Développement, Vieillissement, et Pathologie de la Rétine, Unité 450 Institut National de la Santé et de la Recherche Médicale, Association Claude Bernard, 75016 Paris, France
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ABSTRACT |
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Bovine retinal
pigmented epithelial (RPE) cells express an inducible nitric oxide
synthase (NOS-II) after activation with interferon- (IFN-
) and
lipopolysaccharide (LPS). Experiments were performed to investigate the
effects of tyrosine kinase inhibitors (genistein and herbimycin A) and
antioxidants [pyrrolidine dithiocarbamate (PDTC) and butyl
hydroxyanisol] on NOS-II induction. The LPS-IFN-
-induced nitrite release was inhibited in a concentration-dependent manner by
these compounds. Analysis by Northern blot showed that this inhibitory
effect correlated with a decrease in NOS-II mRNA accumulation. Analysis
by electrophoretic mobility shift assay of the activation of the
transcription factor nuclear factor-
B (NF-
B) involved in NOS-II
induction demonstrated that LPS alone or combined with IFN-
induced
NF-
B binding. NF-
B activation was not changed by the presence of
tyrosine kinase inhibitors but was totally prevented by PDTC
pretreatment. Immunocytochemistry experiments confirmed the reduction
of the nuclear translocation of NF-
B only by PDTC. Our results
demonstrated the existence in retinal pigmented epithelial cells of
different intracellular signaling pathways in NOS-II induction,
since tyrosine kinase inhibitors blocked NOS-II mRNA accumulation
without inhibiting NF-
B activation. Furthermore, the
LPS-IFN-
-induced NOS-II mRNA accumulation was sensitive to
cycloheximide, suggesting that, in addition to NF-
B, transcriptional
factors that require new protein synthesis are involved in NOS-II
induction.
interferon-; lipopolysaccharide; nitric oxide; transcription
factor
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INTRODUCTION |
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THE ENZYME NITRIC OXIDE (NO) synthase (NOS) transforms L-arginine into NO and L-citrulline in the presence of oxygen, NADPH, tetrahydrobiopterin, flavin mononucleotide, and FAD (12, 28). Three isoforms of NOS have been identified. Two isoforms are continuously expressed and are called constitutive NOS: NOS-I (nNOS) is present essentially in neurons of the central and peripheral nervous system (6), whereas NOS-III (eNOS) is originally localized in the plasmic membrane of vascular endothelial cells (12). These two enzymes are calcium and calmodulin dependent. On the other hand, the inducible isoform, NOS-II (iNOS), is calcium and calmodulin independent and is expressed in different cell types only after a transcriptional activation by endotoxins or cytokines (25, 28, 29). Different roles have been attributed to NO, such as vasorelaxation and neurotransmission (6, 28). NO produced by NOS-II plays a role in immunological defenses (28, 29) as antitumoral, antimicrobicidal, and antiviral agents. NO is also considered to be a mediator of autoimmune and inflammatory responses (25).
In the retina, NOS-II is expressed in retinal pigmented epithelial
(RPE) and Müller glial cells in some pathological disorders (10)
as well as in vitro after stimulation by cytokines (13, 15-17). In
this context, we previously demonstrated that, in bovine RPE cells,
NOS-II was induced by treatment with lipopolysaccharide (LPS) and
interferon- (IFN-
) added together, but not individually (14, 17).
NOS-II regulation is dependent on signal transduction activated by
endotoxin, cytokines, and growth factors (13, 28). These signals are
the results of the activation of serine-threonine or tyrosine kinases
(9, 36). It has been established that LPS interacts with its membrane
receptor and activates mitogen-activated protein (MAP) kinases (38) and
reactive oxygen species, which activate the nuclear transcription
factor nuclear factor-B (NF-
B) (20, 32). The activation of
NF-
B has been implicated in the induction of NOS-II in different
cell types, such as murine macrophages (27, 39), vascular smooth muscle
cells (33), or 3T3 fibroblasts (22). IFN-
interacts with its
receptor and uses cytosolic kinases (e.g., Janus kinases) and
transcriptional factors (e.g., STAT proteins), which activate different
genes (9).
In this study we have attempted to elucidate the mechanisms involved in
the induction of NOS-II by investigating the effect of tyrosine kinase
inhibitors and antioxidants on the induction of the NOS-II mRNA by LPS
and IFN-. We have demonstrated that tyrosine kinase inhibitors and
antioxidants decrease nitrite release and NOS-II mRNA accumulation,
whereas only antioxidants reduced the nuclear translocation of NF-
B
and the formation of NF-
B-DNA complexes (or binding of NF-
B to
DNA).
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MATERIALS AND METHODS |
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Cell cultures. Bovine RPE cells were prepared as previously reported (17) and cultured in DMEM supplemented with 10% FCS (GIBCO BRL, Cergy-Pontoise, France), fungizone (2.5 µg/ml), gentamicin (50 µg/ml), and L-glutamine (2 mM). Cultures were homogenous and contained only RPE cells, characterized by immunohistochemistry with anticytokeratin monoclonal antibody KL-1 (4). Cells at passages 1-5 were used for experiments.
Chemicals, cytokines, and antibodies.
LPS from Salmonella typhymurium,
pyrrolidine dithiocarbamate (PDTC), butyl hydroxyanisol (BHA),
actinomycin D, cycloheximide, genistein, and herbimycin A were obtained
from Sigma Chemical. NF-B p65, p50, and C-rel antibodies were
purchased from Santa Cruz Biotechnology. Bovine
recombinant IFN-
was generously provided by Dr. T. Ramp (Ciba-Geigy,
Basel, Switzerland). The rabbit antiliver NOS-II antibody was a
generous gift of Dr. Ohshima [Centre International de Recherche Sur le
Cancer (CIRC), Lyon, France].
Assay for nitrite.
Confluent RPE cells in 12-well culture plates (averaging
105 cells/well) were treated with
LPS and IFN- in the absence or presence of tyrosine kinase
inhibitors or antioxidants in fresh DMEM-10% FCS. After 72 h of
incubation, nitrite concentration was determined in cell-free culture
supernatants with use of the spectrophotometric method based on the
Griess reaction, as previously described (17).
RNA isolation and Northern blot analysis.
Total RNA was extracted from RPE cells in 90-mm petri dishes treated
for 24 h with LPS and IFN-, in the absence or presence of tyrosine
kinase inhibitors or antioxidants, by lysis of cells in guanidinium
isothiocyanate followed by phenol extraction (8). RNA were denatured by
heating (65°C) in 50% formamide-6% formaldehyde, electrophoresed
(25 µg/lane) in 1% agarose gel containing 6% formaldehyde, and then
transferred to a nylon membrane. An
Sma
I/EcoR I digestion of a plasmid
containing the murine macrophage NOS-II cDNA cloned into pGEM (24)
yielded a 950-bp fragment, which was labeled using the random priming
method. Blots were hybridized overnight at 42°C with this
32P-labeled fragment
(106 cpm/ml) and washed at
60°C in 1× NaCl-sodium citrate (0.15 M NaCl-0.015 M sodium
citrate) and 0.1% SDS. Autoradiography was performed by exposure to
Kodak X-omat film at
70°C. Membranes were stripped and
rehybridized with a full-length glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe as a means of checking the amounts of
RNA added for each sample. Northern blot results were quantified with a
scanning densitometer (Biocom). All values obtained with the specific
NOS-II probe were corrected for slight differences in RNA loading by
normalization to the values obtained with the GAPDH probe.
Western blot analysis.
After treatment with LPS and IFN- with or without PDTC, BHA, or
genistein for 72 h, cells were washed with PBS and scraped in lysis
buffer containing protease inhibitors. Samples were centrifuged, and
after one freeze-thaw cycle, 100 µg of supernatant proteins were
subjected to SDS-PAGE. Proteins were then transferred to an Immobilon
membrane (Millipore, Saint Quentin en Yvelines, France) by
electroblotting. Western blot analysis with use of a polyclonal antibody specific for liver inducible NOS was performed as previously described (14). The intensity of the bands was quantified using densitometric measurements with densitometric software (One Descan, Scanalytics, Billerica, MA).
Electrophoretic mobility shift assay.
RPE cells were pretreated for 2 h with or without PDTC (10 µM),
genistein (90 µM), or herbimycin A (2 µM), washed, and treated for
30 min with LPS (1 µg/ml) and IFN- (100 U/ml) in the presence or
absence of antioxidants or tyrosine kinase inhibitors. Cells were
washed three times in cold PBS, and whole cell extracts were prepared
from cells lysed at 4°C in HEPES (10 mM, pH 7.9) containing 0.1 mM
EDTA, 5% glycerol, 0.4 M NaCl, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 0.1 µg/ml leupeptin, 4 µg/ml aprotinin, 1.5 µg/ml pepstatin, 1 µg/ml chymostatin, and 2 µg/ml antipain. After 15 min of centrifugation at 37,000 rpm, the
supernatants were stored at
80°C. Protein concentrations of
extracts were determined by the Bradford analysis (Bio-Rad).
Double-stranded consensus oligonucleotides for NF-
B
(5'-CTAGACAGAGGGGATTTCCGATTCCGAGAGGT-3'; Genset, Paris,
France) were end labeled by Klenow polymerase (Appligene, France) and
-[32P]CTP. The
binding reactions were carried out by incubating extracts (25 µg)
with
-32P-labeled NF-
B
consensus oligonucleotides (104
cpm) in a buffer containing 40% Ficoll, 200 mM HEPES, pH 7.5, 600 mM
KCl, 20 mM dithiothreitol, 0.1% NP-40, 1 mg/ml BSA, and 10 µg of
salmon sperm DNA for 20 min at room temperature. For supershift and
competition binding experiments, antiserum against p50, p65, or C-rel
NF-
proteins or a 100-fold molar excess of unlabeled oligonucleotide
was added to the reaction mixture along with the labeled probe. The
reaction mixture was loaded onto a 5% polyacrylamide gel made in
0.5× Tris borate-EDTA (25 mM Tris, 44 mM borate, 0.5 mM EDTA) and
electrophoresed at 200 V at room temperature.
Immunocytochemistry.
RPE cells were plated onto Labtek slides (Nunc, PolyLabo) and
pretreated for 2 h with or without antioxidants or tyrosine kinase
inhibitors. Fresh medium (DMEM-10% FCS) containing LPS (1 µg/ml) and
IFN- (100 U/ml) was then added to cell cultures. After a 30-min
incubation, coverslips were washed three times with PBS, dried for 15 min at room temperature, and fixed for 30 min in 4% paraformaldehyde.
After they were washed with PBS, the cells were permeabilized with
methanol for 5 min at
20°C, washed, and saturated with
PBS-5% skim milk for 30 min. Coverslips were then incubated for 1 h at
room temperature with the rabbit anti-p65 antibody (diluted 1:50)
in PBS-1% skim milk. Coverslips were washed five times with
PBS-1% skim milk, incubated for 1 h at room temperature with
biotinylated sheep anti-rabbit IgG (Amersham; 1:100 in PBS-1% skim
milk), and then incubated with extravidin-fluorescein isothiocyanate
(1:100 in PBS-1% skim milk). Coverslips were extensively washed in PBS
and, during the last wash, treated for 5 min with the fluorescent
nuclear stain 4,6-diamidino-2-phenylindole. Sections were mounted in
Fluoprep and viewed under a Leitz Aristoplan microscope equipped with
an epi-illuminator mercury vapor arc lamp (HBO) and filters for
rhodamine and 4,6-diamidino-2-phenylindole fluorescence.
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RESULTS |
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Inhibition of nitrite release and NOS-II mRNA accumulation by
antioxidants.
We first studied the effect of two different types of antioxidants,
PDTC and BHA, on nitrite production by LPS-IFN-. PDTC and BHA
decreased the nitrite production after 72 h of stimulation in a
dose-dependent manner with IC50
values of 0.4 and 70 µM and a maximal inhibitory effect of 5-10
and 250 µM, respectively (Fig. 1). Trypan blue viability test
confirmed that the inhibitory effect of PDTC and BHA was not due to a
cytotoxic effect of these compounds (data not shown). The expression of
NOS-II mRNA was investigated by Northern blot to determine whether
inhibition of nitrite release corresponded to a decrease in NOS-II mRNA
accumulation. Total RNA was extracted from RPE cells after 24 h of
treatment, corresponding to the maximal expression of NOS-II mRNA (14).
Only one detectable mRNA signal at 4.4 kb was detected after
LPS-IFN-
treatment, whereas in the unstimulated RPE cells, NOS-II
mRNA was not detectable (Fig. 2), as we
recently reported (14). Figure 2 shows that antioxidant treatment
inhibited to a large extent the expression of NOS-II mRNA in RPE cells
stimulated with LPS-IFN-
. This decreased mRNA accumulation
correlated with NOS activity, which was evaluated by the nitrite level
in the culture medium (Fig. 2).
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Inhibition of nitrite release and NOS-II mRNA accumulation by
tyrosine kinase inhibitors.
To establish the transduction mechanisms involved in NOS-II induction
and, in particular, the role of tyrosine kinases, we have studied the
effect of two different tyrosine kinase inhibitors: genistein and
herbimycin A. Incubation with either compound for 72 h diminished
nitrite production measured after stimulation with LPS-IFN- (Fig.
3). The effect of genistein was superior to
that of herbimycin A: maximal inhibition of LPS-IFN-
-induced nitrite
production was only 40% with 2 µM herbimycin A. Elevating the
concentration of herbimycin A (>5 µM) did not increase inhibition but led to cell toxicity, as evaluated by trypan blue viability test
(data not shown). The presence of genistein with LPS-IFN-
induced a
decrease of NOS-II mRNA accumulation that correlated with the activity
of NOS as determined by release of nitrite into culture media (Fig.
4). The same decrease of NOS-II mRNA could be observed with herbimycin A (data not shown). In the presence of
0.25% DMSO, which was used to make genistein soluble in the aqueous
media, there was no decrease in NOS-II mRNA accumulation due to
LPS-IFN-
(Fig. 4), demonstrating that DMSO did not play a role in
the inhibition due to genistein.
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Inhibition of NOS-II protein accumulation by antioxidants and
tyrosine kinase inhibitors.
The expression of RPE NOS-II protein was investigated by Western blot
analysis after 72 h with LPS-IFN- with or without PDTC, BHA, and
genistein. As we previously reported (14), a band at 130 kDa was
detected in LPS-IFN-
-treated cells but not in untreated cells (data
not shown). Densitometric analysis revealed that simultaneous treatment
with PDTC or BHA greatly decreased the 130-kDa protein signal by 88 and
81%, respectively, compared with LPS-IFN-
(Table 1). Furthermore, a decrease of 84% of the
signal intensity was observed with the addition of genistein (Table 1).
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Activation of NF-B binding activity by
LPS-IFN-
in RPE cells and effect of pretreatment with
PDTC or tyrosine kinase inhibitors.
Because PDTC, described as an inhibitor of the transcription factor
NF-
B (32), inhibited NOS-II mRNA accumulation, we used electrophoretic mobility shift assay (EMSA) to investigate the involvement of NF-
B in the induction of NOS-II. Bovine RPE cells were stimulated for 30 min with LPS alone or LPS-IFN-
with or without tyrosine kinase inhibitors or antioxidants. With LPS alone, two
bands appeared that were absent in unstimulated cells (Fig. 5, lane
2). The addition of excess unlabeled consensus
oligonucleotide completely prevented complex formation
(lane 8), demonstrating the
specificity of the DNA-protein interaction. Stimulation with IFN-
alone did not induce the complexes (lane
3), and the addition of IFN-
did not seem to
increase the intensity of the signal induced by LPS
(lane 4). When cells were pretreated
with PDTC (lane 7) and stimulated
with LPS-IFN-
, a marked decrease of the intensity of both bands was
observed. However, the addition of inhibitors of tyrosine kinases had
no significant effect on NF-
B activation (lanes
5 and 6). The use of
antibody against p65 supershifted both complexes induced by LPS-IFN-
stimulation, whereas antibody against p50 subunit supershifted only the
lower complex (Fig. 6). Antibody directed
against C-rel failed to supershift the complexes (data not shown).
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Differential effects of PDTC and genistein on the nuclear
translocation of p65 subunits induced by LPS-IFN-.
To confirm the translocation of NF-
B to the nucleus after
stimulation of the cells, we performed immunocytochemistry with antibody to NF-
B p65, which was present in both complexes observed by EMSA. In unstimulated cells, cytoplasmic staining was prominent (Fig. 7). When cells were stimulated with
LPS or LPS-IFN-
, which activated NF-
B by EMSA, we observed an
important nuclear staining (Fig. 7). Addition of IFN-
alone, which
failed to activate NF-
B, did not induce translocation of p65 in the
nucleus (data not shown). The presence of genistein did not impair
translocation of p65 induced by LPS-IFN-
. However, pretreatment with
PDTC blocked translocation of p65 to the nucleus (Fig. 7), confirming
its effect on NF-
B binding activity.
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Accumulation of NOS-II mRNA required protein synthesis.
When cycloheximide at 2 µg/ml was added simultaneously with
LPS-IFN-, a marked decrease of NOS-II mRNA was observed (Fig. 8), suggesting that NOS-II mRNA
accumulation was dependent on protein synthesis. This inhibitory effect
was not due to a cytotoxic effect of cycloheximide, as demonstrated by
trypan blue viability test (data not shown).
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DISCUSSION |
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We have demonstrated that induction of NOS-II activity in RPE cells by
LPS-IFN- can be modulated by antioxidants and tyrosine kinase
inhibitors. This study of NOS-II regulation allows elucidation of some
of the different regulatory elements involved in the induction of
NOS-II: tyrosine kinase(s) and oxygen free radicals, which seem to act
at the level of NOS-II transcription.
A decrease of nitrite release and NOS-II mRNA accumulation was observed
with genistein and herbimycin A, as previously observed in different
cell types, such as murine macrophages (11, 25). The use of these
inhibitors demonstrates the important role of tyrosine kinase(s) in the
induction of NOS-II, although they are not specific for a given protein
kinase. Because JA kinases and MAP kinase kinase (MEK)/MAP kinases are
activated by IFN- and LPS, respectively, in different cell types (9,
36), it would be interesting to determine the potential involvement of
these kinases in the induction of NOS-II by LPS-IFN-
in bovine RPE cells. In this context, preliminary experiments showed that PD-98059, a
specific MEK-1 inhibitor, blocked LPS-IFN-
-induced nitrite release,
suggesting the role of the MEK/MAP kinases in NOS-II induction.
Concerning the JAK-STAT pathway, the involvement of STAT-1 and
interferon regulatory factors (IRF-1 and IRF-2) is under investigation
by EMSA studies.
A decrease of nitrite release and NOS-II mRNA production was also
obtained by treatment with two antioxidants (PDTC and BHA). Some
differences between these components were observed, since PDTC was more
potent than BHA. This difference might be related to the dual ability
of PDTC to act as a metal chelator and as a strong antioxidant (34).
Bovine RPE cells react similarly to other cell types in which PDTC is
often described as an inhibitor of NOS-II induction (5, 23, 30, 31,
39). Our results reported some differences in terms of the percentage
of inhibition by antioxidants and genistein between nitrite release or
NOS-II protein level and mRNA accumulation. The uptake of antioxidants and genistein into cells could be a slow process. Because transcription of NOS-II mRNA occurred within a few hours after stimulation with LPS-IFN-, in contrast to NOS-II protein translation and nitrite release, which required >24 h, it would be envisaged that effective concentrations of antioxidants or genistein intracellularly could be
not totally attained. This phenomenon could explain the differences in
the extent of inhibition between mRNA accumulation and NOS-II protein
level or nitrite release. However, we cannot exclude that these
compounds could also have an inhibitory effect on NOS-II induction at a
posttranscriptional level, as suggested in adenocarcinoma EM-T6 cells
with antioxidants (7). Indeed, the large inhibition observed by Western
blot on NOS-II protein level with antioxidants and genistein seems to
confirm an effect of these compounds on NOS-II translation or on NOS-II
mRNA stability.
The presence of NF-B binding sequences in the NOS-II promoter (40)
and the requirement of nuclear translocation of NF-
B for NOS-II
induction (39) indicate the importance of this factor in NOS-II
induction. NF-
B is not synthesized de novo in the cell but is
present in the cytoplasm in an inactivated form complexed by the
protein I
B-
(2). Stimuli activate NF-
B by phosphorylating I
B-
on serines in its regulatory
NH2 terminus, an event leading to
I
B conjugation with ubiquitin and subsequent proteasome-mediated degradation (37). Another possibility is that I
B-
is
phosphorylated on tyrosine, releasing NF-
B, but then not degraded
(18).
In bovine RPE cells we demonstrated that LPS, but not IFN-, induced
the translocation of NF-
B into the nucleus and the formation of
DNA-NF-
B complexes observed by EMSA. Furthermore, IFN-
, which is
required for NOS-II induction, did not seem to potentiate LPS-induced NF-
B activation. By using the tyrosine kinase inhibitors and antioxidants, we demonstrated that nuclear translocation of NF-
B and
formation of DNA-NF-
B complexes involved the action of oxygen free
radicals but that tyrosine kinase(s) was not necessary for NF-
B
activation. This latter result was surprising, because activation of
tyrosine kinases on LPS stimulation (38) has been reported and the
involvement of this pathway in NF-
B activation has been demonstrated
(19). However, Tetsuka et al. (35) recently reported that activation of
tyrosine kinase may not be required for interleukin-1
-induced NF-
B activation in rat mesangial cells. It is likely that, in bovine
RPE cells, LPS can activate NF-
B independently of tyrosine kinase
activation. However, we cannot exclude that the inhibitors used in this
study failed to block activation of some specific tyrosine kinase(s).
An important finding in the present study is that although LPS alone
induced binding of NF-B, it failed to induce NOS-II mRNA and nitrite
production. However, inhibition of NF-
B binding with PDTC partially
prevented NOS-II induction by LPS-IFN-
treatment. These results
indicate that activation of NF-
B may not be sufficient to induce
NOS-II in bovine RPE cells but is required for this process. NF-
B
has been reported to be necessary, but not sufficient, for the
induction of NOS-II in other cell types (1, 3). Our study suggests the
existence of different intracellular signaling pathways in NOS-II
induction in RPE cells, since tyrosine kinase inhibitors blocked NOS-II
mRNA accumulation without affecting NF-
B binding. We propose that
the tyrosine kinase pathway converges with the NF-
B pathway,
downstream of the activation of NF-
B, for NOS-II induction.
Furthermore, the LPS-IFN-
-induced NOS-II mRNA accumulation was
sensitive to cycloheximide. This result is similar to those generally
obtained in cytokine-NOS-II induction models (28) and suggests that, in
addition to NF-
B, transcriptional factors that depend on de novo
protein synthesis are required for NOS-II expression. Effectively,
induction of NO production in RPE cells requires both LPS and IFN-
.
In this context, IRF-1, which is transcriptionally activated on IFN-
stimulation, has been involved in NOS-II induction (26). Furthermore,
in mice genetically deficient in IRF-1, NOS-II induction is markedly
decreased (21). The role of IRF-1 and of its repressor IRF-2 in the
induction of NOS-II by LPS and IFN-
in bovine RPE cells is under
investigation in our laboratory.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. M. Cunningham (Hematology-Oncology Division, Harvard Medical School, Boston, MA) for the kind gift of murine macrophage NOS-II cDNA, Dr. H. Oshima (CIRC, Lyon, France) for the kind gift of antiserum against NOS-II, F. Régnier-Ricard for technical assistance, Dr. S. Michelson for critical reading, and H. Coet for photographic work.
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FOOTNOTES |
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This work was supported by grants from Association Française Retinitis Pigmentosa.
Address for reprint requests: O. Goureau, Développement, Vieillissement, et Pathologie de la Rétine, U450 INSERM, 29 rue Wilhem, 75016 Paris, France.
Received 1 December 1997; accepted in final form 3 April 1998.
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