoreceptor damage in uveitis
toxin-insentitive G protein. J. Biol. Chem. 262, 1685±
1690.
Gery, I., Mochizuki, M. and Nussenblatt, R. B. (1986)
Retinal speci®c antigens and immunopathogenic pro-
cess they provoke. In Osborne N., Chader J. (Eds):
Progress in Retinal Research. Oxford, Pergamon Press,
5, 75-109.
Goto, H., Wu, G.S., Gritz, D.C., Atalla, L.R. and Rao, N.A.
(1991) Chemotactic activity of the peroxidized retinal
membrane lipids in experimental autoimmune uveitis.
Curr. Eye Res. 10, 1009±1014.
Goto, H., Wu, G.S., Chen, F., Kristeva, M., Sevanian, A.
and Rao, N.A. (1992) Lipid peroxidation in experimen-
tal uveitis: sequential studies. Curr. Eye Res. 11, 489±
499.
Goureau, O., Amiot, F., Dautry, F. and Courtois, Y. (1997)
Control of nitric oxide production by endogenous
TNF-alpha in mouse retinal pigmented epithelial and
Muller glial cells. Biochem. Biophys. Res. Commun. 240,
132±135.
Green, W. R. (1985) Uveal tract. In: Spencer, W. H. (Ed.),
Ophthalmic Pathology: an Atlas and Textbook.
Philadelphia, WB Saunders, 1923±1932.
Griscavage, J.M., Wilk, S. and Ignarro, L.J. (1995) Serine
and cysteine proteinase inhibitors prevent nitric oxide
production by activated macrophages by interfering
with transcription of the inducible NO synthase gene.
Biochem. Biophys. Res. Commun. 215, 721±729.
Gritz, D.C., Montes, C., Atalla, L.R., Wu, G.S., Sevanian, A.
and Rao, N.A. (1991) Histochemical localization of
superoxide production in experimental autoimmune
uveitis. Curr. Eye Res. 10, 927±931.
Guy, J., Ellis, E.A., Hope, G.M. and Rao, N.A. (1986)
In¯uence of antioxidant enzymes in reduction of optic
disc edema in experimental optic neuritis. J. Free
Radic. Biol. Med. 2, 349±357.
Guy, J., Ellis, E.A., Hope, G.M. and Rao, N.A. (1989a)
Antioxidant enzymes reduce loss of blood-brain barrier
integrity in experimental optic neuritis. Arch
Ophthalmol. 107, 1359±1363.
Guy, J., Ellis, E.A., Hope, G.M. and Rao, N.A. (1989b)
Antioxidant enzyme suppression of demyelination in
experimental optic neuritis. Curr. Eye Res. 8, 467±477.
Guy, J., Ellis, E.A., Mames, R. and Rao, N.A. (1993) Role of
hydrogen peroxide in experimental optic neuritis. A
serial quantitative ultrastructural study. Ophthalmic
Res. 25, 253±264.
Guy, J., McGorray, S., Fitzsimmons, J., Beck, B., Mancuso,
A., Rao, N.A. and Hamed, L. (1994a) Reversals of
blood-brain barrier disruption by catalase: a serial
magnetic resonance imaging study of experimental
optic neuritis. Invest. Ophthalmol. Vis. Sci. 35, 3456±
3465.
Guy, J., McGorray, S., Qi, X., Fitzsimmons, J., Mancuso, A.
and Rao, N.A. (1994b) Conjugated deferoxamine
reduces blood-brain barrier disruption in experimental
optic neuritis. Ophthalmic Res. 26, 310±323.
Haddad, I.Y., Pataki, G., Hu, P., Galliani, C., Beckman, J.S.
and Matalon, S. (1994) Quantitation of nitrotyrosine
levels in lung sections of patients and animals with
acute lung injury. J. Clin. Invest. 94, 2407±2413.
Halliwell, B. (1981) The biological e€ect of the superoxide
radical and its products. Bull. Europ. Physiopathol.
Respir. 17 (Suppl.), 21±29.
65
Hoey, S., Grabowski, P.S., Ralston, S.H., Forrester, J.V. and
Liversidge, J. (1997) Nitric oxide accelerates the onset
and increases the severity of experimental autoimmune
uveoretinitis through an IFN-g dependent mechanism.
J. Immunol. 159, 5132±5142.
Howes, E. L. Jr. and Rao, N. A. (1996) Basic mechanisms in
pathology. In: Spencer, W.H. (Ed.), Ophthalmic
Pathology, 2935±3044. W. B. Saunders Company, New
York.
Ishimoto, S.I., Wu, G.S., Hayashi, S., Zhang, J. and Rao,
N.A. (1996) Free radical tissue damages in the anterior
segment of the eye in experimental autoimmune uveitis.
Invest. Ophthalmol. Vis. Sci. 37, 630±636.
Jacquemin, E., de Kozak, Y., Thillaye, B., Courtois, Y. and
Goureau, O. (1996) Expression of inducible nitric oxide
synthase in the eye from endotoxin-induced uveitis rats.
Invest. Ophthalmol. Vis. Sci. 37, 1187±1196.
Ja€e, G.J., Roberts, W.L., Wong, H.L., Yurochko, A.D. and
Cianciolo, G.J. (1995) Monocyte-induced cytokine ex-
pression in cultured human retinal pigment epithelial
cells. Exp. Eye Res. 60, 533±543.
Jeon, Y.J., Yang, K.H., Pulaski, J.T. and Kaminski, N.E.
(1996) Attenuation of inducible nitric oxide synthase
gene expression by delta 9-tetrahydrocannabinol is
mediated through the inhibition of nuclear factor-
kappa B/Rel activation. Mol. Pharmacol. 50, 334±
341.
Jun, C.D., Choi, B.M., Kim, S.U., Lee, S.Y., Kim, H.M. and
Chung, H.T. (1995) Down-regulation of transforming
growth factor-beta gene expression by antisense oligo-
deoxynucleotides increases recombinant interferon-
gamma-induced nitric oxide synthesis in murine perito-
neal macrophages. Immunology 85, 114±119.
Khorana, H.G. (1992) Rhodopsin, photoreceptor of the rod
cell. An emerging pattern for structure and function. J.
Biol. Chem. 267, 1±4.
KroÈncke, K.D., Fehsel, K. and Kolb-Bachofen, V. (1997)
Nitric oxide: cytotoxicity versus cytoprotectionÐhow,
why, when and where?. Nitric Oxide 1, 107±120.
Kutty, R.K., Kutty, G., Hooks, J.J., Wiggert, B. and
Nagineni, C.N. (1995) Transforming growth factor-
beta inhibits the cytokine-mediated expression of the
inducible nitric oxide synthase mRNA in human retinal
pigment epithelial cells. Biochem. Biophys. Res.
Commun. 215, 386±393.
Kwon, G., Corbett, J.A., Rodi, C.P., Sullivan, P. and
McDaniel, M.L. (1995) Interleukin-1 beta-induced
nitric oxide synthase expression by rat pancreatic beta-
cells: evidence for the involvement of nuclear factor
kappa B in the signaling mechanism. Endocrinology
136, 4790±4795.
Legrand-Poels, S., Zecchinon, L., Piret, B., Schoonbroodt, S.
and Piette, J. (1997) Involvement of di€erent transduc-
tion pathways in NF-kappa B activation by several
inducers. Free Radic. Res. 27, 301±309.
Limatola, C., Schaap, D., Moolenaar, W.H. and van
Blitterswijk, W.J. (1994) Photphatidic acid activation of
protein kinase C-zeta overexpressed in COS cells.
Comparison with other protein kinase C isotypes and
other acidic lipids. Biochem. J. 304, 1001±1008.
MacMillan-Crow, L.A., Crow, J.P., Kerby, J.D., Beckman,
J.S. and Thompson, J.A. (1996) Nitration and inacti-
vation of manganese superoxide dismutase in chronic
rejection of human renal allografts. Proc. Natl. Acad.
Sci. USA 93, 11853±11858.
66 N. A. Rao and G.-S. Wu
Marak, G.E., Jr. and Rao, N.A. (1982) Retinal S-antigen dis-
ease in rats. Ophthalmic Res. 14, 29±39.
Marak, G.E., Jr., Rao, N.A., Scott, J.M., Duque, R. and
Ward, P.A. (1985) Antioxidant modulation of phacoa-
naphylactic endopthalmitis. Ophthalmic Res. 17, 297±
301.
Marak, G.E., Jr., Rao, N.A., Sevanian, A., Zdravkovich, V.,
Till, G.O. and Ward, P.A. (1987) Modulation of exper-
imental phacoanaphylactic endophthalmitis with the
antioxidants sodium benzoate, and 2,3-dihydroxyben-
zoic acid. Ophthalmic Res. 19, 120±128.
Matsubara, T., Wu, G.S., Numaga, J. and Rao, N.A. (1997)
RPE protective protein production in response to in-
¯ammatory agents. Invest. Ophthalmol. Vis. Sci. 38,
S186.
McKee, C.M., Lowenstein, C.J., Horton, M.R., Wu, J., Bao,
C., Chin, B.Y., Choi, A.M. and Noble, P.W. (1997)
Hyaluronan fragments induce nitric-oxide synthase in
murine macrophages through a nuclear factor kappa
B-dependent mechanism. J. Biol. Chem. 272, 8013±
8018.
McPhail, L.C., Qualliotine-Mann, D. and Waite, K.A. (1995)
Cell-free activation of neutrophil NADPH oxidase by a
phosphatidic acid-regulated protein kinase. Proc. Natl.
Acad. Sci. USA 92, 7931±7935.
Milligan, S.A., Owens, M.W. and Grisham, M.B. (1996)
Augmentation of cytokine-induced nitric oxide syn-
thesis by hydrogen peroxide. Am. J. Physiol. 271((1 Pt
1)), L114±120.
Milligan, S.A., Owens, M.W. and Grisham, M.B. (1998)
Di€erential regulation of extracellular signal-regulated
kinase and nuclear factor-kappa B signal transduction
pathways by hydrogen peroxide and tumor necrosis
factor. Arch. Biochem. Biophys. 352, 255±262.
Moorthy, R.S., Inomata, H. and Rao, N.A. (1995) Vogt±
Koyanagi±Harada syndrome. Sur. Ophthalmol. 39,
265±292.
Nakamura, S. and Nishizuka, Y. (1994) Lipid mediators and
protein kinase C activation for the intracellular signal-
ing network. J. Biochem. 115, 1029±1034.
Newsome, D.A., Miceli, M.V., Liles, M.R., Tate, D.J. and
Oliver, P.D. (1994) Antioxidants in the retinal pigment
epithelium. Prog. Retina Eye Res. 13, 101±123.
Numaga, J., Riono, W.P., Matsubara, T., Wu, G.S. and Rao,
N.A. (1997) E€ects of anti-RPP antibodies on ex-
pression of experimental uveitis. Invest. Ophthalol. Vis.
Sci. 38, S438.
Nussenblatt, R.B., Mital, K.K. and Fuhrmanm, S. (1989)
Lymphocyte proliferative responses of patients with
ocular toxoplasmosis to parasite and retinal antigens.
Am. J. Ophthalmol. 107, 632±641.
Nussenblatt, R. B. and Palestine, A. G. (1989) Uveitis: funda-
mentals and clinical practice. Year Book Medical
Publishers, Inc., Chicago, 21±52.
Nussenblatt, R.B. (1991) Proctor lecture. Experimental auto-
immune uveitis: mechanisms of disease and clinical
therapeutic indications. Invest. Ophthalmol. Vis. Sci. 32,
3131±3141.
Okada, A.A., Sakai, J., Usui, M. and Mizuguchi, J. (1998)
Intraocular cytokine quanti®cation of experimental
autoimmune uveoretinitis in rats. Ocul. Immunol.
In¯amm. 6, 111±120.
Pararajasegaram, G., Sevanian, A. and Rao, N.A. (1991)
Suppression of S antigen-induced uveitis by vitamin E
supplementation. Ophthalmic Res. 23, 121±127.
Percopo, C.M., Hooks, J.J., Shinohara, T., Caspi, R. and
Detrick, B. (1990) Cytokine-mediated activation of a
neuronal retinal resident cell provokes antigen presen-
tation. J. Immunol. 145, 4101±4107.
Planck, S.R., Dang, T.T., Ansel, J.C., Robertson, J.E. and
Rosenbaum, J.T. (1991) Expression of granulocyte-
macrophage colony stimulating factor (GM-CSF) and
interleukin-1 by retinal pigment epithelial (RPE) cells.
Invest. Ophthalmol. Vis. Sci. 32(Suppl), 1056.
Planck, S.R., Dang, T.T., Graves, D., Tara, D., Ansel, J.C.
and Rosenbaum, J.T. (1992) Retinal pigment epithelial
cells secrete interleukin-6 in response to interleukin-1.
Invest. Ophthalmol. Vis. Sci. 33, 78±82.
Radi, R., Beckman, J.S., Bush, K.M. and Freeman, B.A.
(1991) Peroxynitrite-induced membrane lipid peroxi-
dation: the cytotoxic potential of superoxide and nitric
oxide. Arch. Biochem. Biophys. 288, 481±487.
Ramanathan, S., de Kozak, Y., Saoudi, A., Goureau, O.,
Van der Meide, P.H., Druet, P. and Bellon, B. (1996)
Recombinant lL-4 aggravates experimental auto-
immune uveoretinitis in rats. J. Immunol. 157, 2209±
2215.
Rao, N.A., Wacker, W.B. and Marak, G.E., Jr. (1979)
Experimental allergic uveitis: clincopathologic features
associated with varying doses of S antigen. Arch.
Ophthalmol. 97, 1954±1958.
Rao, N.A., Robin, J., Hartmann, D., Sweeney, J.A. and
Marak, G.E., Jr. (1983) The role of the penetrating
wound in sympathetic ophthalmia. Arch. Ophthalmol.
101, 102±103.
Rao, N.A. (1990) Role of oxygen free radicals in retinal
damage associated with experimental uveitis. Trans.
Am. Ophthalmol. Soc. 88, 797±850.
Rao, N.A. and Wong, V. (1981) Etiology of sympathetic
ophthalmitis. Trans. Ophthalmol. Soc. UK 101, 357±
360.
Rao, N.A., Thaete, M.D., Delmage, J.M. and Sevanian, A.
(1985) Superoxide dismutase in ocular structures.
Invest. Ophthalmol. Vis. Sci. 26, 1778±1781.
Rao, N.A., Calandra, A.J., Sevanian, A., Bowe, B., Delmage,
J.M. and Marak, G.E., Jr. (1986a) Modulation of lens-
induced uveitis by superoxide dismutase. Ophthalmic
Res. 18, 41±46.
Rao, N.A., Fernandez, M.A.S., Sevanian, A., Till, G.O. and
Marak, G.E. (1986b) Antiphlogistic e€ect of catalase
on experimental phacoanaphylactic endophthalmitis.
Ophthalmic Res. 18, 185±191.
Rao, N.A., Romero, J.L., Fernandez, M.A., Sevanian, A. and
Marak, G.E. (1986c) E€ect of iron chelation on sever-
ity of ocular in¯ammation in an animal model. Arch.
Ophthalmol. 104, 1369±1371.
Rao, N.A., Bowe, B.E., Sevanian, A., Till, G.O. and
Marak, G.E., Jr. (1986d) Modulation of lens-induced
uveitis by dimethyl sulfoxide. Ophthalmic Res. 18,
193±198.
Rao, N.A., Patchett, R., Fernandez, M.A., Sevanian, A.,
Kunkel, S.L. and Marak, G.E., Jr. (1987a) Treatment
of experimental granulomatous uveitis by lipoxygenase
and cyclo-oxygenase inhibitors. Arch Ophthalmol. 105,
413±415.
Rao, N.A., Romero, J.L., Fernandez, M.A.S., Sevanian, A.
and Marak, G.E., Jr. (1987b) Role of free radicals in
uveitis. Sur. Ophthalmol. 32, 209±213.
Rao, N.A., Fernandez, M.A., Sevanian, A., Romero, J.L.,
Till, G.O. and Marak, G.E., Jr. (1988a) Treatment of
 Free radical mediated photoreceptor damage in uveitis
experimental lens-induced uveitis by dimethyl thiourea.
Ophthalmic Res. 20, 106±111.
Rao, N.A., Wu, G.S. and Pararajasegaram, G. (1994)
Mechanism of tissue injury in uveitis. Reg. Immunol. 6,
95±100.
Rao, N.A., Sultona, C., Gullapalli, V.K., Nenago, J. and
Kalra, V.J. (1997) RPE protective protein (RPP) inhi-
bits p47-phox phosphorylation in in¯ammatory cells.
Invest. Ophthalmol. Vis. Sci. 38, S186.
Robinson, J.M. and Badwey, J.A. (1995) The NADPH oxi-
dase complex of phagocytic leukocytes; a biochemical
and cytochemical view. Histochem. Cell Biol. 103, 163±
180.
Rodenas, J., Mitjavila, M.T. and Carbonell, T. (1995)
Simultaneous generation of nitric oxide and superoxide
by in¯ammatory cells in rat. Free Radic. Biol. Med. 18,
869±875.
Rose, R.C., Richer, S.P. and Bode, A.M. (1998) Ocular oxi-
dants and antioxidant protection (Review). Proc. Soc.
Exp. Biol. Med. 217, 397±407.
Rubbo, H., Radi, R., Trujillo, M., Telleri, R., Kalyanaraman,
B., Barnes, S., Kirk, M. and Freeman, B.A. (1994)
Nitric oxide regulation of superoxide and peroxynitrite-
dependent lipid peroxidation. Formation of novel
nitrogen-containing oxidized lipid derivatives. J. Biol.
Chem. 269, 26066±26075.
Rubbo, H. and Freeman, B.A. (1996) Nitric oxide regulation
of lipid oxidation reactions: formation and analysis of
nitrogen-containing oxidized lipid derivatives. Methods
Enzymol. 269, 385±394.
Sappey, C., Boelaert, J.R., Legrand-Poels, S., Grady, R.W.
and Piette, J. (1995) NF-kappa B transcription fac-
tor activation by hydrogen peroxide can be
decreased by 2,3-dihydroxybenzoic acid and its ethyl
ester derivative. Arch. Biochem. Biophys. 321, 263±
270.
Schmidt, K.N., Amstad, P., Ceruti, P. and Baeuerle, P.A.
(1995) The roles of hydrogen peroxide and superoxide
as messengers in the activation of transcription factor
NF-kappa B. Chem. Biol. 2, 13±22.
Schmidt, K.N., Amstad, P., Cerutti, P. and Baeuerle, P.A.
(1996) Identi®cation of hydrogen peroxide as the rel-
evant messenger in the activation pathway of transcrip-
tion factor NF-kappa B. Adv. Exp. Med. Biol. 387, 63±
68.
Shimizu, K., Zhang, J., Riono, W.P., Wu, G.S. and Rao,
N.A. (1998) Retinal S-antigen and IRBP induce nitric
oxide production in macrophages. Invest. Ophthalmol.
Vis. Sci. 39, S778.
Skinner, K.A., Crow, J.P., Skinner, H.B., Chandler, R.T.,
Thompson, J.A. and Parks, D.A. (1997) Free and pro-
tein-associated nitrotyrosine formation following rat
liver preservation and transplantation. Arch. Biochem.
Biophys. 342, 282±288.
Spink, J., Cohen, J. and Evans, T.J. (1995) The cytokine
responsive vascular smooth muscle cell enhancer of
inducible nitric oxide synthase. Activation by
nuclear factor kappa B. J. Biol. Chem. 270, 29541±
29547.
SzaboÂ, C., Zingarelli, B., O'Connor, M. and Salzman, A.L.
(1996) DNA strand breakage, activation of poly (ADP-
ribose) synthetase, and cellular energy depletion are
involved in the cytotoxicity of macrophages and
smooth muscle cells exposed to peroxynitrite. Proc.
Natl. Acad. Sci. USA 93, 1753±1758.
67
Valentine, J.S., Miksztal, A.R. and Sawyer, D.T. (1984)
Methods for the study of superoxide chemistry in
nonaqueous solutions. Methods Enzymol. 105, 71±
81.
Volpp, B.D., Nauseef, W.M., Donelson, J.E., Moser,
D.R. and Clark, R.A. (1989) Cloning of the
cDNA and functional expression of the 47-kilodal-
ton cytosolic component of human neutrophil res-
piratory burst oxidase. Proc. Natl. Acad. Sci. USA
86, 7195±7199.
Weiss, S.J. and LoBuglio, A.F. (1982) Phagocyte-generated
oxygen metabolites and cellular injury. Lab. Invest. 47,
5±18.
Wiegand, R. D., Jose, J. G., Rapp, L. M. and Anderson, R.
E. (1984) Free radicals and damage to ocular tissues.
In: Armstrong, D. (Ed.), Free radicals in molecular bi-
ology, aging and disease. Raven Press, New York,
317±353.
Wu, G.S., Goto, H., Sevanian, A. and Rao, N.A. (1991)
Generation of chemiluminescence in experimental auto-
immune uveitis. Curr. Eye Res. 10, 909±917.
Wu, G.S., Sevanian, A. and Rao, N.A. (1992) Detection of
retinal lipid hydroperoxides in experimental uveitis.
Free Radic. Biol. Med. 12, 19±27.
Wu, G.S. and Rao, N.A. (1996) A novel retinal pigment epi-
thelial protein suppresses neutrophil superoxide gener-
ation. I. Characterization of the suppressive factor.
Exp. Eye Res. 63, 713±725.
Wu, G.S., Swiderek, K.M. and Rao, N.A. (1996) A novel
retinal pigment epithelial protein suppresses neutrophil
superoxide generation: II. Puri®cation and microse-
quencing analysis. Exp. Eye Res. 63, 727±737.
Wu, G.S., Zhang, J. and Rao, N.A. (1997) Peroxynitrite and
oxidative damage in experimental autoimmune uveitis.
Invest. Ophthalmol. Vis. Sci. 38, 1333±1339.
Wu, G. S. and Rao, N. A. (1999) Activation of NADPH oxi-
dase by docosahexaenoic acid hydroperoxide and its in-
hibition by a novel retinal pigment epithelial protein.
Invest. Ophthalmol. Vis. Sci. 40 (in press).
Xu, H., Rizzo, L.V., Silver, P.B. and Caspi, R.R. (1997)
Uveitogenicity is associated with a Th1-like lymphokine
pro®le: cytokine-dependent modulation of early and
committed e€ector T cells in experimental autoimmune
uveitis. Cell Immunol. 178, 69±78.
Yermilov, V., Rubio, J. and Ohshima, H. (1995a) Formation
of 8-nitroguanine in DNA treated with peroxynitrite in
vitro and its rapid removal from DNA by depurination.
FEBS Lett. 376, 207±210.
Yermilov, V., Rubio, J., Becchi, M., Friesen, M.D., Pignatelli,
B. and Ohshima, H. (1995b) Formation of 8-nitrogua-
nine by the reaction of guanine with peroxynitrite in
vitro. Carcinogenesis 16, 2045±2050.
Yokoi, H., Kato, K., Kezuka, T., Sakai, J., Usui, M., Yagita,
H. and Okumura, K. (1997) Prevention of experimental
autoimmune uveoretinitis by monoclonal antibody to
interleukin-12. Eur. J. Immunol. 27, 641±646.
Zhang, J., Dawson, V.L., Dawson, T.M. and Snyder,
S.H. (1994) Nitric oxide activation of poly (ADP-
ribose) synthetase in neurotoxicity. Science 263,
687±689.
Zhang, J., Wu, G.S. and Rao, N.A. (1993) Role of nitric
oxide (NO) in experimental autoimmune uveitis
(EAU). ARVO abstract. Invest. Ophthalmol. Vis. Sci.
34, 1000.
68 N. A. Rao and G.-S. Wu

Zingarelli, B., O'Connor, M., Wong, H., Salzman, A.L. and
Szabo, C. (1996) Peroxynitrite-mediated DNA strand
breakage activates poly-adenosine diphosphate ribosyl
synthetase and causes cellular energy depletion in
macrophages simulated with bacterial lipopolysac-
chride. J. Immunol. 156, 350±358.
Zweier, J.L., Kuppusamy, P., Williams, R., Rayburn,
B.K., Smith, D., Weisfeldt, M.L. and Flaherty,
J.T. (1989) Measurement and characterization of
postischemic free radical generation in the iso-
lated perfused heart. J. Biol. Chem. 264, 18890±
18895.