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Regular Articles |
From the Department of Ophthalmology,*
University ofAberdeen Medical School, Foresterhill, Aberdeen; and the Division ofOphthalmology,
University of Bristol, BristolEye Hospital, Bristol, United Kingdom
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Experimental models of autoimmune uveoretinitis (EAU) induced by immunization with soluble retinal antigens (REs) closely resemble human disease,8 and are mediated by CD4+ T cells and activated macrophages.9-11 Monocytes/macrophages within the outer retina that are associated with tissue damage have been identified as a major source of nitric oxide synthase (NOS2) during inflammation.12,13 This tissue is the location of the eliciting retinal autoantigens, and the site of infiltrating T cells and major histocompatibility complex class II+ antigen-presenting cells. However, there are conflicting reports of the effects NOS inhibitors and NOS2 gene inactivation have on the incidence and severity of disease in models of autoimmunity such as experimental autoimmune uvcoretintis (EAU) and experimental autoimmune encephalomyelitis (EAE) that target sites of immune privilege.12,14-19
Nitric oxide (NO), when produced in large quantities, usually by
macrophages and neutrophils, provides an important component of the
innate immune system, particularly in the elimination of microorganisms
and parasites.20
This can be achieved through reaction
with superoxide anion to yield peroxynitrite, which can produce the
toxic hydroxyl radical or promote oxidative injury via formation of
peroxynitrous acid.21
More subtle effects of NO include
direct nitrosylation of target proteins, inactivation of membrane ion
channels, and disruption of signaling proteins.22,23
Low
levels of NO have also been implicated in lymphocyte activation and
proliferation, increasing TNF-
production, nuclear factor-
B
binding activity, and enhancing tyrosine kinase p56
activity.24
Conversely, higher levels can suppress
antigen-presenting cell activity and T-cell proliferation by inhibiting
the actions of thiol- and heme-containing enzymes and thus
mitochondrial respiration.25
This has led to the
hypothesis that NOS2 may have different functions in different cells
and that tissue-specific expression may be beneficial and contribute to
the resolution of inflammation through mechanisms such as
activation-induced cell death (AICD).26
In particular,
various models of organ-specific autoimmune disease have indicated that
although NOS2-dependent tissue destruction occurs, protective functions
linked to the presence of interferon (IFN)-
have been demonstrated,
possibly involving suppression of T-cell proliferation and Th1 cytokine
production.27
What then is the role of NOS2 in
inflammation affecting sites of immune privilege such as the eye, and
can modulation of expression be used to restore immunological
homeostasis and reduce tissue damage?
In this histological study, we show that although tissue damage in the inner retina occurs via generation of peroxynitrite anion by monocytes/macrophages, leading to apoptosis of photoreceptors but not the macrophages, NOS also seems to be required for apoptosis of Fas+ T cells infiltrating the tissue. Apoptosis of activated T cells is a recognized mechanism for restoring immunological homeostasis and as elevated Fas and Fas-ligand expression in the retina is a feature of EAU, may play a role in down-regulating EAU.28,29 Thus prevention of membrane nitrosylation, coupled with strategies to trigger apoptosis in infiltrating macrophages while maintaining T-cell AICD may provide a therapeutic advantage in sites of immune privilege vulnerable to damaging inflammatory reactions.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Groups of six, 8- to 10-week-old male, Lewis rats from an inbred strain, housed and treated in accordance with United Kingdom Home Office Regulations, were used in this study. Animals were weighed and selected to give groups an approximately equal average weight. Extract of soluble RE (6.4 mg/ml) was prepared as previously described30 by hypertonic lysis of freshly dissected bovine retinas in the dark, and contained S-antigen and interphoto-receptor retinoid binding protein (IRBP) as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. EAU was induced by intradermal immunization in one hind footpad with 0.1 ml of RE emulsified with an equal volume of complete Freund’s adjuvant (CFA), containing 0.5 mg/ml of Mycobacterium tuberculosis H37RA (Difco, West Molesey, UK). This regime reliably induces a moderate to severe uveoretinitis 10 to 11 days after immunization without the use of Bordetella pertussis toxin.12 Control animals received 0.1 ml of phosphate-buffered saline (PBS) in CFA. Immediately after immunization, test animals were treated with 50 mg/kg of NG-nitro-L-arginine methyl ester (L-NAME) per day (Sigma, Poole, Dorset UK) dissolved in the drinking water as previously described.12 Control animals received normal drinking water. The weight and fluid intake of the rats was monitored before the start of experiments to determine initial dose of L-NAME. Weight and fluid intake was then monitored throughout all experiments and dosages adjusted accordingly.
Clinical and Histopathological Grading of Disease
Ocular examination was performed daily from 7 days after immunization using slit lamp biomicroscopy. The day of onset and course of disease were noted and were graded 0 to 4 according to the degree of cellular inflammatory activity within the anterior chamber of the eye.30 Groups of animals were killed by CO2 asphyxiation on days 9, 10, 11 (early disease stage), or 12. Eyes were removed and either snap-frozen for cryosectioning or fixed in neutral-buffered formalin for paraffin embedding. Tissue sections taken from eyes of untreated immunized control animals and those treated with L-NAME were sampled at each time point, stained with hematoxylin and eosin, and examined. At least three sections from each eye were scored in a masked manner using a semiquantitative scoring system that has been described30 and combines the extent of the inflammatory infiltrate and structural changes or tissue damage in the anterior and posterior chambers of the eye. Results are expressed as mean ± 1 SD. Comparison of the clinical and histological assessment of disease in treated and untreated animals were analyzed using one-way analysis of variance, and P values equal to or less than 0.05 were considered significant. As inflammatory cell infiltration does not always result in tissue damage, the data from each component was also scored independently. Infiltrative scores were measured on a scale of 0 to 7 and structural changes or tissue damage on a scale of 0 to 5.
Ex Vivo NO Production by Splenic Macrophages
Spleens were removed from naïve or immunized control and
immunized L-NAME-treated rats at day 10 after immunization and
macrophages isolated from single cell suspensions by density gradient
centrifugation and adherence to plastic. After 4 hours of incubation in
flasks at 37°C in RPMI 5%/fetal calf serum (Myoclone Super Plus,
endotoxin-free; Life Technologies, Paisley UK) nonadherent cells were
removed by washing with warm medium and further purified on nylon wool
columns to yield T cells. Macrophages were then harvested from the
flasks with cold medium and a cell scraper. Macrophages were seeded at
5 x 106
per ml in 24-well plates in culture
medium alone or with L-NMMA (0.5 µmol/L, Sigma). Cytokines (500 ng/ml
tumor necrosis factor-, 100 U/ml IFN-; R&D Systems, Abingdon,
UK), lipopolysaccharide (1 µg/ml, Sigma), were added and NO
production measured after 72 hours by the Greiss reaction as
described.12
All results are expressed as mean ± 1
SD. Results from each treatment group were compared using Student’s
t-test. P values equal to or less than 0.05 were
considered significant.
Immunohistochemistry and Detection of Apoptosis in Tissue Sections
Serial sections were cut from formalin-fixed and paraffin-embedded eyes for indirect immunoperoxidase, dual immunofluorescence, and terminal dUTP nick-end labeling (TUNEL) methods using the following antibodies: NOS2 (clone 6, 1:100; Transduction Laboratories, Affiniti Research Products, Exeter, UK), Bcl2 (rabbit polyclonal, 1:40; Calbiochem, CN Biosciences UK, Nottingham, UK), BAX (rabbit polyclonal, 1:20; Calbiochem), Fas (rabbit polyclonal,1:20; Calbiochem), Fas ligand (N20, rabbit polyclonal, 1:20; Santa Cruz, Autogenbioclear UK Ltd., Calne, UK), and inducible Hsp70 (rabbit polyclonal, 1:150; ImmunoKontact, AMS Biotechnology Europe Ltd. Abingdon, UK). Leukocyte markers were mouse monoclonal for monocytes/macrophages (ED1, 1:50), tissue macrophages (ED2, 1:50) CD2 (OX34, 1:20), and CD3 (IF4, 1:100) from Serotec, Oxford, UK. Paraffin-embedded sections were treated with proteinase K and for indirect immunoperoxidase, horseradish peroxidase-labeled kits appropriate for mouse monoclonal or rabbit polyclonal antibodies were used according to the manufacturer’s instructions (Vector Laboratories, Peterborough, UK). Nitrotyrosine was detected using mouse monoclonal anti-nitrotyrosine (1:20; Upstate Biotechnology, Lake Placid, NY) with overnight incubation. Secondary antibodies were biotinylated rabbit anti-mouse Ig (DAKO Ltd., High Wycome, UK) or biotinylated swine anti-rabbit Ig (DAKO) followed by streptavidin fluorescein isothiocyanate (FITC) or Texas Red (Amersham, Little Chalfont, UK).
Dual immunofluorescence was performed as previously described.12 Clone OX 34 (IgG2a anti-CD2) was used in dual-immunofluorescence experiments in preference to clone IF4 (IgM anti-CD3) that gave unacceptably high background fluorescence. Serial 10-µm cryostat sections were cut onto poly-L-lysine-coated slides and air-dried overnight for use with Cox-2/PGHS antibody (mouse monoclonal clone 33, 1:25; Transduction Labs) and transforming growth factor (TGF)-ß1 antibody (rabbit polyclonal 1:100; Santa Cruz), in a standard avidin-biotin (alkaline phosphatase complex, APAAP) technique.
Apoptotic cells in the sections were detected using the fluorescence-labeled (FITC) terminal deoxynucleotidyl transferase (TdT) method exactly according to the instructions of the kit manufacturer (Apoptag; Oncor Appligene, Chester-le-Street, UK). For dual labeling, the propidium iodide counterstain was omitted and the sections rinsed in PBS before further incubation with ED1, OX34 (CD2), nitrotyrosine, IHsp70, Fas, Fas ligand, Bax, or Bcl2 antibody. After washing, the appropriate secondary biotinylated rabbit anti-mouse Ig or swine anti-rabbit Ig (both 1:150; DAKO Ltd., Ely, UK) was applied for 30 minutes followed by streptavidin/Texas Red (1:50) conjugate for a further 30 minutes. For triple labeling, OX34 antibody was directly conjugated using a Cy5 mAb-labeling kit (Amersham, Little Chalfont, Bucks, UK) and incubated for 30 minutes on dual-labeled specimens that had been blocked with normal mouse serum. After extensive washing sections were mounted in Citifluor (Agar Scientific, Stanstead, UK) and dual-photographic exposures taken with appropriate filters using an Olympus BH2-RFC microscope, or examined using an MRC 1024 confocal microscope to demonstrate co-localization of signal.
Negative controls included omission of primary or secondary antibodies, addition of preimmune or normal serum Ig in place of polyclonal antibodies, or substitution of irrelevant primary antibodies of the same species isotype for monoclonal antibodies. For NOS2, nitrotyrosine, iHsp70, and TUNEL techniques, control sections from various inflammatory tissues known to be positive were included as positive controls.
Quantification of Positively Stained Cells
Sections of retina from animals taken at days 10 and 11 were examined and lesions selected that displayed equivalent inflammatory infiltrates affecting the full thickness of the retina, including a subretinal exudate (SRE). Apoptosis and nitrotyrosine staining was evaluated from fluorescently labeled sections. Other markers including CD3 and CD2 (T cells), Fas and Fas ligand, and protein products NOS2, iHSP70, Bcl2, and Bax were evaluated using horseradish peroxidase-stained sections using a light microscope. In a separate experiment, the percentage of ED1 (monocytes/macrophages and ED2 (tissue macrophages) within the tissues were calculated by computer-assisted densitometric scanning of individually stained serial sections at time points from day 10 to day 18. Areas were analyzed using one of three methods, and separate data acquired from the inner retina [ganglion cell layer, inner nuclear layer (INL)], outer retina [outer nuclear layer (ONL), photoreceptors including the rod outer segments], or SRE. 1) In fluorescently labeled sections, the total numbers of apoptotic cells present in a minimum of six fields (x20 objective) in retina, SRE, and choroid were counted. 2) For evaluation of membrane markers on inflammatory cells the percentage of positively stained mononuclear cells were counted in a minimum of six fields (x40 objective) per lesion. 3) For evaluation of protein or enzyme expression we used the Aphelion Active X image analysis program from ADCIS (ADCIS SA, Herouville-Saint-Clair, France). The program was adapted using Visual basic to allow analysis of immunostaining within user-defined regions of the image. An average value (percentage of tissue positively stained per x20 field) for each section was obtained from four to six fields. For each technique a minimum of six sections from separate animals were examined in each treatment group. All results are expressed as mean ± 1 SD and were analyzed by the Mann-Whitney U test or using one-way analysis of variance for groups of three or more values. P values equal to or less than 0.05 were considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In our model, L-NAME administered orally in drinking water (50 mg/kg) is well tolerated and reduces EAU disease severity throughout a period of 21 days.12 In the experiments described here, fluid intake was found to be equal between the groups, and although the average weight of L-NAME-treated animals (n = 6) was slightly reduced compared with controls at day 9 (typically 231.7 ± 26.4 g versus 253.2 ± 21.2 g) no other differences in general health were observed.
Nonspecific inhibitors of NOS such as L-NAME given in drinking water
may have effects other than NOS inhibition,26
and in some
models can actually induce compensatory expression of
NOS2.31
This effect is attributed to the dose of inhibitor
used and has led to the idea that the toxicity of NOS2 is restricted to
certain areas or cells within inflammatory lesions in a number of
diseases, and that NOS2 may also have beneficial effects in
inflammation. To assess the systemic effects of L-NAME treatment on
macrophage function we examined NO generation by macrophages isolated
from treated rats. Figure 1a
shows that
splenic macrophages from naïve rats produced basal levels of
17 ± 3.1 µmol/L nitrite ex-vivo, increasing to
33 ± 3.2 µmol/L nitrite in the presence of an NOS2-stimulating
cytokine cocktail containing lipopolysaccharide, tumor necrosis
factor-, and IFN-.32
This was reduced to 11.9
± 1.3 µmol/L nitrite (P < 0.05) with the
addition of 0.5 µmol/L L-NMMA. In contrast, macrophages isolated from
animals 10 days after immunization with RE spontaneously produced much
higher basal levels nitrite levels (194.3 ± 70.2 µmol/L) even
when treated with L-NAME in vivo (130.4 ± 29.4
µmol/L). These cells were not responsive to further stimulation with
the cytokine/lipopolysaccharide cocktail. Nitrite levels were reduced
to 78.5 ± 17.5 µmol/L in macrophages from L-NAME-treated
animals compared with 153.2 ± 40.4 µmol/L by macrophages from
control-immunized animals (P < 0.05). This was
attributed to NO toxicity and macrophage death in culture because phase
dark necrotic cells could be observed in the cultures before harvesting
of supernatants (data not shown). This toxicity may have been because
of the in vivo microenvironment before isolation or to the
additional in vitro stimulation. Although addition of 0.5
mmol/L of L-NMMA reduced nitrite production by macrophages from
untreated RE-immunized rats, no significant reduction in spontaneous or
cytokine-stimulated macrophages from L-NAME-treated RE-immunized rats
was observed. This data indicates that treatment with L-NAME in
vivo did not prevent priming of macrophages for NO production, and
that priming in immunized animals was maximal and irreversible. To
assess the possible impact of in vivo macrophage cell death
on overall macrophage numbers and NO production in the inflamed retina,
we analyzed the numbers of infiltrating macrophages in the choroid and
retina during the course of disease. Figure 1b
shows that although
numbers of ED2 tissue-resident macrophages remained constant throughout
the disease, the numbers of ED1 macrophages increased as the disease
progressed.
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. Histological evaluation of posterior
chamber inflammation showed an infiltrate that was predominantly
mononuclear, with focal lesions within the choroid and retina, and SREs
in both groups, consistent with our previous observations in this
model.12,30
It became apparent during scoring that both
the distribution of mononuclear cells within the lesions and the extent
of photoreceptor disruption differed between the groups (Figure 2b)
.
Further qualitative analysis of these
differences was undertaken using immunostaining techniques (Figures 3 and 4)
and where feasible the differences
were quantified (Table 1)
.
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The target tissue in RE-induced EAU is the photoreceptor and rod
outer segment layers of the outer retina. Immunocytochemical analysis
showed that apoptotic cells were not present in the normal retina, and
correspondingly there was strong Bcl2 expression
by cells in the ONL and rod outer segments without Bax protein
expression (Figure 3
; a to d). In EAU many apoptotic cells were seen in
the retina at day 10 in positive control rats immunized with RE, with
Bax-positive cells present throughout the tissue (Figure 3, e and f)
,
In the normal retina NOS2 is not expressed, and in EAU is expressed
only during the early phases of the inflammation.12
By day
10 many NOS2-positive mononuclear cells were present, particularly in
the ONL, rod outer segments, and SRE, and large infiltrates of
CD3+ T cells were identified, particularly in the
INL of the retina around vessels (Figure 3, g and h)
. In contrast, in
the RE-immunized, L-NAME-treated group, far fewer apoptotic cells were
observed (P > 0.0001; Table 1
) despite
equivalent Bax expression (Table 1
; Figure 3, i and j
). NOS2 expression
was also much reduced (P < 0.01, Table 1
) with
only a few cells staining in the rod outer segments and ONL (Figure 3k)
. Abundant CD3+ T cells were now found
throughout all layers of the retina (Figure 3l)
. Nitrotyrosine staining
to detect free radical production and lipid peroxidation as an end
product of NO production was strong in mononuclear cells within the
retina and SRE of untreated animals (Figure 3m)
, but was primarily
absent in the L-NAME group (P = 0.0006; Table 1
). In both groups Fas ligand was expressed by ciliary body epithelium
(Figure 3n)
with diffuse expression in the retina (Figure 3, n and o)
.
Fas ligand was also expressed by a proportion of infiltrating
mononuclear cells (Figure 3p)
, but no significant difference between
the groups was found (Table 1)
. NOS inhibitors have been reported to
increase PGE2 synthesis,34
but very
little positive staining for COX-2 protein was found, and when present
was primarily restricted to corneal epithelial cells in the L-NAME
group (data not shown). No staining for NOS2 or COX-2 protein could be
found in the retinal pigment epithelium (RPE) or choroid of
either group at any of the three time points examined. The cells of the
outer retina remained strongly Bcl2-positive even
in diseased retinas whereas cells in the inner retina and choroid were
only weakly positive or negative in all sections. No significant
differences in expression of Bax or Bcl2 in the
two groups were found (Table 1)
.
Nitrotyrosine Is Formed by ED1+ Monocytes/Macrophages in EAU, but the Cells Undergoing Apoptosis Are Photoreceptors and Fas+ CD2+ T Cells
Dual immunofluorescence studies revealed that the cells positive
for nitrotyrosine were ED1+ monocytes/macrophages
in the SRE and retina (Figure 4, b and c)
, and although equivalent
infiltrates of ED1+ cells were present in the
L-NAME treatment group, very little nitrotyrosine staining was detected
(Figure 4, a and d
; Table 1
). Nitrotyrosine is a toxic NO reaction
product and evidence of NO formation in vivo, it was
therefore surprising that very few of the cells undergoing apoptosis
were ED1+ (Figure 4e)
. Further investigation
revealed that in addition to the photoreceptors,
CD2+ T cells present in the ONL and rod outer
segments, but not the SRE and choroid were also undergoing apoptosis
(Figure 4, f and g)
. Anti-CD2 antibody (clone OX34; IgG2a) was used
here as anti-CD3 (clone IF4; IgM) gave unacceptably high background in
fluorescence studies. The absence of apoptosis in the choroid and SRE
adjacent to the RPE where infiltrating mononuclear cells were strongly
Bax-positive and where there was little Bcl2
staining (Figure 4, h and i)
, suggests other protective mechanisms
could be operating out/with the target tissue. In the outer retina and
rod outer segments, the majority of apoptotic cells were
CD2+, and dual immunofluorescence showed that
they were also Fas + (Figure 4k)
. Triple
immunofluorescence showed that many CD2+ T cells
also expressed Fas ligand, particularly in the control group although
no difference in Fas-ligand expression by
ED1+ macrophages was observed. The diffuse nature
of Fas-ligand staining within the retina made accurate cell counts
difficult, but image analysis of overall percentage of tissue stained
for Fas ligand did not reveal significant differences between the
groups (Table 1)
. Although some apoptotic cells were also Bax-positive,
the majority of photoreceptors undergoing apoptosis were both Fas- and
Bax-negative (Figure 4n)
.
IHSP70 Is Expressed in the Rod Outer Segments and SRE Cells in L-NAME-Treated Animals
What was preventing apoptosis of monocytes and Bax-positive cells
within the choroid? TGF-ß1 is reported to regulate
apoptosis35
via an increased sensitivity or loss of
resistance to apoptosis in the absence of TGF-ß1. Cells expressing
TGF-ß1 could be found in inflammatory infiltrates in the anterior
chamber of most animals with EAU, but staining in the retina was
restricted to the RPE in some inflamed eyes particularly in the control
group (Figure 4j)
. In the remainder of the posterior chamber, staining
for TGF-ß1 was found only in large granular cells (possibly mast
cells) in the choroid and ciliary body of normal and inflamed eyes from
both L-NAME-treated or control groups.
Expression of the inducible 70-kd heat shock protein (iHsp70) has also
been shown to prevent apoptosis in myeloid cells.36
We
hypothesized that induction of iHsp70 might protect ED1 monocytes or
indeed other cells from apoptotic cell death in the retina. Inducible
iHsp70 could not be detected in eyes from normal or negative control
animals immunized with FCA only (data not shown). In
positive-control animals immunized with RE some diffuse expression was
found in the cytoplasm of the rod outer segments and faint expression
was seen in a very few cells in the SRE (Figure 4m)
. This expression
was markedly up-regulated in L-NAME-treated animals, with strong
expression, both in the rod outer segments, cytoplasm, and inflammatory
cells particularly in the outer retina and SRE (Figure 4n
; Table 1
,
P > 0.01). No expression was found in the anterior
chamber, ciliary body, choroid, or RPE. Confocal analysis revealed that
although a few ED1+ cells expressed inducible
iHsp70, the majority of positive cells were either photoreceptors or
other mononuclear cells within the inflammatory infiltrate of
L-NAME-treated animals (Figure 4, o and p)
.
Treatment with L-NAME Significantly Increases the Percentage of CD2+ and CD3+ T Cells in the Target Organ in EAU Despite Continued High Expression of Fas
The overall numbers of T cells (CD2+ or
CD3+) as a percentage of mononuclear cells in the
inner retina (including ganglion cell layer and INL) was the same in
all animals with EAU. Treatment with L-NAME significantly increased the
T cell infiltrate in the outer retina, including ONL and rod outer
segments that constitute the target organ in EAU and in the SRE (Figure 5a)
. Both CD2+ and
CD3+ cells were scored as CD2 may be
down-regulated in the early stages of apoptosis. Although fewer
CD2+ cells were present, particularly in the
L-NAME group, no significant difference between numbers of
CD2+ or CD3+ cells were
found in any of the tissues. The reduction in
CD3+ T cells in both the ONL and the SRE of
control rats compared to treated rats was highly significant
(P < 0.001). The reduction in
CD2+ T cells in the ONL was less marked compared
with control, individual variation within the group reducing
significance to P < 0.05, but reduction in the SRE was
again highly significant (P < 0.001). Figure 5b
shows that levels of Fas were reduced in the inner retina of
L-NAME-treated animals (P < 0.05) but no
differences were found in the outer retina or SRE. In both groups
levels were significantly higher in the SRE compared with the INL with
up to 100% of inflammatory cells positive (P <
0.01). Reduction in T cell numbers correlated with increased expression
of Fas in the SRE of control animals and it was notable that T cells in
the SRE of L-NAME-treated animals were spared despite equivalent high
Fas expression. This may also be linked to the possible reduction in
Fas-ligand expression by CD2+ T cells in
L-NAME-treated animals but the overall trend was for a slight increase
in Fas-ligand expression in the retina of these animals, but these
changes were not significant.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Apoptosis can be induced by a variety of environmental, physical, or chemical stresses. Although alternative pathways must also exist, two major pathways to cell death by apoptosis can be identified and separated into either the mitochondria-mediated pathway with formation of the apoptosome and activation of caspase 9 or the CD95-CD95L (Fas or membrane death receptor)-mediated pathway that activates caspase 8.37 Both pathways then converge at the level of caspase 3 and are subject to regulation at multiple points. The Fas pathway is generally considered to be independent of the mitochondrion, but caspase 8 can cleave Bid, a proapoptotic member of the Bcl2 family, allowing its translocation to the mitochondrion. The mitochondrial pathway is regulated by proteins of the Bcl2 family, whereas the Fas pathway can be regulated by co-stimulatory signals and cytokines. Heat shock proteins are also important regulators, and induction of iHsp70 in particular has been found to protect cells from apoptosis by preventing formation of the apoptosome and inhibiting caspase 3 processing.38 The data presented here suggests that in EAU NO may contribute to AICD via the Fas or death receptor pathway, and to photoreceptor apoptosis through the mitochondrial pathway, but this interpretation requires further investigation.
Inducible NOS and NO have both regulatory and effector functions in the
immune system, and in organ-specific autoimmunity in particular, may
have pathogenic or protective functions giving rise to the concept of
NO as a "double-edged sword" in
autoimmunity.20,27,39,40
This concept is supported
by the conflicting data emerging from studies in animal models using
specific NOS inhibitors or inactivation or deletion of the NOS2
gene.12,16-19,14
The precise mechanisms are unclear but
it is suggested that tissue-specific expression of NOS is important for
regulation of immune responses in the periphery, with a balance between
toxic and homeostatic functions and production of large and small
quantities of NO, respectively.26
The effectiveness of
nonspecific inhibitors of NOS administered orally has been questioned.
In particular, dose rate and route of administration seems to be
crucial.19,41
Access of inhibitors to peripheral tissues
may be incomplete, but in some models a 60% reduction in inflammation
despite only 15% reduction in NOS2 activity has been
reported31
suggesting that comparatively small reductions
in NOS are physiologically relevant. Alternatively, other, as yet
unappreciated effects of the treatment may influence
inflammation. In our model, L-arginine exacerbates EAU, whereas L-NAME,
given in drinking water at 50 mg/kg is well tolerated, reduces urinary
nitrite indicating access to the systemic circulation, shows no obvious
effects on retinal blood vessel permeability to leukocytes, and affords
some protection from photoreceptor loss12
(Figure 2)
.
Immunohistochemistry indicated that L-NAME was also very effective in
reducing NOS2 protein expression in vivo (Figure 3)
. The
capacity of macrophages isolated from L-NAME-treated animals to release
very large quantities of NO without any further stimulation in
vitro, as demonstrated in this study, was therefore unexpected
(Figure 1a)
. The macrophages appeared to be maximally primed, as
further cytokine stimulation had little effect. The reduction in NO
observed was attributed to increased cell death in these cultures
suggesting that other factors, possibly cytokines or growth factors,
were present in the inflammatory lesions that could provide survival
signals to the macrophages in vivo. Alternatively, necrotic
changes to the cells in vivo were affecting cell viability
in vitro. As ED1+ve macrophage
numbers within the retina remained high until resolution of
inflammation at days 16 to 18 (Figure 1b)
it seems unlikely that
macrophage death by necrosis in vivo during early and peak
disease has a significant impact on disease progression. Despite highly
restricted expression of the enzyme during the early and acute phases
of the disease and effective suppression of NOS2 protein by L-NAME, our
previous studies found that NOS2 mRNA expression persisted in the
ocular tissues throughout the time course studied (9 to 21 days). This
peaked on day 12, and coincided with peak expression of IFN- mRNA in
the retina in untreated animals. As macrophages first exposed to
IFN- are subsequently refractory to functional modulation by other
cytokines42
we can hypothesize that in our EAU model,
monocytes/macrophages are primed for NO release despite the presence of
NOS inhibitors. The possibility then arises, that under the influence
of locally produced IFN-, small quantities of NO may continue to be
produced in the lesions despite the absence of NOS2 enzyme, and
underlines the difficulties of controlling NO release by targeting the
NOS genes alone.
In this study we have focused on the earliest stages of EAU (days 9 to 11) with detailed histological examination of tissue from normal (adjuvant-only immunized) and inflamed eyes. L-NAME treatment given from the time of immunization profoundly reduced both NOS2 enzyme expression and nitrotyrosine deposition in monocytes as an end product of peroxynitrite formation in the outer retina. Oxidative damage and peroxynitrite concentrated in the photoreceptors in EAU together with early and restricted expression of NOS2 in Lewis rat EAU by extravasated ED1+ mononuclear cells has also been observed by Zhang and colleagues13 and Wu and colleagues.43 We now show that inhibition of NOS2 expression and reduction of peroxynitrite formation also reduces apoptosis within the retina. A previous study of apoptosis in EAU in Lewis rats has also shown that apoptosis in the retina peaked 2 days after disease onset,28 coinciding with peak expression of Fas-ligand expression by ciliary body epithelium, other tissue-resident cells being Fas ligandlow.44 Fas ligand is normally expressed only by activated inflammatory cells, principally T cells and macrophages, and mediates AICD primarily in CD4+ T cells.45 In the eye and other sites of immune privilege, however, Fas ligand may be expressed on tissue resident cells.1,46 As RE-specific cells are known to traffic to the retina where they apoptose,47,48 our finding that Fas ligand-positive and apoptotic CD2+ T cells showed the same highly restricted tissue distribution as Fas+ apoptotic cells in the rod outer segments and SRE of untreated animals would suggest that AICD is occurring in EAU.
Nitrotyrosine formation indicated that large quantities of both NO and superoxide (O2-) were generated and reacted to form peroxynitrite (ONOO-) that is highly toxic, particularly to neuronal cells such as photoreceptors. Apoptosis as a result of NO-mediated cytotoxicity is mainly exerted at the level of the mitochondrion, and is regulated by members of the Bcl2 family.37,49 Bcl2 cannot prevent apoptosis under all circumstances. Our study, in part, confirmed this as Bcl2 expression in normal retina remained at apparently high levels throughout retinal inflammation in treated as well as untreated rats, and yet severe apoptotic damage to the photoreceptors was observed. Stable expression of Bax protein in rat EAU has been observed before,50 and in this study we could observe no obvious alterations in balance between proapoptotic Bax and anti-apoptotic Bcl2 that control cytochrome c release from the mitochondrion. Indeed we showed that apoptosis was apparently inhibited, despite continued Bax expression in treated animals, suggesting that regulation by other regulatory proteins of the Bcl family or other mechanisms downstream of the mitochondrion may be involved. One notion is that protection in the treated group may be attributed to induction of Hsp 70. Inducible Hsp70 protects against multiple apoptotic stimuli including DNA damage and growth factor withdrawal by inhibiting caspase processing.51 Activity may occur at more than one point downstream of cytochrome c release from the mitochondrion, but upstream of caspase 3 via regulation of the apoptosome.38
Previous studies have shown that T cell apoptosis driven by cytokine deprivation correlates with reduction in Bcl2 expression relative to Bax.52-54 Using only histological methods we could distinguish no differences in staining intensity or proportion of cells positive in either group suggesting that alterations in cytokine availability were not involved in T cell apoptosis. More sensitive and quantitative molecular or biochemical approaches will be required to confirm this hypothesis. In our study, the most dramatic effect of NOS inhibition by L-NAME was the sparing of T cells within the outer retina and rod outer segments that is the target tissue in EAU. This was despite continued high expression of Fas on all inflammatory cells. Up to 100% of cells were Fas+ in both groups, so many Fas+ T cells in the L-NAME-treated retinas were apparently resistant to AICD despite the presence of 10 to 15% Fas ligand-positive cells. How does inhibition of NOS protect T cells from Fas-driven apoptosis? NO mediated apoptosis may be regulated by COX-255 and macrophage derived TGF-ß1 can protect T cells from AICD35,56 but no histological evidence for significant quantities of either was found in this study. The answer may be related to quantitative levels of NO and the redox state of the cells.57,58 High levels of NO up-regulate expression of Fas and stimulate the release of the soluble form of Fas ligand. This would explain the efficient elimination of activated T cells in the outer retina of untreated rats, and inhibitors of NO have been shown to protect T cells from AICD directly through posttranslational or transcriptional modification of Fas ligand.59 Alternatively, low levels of NO may induce protective stress protein responses such as induction of Hsp70 in T cells as observed in this study.
Treatment with L-NAME profoundly reduced NOS2 expression, and might be predicted to inhibit the constitutive isoforms NOS1 and NOS3 with equal efficiency, however persistent expression of NOS2 mRNA observed in our previous study, priming of monocytes/macrophages to release large quantities of NO despite L-NAME treatment shown here, and evidence of incomplete penetrance of oral L-NAME reported by others31 does not preclude continued NO production in vivo in treated animals. Prevention or reduction of O2- formation and so prevention of ONOO- formation may be of more therapeutic relevance than simply reducing excessive NO production.
In conclusion, our study shows that NO seems to modulate both of the major pathways to apoptosis. The mitochondrial-dependent pathway leading to destruction of photoreceptors and the Fas or death receptor pathway leading to T cell apoptosis. Dysregulation of T cell apoptosis clearly contributes to the chronicity of inflammation and defects in the Fas-Fas-ligand pathway of T cell apoptosis have been suggested as a contributing factor in autoimmune disease particularly through defects in tolerance induction.46 Not only are excess activated T cells killed, their removal by local phagocytic cells generates interleukin-10, and supports immune deviation through development of Th2 cells and control of delayed type hypersensitivity (DTH).60 Therefore, strategies to promote T cell apoptosis while regulating monocyte toxicity to prevent photoreceptor damage because of local production of reactive oxygen species may hold the key to nonantigen-specific strategies to control uveoretinal inflammation.
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Acknowledgements |
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Supported by a grant from The Wellcome Trust.
Accepted for publication November 29, 2001.
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